key: cord- -ikq rm authors: rasmuson, j.; andersson, c.; norrman, e.; haney, m.; evander, m.; ahlm, c. title: time to revise the paradigm of hantavirus syndromes? hantavirus pulmonary syndrome caused by european hantavirus date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: ikq rm hantaviruses have previously been recognised to cause two separate syndromes: hemorrhagic fever with renal syndrome in eurasia, and hantavirus pulmonary syndrome (hps) in the americas. however, increasing evidence suggests that this dichotomy is no longer fruitful when recognising human hantavirus disease and understanding the pathogenesis. herein are presented three cases of severe european puumala hantavirus infection that meet the hps case definition. the clinical and pathological findings were similar to those found in american hantavirus patients. consequently, hantavirus infection should be considered as a cause of acute respiratory distress in all endemic areas worldwide. the present paradigm is that hantaviruses in the americas cause hantavirus pulmonary syndrome (hps), while hemorrhagic fever with renal syndrome (hfrs) is caused by hantaviruses present in eurasia [ ] [ ] [ ] . hantaviruses associated with hfrs and hps are primarily transmitted by inhalation of viral particles shed by infected rodents [ , ] . in both syndromes there is a local immune reaction in the lungs, mainly in terms of a cd + t lymphocyte response [ , , ] . another important feature is endothelial dysfunction and capillary leakage [ ] [ ] [ ] ] . after the prodromal phase, including fever, nausea, myalgia and headache, patients with hfrs commonly develop renal failure whereas in hps the kidneys are often spared and instead the patient frequently presents with severe cardiopulmonary dysfunction [ , , ] . although usually less severe and sometimes overlooked, pulmonary symptoms are common in european hfrs caused by puumala virus (puuv). frequent clinical findings include cough, dyspnoea, interstitial lung infiltrates, pleural effusion and impaired pulmonary function [ ] [ ] [ ] . pronounced lung involvement in hfrs has previously been reported [ ] [ ] [ ] [ ] . however, none of those reported patients had a fatal outcome that could be attributed to the acute infection, and there is no description of histopathological findings. our hypothesis is that also european hantaviruses can cause hps. the aim of this study was to investigate whether hfrs patients with severe cardiopulmonary distress fulfil hps criteria and have similar clinical and pathological features. in a prospective study, during the latest hantavirus outbreak in in the county of västerbotten, sweden [ ] , hfrs patients with cardiopulmonary failure and need of invasive assisted ventilation, i.e. intubation and mechanical ventilation, were selected. we identified three patients, two of which had fatal outcome and one survived. the two patients who died both underwent autopsy three days post-mortem. organ samples were investigated using immunohistochemistry to describe the immunological response and detect presence of viral antigen. quantitative real-time rt-pcr was used to detect viral rna in organ samples, plasma and bronchoalveolar lavage [ ] . for defining hps we used the case definition criteria published by the u.s. centers for disease control and prevention (cdc) [ ] . the project was approved by the research ethics committee of umeå university. informed consent was obtained either from the patient, or a close relative when not possible. a -year-old woman was admitted to the intensive care unit (icu) with acute respiratory distress. she was a lifelong non-smoker living with her husband in a rural area, and had a medical history of hypertension and type ii diabetes. the day prior to admission she fell ill with malaise and fever. nausea, vomiting and pronounced shortness of breath ensued. the initial findings at admission included fever, tachycardia, tachypnoea, hypoxia and somnolence. laboratory findings during hospitalisation indicated coagulopathy, elevated levels of lactate dehydrogenase (ldh), and development of renal failure (table ). there was release of cardiac enzyme troponin t (peak value . ; normal value < . μg/l), indicating myocardial tissue damage. lung computer tomography (ct) on admission revealed pronounced diffuse bilateral interstitial infiltrates with pulmonary oedema, dependant atelectasis, and moderate pleural effusions (fig. ) which were later drained (> ml). echocardiography showed inferior hypokinesia, moderate mitral valve insufficiency, normal sized left ventricle and atrium, and systolic pulmonary arterial pressure estimated at mm hg. despite non-invasive positive pressure respiratory support and furosemide, her respiratory distress progressed and she was intubated and mechanically ventilated on the first hospital day. the preliminary diagnosis was acute respiratory distress syndrome of uncertain cause, and she was treated with broad-spectrum antibiotics and corticosteroids without improvement. vasopressor and inotropic support was required to maintain adequate circulation. repeated echocardiogram showed no overt signs of cardiac failure, though several days into her illness the systolic pulmonary arterial pressure increased to > mm hg. maximal inspiratory pressures ( cm h o) were required to maintain minimally adequate oxygenation. she suffered a pneumothorax, and received a large bore thoracostomy with negative pressure drainage. the patient developed multiple organ dysfunction engaging the central nervous, respiratory, cardiovascular, renal and coagulatory systems, and she died after days of icu care. puuv serology was initially negative, but seroconversion occurred during the first week with development of positive immunoglobulin m (igm) and igg. no puuv rna could be detected in serum or bronchoalveolar lavage fluid, sampled two days after onset of disease. at autopsy puuv rna was detected in lung tissue, but not in samples from heart, brain, spleen and liver. sequencing of the puuv rna from lung tissue showed that it was homologous to puuv strains circulating in northern sweden. no puuv antigen could be detected in the tissue by immunohistochemistry using puuv specific monoclonal antibody. relevant bacterial cultures were all negative. notably, three weeks prior to icu admission the patient had sought medical treatment at our clinic for a urinary tract infection caused by e. coli. in the serum collected at that time puuv rna ( , copies/ml) was later detected. wbc white blood cell count (normal range . - . × /l), tpc thrombocyte particle count (normal range for women - and for men - autopsy revealed oedematous and atelectatic lungs with no normal aerated tissue. pulmonary histopathological features consisted of diffusely oedematous parenchyma with interstitial and intraalveolar fibrosis. the alveoli contained exudates of fibrinous fluid, high number of alveolar macrophages (cd + ), proliferative epithelial cells and thick septa but without characteristic hyaline membranes. interstitial mononuclear cell infiltrates were common, consisting mainly of cd + t lymphocytes whereof a vast majority was cd + . many mononuclear cells were expressing cytotoxic markers granzyme b and t cell restricted intracellular antigen- , tia- (fig. ) . in pulmonary vessels, focal thrombosis was evident. microscopy of the heart showed thrombosis in small vessels and focal massive infiltrates of granulocytes and macrophages. in the brain there was focal vasculitis, perivascular infiltration of cd + t lymphocytes and non-occlusive thrombosis. infarctions were seen in the brain, lungs and spleen. notably, the kidneys had no prominent inflammatory infiltrates. a -year-old woman was admitted to the icu with acute respiratory distress and circulation insufficiency. besides a history of waldenstrom's macroglobulinemia that had not required active treatment, she had no history of health problems, infections or recent hospitalisations. she was a lifelong non-smoker, lived in a rural home with her husband, and handled firewood for home heating. four days prior to admission the patient noted fever, chills, dyspnoea with dry cough and diarrhoea. on the day of admission, these symptoms were more severe, and an ambulance had been called because of syncope. her ability to oxygenate deteriorated progressively during the first hospital day, and she was intubated and mechanically ventilated. chest x-ray on admission showed bilateral diffuse lung infiltrates and signs of interstitial oedema. large bilateral pleural effusions were noted, and > ml were drained. echocardiographic examination identified normal left ventricular wall motion, and no signs of structural abnormalities or of pulmonary hypertension. thin-cut ct images of the lungs on the third icu day showed diffuse bilateral alveolar and interstitial infiltrates with dependent consolidation (fig. ) . the clinical course during the first seven days was dominated by respiratory insufficiency requiring maximal ventilatory support with high levels of inspired oxygen, as well as circulatory shock requiring treatment with vasopressor and inotropic infusions. other important clinical aspects included coagulopathy with diffusely spread petechiae, progression of renal failure with anuria requiring dialysis, and elevated levels of ldh (table ) . she was treated presumptively for bacterial pneumonia and sepsis with a series of broad spectrum antibiotics and corticosteroids, without apparent response. hantavirus infection was verified with the detection of puuv rna in plasma ( , copies/ml) on the day of admission, while igm and igg were negative. seroconversion with positive igm and igg occurred two and seven days later, respectively. consecutive plasma samples were analysed for puuv rna with declining viral copy numbers until negative days post onset of fig. chest ct-scans of two european patients with hantavirus pulmonary syndrome. the examination showed pronounced diffuse bilateral interstitial infiltrates with pulmonary oedema, together with bilateral dependent atelectasis and moderate pleural effusions in patient (a) and diffuse bilateral alveolar and interstitial infiltrates with dependent consolidation in patient (b) fig. immunohistochemistry results in lung sections from two fatal cases of hantavirus pulmonary syndrome. the findings were condensed oedematous pulmonary parenchyma with alveolar fibrinous exudate and infiltrates of mononuclear cells (a), whereof a vast majority were cd + t lymphocytes (b), and many were holding granules containing granzyme b (c) and t cell restricted antigen- , tia- (d); this immunophenotype is characteristic of activated cytotoxic t lymphocytes. in contrast, cd + helper t lymphocytes (e) were uncommon. viral antigen was detected in capillary vascular endothelium (f) and in mononuclear cells, here represented by a monocyte (f; inset), using puumala hantavirus nucleocapsid protein monoclonal antibody (a c , progen biotechnik gmbh, heidelberg, germany). for viral antigen, lung samples from two non-hantavirus patients were used as negative controls (data not shown). panels (patient in a-e; original magnification, x ; and patient in f; original magnification, x ) display lung sections from paraffin embedded material. staining was performed using hematoxylin and eosin (a), and immunoperoxidase technique (b-f) disease (data not shown). puuv rna was found in bronchoalveolar lavage fluid ( , copies/ml) nine days after onset of disease. bacterial cultures were all negative. the patient remained ventilator-dependent for days, but was finally extubated. she developed critical illness myopathy and needed six weeks in-hospital rehabilitation. at follow-up six months later, she complained of muscle ache during exercise and lowered general fitness, but was steadily improving. a -year-old male construction worker was admitted to the icu with acute respiratory distress, confusion and hypotension. he was a current and long-time smoker with a medical history of mild chronic obstructive pulmonary disease and hypertension. three days prior to admission he had fallen ill with fever, chills, diarrhoea, dry cough, and dyspnoea. in the emergency room, physical examination was notable for fever, somnolence, tachycardia and hypotension. arterial blood gas analysis on room air revealed hypoxia and respiratory alkalosis. the patient was taken to the icu and therapy was started with broad spectrum antibiotics due to suspicion of pneumonia and sepsis. as with the other patients, coagulopathy, increased ldh levels, and renal failure were detected (table ) . initial bedside chest x-ray showed no findings to explain the respiratory distress. during the two days icu course, the patient complained mostly of dyspnoea. he received supplemental oxygen and non-invasive ventilator support initially, but was eventually intubated and mechanically ventilated. ct findings which included only the lower lung lobes revealed bilateral pleural effusions and bibasilar atelectasis (data not shown). during the first day blood pressure was maintained with i.v. fluid, but on the second day his circulatory condition rapidly deteriorated. despite massive efforts with i.v. fluid, corticosteroids and vasoactive drugs, the patient died in refractory circulatory shock. puuv serology on the first hospital day was igm positive, while igg was negative. the patient had , copies of puuv rna/ml in plasma. bacterial cultures were all negative. at autopsy, puuv rna was detected in samples from lungs, heart, liver, kidney, brain and spleen. the organs were examined for viral antigen, which was found in the vascular endothelium and in mononuclear cells (fig. ) . post mortem examination showed that pulmonary architecture was preserved but with oedema and focal non-occlusive thrombosis. similar to patient , there were pulmonary infiltrates of lymphocytes with the same immunophenotype as described (data not shown). kidneys showed no prominent inflammation. growing evidence show that there are similarities between hfrs and hps, as both syndromes can give rise to hemorrhagia, renal impairment and cardiopulmonary dysfunction, which have been attributed to thrombocytopenia and capillary leakage [ , , , , , , ] . severe pulmonary involvement has not been generally perceived to be a significant feature of hfrs. previously, respiratory symptoms were commonly attributed to fluid overload as a result of renal failure. however, increasing evidence show that lung and heart involvement is common during the acute phase of hfrs [ , , , ] . concerning the cases of european hantavirus infection in our present report, there was only mild or no renal impairment at the time of admission, whereas the respiratory involvement was early and severe, consistent with acute respiratory distress syndrome (ards), fulfilling criteria of hps according to cdc case definition [ ] . as described in hps, the three reported patients suffered from cardiovascular dysfunction, requiring treatment with inotropic and vasopressor drugs [ ] . autopsy results support the cause of death to be cardiopulmonary distress due to hantavirus infection. the lungs of both the deceased patients were oedematous and contained mononuclear cell infiltrates with predominantly cd + t lymphocytes, as described in hps patients [ , ] . many cells expressed granzyme b and tia- , indicating a cytotoxic activity, which could further be supported by high levels of ldh. we chose to demonstrate the immune response in a non-smoker's lungs (patient ), but a similar picture was seen in patient . in the first two patients, the igm response was delayed, while the quantitative real-time rt-pcr for puuv rna was positive, suggesting it as a valuable tool to hasten the aetiological diagnosis [ ] . notably, in patient puuv rna was found in serum from three weeks prior to the acute onset of disease, that is, in incubation phase and consistent with hantavirus incubation time ranging from one to four weeks [ , ] . pulmonary involvement is well documented in european puumala hantavirus infection. from a single outbreak in our county, we report on three cases of life-threatening hantavirus pulmonary syndrome. arguably, additional cases are likely to either go undiagnosed or unreported. a clinical implication of this observation is that hantavirus infection should be included as a differential diagnosis for patients with febrile illness and acute respiratory distress of uncertain cause in endemic areas in eurasia. conflict of interest the authors declare that they have no conflict of interest. open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. hantavirus pulmonary 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syndrome in chile acknowledgements irene eriksson and ingrid gustafsson are greatly acknowledged for their skilled technical assistance. this work was supported with grants from the swedish heart lung foundation, the heart foundation of northern sweden, the county councils of northern sweden, the county council of västerbotten, and the medical faculty of umeå university. key: cord- -deq tv d authors: bergström, t.; alestig, k.; svennerholm, b.; horal, p.; sköldenberg, b.; vahlne, a. title: neurovirulence of herpes simplex virus types and isolates in diseases of the central nervous system date: journal: eur j clin microbiol infect dis doi: . /bf sha: doc_id: cord_uid: deq tv d herpes simplex virus (hsv) isolates derived from the central nervous system of ten patients with hsv- -induced encephalitis, one patient with multiple sclerosis, and patients with hsv- -induced meningitis were investigated for neurovirulence by assaying the ld( ) after nose and intracerebral (i.c.) inoculation of mice. hsv- encephalitis strains were significantly more virulent after nose inoculation (i.e. neuroinvasive) when compared with hsv- isolates from patients with oral lesions only, whereas hsv- meningitis strains were significantly more virulent after i.c. inoculation when compared with hsv- isolates from patients with genital lesions only. no correlation between high neurovirulence (defined as low ld( ) for both routes of infection) and replication in cell cultures of neuronal and non-neuronal cell lines was found, but the weakly neurovirulent hsv- strain isolated from a patient with multiple sclerosis gave low replication yields. after nose inoculation, a highly neuroinvasive hsv- laboratory reference strain replicated to high titers in nose tissue, the trigeminal ganglia and brainstem, while a strain with low neuroinvasiveness but high i.c. virulence replicated less well in the brainstem. neuroinvasiveness of the virus strain might be one factor of relevance in the pathogenesis of hsv- encephalitis in man. t. bergstr/ m ., k. alestig , b. svennerholm , p. horal , b. sk/ denberg , a. vahlne t herpes simplex virus (hsv) isolates derived from the central nervous system of ten patients with hsv-l-induced encephalitis, one patient with multiple sclerosis, and patients with hsv- -induced meningitis were investigated for neurovirnlence by assaying the lds after nose and intracerebrai (i.c.) inoculation of mice. hsv- encephalitis strains were significantly more virulent after nose inoculation (i.e. neuroinvasive) when compared with hsv- isolates from patients with oral lesions only, whereas hsv- meningitis strains were significantly more virulent after i.c. inoculation when compared with hsv- isolates from patients with genital lesions only. no correlation between high neurovirulence (defined as low lds for both routes of infection) and replication in cell cultures of neuronal and non-neuronal cell lines was found, but the weakly neuruvirulent hsv- strain isolated from a patient with multiple sclerosis gave low replication yields. after nose inoculation, a highly neuroinvasive hsv- laboratory reference strain replicated to high titers in nose tissue, the trigeminal ganglia and brainslem, while a strain with low neuroinvasiveness but high i.c. virulence replicated less well in the brains|era. neuroinvasiveness of the virus strain might be one factor of relevance in the pathugenesis of hsv- encephalitis in man. when infecting the central nervous system (cns) herpes simplex virus type (hsv- ) can cause focal, necrotizing encephalitis, this virus being considered the most important cause of acute encephalitis in immunocompetent patients ( ) ( ) ( ) . the sporadic occurrence of this disease and the fact that as yet no pathogenetic factor has been singled out as playing a decisive role might indicate that in a particular patient a number of unfortunate circumstances concurr. of relevance to the pathogenesis could be predisposing factors in the patient himself, the virus dose, or the site of replication at primary infection, as well as neurovirulent properties of the virus. data have been presented in support of hsv- strain variability of neurovirulence in mouse models ( - ) and in rabbits ( ). attempts have been made to pinpoint the hsv- genes regulating neurovirulence by the use of recombinant strains ( - ) or deletion mutants ( ) volved. however, whether the pathogenesis in hsv- encephalitis in man is influenced by the neurovirulence of hsv- strains is still not known. herpes simplex virus type (hsv- ) is a rare cause of encephalitis in man ( ) , and in most cases reported the patients have been immunocompromised ( , ) . instead, hsv- may sometimes induce a serous meningitis when hsv- infection spreads from the genital tract to the cns ( , ) . symptoms of meningitis are far more common in primary hsv- infection, where the surface area of involvement is larger and the duration of virus shedding longer, as compared with secondary hsv- infection (i.e. the first episode of hsv- infection preceded by an hsv- infection) ( ) . these findings indicate possible relevance of virus load from the periphery in the development of hsv- meningitis, but the potential virulence properties of hsv- strains causing meningitis have not yet been described. we used a mouse model to assay the ld after infection by different routes with cns-derived hsv- isolates from ten patients with encephalitis and one patient with multiple sclerosis, and hsv- isolates from patients with meningitis, in comparison with clinical reference strains isolated from oral lesions (hsv- ) and genital lesions (hsv- ) in patients without neurological symptoms, and with known laboratory reference strains. the mouse is permissive to cns infections with both hsv- and hsv- , also by peripheral routes ( ) . it has been reported that the neurovirulence of recombinant mouseadapted poliovirus strains in this animal is in concordance with neurovirulence in man ( ) . virus isolation. clinical samples of brain material, cerebrospinal fluid (csf), and secretion from oral and genital lesions were inoculated in cell cultures of green monkey kidney (gmk ah- cells) or veto cells. isolates were typed as hsv- or hsv- by eia using monoclonal antibodies ( , ) . virus strains. ten hsv- strains were isolated from brains of patients with encephalitis (hsv- enc). eight of these strains were derived from biopsies (patients no. - , table ) performed during a trial of antiviral treatment of hsv encephalitis ( ). all biopsies were taken before the commencement of antiviral treatment. in three of these patients (no. , and ), serological data were consistent with a probable primary hsv- infection (low level or absence of hsv- igg antibodies and presence of hsv lgm antibodies in the first serum sample) at the time of the encephalitis, while four had serologic evidence of reactivated hsv- infection. in one patient, serological data were missing. two hsv- strains were isolated from brain material obtained at autopsy of two patients who died of encephalitis before receiving antiviral treatment (patients no. and , table i ) and in whom serologic data (high levels of hsv- igg antibodies at onset) indicated reactivation. as controls, we used ten hsv- strains isolated from oral lesions in patients without neurological symptoms seen at the department of dermatology, university of goteborg, these strains are referred to as hsv- ref strains. also included is an hsv- strain which we isolated from the csf of a patient during the first bout of multiple sclerosis. the isolate was designated hsv- ban and was recently described in more detail ( ) . laboratory reference strains (hsv- labref) used were f (b. roizman, chicago), mclntyre (a. nahmias, atlanta) and our own strain kj ( ) . in fourteen patients with meningitis, hsv- was isolated from csf (hsv- men) at either the department of virology, university of g teborg (six strains) or at the department of virology, stockholm county central microbiological laboratory (eight strains). hsv- clinical reference strains (hsv- ref) were isolated from genital lesions in ten patients with hsv- infection without neurological symptoms seen at the department of dermatology, university of g teborg. hsv- laboratory reference strains (hsv- labref) used were b ur and (s. jeansson, g teborg). in all experiments, low (second or third) passages of virus were used, and virus stocks were prepared by limited dilution of hsv strains in gmk ah- cell-tube cultures followed by low multiplicity infection of monolayers of gmk ah- cell in cm glass flasks with eagle's medium and incubated at °c. flasks were frozen at - °c when an almost confluent cytopathic effect was observed. the hsv-infected cell cultures were thawed, homogenized, centrifuged at low-speed, atiquoted in ml vials, and stored at - °c. in connection with the experiments, plaque titrations of the virus strains were performed on gmk ah- ceils grown in cm plastic dishes with % methyl cellulose as overlaying medium, and the virus strains were quantified by counting plaque forming units (pfus). hsv titers were then expressed as pfus/ml. outbred swiss albino mice were used. we preferred an outbred strain, since clear differences in susceptibility of several inbred mouse strains to hsv have been reported ( ) . groups of five mice of to weeks of age were infected by intracerebral (i.c.) injection of . ml in the left temporal region or, when assessing neuroinvasiveness by nose inoculation, by scraping the region of the virbrissae bilaterally, and inoculation ( x . ml) of log dilutions in hank's medium of the hsv strains. virus quantities per mouse given were for i.e. infection - - pfus for hsv- and - - pfus for hsv- , and for • nose infection i ~-i pfus for both hsv- and hsv- . as negative control, medium alone was used. the groups of mice were coded and observed daily for two weeks after i.e. and for three weeks after nose infection for signs of neurological involvement and the number of deaths were recorded. the ldso was calculated for each strain. pfus/ml). on day to , five mice from each group were sacrificed and, after perfusion through the left heart chamber with eagle's medium, nose tissues, trigeminal ganglia and brainstems were collected and frozen at - °c. the samples were thawed simultaneously, homogenized in . ml of eagle's medium containing % penicillinstreptomycin, and centrifuged for minutes at rpm. supernatants were inoculated on plastic dishes with gmk ah- cells for plaque titration as described above. replication assays. twenty-four hour replication of all hsv strains was assayed by inoculating strains on gmk ah- cells in plastic dishes (inoculum . pfu/celi), and on c cells, a mouse neuroblastoma cell line (inoculum . pfu/cell). after adsorption for h, cetl cultures were rinsed four times with eagle's medium and ml eagle's medium was added to each culture. after incubation at o for h, the cultures were frozen for later quantification in log dilutions by plaque titration on gmk ah- cells as described above. statistical methods. the nonparametric wilcoxon's rank sum test was used throughout for comparisons between the cns-derived hsv strains and the oral or genital hsv strains except for the analysis of time to death after nose inoculation of the hsv- strains, and hsv replication in cell cultures, where student's t-test was used. neurovirulence in mice after cns and peripheral infection. the hsv-infected mice died of encephalitis with seizures and/or paresis after both i.c. and nose inoculation of the hsv strains and the log pfu/lds could be calculated for each strain. by definition, death after nose inoculation was characterized as neuroinvasiveness, since this route constitutes replication and transport along the trigeminal pathway before entrance to the cns. neurovirulence for each strain was defined based on the combination of results of neuroinvasiveness and lds after i.c. inoculation (see below), the results from mice infected with hsv- strains are shown in figure . after i.e. inoculation, mean log pfu/lds for hsv- enc strains did not differ significantly from mean log pfu/lds for hsv- ref strains. the hsv- ban strain isolated from a patient with multiple sclerosis showed a higher pfu/lds ratio than did any of the hsv- enc or ref strains. after nose inoculation, one hsv- enc strain and four hsv- ref strains were non-lethal with the inocula used ( figure ) . seven of the data from i.c. and nose inoculation in mice were combined to classify all hsv- strains into three classes of neurovirulence ( table ). the nature of this difference was further investigated by monitoring virus replication at three levels (skin, trigeminal ganglia and brainstem) after nose inoculation with these two strains and the class iii (a ) hsv- labref strain f. as shown in figure , hsv- replication reached its maximum after to days in the periphery, after to days in ganglia, and after to days in the brainstem with all three strains. however, hsv- ban albl aobl "hsv- strain characteristics expressed as a: neuroinvasiveness after nose inoculation (ao indicates > ; -> al > x ; x -> a - x ; and a < x pfu/ld ) and b: neurovirulence after intracerebral inoculation (b~ indicates > ; >-b >_- ; and b < pfu/lds ). class i (highly neurovirulent) is defined as: a + b = or ; class ii (moderately neurovirulent) as: a + b = or ; and class iii (weakly neurovirulent) as: a + b = or . bserological data indicating a probable primary hsv- infection as the cause of the encephalitis. two different patterns of progression of hsv- infection were found: strain f (class iii) replicated well at the periphery but to low titers at both the ganglionic and brainstem levels, whereas strains mcintyre (class ii)and kj (class i) both replicated to relatively high titers in ganglia and brainstems after high-dose inoculation peripherally. when the infectious dose was lowered, although kj replicated better at all levels, strain mcintyre barely replicated in the brainstem, while the highly neuroinvasive strain kj readily progressed to the brainstem level. in figure , results from the assessment of virulence after i.e. or nose inoculation of hsv- strains are depicted. generally, log pfu/lds values were lower for the hsv- strains than for the hsv- strains. after i.c. inoculation, hsv- meningitis strains were significantly (p < . ) more virulent as compared with the hsv- reference strains, but no difference was found after nose inoculation. replication assays. the replication yields in gmk-ah (mean log pfu/ml + sd) and c neuroblastoma cells (mean log pfu/ml +_ sd) of hsv- enc strains ( . + . the pathogenesis of encephalitis caused by hsv- is largely unknown. the sporadic occurrence and scarcity of underlying immunocompromising conditions found in patients ( ) may suggest pathogenetic relevance of viral alteration by recombination ( ) or mutation. although the existence and genomic locations of hsv neurovirulence have been well documented in experimental studies ( , , , ), the link between neurovirulence in humans in the form of encephalitis and neurovirulence in animal models has hitherto not been established. the hsv- brain isolates used in this study were all proven to be neuropathogenic to man by causing encephalitis with either a fatal outcome or sequelae in spite of antivirat treatment. the peripheral route of nose infection, where virus particles reach the brain via the trigeminal pathway by neuron to neuron transmission ( ) , was chosen to imitate the probable route that hsv- travels when this virus causes encephalitis in humans ( ) . the virulence to mice displayed by encephalitits-inducing hsv- strains by this route of infection suggests that invasiveness is a property of relevance for hsv- neurovirulence. furthermore, we have shown that the same encephalitis-inducing strains also were significantly more invasive as compared with the hsv- ref strains in an in vitro model using cultured rat sensory neurons in a dual-chamber system permitting infection of neuritic extensions only ( ) . in experimentally infected animals, spread along sensory neurons has been found to be the main route by which hsv enters the cns ( , ) . hsv strain variability in capacity to invade the cns has been described ( , ), and non-neuroinvasive strains have been found to be unable to progress from sensory ganglia to the cns ( , ) . in our study, results of viral replication at peripheral, ganglionic and brainstem levels after nose inoculation of mice indicate that the neuroinvasive hsv- labref strain kj differs from the non-neuroinvasive strains mc-intyre and f by its greater ability to reach and to replicate in the brainstem. one of the hsv- isolates in this study was derived from a patient who was part of an epidemiotogicat chain of three subjects who all suffered from meningitis after genital herpes infection. this might suggest the existence of hsv- strains that are more prone to induce meningitis, but studies on virulence characteristics of isolates from patients with meningitis are lacking. the finding in this study that cns-derived meningitis-inducing hsv- strains showed enhanced neurovirulence after i.c. inoculation in mice might indicate the existence of such a strain characteristic. this characteristic was not revealed by the peripheral route of nose inoculation used here. the clinical picture of hsv- induced meningitis described earlier ( , ) with local neurological signs in the lumbosacral region but without spread to the brain or higher regions of the spinal cord may suggest that hsv- is a weakly neuroinvasive virus in adults. however, studies on hsv- invasiveness after genital inoculation in animals with meningitis-inducing strains are needed to further address this question, since a difference exists in tropism between hsv- and hsv- ( ). a connection between hsv- neurovirulence in vivo and replicative capacity has been suggested ( ), but conflicting data have been presented. in later studies, neither an avirulent strain ( ) nor a virulent strain (t ) showed any correlation between virulence and replication in different cell lines. the indications of hsv- and hsv- neurovirulence properties of cns-derived isolates found in vivo in our study were not paratlelled by higher replication yields in either a highly permissive non-neuronal (gmk-ah- ) or a mouse neuroblastoma (c ) cell line giving low replication yields. on the other hand, a tendency towards lower replication yields in c cells was seen for the class iii nonneurovirulent strains in comparison with the class i strains (t = . , student's t-test). likewise, the strain hsv- ban from a patient with multiple sclerosis, which displayed weak neurovirulence in vivo, gave a markedly low replication yield in both cell types. restricted viral replication as a pathogenetic factor in demyelination was found in a study of mice infected with mutants of the coronavirus mouse hepatitis virus ( ) , and has earlier been suggested for hsv in humans in a hypothesis on multiple sclerosis ( ) . however, we have not been able to test this hypothesis properly, due to the lack of additional viral isolates from the cns of patients with multiple sclerosis. in man, hsv- induced cns infections such as encephalitis are rare events although this virus is neurotropic and commonly recovered from sensory ganglia at autopsy ( ) . the suggestion derived from our data that the hsv- strain characteristic of neuroinvasiveness is linked to encephalitis indicates the usefulness of welldefined clinical isolates in the further study of hsv neuropathogenesis. infections with herpes simplex virus herpes simplex virus encephalitis: laboratory evaluations and their diagnostic significance herpes simplex virus type encephalitis in two homosexual men with persistent lymphadenopathy herpesvirus type meningoencephalitis following renal transplantation different patterns of neurological involvement with herpes simplex virus types and : isolation of herpes simplex virus type from the bully coat of two adults with meningitis herpes simplex virus type and acute aseptic meningitis genital herpes simplex virus infections: clinical manifestations, course and complications neural spread of herpes simplex virus types and in mice after corneal or subcutaneous (footpad) inoculation a mouse model for poliovirus neurovirulence identifies mutations that attenuate the virus for humans typing of herpes simplex virus by an enzyme-linked immunosorbent assay with monoclonal antibodies characterization of a herpes simplex virus type -specified glycoprotein with affinity for nacetylgalactos-amine-specific lectins and its identification as g k or gg isolation of herpes simplex virus type during first attack of multiple sclerosis subtyping of herpes simplex virus dna restriction-enzyme analysis of herpes simplex virus isolates obtained from patients with encephalitis two avirulent herpes simplex viruses generate lethal recombinants in vivo neuron to neuron transmission of herpes simplex virus the iimbic system and the loealisation of herpes simplex encephalitis neuroinvasion of herpes simplex virus, an in vitro model for characterization of neurovirulent strains pathogenesis of herpes neuritis and ganglionitis in mice: evidence of intraaxonal transport of infection sjiistrandj: spread of herpes simplex virus in peripheral nerves herpes simplex virus type dna sequences which direct spread of virus from cornea to central nervous system functional and molecular analyses of the avirulent wild-type herpes simplex virus type strain kos induction of demyelination by a temperature-sensitive mutant of the coronavirus mhv-a is associated with restriction of viral replication in the brain can a defectwc herpes simplex virus cause multiple sclerosis? recovery of herpes-simplex virus from human trigeminal ganglions this work was financially supported by the gothenburg medical society, the medical faculty, university of goteborg, the swedish medical society and the swedish medical research council. we thank miss ann-sofietyl for skillful technical assistance, and drs marianne forsgren, elisabeth olding-stenquist, monica grandien, jonas blomberg, and gunbritt lowhagen for clinical hsv isolates. key: cord- -s le ugm authors: dimopoulos, g.; karabinis, a.; samonis, g.; falagas, m. e. title: candidemia in immunocompromised and immunocompetent critically ill patients: a prospective comparative study date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: s le ugm the purpose of this study was to compare the risk factors, clinical manifestations, and outcome of candidemia in immunocompromised (ic) and nonimmunocompromised (nic) critically ill patients. data were collected prospectively over a -year period ( / – / ) from patients in a -bed, medical–surgical intensive care unit (icu). eligible for participation in this study were patients who developed candidemia during their icu stay. patients under antifungal therapy and with a confirmed systemic fungal infection prior to the diagnosis of candidemia were excluded. cultures of blood, urine, and stool were performed for all patients in the study, and all patients underwent endoscopy/biopsy of the esophagus for detection of candida. smears and/or scrapings of oropharyngeal and esophageal lesions were examined for hyphae and/or pseudohyphae and were also cultured for yeasts. during the study period, , patients were hospitalized in the icu, % for primary medical reasons and % for surgical reasons. after application of the study’s inclusion and exclusion criteria, patients with candidemia ( ic and nic) were analyzed. total parenteral nutrition was more common in ic than in nic patients ( / [ %] vs / [ %], p = . ). oropharyngeal candidiasis was detected in of ( . %) ic patients and in of ( . %) nic patients (p = . ). esophageal candidiasis was also more common in ic than in nic patients ( / [ %] vs / [ %], p = . ). among the ic patients, all except died, resulting in a crude mortality of %; among the nic patients, died, resulting in a crude mortality of % (p > . ). autopsy was performed in two ic and in six nic patients, with disseminated candidiasis found in one ic patient. oropharyngeal and esophageal candidiasis are frequent in ic patients with candidemia. in contrast, this coexistence is rare in nic critically ill patients with candida bloodstream infections. a high mortality was noted in both ic and nic critically ill patients with candidemia. cases, the yeast's portal of entry is the gut, yet in other patients, especially those with central venous catheters (cvcs), skin is the most likely culprit [ , ] . candidemia may occur in both immunocompromised (ic) and nonimmunocompromised (nic) patients. the development of candidemia, however, strongly implies immunodeficiency, since it often occurs in ic patients and, more specifically, in - % of those with myeloproliferative disorders or leukemia and in up to % of patients with aids [ ] . in immunocompetent patients, candida esophagitis is quite rare and is associated with certain predisposing factors, such as use of h -receptor antagonists, antacids, prior vagotomy producing hypochlorydria, administration of antibiotics and systemic or inhaled corticosteroids, functional or mechanical obstruction of the esophagus, malnutrition, metabolic disorders, and alcoholism [ ] [ ] [ ] . however, it is unclear if candida esophagitis is unusual in the general population because its occurrence in that setting has never been investigated prospectively. no study to date has been specifically designed to compare risk factors, manifestations, and outcome of candidemia in ic and nic critically ill patients. thus, we performed the present study to assess possible clinically significant differences between ic and nic patients with candidemia receiving care in the icu setting. this was a prospective study that was conducted over years (february -january ) in a -bed, medical-surgical icu in a tertiary hospital in greece. the "g. gennimatas" general hospital in athens is a -bed general hospital that mainly serves the north-northeastern part of athens. the study protocol was approved by the hospital's ethics committee. because of the patients' inability to give informed consent for participation in the study, consent was obtained from their next of kin. eligible for participation in the study were patients who had been hospitalized for > h and had developed candidemia during their icu stay. blood cultures were ordered at the discretion of the attending physicians, when clinically indicated. patients with severe thrombocytopenia (platelets < , /μl) or coagulopathy were excluded because our study included a biopsy procedure. patients receiving or who had received prior antifungal therapy the last month before the icu admission and patients with a confirmed systemic fungal infection prior to the diagnosis of candidemia also were excluded because the focus of the study was to be a population with icu-acquired canidemia. one of the authors (g.d.) collected the data in a standard manner and was responsible for the follow-up of the patients during the study period. patients were evaluated in terms of age, acute physiology and chronic health evaluation-ii (apache ii) and sequestrial organ failure assessment (sofa) scores, mean duration of icu stay, main predisposing factors for fungal infection development, and clinical outcome. for the purposes of the study, patients without underlying malignancy or neutropenia (leukocyte count > , /μl and neutrophil count > , /μl) and with human immunodeficiency virus (hiv) seronegative status who had not received corticosteroids orally, intravenously, or by nebulization within the last days prior to icu admission were defined as nonimmunocompromised (nic). patients who received systemic corticosteroids for icu indications, such as sepsis or adult respiratory distress syndrome (ards), after their icu admission were classified in the nic group. the study population did not include transplant patients, since our center is a trauma-oriented general hospital. oral thrush was clinically diagnosed by the presence of typical creamy lesions and whitish plaques or pseudomembranes in the oropharyngeal mucosa and on the tongue. a complete laboratory evaluation, which included a hemogram as well as biochemical and coagulation profiles, urinalysis, serologic testing for hiv, radiological studies, and an electrocardiogram, was performed. blood, urine, and stool cultures, in addition to cultures of scrapings obtained from the oropharynx, were performed for all study patients. oropharyngeal scrapings were also immediately examined microscopically for the presence of yeasts and/or hyphae/pseudohyphae, using % potassium hydroxide. the investigational work-up for invasive candidiasis in our patients (other than blood cultures, esophageal endoscopy, and oral scrapings) included (a) identification of predisposing factors, (b) surveillance cultures to detect possible colonization, (c) eye exam, and (d) ct scans of the suspected site of infection. all endoscopies were performed by the same gastroenterologist. endoscopies were performed during the first h after the diagnosis of candidemia, using a pentax eg videoscope. during endoscopy, brushing specimens and tissue biopsies were obtained for microbiological and histological examination. candida esophagitis was diagnosed by the presence of visible esophageal lesions (white plaques with hyperemia and edema, linear and nodular elevated plaques with ulceration, plaques with increased friability of the mucous membrane) using a modified kodsi method and was verified by biopsy showing hyphae in histological sections and/or candida growth on agar plates [ , ] . additionally, the brushing specimens were evaluated immediately for yeasts and hyphae/pseudohyphae after the addition of % potassium hydroxide and were cultured on sabouraud dextrose agar plates for candida growth. biopsy samples were fixed with % formalin and paraffin followed by acid-schiff staining to detect mucosal invasion by the fungus. cultures were considered positive if there was growth of candida on sabouraud dextrose agar plates. yeasts were identified using the api c aux system (biomérieux, marcy l'etoile, france). candidemia was defined by at least one positive blood culture for candida spp., and candiduria by a urine culture with candida spp. growth of ≥ cfu/ml. candidemia was identified using bactalert (biomérieux, usa). table shows the number of sites (besides blood) colonized by candida spp. disseminated candidiasis was diagnosed when candida was identified by culture and/or biopsy from two or more organs. we used candida id agar (biomérieux) to discriminate between candida albicans, candida non-albicans, and candida tropicalis. additionally, we used the germ-tube method and the api c (assimilation of carbohydrates) for identification of all species of candida. ic and nic patients were compared for certain variables of interest by mann-whitney u test, chi-squared test, and fisher exact test. the level of significance was set at p< . . during the study period, , patients were hospitalized in the icu, % for primary medical reasons and % for primary surgical reasons. twenty-four patients ( ic and nic) met the eligibility criteria. of these, ( %) were ng cultures with no growth medical patients and ( %) surgical patients. their demographic and clinical characteristics are shown in table . among the ic and nic patients, ( %) and ( %) were females, respectively (p> . ). the median age of the ic and nic patients was years (range - ) and years (range - ), respectively (p> . ). the median duration of icu stay for ic and nic patients was . and . days, respectively (p> . ). among the nine ic patients, all had underlying primary medical pathology, since three had leukaemia/lymphoma, three aids, and one each had liver cirrhosis complicated by septic shock, pneumonia following chemotherapy for lung cancer, and rheumatoid arthritis complicated by pneumonia. none of the patients in the study with leukemia/lymphoma received chemotherapy during the study or for weeks prior to the diagnosis of candidemia. the patients with lung cancer and rheumatoid arthritis (patient nos. and in ic group, table ) received treatment with glucocorticosteroids. among the nic patients, the underlying diagnosis was pneumonia/ards in , trauma/head injury in , peritonitis in , pancreatitis in , and pulmonary embolism in . six of the nic patients had surgical pathology: three with trauma/head injury and three with peritonitis. most patients with pneumonia had community-acquired infection but needed icu admission due to respiratory failure. patient numbers and in the nic group (table ) developed ventilator-associated pneumonia. the mean time from icu admission to the isolation of candida from blood specimens was days (range - days). the mean time between drawing blood specimens for culture and isolation of candida was days. the presence of known predisposing factors for development of candidiasis in these patients is shown in table . the ic patients with candidemia had higher mean apache ii ( ± vs ± ) and sofa ( ± vs ± ) scores during icu admission compared to the nic patients (p= . for both scores). in addition, the administration of total parenteral nutrition was more common in ic patients (p= . ). oropharyngeal candidiasis was detected in of ( . %) ic patients and in of ( . %) nic patients (p= . ). in the ic patients, the diagnosis of oral thrush was confirmed prior to the icu admission, while in the single nic patient with oral thrush, the diagnosis was established on the eighth icu day. four of the nine ( %) ic patients and none of the nic patients presented with esophageal candidiasis (p= . ). all patients with esophageal candidiasis manifested symptoms (mainly odynophagia; dysphagia in one patient) before icu admission, but the diagnosis table shows the results of the fungal cultures in ic and nic patients. c. albicans was isolated from the blood in of ( . %) ic patients and in of ( . %) nic patients, while non-albicans candida spp. were isolated in the remaining patients of the two study groups (p> . ). among nic patients, c. tropicalis was isolated from the blood of three patients and candida krusei and candida parapsilosis from one patient each. among ic patients, c. krusei, c. parapsilosis, candida dubliniensis, and candida lusitaniae were isolated from the blood of one patient each. the same candida spp. were isolated in at least two different body sites in eight of ten nic patients and in four of five ic patients with candidemia due to c. albicans. there was one patient (table ) who had concomitant and persistent methicillin-resistant staphylococcus aureus (mrsa) bacteremia during the icu hospitalization. patients with c. albicans candidemia received fluconazole mg every h intravenously and patients with non-albicans candidemia liposomal amphotericin b intravenously (mean duration of therapy, . days; range - days). no statistically significant difference in clinical outcome was detected between nic and ic patients with candidemia. among the nine ic patients, all except two (patient nos. and ; table ) died, resulting in a crude mortality of %. among the nic patients, (patient nos. , , , , , , , , and ; table ) died, resulting in a crude mortality of % (p> . ). when ic and nic patients were combined, the mortality among patients with primary medical pathology and patients with surgical pathology was identical, at %. more specifically, of the patients with medical pathology ( ic and nic), ( ic and nic) died, while of the patients with surgical pathology (all nic) died. autopsy after consent was performed in eight patients (two ic and six nic) and showed disseminated candidiasis in one ic patient (oropharyngeal, esophageal, and gastric candidiasis). the main findings of our study of critically ill patients with candidemia are that ic patients had higher apache ii and sofa scores and were more likely to receive total parenteral nutrition than nic patients. in addition, oropharyngeal and esophageal candidiasis was considerably more common in ic than in nic icu patients with candidemia. of importance, a high mortality was noted in both ic and nic critically ill patients with candidemia. the recent advances in intensive care have improved the survival of critically ill patients at the cost of the emergence of nosocomial infections of the bloodstream and other sites. candidemia is the most common hematogenous fungal infection and the fourth most common bsi overall in the usa, accounting for % of all nosocomial bsis [ ] . in greece, there are no relevant data from large multicenter studies regarding candidal bsi, although there are data from a single-center study reporting that . - . % of bsis during a -year period ( - ) were due to candida spp. [ ] . risk factors for invasive candidiasis in the icu have been defined by several studies and include a prolonged hospital stay; the use of broad-spectrum antibiotics, corticosteroids or other immunosuppressants; the administration of total parenteral nutrition, hemodialysis or multiple blood transfusions; prolonged mechanical ventilation; diabetes mellitus; gastrointestinal perforation or surgery; and pancreatitis [ , [ ] [ ] [ ] [ ] ] . candida spp. colonize the gastrointestinal tract of healthy individuals and, although they are most frequently recovered from the oropharynx, they have also been isolated from the stool of - % of healthy adults [ ] . colonization of the gastrointestinal tract or the oropharynx with candida spp. has been shown to invariably precede hematogenous or systemic disease, although in recent years the improved survival of burn victims and the widespread use of cvcs has allowed direct invasion of the bloodstream by candida spp. that colonize the skin [ , ] . candida colonization as a risk factor is still controversial. it has been studied as a predictor of invasive candidiasis in the icu setting; in fact, a "colonization index" has been proposed to predict invasive candidiasis in icu patients, although guidelines for identifying patients who would best benefit from antifungal prophylaxis are not established [ , ] . it is unclear, however, whether differences exist between ic and nic patients in the way candidemia and/or systemic candidiasis is acquired in relation to the presence of prior mucosal candidiasis. one of the purposes of the present prospective clinical study was to assess a possible correlation between the development of candidemia and/or systemic candidiasis and the pre-existence of oropharyngeal or esophageal candidiasis in critically ill patients. we showed that oropharyngeal and esophageal candidiasis was rare in nic critically ill patients with candidemia, but often the two entities coexisted in ic patients. a prior diagnosis of oral thrush due to candida spp. in conjunction with a candida-positive stool culture was a sensitive marker for the presence of esophageal candidiasis. the development of candida esophagitis is a two-step process that includes colonization of the esophagus and invasion of the epithelial layer. when the intraluminal concentration of candida spp. is high, the fungus is able to transmigrate the wall of the digestive tract and gain access to the circulation, a process known as persorption [ ] . in nic patients, this process, although rare, has been associated with certain predisposing factors and as-yetunknown mechanisms of infection [ ] . the nic patients in our study developed candidemia during their icu hospitalization, and none manifested esophageal candidiasis, although one developed oral thrush. our autopsy findings, which showed that six nic patients had no organ involvement, suggest that candidemia probably resulted from transient fungal translocation across the gastrointestinal tract without affecting the mucosal membranes. however, we performed autopsies in only % of the nic patients. a correlation between oral thrush and esophageal candidiasis has been shown in a number of studies in ic patients with aids or cancer [ ] [ ] [ ] . in our study, the simultaneous diagnosis of oral thrush and a stool culture positive for candida spp. in ic patients was associated with a % specificity and a positive predictive value for development of esophageal candidiasis. the lack of correlation between candidemia/systemic candidiasis and mucosal candidiasis in nic in comparison to ic patients can likely be explained by the defective primary defense mechanisms against tissue invasion and dissemination that are common in ic patients. we would like to emphasize that oral candidiasis that presents as erythematous candidiasis (in the absence of other specific symptoms and signs) could be difficult to diagnose in critically ill patients and might be occasionally misleading because (a) tubes and other devices placed in the oral cavity are themselves able to produce mild inflammatory lesions, and (b) the increased respiratory secretions and, often, the regurgitated enteral nutrition content may cause inflammation of the oral mucosa. the clinical characteristics of the eligible nic and ic patients of the study were well balanced between the groups, except for the more frequent use of parenteral alimentation and the higher apache ii scores in the ic patients. total parenteral nutrition has been a known risk factor for bsi due to candida spp., and its use has recently been associated with candidemia due to c. parapsilosis in both children and adults [ , ] . interestingly, candidemia due to c. parapsilosis in non-neutropenic patients with an intravenous hyperalimentation catheter has a low mortality rate and a good prognosis [ ] . in our study, c. parapsilosis was isolated from the blood cultures of two patients, one nic and one ic, making it the third most common candida sp. encountered, after c. albicans ( blood isolates total; in nic patients) and c. tropicalis ( blood isolates, all in nic patients). the nic patient with c. parapsilosis candidemia in our series had charcot disease complicated by pneumonia, but she survived. the other patient with c. parapsilosis candidemia had aids and succumbed to his disease. the higher apache ii scores in our ic patients are suggestive of an increased severity of the underlying critical illness. lower (< points) apache ii scores have been associated with a higher probability of survival in a multicenter study of non-neutropenic critically ill patients with candidemia [ ] , and higher apache iii scores have been associated with increased mortality in cancer patients with candidemia in a study of episodes at the m.d. anderson cancer center [ ] . c. albicans was the candida sp. most commonly isolated from the blood of our patients ( of isolates, or . %), followed by c. tropicalis ( isolates, or . %), c. parapsilosis ( isolates, or . %), c. krusei ( isolates or . %), c. dubliniensis, and c. lusitaniae ( isolate each, or . %). in one patient (table ) , persistent mrsa bacteremia was observed concomitantly with candidemia. there are sporadic reports indicating a small but significant proportion of patients who have icu-acquired candidemia concomitantly with bacteremia [ ] . in our patients, fungemia due to c. tropicalis was not preceded by colonization with the same species. it is well known that c. tropicalis, part of the normal human mucocutaneous flora, is a major cause of septicemia/disseminated candidiasis, mainly in patients with lymphoma, leukemia, and diabetes [ ] . accordingly, a possible explanation for the development of c. tropicalis candidemia in our patients could be the uncontrolled diabetes combined with the severe septic syndrome. we did not have any cases of candidemia due to candida glabrata, a common pathogen in the usa and israel [ , ] . this finding is in accordance with the data of the sentry antimicrobial surveillance program, which surveyed episodes of bsi due to candida spp. in patients hospitalized in european icus. overall, % of the bsis due to candida spp. were attributable to c. albicans, followed by c. parapsilosis ( %), c. glabrata ( %), c. tropicalis ( %), c. famata ( %), c. krusei ( %), and candida inconspicua ( %) [ ] . with regards to c. albicans, our figures are very close to both european [ ] and american studies. for example, trick et al. [ ] , who described the secular trend of hospitalacquired candidemia among icu patients in the usa during - , found that among cases of monomicrobial and polymicrobial candidemia, c. albicans was isolated in and %, respectively. the high crude mortality among our patients ( % for nic and % for ic) was not unexpected, given the severity of their underlying diseases, and is comparable to the mortality rate for adults with candidemia reported by lark et al. [ ] ( %), gudlaugsson et al. [ ] ( %), karlowsky et al. [ ] ( %), colombo et al. [ ] ( %), and pappas et al. [ ] ( %). although we saw no difference in outcome between medical and surgical patients, others have described a poorer outcome for medical patients with candidemia [ ] . in our study population, patients with ards and sepsis were classified in the nic group because we believe that these patients have a temporary immunosuppression (if they survive), while the patients classified in the ic group had a certain degree of chronic immunosuppression, despite the differences in the underlying disease. the patient with alcoholic cirrhosis and sepsis (table ) was classified in the ic group because he had received glucocorticosteroids due to exacerbations of chronic obstructive pulmonary disease for months before admission to the icu. the design of the study did not allow for all patients admitted to the icu to be evaluated for the presence of oropharyngeal and esophageal candidiasis. additionally, we focused on a subset of patients with icu-acquired candidemia without prior systemic fungal infection who had not been exposed to prophylactic antifungal drugs. since, by definition, the term "icu-acquired candidemia" means that the fungal infection is acquired in the icu, we excluded from our study those patients with previous invasive fungal infection . we should acknowledge that our study has several limitations. first, we were unable to pinpoint the exact time of onset of the esophageal candidiasis, especially since oral thrush was already present before admission to the icu. due to obvious ethical reasons, we evaluated our patients for esophageal candidiasis with endoscopy only once. thus, we do not know if the esophagus was involved before or after the icu admission. second, it is unclear if the esophageal candidiasis resulted from hematogenous spread of the fungi in the esophagus, or if the bloodstream involvement was directly related to the oral thrush or the esophageal candidiasis. third, we did not focus on the treatment administered to patients with candidemia, although we did report that patients with c. albicans and non-albicans candidemia received fluconazole and liposomal amphotericin b intravenously, respectively. fourth, we do not have readily available data on the length of stay of all patients who received care in the icu or the total number of cases of candidemia in the icu, including those excluded, to know how common candidemia is among icu patients. fifth, there are no readily available data regarding the type of therapy and the removal of cvcs and the influence of either factor on outcome in the two patient groups studied. sixth, the sample of our study population is relatively small (study population from a single center) and thus does not permit a powerful statistical analysis; unfortunately, various practical reasons prevented us from incorporating data from other large icus into our study. finally, we do not have readily available data on mics because the laboratory usually reported the results for an isolate as either "susceptible" or "not susceptible" to fluconazole. in conclusion, we compared various characteristics, including risk factors, manifestations, and outcome of ic and nic icu patients with candidemia. we found that oropharyngeal and esophageal candidiasis frequently coexisted with candidemia in ic patients. prospective, clinical studies are needed to better define the value of avoiding colonization and/or of treating oropharyngeal and esophageal candidiasis for the prevention of systemic disease in selected critically ill patients. nosocomial bloodstream infections in united states hospitals: a three-year analysis system report, data summary from predominant pathogens in hospital infections new approaches to the risk of candida in the intensive care unit risk factors for candidal bloodstream infections in surgical intensive care unit patients: the nemis prospective multicenter study. the national epidemiology of mycosis survey nosocomial fungal infections: candidemia candidemia in 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presumptive clinical criteria versus endoscopy in the diagnosis of candida esophagitis at various hiv- disease stages hematogenous infections due to candida parapsilosis: changing trends in fungemic patients at a comprehensive cancer center during the last four decades emergence of candida parapsilosis as the predominant species causing candidemia in children for the study group of fungal infection in the icu ( ) candidemia in nonneutropenic critically ill patients: analysis of prognostic factors and assessment of systemic antifungal therapy risk factors and predictors of outcome in patients with cancer and breakthrough candidemia infections with candida spp. in critically ill patients are primarily related to the length of stay in the intensive care unit candidiasis: pathogenesis, diagnosis and treatment secular trend of hospital-acquired candidemia among intensive care unit patients in the united states during - epidemiology of candidemia-a nationwide survey in israel international surveillance of blood stream infections due to candida species in the european sentry program: species distribution and antifungal susceptibility, including the investigational triazole andechinocandin agents. sentry participant group (europe) four-year prospective evaluation of nosocomial bacteremia: epidemiology, microbiology, and patient outcome attributable mortality of nosocomial candidemia, revisited candidemia in a canadian tertiary care hospital from to high rate of nonalbicans candidemia in brazilian tertiary care hospitals a prospective observational study of candidemia: epidemiology, therapy, and influences on mortality in hospitalized adult and pediatric patients candidemia in critically ill patients: difference of outcome between medical and surgical patients key: cord- - cwwi authors: hsueh, p.-r.; huang, h.-c.; young, t.-g.; su, c.-y.; liu, c.-s.; yen, m.-y. title: bacteria killing nanotechnology bio-kil effectively reduces bacterial burden in intensive care units date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: cwwi a contaminated hospital environment has been identified as an important reservoir of pathogens causing healthcare-associated infections. this study is to evaluate the efficacy of bacteria killing nanotechnology bio-kil on reducing bacterial counts in an intensive care unit (icu). two single-bed rooms (s- and s- ) in the icu were selected from april to may . ten sets of new textiles (pillow cases, bed sheets, duvet cover, and patient clothing) used by patients in the two single-bed rooms were provided by the sponsors. in the room s- , the sets of new textiles were washed with bio-kil; the room walls, ceiling, and air-conditioning filters were treated with bio-kil; and the surfaces of instruments (respirator, telephone, and computer) were covered with bio-kil-embedded silicon pads. room s- served as the control. we compared the bacterial count on textiles and environment surfaces as well as air samples between the two rooms. a total of , samples from different sites in each room were collected. the mean bacterial count on textiles and environmental surfaces in room s- was significantly lower than that in room s- ( . vs . colony-forming units [cfu]/ cm( ); p < . ). room s- had lower bacterial counts in air samples than room s- ( . – . vs . – . cfu/hour/plate; p < . ). the density of microbial isolations was significantly greater among patients admitted to room s- than those to room s- ( . vs . isolates per patient-days, p < . ). bio-kil can significantly reduce bacterial burden in the environment of the icu. hospitals face higher costs as the number of patients with healthcare-associated infections (hai) and the duration of hospital stay increase [ , ] . transmission of healthcareassociated pathogens takes place through air, droplets, and hand contact by medical staff [ ] [ ] [ ] [ ] . contamination of equipment has been identified as a major reservoir of pathogens associated with hai [ ] [ ] [ ] [ ] . relevant studies have shown that surfaces in the rooms of hospitalized patients who have been infected with or colonized by methicillin-resistant staphylococcus aureus (mrsa) or vancomycin-resistant enterococci (vre) are more likely to be contaminated with these bacteria than surfaces in rooms housing patients without mrsa or vre infection [ , ] . this indicates that pathogens like mrsa or vre can be transmitted to the environment by patients. although hand hygiene has already become a focal point at various hospitals worldwide and is expected to have a major effect on reducing the rates of hai, other measures are needed to reduce the colonization density in the hospital environment so that the chain of infection can be disrupted [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . bio-kil (cargico group, taiwan) is an antimicrobial agent comprising inorganic metal components and organic quaternary ammonium compounds (qacs) [ ] . bio-kil molecules have a high-affinity structure and a strong electric field that effectively attracts pathogens [ ] . their strong electrical charge damages the membrane proteins of microorganisms, thereby killing the pathogens [ ] . bio-kil forms a permanent, covalent bond with the surface of the textile fibers. even after the textiles have been washed times, the product still retains more than % of its bacteria-killing power [ , ] . depending on the frequency with which the textiles are washed, treatment only needs to be repeated every to months in order to maintain a long-term bactericidal effect [ , ] . in the same icu environment and its instruments, such as nursing station desktops, workbenches, telephones, computer keyboards, and surfaces close to the patients, the catalyst will directly form a covalent bond with the surface of these objects, where it will have a long-term, bactericidal effect [ ] . as it reduces the possibility of contamination of textiles, environments, and instruments, it lowers the environmental bacterial count in the hospital, thus reducing hai due to bacterial colonization [ ] . in the air filter system in the icu, air circulates approximately - times per hour through an indoor air conditioning system, and each time the air passes through a system in which bio-kil has been applied, it comes into contact with the bio-kil platform where a catalyst reaction will take place, killing the bacteria [ , ] . this way, as the air continues to circulate, it is continuously disinfected, thus reducing the amount of bacteria in the indoor air flow. although bio-kil has been shown to be a highly effective anti-microbial agent, no comprehensive studies have been performed on its effectiveness in clinical settings. in this study, we analyzed whether bio-kil when applied to different materials in the icu, such as sheets, bedding, and clothing, desktops and the surfaces of instruments and equipment, reduces or eliminates bacteria in the environment, on the surfaces of surrounding items, and in the air. this study was conducted during the period april to may in two adjacent single-bed rooms (s- [control bed] and s- [experimental bed]) in a surgical intensive care unit of the taipei city hospital, a , -bed regional hospital in taipei, taiwan. during the study period, patients were admitted to room s- and patients were admitted to room s- . routine textile washing and replacement as well as infection control practices for nurses, physicians, visitors, and disinfection of environments and instruments in these two rooms were performed according to hospital regulations. during the study period, ten sets of new textiles (pillow cases, bed sheets, duvet covers, and patient clothing) were provided by the sponsors for use by the patients who stayed in rooms s- and s- . clothing for physicians and nurses who worked in the two rooms and for people who visited the patients was provided by the hospital. the surfaces of the environment and the instruments were cleaned and disinfected (by ppm sodium hypochlorite) every morning ( : - : am) as part of routine cleaning practice. textiles were cleaned and replaced every day. embedding of textiles, environment, and instrument surfaces with bio-kil in room s- the ten sets of textiles to be placed in room s- were first washed for min in °c water with neutral detergents, rinsed with tap water, and then spin dried. the dry textiles were then soaked in bio-kil solution for minutes and dried in a °c oven (fig. a) . bio-kil solution was also treated evenly on the walls (cement and glass walls), ceiling, and in the air-conditioning filters of room s- (fig. b, c) . in addition, bio-kil antibacterial silicon pads ( cm× cm) were placed over the instrument panel (respirator), computer keyboard, and telephone keypads in the nursing station associated with room s- (fig. d ). samples were collected from surfaces of the environment and instruments with which medical staff and patients frequently come into physical contact. moistened sterile cotton swabs were used to evenly wipe designated areas measuring cm× cm ( cm ; fig. a -c). the samples were then inoculated into trypticase soy agar and placed in a °c incubator for h. the number of colonies was recorded as colony-forming units (cfus)/ cm . textiles and the surface of the environment and instruments in rooms s- and s- were sampled in the morning starting on the third day after the placement of new textiles and then every - days prior to routine cleaning practice by hospital cleaning staff. culture media (lb agar plates) were placed at four locations (right-and left-hand side of the entrance and head boards) in rooms s- and s- for min to collect bacteria in the air (table and fig. d ). the plates were then placed in a °c way, when the bio-kil® anti-bacterial catalyst is applied to the incubator for h. the bacterial colonies were counted and were recorded as cfu/hour/plate. air samples in rooms s- and s- were taken in the morning along with sampling from textiles and environments. bacterial culture results for patients admitted to the rooms during the study period bacteria grown from textiles, the environment, and air samples were not identified to the species level and testing of susceptibility to antimicrobial agents was not performed. in order to evaluate the efficacy of bio-kil on microbial infections or colonization among patients admitted to the two rooms, all bacterial culture results and the resistant profiles of the isolates, particularly those resistant to extended-spectrum cephalosporins (esc, ceftazidime or cefepime) or carbapenems (imipenem or meropenem), from all clinical specimens of all patients were evaluated during the study period. the density (per patientdays) of microbial infections or colonization among patients admitted to rooms s- and s- was determined. the independent-sample t test was used to compare the mean bacterial count of the two groups. differences in mean bacterial count between the two groups were tested by the student's t test and checked by one-way analysis of variance (anova). a p value of < . was considered to indicate statistical significance. all statistical analyses were performed on a personal computer using the statistical package spss for windows (version , spss, chicago, il, usa). a total of , samples were taken from sampling areas, including environmental surfaces and four air samples in the control (s- ) and experimental (s- ) rooms. the ranges and means of bacterial counts from each sampling site are summarized in table . with the exception of bacterial counts from the walls, the mean bacterial count from other sampling sites from room s- was higher than that from sampling sites from room s- . the mean and % confidence values of bacterial counts on the environment surfaces at five different time periods are illustrated in fig. . the mean bacterial count from textiles and environment surfaces in room s- ( . cfu/ cm ) was significantly lower than that in room s- ( . cfu/ cm ; p < . ). the mean bacterial count from air samples was significantly higher in room s- than in room s- (p < . ), although there was no significant difference between the four sampling areas in the two rooms (p = . ). a total of isolates were reported among the three patients hospitalized in room s- . the isolates included methicillinresistant coagulase-negative staphylococci (n = ), klebsiella pneumoniae (n = ), including isolates resistant to esc (n = ); ceftazidime-resistant enterobacter cloacae (n = ); proteus mirabilis (n = ), susceptible to esc; pseudomonas aeruginosa (n = ), both susceptible to carbapenems; acinetobacter baumannii (n = ), all carbapenem-resistant; and stenotrophomonas maltiphilla (n = ). as for the patients admitted to room s- , a total of isolates were reported. they included methicillin-resistant coagulasenegative staphylococci (n = ), escherichia coli (n = ), susceptible to esc; k. pneumoniae (n = ), susceptible to esc; e. cloacae (n = ), susceptible to esc; citrobacter freundii (n = ), susceptible to esc; p. aeruginosa (n = ), were susceptible to carbapenems and was resistant to carbapenems; a. baumannii (n = ), both susceptible to carbapenems; s . maltiphilia (n = ); and candida albicans (n = ). the density of microbial infections or colonization was significantly greater among patients admitted to room s- ( . isolates per patient-days) than among patients admitted to room s- ( . isolates per patient-days; p < . ). in this study, we found that bio-kil significantly reduced the bacterial burden in the icu. in health care settings, it is necessary to provide a safe environment and implement infection control measures to prevent hai [ ] . the majority of hai are caused by colonization and subsequent infections due to endogenous flora and exogenous organisms from the environment have long been ignored as insignificant risk factors for hai [ , ] . since cross transmission due to direct physician-patient contact plays a major role in hai caused by exogenous pathogens [ ] , handwashing to break the chain of transmission has remained the most critical and preventing intervention in hai control ever since the era of semmelweis [ ] . however, an average compliance rate of % has always been a problem in real-world practice [ , ] . of the numerous infection control bundles and policies that were enacted in the era of zero tolerance after sars [ , ] , the hand hygiene campaign has been shown to be the most cost-effective and widely accepted practice and has led to a trend toward reduction of mrsa nosocomial infections [ ] . however, the incidence of mdr outbreaks such as carbapenem-resistant or pandrug-resistant acinetobacter baumannii , or vancomycin-resistant enterococci has increased recently [ , , , , ] . a series of studies have demonstrated that physician-environment-patient transmission of hais was the result of indirect contact with the hospital environment or the patient's surroundings [ , ] . ohl et al. suggested a potential link between transmission of pathogens to patients and healthcare workers (hcws) who do not perform hand hygiene after touching the curtains [ ] . as such, environmental disinfection has been emphasized in recent years to compensate for inadequate adherence to hand hygiene. in addition to cleaning on patient's discharge to reduce the chances of mdro transmission from a previous occupant to a new occupant [ , ] , environmental disinfection of a patient's bed and surroundings should be performed to control mdr [ , ] . however, for manual surface cleaning and disinfection there has always been a risk of quality instability due to poorly controlled processes or, arguably, the possibly undertrained and incompetent hands of housekeeping staff [ , ] . a systematic renovation of automated environmental control may have to be considered to make up for the shortfalls of hcws, whose behavior ultimately determines the exogenous factor responsible for hai. several environment disinfection control models have been developed, including surface coating and photocatalyzing or ultraviolet germicidal irradiation (for surface and air sterilization), and copper and silver nanofilms [ - , , , ] . yet, there are still barriers to overcome before they are adopted as standard clinical applications, some with long d-values and repopulation of bacteria in a short time. instead, as we observed in present study, bio-kil reacts within seconds and maintains a constant effect for up to months depending on the surfaces to which it is applied. airborne transmission of hospital pathogens and their contribution to the burden of hai has been well described [ , , , ] . a novel hydroxyl radical air disinfection system (inov unit) has been reported to improve air quality and reduce environmental contamination in health care settings [ , ] . a previous study in a regional hospital in taiwan showed that the addition of bio-kil apparently reduced the bacteria count by up to % (from . cfu/h/plate to . cfu/h/plate) [ ] . similar findings were also observed in the present study. the main limitation of this study is the lack of assessment of the effect of bio-kil on other emerging health careassociated pathogens, including multidrug-or pandrugresistant pathogens, clostridium difficile , fungi (candida and aspergillus), and viruses in the hospital environment. these organisms were associated with an increasing incidence of life-threatening infections in hospitalized patients [ , , [ ] [ ] [ ] . furthermore, the low sample size (only two rooms) and the lack of detailed clinical characteristics of patients admitted to these two rooms (infections or contamination) and the following further infection prevention for the next patients admitted to these two rooms were also the limitations of this study. further work is also required to validate whether reducing the burden of these organisms in the environment will minimize the risk of hai. in conclusion, we found that bio-kil nanotechnology can significantly reduce the bacterial burden in the environment (textiles, environmental surfaces, and air) and the bacterial density of microbial infections or colonization among patients in the icu. update from the senic project. hospital infection control: recent progress and opportunities under prospective payment the financial incentive for hospitals to prevent nosocomial infections under the prospective payment system. an empirical determination from a nationally representative sample association between isolation sites of methicillin-resistant staphylococcus aureus (mrsa) in patients with mrsa-positive body sites and mrsa contamination in their surrounding environmental surfaces a study of the relationship between environmental contamination with methicillin-resistant staphylococcus aureus (mrsa) and patients' acquisition of mrsa the improvement of air bacteria counts among out-patient department areas at a regional hospital environmental contamination makes an important contribution to hospital infection doctors and hand washing: instilling semmelweis' message the efficacy of infection surveillance and control programs in preventing nosocomial infections in us hospitals equal efficacy of glucoprotamin and an aldehyde product for environmental disinfection in a hematologic transplant unit: a prospective crossover trial quantification and analysis of airborne bacterial characteristics in a nursing care institution environmental contamination and airborne microbial counts: a role for hydroxyl radical disinfection units? an outbreak of multidrug-resistant acinetobacter baumnnii due to contaminated bathing water at a burn center pandrug-resistant acinetobacter baumannii causing nosocomial infections in a university hospital association between contaminated faucets and colonization or infection by nonfermenting gram-negative bacteria in intensive care units in taiwan hospital privacy curtains are frequently and rapidly contaminated with potentially pathogenic bacteria risk of acquiring multidrug-resistant gram-negative bacilli from prior room occupants in the intensive care unit role of hospital surfaces in the transmission of emerging health care associated pathogens: norovirus, clostridium difficile , and acinetobacter species reducing healthcare-associated infections caused by multidrugresistant acinetobacter baumannii by implementing hospital-wide environmental cleaning measures feasibility study for epidemic prevention and control in a regional hospital the effect of applying quality control circle in reducing nosocomial infections an evaluation of environmental decontamination with hydrogen peroxide vapor for reducing the risk of patient acquisition of multidrug-resistant organisms taiwan's traffic control bundle and the elimination of nosocomial severe acute respiratory syndrome among health care workers combating antimicrobial resistance: antimicrobial stewardship program in taiwan antimicrobial activity of copper and silver nanofilms on nosocomial bacterial species bioaerosol deposition in single and two-bed hospital rooms: a numerical and experimental study effect of a novel air disinfection system on airborne micro-organisms in a hospital outpatient clinic national healthcare safety network (nhsn) team and participating nhsn facilities ( ) antimicrobial-resistant pathogens associated with healthcareassociated infections: summary of data reported to the national healthcare safety network at the centers for disease control and prevention seven-year surveillance of nosocomial invasive aspergillosis in a french university hospital fungal infections in icu patients: epidemiology and the role of diagnostics acknowledgments we would like to thank mr. yen-kuen shiau, technical director of the cargico group, taiwan for providing the sterilization materials that this study required, as well as mr. liao-kuo lu, researcher at the cargico group, for providing his assistance, enabling the completion of our experiment.potential conflicts of interest the authors declare that they have no conflict of interest. the study was supported in part by cargico group, taiwan. cargico group had no influence on the design and analysis of the study.ethical approval this study was approved by the taipei city hospital institutional review board (tchirb- -e). key: cord- -si pb authors: jouneau, s.; poineuf, j.-s.; minjolle, s.; tattevin, p.; uhel, f.; kerjouan, m.; le hô, h.; desrues, b. title: which patients should be tested for viruses on bronchoalveolar lavage fluid? date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: si pb bronchoalveolar lavage (bal) is a major diagnostic tool in lung diseases, including viral respiratory infections. we aimed to better define the situations where viral tests should be performed on bal fluid (balf). we retrospectively studied all cases where viral tests [immunofluorescence, immunocytochemistry, viral culture, and/or polymerase chain reaction (pcr)] were performed on balf during a period of year ( ) in our institution. we compared the characteristics of patients with virus-positive versus virus-negative balf. of the balf samples sent to the microbiology laboratory, underwent viral tests. of these, ( %) were positive and identified viruses: herpes simplex virus (hsv)- (n = ), cytomegalovirus (cmv, n = ), epstein–barr virus (ebv, n = ), human herpesvirus (hhv)- (n = ), respiratory syncytial virus (rsv, n = ), rhinovirus (n = ), and adenovirus (n = ). the variables associated with positive viral tests on univariate analysis were immunosuppression [human immunodeficiency virus (hiv), corticosteroids > mg/day for ≥ weeks, or other immunosuppressive therapy], ground-glass attenuations on computed tomography (ct) scanning, late-onset ventilator-associated pneumonia (vap), and durations of (i) hospital stay, (ii) intensive care unit (icu) stay, and (iii) mechanical ventilation before bal (p < . for each comparison). on multivariate analysis, only immunosuppression [odds ratio (or) . , % confidence interval (ci) [ . – . ], p < . ] and ground-glass attenuations (or . , % ci [ . – . ], p = . ) remained associated with virus-positive bal. none of the viral tests performed on balf for the initial assessment of diffuse infiltrative lung disease (n = ) was positive. pcr improved the diagnostic yield of viral tests on balf by %. testing for viruses on balf should be mostly restricted to immunocompromised patients with acute respiratory diseases and/or patients with unexplained ground-glass attenuations on ct scanning. bronchoalveolar lavage (bal) is a major diagnostic tool for infectious lung diseases, especially in immunocompromised patients [ ] . viral agents play an important role as etiologies of pneumonia in immunocompetent and immunocompromised hosts [ ] [ ] [ ] [ ] , and may be involved in a substantial proportion of asthma and chronic obstructive pulmonary disease (copd) exacerbations [ ] [ ] [ ] [ ] . the development of molecular biology techniques, including polymerase chain reaction (pcr), has dramatically increased the yield of viral tests for various respiratory diseases [ , , , ] . however, most studies have been performed in specific settings, with highly selected patients. hence, the yield of viral tests on bal fluid (balf) remains poorly characterized in routine practice. a key dilemma with the development of new tests is the relatively high cost associated with their non-discriminated use. to better inform the use of viral tests in patients with respiratory diseases, we performed an observational, retrospective study in our institution, with three aims: (i) to assess the diagnostic value of viral tests on balf in routine practice; (ii) to analyze the characteristics of patients with virus-positive balf; and (iii) to identify the factors predictive of positive viral tests in balf. pontchaillou university hospital is a , -bed tertiary-care hospital which serves as a referral center for the area of rennes, france. all adults (≥ years of age) who had viral test(s) performed on balf during the year were included. cases were identified through the computerized database in the virology department. data were extracted from this database and from the medical records using a standardized questionnaire. the protocol was approved by our institutional review board (rennes university hospital ethics committee, project approval number . ), and informed consent was waived. the data collected included demographic information and co-morbidities. alcohol abuse was defined as daily consumption > units/day for men or > units/day for women. patients were classified as immunocompromised if they were infected with human immunodeficiency virus (hiv), were on systemic corticosteroids ≥ mg/day for at least weeks [ ] , or were on any immunosuppressive drug on admission. medical charts were checked for the following signs: fever > °c, cough, purulent sputum, hemoptysis, chest pain, dyspnea, and crackles on chest examination. radiographic patterns prior to bal were defined by the type of consolidation (alveolar or interstitial, nodules, micronodules, ground-glass attenuations, bronchiectasis, or excavation). other data collected included any antimicrobial treatment received during the week preceding bal, mechanical ventilation, intensive care unit (icu) admission, and in-hospital mortality. bal was performed according to the guidelines [ , ] , under local anesthetic (lidocaine spray): - ml of sterile saline solution was instilled - times into the distal bronchial tree, either at the site where radiographic abnormalities predominated or in the middle lobe in cases with diffuse radiographic pattern. balf specimens were aliquoted and immediately transported to laboratories. appropriate staining was carried out for the direct identification of bacteria, mycobacteria, fungi, and parasites. cultures for bacterial identification were inoculated under standard aerobic conditions on four different media, as well as on specific media for mycobacterium spp., when indicated. for the usual respiratory pathogens, the bacterial count was considered to be significant when quantitative culture yielded > cfu/ml of specimen. in addition, tests for atypical pneumonia agents were performed at the request of the physician in charge, on balf (i.e., pcr, immunofluorescence, specific staining, and culture on appropriate media) and by serology. the first sample of balf was used for viral tests, as it is the most likely to contain significant numbers of epithelial cells. the sample was mixed (volume : ) with viral transport medium, i.e., minimum essential medium enriched with sorbitol ( %), bovine serum albumin, streptomycin, vancomycin, colimycin, and amphotericin b. balf was first analyzed by immunofluorescence (if) with a panel of monoclonal antibodies against respiratory syncytial virus (rsv), a and b influenza viruses, - parainfluenza viruses, and adenovirus, as well as herpes simplex viruses (hsv)- and - , in immunocompromised patients. in parallel, balf was inoculated onto mrc- and llc-mk monolayer cells, and also onto mdck cells during the influenza season. in immunocompromised patients, an immunocytochemistry (icc) for cytomegalovirus (cmv) was performed. the detection of viral genomes was carried out according to the clinical context and a request from the physician in charge. hsv- , hsv- , cmv, varicella zoster virus (vzv), epstein-barr virus (ebv), and human herpesvirus (hhv)- were detected using a commercial method (herpes consensus®, argène, france). in addition, cmv, hsv- , hsv- , ebv, hhv- , and influenza a and b were detected with in-house pcr, adapted from previously described methods [ ] . neither pcr testing on nasopharyngeal swab/aspirates nor viral serology tests were routinely performed during the study period in our institution. statistical analyses were performed using sas software . (sas institute inc., cary, nc, usa). the results are presented as the mean ± standard deviation, with the range in parentheses. we compared the characteristics of patients with viruspositive versus virus-negative balf using student's t-test (n≥ ) or the mann-whitney test (n< ) for quantitative variables, and fisher's exact test for categorical variables. multiple comparisons were performed using analysis of variance with a bonferroni post hoc correction. multivariate analysis was performed using logistic regression models, after adjustment for the duration of stay before balf. variables with a p-value < . in univariate analysis were entered into the multivariate analysis. values of p< . were regarded as significant. in , balf samples were sent to the microbiology department in our institution. of these, viral tests were ordered in balf, from patients (fig. ) . the mean age of the patients was . ± . years (range, . - . ) and the male-to-female ratio was . ( / ). symptoms on admission included dyspnea ( . %), cough ( . %), fever ( . %, with a mean body temperature of . ± . °c (range, . - . ), purulent sputum ( . %), and hemoptysis ( . %). chest radiographic findings included consolidations ( . %), nodules ( . %), and excavation ( . %). chest computed tomography (ct) scanning, performed in patients, indicated ground-glass attenuations ( . %), micronodules ( . %), and bronchiectasis ( . %). for procedures ( . %), bal was performed in an icu, including procedures performed under mechanical ventilation. the mean volume of sterile saline serum instilled during the bal procedure was ± ml (range, . the use of viral transport medium was adequate for % of samples. of the bal investigated for viruses, ( . %) identified at least one virus, for a total of viruses (mean, . viruses per virus-positive balf). viral species identified, and the yields of the four techniques used, are detailed in table . of note, . % of viruses were members of the herpesviridae family. the most frequent virus associations were hsv- +hhv- (n ) and cmv + ebv (n ). one hiv-infected patient with interstitial pneumonia had three herpes viruses identified in the bal: cmv (icc), hsv- (viral culture), and ebv (pcr). pcr, performed in balfs, was positive in cases ( . %). of these, pcr was the only test positive for virus in cases, confirmed the diagnosis documented by other techniques in cases (hsv- , n ; cmv, n ; ebv + cmv, n ) and identified a virus different to that documented by other techniques in eight cases. had pcr not been used, the diagnostic yield would have been %. hence, pcr improved the diagnostic yield of viral tests on balf by %. testing for bacteria and mycobacteria was performed in balfs ( . %) and was positive in balfs comparisons between virus-positive and virus-negative balf are detailed in table (univariate analysis). the variables significantly associated with positive viral tests on univariate analysis were immunosuppression (i.e., hiv infection, corticosteroids > mg/day for ≥ weeks, and/or other immunosuppressive therapy), ground-glass attenuations on chest ct scans, late-onset ventilator-associated pneumonia (vap), and durations of (i) hospital stay, (ii) icu stay, and (iii) mechanical ventilation before bal was performed (p< . for each comparison). on multivariate analysis, after adjustment for the duration of stay before bal, only immunosuppression [odds ratio (or) . , % confidence interval (ci) [ . - . ] , p< . ) and ground-glass attenuations (or . , % ci [ . - . ], p . ) remained significantly associated with virus-positive bal. retrospective analysis of medical charts allowed us to classify indications for the viral analysis of balf into eight categories (table ) and to estimate the diagnostic yield of viral tests in each subgroup. striking differences were observed: for example, the proportion of virus-positive balf was . % in immunocompromised patients, as compared to . % in immunocompetent patients (p< . ). none of the bal performed for the initial assessment of diffuse infiltrative lung disease was virus-positive. associations between viral analysis of bronchoalveolar lavage (bal) and outcomes ( . %) received an antiviral agent: aciclovir (n ), ganciclovir (n ), valganciclovir (n ), or foscarnet (n ). icu patients with virus-positive balf were more likely to be treated than non-icu-patients with virus-positive balf ( % vs. %, p . ). there was no significant association between antiviral treatment and outcome in patients with virus-positive bal. this observational study evaluated the diagnostic yield of viral tests on balf when requested by the physician in charge. of the consecutive balf tested for viruses, ( %) were positive. previous studies have estimated the diagnostic yield of viral tests from balf in different settings, and the proportion of virus-positive balf ranged from to . %, depending on the population studied (e.g., immunocompromised, icu patients) and the viral techniques used (e.g., pcr, icc, if, culture) [ - , , ] . a broad range of respiratory diseases have been associated with viral infections. hence, testing for viruses in balf may be considered in patients with a wide spectrum of clinical and radiological abnormalities [ , ] . however, our study suggests that viral tests are unlikely to return positive except in two, nonexclusive, situations: (i) immunocompromised patients; (ii) bilateral ground-glass attenuations on ct scan. in this study, the vast majority of viruses detected belong to the herpesviridae family, mainly hsv- ( . % of all [ ] . cmv replication was observed in the plasma of onethird of cmv-seropositive patients with vap after - days of mechanical ventilation, and lung involvement was documented by cmv-related cyto-pathogenic effect in - % of patients [ ] [ ] [ ] . all these studies found that hsv and cmv in balf are associated with increased morbidity and/ or mortality. however, the causal relationship between hsv or cmv in balf and patient outcomes cannot be ascertained from these observational studies. among the rapid tests currently available, pcr is one of the most valuable, the results being available within hours with high sensitivity, especially in immunocompromised patients [ ] . multiplex pcr tests, with their ability to detect several viruses in one set, may be particularly interesting in the diagnostic workup of acute respiratory diseases suspected to be of viral origin [ ] [ ] [ ] . in our study, the diagnostic yield of viral tests would have dropped from to %, had pcr tests not been performed. this advocates for the systematic use of pcr techniques for viral tests in balf, in accordance with previous studies [ , ] , in the situations where viruses may reasonably be suspected (i.e., acute lower tract respiratory disease in immunocompromised patients and/or patients with unexplained bilateral ground-glass attenuations on ct scan). on the other hand, the initial assessment of immunocompetent patients with interstitial lung disease should not include any viral test on balf, as previously reported in patients with interstitial fibrosis [ ] . during the exacerbation of idiopathic fibrosis, viral tests on balf may be of higher value, although recent papers have questioned their clinical significance [ , ] . this study has limitations related to its retrospective, monocentric, and observational design, as investigations on balf were not protocolized, and the request for viral testing was left to the discretion of the physician in charge. firstly, the retrospective review of medical charts identified a significant proportion of patients who were unlikely to suffer from viral infections, and for whom viral tests should not have been performed. in contrast, among patients not included in this study as no viral test was requested, there probably were patients who would have benefited from viral tests on balf. however, the comparison of the patients who had balfs tested for viruses in and a random selection of patients who had balf samples not tested for viruses during the same year found that the only significant differences were immunodepression ( % vs. %, p< . ) and ground-glass attenuations on ct scan ( % vs. %, p< . ). this suggests that clinicians are aware of the situations most likely to be associated with the presence of viruses, and that they appropriately select the patients in whom viral tests are more likely to return positive. lastly, pcr was performed in only of balf investigated for viruses. more systematic use of pcr tests in these patients may have increased the proportion of viruses identified. secondly, the identification of virus in balf during the diagnostic workup of a respiratory disease does not systematically imply that the virus is responsible for the disease and that the patient will improve with appropriate antiviral treatment. despite these limitations, this observational study allowed us to identify categories of respiratory diseases where viral tests are very unlikely to return positive (e.g., initial assessment of immunocompetent patients with interstitial pneumonia or pulmonary micronodules). as a consequence, indications for viral tests in balf were dramatically reduced for these patients in our institution. in conclusion, testing for viruses on balf should be mostly restricted to acute lower tract respiratory disease in immunocompromised patients and/or patients with unexplained groundglass attenuations on ct scan, especially when pcr tests are used. acknowledgments the authors thank jean-marc malecot for his statistical advice. the authors declare that they have no conflict of interest. ards acute respiratory distress syndrome a patients were classified as immunocompromised if they were infected with human immunodeficiency virus (hiv), were on systemic corticosteroids ≥ mg/day for at least weeks, or were on any immunosuppressive therapy b clinical or radiological deterioration in immunocompetent patients with infiltrative lung disease (n ), chronic cough (n ), post-surgical atelectasis (n ), pulmonary abscess (n ), unexplained hyperleukocytosis under mechanical ventilation (n ), initial assessment for pulmonary nodules (n ), systemic granulomatosis (n ), or screening before bone marrow allograft in a patient with ill-defined pulmonary abnormalities (n ) a persistent challenge: the diagnosis of respiratory disease in the non-aids immunocompromised host virological diagnosis in community-acquired pneumonia in immunocompromised patients viral community-acquired pneumonia in nonimmunocompromised adults lower respiratory viral illnesses: improved diagnosis by molecular methods and clinical impact respiratory viruses in bronchoalveolar lavage: a hospital-based cohort study in adults acute exacerbations of copd: identification of biological clusters and their biomarkers role of viral respiratory infections in asthma and asthma exacerbations importance of viral and 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prospective study monitoring of herpes simplex virus in the lower respiratory tract of critically ill patients using real-time pcr: a prospective study herpes simplex virus lung infection in patients undergoing prolonged mechanical ventilation cytomegalovirus reactivation in critically ill immunocompetent patients cytomegalovirus infection in critically ill patients: a systematic review active cytomegalovirus infection is common in mechanically ventilated medical intensive care unit patients simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays rapid identification of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-pcr: feasibility study evaluation of a multiplex reverse transcriptase pcr elisa for the detection of nine respiratory tract pathogens role of flexible bronchoscopy in immunocompromised patients with lung infiltrates herpesviruses detection by quantitative real-time polymerase chain reaction in bronchoalveolar lavage and transbronchial biopsy in lung transplant: viral infections and histopathological correlation viral infection in acute exacerbation of idiopathic pulmonary fibrosis acute exacerbations of idiopathic pulmonary fibrosis key: cord- -de dhv b authors: seitsonen, e.; hynninen, m.; kolho, e.; kallio-kokko, h.; pettilä, v. title: corticosteroids combined with continuous veno-venous hemodiafiltration for treatment of hantavirus pulmonary syndrome caused by puumala virus infection date: - - journal: eur j clin microbiol infect dis doi: . /s - - -z sha: doc_id: cord_uid: de dhv b reported here are two cases of hantavirus pulmonary syndrome caused by puumala virus infection, which rapidly resolved after initiation of corticosteroid treatment combined with continuous veno-venous hemodiafiltration. these cases emphasize the role of the inflammatory response in the pathogenesis of hantavirus pulmonary syndrome. puumala virus (puu) is a hantavirus that is endemic in northwestern europe, the balkans, and western russia [ ] . it is carried and spread by the bank vole. puumala virus infection is usually either asymptomatic or causes a mild form of hemorrhagic fever with renal syndrome. the incidence in finland is / , . the mortality rate is low ( . - . %) [ ] . the most common signs and symptoms are fever, headache, myalgia, back pain and gastrointestinal symptoms, often followed by an oliguric phase with proteinuria and hematuria, and later a polyuric phase. transient visual impairment (myopia) is a pathognomonic sign. hemorrhagic manifestations are present in about % of cases. however, mild mucosal bleeding is very common [ ] . spontaneous recovery usually takes place in - weeks [ ] . of the hospital-treated patients, about % require transient hemodialysis [ ] , and over % have pulmonary abnormalities. most often these relate to both the inflammation-associated capillary permeability disorder and fluid retention caused by renal failure [ ] . however, a few cases of acute non-cardiogenic pulmonary edema, similar to the hantavirus pulmonary syndrome (hps) encountered on the american continent, have been described in europe in association with puu and other hantavirus species [ ] [ ] [ ] [ ] . we describe two cases of puu-associated hps, in which administration of intravenous corticosteroids combined with continuous veno-venous hemodiafiltration (cvvhdf) was followed by rapid clinical improvement. both patients gave their consent to publication of the data. the first day of fever is defined as day post-onset of symptoms (pos). a -year-old male patient was admitted to a district hospital in southern finland in february with a -day history of fever, upper abdominal pain and melena. an infectious infiltrate was suspected in the right lung, and an enlarged spleen was detected on abdominal ultrasound. creactive protein (crp) level was mg/l, platelet count × /l, leukocyte count . × /l, and hematocrit %. the qualitative urinalysis showed proteinuria. intravenous antimicrobial agents were started for suspected community-acquired pneumonia. urine output began to decrease, and serum creatinine increased from to μmol/l. two blood cultures on consecutive days were negative. the patient became hypotensive, and oxygenation deteriorated. the patient was transferred to a university hospital on day pos. echocardiography showed normal left ventricular function. the patient needed continuous ventilation with a positive airway pressure mask and % inspiratory oxygen fraction, and he was transferred to the intensive care unit (icu). diffuse bilateral perihilar infiltrates were present on chest radiograph (fig. ) . crp level was mg/ l, hematocrit %, platelet count × /l, leukocyte count × /l, creatinine μmol/l, blood urea nitrogen . mmol/l, potassium . mmol/l, and lactate . mmol/l. troponin-t and creatine-kinase mb mass were within normal limits. due to poor oxygenation and oliguria, the patient required endotracheal intubation and cvvhdf. initially, the ratio of arterial oxygen tension to inspired oxygen fraction (pao /fio ratio) was mmhg, and intermittent prone positioning was used for days to improve gas exchange. ceftriaxone ( g/day) and fluconazole ( mg/day) were given as antimicrobial treatment. the blood morphological examination showed both neutrophilia and lymphocytosis, including atypical lymphocytes, and a variable degree of maturation. on day pos, puu-igm was positive by μ-capture enzyme-linked immunosorbent assay based on baculovirus-expressed rn-antigen [ , ] . puu-igg was detected with fluorescein isothiocyanate-conjugated sheep antihuman igg, and demonstrated a granular fluorescence pattern associated with antibodies against the puu nucleocapsid protein, typical of a recent infection [ , ] . on day pos, the pao /fio ratio was still less than mmhg with mmhg pulmonary artery occlusion pressure, and intravenous methylprednisolone ( mg/day in three doses) was started. on the following days, the pao /fio ratio rapidly stabilized at over mmhg. the evolution of the pao /fio ratio and the lung injury score is shown in fig. a ,b. the patient required vasoactive support for days. the minimum urine output was ml on day pos. cvvhdf was discontinued on day pos. the calculated dialysis and total fluid balances are shown in table . the lowest platelet count was × /l on day pos, the highest leukocyte count . × /l on day pos, and the highest crp mg/l on day pos. the evolution of crp values and platelet counts is shown in table . blood cultures taken on days and pos were negative. the patient was extubated on day pos, and discharged from the icu on day pos. methylprednisolone was tapered off and switched to oral prednisone. on day pos, left laterobasal infiltration was still visible on chest radiograph. on that day, proteinuria was . g/ h. the levels of c and c were normal, and there was no indication of c subtype insufficiency. the patient's hla genotype was b drb * . the patient confirmed there were signs of vole infestation in the yard and shed of his home. he had not recently traveled abroad. a -year-old male patient came to a university hospital in southern finland in august for treatment of upper abdominal pain. his symptoms had started days previously with high fever, fatigue, and myalgia. he had vomited once and had diarrhea in which he detected traces of blood. on day pos, his crp level was mg/l, platelet count × /l, leukocyte count . × /l, hematocrit %, sodium mmol/l, and alanine aminotransferase u/l. the chest radiograph showed bilateral perihilar infiltrates, and the patient was started on ceftriaxone ( g/day) and levofloxacin ( g/day). he received supplemental oxygen via nasal cannulae. abdominal radiograph and ultrasound examinations were normal. on day pos the lung infiltrates had increased considerably (fig. a,b) , and the patient required continuous ventilation with a positive airway pressure mask. he became oliguric, proteinuria . g/ h was detected, and his creatinine level increased from to μmol/l. the patient's platelet count decreased to × /l, and he experienced visual disturbances. he confirmed that days before the onset of symptoms he had visited a summer cottage, where exposure to vole secretions was possible. he had not recently traveled abroad. the patient was transferred to the icu due to respiratory insufficiency and renal failure. oxygenation deteriorated rapidly, and he required endotracheal intubation and mechanical ventilation. the lowest pao /fio ratio was mmhg on day pos, and intermittent prone positioning was continued until day pos. the patient required a low dose of vasopressor support for days. on day pos, puu-igg-and puu-igm-antibodies were positive by the same methods described for case , and igg-ifa showed the typical granular fluorescence pattern seen in the acute phase of immunity. metabolic acidosis was detected, the patient's creatinine level increased to μmol/l and blood urea nitrogen to . mmol/l. cvvhdf was started on day pos. as the patient was severely ill, and a concomitant bacterial infection could not be ruled out, antibiotics were continued until crp began to decrease on day pos. the evolution of crp values and platelet counts is shown in table . blood cultures taken on days and pos were negative. on day pos the pao /fio ratio was still - mmhg with mmhg pulmonary artery occlusion pressure, and intravenous methylprednisolone ( mg/day in three doses) was started. the evolution of the pao /fio ratio and the lung injury score is shown in fig a,b . the minimum urine output was ml on day pos. cvvhdf was discontinued on day pos. the calculated dialysis and total fluid balances are shown in table . the patient was extubated on day pos. he was discharged from the icu on day pos. on that day, left mediobasal and right basal atelectasis and slight bilateral pleural effusion were visible on chest radiograph. the methylprednisolone dose was tapered, and an oral preparation was substituted on day pos. the patient was discharged home on day pos. the patient lacked one of the c b-type genes. his hla genotype was b , drb * , . we describe two cases of puu-infected patients who presented with both renal and respiratory failure requiring renal replacement therapy and mechanical ventilation. radiologically, both patients had alveolar infiltrates separate from pulmonary vascular congestion, and no prominent cardiomegaly. nevertheless, fluid inevitably accumulated in the lung during the disease process. most likely, both the corticosteroid treatment and the negative fluid balance achieved during cvvhdf treatment and recovery of renal values in bold type indicate the day when corticosteroid treatment was started (days and pos for cases and , respectively) function contributed to the subsequent improvement in gas exchange. in hps, a febrile prodrome is followed by a severe increase in pulmonary vascular permeability and myocardial depression. the syndrome progresses very rapidly from interstitial pulmonary edema to a clinical picture resembling adult respiratory distress syndrome (ards). the alveolar infiltrates are typically central or bibasilar, and pleural effusion is common [ ] . the hantaviruses are not cytopathic, and the increased capillary permeability is likely caused by immunologic factors [ ] . in pathological specimens of hps-afflicted lungs, intra-alveolar edema and interstitial lymphoid cell infiltration are observed, the pneumocytes are preserved, and neutrophils are notably scarce [ , ] . the endothelial cells are also largely intact, and the alveolar walls are thickened due to extravasation [ ] . activation of t-lymphocytes and cytokine production appear to be involved in both hps and hemorrhagic fever with renal syndrome [ , ] . terajima et al. [ ] demon-strated that at the onset of pulmonary edema, hps patients have abundant viremia which clears rapidly after the resolution of fever, while respiratory distress may persist. the increase in alveolocapillary permeability in acute lung injury results from the inflammatory response [ , ] . cortisol acts in concert with catecholamines to maintain vascular tone and endothelial integrity, and it suppresses the inflammatory cascade by down-regulating the production of proinflammatory molecules [ ] . animal studies on the efficacy of corticosteroids in acute lung injury have produced mixed results [ , ] . the american-european consensus conference on ards stated that corticosteroids are not generally useful in the early management of sepsis and ards [ ] . early or prophylactic use of high-dose corticosteroid therapy in septicemia and ards may even be harmful [ ] . the consensus conference acknowledged, however, that corticosteroids may be of value in treating certain ards variants [ ] . currently, there is no universally accepted drug treatment for the high permeability edema associated with acute lung injury/ards in humans [ ] . an overaggressive inflammatory response may be an important factor in the outcome of ards patients [ ] . late corticosteroid treatment in sustained lung injury has been shown to inhibit proinflammatory activity and to improve lung function and possibly outcome [ ] . in the series of seven patients with puu-associated hps described by clement et al. [ ] , only one patient required mechanical ventilation. the course of mechanical ventilation was prolonged ( days), but the patient made a full recovery. the treatment of the european hps cases described by launay et al. [ ] and klempa et al. [ ] was also mainly supportive (i.e., no mechanical ventilation was required), and the patients recovered without sequelae. the patient with dobrava virus-associated hps reported by schütt et al. [ ] received both cvvhdf and prolonged mechanical ventilation ( days), and eventually recovered. in a series of andes virus-associated hps cases in chile, five patients, all of whom were among the survivors, received corticosteroid treatment along with supportive treatment, but the efficacy of corticosteroid treatment could not be determined due to the low number of cases [ ] . in the series of hps cases described by hallin et al. [ ] , antimicrobial and supportive treatment was given. the two table platelet counts (pc, × /l) and c-reactive protein values (crp, mg/l) in cases and on consecutive days pos during the period of icu treatment (days - pos and - pos for cases and , respectively) survivors who required mechanical ventilation were extubated after days and made a full recovery. renal histology in nephropathia epidemica is characterized by tubulointerstitial nephritis with mixed interstitial cellular infiltration and edema, tubular epithelial changes and occasional medullar hemorrhages [ ] . in a recent retrospective survey of mainly drug-induced acute interstitial nephritis, no effect of corticosteroid treatment on renal recovery was found as compared with supportive therapy alone [ ] . the haplotype hlab dr dq is associated with a severe form of nephropathia epidemica [ ] . this haplotype is also associated with a high level of tnf-α production [ ] and deletion of the c a gene [ ] . the hlab dr haplotype is known to be associated with several chronic autoimmune diseases and immunologic abnormalities. neither of our patients had that hla haplotype, but one of them lacked one of the c b genes, leading to lowered levels of c b and possibly altered immune function. as far as we know, this report is the first to describe the effects of corticosteroid treatment for puu infection complicated by severe hypoxemia, consistent with the ards criteria. in our two patients, the implementation of intravenous corticosteroids combined with cvvhdf was associated with resolution of both respiratory failure and shock. we suggest that corticosteroid therapy is safe and may hasten recovery in severe cardiopulmonary forms of puu infection. hantavirus infections in europe pulmonary involvement in nephropathia epidemica: radiological findings and their clinical correlations hantavirus pulmonary syndrome in new england and europe pulmonary-renal syndrome due to hemorrhagic fever with renal syndrome: an unusual manifestation of puumala virus infection in france life-threatening dobrava hantavirus infection with unusually extended pulmonary involvement occurrence of renal and pulmonary syndrome in a region of northeast germany where tula hantavirus circulates antigenic properties and diagnostic potential of puumala virus nucleocapsid protein expressed in insect cells evaluation of puumala virus igg and igm enzyme immunoassays based on recombinant baculovirus-expressed nucleocapsid protein for early nephropathia epidemica diagnosis human b-cell epitopes of puumala virus nucleocapsid protein, the major antigen in early response human immune response to puumala virus glycoproteins and nucleocapsid protein expressed in mammalian cells viral pneumonias in adults: radiologic and pathologic findings spectrum of hantavirus infection: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome hantavirus pulmonary syndrome: a clinical description of patients with a newly recognized disease. the hantavirus study group cardiopulmonary manifestations of hantavirus pulmonary syndrome hantavirus pulmonary syndrome: radiographic findings in patients high levels of cytokineproducing cells in the lung tissues of patients with fatal hantavirus pulmonary syndrome high levels of viremia in patients with the hantavirus pulmonary syndrome ventilatory, pharmacologic, supportive therapy, study design strategies and issues related to recovery and remodeling vascular pharmacology of acute lung injury and acute respiratory distress syndrome anti-inflammatory therapy for acute lung injury. a review of animal and clinical studies dexamethasone and pentastarch produce additive attenuation of ischaemia/ reperfusion lung injury early methylprednisolone treatment for septic syndrome and the adult respiratory distress syndrome persistent elevation of inflammatory cytokines predicts a poor outcome in ards. plasma il- β and il- levels are consistent and efficient predictors of outcome over time effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome. a randomized controlled trial hantavirus pulmonary syndrome due to andes virus in temuco, chile: clinical experience with adults cytokines, adhesion molecules, and cellular infiltration in nephropathia epidemica kidneys: an immunohistochemical study acute interstitial nephritis: clinical features and response to corticosteroid therapy genetic susceptibility to severe course of nephropathia epidemica caused by puumala hantavirus highproducer allele of tumour necrosis factor-alpha is part of the susceptibility mhc haplotype in severe puumala virus-induced nephropathia epidemica acknowledgement this report was supported by a hus research grant. key: cord- -qkw ik e authors: blahová, j.; králiková, k.; krčméry sr., v.; bartoníková, n. title: high-frequency transduction of antibiotic resistance in pseudomonas aeruginosa by a wild-type bacteriophage with restricted specificity for recipient strains date: journal: eur j clin microbiol infect dis doi: . /s sha: doc_id: cord_uid: qkw ik e nan pseudomonas aeruginosa by a wild-type bacteriophage with restricted specificity for recipient strains j. blahová, k. králiková, v. krčméry sr., n. bartoníková antibiotic resistance can be transferred among strains of pseudomonas aeruginosa by conjugation [ ] , transduction by wild-type phages as well as by generalised transducing phages, such as f- or g- [ ] [ ] [ ] [ ] , or by transposition of integrons [ ] . while determinants of resistance can be transferred by conjugation from pseudomonas aeruginosa to, for example, escherichia coli or proteus mirabilis recipient strains, transduction by bacteriophages is a species-specific event. nevertheless, it could be also an important factor contributing to the dissemination and spread of antibiotic resistance among pseudomonas aeruginosa in clinical settings [ ] . in this report we describe several properties of a new wild-type bacteriophage (no. ap- ) from a ceftazidime-resistant strain of pseudomonas aeruginosa (no. ) isolated from the urine of a patient hospitalised in the intensive care unit of a large teaching hospital (bata's hospital) in zlín, czech republic. this phage, in contrast to bacteriophages described previously, exhibited two rather peculiar properties: the frequency of transduction was unusually high ( - to - in comparison with, for example, - to - for phages , , and [ ] ), and the phage ap- was lytic to a single recipient strain, i.e., the pao strain, and showed no evidence of a lytic reaction to ml recipient strains, i.e., to pseudomonas aeruginosa ml- , ml- , or ml-m- . nevertheless, it transduced genes of antibiotic resistance to both pao and ml series of strains. plaques of phage lysis were detected on the surface of antibiotic-containing media during testing for multiple drug resistance in a donor strain of pseudomonas aeruginosa . isolation of the bacteriophage (ap- ) and preparation of a wild-type phage lysate were performed as described previously [ , , ] . transduction procedures were performed as described in detail previously [ , , ] . four auxotrophic recipient strains of pseudomonas aeruginosa were used in transduction systems: pseudomonas aeruginosa pao- (ade-leu-rifc), pseudomonas aeruginosa ml- (trp-leu-arg-ile-val-his-rifc), pseudomonas aeruginosa ml- (trp-met-ile-val-his-), and pseudomonas aeruginosa ml-m- (leu-trp-strc). auxotrophic recipient strains were obtained courtesy of prof. s. mitsuhashi, maebashi, japan. all four recipient strains were highly susceptible to all antibiotics used for selection of transductants (e.g., mic of cefotaxime, . mg/ml; mic of ceftazidime, . to . mg/ml), and they were used as h shake cultures in nutrient broth (difco, usa) (eventually adjusted by centrifugation to a density of ! cfu/ml). the sterile phage lysate ap- was added to each recipient strain in an amount sufficient to obtain a multiplicity of infection (moi) of . pfu/cell. the time allowed for phenotypic expression of transduced determinants was min. the surface of antibiotic-containing plates (with mg/ml of streptomycin, kanamycin, or carbenicillin, or mg/ml of cefotaxime, ceftazidime, or aztreonam) was then inoculated with a mixture of bacteria plus the phage. identical plates were inoculated in parallel with the control (recipient bacteria only, without phage added). antibiotic-containing plates were then incubated at c for h and h, and number of transductants was recorded. transductants were then picked from each experimental plate and examined by an indirect selection procedure for their complete spectra of transduced resistance. in contrast to other wild-type phages isolated from multiple-drug-resistant nosocomial strains of pseudomonas aeruginosa [ , , ] , the phage lysate of pseudomonas aeruginosa showed a lytic reaction when added, in dilution of up to - , to the recipient strain pseudomonas aeruginosa pao (figure ) but not to the other strains. this phage was not lytic for ml strains, but we thought it might be a good transducing phage for this series of strains. transduction experiments confirmed this hypothesis. a high frequency of transduction of resistance to all blactam antibiotics tested and to kanamycin was observed for all four recipient auxotrophic mutants of pseudomonas aeruginosa pao and ml examined. genes coding for resistance to ceftazidime, aztreonam, and cefotaxime were transduced to pseudomonas aeruginosa pao- , ml- , and ml-m- at frequencies of - to - ( figure ) , which was two logarithms higher than the highest frequency obtained with the phages ap- , ap- , and ap- ( - for transduction of carbenicillin or imipenem resistance to pseudomonas aeruginosa m- ) and four logarithms higher than the frequency of transduction of imipenem resistance to pseudomonas aeruginosa pao- [ ] . transfer of resistance to kanamycin was found to occur at a frequency of - to - . analysis of transductants, selected on any of the single antibiotic media used, showed that all transductants were co-resistant to all five antibiotics, i.e., ceftazidime, aztreonam, cefotaxime, carbenicillin, and kanamycin. the presence of various bla genes on a single integron with genes coding the resistance to kanamycin or other antibacterial agents was demonstrated in nosocomial strains of pseudomonas aeruginosa and of other gramnegative bacteria [ , , ] . in conclusion, the results obtained in transduction experiments with the wild-type bacteriophage ap- indicate a relation between the lytic and the transducing capacities of a phage preparation. if such a relation is confirmed, the possibility exists that some phages might actually remain undetected in nosocomial strains of pseudomonas aeruginosa. furthermore, it could be demonstrated that some phages, including phage ap- described here, transduce genes of antibiotic resistance, mainly in a block of resistance determinants, to several recipient strains at an unexpectedly high frequency, which might play an important role in the dissemination of antibiotic resistance among nosocomial strains of pseudomonas aeruginosa. transferable resistance to specific antibiotics in nosocomial strains of pseudomonas aeruginosa from two teaching hospitals cross-resistance to meropenem, cephems and quinolones in pseudomonas aeruginosa ceftazidime resistance in pseudomonas aeruginosa: transduction by a wild-type phage transduction of imipenem resistance by the phage f- from a nosocomial strain of pseudomonas aeruginosa in slovakia transduction of antibiotic resistance including imipenem resistance in nosocomial pseudomonas aeruginosa strains by wild-type phages insertion of a carbapenemase gene casette into an integron of a pseudomonas aeruginosa plasmid the genetic organisation of arginine biosynthesis in pseudomonas aeruginosa resistance of pseudomonas aeruginosa pao to nalidixic acid and low-levels of beta-lactams: mapping of chromosomal genes transduction of imipenem resistance by wild-type bacteriophages from three pseudomonas aeruginosa strains from a single clinical source plasmid-mediated dissemination of the metallo-blactamase gene bla imp among clinically isolated strains of serratia marcescens molecular characterization of an enterobacterial metallo-b-lactamase found in a clinical isolate of serratia marcescens that shows imipenem resistance akan department of microbiology, faculty of medicine - % of acute and chronic hepatitis cases are of unknown etiology. recently, two novel hepatitis viruses, the hepatitis g virus (hgv) and the gb virus c (gbv-c), were independently isolated by two different research groups [ ]. in fact, hgv and gbv-c are separate isolates of the same virus hgv is a single-stranded, positive-sense rna virus that belongs to the flaviviridae family hgv has been found in % of patients with acute non-a-e hepatitis, in - % of those with chronic non-a-e hepatitis, in - % of those with fulminant hepatitis, and in - % of those with cryptogenic cirrhosis this study was performed to determine the prevalence of hgv infection among patients with chronic hepatitis in our area using the nested reverse transcriptase polymerase chain reaction eighty-seven patients with chronic hepatitis were included in the study. the diagnosis of chronic hepatitis key: cord- -j hnoag authors: clement, j.; maes, p.; lagrou, k.; van ranst, m.; lameire, n. title: a unifying hypothesis and a single name for a complex globally emerging infection: hantavirus disease date: - - journal: eur j clin microbiol infect dis doi: . /s - - -y sha: doc_id: cord_uid: j hnoag nan in the january issue of this journal, swedish authors described three severe cases of predominantly pulmonary infection with the european hantavirus puumala virus (puuv), leading to death due to refractory shock in two of these patients [ ] . of note, the kidneys in these two fatal cases had, on autopsy, no prominent inflammatory infiltrates or haemorrhages, in contrast with the lungs which showed extensive interstitial oedema and mononuclear cell infiltrates, mainly cd + t lymphocytes. as the authors justly point out, even in the title of their communication, it is now, perhaps, time to revise the sacro-saint paradigm existing since of two "different" infectious diseases caused by the same genus of rodent-borne hantaviruses of the old and the new world, respectively. the former would target mainly the human kidney, and the latter mainly the human lung, resulting in the so-called "haemorrhagic fever with renal syndrome" (hfrs) [ ] or the "hantavirus pulmonary syndrome" (hps) [ ] . when it appeared that the failing heart was, in fact, the most direct cause of death in refractory shock in hps, yet another name, "hantavirus cardio-pulmonary syndrome" (hcps), was added to the already bewildering list of over mostly exotic synonyms for a disease described already in by russian doctors in vladivostok. on the other side of the eurasian landmass and in , swedish doctors described an epidemic renal affection which they called "nephropathia epidemica" (ne), which was proven in to be, in fact, a milder variant of hfrs, caused by puuv [ ] . to date, distinct species of hantaviruses have been recognized, each carried by their more or less specific rodent or insectivore reservoir. the most important pathogens are hantaan (htnv) and seoul virus (seov) in the far-east (where > % of worldwide hantavirus infections occur), puuv and dobrava-belgrade virus (dobv) in europe and russia, and sin nombre (snv) and andes virus (andv) in the americas [ ] . the first hps report that appeared in [ ] described "a newly recognized disease" which was of paramount importance for a better understanding of an emerging viral infection in the new world, and generated an explosive surge in hantavirus research. however, h(c)ps was, and remains, a rare disease, with only confirmed cases in the usa during the - period [ ] (meaning cases/year) and about , cases in the entire americas, albeit with a much higher case fatality rate (cfr) of % [ ] . this is in stark contrast with the current . - . % cfr for puuv infections with high morbidity in the old world, with now over , registered ne cases in europe, and even over , in western russia. in some peak years, russia witnessed more than , cases/year (e.g. , in ) , whereas registered ne numbers in western europe recently took epidemic proportions (e.g. , in finland, , and , in germany, ), probably as a result of global warming [ ] . thus, it should not come as a surprise that russian or european clinicians had or have now identified clinical characteristics claimed as being specific for "american hps". the centers for disease control and prevention (cdc), atlanta, ga, usa, propose a total of eight distinctive clinical criteria for its "hantavirus pulmonary syndrome case report form" [ ] : ( ) fever, ( ) thrombocytopaenia, ) ) elevated haematocrit (hct), ( ) elevated serum creatinine, ( ) left shift leukocytosis with a high percentage of "atypical lymphocytes", ( ) need for supplemental oxygen, ( ) need for intubation (and mechanical ventilation), and ( ) chest rx showing unexplained bilateral infiltrates or being suggestive of acute respiratory distress syndrome (ards). it is gratifying to see that at least one pivotal renal parameter is now used for the diagnosis of a "pulmonary syndrome". indeed, hps was originally announced as having little or no renal involvement [ , ] , but increased levels of serum creatinine are now confirmed as being present in % of all hps cases in the usa and even in % of fatal cases [ ] . furthermore, when it is generally agreed that cdc criteria to are encountered in virtually all moderate to severe hantavirus infections worldwide, it can be questioned, as was done already years ago [ ] , whether criteria to really are to be considered as truly unique to the american situation. russian authors reported proven puuv infections in five young and previously healthy patients, without any elevated creatinine levels or even proteinuria, without oliguria or polyuria, but with bilateral infiltrates on rx chest in / cases and pleural effusion in / cases. these authors, working in one of the hotspots of puuv endemicity in western russia, suggested already in that different clinical entities may exist, one of these being (we quote) "a quite severe febrile condition without renal involvement" [ ] . while it seems acceptable that old, respectively new world pathogens might have a somewhat different "organ tropism", and, hence, a somewhat different clinical picture, it is unlikely that these genetically related viruses should be radically different pathogens, as originally stated [ , ] . moreover, hantavirus pathogenicity is not determined by the negligible viral tissue damage, but, rather, by the immunological response of the human host, the so-called "cytokine storm" [ , ] . in a series of clinical observations and in vitro experiments, hayasaka et al. and terajima and ennis suggested that increased capillary permeability observed in both h(c)ps and hfrs may be caused by a common immunopathological mechanism, i.e. hantavirus-specific cytotoxic cd + t lymphocytes, attacking endothelial cells presenting viral antigens on their surface [ , ] . in hindsight, it may now seem a bit premature that some authors, without having seen the patients but confronted in with the first reports of "european hps", should dismiss these forms merely as a complication of oligo/ anuria, leading to lung oedema secondary to fluid overload, and, hence, a form of right-handed cardiac failure [ ] . first, ne cases sometimes presented with clear lung symptoms well before oligo/anuria and fluid overload were properly initiated. one of the earliest recorded ( ) belgian ne cases involved a -year-old female presenting with °c fever, cough, dyspnoea, bibasilar lung infiltrates, hypoxaemia (partial pressure of arterial oxygen [pao ] of mm hg), hypocapnia and a severely restricted lung diffusion capacity (carbon monoxide diffusion capacity, % of the predicted value), necessitating o supplementation. this form of symptomatic viral ards without oliguria could not be clinically explained by fluid overload, nor by the very mild degree of subsequent renal involvement (serum creatinine level at admission . , peak value only . mg/dl), and showed a spontaneous remission [ ] . second, during the first ( ) ne epidemic in belgium, a total of seven cases suffered from hypoxia (spread pao to mm hg) and hypocapnia (paco to mmhg). in all of these cases, fluid overload was excluded on the basis of normal values of continuously monitored central venous pressure (cvp) [ ] . in the most severe case (pao and paco mm hg), needing both dialysis and life-saving mechanical ventilation, signs of cardiac decompensation were excluded by swan-ganz catheter monitoring (pulmonary capillary wedge pressure mm hg, pulmonary artery pressure / mm hg) [ ] . third, this same patient had a low cardiac index ( . l/min/m ), a systemic vascular resistance of . dyn s cm - and extreme thrombocytopaenia ( , /mm ), followed by disseminated intravascular coagulation (dic), all of which are signs considered as typical for hps according to cdc guidelines [ , ] . other early european reports documenting more severe pulmonary rather than renal involvement, and implicating hantaviruses distinct from puuv, are available as well. the first isolation in europe of a murine htnv-like hantavirus (in hindsight, probably dobv) and dated , was from the urine of a -year-old greek soldier, infected during manoeuvres in porogia (northern greece) and suffering from acute pulmonary oedema, and later from acute renal failure (arf), prompting both life-saving ventilation and acute dialysis [ ] . on admission, this case fulfilled all eight current cdc criteria for hps, with fever of °c, severe thrombocytopaenia and even dic, hct of %, wbc of , /mm with % "large lymphocytes" and pulmonary oedema initially even resistant to massive fluid extraction during dialysis. this patient had an admission cvp of only cm of h o, making the hypothesis of secondary overwhelming fluid overload highly improbable. a -year-old british soldier stationed in bosnia as a member of the united nations protection force (unprofor) was reported as a sudden severe case of wild rat-transmitted seov (though, in hindsight, probably dobv)-induced ards followed by arf, urgently needing intubation and ventilation in a local military field hospital, emergency repatriation by air while on positive-pressure o supplementation, followed by intensive care therapy in london, uk [ ] . another dobv case in northern germany developed life-threatening pulmonary oedema days before any renal impairment [ ] . a first hfrs case associated with tula virus infection, presenting both renal and pulmonary involvement, was documented in northern germany [ ] . thus, and as predicted [ ] , there is increasing evidence of a considerable overlap of symptoms in both syndromes. moreover, symptoms are rarely complete: in puuv infections, since only a tiny minority (probably less than %) develops symptoms severe enough for a hospitalisation [ ] , whereas for a local h(c)ps agent like choclo virus in panama, pauci-or asymptomatic seroconverters outnumbered full-blown h(c)ps cases by to in a recent study [ ] . should we continue to call symptomatic new world hantavirus infections without any pulmonary involvement [ , ] "h(c)ps", or the many old world infections without much renal involvement still "hfrs"? and how to name the fatal cases with both severe arf and hypoxia, prompting ventilation and dialysis, as recently found in india, and probably caused by a "new" hantavirus [ ] ? these denominations become utterly paradoxical if we learn, for instance, that % of south-american andv-induced "pulmonary syndromes", in fact, need dialysis for acute treatment [ ] , whereas only % or even less of european "nephropathia" cases needs the same [ ] . moreover, russian scientists, reluctant to use the european denomination ne for puuv infections in their own country, prefer to stick to the official world health organization (who) name hfrs, thus, adding to the semantic confusion of non-initiated readers. worse, the definition of what exactly a "renal syndrome" should be has never been given, not even when the name was officially coined by the who in [ ] . if a "renal syndrome" is clear proteinuria, then virtually all old world hantavirus infections fully comply [ ] , but so do most new world infections. it should be remembered that, even amongst the hps patients originally described in , already % had ≥ + proteinuria on admission [ ] , a figure later increasing to %, at least for all h(c)ps cases other than those caused by snv [ ] . for a full assessment of this distinctive feature, it can only be regretted that the cdc did not simply include proteinuria amongst its criteria for "h(c)ps" as the easiest, quickest and cheapest test available. moreover, proteinuria is minimal or absent in other febrile affections that might initially mimic h(c)ps, such as plague, tularaemia and rickettsioses. as for "renal impairment" or arf, the definitions become increasingly unclear: when starts the "failure" of renal function? up to , several definitions had been used to define arf [ ] . acute kidney injury (aki) has recently become the preferred term to describe acute renal impairment, with "failure" or "arf" restricted to patients needing renal replacement therapy (rrt). in , a workgroup of experts proposed a multi-level and evidence-based classification system for aki, identified by the acronym rifle (risk, injury, failure, loss of kidney function, and end-stage kidney disease). aki was first defined on the basis of either a . -fold increase in serum creatinine levels, a decrease in estimated glomerular filtration rate by > % or a decline in the urine output to < . ml/kg/h over h [ , ] . the acute kidney injury network (akin) group further modified this definition by adding an increase in the creatinine level by ≥ . mg/dl as criterion [ ] . while these new criteria might not always be fully applicable in hantavirus infections in view of the rapid and spontaneous self-remittance without sequelae of renal symptoms, they have at least the benefit of clarity and current general agreement, even when the decision of when best to start rrt should remain a purely clinical one [ ] . nowadays, virtually each major hantavirus research team handles its own criteria for distinguishing "mild", "moderate" and "severe" renal involvement. for this emerging zoonosis, the only one (together with leptospirosis) with a global distribution, and probably the most underestimated cause worldwide of infectious aki, a more streamlined approach is really needed for classification, and even for research [ , ] . for instance, no conclusive mechanism has been forwarded so far to explain the sudden initial proteinuria. american authors found, however, that, due to extreme capillary leaking, pulmonary effusions in h(c)ps can have almost the same protein composition as plasma [ ] . bearing in mind that hantavirus-induced proteinuria, in spite of a puzzling absence of glomerular lesions that can be detected by light microscopy, immunofluorescence or electron microscopy, often reaches nephrotic levels and is always unselective [ ] , the most straightforward explanation is, again, the temporary leakage of a proteinrich filtrate through hyper-permeable endothelium in the glomerular capillary loops, together with varying degrees of initial tubulopathy. if confirmed by comparative studies, this hypothesis could further underline similarities between h(c)ps and hfrs instead of differences, thus, corroborating a common, rather than a divergent pathogenesis. in such a case, maintaining disease names based on varying or overlapping symptoms makes little sense, and a unifying name becomes even more mandatory. with the advent of biomolecular techniques such as reverse transcription polymerase chain reaction (rt-pcr), pinpointing the exact nature of infecting hantavirus species and discovering even new pathogens without a proper virus isolation [ , ] , it is, perhaps, time now to refine also the old denominations of this global disease. a good start would be a simple, short name like "hantavirus disease", having the additional merit of fulfilling at least part of koch's postulate, which names of various symptoms never can do. this way, the proposition of desmyter et al. [ ] , after having unravelled the first documented european (laboratory) hantavirus outbreak outside of scandinavia [ ] , could finally be accepted. time to revise the paradigm of hantavirus syndromes? hantavirus pulmonary syndrome caused by european hantavirus world health organization (who) ( ) haemorrhagic fever with renal syndrome: memorandum from a who meeting hantavirus pulmonary syndrome: a clinical description of patients with a newly recognized disease aetiological relation between korean haemorrhagic fever and nephropathia epidemica hantaviruses: immunology, treatment, and prevention notes from the field: hantavirus pulmonary syndrome-maine hantavirus pulmonary syndrome global warming and epidemic trends of an emerging viral disease in western-europe: the nephropathia epidemica case. in: casalegno s (ed) global warming impacts-case studies on the economy, human health, and on urban and natural environments hantavirus pulmonary syndrome (hantavirus disease) (hps), case definition hantavirus pulmonary syndrome in new england and europe, author reply hantavirus pulmonary syndrome in new england and europe puumala virus infection without signs of renal involvement hantavirus pulmonary syndrome: the new american hemorrhagic fever increased permeability of human endothelial cell line ea.hy induced by hantavirus-specific cytotoxic t lymphocytes t cells and pathogenesis of hantavirus cardiopulmonary syndrome and hemorrhagic fever with renal syndrome hantavirus nephropathy in belgium: description of new cases originating in southern provinces isolation of a hantavirus from a severely ill patient with hemorrhagic fever with renal syndrome in greece a soldier in respiratory distress life-threatening dobrava hantavirus infection with unusually extended pulmonary involvement occurrence of renal and pulmonary syndrome in a region of northeast germany where tula hantavirus circulates the hantaviruses of europe: from the bedside to the bench incidence rate for hantavirus infections without pulmonary syndrome acute sin nombre hantavirus infection without pulmonary syndrome, united states first evidence of fatal hantavirus nephropathy in india, mimicking leptospirosis acute kidney injury in emerging, non-tropical infections acute renal failure definitions and classification: time for change? developing a consensus classification system for acute renal failure acute renal failuredefinition, outcome measures, animal models, fluid therapy and information technology needs acute kidney injury network: report of an initiative to improve outcomes in acute kidney injury when to start dialysis in patients with acute kidney injury? when semantics and logic become entangled with expectations and beliefs severe crescentic glomerulonephritis linked to an acute hantaan virus infection? laboratory rat associated outbreak of haemorrhagic fever with renal syndrome due to hantaan-like virus in belgium key: cord- - no e authors: jeannoël, m.; lina, g.; rasigade, j. p.; lina, b.; morfin, f.; casalegno, jean sebastien title: microorganisms associated with respiratory syncytial virus pneumonia in the adult population date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: no e respiratory syncytial virus (rsv) has been recognized as responsible for severe respiratory illness in adults, especially in the elderly. while pneumonia is commonly observed during rsv infection, the burden and epidemiology of bacterial superinfection is poorly understood. the aim of this study was to identify microorganisms associated with rsv-positive pneumonia in adults. a retrospective study was conducted during three consecutive winters (october to april – ) in the university hospital of lyon, france. during rsv circulation periods, a systematic rsv screening was performed by reverse-transcription pcr on all respiratory samples collected from adults. records of rsv-positive patients were subsequently analyzed to identify radiologically confirmed pneumonia cases. bacteria were identified by standard bacteriology cultures or urinary antigen screening and classified as potentially causative of pneumonia if quantification was above the specific threshold as defined by the european manual of clinical microbiology. overall, , adult respiratory samples were screened for rsv detection by pcr. in total, had a positive rsv detection ( . %) among which presented with pneumonia including bacterial superinfections ( . %) with streptococcus pneumonia, haemophilus influenza, staphylococcus aureus, pseudomonas aeruginosa, and moraxella catarrhalis. most patients were elderly ( . %) and patients with comorbidities ( . %). a more severe outcome was observed for rsv-bacteria-associated pneumonia compared with rsv pneumonia: length of stay was significantly longer ( days vs days) and icu hospitalization more frequent ( . % vs . %) (p < . ). in conclusion, we did not observe major differences in the epidemiology of bacterial superinfections in rsv-positive pneumonia compared to reports on post-influenza pneumonia. respiratory syncytial virus (rsv) is now recognized as an important cause of respiratory illness in adults, especially in the elderly [ ] . secondary bacterial pneumonia after or during an rsv infection is among the more severe complications reported in this population [ ] . few studies have described superinfection or coinfection with bacteria among adults with rsv-positive pneumonia in inpatient setting [ , ] . diagnostic testing for rsv is usually not routinely performed in this population. however burden and epidemiology of rsv-positive pneumonia in adults is important to assess in regards of the current antibiotic therapy recommendations [ ] . the aim of our study was to gain insights in the epidemiology of bacterial superinfections associated with rsv pneumonia in adults. we conducted a retrospective, monocenter, and observational study on three successive winter periods (october to april, april, - . in the university hospital of lyon (hospices civils de lyon, france), upper and lower respiratory tract samples from patients with respiratory symptoms are systematically screened for rsv and metapneumovirus (hmpv) using a reverse transcriptase polymerase chain reaction (rt-pcr). we included all adult patients (≥ years old) positive for rsv with radiologically confirmed pneumonia as diagnosed in the medical chart. hospital-acquired pneumonia (hap) was defined as radiologically confirmed pneumonia that manifests ≥ h after the patient's admission to the hospital, and not present at the time of intubation. data collected included laboratory results, demographics (age, sex), underlying conditions (cardiovascular disease, pulmonary disease, diabetes mellitus, and immunodeficiency), as well as clinical outcomes (length of stay, acute respiratory distress syndrome (ards), intensive care unit (icu) admission, in-hospital death). for virus detection, clinical specimens comprised nasopharyngeal swabs, endotracheal aspirates, and broncho-alveolar lavages. respiratory specimens were extracted using easymag (biomerieux, marcy l'étoile, france). rsv detection was performed using a rt-pcr (abi ; thermofisher, illkirch, france), or duplex rt-pcr detecting rsv and hmpv (r-gene detection kit, biomérieux-argene, france) as previously described [ ] . for bacteria detection, pathogens were classified as potentially causative of pneumonia if identified in pleural fluid or blood, or if identified within a sputum specimen, endotracheal aspirate, and broncho-alveolar lavage with good-quality criteria and if quantification was above the appropriate emcm decision threshold. semi-quantitative bacterial culture process and interpretation was performed following the european manual of clinical microbiology (emcm) guidelines [ ] . maldi-tof (vitek-ms) was used for species identification. statistical analyses were performed using graphpad prism software (version . , graphpad software, la jolla, ca, usa). univariate analysis was used to compare rsvbacteria-associated pneumonia and rsv-positive pneumonia. quantitative variables (age and length of stay) were expressed as median and interquartile range (iqr). difference between the age groups was assessed by student's t test. difference between lengths of stay was assessed by non-parametric mann-whitney test. qualitative variables were expressed as number and percentage. a fisher's exact test was used for univariate comparisons. a p value < . was considered significant. during the study period, viral rt-pcr was performed on , respiratory samples collected from adult patients. influenza was detected in patients ( . % of all respiratory samples), rhinovirus in patients ( . % of all respiratory samples), rsv in patients ( . % of all respiratory samples), and hmpv in patients ( . %). the median age of patients tested positive for rsv was years (interquartile range to ), with a sex ratio of ( . % were men). among rsv-positive patients, . % of case records were retrieved (n = / ). primary discharge diagnoses in patients without respiratory symptoms were as follows: . % (n = / ) of systematic sampling without acute respiratory infection in immunosuppressed patients, . % (n = / ) of acute heart failure, . % (n = / ) of sepsis, and . % (n = / ) of neurological disorders. . % (n = / ) patients presented with respiratory disorders, including . % (n = / ) of respiratory failure, . % (n = / ) of acute respiratory infection, . % (n = / ) of acute respiratory distress syndrome, and . % (n = / ) radiologically confirmed pneumonia. among these pneumonia, cases were associated with bacteria classified as potentially causative (rsv-bacteria-associated pneumonia) and cases had no bacterial documentation (rsv-positive pneumonia). among these rsvpositive pneumonia, cases were associated with another virus ( rsv and influenza coinfections, rsv and picornavirus coinfection, rsv and bocavirus coinfection, and rsv and cmv coinfection). among the rsv-bacteria associated pneumonia, hospital-acquired pneumonia (hap) was observed mostly in adults ≥ years old (n = / ) whereas community-acquired pneumonia (cap) occurred mostly in to -year-old adults (n = / ). most of the patients presenting a pneumonia were elderly (median age years (interquartile range to ) and had underlying conditions ( . %) ( table ). only . % were resident of nursing homes. demographic characteristics did not differ between patients with an rsv-bacteria-associated pneumonia and patients with an rsv-positive pneumonia. among these pneumonia, patients received an empiric antibiotic treatment and immunosuppressed patients received an antiviral treatment: patient received ribavirin, patient received polyvalent immunoglobulins, and patient received ribavirin and polyvalent immunoglobulins. in total, patients presented an ards, patients were admitted to icus and in-hospital mortality occurred in adults of which all were ≥ years old (n = / ) or immunosuppressed (n = / ). rsv and bacteria coinfection was statistically associated with a more severe outcome than rsv-positive pneumonia as length of stay was significantly longer ( days vs days) and icu hospitalization more frequent ( . % vs . %) (p < . ). however no significant difference was observed regarding in-hospital mortality ( table ) . among the cases of rsv-positive pneumonia with superinfection, the most frequent bacteria were streptococcus pneumoniae, enterobacteriaceae ( kleb siella p neu monia , en teroba cte r cloa ca e , citrobacter koseri), pseudomonas aeruginosa, haemophilus influenza, staphylococcus aureus, and moraxella catarrhalis. in two patients, more than one bacterial pathogen was detected. in patients hospitalized f o r c a p, t he m o s t f r e qu en t ba ct er i a w e re s . pneumoniae, h. influenzae, and s. aureus whereas in hap the most frequent bacteria were p. aeruginosa and enterobacteriacae (table ). in this study, detection rates of the screened viruses were consistent with previous reports describing the epidemiology of respiratory viruses in hospitalized adults [ ] [ ] [ ] . rsv detection in our study was low ( . %) compare to rates of rsv detection reported in previous studies ranging from % of hospitalized adult patients to % of hospitalization for acute respiratory illness in an elderly population [ ] [ ] [ ] . it is probably due to the systematic testing strategy associated to a species distribution of pathogenic bacteria involved in rsv-positive pneumonia (cap) and hospital-acquired pneumonia (hap) sampling bias toward influenza-like illness. those symptoms are indeed poorly predictive of an rsv infection [ ] . among rsv infected patients, . % presented a radiologically confirmed pneumonia and . % had an rsv-bacteriadocumented pneumonia. despite the large number of rsv testing among the adult population, we identified only a few rsv radiologically confirmed pneumonia. although this is a low prevalence, it is in agreement with the current literature in which to % of rsv-bacteria co-infections were reported [ ] . in our study, a higher prevalence was noted in the elderly ( / ), immunosuppressed, and patients with comorbidities ( / ). as it has been previously described, we observed that rsv-bacteria-associated pneumonia have a more severe outcome than rsv-positive pneumonia [ ] . enterobacteriaceae and pseudomonas aeruginosa were the most frequently detected pathogens in hap and did not differ from the pathogens usually responsible for hap in icu [ ] . s. pneumonia and h. influenza were the most frequently detected pathogens in cap. in a previous study in adults with cap, s. pneumonia and h. influenza were also mainly reported in rsv-bacteria coinfections [ ] . we did not observed major differences to influenza-bacterial coinfections [ ] . those results suggest that there is no main difference between the microbiological epidemiology of rsv-bacterial coinfections and influenza-bacterial coinfections. this observation needs to be confirmed by other studies. further research should focus on estimating the rsv cap burden in adult as well as understanding the underlying mechanisms of copathogenesis. conflict of interest the authors declare that they have no conflict of interest. ethical approval this article does not contain any studies with human participants performed by any of the authors. informed consent for this type of study formal consent is not required. risk of mortality associated with respiratory syncytial virus and influenza infection in adults viral-bacterial coinfection affects the presentation and alters the prognosis of severe community-acquired pneumonia respiratory syncytial virus infection in elderly and high-risk adults incidence and characteristics of viral community-acquired pneumonia in adults community-acquired pneumonia genetic characterization of respiratory syncytial virus highlights a new ba genotype and emergence of the on genotype in lyon european manual of clinical microbiology. basel: european society for clinical microbiology and infections diseases : société française de microbiologie can analysis of routine viral testing provide accurate estimates of respiratory syncytial virus disease burden in adults? prevalence of respiratory viruses among adults, by season, age, respiratory tract region and type of medical impact of viral multiplex real-time pcr on management of respiratory tract infection: a retrospective cohort study respiratory syncytial virus hospitalization in middle-aged and older adults rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults respiratory syncytial virus infection in adults clinical characteristics and outcome of respiratory syncytial virus infection among adults hospitalized with influenza-like illness in france elucidation of bacterial pneumonia-causing pathogens in patients with respiratory viral infection hospital-acquired pneumonia in icu the co-pathogenesis of influenza viruses with bacteria in the lung key: cord- -xrhqisii authors: lópez-rodríguez, m.; herrera-ramos, e.; solé-violán, j.; ruíz-hernández, j. j.; borderías, l.; horcajada, j. p.; lerma-chippirraz, e.; rajas, o.; briones, m.; pérez-gonzález, m. c.; garcía-bello, m. a.; lópez-granados, e.; rodriguez de castro, f.; rodríguez-gallego, c. title: ifitm and severe influenza virus infection. no evidence of genetic association date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: xrhqisii influenza virus infection (ivi) is typically subclinical or causes a self-limiting upper respiratory disease. however, in a small subset of patients ivi rapidly progresses to primary viral pneumonia (pvp) with respiratory failure; a minority of patients require intensive care unit admission. inherited and acquired variability in host immune responses may influence susceptibility and outcome of ivi. however, the molecular basis of such human factors remains largely elusive. it has been proposed that homozygosity for ifitm rs -c is associated with a population-attributable risk of . % for severe ivi in northern europeans and . % for severe h n pdm infection in chinese. a total of patients with confirmed ivi were considered for recruitment; spanish patients ( of them hospitalized with pvp) and healthy spanish individuals were finally included in the statistical analysis. pcr-rflp was used with confirmation by sanger sequencing. the allele frequency for rs -c was found to be . % among the general spanish population. we found no rs -c homozygous individuals in our control group. the only spanish patient homozygous for rs -c had a neurological disorder (a known risk factor for severe ivi) and mild influenza. our data do not suggest a role of rs -c in the development of severe ivi in our population. these data may be relevant to recognize whether patients homozygous for rs -c are at risk of severe influenza, and hence require individualized measures in the case of ivi. influenza is mainly caused by h n and h n influenza a virus (iav) and influenza b virus. most individuals, including those infected with the pandemic h n iav (h n pdm), experience an uncomplicated influenza. up to % of infections with seasonal influenza or h n pdm are estimated to be subclinical [ ] [ ] [ ] [ ] . however a low percentage of h n pdm-infected patients, many of them without known risk factors, develop primary viral pneumonia (pvp) and a minority even develop acute respiratory distress syndrome (ards), requiring admission to an intensive care unit (icu) [ , ] . inherited variability in host immune responses may influence susceptibility and outcome of iav infection, but the molecular nature of such factors remains largely elusive [ ] [ ] [ ] [ ] . ifitm was found to restrict the entry and replication of pathogenic viruses, including iav, in both mice and humans [ ] [ ] [ ] . the variant ifitm rs -c alters a splice acceptor site and encodes an aberrant form of the protein lacking the nterminal amino acids (Δ ifitm ). conflicting results about the impact of the ifitm rs -c variant, coding for the Δ ifitm protein, in controlling iav restriction in vitro have been reported [ , ] . it has been proposed that homozygosity for the rs -c allele is associated with development of severe influenza in northern europeans and severe iav infection in asians [ , , ] . however, these results were not replicated in a recent report from the united kingdom [ ] . in the present study, we assessed whether the ifitm rs -c allele is associated with susceptibility to and severity of influenza virus infection (ivi) among a white spanish population. we performed a case-control genetic association study aimed to analyze the role of the ifitm rs polymorphism in the susceptibility and severity of ivi. from july to march , patients with confirmed ivi from five tertiary spanish hospitals and primary care centers from gran canaria (spain), were considered for recruitment. data and samples from ambulatory patients and from % of the hospitalized patients were retrospectively obtained; in the remaining patients, data were obtained prospectively. ethnicities other than white spanish were excluded. all patients were treated with oseltamivir, and only one patient had been previously vaccinated against h n pdm. the general spanish population group consisted of (blood and bone marrow donors, as well as hospital staff) unrelated white spanish volunteers (age . ± . years; . % females). no data about ivi were available in this group, and none of these individuals had previously developed any severe respiratory illness. influenza a virus h n pdm was detected in nasopharyngeal swabs using the real-time polymerase chain reaction (rt-pcr). infection by influenza viruses other than h n pdm was confirmed by either a fourfold increase in complement fixation titers between acute and convalescent phases ( out of the patients) or rt-pcr (n = ). genomic dna was extracted from μl of peripheral blood using iprep™ purelink™gdna blood kit in the iprep™purification instrument (invitrogen by life technologies, carlsbad, ca). extracted dna integrity was checked by nanodrop nd- (nanodrop technologies, wilmington, de). dna samples were conserved at − °c. genotyping was performed at the department of immunology of the hospital universitario de gran canaria dr. negrín (las palmas de gran canaria, spain). analysis of the ifitm rs polymorphism was performed by means of pcr-rflp. pcr was carried out with the pair of primers ′-aatttgttccgccctcatct- ′ and ′-atgt cgtctggtccctgttc- ′ in a geneamp® pcr system (applied biosystems, foster city, ca, usa). amplification was carried out with a first heating at °c for min in order to pre-denature the template dna, followed by cycles of denaturation at °c for s, annealing at °c for s and extension at °c for s. the final extension was completed at °c for min. pcr products were digested with one unit of bali (promega co., wi, usa) for h at °c, and then separated on % agarose gel and visualized under uv with etidium bromide. after restriction enzyme digestion, fragments, in function of the length of , and/or bp, were classified in accordance with the genotype. in order to confirm genotypes, % of the samples were sequenced (including all individuals homozygous for rs -c). pcr products were purified with exosap-ittm (amersham biosciences), according to manufacturer's protocol. purified pcr products were sequenced using the abi prism bigdye® terminator v . cycle sequencing kit (applied biosystems) on the abi prism xl genetic analyzer (applied biosystems, california, usa). to ensure the removal of bigdye® terminator v . along with dntps, salts and other low molecular weight materials, performa® dtr gel filtration cartridges (edgebio, gaithersburg, usa) were used to minimize the potential for interference with sequencing applications. both sense and antisense strands of pcr products were directly sequenced using the same primers used for the pcr amplification. the number of individuals with each genotype was calculated by direct counting. hardy-weinberg equilibrium (hwe) and allele frequencies were calculated using the plink software (http://pngu.mgh.harvard.edu/purcell/plink/). statistical analysis of the data was performed with spss . (spss, chicago, il, usa). χ test or fisher exact test, when needed, and odds ratio with % confidence intervals were calculated. a p value < . was considered statistically significant. this study was approved by the clinical research ethics committees of the hospitals involved. informed consent, provided according to the declaration of helsinki, was obtained from all patients and/or from their legal representatives. among the patients with confirmed ivi initially recruited, patients were excluded from the analysis due to ethnicities other than white spanish, and in three individuals genotyping was unsuccessful. the study group comprised spanish caucasian patients. sixty out ( %) of these patients were hospitalized and had radiographic evidence of pvp; were attended at primary care centers, and patients were hospitalized with no evidence of pvp. thirty-four patients required icu admission ( [ %] with ards) (fig. ) . clinical and demographic characteristics of the patients are summarized in table . the allele frequency for rs -c was found to be . % among the general spanish population, a frequency similar to that reported for european populations in the genomes project data source (www. genomes.org). we found no individuals homozygous for rs -c allele in our control group, nor on the available data from individuals in the european population from the genomes project. the ifitm variant rs was in hardy-weinberg equilibrium (p > . ) in all the studied groups. among our spanish patients with influenza, homozygosity for rs -c was identified only in one patient with mild h n pdm infection. nevertheless, two cc homozygous individuals with severe ivi among the patients who did not satisfy the criteria of inclusion were found. both patients were of asian ethnicity, where the prevalence of homozygosity for the variant rs -c is much higher ( - %) [ , ] . we performed a case/control study to compare the allele and genotype frequencies between influenza-infected patients and our control group. these comparisons did not yield significant results ( table ). the same comparison was performed with data obtained from the ibs population (iberian population in spain, n = ) in the genomes project, and also with combined data from the control group of our study and the ibs population (n = ). this last comparison showed a near significant association of the rs -c allele with susceptibility to influenza (p = . ; or = . , . - . ). mills et al. [ ] did not find rs -c homozygotes in a group of patients from the united kingdom who required icu admission due to severe h n ivi. however, the authors found two rs -c homozygous individuals in a group of caucasian patients with mild influenza, and four in a group of , caucasian controls who had never required hospital admission. since allele frequencies were also similar in our population and in the study from mills et al. [ ] , we combined our data with the data from their study. this comparison showed an increase of rs -c homozygotes in patients with mild ivi compared with control individuals: three out of patients with mild influenza versus four out of , controls were homozygous for the rs -c allele (p = . by fisher exact test; or = . , - . ). no significant differences were observed when patients with severe influenza (patients hospitalized with pvp or those requiring icu admission) and the general population were compared ( table ). no differences were either observed when only patients with a/h n pdm infection were compared separately (data not shown). when the different groups of patients were compared according to severity of infection (development of pvp, ards or acute respiratory failure, need of icu admission or hospital mortality), no significant associations were found (data not shown). in , everitt et al. [ ] found three homozygous individuals for the variant rs -c among patients from the united kingdom hospitalized with influenza ( . % h n pdm), resulting in a significant overrepresentation when compared with europeans. later, zhang et al. [ ] found a significant overrepresentation of rs -c homozygotes among chinese patients with severe h n pdm infection compared with individuals with mild disease or population controls. these studies suggested that homozygosity for the ifitm rs -c allele was associated with a populationattributable risk for severe influenza of . % in northern europeans and . % for severe h n pdm infection in chinese [ , ] . both studies included small sample sizes. moreover, the association found by everitt et al. [ ] relied on imputed control genotypes, which may bias the frequencies when rare snps, such as rs , are studied. in addition, not all of their three rs -c homozygous patients have undergone population stratification analysis, thus not ensuring only caucasian ancestors. a more recent study did not find any rs -c homozygous in a group of patients from the united kingdom who required icu admission due to severe h n ivi [ ] . by contrast, two rs -c homozygous individuals in a group of caucasian patients with mild influenza, and four in a group of , caucasian controls who had never required hospital admission, were identified. the authors found a recessive association of the rs -c variant with mild influenza, but not with severe influenza. these data contrast with those of zhang et al., who found an association with severe but not mild influenza [ ] . among our patients with ivi, the only patient homozygous for rs -c was a -yearold girl who had a severe neurological disorder (west syndrome), a risk factor for severe influenza. however, after infection with h n pdm, she presented with mild influenza. hence, no association with severe ivi was observed in our study. by contrast, we found a nonsignificant increase of the rs -c allele, but not genotypes, in patients with ivi compared to control spanish individuals. in addition, when data from our study and those from mills et al. [ ] were analyzed together, a marginal significant association of homozygosity for rs -c with mild ivi was observed. these data suggest that, at least in populations of european origin, homozygosity for rs -c could confer an increased risk for ivi, but not for severe influenza. since no patients with mild influenza were included in the study of everitt et al. [ ] , their results might also arise as a consequence of the association of rs with susceptibility to, rather than severity of, ivi. it was suggested that ifitm plays a pivotal role in limiting the entry and replication of several viruses, including iav, in humans and mice [ ] [ ] [ ] . however, the impact of the rs -c polymorphism in viral restriction in vitro is also conflicting. everitt et al. [ ] reported that the ifitm rs -c polymorphism, coding for the Δ ifitm isoform, has reduced influenza virus restriction in vitro. however, no effect of Δ ifitm on the control of influenza virus restriction in vitro has been recently reported by williams et al. [ ] . although our study includes the highest number of patients with severe ivi analyzed for the rs polymorphism to date, it is limited by a relatively small sample size. our control group may be considered a representative sample of the spanish population rather than a representation of noninfluenza infected individuals, since no data about ivi was available. in any event, this limitation would probably lead to underestimation of the association of the variant with susceptibility to ivi. no stratification analysis was performed in our study. however, it does not undermine the fact that, those patients homozygous for rs -c suffered from a mild influenza. overall, both the results of our study and those obtained by other authors suggest that homozygosity for the rs -c allele may predispose to mild, but not to severe, influenza, at least in patients of european origin. in view of the contradictory results observed in asian and european populations, and the sample sizes of the groups of patients with influenza analyzed to date, further large-scale collaborative studies are required to clarify the role of ifitm rs in ivi. a major issue is to know whether patients homozygous for rs -c should be actually considered at risk of severe influenza, and if they would require individualized measures in the case of ivi. clinical aspects of pandemic influenza a (h n ) virus infection seroprevalence to influenza a (h n ) virus-where are we? mortality associated with influenza and respiratory syncytial virus in the united states comparative community burden and severity of seasonal and pandemic influenza: results of the flu watch cohort study evidence for a heritable predisposition to death due to influenza an updated systematic review of the role of host genetics in susceptibility to influenza. influenza other respir viruses the role of polymorphisms in host immune genes in determining the severity of respiratory illness caused by pandemic h n influenza surfactant protein a genetic variants associate with severe respiratory insufficiency in pandemic influenza a virus infection ifitm restricts the morbidity and mortality associated with influenza ifitm polymorphism rs -c restricts influenza a viruses ifitm limits the severity of acute influenza in mice interferon-induced transmembrane protein- genetic variant rs -c is associated with severe influenza in chinese individuals early hypercytokinemia is associated with interferon-induced transmembrane protein- dysfunction and predictive of fatal h n infection ifitm and susceptibility to respiratory viral infections in the community acknowledgments we would like to thank the patients and their families for their trust. we also thank nereida gonzález-quevedo and yanira florido for technical support and consuelo ivañez for their invaluable help. . the sponsors of the study had no role in designing the study, collecting, analyzing and interpreting the data, or writing the paper. the authors declare that they have no conflict of interest.ethical approval this study was approved by the clinical research ethics committees of the hospitals involved.informed consent informed consent was obtained from all individual participants included in the study. key: cord- -pnnkfid authors: ioakeimidou, a.; pagalou, e.; kontogiorgi, m.; antoniadou, e.; kaziani, k.; psaroulis, k.; giamarellos-bourboulis, e. j.; prekates, a.; antonakos, n.; lassale, p.; gogos, c. title: increase of circulating endocan over sepsis follow-up is associated with progression into organ dysfunction date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: pnnkfid how circulating inflammatory mediators change upon sepsis progression has not been studied. we studied the follow-up changes of circulating vasoactive peptides and cytokines until the improvement or the worsening of a patient and progression into specific organ dysfunctions. in a prospective study, concentrations of tumor necrosis factor-alpha (tnfα), interleukin (il)- , il- , il- , interferon-gamma (ifnγ), endocan and angiopoietin- (ang- ) were measured in serum by an enzyme immunoassay in patients at baseline; this was repeated within h upon progression into new organ dysfunction (n = ) or improvement (n = ). endocan and ang- were the only parameters that were significantly increased among patients who worsened. any increase of endocan was associated with worsening with odds ratio . (p < . ). this increase was independently associated with progression into acute respiratory distress syndrome (ards) as shown after logistic regression analysis (odds ratio . , p: . ). changes of circulating cytokines do not mediate worsening of the critically ill patients. instead endocan and ang are increased and this may be interpreted as a key-playing role in the pathogenesis of ards and septic shock. any increase of endocan is a surrogate of worsening of the clinical course. despite progress made in early recognition and management, sepsis remains the leading cause of death accounting for more than , deaths every year in the united states [ ] . sepsis is nowadays defined as a life-threatening organ dysfunction induced from the dysregulated response of the host to an infection [ ] . this definition is coming from our better understanding of the complex pathophysiology of sepsis acquired in recent years. we have managed to understand that when sepsis develops not all patients react in a similar way; in some hosts a aikaterini ioakeimidou and evgenia pagalou contributed equally to this submission. pro-inflammatory response prevails whereas in others an antiinflammatory response emerges [ ] . however, this distinction is not clear-cut in all patients. everyday practice teaches that unfavourable outcome is due to the deterioration of the initial host response to progressive organ dysfunction. it is highly probable that the mechanisms underlying this deterioration are individualized from patient to patient. these mechanisms are poorly studied and their knowledge is mandatory for future immunotherapy studies. deterioration of the host from an initial septic state to multiple organ dysfunctions most probably results from the dynamic interaction of circulating cytokines and peptides secreted from endothelial cells. vascular endothelium is s t i m u l a t e d b y c y t o k i n e s f o r t h e p r o d u c t i o n o f angiopoietin- (ang- ) and endocan. ang- increases vascular permeability and mediates progression into shock [ ] whereas endocan inhibits leukocyte migration in the lungs and kidneys [ ] . our aims were to monitor the changes of circulating levels of pro-inflammatory and antiinflammatory cytokines and of vasoactive peptides of critically ill patients at well-defined time-points of the clinical course and to understand how these changes mediate progression to organ dysfunction in an individualized way. this is a prospective study that was conducted in ten intensive care units (icus) participating in the hellenic sepsis study group from january until december . the study protocol was approved by the ethics committees of the participating hospitals. a patient was enrolled after written informed consent provided by his first-degree relatives. inclusion criteria were: (a) written informed consent, (b) age equal to or above years, (c) both genders, (d) at least two signs of the systemic inflammatory response syndrome (sirs) as defined elsewhere [ ] , (e) one of the following infections: acute pyelonephritis, community-acquired pneumonia, acute intraabdominal infection, primary bacteremia or ventilatorassociated pneumonia. the diagnosis of infections was done by international definitions [ ] , (f) first blood sampling within the first h from the presentation of the first two signs of sirs, and (g) repeat blood sampling at a follow-up time point. the follow-up time point could be h from the first blood sampling or within the first h from the onset of any new organ dysfunction provided that this was at least h apart from the first blood sampling. this -h time difference between the two time-points of sampling was selected in order to increase the likelihood that the changes described over-time of the studied mediators were associated with the new organ dysfunction and not with the existing baseline inflammatory process. exclusion criteria were: (a) infection by the human immunodeficiency virus − , (b) neutropenia defined as an absolute neutrophil count less than /mm unless due to sirs per se, and (c) intake of corticosteroids at a daily dose of more than . mg/kg of equivalent prednisone for at least consecutive days. five ml of blood was sampled after venipuncture of one forearm vein under sterile conditions. blood was poured into pyrogen-free tubes (vacutainer, becton dickinson, cockeysville, md) and centrifuged at room temperature. the supernatant serum was immediately aliquoted and aliquots were shipped in dry-ice on the same day into the central lab at the laboratory of immunology of infectious diseases as the th department of internal medicine of attikon university hospital. aliquots were stored there at − °c until measurement. patients were then followed-up daily for days. patients who progressed into new organ dysfunctions less than h from the first blood sampling were excluded from the study. a second blood sample was collected from the remaining patients h after the first blood sampling. patients were then followed daily for days and sequential organ failure assessment (sofa) score was measured daily. if the patient was presenting with a new organ dysfunction, blood sampling was done within h and the blood sample drawn at h was discarded. if not, the blood sample originally collected at h was processed for analysis to be used as comparator. organ dysfunctions were defined as follows: (a) acute respiratory distress syndrome (ards) as diffuse shadows in chest x-ray and ratio of partial oxygen tension to fraction of inspired oxygen below [ ] , (b) acute kidney injury (aki) as less than . mg/kg/min of urine output for at least two consecutive hours provided that the negative fluid balance was restored [ ] , (c) disseminated intravascular coagulation (dic) as absolute platelet count below , /mm accompanied by an increase of the concentrations of d-dimers and decrease of circulating fibrinogen [ ] , and (d) septic shock as decrease of systolic arterial pressure below mmhg not responding to fluid resuscitation and requiring the administration of vasopressors [ ] . for all patients retrospective evaluation was done using the recently published sepsis- definitions [ ] . those who did not meet the new definitions at study enrolment were excluded from analysis. concentrations of tumor necrosis factor-alpha (tnfα), of interleukin (il)- , il- , il- , interferon-gamma (ifnγ), angiopoietin- (ang- ) and of endocan were measured by an enzyme immunoassay. reagents for tnfα, il- , il- , il- and ifnγ were purchased from ebiosciences (san diego, usa); those for ang- by r&d (minneapolis, usa); those for endocan were a kind donation by lunginnov s.a.s (campus de l'institut pasteur de lille, , lille, france). the lower limits of detection were pg/ml for tnfα, il- , il- , il- and ifnγ; pg/ml for ang- ; and . ng/ml for endocan. the primary study endpoint was the correlation between the follow-up changes of each measured parameter from baseline until the improvement or the worsening of a patient. a patient was considered worsened if his total sofa score was increased on the follow-up time point by at least two points from the baseline sofa score on study enrolment. a patient was considered improved if his total sofa score was decreased on the follow-up time point by at least two points from the baseline sofa score on study enrolment. the secondary endpoint was the comparative changes of the measured parameters for specific organ dysfunctions. results of measured parameters were expressed as median and % confidence intervals. for the primary endpoint, pair-wise comparisons were done between the two time points of sampling separately for patients worsening and for patients improving by the wilcoxon test. the percent of change of parameters from baseline was analyzed by receiver operating characteristic curve (roc) analysis. using co-ordinate points of the curve, the cut-off with specificity greater than % for worsening was chosen. the sensitivity, specificity, positive and negative predictive values of that cut-off were calculated. the odds ratio (or) and % confidence interval (ci) for worsening at the depicted cut-off point were calculated by mantel and haenzel's statistics. for the secondary endpoint, pair-wise comparisons between baseline and follow-up measurements were done within the subgroups of patients developing specific new organ dysfunctions. the association of changes of circulating peptides with the development of a specific organ failure was further analyzed by logistic regression analysis; the presence of at least one comorbidity and baseline severity entered the equation as co-variates. any value of p below . after correction for multiple comparisons was considered significant. the study flow-chart is shown in fig. . a total of patients were screened. the average number of beds per participating icu was ten. taking into consideration that the median patient pair-wise comparisons showed that the only parameters that were significantly increased upon worsening of the patients were endocan and ang- (fig. ) . endocan significantly decreased among patients who improved. roc curves of the percent changes of endocan and ang- from the baseline were designed to explore the use of endocan and of ang- as surrogate markers of worsening. only the roc curve of the change of endocan provided a significant area under the curve (fig. a) . using the coordinate points of roc curve, it was found that any increase of endocan from baseline could be associated with sepsis worsening with . % sensitivity and . % positive predictive value (fig. b) . any baseline increase of endocan was associated with or . ( % cis: . - . , p: × − ) for worsening. when pair-wise comparisons between baseline and follow-up measurements were done within the subgroups of patients developing new organ dysfunctions, it was found that the only parameters significantly changing were endocan and ang- . more precisely, both were increased on development of ards and septic shock; ang- was also increased on development of aki (fig. ) . no changes were found upon development of dic (data not shown). logistic regression analysis showed that any increase of endocan from baseline was independently associated with the progression into ards. a similar association was shown for the progression into septic shock with a trend towards statistical significance (table ). the present study measured the concentrations of a range of circulating protein molecules in the sera of patients at icu admission and upon progression into a new organ dysfunction. results showed that only vasoactive and endothelialderived molecules like endocan and ang- are increased upon deterioration of the patient into a more severe stage as this is defined by any increase of the total sofa score by at least two points. increase of endocan and ang- were mainly found on progression into ards and septic shock whereas ang- was also increased among patients who progressed into aki. any increase of endocan was associated with increased likelihood for the deterioration of a patient. in a recently published single-center prospective study, patients with sirs, nine patients with sepsis, patients with severe sepsis and patients with septic shock were enrolled. serum levels of endocan were measured on days , and . results showed that concentrations of endocan were greater among patients at septic shock, and that day levels of endocan could prognosticate both -day and six-month outcome [ ] . although at first glance our study appears similar in design, it presents with some clearly distinctive characteristics in the design: (a) serial sampling at clinically indicative time points of progression into a new organ dysfunction or improvement, (b) analysis based on the new sepsis- definitions, and (c) correlation of changes of baseline endocan with progression into specific organ dysfunctions. endocan is a proteoclycan located on endothelial cells of the lungs and kidneys. at the event of a systemic inflammation, the molecule is cleaved through the activity of cathepsin g of neutrophils and a kda polypeptide is generated at greater concentrations in patients with sepsis than in healthy volunteers [ ] and also in patients with severe sepsis and septic shock than in patients with sirs [ , ] . former studies have shown that this glycoprotein may be involved in the pathophysiology of an infection without its contribution been clearly defined. in a prospective study of patients, serum endocan greater than . ng/ml was associated with the presence of bacteremia [ ] . our overall findings are in general agreement with those of recent studies enrolling a lower number of patients. these studies have explored the probable associations of admission endocan levels with the development of organ failures within two to five days. in a study of patients with multiple injuries, developed acute lung injury (ali) and did not. admission levels of endocan in the icu were lower among patients who developed ali compared to those who did not [ ] . a similar finding was described in critically ill patients; admission serum endocan was lower among those who developed respiratory failure on day [ ] . their findings may at first appear contradictory to ours. however follow-up measurements were missing so it could be hypothesized that serum endocan might increase on the day of respiratory failure. the first study on critically ill patients showed that endocan was greater among patients who developed organ failures and mods within the first h post admission [ ] . the second study on patients with ards showed that admission endocan levels were greater in nonsurvivors than in survivors [ ] . our study is not using the traditional design that compares the concentrations of circulating peptides between patients with infection and patients with sepsis. instead, all enrolled patients had sepsis at baseline and the changes of circulating peptides between those improving and those worsening were compared. our findings suggest that changes of circulating cytokines do not mediate progression of the clinical course of sepsis. probably this is explained by the fact that upon deterioration of sepsis the patient is already at the state of immunosuppression where further cytokine production does not take place [ , ] . instead endocan and ang are increased and this may also be interpreted as a key-playing role in the pathogenesis of ards and septic shock. from the practical aspect, it becomes evident that any increase of endocan is a surrogate of prediction for the overall deterioration of the patient. our findings clearly show that endocan and angiopoietin- are mediators increased upon deterioration of the clinical course of sepsis. any baseline increase of endocan is a specific surrogate marker to indicate worsening of the patient. increases of endocan are independently associated with progression into adult respiratory distress syndrome. the findings require validation by an independent cohort where changes of endocan are correlated with respective changes of the sofa score. compliance with ethical standards the study protocol was approved by the ethics committees of the participating hospitals. funding the study was funded by the hellenic institute for the study of sepsis. immunoassay reagents for endocan were a kind donation by lunginnov s.a.s (campus de l'institut pasteur de lille, , lille, france). conflict of interest none of the authors has any conflict of interest related to this submission. ethical approval the study protocol was approved by the ethics committees of korinthos general hospital, korgialeneion benakeion general hospital, attikon university hospital, bg.gennimatast hessaloniki general hospital, sotiria general hospital,baghios pavlos^thessaloniki general hospital, and tzaneion general hospital. informed consent a patient was enrolled after written informed consent provided by his first-degree relatives. community-and healthcareassociated infections in critically ill patients: a multicenter cohort study the third international consensus definitions for sepsis and septic shock (sepsis- ) sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy angiopoietins in angiogenesis and beyond endocan is a novel chondroitin sulfate/dermatan sulfate proteoglycan that promotes hepatocyte growth factor/scatter factor mitogenic activity sis international sepsis definitions conference the international sepsis forum consensus definitions of infections in the intensive care unit diagnosis and treatment of disseminated intravascular coagulation (dic) according to four dic guidelines endothelial cell-specific molecule- /endocan: diagnostic and prognostic value in patients suffering from severe sepsis and septic shock identification of a kda fragment generated by cathepsin g, a novel circulating biomarker in sepsis endocan, a new endothelial marker in human sepsis characteristics of serum endocan levels in infection lower serum endocan levels are associated with the development of acute lung injury after major trauma evaluation of endothelial biomarkers as predictors of organ failures in septic shock patients endocan is a useful biomarker of survival and severity in sepsis endocan levels in peripheral blood predict outcomes of acute respiratory distress syndrome inhibition of caspase- activation in gram-negative sepsis and experimental endotoxemia reversal of immunoparalysis in humans in vivo: a double-blind, placebo-controlled, randomized pilot study key: cord- - w r l authors: lalueza, a.; trujillo, h.; laureiro, j.; ayuso, b.; hernández-jiménez, p.; castillo, c.; torres, m.; folgueira, d.; madrid, o.; díaz-pedroche, c.; arrieta, e.; arévalo, c.; lumbreras, c. title: impact of severe hematological abnormalities in the outcome of hospitalized patients with influenza virus infection date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: w r l although hematological abnormalities have been described among patients with influenza virus infection, little is known about their impact on the outcome of the patients. the aim of this study was to assess the frequency and clinical impact of severe hematological abnormalities in patients with confirmed influenza virus infection. this was an observational retrospective study including all adult patients with diagnosis of influenza virus infection hospitalized from january to may in our institution. influenza virus infection was diagnosed by means of rrt-pcr assay performed on respiratory samples. poor outcome was defined as a composite endpoint in which at least one of the following criteria had to be fulfilled: (a) respiratory failure, (b) sofa ≥ , or (c) death. two hundred thirty-nine patients were included. applying the hlh- criteria for the diagnosis of hemophagocytic syndrome, cytopenias (hemoglobin ≤ g/dl, platelets < , /μl or neutrophils < , /μl) were present in patients ( %). patients with hematological abnormalities showed higher sofa scores, respiratory failure, septic shock and in-hospital mortality than the remaining patients. the composite endpoint was present in . % in the cytopenias group vs. . % in the group without cytopenias (p= . ). in a multivariate analysis, variables associated with the composite endpoint were: use of steroids prior to present admission (or: . ; % ci: . – . , p= . ), presence of any hematological abnormality (or: . ; % ci: . – . , p= . ), and ldh> u/l (or: . ; ci: – . , p= . ). hematological abnormalities are not uncommon among hospitalized patients with influenza virus infection, and they are associated with a poorer outcome. hematological abnormalities have been previously described among patients with influenza virus infection [ , ] . moderate thrombocytopenia [ , , ] and lymphopenia [ ] [ ] [ ] [ ] [ ] [ ] are the most common findings, whilst anemia and neutropenia are rarely described [ ] . as an example, the incidence of leukopenia and thrombocytopenia in patients who were hospitalized for the treatment of influenza a (h n ) pdm early in the u.s. epidemic was % and %, respectively [ ] . most studies directed toward identifying mortality risk factors in influenza virus infection mainly take into consideration clinical variables, especially those related to comorbidities [ , ] . little is known about the impact of hematological abnormalities in the prognosis of this infection [ , ] . lymphopenia and thrombocytopenia are more frequently seen in patients with respiratory failure and shock [ , ] . additionally, a tendency toward increased mortality has been observed in the presence of lymphopenia or thrombocytopenia. however, these findings have not been confirmed in large multivariate analyses [ ] . moreover, hemophagocytic syndrome (hps) secondary to influenza virus infection has been rarely described in immunocompetent and immunocompromised patients, with a high mortality rate [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nevertheless, the real incidence of this severe complication in patients with influenza is not known. to the best of our knowledge, there is only one prospective observational study carried out in critically ill patients with respiratory failure and hps secondary to influenza a (h n ) pdm infection. hps was found in patients ( %), with a high mortality rate ( %) compared to those without hps ( %) [ ] . the aim of the present study was to assess the frequency and clinical impact of hematological abnormalities in the range of those accepted by the histyocite society for the suspicion of hps [ ] in patients who were admitted to the hospital with a confirmed influenza virus infection. we conducted an observational retrospective study including all adult patients with a diagnosis of influenza virus infection hospitalized from january to may in a -bed tertiary teaching hospital in madrid, spain. the study protocol was approved by the university hospital de octubre review board. a confirmed case was defined by a positive result of a real-time reverse-transcriptase-polymerase-chain-reaction (rrt-pcr) assay performed at the local laboratory performed on respiratory samples [nasopharyngeal swabs (flocked swabs in utm™ viral transport medium, copan, brescia, italy)] from adult patients with respiratory tract symptoms. for the molecular diagnosis, rna was extracted from μl of the specimen using nuclisens®easymag instrument (biomérieux diagnostics, marcy l'etoile, france) and eluted in μl. five μl of the elution were employed to perform each rt-pcr reaction. the modular duplex rrt-pcr for influenza a/influenza b detection (influenza a/b r-gene ™, biomérieux) was run in the lightcycler instrument (roche) [ ] . all samples testing positive for influenza awere subtyped using rrt-pcr previously described [ ] to detect specific regions of subtypes h and h hemagglutinin. for the detection of influenza a (h n ) pdm subtype commercially available primers and probe (realtime ready infa/h n detection set, roche) [ ] were used. hematological abnormalities secondary to influenza virus infection were only considered when they are in the range of the hlh- updated criteria proposed by the histiocyte society [ ] for the diagnosis of hematophagocytic syndrome (hemoglobin ≤ g/dl, platelets < , /μl, neutrophils < /μl). a diagnosis of hemophagocytic syndrome needed to fulfill at least five of the clinical and non-clinical findings included in the previously mentioned hlh- updated criteria [ ] : (a) fever of . °c or more, (b) splenomegaly, (c) cytopenias affecting at least two of three cell lineages in peripheral blood (hemoglobin ≤ g/dl, platelets < , /μl, neutrophils < /μl), (d) hypertriglyceridemia (≥ mg/dl) and/or hypofibrinogenemia (≤ mg/dl), (e) hyperferritinemia (≥ ng/ml), (f) hemophagocytosis in bone marrow, spleen, lymph nodes, or liver, (g) low or absent nk cell activity or (h) increased soluble il- receptor concentration. comorbidity was defined using the charlson index. immunosuppression was defined as the presence of any the following: active malignant neoplasia, autoimmune disease, solid organ transplantation, hiv infection, use of steroids or chemotherapy. use of steroids was defined as: ( ) more than mg/day of oral prednisone for days or longer or ( ) less than mg/day over a minimum of months [ ] . respiratory failure was defined as the need for mechanical ventilation, either non-invasive positive pressure ventilation or invasive mechanical ventilation, including those patients who had a clinical indication for ventilatory support but were finally not ventilated. sepsis, septic shock and organ dysfunction were defined according to the terms proposed recently by the third international consensus definitions for sepsis and septic shock [ ] using for this purpose the sofa score and the qsofa score. acute respiratory distress syndrome (ards) was defined according to the american-european consensus conference on ards [ , ] . poor outcome was defined as a composite endpoint in which at least one of the following criteria had to be fulfilled: (a) respiratory failure, (b) sofa ≥ , or (c) death (related or not related to influenza infection). a descriptive analysis of patients was initially performed, comparing those who had cytopenias with those who did not have cytopenias. descriptive analysis was performed using means (±sd) or medians with interquartile ranges (iqr). student's t-test for independent samples was used to compare continuous variables. mann-whitney u test was used to compare continuous variables with a non-normal distribution, and the fisher exact test to compare proportions. we further analyzed the risk factors associated with poor outcome by means of a logistic regression model that included those variables found to be statistically significant at the univariate level or those deemed clinically relevant. associations were expressed as odds ratios (ors) with % confidence intervals ( % ci). seventeen independent variables were initially selected based on clinical judgment and published literature. prior to model fitting, cluster analysis was used to reduce the number of candidate variables. backward selection with a type i error rate of . was used to reach a final reduced model containing three predictor variables. discrimination of the final model was quantified via a c index (roc area). all statistical tests were -tailed and the threshold of statistical significance was p < . . statistical analysis was performed with computer software ( during the study period cases of influenza virus infection were confirmed by rt-pcr at our institution, of which were included in the present study (fig. ). all cases were diagnosed between january and may . patient's demographic and clinical characteristics are shown in table . only ( . %) patients were living in a nursing home at the time of admission, and three patients were hiv positive. only % of the cases were vaccinated against influenza with a mean time between vaccination and confirmed infection of . ± . days. symptoms initiated a median of days (range - days) before influenza diagnosis. dyspnea was present in . % of the patients, and bronchospasm was described in % of them. laboratory parameters of the study population are presented in table . both baseline and nadir values were significantly lower in all cell lineages in patients who developed cytopenias during admission (fig. ). the median time to nadir of leukocytes, neutrophils, lymphocytes, platelets and hemoglobin was day (range, - ), days (range, - ), days (range, − to ), days (range, − to ), and day (range, - ), respectively. when applying the hlh- criteria, we observed that % (n = ) of the patients had at least one hematological abnormality, while . % (n = ) had cytopenias affecting at least two of three cell lineages. as is shown in table , neutropenia (< neutrophils/μl) was present in . % of the cases (n = ), thrombocytopenia (< , platelets/μl) in . % of the cases (n = ), and anemia (hb < g/dl) in . % (n = ) of the cases. in the group of patients with at least one hematological abnormality, neutropenia was present in . %, thrombocytopenia in . %, and anemia in %, respectively. only one patient fulfilled the five criteria required for the diagnosis of hps. one patient met three criteria for the diagnosis of hps, six patients met two criteria, and cases met one criterion. overall, of the patients with at least one hematological abnormality did not meet any of the hlh- criteria. however, it is noteworthy that fibrinogen levels were only available in eight patients (> mg/dl in all cases), triglyceride levels in patients (≥ mg/dl in one case), and ferritin levels in patients (> ng/dl in cases). nk cell activity was not available in any case, while increased soluble il- receptor concentration was observed in one of the two patients from whom it was available. bone marrow biopsy was performed in one patient, which showed % of hemophagocytosis. splenomegaly was not reported in any patient. of patients in which chest x-ray was performed, . % (n = ) had abnormal findings ( . % in the cytopenias group vs. . % in the group without cytopenias, p = . ). the most common radiologic findings were patchy infiltrates ( . %) and interstitial infiltrates ( . %). thoracic computed tomography was performed in cases, of which . % (n = ) had abnormal findings ( . % in the cytopenias group vs. . % in the group without cytopenias, p = . ). the most common abnormalities were patchy infiltrates ( . %) and ground-glass opacities ( . %). as is shown in table , patients ( . %) developed an associated bacterial pneumonia in the course of admission (p = ns between groups) with staphylococcus aureus being the causative microorganism in two cases ( . %), both in the cytopenias group. antiviral treatment with oseltamivir was started in . % (n = ) of the patients and it was always initiated early upon confirmation of influenza diagnosis. empiric antibiotic treatment was started in . % (n = ) of the cases, with a median duration of treatment of days (range . - ). in [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in the group without cytopenias, p = . ). overall in-hospital mortality was . %, with . % influenza related mortality, and a -day in-hospital mortality of . %. as is shown in table , patients with hematological abnormalities presented with higher sofa and qsofa scores, had longer hospital and icu stays, and more frequently presented respiratory failure and septic shock that required results are expressed as mean ± standard deviation or median with interquartile range (iqr) or as absolute value (percentage). values in bold text are considered statistically significant a upper limit of ldh in the local laboratory is mg/dl. b nk cell activity was not determined in any case, splenomegaly was not reported in any patient, bone marrow biopsy was performed in only one patient, and soluble il- receptor concentration was determined in only two patients. c dexamethasone ( mg/m ), observing a prompt clinical improvement icu admission. as a consequence, we did notice significant differences between the two groups in the poor outcome composite endpoint ( . % vs. . %, p = . ). patient characteristics with respect to the poor outcome composite endpoint are presented in table . no statistically significant differences between active smokers ( . % in the good outcome group vs. . % in the poor outcome group) or ex-smokers ( . % in the good outcome group vs. . % in the poor outcome group) were observed. in the favorable outcome group, of the cases ( . %) presented complete recovery of the episode and nine patients ( . %) presented sequelae, while in the poor outcome group only of the cases ( . %) presented complete recovery and five cases ( . %) presented sequelae. twelve patients died during hospitalization. thirty-day related mortality was . % in the good outcome group vs. . % in the poor outcome group (p < . ). a reduced model to identify factors associated with poor outcome was generated. the three following variables remained significantly associated with the presence of the composite endpoint: use of steroids prior to present admission (or: . ; % ci: . - . , p = . ), presence of any hematological abnormality (or: . ; % ci: . - . , p = . ), and ldh > u/l (or: . ; ci: - . , p = . ) ( table ). this observational retrospective study seeks to address mainly two issues. on the one hand, we seek to identify the frequency of hematological abnormalities (cytopenias) secondary to influenza virus infection in patients who required in-hospital treatment. on the other hand, we seek to determine if these hematological abnormalities are associated with a poor outcome defined as a composite endpoint that included the presence of respiratory failure, sofa score ≥ , and mortality. values are expressed as the mean percentage difference (or median) in both groups (cytopenias vs no cytopenias) between initial laboratory findings and nadir. the mean had been used for the hemoglobin and the median for the rest of the variables. ( ) group with cytopenias, ( ) group without cytopenias) in the present study, hematological abnormalities were defined according to the criteria proposed by the hlh- for the diagnosis of hps [ ] . one theoretical disadvantage of these criteria is that lymphocyte count is not considered as part of the diagnosis and lymphopenia is frequently seen in patients with influenza virus infection. additionally, more severe cytopenias are considered in the hlh- definition than those frequently reported in patients with influenza virus infection [ ] [ ] [ ] [ ] [ ] [ ] . however, it is noticeable that in the present study we observed that . % of the patients presented with at least one cytopenia in the hlh- range, while . % of the cases presented with cytopenias affecting at least two of three cell lineages. thrombocytopenia ( % of all patients) was the most common hematologic abnormality. as is shown in table , these hematological abnormalities were already present in baseline laboratory data at admission. additionally, the median time to nadir was between and day depending on the affected cell lineage. these observations suggest that cytopenias present early in the course of the infection. we therefore believe that the presence of hematological abnormalities is an early predictive marker of poor outcome in patients with influenza virus infection who required hospitalization. interestingly, we observed a tendency of a decrease in peripheral blood cell values even in patients without cytopenias, with a median time to nadir similar to those patients with cytopenias. we did not randomly choose the hlh- criteria for defining the hematological abnormalities in the present study. we suspect that hemophagocytic syndrome triggered by influenza virus infection was more frequent than previously reported. unfortunately however, no specific disease markers were ordered in most cases, making it impossible for us to retrospectively know the exact incidence of this disorder in our present cohort. in fact, albeit patients presented with at least one cytopenia according to the hlh- criteria [ ] , fibrinogen levels were available in only eight cases, triglycerides and ferritin levels in cases, and soluble il- receptor concentration in only two cases. although rarely, hemophagocytic syndrome has been described in patients with severe influenza virus infection, usually associated with a poor outcome and a high mortality rate [ , ] . in beutel's study of critically ill patients with influenza a (h n ) pdm virus associated hemophagocytic syndrome, the absence of steroid therapy in the early phase of the infection might have contributed to the high incidence of hps ( out of patients) and the rather poor outcomes [ ] . we consider that a low clinical index of suspicion for influenza associated hemophagocytic syndrome is the main factor for delayed initiation of immunomodulatory therapy, which could have improved the outcome in these patients [ ] . in the present study, a multivariate model was conducted to identify risk factors associated with a poor outcome. presence of hematological abnormalities and ldh levels > u/l remained significantly associated with a worse outcome in patients with influenza virus infection, while previous use of steroids was identified as a favorable prognostic factor. although no conclusive evidence has been reached, thrombocytopenia has been associated with a worse outcome in previous studies [ , ] . in our univariate analysis, both thrombocytopenia and anemia were significantly associated with poor outcome. to our knowledge, anemia as a poor prognostic marker in patients with influenza virus infection had not been previously described. indeed, the two most notable aspects of our model are the presence of any hematological abnormality as a marker of poor outcome and the potential beneficial role of steroids in these patients. an interesting finding in our study was the better outcome observed in patients who were already using steroids before admission, while the use of steroids during admission was associated with poor outcome. this paradox could be explained by two facts. first, as in other series [ , ] , steroids were mostly prescribed for copd exacerbations, asthma exacerbations or bronchospasms, but not as immunomodulatory therapy; therefore, time to treatment initiation, duration of treatment, and prescribed doses, were not always optimal. in fact, only one patient in our series received high.dose steroids (dexamethasone mg/m ) and that was for the treatment of hemophagocytic syndrome triggered by influenza virus infection with marked hemophagocytosis in bone marrow. second, it is known that onset of influenza a viruses infections is very acute by triggering a cascade of immune responses and switching on almost all parts of the immune defense system [ , ] . in fact, in the present study, hematological abnormalities presented early in the course of the infection in a large number of patients. we believe that steroid therapy before admission could have modulated the acute inflammatory response triggered by influenza virus infection in the early course of the disease, and with this, prevented an uncontrolled immune response [ ] . this finding, if confirmed in properly prospective designed studies, may establish the utility of early use of steroids in some subgroup of patients with influenza virus who required hospitalization. by using more restrictive criteria for the definition of cytopenias as compared to other series, our study might have underestimated the percentage of patients who presented with hematological abnormalities. additionally, the observational retrospective nature of the study complicates the accurate assessment of the role of steroid therapy during admission, especially since it was mostly prescribed for other indications rather than the infection itself. this wide range of indications, although similar to other series, precludes a correct assessment of steroid therapy as a poor prognostic factor. the potential benefit of steroid therapy in patients with influenza virus infection with risk factors for poor outcome should be further studied. unfortunately, an active pursuit of hemophagocytic syndrome secondary to influenza virus infection was conducted only in a few cases, which precluded us to confirm the real incidence of this severe complication in the subgroup of patients with hematological abnormalities. since a complete blood count is an easily accessible test, we believe that it should be the cornerstone in the screening of a possible underlying hps secondary to influenza virus infection. if severe cytopenias were observed, we consider that a systematic determination of ferritin, triglycerides and fibrinogen levels would be beneficial. in the case of abnormal levels of any of these three hps markers, determination of nk cell activity and soluble il- receptor concentration should be ordered to confirm the diagnosis. significant hematological abnormalities are frequently seen in patients with influenza virus infection who required hospital admission and are associated with a poor outcome. the ongoing use of steroids upon the start of influenza was associated with a better prognosis suggesting that an early immunomodulatory therapy could improve the outcome of these patients. funding information this study has not received any funding. predictors and outcomes of respiratory failure among hospitalized pneumonia patients with h n influenza in taiwan severe influenza in us hospitals, - : complications and risk factors for death in patients risk factors for death from influenza a(h n )pdm , state of sao paulo, brazil hospitalized patients with pandemic influenza a (h n ) virus infection in the united states clinical features of the initial cases of pandemic influenza a (h n ) virus infection in china diagnostic importance of relative lymphopenia as a marker of swine influenza (h n ) in adults hospitalized patients 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coordination the distinct clinical profile of chronically critically ill patients: a cohort study infections associated with haemophagocytic syndrome cytotoxic therapy for severe avian influenza a (h n ) infection induction of innate immunity and its perturbation by influenza viruses the host immune response in respiratory virus infection: balancing virus clearance and immunopathology conflict of interest the authors declare no conflicts of interest.ethical approval the study protocol was approved by the university hospital de octubre review board. key: cord- -f o z authors: gizzi, m.; delaere, b.; weynand, b.; clement, j.; maes, p.; vergote, v.; laenen, l.; hjelle, b.; verroken, a.; dive, a.; michaux, i.; evrard, p.; creytens, d.; bulpa, p. title: another case of “european hantavirus pulmonary syndrome” with severe lung, prior to kidney, involvement, and diagnosed by viral inclusions in lung macrophages date: - - journal: eur j clin microbiol infect dis doi: . /s - - -x sha: doc_id: cord_uid: f o z puumala virus (puuv) is considered a classic old world etiologic agent of nephropathia epidemica (ne), or hemorrhagic fever with renal syndrome (hfrs). hfrs is considered to be distinct from hantavirus (cardio-)pulmonary syndrome (hps or hcps), described in the new world. here, we report a severe case, which fulfilled most, if not all, centers for disease control and prevention (cdc) criteria for hps, needing non-invasive ventilation and subsequent acute hemodialysis. however, the etiological agent was puuv, as proved by serological testing, real-time polymerase chain reaction (pcr), and sequencing. viral antigen was detected by specific anti-puuv immunostaining, showing, for the first time, greenish intracytoplasmic inclusions in bronchoalveolar lavage (bal) macrophages. this case definitely confirms that hps can be encountered during puuv infections. interestingly, special findings could render the diagnosis easier, such as greenish homogeneous cytoplasmic inclusions, surrounded by a fine clear halo in bal macrophages. therefore, although the diagnosis remains difficult before the onset of renal involvement, the occurrence of severe respiratory failure mimicking community-acquired pneumonia must alert the clinician for possible hps, especially in endemic areas. members of the genus hantavirus comprise the only nonarthropod-borne viruses in the family bunyaviridae. hantaviruses are primarily transmitted by the inhalation of aerosolized viral particles shed by rodents, which serve as chronically infected but asymptomatic reservoirs. recent overviews [ , ] have listed up to named hantaviruses, of which at least were described as human pathogens, mostly in the americas. this is in contradiction, however, with the official standpoint of the international committee of taxonomy of viruses (ictv) having recognized, so far, only lineages as separate hantavirus species, of which not all are known pathogens [ ] . each hantavirus species is multiplied and spread by a mostly unique rodent species. the most important pathogens are hantaan virus and seoul virus in the far east (> % of worldwide infections), puumala virus (puuv) and dobrava-belgrade virus in europe and russia, and, finally, sin nombre virus and andes virus in the americas [ , ] until recently, two clinical syndromes were classically described: hantavirus (cardio-)pulmonary syndrome (hps or hcps) described since in the americas [ ] and hemorrhagic fever with renal syndrome (hfrs) or its milder form nephropathia epidemica (ne), known in eurasia since the early s. however, a considerable overlap of symptoms was noted from onwards [ ] , and recent evidence has shown yet more pathogenic similarities between the syndromes caused by the two groups of genetically related viruses [ , , ] . the case described herein adds to the mounting evidence that hps can be seen in europe, and be caused by puuv, as reported previously [ , [ ] [ ] [ ] . a -year-old belgian woman was admitted in the emergency room with fever of °c, thoracic pain, and progressive dyspnea lasting for days. she also complained of rhinorrhea, blurred vision, headache, unusual lumbar discomfort, and nausea, with two episodes of vomiting. her general practitioner started ibuprofen ( mg × /d), as well as a quinolone (levofloxacin mg/d) days before her hospital admission. she had smoked since her youth ( pack-years) and her medical history was characterized by mild asthma, fallopian tube ligature, and a subarachnoid hemorrhage treated by endovascular therapy, resulting in complete recovery. carbamazepine, prescribed to prevent seizures, was her only drug. she lived with her family in doische, a rural area in the ardennes, the densely forested south of belgium, a region highly endemic for ne [ ] . she had not traveled recently and had no pets. her job consisted on delivering newspapers by car or on foot. she also took a stroll through the woods near doische about weeks before the onset of symptoms. physical examination revealed bibasal lung crackles with bilateral lumbar pain and conjunctivitis. her heart rate was /min and blood pressure / mmhg. arterial blood gases obtained while the patient was breathing room air on admission showed hypoxemia with respiratory alkalosis (ph . , po mmhg, pco mmhg). other significant laboratory findings were: crp mg/dl, thrombocytes , /μl, leukocytes , /μl (atypical activated lymphocytes %), hyponatremia meq/l, hypokalemia . meq/l, asat ui/l [normal value (nv) < ], alat ui/l (nv< ), ldh ui/l (nv< ), but a strictly normal renal function on admission, with creatinine . mg/dl and urea mg/dl. at admission, i.e., days post onset of symptoms (pos), chest x-ray showed perihilar infiltrates. intravenous amoxicillin/clavulanate ( g/day) and clarithromycin ( g/day) were started for suspected severe community-acquired pneumonia. due to the increase of hypoxemia and the deterioration of pulmonary infiltrates (fig. ) , the patient was eventually admitted to the intensive care unit (icu) h later, i.e., days pos. at admission, she was normotensive ( / mmhg) and tachypneic (respiratory rate /min), tachycardic (heart rate /min), and had an oxygen saturation of only % under l/min of oxygen. arterial blood analysis with fio at % showed ph: . ; po : mmhg; pco : mmhg. anomalies in laboratory values progressed: at day pos, neutrophils had increased to . /μl, sodium was meq/l, osmolarity mosm/kg, ldh , ui/l, ferritin . μg/l, total cholesterol mg/dl, hdl cholesterol mg/dl, and fasting triglycerides were mg/dl (nv< mg/dl). protein electrophoresis, fan, anca, and troponin i were normal, as well as renal function. a urine spot, however, showed significant proteinuria of . g/l, with an increased urinary sodium of meq/l. despite antibiotics, her respiratory distress worsened and initiation of non-invasive ventilation with high oxygen level was required, but orotracheal intubation could be avoided. blood cultures and sputum remained negative. blood serology was negative for epstein-barr virus, human cytomegalovirus (cmv), human immunodeficiency virus (hiv), hepatitis b virus, hepatitis c virus, chlamydia pneumoniae/trachomatis, and mycoplasma pneumoniae, as well as urinary legionella antigen. at day pos, a thoracic computed tomography (ct) scan exhibited bilateral ground glass opacities, predominantly in inferior lobes (fig. ) and transthoracic echocardiography showed normal left ventricular function without pericardial effusion. at day pos, a bronchoalveolar lavage (bal) was performed in the lingula. the tracheobronchial mucosa appeared slightly inflammatory. the bal fluid was showing bilateral pulmonary interstitial infiltrates macroscopically gray, and contained white cells/mm , with % of them macrophages, and and % of neutrophils and eosinophils, respectively. lymphocyte subtype revealed % of cd and % of cd t cells. interestingly, macrophages contained isolated or multiple greenish homogeneous cytoplasmic inclusions of variable size that were surrounded by a fine clear halo (fig. ) . this pattern suggested a viral infection, and no bacteria could be identified at direct examination. hemosiderin was abundant in the fluid. bal cultures were negative for bacteria, fungi, mycobacteria, and virus (including respiratory syncytial virus, adenovirus, influenza, or hcmv). only at day pos oliguric renal failure developed, suggesting, finally, the possibility of a hantavirus infection. serum creatinine rose to . mg/dl from . mg/dl the day before, and reaching a peak of . mg/dl at day pos. puuv infection was confirmed by serology [enzyme-linked immunosorbent assay (elisa) technique] at day pos, using puuv strain cg - antigens, with igm optical density at . (nv below . ) and igg at . (nv below . ), using a -fold diluted serum sample. consequently, antibiotics were discontinued. the course of disease was complicated by anuria and hypotensive shock, responding well to colloid challenge and vasopressors therapy. a total of five hemodialyses were required to treat uremia (> mg/dl), without signs of fluid overload. finally, the patient was discharged from the icu days pos without renal assistance, and from hospital on day pos with transient oxygen support. bal fluid was subsequently analyzed by immunohistochemistry. macrophage inclusions stained positively with a specific puuv antibody (fig. a, b) . the acute serum at days pos was also tested by real-time polymerase chain reaction (pcr) amplification, and an amplicon of bp was obtained. sequencing showed a puuv belonging to the same clade of four other puuv lineages, found previously in the geographically direct vicinity, i.e., the belgian ardennes (fig. ) [ ] . contrary to the paradigm that puuv could only induce ne or hfrs, there is now increasing evidence that a clinical picture showing that most, if not all, features of hps might also be encountered in european ne cases, as reported previously [ , [ ] [ ] [ ] [ ] ] . in fact, one of the very first puuv cases in belgium, described in , presented likewise with manifest pulmonary involvement and arterial desaturation, before any renal deterioration was detected, and noteworthy, was diagnosed also in our hospital [ ] . the history of our patient fulfills most of the eight clinical findings specified in the "hantavirus pulmonary syndrome case report form", as issued by the centers for disease control and prevention (cdc), atlanta, ga [ ] : ( ) fever, ( ) thrombocytopenia, ( ) elevated hematocrit, ( ) elevated serum creatinine, ( ) left shift leukocytosis with "atypical lymphocytes", ( ) need for supplemental oxygen, ( ) need for intubation (and mechanical ventilation), and ( ) chest radiograph showing unexplained bilateral infiltrates or being suggestive of adult respiratory distress syndrome (ards). the level of atypical lymphocytes seen in new world hps cases is higher (rarely if ever less than %) than the % observed in our patient [ ] . moreover, and typical for hps, rather than for classic hfrs, is the initial clinical and radiological presentation with pronounced pulmonary involvement, but without any initial renal injury on admission, as described recently in three severe swedish ne cases, two of which were fatal [ ] . pulmonary edema secondary to right heart decompensation after overhydration due to oliguria as a suggested mechanism in hfrs [ ] could clinically and radiologically be excluded in cases described in belgium [ , ] , france [ ] , and sweden [ ] , as in this case with normal echocardiography. other biological markers mainly noted so far in hfrs were also present: marked proteinuria, fasting hypertriglyceridemia contrasting with low levels of total cholesterol, and very low hdl levels [ , , ] . finally, initial blurred vision due to transient shallowing of the anterior eye chamber is an often overlooked but very specific clinical sign in ne and, to a lesser degree, hfrs cases [ , , ] , but have not been reported so far in hps cases from the americas. in the herein described hps case, puuv infection was confirmed serologically, and corroborated by positive blood reverse transcription (rt)-pcr, confirming the considerable clinical overlap between the new and old world syndromes. on the other hand, predominance of cd lymphocytes in bal has been considered so far as typical for hps [ , ] . this case revealed the presence of greenish homogeneous cytoplasmic inclusions of variable size surrounded by a fine clear halo in bal macrophages, a new distinctive sign that is particularly useful. with specific immunostaining, it was possible to prove that these images corresponded to puuv viral inclusions. puuv viral rna was demonstrated by real-time pcr in bal at day pos in a swedish ne case [ ] , but the detection of viral antigen with immunohistochemistry has been described so far only on autopsy lung specimens [ ] . our case further supports the value of fiberoptic bronchoscopy in outpatients suffering from severe respiratory failure with bilateral pulmonary infiltrates of unknown origin. hantavirus pathological mechanisms are not well understood, especially the reason why some puumala viruses had a more pronounced renal or pulmonary feature. since direct viral tissue damage is negligible, a so-called "cytokine storm" as an immunopathological response of the human host seems to be of core importance [ ] . recent publications suggested that the increased capillary permeability observed in both hps and hfrs might be caused by a common immunopathological mechanism, i.e., hantavirus-specific cytotoxic cd + t lymphocytes, attacking endothelial cells presenting viral antigens on their surfaces [ ] . it is also possible that direct viral toxicities or toxicities due to mediators should be considered as well, since t cells apparently cannot be implicated in pathogenesis in an hps animal model [ ] . in conclusion, this case firmly confirms that puuv is also an etiologic agent of hps. additionally, special features could help the diagnosis, such as greenish homogeneous cytoplasmic inclusions, surrounded by a fine clear halo in bal macrophages. therefore, diagnosis clinical suspicion of hfrs remains difficult on admission, before the onset of renal function impairment. however, the combination of an episode of fever with blurred vision, early and massive proteinuria as in this patient, followed by severe lung involvement looking like a community-acquired pneumonia, elevated ldh, hyponatremia, and thrombocytopenia must alert the clinician for a possible hantavirus infection with hps features, especially in endemic areas. a global perspective on hantavirus ecology, epidemiology, and disease hantaviruses in the americas and their role as emerging pathogens a unifying hypothesis and a single name for a complex globally emerging infection: hantavirus disease hantavirus pulmonary syndrome: a clinical description of patients with a newly recognized disease. the hantavirus study group hantavirus pulmonary syndrome in new england and europe time to revise the paradigm of hantavirus syndromes? hantavirus pulmonary syndrome caused by european hantavirus plasma exchange-associated immunoglobulin m-negative hantavirus disease after a camping holiday in southern france pulmonary-renal syndrome due to hemorrhagic fever with renal syndrome: an unusual manifestation of puumala virus infection in france hantavirus nephropathy in belgium: description of new cases originating in southern provinces epidemic of hantavirus disease in entre-sambre-et-meuse: year - . clinical and biological aspects hantavirus nephropathy as a pseudo-import pathology from ecuador hantavirus infections in europe and their impact on public health hantavirus pulmonary syndrome (hps) case definition rapid presumptive diagnosis of hantavirus cardiopulmonary syndrome by peripheral blood smear review acute kidney injury in emerging, non-tropical infections hantaviruses: immunology, treatment, and prevention presence of activated airway t lymphocytes in human puumala hantavirus disease t cells are not required for pathogenesis in the syrian hamster model of hantavirus pulmonary syndrome the authors declare that they have no conflict of interest. key: cord- -jo dhtb authors: maillet, m.; maubon, d.; brion, j. p.; françois, p.; molina, l.; stahl, j. p.; epaulard, o.; bosseray, a.; pavese, p. title: pneumocystis jirovecii (pj) quantitative pcr to differentiate pj pneumonia from pj colonization in immunocompromised patients date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: jo dhtb conventional polymerase chain reaction (pcr) in respiratory samples does not differentiate between pneumocystis pneumonia (pcp) and pneumocystis jirovecii (pj) colonization. we used pj real-time quantitative pcr (qpcr) with the objective to discriminate pcp from pj colonization in immunocompromised patients. all positive pj qpcr [targeting the major surface glycoprotein (msg) gene] obtained in respiratory samples from immunocompromised patients presenting pneumonia at the grenoble university hospital, france, were collected between august and april . diagnoses were retrospectively determined by a multidisciplinary group of experts blinded to the pj qpcr results. thirty-one bronchoalveolar lavages and four broncho aspirations positive for the pj qpcr were obtained from immunocompromised patients. diagnoses of definite, probable, and possible pcp, and pneumonia from another etiology were retrospectively made for , , , and patients, respectively. copy numbers were significantly higher in the “definite group” (median , copies/ml) than in the “probable group” (median , copies/ml), the “possible group” (median , copies/ml), and the “other diagnosis group” (median copies/ml). with the value of , copies/ml, the sensitivity and specificity of qpcr for the diagnosis of pcp were % and %, respectively. with the value of , copies/ml, the sensitivity and specificity were % and %, respectively. the positive predictive value was % for results with more than , copies/ml and the negative predictive value was % for results with fewer than , copies/ml. qpcr targeting the msg gene can be helpful to discriminate pcp from pj colonization in immunocompromised patients, using two cut-off values, with a gray zone between them. the incidence of pneumocystis pneumonia (pcp) in human immunodeficiency virus (hiv)-infected patients has decreased since the introduction of chemoprophylaxis and antiretroviral therapy [ ] ; meanwhile, the incidence of pcp in non-hiv immunocompromised patients is increasing [ ] . the standard laboratory method to diagnose pcp remains the microscopic identification of pneumocystis jirovecii (pj ) by staining methods in respiratory samples. however, studies highlighted the low burden of pj in non-hiv immunocompromised patients [ ] and the lack of sensitivity of microscopic methods [ ] . this justifies the increasing use of polymerase chain reaction (pcr) methods for diagnosis [ , ] . the detection of pj in individuals presenting without pneumonia or with pneumonia from another etiology has been defined as colonization or "carriage" [ ] . conventional pj pcr is qualitative and very sensitive, but does not differentiate between active pcp and pj colonization. the aim of this study was to use pj real-time quantitative pcr (qpcr) in order to differentiate pcp and pj colonization in immunocompromised patients. all positive pj qpcr obtained in respiratory samples from immunosuppressed patients presenting with pneumonia at the grenoble university hospital, france, were collected between august and april . we retrospectively studied the clinical histories of the concerned patients. the collected clinical data included: age, sex, underlying diseases, immunosuppression therapy during the previous months, pcp prophylaxis, clinical symptoms, routine laboratory data, anti-pneumocystis treatment, stay in a critical care unit, mechanical ventilation, hypoxemia (pao < mmhg in room air or requirement for supplemental oxygen), use of other anti-infectious agents, and outcome. the bronchoalveolar lavage (bal) and broncho aspirations samples were stained and examined by a qualified microscopist at the laboratory of parasitology-mycology, with μl of fluid analyzed by fast giemsa and μl observed after gomori-grocott staining. a dna extract solution from each sample was tested with a pj qpcr targeting the major surface glycoprotein (msg) gene of pj as previously described [ , ] . the results were expressed as the number of msg copies/ml of bal fluid. a group of six experts, including three infectious diseases specialists, one internist, one hematologist, and one mycologist, classified the clinical histories of these patients in one of the following categories: definite, probable, and possible pcp, or other diagnosis (case definitions in table ). the experts were blinded to the pj pcr results. statistical analyses were performed with the statview software (for windows, sas institute inc., cary, nc, usa). categorical variables were compared using the fisher's exact test. continuous variables were compared using the kruskal-wallis test. a receiver operating characteristic (roc) curve for copy numbers was constructed and used to define cut-off values in order to discriminate the definite and probable pcp groups from the colonized group (possible pcp group and other diagnosis). a p-value< . was considered statistically significant. the youden's index (se + sp − ) was calculated for each cut-off value, as well as the number of correctly classified patients, in order to determine an optimal cut-off value. thirty-one bal samples and four broncho aspirations with positive pj qpcr were obtained from immunocompromised patients. broncho aspirations were used when bal could not be performed. all of these patients had clinical presentations such as fever, cough, sputum, or dyspnea, and suspected pneumonia. seven patients met all the criteria for definite pcp, four patients for probable pcp, and five for possible pcp; another diagnosis was finally made for patients. prophylaxis therapy was used in one of the patients ( %). all definite, -clinical signs of progressive pneumonia and: -either compatible radiological signs -or complete resolution of symptoms after anti-pcp treatment -absence of microscopical identification of pj with staining methods other diagnosis none of the above criteria the "definite pcp" and "probable pcp" groups were then re-assigned to the "final diagnosis of pcp" group, and the "possible pcp" and "other diagnosis" groups were re-assigned to the "non-pcp" group in order to construct a receiver operating characteristic (roc) curve possible, and probable patients had received anti-pcp treatment. microscopic examination was positive in of samples of bal fluid samples (one patient's retained diagnosis was allergic bronchopulmonary aspergillosis). the clinical features for each group are described in table . there was no statistical difference between the four groups. real-time qpcr qpcr copy numbers were significantly higher in the definite group (median , copies/ml, range , - , , ) than in the probable group (median , copies/ml, range , - , ) and the possible group (median , copies/ml, range - , ) or the other diagnosis group (median copies/ml, range - , ) (fig. ) . the difference was significant between the four groups (p < . ). roc curve analysis gave an area under the curve of . (fig. ) , confirming the ability of qpcr to discriminate pcp from colonization. the sensitivity and specificity of qpcr on bal fluids for discrimination between pcp and colonization were estimated with different qpcr cut-off values. two cutoff values were determined to give % positive and negative predictive values, with a gray zone between them. the estimated cut-off values were , and , copies/ml, respectively. the sensitivity and specificity of qpcr for the diagnosis of pcp are specified in fig. for each cut-off value. the youden's index and the rate of correctly classified patients for each cut-off value provided an optimal cut-off value of , copies/ml for the diagnosis of pcp (fig. ) . the "other diagnoses" group included bacterial pneumonia (n = ), atypical pneumonia (n = ), influenza virus pneumonia (n = ), acute respiratory distress syndrome (ards) due to sepsis (n = ), candidemia (n = ), bronchiolitis obliterans organizing pneumonia (n = ), drug-induced pneumonia (n = ), bronchial hamartoma (n = ), multifactorial pulmonary fibrosis (n = ), allergic bronchopulmonary aspergillosis (n = ), pulmonary lymphoma (n = ), and undetermined (n = ) the diagnostic performance of the microscopic visualization of pj is dependent on the quality and type of sample, the number of organisms, and the experience of the microscopist [ ] . the higher sensitivity of pcr for pj detection has been demonstrated previously [ ] . the main risk of using conventional pcr for the diagnosis of pcp is a positive result for colonized patients having pneumonia from another etiology [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in this study, we observed that the qpcr results in the probable pcp group were significantly higher than those in the possible pcp group and in the group with another etiology, and were significantly lower than those in the definite pcp group. these results suggest that qpcr can help discriminate pcp from colonization and are in concordance with the hypothesis that the burden of pj is lower in colonized people than in patients with pcp [ ] . this is particularly interesting for non-hiv-infected immunocompromised patients, who often present with pcp with a low burden of pj organisms and may not be diagnosed using microscopic examination [ ] . qpcr values between , and , copies/ml fall within a gray zone, in which curative or prophylactic treatment should be discussed individually. it included nine out of the ( %) patients. nevertheless, the youden's index and the rate of correctly classified patients allowed determining the optimal cut-off value of , copies/ml, maximizing the accuracy for the diagnosis of pcp. a recent study concluded that early diagnosis and treatment are crucial for the survival of pcp patients without hiv infection [ ] , suggesting that a rapid and accurate pcp diagnosis method such as qpcr should be used in these patients with a low burden of pj . in our study, the cut-off value of , copies/ml would have avoided of ( %) treatments prescribed to patients whose final diagnosis was not pcp. this cut-off would allow physicians to stop probabilistic treatment, and, therefore, limit adverse effects, cost of treatment, and development of resistances [ , ] . a meta-analysis showed that pcr has good diagnosis accuracy and may be a useful tool for the diagnosis of pcp in immunocompromised patients [ ] . previous studies determined various cut-offs of qpcr targeting different genes to discriminate pcp from colonization: for msg gene, larsen et al. proved that a cut-off value of copies/tube for qpcr in oral-wash samples increased the specificity for diagnosis of pcp to % [ ] . flori et al.'s study established that a cutoff of copies of dna per capillary of bal (pcr targeting the msg gene) increased the specificity from . to . % with % sensitivity [ ] . other studies highlighted the usefulness of qpcr in discriminating colonization from pcp: alanio et al. proposed two cut-off values ( and , trophic forms equivalents/ml) with a qpcr targeting the mitochondrial large subunit ribosomal rna gene [ ] ; for matsumura et al., the value of , copies/ml distinguished pcp from colonization with a sensitivity of % and a specificity of %, using qpcr targeting the dhps (dihydropteroate synthase) gene [ ] . we need to consider limitations when interpreting our results: this is a single-center study, the number of patients is relatively small, and we combined the data from the bal and the broncho aspiration samples to evaluate the qpcr. without a test capable of confirming or excluding the diagnosis of pcp, the classification of patients is uncertain. however, one quality of our study is that patients were classified by a multidisciplinary group of experts, ruling by consensus in view of all the clinical and complementary data. the fact that the experts did not know the qpcr results avoids classification bias. in spite of the small number of patients, the observed differences between the groups are statistically significant, which suggests that these differences are important. in our study, we used qpcr targeting the msg gene, which is appropriate in discuss pcp diagnosis: the sensitivity is excellent, and it has been proved that qpcr analysis and the msg target have the highest specificity compared with other pcr assays [ ] . we can note that the rate of anti-pneumocystis prophylaxis was very low in our population. there is no current recommendation for anti-pcp prophylaxis or empirical therapy for non-hiv immunocompromised patients. it is known that colonization can sometimes lead to the development of pcp [ ] . we propose to treat patients with a high probability of pcp and pj burdens in the gray zone, and to monitor patients with low burdens and low probability of pcp with biological markers such as β-d-glucan, while prescribing a prophylactic treatment [ , , ] . the proportion of patients having a hematological malignancy was very high in our population. we hypothesize that cut-off values may differ with the underlying diseases and may vary from one center to another. in conclusion, our study shows that our qpcr targeting the msg gene in respiratory samples can help discriminate pcp from pneumonia with pj colonization in immunocompromised patients, using two cut-off values of , and , copies/ml, with a gray zone between them. cut-off values should be determined for each laboratory and each population of patients. trends in hospitalizations for aids-associated pneumocystis jirovecii pneumonia in the united states management and outcome patterns for adult pneumocystis carinii pneumonia, to : comparison of hiv-associated cases to other immunocompromised states pneumocystis pneumonia comparison between realtime pcr, conventional pcr and different staining techniques for diagnosing pneumocystis jiroveci pneumonia from bronchoalveolar lavage specimens clinical significance of quantifying pneumocystis jirovecii dna by using real-time pcr in bronchoalveolar lavage fluid from immunocompromised patients molecular diagnosis of pneumocystis pneumonia colonization by pneumocystis jirovecii and its role in disease characteristics and clinical relevance of the quantitative touch-down major surface glycoprotein polymerase chain reaction in the diagnosis of pneumocystis pneumonia update on the diagnosis and treatment of pneumocystis pneumonia epidemiology and clinical significance of pneumocystis colonization pneumocystis colonization is highly prevalent in the autopsied lungs of the general population epidemiology of pneumocystis colonization in families pneumocystis infections: the iceberg? pneumocystis infection: unraveling the colonization-to-disease shift pneumocystis jirovecii colonization in patients with systemic autoimmune diseases: prevalence, risk factors of colonization and outcome pneumocystis jirovecii in general population asymptomatic carriage of pneumocystis jiroveci in subjects undergoing bronchoscopy: a prospective study development and evaluation of a quantitative, touch-down, real-time pcr assay for diagnosing pneumocystis carinii pneumonia early diagnosis and treatment are crucial for the survival of pneumocystis pneumonia patients without human immunodeficiency virus infection current insights into the biology and pathogenesis of pneumocystis pneumonia use of real-time polymerase chain reaction for the diagnosis of pneumocystis pneumonia in immunocompromised patients: a metaanalysis real-time pcr assay-based strategy for differentiation between active pneumocystis jirovecii pneumonia and colonization in immunocompromised patients quantitative real-time pcr and the ( → )-β-d-glucan assay for differentiation between pneumocystis jirovecii pneumonia and colonization pcr diagnosis of pneumocystis pneumonia: a bivariate meta-analysis contribution of the ( → )-β-d-glucan assay for diagnosis of invasive fungal infections diagnostic accuracy of serum , -β-d-glucan for pneumocystis jiroveci pneumonia, invasive candidiasis, and invasive aspergillosis: systematic review and meta-analysis the authors declare they have no conflict of interest. key: cord- -elfroq f authors: hakim, f. a.; tleyjeh, i. m. title: severe adenovirus pneumonia in immunocompetent adults: a case report and review of the literature date: - - journal: eur j clin microbiol infect dis doi: . /s - - -z sha: doc_id: cord_uid: elfroq f adenovirus is a frequent cause of mild self-limiting upper respiratory tract infection, gastroenteritis, and conjunctivitis in infants and young children. fatal infections (severe pneumonia progressing to respiratory failure, septic shock and/or encephalitis) are rare among immunocompetent adults. we report a case of severe adenovirus pneumonia in a young immunocompetent male who presented with sudden onset respiratory distress that progressed rapidly to respiratory failure and made a successful recovery on supportive measures. systematic review of the literature identified cases of severe adenovirus pneumonia (defined as respiratory failure requiring ventilatory support at any point during the course of illness) in otherwise healthy immunocompetent adults both in epidemic and community settings. we describe the clinical characteristics, radiological features, and outcome of identified cases. adenovirus is a frequent cause of mild self-limiting upper respiratory tract infection, gastroenteritis, and conjunctivitis in infants and young children. epidemics of self-limited infections by adenovirus (especially serotype ) have been reported from time to time in military recruits. severe adenovirus infections causing significant morbidity and mortality are well described in immunocompromised patients but are extremely rare in healthy immunocompetent adults [ ] . among the known serotypes of human adenoviruses, serotypes and account for most of these severe infections. such infections first gained attention during the epidemics of the s and early s in military trainees, when no vaccine was available [ , ] . occurrence of such infections among the military recruits dropped significantly with the introduction of a vaccine in . however, severe adenovirus infections among immunocompetent adults raised concern when ryan et al. reported related deaths in two military recruits in [ ] . sporadic cases of severe infection have been reported from the community as well [ , ] . we report a case of severe adenovirus pneumonia in a previously healthy immunocompetent male who presented to us with rapidly developing respiratory failure and made a successful recovery on supportive measures. we also summarize the clinical features, radiological findings, and outcome of severe adenovirus pneumonia cases that have been reported in the literature. immunodeficiency virus (hiv). there was no history of travel outside riyadh city in the recent past. he had no exposure to birds or contact with sick persons. he had no pets at home. past medical history and family were unremarkable. on his arrival to the er, the patient was febrile (temperature . °c), tachypneic (respiratory rate /min), and tachycardic (pulse rate /min). his blood pressure was / mmhg and oxygen saturation was % by pulse oximetry while breathing room air. there was mild pharyngeal and tonsillar erythema but no conjunctival injection, rash, or lymphadenopathy. there was no lower limb edema or jugular venous distension. cardiovascular examination was normal and auscultation of the chest revealed bilateral scattered rhonchi. the rest of the systemic examination was unremarkable. laboratory investigations at admission revealed polymorpholeukocytosis (white cell count . × /μl with . % neutrophils). hemoglobin ( . g/dl), platelets ( × /μl), sedimentation rate ( mm st h), and urinalysis were normal. chest x-ray was normal and arterial blood gases revealed hypoxemia and mild respiratory alkalosis. serum biochemical panel including random glucose, urea/electrolytes, liver function tests, and amylase were normal. serum cardiac enzymes and coagulation profile were normal. doppler ultrasound of leg veins was negative for venous thrombosis and ventilation perfusion scan revealed multiple bilateral ventilation perfusion defects. computed tomogram (ct) scan of the chest showed no parenchymal lung disease and spiral ct angiogram scan did not reveal pulmonary embolism. six hours after presentation, the patient developed worsening respiratory distress and was transferred immediately to the intensive care unit (icu) and mechanically ventilated. given the septic shock picture, empiric antimicrobial therapy was started. over the next h the patient's respiratory failure worsened and he developed fluidresistant shock. inotropes and vasopressors were used to maintain his mean arterial pressure between and mmhg. he continued to have low-grade fever, developed new conjunctival congestion, and had coarse crackles on chest auscultation. repeat portable chest x-ray showed bilateral fluffy and confluent air space shadowing in the mid and lower zones. ct scan of the chest was consistent with bilateral consolidation and bilateral pleural effusion. transesophageal echocardiography was normal. cultures drawn at admission were negative. legionella urine antigen, mycoplasma serology, and hiv serology were negative. gram staining, acid fast staining, mycobacterium tuberculosis dna probe, cytomegalovirus, and pneumocystis carinii performed on bronchoalveolar lavage were negative. repeat cultures and urine drug screen were negative. the patient developed nonoliguric acute renal failure. urinalysis was negative for active urine sediment and proteins and renal ultrasound was normal. immunologic tests including ana, anti-dsdna, c-anca, and p-anca and cryoglobulins were negative. interestingly, the patient developed bilateral conjunctival chemosis and hemorrhages with mild watery discharge on day of illness ( fig. ). tracheal and nasopharyngeal aspirates sent for immunofluorescent viral studies were reported as strongly positive for adenovirus (magen direct antibody fluorescence). adenovirus serology and serotyping were not performed. droplet precautions were observed to prevent infection of the caring staff. ophthalmological evaluation revealed hemorrhagic conjunctivitis. the systemic empirical antibiotics were withdrawn and supportive measures for respiratory and renal failure continued with continuous monitoring of vitals and serum biochemical panels. no antiviral therapy was used. inotropes and vasopressors were gradually withdrawn. the patient's respiratory failure and renal failure improved over the next days and he was successfully weaned off the ventilator and extubated. he was discharged home on day after admission. he was doing very well at the -month follow-up. we reviewed and analyzed the clinical features, radiological findings, and outcome of severe adenovirus pneumonia in immunocompetent patients reported between and by searching medline for english reports and using "severe," "adenovirus," "pneumonia," and "immunocompetent" as search terms. we defined severe adenovirus pneumonia if associated with respiratory failure requiring ventilatory support at any point during the course of illness and immunocompetent adults as individuals with no acquired or congenital immunodeficiency state with or without associated premorbid conditions. we identified cases. seven of these cases occurred in epidemics among fig. photograph of the patient showing bilateral hemorrhagic conjunctivitis military recruits and a mental health care center and the remaining seven were reported from the community. table summarizes age, sex, presenting complaints, physical findings, radiological findings, and outcome of each patient. adenovirus serotype (wherever performed), time to ventilation, and time to death from onset of illness are also included when available. the majority of the patients ( / , . %) were males and the mean age was . years (range: - years). most patients had no premorbid conditions. respiratory symptoms were present in all patients; three had associated diarrhea. radiograph of the chest revealed focal or diffuse infiltrate or consolidation. serotyping was done in six patients: three had serotype , three had serotype , and one had both serotypes and . mean time from onset of symptoms to admission to icu for respiratory failure was . days (range: - days). of the patients identified, ( . %) died due to worsening respiratory failure. three of the deceased patients had associated renal failure and disseminated intravascular coagulopathy (dic) and one had meningitis. an antiviral agent (cidofovir) was used in one patient. most of these patients developed hypotension during the course of their disease and six of these progressed to fluid-resistant shock. adenovirus is a frequent cause of mild upper respiratory tract illness. severe adenovirus infection is rare in immunocompetent adults. outbreaks of fulminant respiratory illness due to adenovirus have been reported in military camps in and [ , ] , in hospitalized patients in [ ] , in a mental health care center in [ ] , and among civilians in [ ] . several sporadic cases of adenovirus infections in healthy immunocompetent individuals with case fatalities have also been reported [ , ] . our review suggests that most patients have upper respiratory symptoms prior to the onset of respiratory distress and progression to respiratory failure is unpredictable, occurring over hours to days. the presence of flu-like symptoms, diarrhea, and conjunctival infection may be a clue to the viral etiology of the illness. hemorrhagic conjunctivitis was present in only one of the identified patients; when present it is strongly associated with adenovirus infection. however, it may not be apparent early in the course of the disease as is illustrated in our case. the majority of such patients are febrile and tachycardic at presentation. chest auscultation may be normal or may reveal localized or bilateral rales, rhonchi, or signs of consolidation. fluid responsive hypotension and a transient drop in leukocyte count (relative leukopenia and lymphopenia) are common during the course of illness. initial chest x-ray may be normal but reveal focal reticulonodular infiltrate or a lobar consolidation with the progression of the infection. lobar consolidation is rare in other types of viral pneumonia [ ] [ ] [ ] [ ] . the majority of patients with severe adenovirus infection died due to respiratory failure and shock. disseminated intravascular coagulopathy, renal failure, and fluid-resistant shock are also common causes of death and should be always looked for in such patients. meningitis is a rare but fatal complication of severe adenoviral pneumonia. the whole clinical scenario may be mistaken for bacterial sepsis. whenever considered, the diagnosis of adenovirus infection is established by isolating the virus on cultures or detecting viral particles on fluorescence antibody testing from material obtained from nasopharyngeal and tracheal aspirate, bronchoalveolar lavage, or lung biopsy. a fourfold or more rise in adenovirus-specific antibody titers during the course of illness suggest acute infection and alternatively establishes the diagnosis. treatment of severe adenovirus infection is supportive and no antiviral agent has been shown to alter the unpredictable course of the disease. cidofovir and intravenous immunoglobulin have been used with some success in patients with liver and bone marrow transplant [ ] . in our review, it was used in only one patient who did not survive. it appears that severe adenovirus infections even in immunocompetent patients are associated with high mortality and morbidity. the spectrum of presentation and characteristics of severe adenovirus infections could be affected by publication bias. adenovirus produces cytolysis which accounts for tissue damage and subsequent dysfunction of the affected organs. it also induces interleukin (il- ) production, which causes a severe systemic inflammatory response causing systemic vasodilatation and triggers dic. damage to pulmonary capillaries also contributes to respiratory failure. cellular immunity is important in viral infection in general. hence, severe adenovirus infections are common in patients with defective or deficient t-cell immunity such as those with allogenic stem cell transplantation, solid organ transplants, and hiv infection. infants and young children also are at risk of severe adenovirus infections due to immature t-cell function. why severe adenovirus infection should occur in immunocompetent individuals is not well understood at this point in time. a unique interplay between viral factors (virulence) and host factors (genes) have been well described in adenovirus infections. virulent strains of adenovirus (serotypes and , etc.) in a genetically susceptible host impair cytokine, t-cell function, and expression of major histocompatibility complex expression [ , ] . however, the relationship between virulence and genotype needs to be explored. to conclude, the clinical scenario of severe adenovirus pneumonia resembles severe bacterial pneumonia and the two may be indistinguishable on clinical and radiological grounds. diagnosis of adenovirus infection should be always considered in patients with severe pneumonia with negative cultures and failure to respond to antibiotics. since this can rapidly progress to respiratory failure, the diagnosis should be looked for early in the course to avoid unnecessary drugs and to apply aggressive supportive measures in the icu setting which might improve outcome in such patients. more work needs to be done to identify viral determinants and genetic factors triggering severe adenovirus infection in immunocompetent individuals and to better understand the pathophysiology and immunology of such infections. we also need to identify subsets of patients with severe adenovirus who could benefit from antiviral drugs. adenovirus infections in immunocompromised patients association of type adenovirus with acute respiratory illness in military recruits fatal pneumonia associated with adenovirus type in three military trainees two fatal cases of adenovirus-related illness in previously healthy young adults--illinois fatal type adenoviral pneumonia in immunocompetent adult identical twins severe adenovirus infection in an immunocompetent adult-a case report type adenoviral pneumonia occurring in a respiratory intensive care unit multiple cases of life-threatening adenovirus pneumonia in a mental health care center severe adenovirus pneumonia in an adult civilian successful treatment of adenovirus disease with intravenous cidofir in an unrelated stem cell transplant recipient molecular basis of immune evasion strategies by adenovirus adenovirus-pulsed dendritic cells stimulate human virus-specific t-cell responses in vitro key: cord- -w s k p authors: fink, d.; barakat, f.; ellis, j.; lakra, c.; bodhani, r.; creer, d.; elsaghier, a. title: blood culture fluorescence rates predict severity and mortality of invasive pneumococcal pneumonia date: - - journal: eur j clin microbiol infect dis doi: . /s - - -x sha: doc_id: cord_uid: w s k p invasive pneumococcal pneumonia is associated with high rates of mortality. clinical assessment tools have poor sensitivity for predicting clinical outcomes. molecular measurements of bacterial load correlate closely with clinical outcome but require specialist facilities and expertise. this study describes how routine blood culture testing can estimate bacterial load and predict clinical outcome for invasive pneumococcal pneumonia. between december to march , clinical and laboratory data were collected for patients with streptococcus pneumoniae bacteraemia secondary to community-acquired pneumonia. fluorescence rates (fr) were calculated from growth curves generated by bactec blood culture analysers by dividing change in fluorescence units (fu), measured at the first point of detectable fluorescence and at the point of automated bactec positivity, by time in hours. the mean age of the patients was . years ( . – . ). forty patients survived invasive pneumococcal disease and ten patients died. these two groups did not significantly differ by demographic or clinical characteristics. the mean fr for the non-survival group ( . × (− ) fu/h) was significantly higher (p < . ) than that of the survival group ( . × (− ) fu/h). fr did not vary by serotype. we determined that an fr of . × (− ) fu/h might represent a useful threshold for predicting high mortality risk with a sensitivity of % and a specificity of %. our fr calculation uses cheap and accessible routine blood culture techniques to predict mortality in a small retrospective cohort study. in patients admitted to hospital with pneumococcal bacteraemia and, potentially, other organisms, this single tool could guide early escalation of clinical care. streptococcus pneumoniae (pneumococcus) is the main cause of community-acquired pneumonia worldwide [ ] . bacteraemia secondary to pneumococcal pneumonia is the most common presentation of invasive pneumococcal disease (ipd) and is associated with high mortality rates, particularly in elderly populations [ ] [ ] [ ] [ ] [ ] [ ] . mortality rates are highest during the first days of admission [ , ] . during this critical period, precise diagnostic evaluation, combination antibiotic therapy [ , ] , adjunctive treatments [ ] and early admission to the intensive care unit (icu) [ ] may all reduce inpatient mortality from ipd. the accurate identification of individual patients who could benefit from these interventions remains inexact and challenging. prognostic scoring systems [such as the pneumonia severity index (psi) [ ] and the confusion, elevated blood urea nitrogen level, respiratory rate and blood pressure plus age ≥ years (curb score) [ ] ] stratify patients in large cohorts according to mortality risk in order to identify those who can be safely managed in an outpatient setting. however, both of these clinical scores and consensus guidelines do not reliably identify individual patients requiring icu escalation [ , [ ] [ ] [ ] [ ] . in contrast, there are increasing data that correlate pneumococcal bacterial load measured by molecular assays with clinical outcome [ , ] : detection of high bacterial load has specificity exceeding % for septic shock and mechanical ventilation [ ] [ ] [ ] . thus, besides diagnostic utility in organism identification, the quantification of bacterial load may be useful in stratifying individuals with ipd to determine the site and intensity of inpatient care [ , ] . dna assays have increasing sensitivity and utility in the diagnosis of ipd in patients with negative blood cultures [ , ] . however, dna assays do have limitations: they may be only - % sensitive in patients with positive blood cultures, do not routinely yield antibiotic sensitivity data and require potentially expensive specialist facilities and expertise [ , ] . culture of viable pathogens from the bloodstream is intrinsically superior to the detection of pathogenic dna and remains fundamental to the initial diagnostic work-up for sepsis [ ] . the aim of this study was to determine the relationship of bacterial load to clinical outcome for patients with invasive pneumococcal pneumonia by using an affordable and sensitive method in a routine diagnostic microbiology laboratory. this was a retrospective study of adult patients admitted via the emergency department (ed) to hospital with invasive pneumococcal community-acquired pneumonia (cap). during the study period (december to march ), patients were identified with s. pneumoniae bacteraemia secondary to cap. cap was defined as new or worsening cough, with or without sputum production, associated with an abnormal temperature (t < . °c or > . °c) or abnormal serum inflammatory markers [leucocytosis, leucopaenia, elevated c-reactive protein (crp)], or as new pulmonary infiltrates on chest radiography associated with an abnormal temperature or abnormal serum inflammatory markers. the hard-copy notes for a single patient were not available, despite the availability of laboratory and radiology records, and, thus, the antibiotic history and psi score could not be recorded for that individual. patients receiving antimicrobial therapy prior to blood cultures were excluded from the study. the study cohort was organised into two groups: survived or died based on the mortality outcome of the pneumococcal cap episode. a retrospective review of hard-copy and computer-based notes and letters, and pathology and radiology data was undertaken. characteristics recorded were: demographics (age, gender, ethnicity), clinical characteristics [co-morbidities: immunocompromise, diabetes mellitus, chronic obstructive pulmonary disease (copd), smoking history, chronic liver, heart, renal and neurological diseases, active malignancy, human immunodeficiency virus (hiv) infection, steroid use], clinical outcomes at any time during admission [acute respiratory distress syndrome (ards), radiological progression, severity score according to the psi [ ] , septic shock, acute kidney injury (aki), icu admission, intubation, noninvasive ventilation, video-assisted thoracoscopy (vats) for empyaema] and microbiological characteristics [sputum culture, endotracheal aspiration culture, bronchoalveolar lavage (bal) culture, pleural fluid culture, pneumococcal urinary antigen]. comorbidity was defined as the presence of one of the following chronic conditions: immunocompromise (defined as primary immunodeficiency or secondary immunodeficiency due to splenectomy, haematological malignancy, autoimmune disorder, chemotherapy or radiotherapy within weeks prior to admission), diabetes mellitus (defined as receiving any oral or subcutaneous therapy for diabetes mellitus), copd (defined as the presence of airflow limitation due to chronic bronchitis or emphysema), current smoker (any number of cigarettes/day), heart disease (defined as prior elective or emergency percutaneous coronary intervention or coronary artery bypass grafting, or new york heart association classes iii or iv symptoms of heart failure), liver disease (defined as biopsy-proven cirrhosis or past medical history of complications of portal hypertension, including upper gastro-intestinal bleeding, encephalopathy, spontaneous bacterial peritonitis or more fig. the standard curve for streptococcus pneumoniae (nctc strain) was generated by plotting the mean of at least four samples of each ten-fold dilution (log cfu/ml) against the corresponding fluorescence rate (fr). the generated equation of the logarithmic regression curve, y= . ln(× − fu/h)+ . , was used to estimate the bacterial load in all blood culture samples than one episode of paracentesis), chronic kidney disease [ckd stages ii to v inclusive defined by the renal association uk as estimated glomerular filtration rate (egfr) less than ml/min/ . m on two serum samples at least days apart [ ] ], neurological condition (defined as any documented neurological condition, including dementia, on the initial medical clerking or available clinic letters), active malignancy, hiv, corticosteroid use (equivalent of mg prednisolone or more for at least weeks prior to admission). ards diagnosed according to the criteria of the american-european consensus conference committee [ ] . rapid radiological progression was defined as an increase in the size of pulmonary infiltrates of chest radiography by % or more at h after presentation. septic shock was defined as [ ] . blood sample collection and processing whole blood samples ( - ml) were collected from the patients and inoculated into each vial of the blood culture set, which includes an aerobic bottle (bd bactec plus aerobic/f medium) and an anaerobic bottle (bd bactec lytic/ anaerobic/f), according to the hospital policy for taking blood samples for blood culture. blood culture vials were incubated into the bactec blood culture analyser (bd bactec fx) within h of taking blood samples. bacterial identification and susceptibility testing were performed using standard microbiological methods. the bactec blood culture analyser detects bacterial growth in the blood culture vials by detecting co produced during cell growth, which is translated into reflectometric (fluorescence) units by the automated fluorescence detector [ ] . the computer generates growth curves based on plots of fluorescence units (fu) recorded every min versus time. by analysing the generated growth curves, we have learned that, at approximately to min prior to the detection time, the fu start to increase (the log phase) after a period of unchanging values (the lag phase). the shape of the growth curve from the end of the lag phase to the time to detection (ttd) satisfies the following differential equation: where Δf is the difference in the fluorescence readings between that at the end of the lag phase (f ) and that at the ttd (f x ), and Δt is the difference in time between that at the end of the lag phase (t ) and that at the ttd (t x ). the above equation was calculated according to the following equation: where k is the rate constant. the rate constant was calculated for each isolate according to the following equation: ten-fold dilutions were performed using the nctc strain of s. pneumoniae. known volumes were inoculated into bactec lytic/ blood culture vials and placed into the blood culture analyser. the fluorescence rate (fr) was calculated for each dilution and plotted against bacterial concentration (log cfu). the logarithmic regression standard curve (cfu vs. fr) for s. pneumoniae strain nctc ( fig. ) was generated by plotting the mean of at least four samples of each ten-fold dilution (log cfu/ml) against the corresponding fr. the generated equation of the logarithmic regression curve y= . ln(× − fu/h)+ . was used to estimate the bacterial load in all blood culture samples. the bacterial load for each of the clinical isolates was measured by exploiting the fr to the corresponding bacterial concentration. categorical data are presented as frequency and percentages, while continuous data are presented as mean and standard deviation or median and interquartile range, depending on whether the variable conforms to a near normal distribution or not, respectively. each variable is stratified by mortality and a test is performed comparing the difference between the two categories. the chi-squared test is used for categorical data, and either the t-test or mann-whitney u-test is performed for continuous data, also dependent on whether they conform to a near normal distribution or not. univariable logistic regression models were built looking at the association between each predictor variable and mortality. corresponding multivariable logistic regression models were constructed after age and gender adjustments. values of p< . are considered to be statistically significant. statistical analysis was performed using stata . statistical analysis software (statacorp, college station, tx). fifty patients were included in the study, having a mean age of . years ( . - . ). forty patients survived and ten patients died. we identified no statistically significant differences in the demographic or clinical features between the cohort which survived and the cohort which died ( table ) . the presence of aki was significantly more common in the patients who did not survive the pneumonia (p< . ; table ). the presence of rapid radiological progression and mean psi score differed significantly between the group that survived and the group that did not (p= . ; table ). despite reaching statistical significance, the crude odds ratio for the mean psi score between the groups was only . (p= . ). the mean fr for the non-survival group ( . × − fu/h) was significantly higher (p< . ) than that of the survival group ( . × − fu/h; table and fig. ) . for the estimated bacterial load in the patients' blood samples at the time of taking the blood culture, the mean for the non-survival group ( . ± . log cfu/ml) was significantly higher (p< . ) than that of the survival group ( . ± . log cfu/ml; fig. ). the ttd did not predict mortality. fig. ). there was no correlation between the fr and the ttd (fig. ) . the relationship between the fr and the probability of mortality is described in fig. . logistic regression analysis showed a small but statistically significant association of age with mortality (table ) . however, the fr is not independently associated with key clinical components of the psi score ( table ). the discriminative ability of the fr to differentiate between survivals and non-survivals was evaluated with area under the receiver operating characteristic (auroc) curve analysis. the fr was an excellent marker for the determination of the risk of mortality (auroc . ). we determined that the optimal cut-off for high risk of mortality is . × − fu/h (figs. and ), with a sensitivity of % (ci, to %) and a specificity of % (ci, to %). our small retrospective cohort study describes a novel application of the bactec blood culture analyser to predict mortality in patients with invasive pneumococcal pneumonia. we found that the fluorescence rate (fr), calculated from routine blood culture analysis, correlated closely with the probability of mortality in this patient group. our data suggest that, across different strains of streptococcus pneumoniae, the fr is an accurate surrogate for bacterial load. these findings concord with existing data that describe the strong relationship between the bacterial load of invasive pneumococcal infections and clinical outcome [ , ] . our results suggest that calculation of the fr as part of routine blood culture work-up for suspected pneumococcal sepsis may help stratify the severity of illness, thus guiding appropriate escalation of clinical care. it is well recognised that current clinical and microbiological tools fail to achieve this reliably in the setting of acute medical admissions [ , [ ] [ ] [ ] [ ] . our extrapolated analysis of existing culture techniques is cheap, widely available and can be integrated into the automated process of the bactec blood culture analyser. the software required to optimise the bactec system adds negligible cost and an fr report can be generated instantaneously. manual calculation of the fr, without additional software, takes less than min. our innovative approach may have a role in other invasive pneumococcal infections, such as meningitis, but also in sepsis caused by different organisms, where objective and early assessment of disease our study is necessarily limited by the small sample size and its retrospective design. bacterial load based on blood culture inoculation, and, consequently, the fr, is directly affected by preceding antibiotic administration. as far as possible in our study, we have excluded patients receiving antibiotic therapy prior to blood culture collection. however, owing to individual variation in the documentation of clinical notes, we cannot definitively report that all patients did not receive oral antibiotic preparations prior to hospital admission. in contrast, the sensitivity of dna assays does not significantly change over days after antibiotic administration [ ] . six patients out of the ten patients who died due to invasive pneumococcal pneumonia were intentionally not escalated to intensive care or to intubation as part of the clinical plan. it may be the case that death may be preventable in patients where escalation is clinically appropriate. however, we acknowledge that this may have a compromising effect on the correlation of the fr to mortality. alternatively, there may be unrecognised clinical or nonclinical parameters, not identified by our linear regression analyses, which predispose to the high mortality in this specific group of patients. however, the survival and non-survival groups demonstrated no statistically significant variation in the demographic or clinical profiles. with the two groups matched in this way, it suggests that the fr remains a prognostic tool of high utility. similarly, while delays in presentation to hospital might also predict poor prognosis, owing to the untreated complications of sepsis, we could not accurately capture these data from the clinical notes audit. however, at presentation, the bacterial load and fr will also increase with prolonged and uninhibited bacterial growth. thus, these analyses will still likely provide a clinically useful snapshot of disease severity at the time of blood culture sampling. the ideal diagnostic tool in the setting of any presentation of sepsis provides organism identification and measure of disease severity as early as possible during admission when mortality is highest [ ] . one limitation of the fr is that it provides discriminating results only as quickly as blood culture, which is slower than potential future molecular tests. we found that the time to detection (ttd) did not correlate with clinical outcomes in our cohort. in data not shown, we found that the ttd varied significantly according to isolate serotype, whereas the fr did not. the fr does not appear to be strain-dependent and provides a consistent measure of bacterial load. in conclusion, bacterial load is known to predict clinical outcome in invasive pneumococcal pneumonia. our innovation of fr calculation using routine blood culture techniques correlates with bacterial load and predicts mortality in a small retrospective cohort study. the implications for this simple and universal tool may extend beyond stratifying severity in patients with invasive pneumococcal disease (ipd) to support clinical decisions for all causes of sepsis with detectable bacteraemia. estimating the burden of pneumococcal pneumonia among adults: a systematic review and meta-analysis of diagnostic techniques active bacterial core surveillance team ( ) changing epidemiology of invasive pneumococcal disease among older adults in the era of pediatric pneumococcal conjugate vaccine active bacterial core surveillance (abcs)/emerging infections program network ( ) epidemiology of invasive streptococcus pneumoniae infections in the united states, - : opportunities for prevention in the conjugate vaccine era outcome of community-acquired pneumonia: influence of age, residence status and antimicrobial treatment regional epidemiology of invasive pneumococcal disease in asian adults: epidemiology, disease burden, serotype distribution, and antimicrobial resistance patterns and prevention impact of the emergence of non-vaccine pneumococcal serotypes on the clinical presentation and outcome of adults with invasive pneumococcal pneumonia clinical and economic burden of community-acquired pneumonia among adults in europe pneumococcal bacteremia with especial reference to bacteremic pneumococcal pneumonia new perspectives on community-acquired pneumonia in patients. results from a nationwide mandatory performance measurement programme in healthcare quality combination antibiotic therapy lowers mortality among severely ill patients with pneumococcal bacteremia a survival benefit of combination antibiotic therapy for serious infections associated with sepsis and septic shock is contingent only on the risk of death: a meta-analytic/meta-regression study bacteraemic pneumococcal pneumonia: current therapeutic options late admission to the icu in patients with communityacquired pneumonia is associated with higher mortality a prediction rule to identify low-risk patients with community-acquired pneumonia defining community acquired pneumonia severity on presentation to hospital: an international derivation and validation study severe community-acquired pneumonia: use of intensive care services and evaluation of american and british thoracic society diagnostic criteria pneumonia severity index class v patients with community-acquired pneumonia: characteristics, outcomes, and value of severity scores value of severity scales in predicting mortality from communityacquired pneumonia: systematic review and meta-analysis severity assessment tools to guide icu admission in community-acquired pneumonia: systematic review and meta-analysis high pneumococcal dna loads are associated with mortality in malawian children with invasive pneumococcal disease severity of pneumococcal pneumonia associated with genomic bacterial load streptococcus pneumoniae dna load in blood as a marker of infection in patients with community-acquired pneumonia diagnostic value of serum pneumococcal dna load during invasive pneumococcal infections why should we measure bacterial load when treating community-acquired pneumonia? association between pneumococcal load and disease severity in adults with pneumonia multicenter clinical evaluation of a continuous monitoring blood culture system using fluorescent-sensor technology (bactec ) the renal association ( ) ckd stages. available online at report of the american-european consensus conference on acute respiratory distress syndrome: definitions, mechanisms, relevant outcomes, and clinical trial coordination. consensus committee acute kidney injury acknowledgements the authors declare that they have no acknowledgements. the authors declare no potential conflicts of interest.compliance with ethical standards this study is compliant with ethical standards and did not require informed consent. key: cord- -kox uah authors: wong, s. f.; chow, k. m.; shek, c. c.; leung, y. p.; chiu, a.; lam, p. w. y.; ho, l. c. title: measures to prevent healtcare workers from contracting severe acute respiratory syndrome during high-risk surgical procedures date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: kox uah nan severe acute respiratory syndrome (sars) is a contagious viral disease caused by a novel coronavirus (sars-cov) and transmitted through droplets and close contact [ ] . healthcare workers (hcws) are particularly at risk when looking after sars patients. as reported by the world health organization [ ] more than % ( ) of those infected with sars in hong kong were hcws. described here are the infection control procedures undertaken at a hospital in hong kong when three caesarean sections were performed on women with active maternal sars infection. sars-cov infection had been confirmed by reverse transcriptase polymerase chain reaction in all three mothers prior to the time of operation. the operations were performed between april and april . none of the hcws (> ) involved in the three separate operations developed fever or clinical sars within days of exposure. when the operations were performed, the centers for disease control and prevention (cdc; atlanta, ga., usa) had not yet prepared guidelines for the prevention of sars transmission during caesarean sections. since these procedures are associated with the use of suction irrigation and diathermy, it was likely that body fluid and blood would spill and evaporate into the room during the operation. therefore, the procedures were assumed to be aerosol generating, and previously described precautions to prevent contact and airborne infection were undertaken to prevent nosocomial sars infection [ ] . although the cdc provided guidelines for the prevention and control of sars infection on may [ ] , those guidelines have, to the best of our knowledge, never been tested for the procedure described here. following are the steps and procedures we undertook to prevent hcws in our hospital from contracting sars. for the three caesarean sections performed on mothers with sars, the number of healthcare workers was limited to a minimum, with only those personnel essential to carry out the operation, neonatal resuscitation, and cleanup being involved (i.e., senior obstetricians, senior neonatologists, senior anaesthetist, theatre assistant, a team of senior midwives, and cleansing staff). a nurse supervisor was designated as in-charge and was responsible for monitoring the other hcws with regard to the use of personal protective equipment (ppe), cleansing of the room, sterilising the equipment and transferring the patients. she ensured all hcws had fit-tested their respirators. she was also responsible for testing the portable high-efficiency particulate air filter (hepa) units (air-mate papr; usa), checking the batteries, and training other hcws on the proper use of the hepa units. all unnecessary instruments were removed from the operating theatre. extra laparotomy instruments were prepared to deal with possible intra-operative complications. two bags of blood were made available in the theatre for the operation. disposable instruments were used if available. to avoid spillage of blood, drapes with plastic bags on the sides were used. an operating theatre separate from the main theatre block was designated for this type of surgery in order to minimise contamination of the main theatre. the door of the operating theatre was kept closed during the operation, except when participating personnel entered or left the room. in order to keep the number of entrances and exits to a minimum during the operation, most of the equipment was prepared before the patient arrived. the air circulation was adjusted to exchanges per minute. negative pressure was created within the operating theatre relative to the adjacent room or hallway; thus, recirculation of the room air was avoided. the hospital engineer inspected and checked the air circulation prior to each operation. the participating hcws wore appropriate ppe according to the hospital's guidelines prior to the arrival of the patient from the intensive care unit. every hcw wore a disposable cap, a protective waterproof gown, gloves, n respirators, goggles/face shield and waterproof boot cover. in addition, all hcws working inside the operating theatre wore hepa units. the hepa units each had a hood that covered the face and shoulders, and this was connected to a breathing tube on the portable hepa unit. a non-sterile waterproof protective gown was then put on. after donning this protective gear, the surgeons performed standard scrub then put on sterile waterproof gowns and two pairs of sterile gloves. hcws were advised to avoid touching their faces and the ppe on their faces with contaminated gloves. they were also advised to avoid contaminating surfaces around the room. before leaving the operating theatre, the soiled gowns, gloves and boot covers were removed. an assigned hcw wearing appropriate ppe then wiped the remaining external outfits of the theatre personnel with a diluted sodium hypochlorite solution (a disinfectant, diluted in ). the remaining outfit was then removed carefully preventing contamination of skin, mucous membranes and clothing. all hcws washed their hands with chlorhexidine solution, (hibiscrub; astrazeneca plc, uk) and took full-body showers after the operation. all three patients had been intubated and ventilated prior to the decision for caesarean section being made. however, in the absence of an effective filtering valve, they were likely to disseminate virus through the endotracheal tube while being transferred from the intensive care unit. to avoid this, a bacterial/viral filter on an exhalation valve was fitted to the endotracheal tube prior to transfer to the operating theatre. the neonatal resuscitator was placed outside the operating theatre to (i) reduce the number of hcws inside, (ii) reduce the number of entries to the theatre, and (iii) reduce the exposure of the newborn to the viral load in the environment. neonatologists wore the ppe described above, including hepa units with face masks, but their ears were exposed to enable auscultation with a stethoscope during the resuscitation procedure. after birth, thorough suctioning of the newborn's nasopharyngeal tract was performed prior to the first breath. the newborns were wiped dry of blood and amniotic fluid with a sterile cloth before being transferred to the resuscitator. routine suction of the mouth and nose was not performed. if suction was required, it was performed using the vacuum suction system on the wall with a sealed system. suction attached to the resuscitator was avoided, since the gas aspirated would be released directly into the adjacent environment. bagging with a face mask was kept to a minimum and direct intubation, using a closed suction system, was performed more readily. after stabilisation, the newborns were wrapped in sterile linens and transferred to the isolation nursery using transport incubators. the newborns were kept in the isolation nursery until proven non-contagious. none of the three newborns had any clinical evidence of sars and paired sars-cov antibody titres were not raised. after the operation, sodium hypochlorite diluted : was used to clean the floor, the operating table and environmental surfaces as soon as possible. the cleaners all wore appropriate ppe as described above. all waste materials and disposable instruments were treated as highly contagious and were disposed of appropriately according to the hospital's guidelines. samples collected for pathology and microbiology were placed in two clean plastic bags. if there was evidence of soiling, an extra plastic bag was to be used. maternal sars infection was clearly labeled on the laboratory forms. the optimal ppe for preventing transmission of sars during such ultra-high-risk procedures has not yet been determined. however, ppe should be used that covers all exposed areas of skin including the arms, torso, eyes, nose and mouth. n or higher standard respirators are also necessary. a fit-check should be performed each time a respirator is put on. for better protection of staff working inside operating theatres, we used hepa units with hoods completely covering the head and neck and portions of the shoulders and torso. factors to consider when choosing respirators in this setting include availability, impact on mobility, comfort, duration of use and recharge power supply. with the units we used, the hcws did not encounter any major difficulty, apart from slightly reduced mobility and difficulty in communicating while carrying out the operation. some problems known to be associated with hepa units include partially charged batteries, ineffective filters, and contamination of hcws during disrobing. proper training prior to use may reduce such problems. negative-pressure air circulation helps to reduce the viral load within the operating theatre. the main con-cern with this practice, however, is that by drawing air from the hallway a high flow of unfiltered air is created, which may increase the risk of wound infection. two of our three patients who underwent surgery developed wound infections, but it is difficult to attribute these infections with certainty to either the effect of the high-dose steroid given and/or the negative-pressure air circulation. in conclusion, the procedures described above were sufficient to prevent our healthcare workers from contracting sars while performing these very high-risk operations. therefore, we advocate the use of such measures or those with higher levels of precaution. we cannot comment on the effectiveness of less stringent measures in minimising sars transmission. coronavirus as a possible cause of severe acute respiratory syndrome guidelines for preventing the transmission of mycobacterium tuberculosis in health-care facilities key: cord- -u go xi authors: lepur, d.; kutleša, m.; baršić, b. title: prospective observational cohort study of cerebrovascular co( ) reactivity in patients with inflammatory cns diseases date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: u go xi the purpose of this study was to evaluate the significance of cerebrovascular co( ) reactivity (co( ) r) in the course and outcome of inflammatory central nervous system (cns) diseases. sixty-eight patients with inflammatory cns diseases and healthy volunteers were included in this prospective observational cohort study. the observational period was between january and may . the co( ) r was measured by transcranial doppler (tcd) ultrasound using the breath-holding method. we compared patients with normal co( ) r (breath-holding index [bhi(m)] ≥ . = bhi(n) group) with patients who showed impaired co( ) r (bhi(m) < . = bhi(r) group). we also analyzed the association of impaired co( ) r with the etiology, severity, and outcome of disease. when compared to the bhi(n) group, the patients from the bhi(r) group were older, had a heavier consciousness disturbance, experienced more frequent respiratory failure, and, subsequently, had worse outcomes. there were no fatalities among the patients in the bhi(n) group. the comparison of subjects with bacterial and non-bacterial meningitis revealed no significant differences. the unfavorable outcome of disease (glasgow outcome scale [gos] score – ) was significantly more common in subjects with impaired co( ) r ( . % vs. . %). logistic regression analysis was performed in order to establish the prognostic value of bhi(m). the outcome variable was unfavorable outcome (gos – ), while the independent variables were age, glasgow coma scale (gcs) score, and bhi(m). the age and bhi(m) showed the strongest influence on disease outcome. a decrease of bhi(m) for each . unit increased the risk of unfavorable outcome by %. our study emphasizes the importance of co( ) r assessment in patients with inflammatory cns diseases. despite advances in the treatment of bacterial meningitis (bm), the overall mortality as well as long-term neurological sequelae are still high. this is particularly true with pneumococcal meningitis [ ] [ ] [ ] [ ] . the most common factors associated with poor outcome in bm are seizures, advanced age, disturbed consciousness, the presence of multipleorgan dysfunction, hypotension, apache ii score > , pneumococcal etiology, and delay in antimicrobial treatment [ ] [ ] [ ] [ ] [ ] [ ] . cerebrovascular dysregulation represents one of the most deleterious consequences of neuroinflammation [ ] . the relevance of impaired cerebral blood flow (cbf) chemoregulation relies on subsequent hypoperfusion or global 'luxury' perfusion. in addition, although not caused by co reactivity (co r) loss, severe blood-brain barrier disruption frequently occur in these patients. therefore, the aggravation of brain edema with the use of mannitol infusion due to heavy capillary leak can be expected in such patients. beside osmotic diuretics and steroids, the usual treatments for increased intracranial pressure include thiopental infusion and hyperventilation [ ] . the latter often has limited effectiveness if the cerebrovascular co r is impaired. the alternative and co r-independent symptomatic treatments, such as therapeutic hypothermia (th), in such patients hold promise. the effects of mild therapeutic hypothermia may target the pathophysiological mechanisms in bm because the majority of them are temperature-dependent [ , ] . even with this knowledge, the co r has only been studied in very few patients with bm [ , ] . transcranial doppler (tcd) ultrasound is widely accepted and considered to be an appropriate technique for the noninvasive assessment of cerebral arterioles' reactivity because the changes in the mean blood flow velocities (mbfv) correlate with the changes in cbf [ , ] . the aim of this study was to assess the cerebrovascular co r measured by tcd using the breath-holding method in patients with inflammatory central nervous system (cns) diseases. the study was approved by the hospital ethics committee and informed consent was obtained from all examinees or their next of kin. this prospective study was performed between january and may at the university hospital for infectious diseases in zagreb, croatia. the following parameters were recorded in a database: age, gender, physical and neurological signs, mechanical ventilation (mv), cerebrospinal fluid (csf) characteristics, microbiological findings in csf and blood, mean arterial pressure (map), glasgow coma scale (gcs) score, glasgow outcome scale (gos) score, and the mean breath-holding index (bhi m ). data were collected during the first h after admission to the department and within the first days of illness in all patients. the patients were eligible for the study if they were years of age or older and had inflammatory cns disease. patients were excluded if they had brain abscess, subdural empyema, nosocomial and shunt meningitis, spinal meningitis or myelitis, stroke, transitory ischemic attacks, carotid artery disease, chronic obstructive pulmonary disease (copd), acute respiratory distress syndrome (ards), diabetic microangiopathy, septic shock, death not related to meningitis, temporal bone 'acoustic window' absence, or had been treated with sodium nitroprusside. during the same time period, healthy volunteers were studied following written informed consent. all volunteers were examined and found to be healthy and without infection. the purpose of the control group was to establish the lowest value of the bhi m in healthy volunteers. definitions bm was diagnosed on the basis of clinical picture (fever, headache, neck stiffness, and consciousness disturbance with or without seizures and neurologic deficits), supportive csf findings (pleocytosis, increased protein concentration, and decreased csf-blood glucose ratio), and a positive csf culture; a negative csf culture with a positive csf polymerase chain reaction (pcr) assay, a positive blood culture, or a positive gram stain of a csf sample. non-bacterial inflammatory cns disease (nbm) was diagnosed on the basis of present encephalopathy with at least one of the following: fever, seizures, focal neurological findings; pleocytosis with increased protein concentration in the csf; and a positive csf or blood culture (fungal meningitis); virus detection by pcr assay in csf samples; proved intrathecal antibody production and electroencephalographic or neuroimaging findings consistent with encephalitis or acute disseminated encephalomyelitis (adem). the patients' mental status was assessed using the gcs score. recorded scores were the lowest during the first h after admission to the hospital. altered mental status was defined as gcs < . the patients' clinical outcome was assessed by using the gos score at the time of discharge from the hospital. unfavorable clinical outcomes included death (gos ), vegetative state (gos ), and severe neurological deficit (gos ). gos (mild or no disability) but also gos (moderate disability) were considered to be favorable outcomes because of the extremely detrimental nature of cns infections. according to the disease etiology, patients were divided into bacterial (bm) and non-bacterial (nbm) groups. thirty healthy volunteers represented the control group. patients were stratified according to co r into 'bhi n ' (normal co r defined with bhi m ≥ . ) and 'bhi r ' (impaired co r defined with bhi m < . according to bhi values in healthy volunteers) groups. tcd measurements of co r were performed during the first h after admission to the hospital by using a multidop x (dwl, sipplingen, germany) with two -mhz pulsed-wave probes . cm in diameter. the software used was tcd- for mdx (version . , aaslid rune). normal basic hemodynamic parameters (mean arterial pressure and heart rate) and paco [ - torr ( . - . kpa)] were confirmed in all examinees before measurements. direct blood pressure monitoring was performed by cannilation of the radial artery. thus, we were able to note every change in the arterial pressure which could potentially interfere with the measurements. the left and right middle cerebral arteries (mcas) were insonated simultaneously through the temporal bone windows at a depth of - mm. the probes were secured to the head of the patient with a specially designed spectacle frame that permitted a constant angle of insonation. the mean blood flow velocities (mbfv) were continuously recorded during normal ventilation and during breath-holding. the co r was assessed using the breath-holding method. spontaneously breathing and compliant examinees were asked to hold their breath after normal inspiratory breath for s or less if it becomes uncomfortable. mechanically ventilated patients were sedated and relaxed before the procedure using midazolam and vecuronium bromide, respectively. the administered drugs have no significant effect on the cerebral vasoreactivity and hemodynamics [ , ] . in that case, the patients were disconnected from the ventilator for s. assisted controlled ventilation (acv) mode was used in all mechanically ventilated patients. mbfv at the start and at the end of the breath-hold period was recorded. the procedure was repeated after a min rest period and the mean values from both mcas were taken for calculation. the bhi was calculated by dividing the percentage of mbfv increase during breath-holding by the time (in seconds) of apnea. the bhi m was calculated from both mca (left and right mcas) breath-holding indexes. univariate statistics included calculation of the mean value, standard deviation, median, and interquartiles for continuous variables. values for categorical variables were presented as frequencies. bivariate statistics assessed the differences between compared groups. the mann-whitney test was used to estimate the difference amongst continuous variables. for categorical data, the chi-square test and fisher's two-tailed exact test were used when appropriate. all relevant demographic and clinical variables of the patient groups were compared. the outcome variable was the gos scores. the correlation between bhi m and the severity of disease was assessed using multivariate tests after bivariate analysis. logistic regression analysis was performed to ascertain the independent predictive potential of co r, after adjustment for potential confounding variables. the criteria for the inclusion of variables in the logistic regression models were based on the evidence of an association in the bivariate analysis. p-values less than . were considered to be statistically significant. the data were analyzed using the sas . software (sas institute inc., cary, nc). sixty-eight patients and healthy volunteers were included in the study. there were no patients requiring vasopressor or inotrope support. the baseline demographics and patient characteristics are presented in table . thirty-four patients with bm comprised the bm group. the etiology was confirmed in ( . %) patients. the most common pathogen was streptococcus pneumoniae ( patients), followed by neisseria meningitidis ( patients), listeria monocytogenes ( patients), klebsiella pneumoniae ( patient), enterococcus faecalis ( patient), and s. agalactiae ( patient). in the nbm group, there were patients with non-bm and meningoencephalitis. the disease etiology in patients with viral meningoencephalitis ( . %) was not confirmed. the most common pathogen in the remaining patients was herpes simplex virus ( patients), followed by tick-borne encephalitis virus ( patients), cryptococcus neoformans ( patients), influenza a virus ( patient), mycobacterium tuberculosis ( patient), epstein-barr virus ( patient), enterovirus ( patient), and borrelia burgdorferi ( patient). five patients suffered from postinfectious meningoencephalitis and two patients had acute disseminated encephalomyelitis (adem). the breath-hold period in the control group was . ± . s (median ). we found no correlation between bhi m and the duration of breath-holding (spearman r . ; p= . ). the possible effect of a too short breath hold to bhi values was, thus, ruled out. advanced age and higher incidence of respiratory failure that required mechanical ventilation were found in the bm group ( vs. years and . % vs. %). the most prominent difference between bm and nbm patients was the difference in the level of consciousness disturbance (gcs): bm patients had worse scores (gcs vs. . ). therefore, the on-admission gcs score was included in the multivariate model analyzing the predictivity of bhi for the disease outcome. adjuvant steroid treatment was applied in % patients with bm according to the current guidelines [ ] . six patients in the nbm group received short-term high-dose steroids because of severe brain edema ( patients) and acute disseminated encephalomyelitis ( patients). the patients were stratified according to co r and additional analysis was made. the normal co r group (bhi n ) was defined by bhi m ≥ . . the impaired co r group (bhi r ) was defined by bhi m < . according to breath-holding indexes in healthy volunteers. the bhi r group was characterized by advanced age, heavier consciousness disturbance, frequent respiratory failure, and, often, unfavorable outcome in contrast to the bhi n group (table ). there were no lethal outcomes amongst the patients in the bhi n group. in patients with bm and normal co r, we noted a higher incidence of mechanical ventilation, advanced age, and heavier consciousness disturbance compared with patients with nbm and preserved co r (table ). these findings indicate that, in patients with bm, neither severe consciousness disturbance nor respiratory failure necessarily imply co r loss. in contrast, unconscious patients with nbm who are mechanically ventilated and have normal co r are rare ( . % vs. %). there was no significant difference in the bhi between patients with bm that received dexamethasone treatment and those who did not. normal co r was noted in . % ( / ) of patients with steroid treatment compared with . % ( / ) in the non-steroid group (p= . ). the major indicator of disease severity in patients with cns infections is gcs [ ] . because of that, the correlation of bhi m and gcs was analyzed together with other variables which may influence bhi m . we found that . % ( / ) of all patients with gcs ≤ had impaired co r. in the bm and nbm groups, that was % ( / ) and . % ( / ), respectively. univariate regression analysis revealed a significant correlation of bhi m with gcs (β= . ; p< . ), and bhi m with age (β=− . ; p= . ). the unfavorable outcome of disease (gos - ) was noted in patients ( / ; . %) ( table ) . nine patients died (five patients in the bm and four patients in the nbm groups) and the overall mortality was . %. the mortality in pneumococcal meningitis was . % ( / ). the bhi m was almost identical in the gos - groups, regardless of the etiology (bhi m median . in the bm versus . in the nbm groups). the comparison of the bm and nbm groups according to disease outcome and related bhi m revealed no signifi- (table ). such findings confirmed the bhi m etiology independence. however, significant differences in bhi m were noted according to the gos (fig. ) . the bhi m in the favorable group (gos - ) was significantly higher ( . vs. . ) than that in the unfavorable group (gos - ) ( table ). the unfavorable outcome of disease (gos - ) was significantly more frequent in patients with impaired co r ( . vs. . %) ( table ) . logistic regression analysis was performed in order to establish the prognostic value of bhi m . the outcome variable was unfavorable outcome (gos - ), while the independent variables were age, gcs, and bhi m . the appropriateness of the fitted model and its predictive utility was confirmed. patient age and bhi m showed the strongest influence on disease outcome. a decrease of bhi m for each . unit increases the risk of unfavorable outcome by %. an increase of age for one year increases the risk of unfavorable outcome by % (table ). these results indicate that bhi m changes might have a better predictive value than gcs. the receiver operating characteristic (roc) curve showed that bhi m has better explanatory value than gcs (area under the curve [auc] . vs. . ). this prospective study assessed cerebrovascular reactivity (co r) by tcd using the breath-holding method in patients with inflammatory cns diseases. we found that impaired co r is independently associated with the severity and outcome of disease. the etiology of cns infection did not show a significant association with chemoregulation loss. however, the etiology was associated with the severity of disease and mechanical ventilation requirement. in patients with bm, we found a higher incidence of respiratory failure, advanced age, and heavier consciousness disturbance. this finding, however, does not imply obligatory chemoregulation loss in these patients. their grave condition can be explained with reasons outside the cns, such as multipleorgan dysfunction/failure due to severe systemic inflammatory reaction and other co-morbidities in older patients. in addition, there were no lethal outcomes in mechanically ventilated patients with normal co r, regardless of the in contrast to previous reports, we found no significant influence of adjuvant dexamethasone treatment on cbf chemoregulation recovery [ ] . the analysis of bhi according to disease outcome ascertained the association of impaired co r with unfavorable outcome (gos - ) . according to the literature review, the impaired cbf chemoregulation is almost exclusively confined to bm [ ] . that is probably because of particular interest in pneumococcal meningitis pathophysiology and the great number of experimental studies [ ] [ ] [ ] [ ] [ ] . the lipid peroxidation and effects of matrix metalloproteinases with consequent endothelial dysfunction seems to be a plausible explanation. according to our results, the most severe cns infections resulted in co r impairment or complete co r loss, which was proved to be etiology-independent. the possible mechanisms which could explain the chemoregulation loss in viral and other non-bacterial cns infections are not known. intracranial hypertension alone, regardless of cause, cannot explain impaired co r. for example, most patients with extreme intracranial hypertension due to cerebral abscess or tumors have normal co r, and the use of hyperventilation as well as mannitol infusion results in obvious short-term improvement. a similar response could be seen in patients with bm and preserved co r. we chose the breath holding method because it proved to be non-aggressive, well-tolerated, real-time, reliable, and reproducible [ , ] . the lowest bhi m in our healthy volunteers was similar to a previously reported bhi m by zavoreo and demarin ( . vs. . ) [ ] . other methods such as inhalation of % co or acetazolamide injection strongly stimulate the vasodilatation of cerebral arterioles [ , ] . because they also carry the risk of cerebral hyperemia and intracranial hypertension aggravation, these methods are not appropriate for the evaluation of cerebral vasoreactivity in patients with severe acute cns infections. more advanced techniques to assess the smalland medium-sized vessel reactivity such as arterial-spin labeled magnetic resonance imaging (asl-mri) and blood oxygenation level-dependent (bold) imaging are usually unavailable. a major disadvantage of the breath holding method is the necessity for the patients' full cooperation if they are not mechanically ventilated. besides disease severity, the inability of compliance in a proportion of patients is the reason for a relatively high proportion of mechanically ventilated patients in our study ( . %; / ). that is the recognized limitation of our study. the second limitation of the study is a relatively small patient population. this occurred because of strict exclusion criteria and that may have resulted in selection bias. despite the limitations of our study, our results confirmed the importance of co r assessment in patients with cns infections. co r showed good correlation with the severity and outcome of bacterial as well as non-bacterial meningitis. however, the most important value of co r is the capability to define patients with chemoregulation loss immediately upon admission to the hospital. it should be stressed that it is not possible to distinguish these patients by clinical examination or with computed tomography (ct) brain scan. the effects of conventional symptomatic treatment (osmotic diuresis, thiopental, hyperventilation) are greatly dependent (or associated with) on cerebral arterioles' vasoreactivity [ ] . patients with impaired co r are candidates for therapeutic hypothermia (th). this life-saving procedure should be started as soon as possible because conventional symptomatic treatment failure and poor outcome of disease in these patients should be anticipated. based on the results of this study, we made an internal guideline for th in patients with severe cns infections. the major criterion for th is impaired co r assessed by tcd. in patients without temporal bone 'acoustic window', minor criteria [optic nerve sheath diameter ≥ . , gcs ≤ , and sjo (jugular bulb venous saturation) < % or > %] are required. therapeutic hypothermia during cns infections may assist with the reduction in cerebral metabolism, cerebral blood volume (cbv), lowering of the intracranial pressure, and suppression of the inflammatory host response, allowing the maintenance of adequate cerebral perfusion pressure. according to our experience, the recovery of co r cannot be expected before the fourth day of treatment. we recommend the use of th as soon as possible and at least during the first three days after presentation. the first results of that therapeutic concept in patients with bm are very promising [ ] . therapeutic hypothermia halved the overall mortality in patients with bm and significantly decreased the mortality rates in patients with viral meningoencephalitis at our hospital during the last two years ( % vs. % and % vs. %, respectively; unpublished data). our study emphasizes the importance of co r assessment in patients with cns infections, regardless of etiology. a great predictive value as well as reliable stratification criteria in treatment strategy decisions make this method a very promising tool. community-acquired bacterial meningitis in adults: the epidemiology, timing of appropriate antimicrobial therapy, and prognostic factors management of bacterial meningitis in adults: algorithm from the british infection society represents current standard of care community-acquired bacterial meningitis in adults: antibiotic timing in disease course and outcome community-acquired bacterial meningitis in adults acute community-acquired bacterial meningitis in adults admitted to the intensive care unit: clinical manifestations, management and prognostic 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vasoreactivity cerebral co vasoreactivity evaluation by transcranial doppler ultrasound technique: a standardized methodology cerebral hemodynamics in patients with acute severe head trauma induced hypothermia in adult community-acquired bacterial meningitis-more than just a possibility? financial support there was no financial support for this study. key: cord- -nmgtan e authors: hu, zhigang; li, sijia; yang, ailan; li, wenxin; xiong, xiaoqi; hu, jianwu; jiang, jun; song, xinyu title: delayed hospital admission and high-dose corticosteroids potentially prolong sars-cov- rna detection duration of patients with covid- date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: nmgtan e coronavirus disease (covid- ) with the infection of sars-cov- has become a serious pandemic worldwide. however, only few studies focused on risk factors of prolonged sars-cov- rna detection among patients with covid- . we included adult patients with laboratory-confirmed covid- from two hospitals between jan and april . least absolute shrinkage and selection operator (lasso) analysis was used to screen out independent risk factors of sars-cov- rna detection. by multivariate binomial logistic regression analysis and cox regression analysis, we further determined the associations between sars-cov- rna detection and potential risk factors. all patients had two negative sars-cov- tests with days of median duration of sars-cov- rna detection (interquartile range: . – days). lasso and binomial logistic regression analyses suggested that delayed hospital admission (adjusted or = . , % ci: . – . ), hypokalemia, and subpleural lesion (adjusted or = . , % ci: . – . ) were associated with prolonged sars-cov- rna detection. by lasso and multivariate cox regression analyses, we observed that delayed hospital admission, subpleural lesion, and high-dose corticosteroid use were independent risk factors of prolonged sars-cov- rna detection. early hospital admission shortened . days of mean duration of sars-cov- rna detection than delayed hospital admission after adjusting confounding factors. our study demonstrated that delayed hospital admission and subpleural lesion were associated with prolonged sars-cov- rna detection among patients with covid- . the use of high-dose corticosteroids should be interpreted with extreme caution in treating covid- . coronavirus disease (covid- ) with the infection of novel severe acute respiratory syndrome coronavirus (sars-cov- ) was initially reported in wuhan, china [ ] . as of april, there were , laboratory-confirmed covid- patients and death ( . %) in china [ ] . compared with previous coronavirus diseases caused by the sars and the middle east respiratory syndrome coronavirus (mers-cov), covid- for sars-cov- was associated with lower overall mortality and stronger transmissibility [ ] . few studies focused on risk factors of sars-cov- rna detection duration among patients with covid- [ ] [ ] [ ] [ ] . in a retrospective study from china, covid- survivors have a median duration of about days of sars-cov- rna detection after illness onset, but deceased covid- patients have continuously detectable sars-cov- shedding until their death [ ] . the study of xu and colleagues [ ] estimated the risk factors of delayed viral shedding (≥ days after illness onset) and found that male, delayed hospital admission, and invasive mechanical ventilation were positively associated with prolonged sars-cov- rna detection duration. another study for patients with severe covid- found no significant effect of sex and age on sars-cov- rna detection duration [ ] . overall, the current studies related to sars-cov- rna detection duration were relatively few and reported inconsistent results. here, we performed a retrospective study to describe the characteristics of patients with covid- outside of wuhan in hubei province. least absolute shrinkage and selection operator (lasso) analysis and binomial logistic regression analysis with a generalized additive model were used to determine the independent risk factors of sars-cov- rna detection. we also estimated the median duration of sars-cov- rna detection and identified its independent risk factors by lasso analysis, multivariate cox regression analysis, and restricted mean survival time analysis. our study population came from two designated hospitals for treating patients with covid- , which included yichang central people's hospital and yichang third people's hospital. a total of adult patients with laboratoryconfirmed covid- were included into this retrospective study between jan and april . all patients had two negative sars-cov- testing results and were discharged from the hospital. all patients gave written informed consent and the retrospective study was approved by the ethics committee of yichang central people's hospital. two physicians extracted all data about epidemiological, demographic, clinical symptoms, laboratory findings, chest imaging, treatment, and outcome from electronic medical records using a standardized data collection. the third physician checked all relevant data and adjudicated any difference in interpretation between the two primary physicians. the demographic and epidemiological data included the following variables: sex, age, smoking, comorbidity, exposure history, and family clustering occurrence. we also collected some clinical symptoms as potential risk factors, including fever, cough, sputum, fatigue, diarrhea, nausea or vomiting, muscle soreness, and dyspnea. a large amount of laboratory findings were incorporated into our study, including complete blood counts, liver and renal function, d-dimer, c-reactive protein, procalcitonin, lactate dehydrogenase and creatine kinase, and blood chemistry (serum potassium and sodium). the samples of sputum or nasopharyngeal swab were tested by realtime reverse transcriptase polymerase chain reaction (rt-pcr) with the chinese center for disease control and prevention (cdc)-recommended kit (shengxiang, hunan, china). all specimens were performed at the clinical laboratory of yichang central people's hospital for further testing. laboratory findings were stratified according to previous studies [ , ] . all patients received chest computed tomography scan. the following radiological manifestations were considered potential risk factors of sars-cov- rna detection: ground-glass opacities, consolidation, interlobular septal thickening, fibrosis, the site of involving pulmonary lobe, the number of involving pulmonary segments, and subpleural lesion. the severity of covid- at admission was evaluated according to the guidelines of the national health commission of the people's republic of china [ ] . to simplify the analysis process, all patients were categorized into mild, severe, and critical groups with reference to the study of xu and colleagues [ ] . considering the lack of specific drugs for covid- treatment, all patients received different therapeutic options based on the recommendation of the national health commission of the people's republic of china and brainstorming of senior physicians [ ] . we regarded the time from illness onset to admission as hospital admission (≤ days vs > days) and the time from admission with first positive sars-cov- to two negative sars-cov- testing as the duration of negative sars-cov- rna detection. in our study, all patients were classified into short-term (< days) and long-term (≥ days) positive sars-cov- groups according to the duration of sars-cov- rna detection. all relevant data were merged and compared using microsoft excel and spss. categorical variables were presented as counts and percentages (%) and differences between two groups were estimated through a chisquare test. means and standard deviations were used to describe continuous variables with the mann-whitney u test for skewed continuous variables and student's t test for normally distributed continuous variables. chi-square goodness of fit and kolmogorov-smirnov tests were used to examine the normality of distribution of the data. our study included more than variables as the potential risk factors of sars-cov- rna detection among patients with covid- . for high-dimensional data, the least absolute shrinkage and selection operator (lasso) method is more available to screen the predictive features from the primary data set compared with conventional regression analysis [ ] [ ] [ ] [ ] . tuning parameter (lambda) selection in the lasso model uses -fold cross-validation [ , ] . by shrinking down to zero coefficient weights, lasso regression analysis has the ability to eliminate exposures that are non-related to the outcome [ , ] . in the first step of our study, we identified the independent risk factors of long-term positive sars-cov- rna detection by using lasso logistic regression analysis and sars-cov- rna detection duration by using lasso cox regression analysis. a generalized additive model with a binomial logistic regression model was used to further determine the associations between long-term sars-cov- rna detection and the independent risk factors obtained by lasso analysis. we further examined the associations between the independent risk factors obtained by lasso analysis and sars-cov- rna detection duration through multivariate cox regression analysis with a proportional hazards model. moreover, we applied restricted mean survival time analysis to better assess the effect of the independent risk factors on sars-cov- rna detection duration. a valid hazard ratio (hr) in conventional cox regression analysis requires the proportional hazards assumption [ ] . when proportional hazards are not met, hr may lack statistical power to detect a true treatment effect [ ] . restricted mean survival time analysis is an alternative robust and clinically interpretable summary measure of time-to-event outcome, which does not depend on the proportional hazards assumption [ ] . restricted mean survival time analysis also provides crude and adjusted differences between two groups. all statistical analyses were performed by using empower(r) (www. empowerstats. com; x&y solutions, inc., boston, ma) and r software, version . . (http: //www. r-project. org). the odds ratio (or) for binomial logistic regression analysis and hr for multivariate cox regression analysis with % confidence intervals (cis) were used to estimate the differences, and a two-tailed p < . was considered statistically significant. of the adult patients with covid- , there was predominantly female ( . %) and . ± . years of mean age that ranged from to years. there were patients with exposure history of sars-cov- and patients with family clustering occurrence. fever ( . %) was the most common symptom of covid- , followed by cough ( . %), fatigue ( . %), dyspnea ( . %), sputum ( . %), muscle soreness ( . %), diarrhea ( . %), and nausea or vomiting ( . %). in total, . % of patients with covid- received early hospital admission (the time from illness onset to admission ≤ days). in terms of laboratory findings, lymphocytopenia and eosinophilia were presented in patients and patients, respectively. the median duration of sars-cov- rna detection was days (interquartile range (iqr): . - days). about . % of patients were classified into the long-term positive sars-cov- group. the shortest duration of sars-cov- rna detection was days, whereas the longest was days. the most common lobe involved by sars-cov- infection was the right lower ( . %), followed by the left lower ( . %), left upper ( . %), right upper ( . %), and right middle ( . %). there were patients involving > pulmonary segments and patients with subpleural lesion. the majority of patients received combined antiviral treatment, including lopinavir/ritonavir, interferon-α, oseltamivir, and arbidol. a total of patients ( . %) received antibiotics and patients ( . %) received intravenous immunoglobulin. corticosteroids (methylprednisolone) and thymosin were used in . % and . % of patients with covid- , respectively. fifteen patients with severe and critical covid- ( . %) required high-flow nasal cannula oxygen therapy. five patients underwent non-invasive mechanical ventilation treatment and patients with invasive mechanical ventilation (more detailed information are shown in table ). lasso analysis with binomial logistic regression model screened out risk factors of long-term sars-cov- rna detection, which included dyspnea, delayed hospital admission, hypokalemia, subpleural lesion, right upper lesion, the use of methylprednisolone, and the use of thymosin. the area under the receiver operating characteristic curve (auc) was . . delayed hospital admission, hypokalemia, and subpleural lesion were still the independent risk factors of long-term sars-cov- rna detection in multivariate binomial logistic regression analysis with a generalized additive model. early hospital admission was associated with less probability of long-term sars-cov- rna detection compared with delayed hospital admission ( % vs %, adjusted or = . , % ci: . - . , p < . ; see fig. ). patients with hypokalemia (hypokalemia vs normal potassium, adjusted or = . , % ci: . - . , p = . ) and subpleural lesion (no vs yes, adjusted or = . , % ci: . - . , p = . ) seemed to prolong sars-cov- rna detection. lasso analysis with cox regression model found six independent risk factors of prolonged sars-cov- rna detection duration, including cough, dyspnea, delayed hospital admission, subpleural lesion, the use of methylprednisolone, and the use of thymosin. the value of auc was . . multivariate cox regression analysis further suggested that delayed hospital admission (adjusted hr = . , % ci: . - . , p < . ; see fig. ) and subpleural lesion (adjusted hr = . , % ci: . - . , p < . ) were still the independent risk factors of prolonged sars-cov- rna detection duration. in addition, patients with the use of high-dose ( mg/day vs no, adjusted hr = . , % ci: . - . , p = . ) but not low-dose ( mg/day vs no, adjusted hr = . , % ci: . - . , p = . ) methylprednisolone seemingly were associated with longer sars-cov- rna detection duration than those without methylprednisolone. when we restricted the time point to days, crude mean duration of sars-cov- rna detection in the early hospital admission group differed by . days ( % ci: . - . days) from delayed hospital admission. after adjusting potentially confounding factors, the adjusted mean duration of sars-cov- rna detection in the early hospital admission group was . days ( % ci: . - . days) less than that in the delayed hospital admission group (see fig. ). the majority of current studies about covid- focused on diagnosing this disease, estimating its transmission, assessing its severity, and identifying the risk factors of death [ ] [ ] [ ] . our study provided more detailed information about the epidemiological, demographic, clinical symptoms, laboratory, chest imaging, treatment, and outcome among patients with covid- . compared with patients in wuhan, our study population had more patients with mild covid- . our study suggested that early hospital admission seemingly had the ability to shorten sars-cov- rna detection duration. during days after hospital admission, early hospital admission reduced approximately . days in mean duration of sars-cov- rna detection. in addition, we also observed that high-dose ( mg/day) but not low-dose ( mg/day) methylprednisolone use potentially prolonged the mean duration of sars-cov- rna detection. our study provided another reason for early diagnosis and treatment of covid- . compared with delayed hospital admission, early hospital admission was associated with lower probability of long-term positive sars-cov- rna detection and shorter mean duration of sars-cov- rna detection. a shorter duration of sars-cov- rna detection also means less consumption of medical resources. a possible fig. the association between hospital admission and long-term positive sars-cov- rna detection in multivariate binomial logistic regression analysis with a generalized additive model fig. the association between hospital admission and sars-cov- rna detection in multivariate cox regression analysis with a proportional hazards model and restricted mean survival time analysis explanation of this phenomenon is that early hospital admission potentially decreases the probability of the convention of mild to severe covid- and improves the physical conditions to confront sars-cov- [ ] . the study of xu et al. demonstrated that early hospital admission was associated with a lower probability of severe patients at admission and less frequency of critically severe illness during hospitalization compared with delayed hospital admission [ ] . in our study, . % of covid- patients were observed with the involvement of the subpleural area in chest imaging, similar to the result of zhao et al. [ ] . subpleural lesion was identified to be positively associated with longterm positive sars-cov- and duration of sars-cov- rna detection in our study. hypokalemia seemingly was associated with higher probability of long-term positive sars-cov- rna detection than normal serum potassium. the maintenance of serum potassium balance involves three key elements: gastrointestinal losses, renal excretion, and cellular shifts [ ] . sars-cov- can attack the kidney, gastrointestinal tract, and liver by attaching to angiotensin-converting enzyme receptors of human organs [ ] . the occurrence of hypokalemia indicates that multiple human organs may suffer from sars-cov- viral infection and require long-term recovery. considering the effect of the cytokine storm syndrome, some patients with covid- received systemic corticosteroids and were recommended - mg/kg in china [ ] . however, the use of systemic corticosteroids in treating coronavirus infection is still controversial. a recent systematic review and meta-analysis showed that the use of systematic corticosteroids is associated with higher mortality and longer length of stay [ ] . several previous studies also suggested that corticosteroid use prolonged the duration of viral rna shedding in patients with sars [ ] and mers [ ] . fan et al. [ ] reported that the treatment of low-dose corticosteroids does not delay viral shedding in patients with covid- , similar to the finding of xu et al. [ ] . our study demonstrated that the prolonged duration of sars-cov- rna detection was shown in high-dose ( mg) methylprednisolone treatment (adjusted hr = . , % ci: . - . , p = . ), but not in low-dose ( mg) methylprednisolone treatment (adjusted hr = . , % ci: . - . , p = . ). high-dose but not low-dose corticosteroid treatment was reported to be associated with the increase of mortality in patients with severe covid- [ ] . therefore, the use of high-dose corticosteroids should be interpreted with extreme caution and low-dose corticosteroid use may be considered only for patients with severe covid- . there were the following strengths in our study. this study provided more detailed information about patients with covid- . in comparison to the study of xu et al. [ ] , we included more than variables as potential risk factors and screened the independent risk factors through comprehensive statistical analyses. using conventional logistic regression analysis to accurately handle high-dimensional data sets and correlated features is difficult, but lasso analysis can effectively overcome this obstacle [ ] . restricted mean survival time analysis was initially used to study covid- and confirmed the clinical benefit of early hospital admission in shortening the mean duration of sars-cov- rna detection. the major limitation of this work was it being a retrospective study with a relatively small sample size, which can produce selective bias. however, the clinical benefit of early hospital admission in shortening the duration of sars-cov- rna detection has been shown in the study from zhejiang province [ ] , located km east of wuhan. thus, we believe that our conclusions are valid. in conclusion, our study suggested that early hospital admission seemed to shorten mean duration of sars-cov- rna detection among patients with covid- , which potentially reduced the severity of covid- and the consumption of medical resources. moreover, high-dose corticosteroids should be used with extreme caution for treating covid- . a recent study showed that the use of dexamethasone ( mg) potentially decreased -day mortality among covid- patients who were receiving either invasive mechanical ventilation or oxygen alone at randomization but not among those without respiratory support [ ] . therefore, low-high corticosteroids can be considered for eligible patients with covid- . large-sample multicenter studies are warranted to further support our conclusions. estimating clinical severity of covid- from the transmission dynamics in wuhan, china national health commission of the people's republic of china. the outbreaks of covid- the sars, mers and novel coronavirus (covid- ) epidemics, the newest and biggest global health threats: what lessons have we 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corticosteroid therapy for critically ill patients with middle east respiratory syndrome low-dose corticosteroid therapy does not delay viral clearance in patients with covid- risk factors for severity and mortality in adult covid- inpatients in wuhan dexamethasone in hospitalized patients with covid- -preliminary report publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments we thank all our colleagues who helped us during the current study. we are also grateful to the many front-line medical staff for their dedication in the face of this outbreak, despite the potential threat to their own lives and the lives of their families.authors' contributions all authors contributed to data analysis and drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work. key: cord- -ghudhac authors: eichenberger, emily m.; dagher, michael; ruffin, felicia; park, lawrence; hersh, lisa; sivapalasingam, sumathi; fowler, vance g.; prasad, brinda c. title: complement levels in patients with bloodstream infection due to staphylococcus aureus or gram-negative bacteria date: - - journal: eur j clin microbiol infect dis doi: . /s - - -z sha: doc_id: cord_uid: ghudhac the complement system is a vital component of the innate immune system, though its role in bacteremia is poorly understood. we present complement levels in staphylococcus aureus bacteremia (sab) and gram-negative bacteremia (gnb) and describe observed associations of complement levels with clinical outcomes. complement and cytokine levels were measured in serum samples from hospitalized patients with sab, hospitalized patients with gnb, non-infected hospitalized patients, and community controls. c a levels were significantly higher in patients with sab as compared to patients with gnb. low c and c levels were associated with septic shock and -day mortality in patients with gnb, and elevated c was associated with a desirable outcome defined as absence of ( ) septic shock, ( ) acute renal failure, and ( ) death within days of bacteremia. low levels of c were associated with septic shock in patients with gnb but not sab. elevated il- was associated with increased -day mortality in patients with sab. complement profiles differ in patients with sab and those with gnb. measurement of il- in patients with sab and of c , c , and c in patients with gnb may help to identify those at higher risk for poor outcomes. electronic supplementary material: the online version of this article ( . /s - - -z) contains supplementary material, which is available to authorized users. bloodstream infections are associated with substantial morbidity and mortality worldwide [ , ] . it is estimated that approximately million episodes and , deaths from bloodstream infections occur annually in europe and north america combined [ ] . a consistent predictor of mortality for patients with s. aureus bacteremia (sab) [ , ] and gram-negative bacteremia (gnb) is the presence of septic shock [ ] . septic shock is thought to involve a dysregulated immune response to infection, the mechanisms of which are incompletely understood. the complement system is a critical component of innate immunity and has been implicated as a significant player in the pathogenesis of sepsis and septic shock [ ] [ ] [ ] . acute phase cytokine expression has also been explored in sepsis in hopes of understanding the immune dysregulation during infection [ ] . despite these studies, significant gaps remain in understanding the immune dysregulation that occurs during bacteremia and septic shock. the prognostic implications of acute phase inflammatory markers in patients with bacteremia are also poorly understood. in the current report, we undertook an exploratory study to address these unknowns. we evaluated complement and cytokine levels in patients with staphylococcus aureus and gram-negative bacteremia as compared to non-bacteremic hospitalized patients and community controls and explored potential associations between levels of these immune components and clinical outcome of the source patients. electronic supplementary material the online version of this article (https://doi.org/ . /s - - -z) contains supplementary material, which is available to authorized users. this prospective cohort study was conducted at two medical centers in north carolina: duke university hospital (a tertiary referral center with beds) and duke regional hospital (a community hospital with beds). the patient sample was identified using an existing protocol active at both sites known as the bloodstream infections registry (bsir). the bsir collects and stores both clinical data and biological specimens from non-neutropenic hospitalized patients aged years or older who have culture-confirmed monomicrobial bloodstream infections caused by either staphylococcus aureus or gram-negative bacteria. all patients with either monomicrobial s. aureus bacteremia (sab) or gram-negative bacteremia (gnb) enrolled in the bsir during a -month period between august and may were eligible for inclusion in the current study. the study was approved by the duke institutional review board, and written informed consent was obtained from all participants or their legally authorized representatives prior to their enrollment in this study. patients were classified as sab or gnb based on the results of their index microbiological cultures. participants from each of these two groups were then randomly selected to form the final study sample. a third group of hospitalized patients was comprised of patients who did not have a bloodstream infection but met all other bsir inclusion criteria from april to may . these controls were approached and enrolled sequentially until a target of patients, from critical care units and from non-critical care units, was met. an additional samples from non-infected, non-hospitalized adult community controls were added by the sponsor as a quality control measure. clinical data were collected from the electronic medical record of each participant using a standardized case report form (crf). information including demographics, comorbidities, hospitalization, and clinical outcomes was obtained for all in-patient participants. sepsis was defined as having a positive blood culture and having met at least two of the sirs criteria: [ ] temperature > °c or < °c, [ ] heart rate > beats per minute, [ ] respiratory rate > or partial pressure of carbon dioxide < , [ ] white blood cell count > , cells/mm or < cells/mm or having > % immature (neutrophil bands) forms [ ] . septic shock was defined as sepsis with hypotension (systolic blood pressure ≤ mmhg) and perfusion abnormalities as previously described [ ] . acute renal failure was defined as serum creatinine > . higher than baseline or increasing by > . mg/dl within hours [ ] . persistent bacteremia was defined as the presence of repeat positive blood cultures following appropriate antimicrobial therapy after ≥ days for sab patients [ ] and ≥ days for patients with gnb [ ] . a patient was classified as taking an immunosuppressive agent if taking a medication from any one or more of the following pharmacologic classes: calcineurin inhibitors, mtor inhibitors, antiproliferative agents, monoclonal antibodies, or corticosteroids. a desirable outcome was achieved if a patient did not experience septic shock or acute renal failure and was alive at days after bloodstream infection. blood samples from were collected for all participants at a single time point, within days following the index culture. cell-free plasma and serum from each sample, including controls, were obtained and stored at − °c. complement and cytokine levels were then measured as outlined below. c q, c , c , and c concentrations were measured by radial immunodiffusion with their respective polyclonal anticomplement molecules (complement laboratory, national jewish hospital). plasma specimens were aliquoted into duplicate wells in agarose and allowed to diffuse into the gel to form immunoprecipitation rings around the application well. the gels were washed to remove unprecipitated proteins, dried and stained. the outside diameter of the ring precipitated antigen was measured and used to calculate the area of the outer ring. then the diameter of the inner ring was measured and used to calculate the area of the inner ring. the total area of the precipitation was determined by subtracting the area of the inner ring from the area of the outer ring: (a precipitation ) = (a outer ring ) − (a inner ring ). c a concentrations were determined using the c a microvue elisa assay (c a quidel elisa, san diego). methods were carried out according to manufacturer's instructions. briefly, plasma specimens were added to a -well plate and coated with a murine monoclonal antibody to c a, incubated, and then washed. horseradish peroxidase conjugated anti-c a was added to each well to bind immobilized c a captured in the first step, incubated, and then washed. last, the chromogenic substance , ′, , ′ tetramethylbenzidine (tmb) was added to the wells and incubated. spectrophotometry at a was used to determine the color intensity, which was proportional to the concentration of c a present in the specimens. c and c concentrations were determined by using the human c kit and c kit, respectively, for use on the binding site spa plus analyzer (binding site, san diego), a turbidity analysis, according to the manufacturer's instructions. briefly, human c antiserum or c antiserum was added to the serum samples to form insoluble complexes. the turbidity of the specimens was measured using the spa plus analyzer. concentrations were calculated automatically by reference to a calibration curve stored within the instrument. to determine the normal range of complement levels, edta plasma samples from self-reported healthy individuals were collected and measured in accordance with the laboratory standard operating procedures and national jewish health irb policies. the measurements were made on seven different assays by two different technologists. the normal range was defined as the mean for the normal distribution of the donor samples plus or minus two standard deviations. the normal ranges were determined as follows; c q ( - μg/ml), c ( . - . μg/ml), c ( . - . mg/ml), c ( . - . mg/ml), c ( - μg/ml), c a ( . - . ng/ ml), and c ( - μg/ml). cytokines including il- , il- , il- , il- , il- , il- p , ifnγ, and tnfα were determined using milliplex® map based on the luminex® xmap® technology according to the manufacturer's instructions. briefly, cytokine levels were determined using immunoassays on the surface of magplex® -c microspheres, fluorescent-coded magnetic beads, which were coated with specific capture antibodies. after analyte from each test sample was captured by the bead, the mixture was incubated with the reporter molecule streptavidin-pe conjugate, and the fluorescent reporter signals were analyzed on the luminex® analyzer (magpix®) to determine the concentration of cytokine present in each sample. the normal ranges for cytokines were validated per the clinical laboratory standards institute (clsi) guidelines [ ] and were determined as follows: il- ( . - . pg/ml), il- ( . - . pg/ml), il- ( . - . pg/ ml), il- ( . - . pg/ml), il- ( . - . pg/ml), il- p ( . - . pg/ml), ifnγ ( . - . pg/ml), and tnfα ( . - . pg/ml). the lower limit of quantification (lloq) for all above cytokines was . pg/ml. values below the lloq were reported as < . pg/ml. the distributions of continuous measures are presented as medians and quartiles and categorical variables are evaluated using counts and percentages. statistical comparisons between groups were made with the kruskal-wallis test for continuous variables and fisher's exact test for categorical variables. statistical significance was set at p < . . a total of patients were included in the study: hospitalized patients with sab, hospitalized patients with gnb, hospitalized non-infected patients, and non-infected, nonhospitalized community controls. of the patients with sab, ( %) were due to methicillin resistant s. aureus (mrsa). of the patients with gnb, ( %) had escherichia coli, ( %) had klebsiella pneumoniae, ( %) had serratia marcescens, ( %) had pseudomonas aeruginosa, ( %) had morganella morganii, and ( %) had enterobacter cloacae. patient demographics are reported in table . for the non-infected, non-hospitalized community controls, only demographic data were available. median age of patients did not differ significantly between any of the four study groups ( ; p = . ). sex and race differed significantly between the four groups (p = . and p < . , respectively). clinical characteristics of the bacteremic patients and hospitalized controls are reported in table . there was a greater incidence of neoplasm in the gnb group than the sab group and hospitalized, noninfected control group ( % vs % vs %, p = . ). median creatinine was higher in the sab group than the gnb group (median [iqr]: gnb . [ . - . ] vs sab . [ . - . ], p = . ); however, incidence of acute renal failure was not significantly different (gnb % vs sab %, p = . ). nine ( %) of the patients with sab and ( %) patient with gnb had persistent bacteremia (p = . ). thirty-day all-cause mortality, septic shock, and acute respiratory distress syndrome were not significantly different between patients with gnb vs sab (p = . , p = . , and p = . , respectively). complement and cytokine levels were compared across each group (fig. a-e) . the median level of c in the gnb group was . μg/ml and was significantly greater than the sab and community control groups where the median levels were . μg/ml and . μg/ml, respectively (p = . and p = . ). levels of c in the sab group did not differ significantly from that of either control group (non-infected, hospitalized controls: p = . and community controls: the median level of c in the gnb group was . mg/ ml and was significantly greater than in the sab group where the median level was . mg/ml (p = . ), but was not significantly different from median c levels in either the hospitalized control or community control groups ( . mg/ ml, p = . and . mg/ml, p = . , respectively). the median level of c in gnb was . μg/ml and was greater than median c level in sab, hospitalized controls and community controls where the median levels of c were . μg/ml (p = . ), . μg/ml (p = . ), and . μg/ml (p = . ), respectively. median level of c in the gnb group was . μg/ml and was significantly greater than the median c levels in the hospitalized controls and community controls which were μg/ml (p = . ) and . μg/ml (p = . ), respectively. median level of c in the sab group was . μg/ml and was also significantly greater in the sab group as compared to the community controls (p = . ). the median c a level in the sab group was . ng/ml, and was significantly higher than the median c a levels in the gnb, hospitalized control and community control groups which were . ng/ml (p < . ), . ng/ml (p < . ) and ng/ml (p < . ), respectively. the median c a levels were also significantly higher in the gnb group as compared to the hospitalized controls and community controls (p = . and p = . , respectively). median levels of c q and c did not significantly differ between any of the four groups. complement levels did not significantly differ by immunosuppressive therapy. the median il- level in the sab population was . pg/ml, which was significantly higher than the median il- level in the gnb group, hospitalized controls, and community controls, which were . pg/ml (p = . ), . pg/ml (p = . ), and . pg/ml (p = . ), respectively. all other cytokine levels were similar among the bacteremic groups and are reported in the supplementary data. median levels of il- , il- , il- ra, and tnf-α were significantly higher in the gnb group as compared to the community controls (il- . pg/ml vs . pg/ml, p < . ; il- . pg/ml vs . pg/ml, p = . ; tnf-α . pg/ml vs . pg/ml, p < . ; il- ra . pg/ ml vs . pg/ml, p = . ) (supplemental figure) . median levels of il- , il- and tnf-α in the sab group were . pg/ml, . pg/ml, and . pg/ml, respectively, and were significantly higher than the median levels in the community control group (p = . , p = . and p = . , respectively). in patients with gnb, low levels of c ( figs. a and a) and c (figs. b and b) were associated with day mortality and septic shock while decreased c (fig. c) was associated with septic shock alone. c levels were similar among sab patients with or without acute renal failure, but c levels were significantly lower in patients with gnb with acute renal failure compared to those without renal failure. patients with gnb who experienced desirable outcomes had elevated c levels. none of the complement molecules were associated with the development of sepsis, septic shock, or -day mortality in patients with sab (fig. ) . levels of il- ( fig. c ) were significantly higher in sab patients who experienced mortality at -days as compared to those who survived. none of the complement or cytokine levels were associated with persistent bacteremia in the gnb or sab groups. in this exploratory study our findings suggest that complement profiles among patients with sab differ from patients with gnb. during a bacterial infection, the complement cascade may be activated through either the classical, alternative, gnb gram-negative bacteremia, sab s. aureus bacteremia, iqr interquartile range or mannose-binding lectin (mbl) pathway. upon initiation of the pathway through engaging c , c , or mbl, a serine proteolytic cascade leads to cleavage of complement proteins into smaller active compounds [ ] , ultimately converging on c activation and downstream target deposition of the membrane attack complex and release of anaphylatoxins c a and c a [ ] . these anaphylatoxins have garnered the most attention in animal models and observational human studies of sepsis, with several studies reporting increased levels of c a and c a in sepsis and shock [ ] [ ] [ ] [ ] . in the present study, c a levels were significantly higher in the bacteremic groups as compared to the non-infected and community control groups, likely reflecting the greater degree of complement cascade activation in bacteremic patients, with ongoing cleavage of c in the terminal pathway to produce c a and c b [ ] . interestingly, c a levels were significantly higher in the sab cohort as compared to gnb, indicating that there are marked differences in the way s. aureus and gram-negative pathogens [ ] . we postulate that the elevated c a levels in our sab cohort may reflect high levels of unbound c a in the serum due to the competitive binding of chips to the c ar, and may provide an opportunity for further understanding of how s. aureus manipulates the innate immune system. notably, there was no association of elevated c a with septic shock or increased -day mortality in any of the patients with either sab or gnb. this was an unexpected finding as elevated c a in experimental models of sepsis leads to increased mortality due to neutrophil dysfunction and damage to the vasculature [ ] . it is possible that this association was not detected due to the limitations of sample size. alternatively, perhaps chips blocking c ar may prevent the anaphylatoxic effects of c a that can lead to shock during sab. some studies also suggest a protective role for c a in animal models of sab [ , ] . larger studies are needed to confirm these findings. conversely, there is no evidence to support a protective role of c a during gnb. in fact, the presence of c a has been directly correlated with the development of shock, multi-organ failure, and death in murine models undergoing cecal ligation and puncture (clp) [ ] . it may be that our gnb cohort was too small to detect such a difference, or perhaps the effects of c a are not quite as detrimental in humans as they are in mice. we conclude that a b c fig. a-c biomarkers of mortality in bacteremic patients. gnb gram-negative bacteremia, sab s. aureus bacteremia; the asterisk denotes statistically significant difference. the horizontal lines compare medians across groups fig. a-e complement and cytokine concentrations during bacteremia vary by pathogen. gnb gram-negative bacteremia, sab s. aureus bacteremia; the asterisk denotes a statistically significant difference. the horizontal lines compare medians across groups the role of c a in bacteremia is controversial, incompletely understood, and needs further investigation. low levels of c and c were significantly associated with septic shock and with -day mortality in patients with gnb in our sample. these findings suggest a protective role of c and c . in murine models of cecal ligation and puncture (clp), c and c deficient mice are more likely to experience septic shock, and the absence of c is associated with increased mortality in sepsis [ ] . low c has also been associated with mortality in infected humans in a handful of small observational studies [ ] [ ] [ ] . to our knowledge, this is the first study to report an association between low c levels and mortality during gram-negative bloodstream infection, and further studies are needed to validate this finding. importantly, c and c levels were not associated with any adverse outcomes in patients with sab. collectively, these observations provide insight into possible differences in immune dysregulation that occur during gnb versus sab. we submit that low levels of these c and c are in large part due to lps mediated overconsumption of these key molecules in gnb, as supported by murine models of c -and c -deficient mice challenged with endotoxin [ ] . we conclude that depletion of c and c may lead to uncontrolled infection, septic shock and subsequently death. low levels of c were significantly associated with septic shock in our patients with gnb. to our knowledge this association has also not previously been established. c is a key component of the membrane attack complex that inserts on gnb outer membrane and leads to bacterial lysis. it is recognized that neonates are deficient in c and are highly susceptible to e. coli sepsis. supplementation of c to neonatal serum enhances the capacity of neonatal serum to kill e. coli [ ] . hence, the association of low c with septic shock in gnb patients is plausible in this regard, with low levels likely reflecting over-consumption of this key complement protein. levels of c and c were significantly higher in gnb as compared to hospitalized, non-infected controls and community controls, and levels of c were significantly higher in a b c fig. a-c biomarkers of septic shock in patients with bloodstream infections. gnb gram-negative bacteremia, sab s. aureus bacteremia; the asterisk denotes a statistically significant difference. the horizontal lines compare medians across groups sab patients as compared to community controls. these were unexpected results as c and c , members of the terminal complement pathway, were expected to be cleaved and consumed, respectively, during an active infection [ ] . however, a cross-sectional study investigating complement levels in healthy adults recently reported that aging may be associated with enhanced functioning of the alternative and classical pathways, leading to increased levels of the terminal pathway components [ ] . we hypothesize that the aforementioned elevated levels of c and c in the bacteremic groups may in part be a function of the older age in the gnb and sab groups (i.e. > years). indeed, the median age > years in our sab and gnb groups should be recognized as a potential confounder and a possible explanation for the elevated c in both sab and gnb, and c in gnb, as compared to community controls. we found that c levels were significantly lower in patients who develop acute kidney injury (aki) during gnb as compared to those who did not develop aki. while the role of complement in glomerulonephropathies such as anca vasculitis, hemolytic uremic syndrome and anti-glomerular basement membrane syndrome is well characterized [ ] , the role of complement in sepsis-related aki is poorly understood. in porcine models of sepsis, terminal complement complexes (c b-c ) concentrate in the kidney [ ] . perhaps lower levels of c in patients with gnb are indicative of excessive terminal complement system activation with c b- renal deposition and subsequent aki. alternatively, lower levels of c may instead reflect renal ischemia during gnb septic shock. whether c is involved in the pathogenesis of aki during gnb or whether it is simply a harbinger of end-organ damage during sepsis is yet to be determined. as an important corollary, we found that elevated levels of c were associated with a desirable outcome in patients with gnb. due to the small gnb sample size and the highly variable measurements of c within this sample, further studies are needed to validate this finding. however, based on our results, we submit that measurement of c in patients with gnb to assist in illness severity stratification may prove to be an informative subject for future studies, eventually providing a platform to begin investigating the role of recombinant c as an adjunctive therapy in patients with gnb septic shock. patients with sab had greater levels of il- than patients without sab (i.e., controls, gnb). this may be because staphylococcal cell wall peptidoglycan lipopeptides and glycopolymers employ toll-like receptor signaling (tlr ) on antigen-presenting cells to generate increased il- expression [ ] . notably, within the sab group, elevated levels of il- were associated with mortality. our findings validate those by rose et al. who found that an elevated level of il- is independently associated with mortality in patients with sab (or, . ; p = . ) [ ] . il- is an anti-inflammatory cytokine which regulates the pro-inflammatory response during infection by downregulating the expression of proinflammatory cytokines and induces apoptosis of antigen presenting cells and inhibits cd + t cell activation. we hypothesize that elevated il- in patients with sab reflects an overly suppressed inflammatory state, or "immunoparalysis" as suggested by rose et al. and chau et al. [ , ] , whereby the host immune system cannot control the infection. this association was not found in the gnb population. we suspect this association of elevated il- and mortality is unique to s. aureus and alludes to a unique mechanism employed by s. aureus to exploit the host immune system. measuring il- levels in patients with sab may assist in identifying patients at increased risk for mortality from their bloodstream infection, and raises the question of whether an il- monoclonal antibody can aide in the treatment of patients with sab and high il- levels. lastly, patients with gnb and sab had greater levels of il- , il- and tnf-α than community controls. all three of these pro-inflammatory cytokines are critical components of acute phase response to infection [ ] , and have been reported to be elevated in bacteremic individuals [ ] [ ] [ ] . as anticipated, complement levels in bacteremic patients did not differ by immunosuppressive therapy use. medications targeting t-cells (i.e., calcineurin inhibitors, belatacept), b-cells (i.e., rituximab, atacicept), and specific cytokine inhibitors (i.e., il- receptor antagonist basiliximab, il- receptor antagonist tocilizumab), do not appear to affect the complement cascade [ ] . similarly, despite their wide range of effects on the immune system, corticosteroids do fig. high c is associated with a desirable outcome in gnb. gnb gram-negative bacteremia, sab s. aureus bacteremai, arf acute renal failure; the asterisk denotes statistically significant difference. the horizontal lines compare medians across groups. desirable outcome is defined as the absence of acute renal failure, septic shock, or death within days of first positive blood culture not appear to affect the complement cascade [ ] . hence, use of immunosuppressive agents in % of the bacteremic population would be unlikely to affect the complement concentration. the power of our study is limited owing to the small sample size, the heterogenous gnb group that includes six different pathogenic organisms, and the single time point of collection of biological specimens. while all biological specimens were collected within hours of initial positive blood culture, it is possible that there is variability of complement and cytokine concentrations within this initial hour time interval, and that timing of antimicrobial therapy may influence concentrations. another limitation is that, given the substantial number of markers we examined, the use of p < . for statistical significance can be called into question. however, as this is an exploratory analysis, this limitation is of less concern than would be the case for an inferential study. our results indicate that the immunologic biosignature of sab is fundamentally distinct from that of gnb. additionally, our findings add to the limited data regarding complement levels and the corresponding immune dysregulation in sepsis. we hypothesize that measurement of c , c and c levels at the time of admission in patients with gnb may help to risk stratify patients who are at increased risk for septic shock, and measurement of c and c specifically may help to stratify those gnb patients at increased risk for mortality. similarly, we suspect that measurement of il- levels in sab may stratify those at increased risk for mortality. finally, we postulate that our observations that normal to elevated c levels in patients correlate with a desirable outcome could provide the rational for an interventional trial of c supplementation in c deplete patients with gnb. the cost-effectiveness of rapid diagnostic testing for the diagnosis of bloodstream infections with or without antimicrobial stewardship overall burden of bloodstream infection and nosocomial bloodstream infection in north america and europe predictors of mortality in staphylococcus aureus bacteremia adequacy of antimicrobial treatment and outcome of staphylococcus aureus bacteremia in western european countries diagnosis and 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bloodstream infections in febrile neutropenic pediatric cancer patients: microbiological and sepsis biomarkers insight the diagnostic value of interleukin- for the detection of bacteremia in pediatric hematopoietic stem cell recipients with febrile neutropenia the role of tumor necrosis factor in sepsis immunosuppressive medications publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements the authors would like to thank alida coppi for help with the cytokine data and reading the manuscript.funding information this work was funded by a grant from regeneron to dr. vance g. fowler jr. dr. fowler was supported by nih grant k -ai . dr. eichenberger was supported by nih grant t -ai . conflicts of interest dr. vance fowler reports personal fees from novartis, novadigm, durata, debiopharm, genentech, achaogen, affinium, medicines co., cerexa, tetraphase, trius, medimmune, bayer, theravance, basilea, affinergy, janssen, xbiotech, contrafect, regeneron, basilea, destiny, and amphliphi biosciences. integrated biotherapeutics; c j, grants from nih, medimmune, cerexa/forest/ actavis/allergan, pfizer, advanced liquid logics, theravance, novartis, and cubist/merck; medical biosurfaces; locus; affinergy; contrafect; karius; genentech, regeneron, basilea, and janssen, from green cross, cubist, cerexa, durata, theravance; debiopharm, royalties from uptodate; and a patent sepsis diagnostics pending.ethical approval the study was approved by the duke institutional review board.informed consent written informed consent was obtained from all participants or their legally authorized representatives prior to their enrollment in this study key: cord- -q wy e authors: delannoy, p.-y.; boussekey, n.; devos, p.; alfandari, s.; turbelin, c.; chiche, a.; meybeck, a.; georges, h.; leroy, o. title: impact of combination therapy with aminoglycosides on the outcome of icu-acquired bacteraemias date: - - journal: eur j clin microbiol infect dis doi: . /s - - -z sha: doc_id: cord_uid: q wy e pharmacodynamic studies report on the rapid bactericidal activity of aminoglycosides, conferring them as being of theoretical interest for bacteraemia treatment. we assessed this issue in a retrospective study of patients with intensive care unit (icu)-acquired bacteraemias. to determine the impact of aminoglycosides in antimicrobial combination on the outcome of patients with bacteraemia, we performed a monovariate analysis and a logistic regression analysis comparing patients treated with or without aminoglycosides. forty-eight bacteraemias in patients were included. eighteen patients received aminoglycosides. baseline characteristics as well as adaptation and adequation of antibiotherapy did not differ in patients who did or did not receive aminoglycosides. patients who received aminoglycosides had longer time alive away from the icu ( . ± . ( [ – ]) vs. . ± . ( [ – ] days; p = . ) and free from mechanical ventilation ( . ± . ( [ – ] vs. . ± . ( [ – ] days; p = . ) on day . the icu mortality was % in the aminoglycoside group versus % (p = . ). in the multivariate analysis, patients treated with aminoglycosides were times less likely to die than those treated without aminoglycosides (confidence interval [ci] = [ . – . ]; p = . ). our study supports the hypothesis that combination short-term antibiotherapy with an aminoglycoside for icu-acquired bacteraemias could increase survival. combination antimicrobial therapy with beta-lactams plus aminoglycosides (ags) was widely used in the s and s. the advent of broader spectrum beta-lactams and reports on ag toxicity were the basis of the current controversy on combination therapy. meta-analysis failed to demonstrate improved outcomes in patients treated with antibiotic combinations over those receiving monotherapy [ ] [ ] [ ] [ ] and resulted in a decreased use of combination therapy. however, it remains frequently used in intensive care units (icus) due to the theoretical advantages of rapid bacterial killing and extending the spectrum of activity in the era of multidrugresistant microorganisms. bacteraemia is a severe infection associated with increased morbidity and mortality [ ] [ ] [ ] . hospital-acquired bacteraemias are more often due to drug-resistant organisms. a delay in the administration of active antibiotics has been linked to an increased risk of death. ags exhibit concentration-dependant activity, allowing for maximum efficacy with once-daily, short-duration therapy, which offers the added benefit of decreased toxicity. moreover, the pharmacologic properties of ags could be beneficial in the specific subgroup of critically ill patients presenting with icu-acquired bacteraemia. we performed a retrospective study to evaluate the impact of ags in antimicrobial combination on icu-acquired bacteraemia in our universityaffiliated icu [ ] [ ] [ ] [ ] . eligible patients were those who developed icu-acquired bacteraemia from january to july during their stay in the icu of tourcoing hospital, france. patients were recruited though the icu ongoing database and the microbiology laboratory records. neither approval of the ethics committee nor informed consent was required, considering our study was retrospective. the aim of the study was to evaluate the impact of ags in antibiotic combination on the outcome of patients with icu-acquired bacteraemia. for each bacteraemia, the following covariates were collected: age, gender, comorbidities (cardiac disease, diabetes mellitus, chronic obstructive pulmonary disease [copd], chronic hepatic failure, chronic renal failure, haematologic malignancies, non-haematologic malignancies). patients were considered as being on immunosuppressive treatment if they had received corticosteroids ( mg/d for at least two weeks), cytotoxic therapies or radiotherapy in the last three months. organ transplant patients, human immunodeficiency virus (hiv)positive patients and patients with splenectomy or neutropaenia (granulocyte count < / mm ) were considered to be immunocompromised. we collected all antibiotics administered during the month before bacteraemia. icu-acquired bacteraemia was defined as any bacteraemia occurring in our unit more than h after admission. coagulase-negative staphylococci were considered if two successive blood cultures were positive with the same antibiogram. the source of infection was classified as follows: lower respiratory tract, intra-abdominal, genitourinary tract, catheter-related infection, endocarditis, meningitis, cutaneous infection, primary bacteraemia or other. secondary bacteraemia was defined as an episode developing after a documented infection with the same microorganism at another body site [ ] . the severity of illness was evaluated with the simplified acute physiology score (saps ii) [ ] and the sepsisrelated organ failure assessment (sofa) score [ ] . we considered organ dysfunction if the score was greater . haemodynamic instability was defined as a need for vasopressive drugs to maintain adequate blood pressure and respiratory failure was noted when pao /fio < with ventilatory support was required. acute renal dysfunction was defined by a serum creatinine > mg/l or urine output < ml/d, acute hepatic dysfunction by an increase in bilirubin level > mg/l, neurologic failure by glasgow coma scale score < . platelet count < , /mm was considered as a coagulopathy. we collected biological data including leukocyte and platelet counts, ph, urea, creatinine, pao /fio , prothrombin time, bilirubin and c-reactive protein (crp). variables collected on antibiotherapy were as follows: time from the first positive blood culture to first antibiotic administration and antimicrobial agents used. the administration schedule of ags was recorded. following local guidelines, we performed peak serum dosages and trough dosages for patients with initial renal impairment. a trough over μg/ml was considered to be toxic. antibiotic therapy was considered as adapted if it followed the recommendations and adequate if it included at least one antibiotic active in vitro on the isolated pathogen [ ] . we also recorded the use of activated protein c, hydrocortisone, intensive insulin therapy, and need for mechanical ventilation or renal replacement therapy (rrt). the sofa score evolution was recorded on days and following bacteraemia. we investigated whether the patient had developed infection-related complications, such as septic shock, acute respiratory distress syndrome (ards), renal failure, disseminated intravascular coagulopathy or hepatic dysfunction. to evaluate the renal function, we used the risk, injury, failure, loss of kidney function and end-stage kidney disease (rifle) criteria [ ] . patients were considered as having acute kidney injury when the rifle class increased to injury or failure after bacteraemia. we also searched for any other hospital-acquired infection and bacteraemia recurrence. the primary endpoint was -day survival. secondary endpoints were number of days without mechanical ventilation, without vasopressive drugs, without rrt and with icu discharge on day from bacteraemia. to evaluate the impact of ags on patients' outcomes, we compared patients with or without ags in their empirical antibiotic regimen for bacteraemia through a monovariate analysis. comparisons between groups were performed using the chi-square test or fisher's exact test for categorical parameters. continuous variables were analysed using wilcoxon's test. differences between groups were considered to be significant for variables yielding a p-value less than . . all significant parameters in the monovariate analysis were evaluated in the multivariate logistic regression analysis. all analyses were performed using sas software version . . between january and july , we identified icuacquired bacteraemias in patients. of the overall population, % were male, the mean age was years ± , the mean saps ii on the bacteraemia day was ± and the mean sofa score was . ± . . the baseline characteristics did not differ in patients who did or did not receive ags (table ) . the most frequent portal of entry was a catheter-related bacteraemia ( %). the most frequent microorganisms were staphylococcus aureus ( %) and enterobacteriaceae ( %). no polybacterial bacteraemia was observed. the bacteriological data are presented table . all but three patients received a beta-lactam as the primary therapy. eighteen patients ( . %) received combination therapy with an ag ( with amikacin and six with gentamicin). patients with ags received a mean of . ± . ag injections. ags were more frequently administered in gram-negative than in gram-positive bacteraemias ( vs. %, p . ). two patients developed acute renal failure in the ag group versus three in the non-ag group (p ). in the ag group, % of patients received at least two antibiotics active on the isolated pathogen, whereas this was only % in the other group (p< . ). the most frequent combination in the ag group was beta-lactam with ags (n ) for eight enterobacteriaceae, for five methicillin-susceptible s. aureus and for one coagulase-negative staphylococcus bacteraemias. the other combination in the ag group was glycopeptides plus ag for one coagulase-negative staphylococcus bacteraemia. in the non-ag group, combinations were beta-lactam plus quinolone (n ) and beta-lactam plus colistin (n ) for enterobacteriaceae bacteraemias, and one association of beta-lactam with linezolid for methicillin-susceptible staphylococcus. no difference was found between the two groups in the management and the occurrence of complications (table ). antibiotherapy delay was not different between the two groups and the adaptation and adequation of antibiotherapy reached % in both groups. patients who received ags had longer time alive away from the icu ( . ± . ( [ - ]) vs. . ± . ( [ - ]); p . ) and free from mechanical ventilation ( . ± . ( [ - ]) vs. . ± . ( [ - ]); p . ) on day . the icu mortality was % in the ag group and % in the non-ag group (p . ). the occurrence of another nosocomial infection, complications of bacteraemia and sofa score evolution did not differ between the two groups. the univariate analysis identified five variables associated with a higher mortality: -hepatic failure (p . ) -oliguria (p . ) -no ag (p . ) -no vancomycin (p . ) -sofa score ≥ (p . ) these variables were included in a logistic regression multivariate analysis model. two independent risk factors for mortality were identified: we found a survival benefit with the use of combination therapy with ags for icu-acquired bacteraemias. ags have unique characteristics among antimicrobial agents. they exhibit extremely rapid bacterial killing, have a demonstrated synergy with beta-lactams on some bacteria and have a prolonged post-antibiotic effect [ ] . in the icu, inadequate empirical antibiotic therapy is associated with an increased mortality risk in patients with ventilator-associated pneumonia and bacteraemia. with the increasing occurrence of drugresistant microorganisms, both community-and hospitalacquired, icu physicians often use empirical combination therapy to increase the initial spectrum coverage. in our study, the rates of appropriate antibiotic treatment in the two groups were not statistically significant and do not explain our main result [ ] [ ] [ ] [ ] . the bacteraemias' portals of entry, similarly, could not explain our results. catheter-related bacteraemias were usually associated to a low mortality rate and were more frequent in the group without ags. the major adverse effects of ags are dose-dependent nephrotoxicity and ototoxicity. in our study, the rates of adverse events were not increased by ags. ag nephrotoxicity is influenced by the administration schedule, the duration of treatment and individual variability. the risk of nephrotoxicity is lower for once-daily ag dosing than for traditional twiceor thrice-daily dosing. our dosing regimen, drug level monitoring and duration of treatment are strictly adherent to our institution's guidelines. we only use once-daily high dosing, with a maximum of days treatment duration. this might explain the lack of observed adverse events [ , ] . icu patients with or without bacteraemia have an increased volume of distribution. the usual dosages of betalactams might be less effective. these patients would be the most likely to benefit from the enhanced bactericidal activity offered by a beta-lactam-ag combination therapy. most studies do not support a benefit for combination therapy. the comparison of ag/beta-lactam combination with betalactam monotherapy has been the subject of numerous studies and meta-analysis. paul et al., in , reviewed , patients from clinical trials [ ] . combination therapy did not prevent the emergence of antimicrobial resistance and did not affect patient outcomes. acute kidney injuries were mostly found in the combination group. paul et al. found the same conclusions in another meta-analysis published in [ ] . furno et al., in , performed a similar meta-analysis focussing mostly on immunocompromised patients [ ] . among , septic episodes with , bacteraemias, their conclusions were similar. safdar et al. found a significant survival advantage with ags for bacteraemia caused by pseudomonas aeruginosa in comparative non-randomised studies [ ] analysed in a meta-analysis. the meta-analysis by bliziotis et al. concluded that combination therapy lacks benefit with respect to the selection of drug-resistant organisms, superinfection, treatment failure and mortality [ ]. leibovici et al., reviewing all of the previously mentioned meta-analyses, concluded to a lack of interest of the beta-lactam ag combination [ ] . however, it has been objected [ ] that most studies included suffered from limitations weakening this interpretation: the studies were heterogeneous and rarely compared the same beta-lactam in both arms. the latter means that it is difficult to relate the difference in efficacy to the ag and not the betalactam. furthermore, the studies mainly used outdated ag administration schedules, such as thrice-daily administration, long treatment duration and lack of serum monitoring. these are related to treatment toxicity, which was counted as treatment failure in many studies. finally, bliziotis et al. suggested that once-daily ag therapy combined with a beta-lactam could have a beneficial effect on the development of resistance compared with beta-lactam monotherapy and should be the subject of further research. recently, a systematic review of randomised trials focussing on the clinical implications of beta-lactam-ag synergy recommended avoiding the routine use of beta-lactam-ag combination therapy. in a subgroup of bacteraemic patients, they did not find any survival advantage for combination therapy [ ] . our study focussed on the most severe patients, i.e. icu patients with hospital-acquired bacteraemia who need more rapidly active treatment than less severe patients. our study has several limitations that should be taken into account. first, this study is retrospective and low-powered. second, our groups were statistically homogeneous, except for greater hepatic failure in patients without ag. this failure, a major prognostic factor associated with a high mortality, could have biassed our study. third, we considered a very specific clinical scenario: icu-acquired bacteraemias. fourth, except for methicillin-resistant s. aureus, we observed only rarely drug-resistant organisms. however, considering the susceptibility pattern of drug-resistant bacteria now observed in our icu, this would probably enhance our results. mostly, treatment with ag permitted to reach an % rate of effective bi-therapy instead of only % without ag. the debate about aminoglycoside (ag) interest is not closed. our study suggests that short-term combination beta-lactams plus ags therapy in intensive care unit (icu)-acquired bacteraemia could reduce mortality. this should be confirmed in future prospective randomised trials. effect of aminoglycoside and beta-lactam combination therapy versus beta-lactam monotherapy on the emergence of antimicrobial resistance: a meta-analysis of randomized, controlled trials does combination antimicrobial therapy reduce mortality in gram-negative bacteraemia? a meta-analysis monotherapy or aminoglycoside-containing combinations for empirical antibiotic treatment of febrile neutropenic patients: a meta-analysis community-acquired bloodstream infection in critically ill adult patients: impact of shock and inappropriate antibiotic therapy on survival nosocomial bacteremia in critically ill patients: a multicenter study evaluating epidemiology and prognosis. spanish collaborative group for infections in intensive care units of sociedad espanola de medicina intensiva y unidades coronarias (semiuc) antibiotic use and impact on outcome from bacteraemic critical illness: the bacteraemia study in intensive care (basic) beta-lactams with or without aminoglycosides a prospective randomized study comparing once-versus twice-daily amikacin dosing in critically ill adult and paediatric patients once-daily dosing of aminoglycosides: review and recommendations for clinical practice prospective evaluation of the effect of an aminoglycoside dosing regimen on rates of observed nephrotoxicity and ototoxicity a new simplified acute physiology score (saps ii) based on a european/north american multicenter study the sofa (sepsis-related organ failure assessment) score to describe organ dysfunction/failure. on behalf of the working group on sepsis-related problems of the european society of intensive care medicine promoting appropriate antimicrobial drug use: perspective from the centers for disease control and prevention acute renal failuredefinition, outcome measures, animal models, fluid therapy and information technology needs management of infections caused by gram-negative bacilli: the role of antimicrobial combinations mortality and morbidity attributable to inadequate empirical antimicrobial therapy in patients admitted to the icu with sepsis: a matched cohort study impact of inappropriate antibiotic therapy on mortality in patients with ventilator-associated pneumonia and blood stream infection: a meta-analysis benefit of empirical appropriate antibiotic treatment: thirty-day mortality and duration of hospital stay impact of adequate antibiotic empirical therapy on the outcome of patients admitted to the intensive care unit with sepsis aminoglycoside nephrotoxicity: modeling, simulation, and control aminoglycoside nephrotoxicity: do time and frequency of administration matter? beta lactam antibiotic monotherapy versus beta lactamaminoglycoside antibiotic combination therapy for sepsis aminoglycoside drugs in clinical practice: an evidence-based approach clinical implications of β-lactam-aminoglycoside synergism: systematic review of randomised trials key: cord- -nsrs dmc authors: waldeck, frederike; boroli, filippo; suh, noémie; wendel garcia, pedro david; flury, domenica; notter, julia; iten, anne; kaiser, laurent; schrenzel, jacques; boggian, katia; maggiorini, marco; pugin, jérôme; kleger, gian-reto; albrich, werner christian title: influenza-associated aspergillosis in critically-ill patients—a retrospective bicentric cohort study date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: nsrs dmc influenza was recently reported as a risk factor for invasive aspergillosis (ia). we aimed to describe prognostic factors for influenza-associated ia (iaa) and poor outcome and mortality in critically ill patients in switzerland. all adults with confirmed influenza admitted to the icu at two swiss tertiary care centres during the / influenza season were retrospectively evaluated. iaa was defined by clinical, mycological and radiological criteria: a positive galactomannan in bronchoalveolar lavage or histopathological or cultural evidence in respiratory specimens of aspergillus spp., any radiological infiltrate and a compatible clinical presentation. poor outcome was defined as a composite of in-hospital mortality, icu length of stay (los), invasive ventilation for > days or extracorporeal membrane oxygenation. of patients with influenza in the icu, ( %) were diagnosed with iaa. all patients with iaa had poor outcome compared to ( %) patients without iaa (p < . ). median icu-los and mortality were vs. days (p < . ) and / ( %) vs. / ( %; p = . ) in patients with vs. without iaa, respectively. patients with iaa had significantly longer durations of antibiotic therapy, vasoactive support and mechanical ventilation. aspergillus was the most common respiratory co-pathogen ( / , %) followed by classical bacterial co-pathogens. iaa was not associated with classical risk factors. aspergillus is a common superinfection in critically ill influenza patients associated with poor outcome and longer duration of organ supportive therapies. given the absence of classical risk factors for aspergillosis, greater awareness is necessary, particularly in those requiring organ supportive therapies. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. influenza infection has been recently defined as a risk factor of invasive aspergillosis [ , ] . influenza can cause severe pneumonia and acute respiratory distress syndrome (ards) [ , , ] . respiratory bacterial superinfection of influenza represents a common complication with high mortality [ , , , , ] . bacterial pathogens superinfecting influenza pneumonia have changed over time probably as a function of both different hosts and influenza strains. these include haemophilus influenzae, streptococcus pyogenes, staphylococcus aureus and streptococcus pneumoniae [ ] . s. pneumoniae was the most common pathogen in the pandemic [ ] and s. aureus emerged as another frequent co-pathogen during the / h n pandemic [ , ] . high prevalence of pseudomonas aeruginosa superinfection has been reported in intensive care unit (icu) patients [ , ] . superinfection with aspergillus spp. has been increasingly described since the electronic supplementary material the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. / influenza pandemic [ , , , ] associated with even higher mortality ( - %) [ , , , , ] . iaa is independent of influenza type (a or b [ , ] ) and also affects immunocompetent hosts. between - % of influenza patients in the icu [ , , ] , less frequently in north america ( . %) [ ] and % of immunocompromised influenza patients have been reported to have iaa [ ] . predictors of iaa, poor outcome and mortality in influenza patients in the icu remain unknown. since no data on iaa was available from switzerland, we retrospectively analysed all patients with severe influenza infection needing treatment in two large swiss icus during the / influenza season with regard to predictors of iaa, mortality and poor outcome. in this retrospective cohort study, sixteen icus of tertiary hospitals in switzerland were asked if they had observed cases of iaa and severe influenza and routinely looked for iaa based on clinical suspicion with galactomannan and fungal cultures in bal; only two of them met the criteria (cantonal hospital of st. gallen and university hospital of geneva). all adults (≥ years) with confirmed influenza infection during the / influenza season (december -april ) admitted to the icu for ≥ h of those centres were included. patients whose influenza diagnosis occurred after the discharge from the icu were excluded. influenza infection was diagnosed by polymerase chain reaction (pcr) from nasopharyngeal swab, sputum or bronchoalveolar lavage (bal). patients were identified from the icu databases, infectious diseases and hospital epidemiology databases in order to improve identification of patients and reduce reporting bias. the study was approved by the local ethics committees (ekos - ). there was no funding. the primary aim was to identify predictors of iaa in critically ill patients with influenza infection. secondary aims were to detect predictors of mortality and poor outcome of severe influenza infection. poor outcome was defined as a composite of icu length of stay (los) ≥ days, need of extracorporeal membrane oxygenation (ecmo), invasive ventilation for ≥ days or in-hospital death. ards was diagnosed according to the berlin criteria [ , ] . a. definitions iaa was defined by clinical, radiological and mycological criteria according to the modified criteria of iaa by schauwvlieghe et al. and blot et al. (supplementary table ) [ , ] . records of all iaa patients were reviewed and consensus was achieved by investigators from both centres whether criteria of iaa were fulfilled. the platelia assay "aspergillus ag" (biorad) was used to detect gm. respiratory superinfection was defined as (i) detection of clinically relevant bacterial or fungal pathogen in respiratory specimens or positive blood cultures that was treated with antibiotics or antifungals, respectively, (ii) a positive pneumococcal urinary antigen, pneumocystis jirovecii antigen in bal or positive gm in serum or bal ( table in the appendix). other superinfection included all respiratory superinfections, bacteraemia, catheter-associated infections and clostridioides difficile colitis. organ supportive therapies were defined as need of renal replacement therapy, vasoactive support, invasive mechanical ventilation and ecmo. continuous variables were assessed by t tests or mann-whitney u tests as appropriate. for categorical values, comparison was done by fisher's exact test. missing data were not imputed. a two-sided p value < . was considered as statistically significant. given the small number of each outcome, only the univariate but not the multivariable analyses are reported. data analysis was performed with sas (version . , sas institute inc., cary, nc, usa) and r core team ( , r foundation for statistical computing, vienna, austria). of patients identified from the databases, were excluded ( patients per centre) and were included and analysed. median follow-up time was days. nine patients had iaa ( %). there were no statistically significant differences in baseline characteristics between patients with and without iaa ( table in the appendix). immunosuppressive diseases were uncommon in both groups. during hospitalization, corticosteroids were initiated in half of all influenza patients ( % vs. % in iaa and non-iaa, p = . ). / ( . %) patients with influenza a had iaa and / ( %) with influenza b (p = . ). duration of influenza symptoms before diagnosis of influenza was significantly longer in those with iaa (median duration days (interquartile range (iqr) - ) vs. days (iqr - ) in non-iaa, p = . ), whereas there was no difference in duration of symptoms until hospitalization (median duration days (iqr - ) in iaa and days (iqr - ) in non-iaa, p = . ). iaa symptoms started after a median of days after icu admission. iaa was diagnosed after a median of days after icu admission and days after first symptoms of influenza (iqr - and - days respectively). gm was measured in % of all patients ( % in iaa and % in non-iaa, p < . ). gm was positive in of ( . %) patients in non-iaa and of ( %) samples with iaa (p < . ). all patients with positive gm had antibiotics prior to gm testing, as had of ( . %) patients with a negative gm (p = . ). two patients with iaa and negative gm had therapy with voriconazole started on the same day as gm testing was performed. it cannot be excluded that treatment was started before bal was performed which could have caused in a false-negative test result. eight of patients with iaa had radiologic infiltrates and nodules. there were no positive cultures of aspergillus spp. in non-iaa patients but growth of aspergillus spp. in ( %) patients with iaa, mainly aspergillus fumigatus ( of patients). organ supportive therapies and complications were more frequent in iaa patients than in non-iaa patients ( table in the appendix). median duration of antibiotic treatment ( vs. . days), invasive mechanical ventilation ( vs. days), vasoactive support ( vs. days), hospital-los ( vs. days) and icu-los ( vs. days) were significantly extended in iaa compared with non-iaa patients (fig. ) . the length of stay in the icu was significantly elevated in iaa patients (fig. ) . treatment with antivirals (oseltamivir with or without zanamivir) was initiated in all patients with iaa and % of non-iaa patients (p = . ). mold-active antifungal treatment was initiated in patients ( patients each with iaa and non-iaa because of presumed iaa during hospitalisation). median duration of antifungal treatment was days (iqr . - . ) in iaa. antifungal treatment was stopped when diagnostics were negative in those without iaa. corticosteroids were used during hospitalisation in . % of patients ( % in iaa vs. . % in non-iaa, p = . ) and before influenza diagnosis in of of patients with iaa and in of of patients without iaa ( % vs. %, p = . ). all patients with iaa had poor outcome compared with % of non-iaa patients (p < . ). iaa patients had more bacterial or fungal (other than due to aspergillus spp.) respiratory superinfections ( % vs. %, p = . ). other infectious complications were catheter-related bloodstream infections and oropharyngeal and oesophageal candidiasis. aspergillus (n = ) was the most frequent respiratory co-pathogen in influenza patients. other common pathogens were s. aureus, s. pneumoniae, s. pyogenes and gramnegative bacteria (fig. ) . invasive mechanical ventilation, vasoactive support, ecmo, any complication, respiratory and any superinfection were significantly associated with mortality and poor prognosis ( table in the appendix). our study has several main findings. iaa is a severe and relatively frequent complication affecting % of patients with influenza treated in two swiss icus. aspergillus represented the most frequent respiratory co-infection of influenza in this cohort. patients with influenza were most commonly superinfected with aspergillus spp. despite lacking classical risk factors of invasive aspergillosis. in our study, all patients with iaa had poor outcome and needed more frequently and longer organ supportive therapies such as invasive mechanical ventilation, renal support and vasopressors. further risk factors for poor outcome were respiratory superinfections. iaa patients in our study did not have classical risk factors for aspergillosis such as underlying immunosuppressive disease, hematologic malignancies, solid organ or haematopoietic stem cell transplantation or immunosuppressive medications before hospitalisation. instead, severe influenza infection was frequently their unique risk factor. the occurrence of iaa independent of classical risk factors of invasive aspergillosis has been previously reported [ , , , ] . one study showed higher mortality in immunosuppressed patients with iaa in comparison to immunocompetent patients [ ] . reported risk factors for iaa are male sex and corticosteroid therapy prior to the influenza infection [ , ] . the latter might explain the relatively high number of patients with obstructive lung diseases [ ] . we identified the need for prolonged organ supportive therapies in the icu as a predictor for iaa. the length of icu was significantly elevated. because of small sample size, a competitive risk analysis was not performed. since iaa can occur in the absence of underlying diseases, requirement of vasoactive therapy, rapidly deteriorating respiratory failure and progressive multiple organ failure might be useful alerts for icu physicians of iaa in severely ill patients with influenza. surprisingly, in our cohort, aspergillus was identified as the most common respiratory superinfecting pathogen in influenza infection. aspergillus was seen in % of all respiratory copathogens in our analysis in contrast to . - . % of positive cultures from respiratory specimen reported in the literature [ , ] . bacterial pathogens are more frequently reported as influenza co-pathogens than aspergillus [ , ] . bacterial coinfections in influenza showed increasing trends over the last years (from . % in to % in ) while aspergillus isolation from culture was relatively stable at~ % in a study of spanish icus [ ] . bacterial respiratory superinfection was even more common in our analysis ( %). concurrently, s. aureus and s. pneumoniae were frequently observed in our analysis as were gram-negative bacteria including p. aeruginosa. respiratory co-infections were significantly associated with duration of antibiotic use, organ supportive theraphies and los in patients with iaa (light green) and without iaa (dark green). boxplots show th and th percentile with horizontal bar indicating median and whiskers the th and th percentile. outliers are shown with dots. *only vv-ecmo-treated patients were analysed for duration of ecmo. niaa = patients without influenza-associated invasive aspergillosis, iaa = patients with influenza-associated aspergillosis, ecmo = extracorporcal membrane oxygenation, los -lenght of stay, icu -intensive care unit, vent -ventilation kaplan-meier curve on the probability and lenght of icu stay in iaa (light green) and non-iaa patients (dark green). probability to stay on the icu is shown on x-axis, days after admission to icu are shown on y-axis. iaa = influenza-associated aspergillosis, niaa = patients with influenza without aspergillosis fig. kaplan meier curve on the probability of icu stay in iaa and non-iaa patients mortality in our univariate analysis. association of co-infection with s. aureus, p. aeruginosa or aspergillus spp. with mortality has been described [ ] . therefore, influenza patients at risk for respiratory superinfection need to be identified and early diagnostics and treatment need to be implemented. the increasing reports of iaa could be due to greater awareness and lower threshold for more and more sensitive diagnostics or indeed an increasing prevalence of this fungal disease. iaa was first reported in [ ] but only received attention of a wider audience in the last decade after the influenza h n pandemic [ , , , ]. an influencing factor could be the wide use of corticosteroids [ ] and neuraminidase inhibitors for influenza infections in the icu. in our analysis, many influenza patients received corticosteroids during hospitalization ( . %) with a non-significant difference between patients with or without iaa, despite data showing increased mortality of influenza pneumonia with corticosteroid use [ , ] . corticosteroid use has been associated with aspergillosis independently of influenza infection [ , , , ] and corticosteroid use prior to influenza infection has been associated with iaa [ , ] . furthermore, experimental in vitro studies and studies in mice indicate that neuraminidase inhibition decreases immune response by impaired cytokine production in response to aspergillus spp. this effect is also seen with corticosteroid and neuraminidase treatment [ ] . this implies that therapy of influenza with neuraminidase inhibitors and corticosteroids might increase susceptibility and predispose to mold infection in influenza. we hypothesize that a greater use of neuraminidase inhibitors since the influenza pandemic could have contributed to the emergence of iaa. alternatively, as recently reported in a retrospective cohort study from alberta, canada, from to , different influenza seasons may be associated with varying rates of iaa. in their study, schwartz et al. identified a considerably higher rate in the most recent / season [ ] . gm values in bal in our study were slightly lower than in previous reports ( - % [ , , ] or even % among patients with underlying respiratory diseases [ ] ), which may have been in part due to the possibility that patients with negative bal gm might have received voriconazole prior to diagnostic testing. interestingly, two patients, who met our criteria for iaa, were not or only briefly treated with antifungal therapy. both patients survived. we cannot definitely exclude colonisation with aspergillus because no histological examination was done. elevated gm, long duration of invasive ventilation ( and days) and icu los ( and days) argue in favour of iaa in these two patients. these two patients could have had a less severe form of iaa. importantly, of patients had cultural evidence of aspergillus spp., arguing against false-positive gm results. iaa is a relatively new field of research with few clinical studies and no prospective data. because iaa patients do not fulfil eortc criteria for invasive fungal disease [ ] new diagnostic criteria were proposed [ , , ] . these criteria do not allow for graduation of classification as with eortc criteria (possible, probable, confirmed ia), have not been evaluated prospectively and diagnosis of iaa remains difficult. furthermore, optimal treatment duration remains a matter of debate. iaa likely represents a heterogeneous entity and a benign course of disease could be possible although previous studies suggest otherwise with reported mortality of - %, and % in taiwan [ , , , , ] . mortality in our cohort was in concordance with previous reports in non-immunocompromised patients ( %) [ ] . our study has a few limitations. firstly, it is limited by the retrospective design. we cannot rule out that cases of aspergillosis have been missed but the rarity of positive gm tests or growth of aspergillus in cultures in the non-iaa group and the poor outcome in iaa which would lead to diagnostic work-up suggest otherwise. however, the true incidence of iaa might have been even higher. secondly, two patients without iaa did not fulfil mycological iaa criteria but received long antifungal therapy for presumed iaa during hospitalisation and had extended los on the icu. therefore, differences concerning outcomes might have been underestimated between groups. furthermore, the generalisability is limited by small sample size. however, respiratory co-pathogens in influenza. pathogens were isolated from bal or blood cultures. *other gram-negative pathogens include escherichia coli, morganella morganii, haemophilus influenzae and proteus mirabilis ( isolate each) fig. number of isolated pathogens in patients with influenza patients' characteristics and mortality are concordant with previous studies on iaa. finally, owing to the retrospective design, it was not always possible to distinguish whether a complication was predisposing to iaa or a complication of iaa. of note, both hospitals are the only major tertiary care centres in their area. since all patients who fulfilled the listed criteria were included in the study, we consider recruitment bias unlikely. in conclusion, iaa represents an underappreciated complication of influenza infection with severe morbidity and mortality in swiss icus. only out of swiss icus participated in the study because screening and diagnosis of iaa were rarely done which is evidence for the poor awareness of this disease. while bacterial superinfection was frequent in influenza patients in the icu, aspergillus was even more common. the need for organ support therapy might serve as a predictor of iaa. because of frequency and severity of disease, greater awareness of iaa is needed and lower thresholds for diagnostic testing (gm, bacterial and fungal cultures from bal) and treatment should be implemented in the icu, especially in patients requiring organ support therapies. a multicentre study including all university hospitals in switzerland and two tertiary care centres is on its way to confirm our study outcome and raise awareness of this severe entity. prospective studies are urgently needed to evaluate proposed diagnostic criteria, characterize clinical outcomes, identify patients at risk of iaa and define optimal antiviral and antifungal treatment and duration. authors' contributions w.f. and a. w.c. had the idea and initiated the study and wrote the protocol. w. f., b. f., s. n., w. g. p. d., a. w. c., m. m. and s. j. managed the study and collected the data. w. f., k. g. r. and a. w. c. were responsible for and performed the statistical analysis. w. f., b. f., i. a., b. k., k. g. r., a. w. c., k. l. and j. p. interpreted the data. w. f., k. g. r. and a. w. c. drafted the manuscript. all authors amended and commented on the final manuscript. conflict of interest the authors declare that they have no conflict of interest. y years, iqr interquartile range, copd chronic obstructive pulmonary disease, iaa influenza-associated aspergillosis, non-iaa influenza infection without aspergillosis, gm galactomannan, bal bronchoalveolar lavage, ta tracheal aspirate saps ii simplified acute physiology score, estimates mortality in icu patients [ ] . neutropenia is neutrophil count ≤ . g/l and lymphopenia is lymphocyte count ≤ g/l *any immunosuppressive condition included solid organ or haematologic stem cell transplantation, haematologic malignancy or any immunosuppressive drugs including corticosteroids before diagnosis of influenza **total of patients with gm measured ( cases had gm measured in bal (cut-off, optical density (od) ≥ . ) and serum (cut-off, od ≥ . 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epidemiology, diagnosis and treatment. curr o p i n i n f e c t d i s the acute respiratory distress syndrome invasive pulmonary aspergillosis is a frequent complication of critically ill h n patients: a retrospective study do corticosteroids reduce the mortality of influenza a (h n ) infection? a meta-analysis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -nnxa ant authors: guedez-lópez, gladys virginia; alguacil-guillén, marina; gonzález-donapetry, patricia; bloise, ivan; tornero-marin, carolina; gonzález-garcía, juan; mingorance, jesus; garcía-rodríguez, julio title: evaluation of three immunochromatographic tests for rapid detection of antibodies against sars-cov- date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: nnxa ant lateral flow immunoassays (lfia) for rapid detection of specific antibodies (igm and igg) against sars-cov- in different human specimens have been developed in response to the pandemic. the aim of this study is to evaluate three immunocromathographic assays (sienna®, wondfo® and prometheus®) for detection of antibodies against sars-cov- in serum samples, considering rt-qpcr as a reference. a total of serum samples from patients with clinical suspicion of covid- were collected: all of the samples were tested with sienna®, with wondfo® and with prometheus®. the overall results of sensitivity, specificity, positive predictive value and negative predictive value obtained were as follows: . %, %, . % and . % with sienna®; . %, . %, . % and . % with wondfo® and . %, . %, . % and . % with prometheus®. the accuracy of the test for sienna®, wondfo® and prometheus® was . %, % and . %, with a prevalence of covid- of . %, . % and . % respectively. sensitivity of the three tests (sienna®, wondfo® and prometheus® respectively) along the three different stages was . %, . % and . % in the early stage (first week); . %, . % and . % in the intermediate stage (second week) and %, . % and % in the late stage (third week). the results demonstrate that even though prometheus® presented a high sensitivity, the specificity was notably lower than the other two tests. sienna® showed the greatest contrast between sensitivity and specificity, achieving the best accuracy, followed by wondfo®. the sensitivity of the three ict assays was higher in late stages of the disease. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. in late december , a novel coronavirus was identified as the etiological agent of anew pneumonia [ , ] . the etiological agent, named as sars-cov- , rapidly spread to other cities in china and to other countries worldwide and on march world health organization (who) declared the outbreak as a pandemic. this situation has forced many countries to adopt firm measures in order to promote early detection of covid- and early isolation of the cases, track contacts and encourage distancing measures. real-time reverse transcription quantitative polymerase chain reaction (rt-qpcr) has been established as the gold standard for microbiological diagnostic of sars-cov- infection, targeting at least two different regions of sars-cov- genome in order to avoid cross-reactivity with other coronavirus and the potential genetic drift of sars-cov- [ ] [ ] [ ] [ ] . gladys virginia guedez-lópez and marina alguacil-guillén contributed equally to this work. electronic supplementary material the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. rt-qpcr tests presented a high specificity with a low probability of false positive; however, sensitivity relies on different factors as specimen site, method of collection, viral load and time from the onset of symptoms [ , ] . an increasing number of cases with negative rt-qpcr and clinical features consistent with covid- pneumonia has been reported [ ] [ ] [ ] .therefore, supplementary diagnostic approaches are needed to reduce the number of false-negative cases, which is essential for the epidemiologic control of the disease [ ] . several studies focused on antibody response against sars-cov- suggested that igm can be detected during the first week since the onset of the symptoms, although the igm detection rate is highly variable at this early stage [ ] [ ] [ ] . igg can be detectable after days since the onset of the symptoms, and after days, over % of cases present antibodies against sars-cov- [ ] [ ] [ ] . however, the strength of antibody response depends on several factors (nutritional state, severity of disease, immune status...) and it has been observed that some patients do not produce detectable levels of antibodies [ , , ] . therefore, serological methods had limited utility for early diagnosis of covid- , since the sensitivity of the assay is low during the first days and increases with time. however, serologic tests could play an important role in confirmation and late diagnostic of covid- , mainly in patients with repetitive negative rt-qpcr, as well as giving information about the immune status of asymptomatic patients and contributing to the determination of the prevalence and mortality rate [ ] . combination of rt-qpcr and igm/igg detection methods could provide a suitable approach to covid- diagnosis [ ] . several studies suggested that acquired immunity is protective against sars-cov re-infection [ , ] , though some reports described cases of post-recovery positive nasopharyngeal rt-qpcr [ ] [ ] [ ] . currently, is being discussed if the recurrence of positive sars-cov- rt-qpcr in recovered patients with subsequent negative rt-qpcr is due to prolonged viral shedding with false negative results of rt-qpcr, or a possible re-infection [ , , ] . in this context, the use of serologic test for the follow-up of the immune response in those cases may be useful to a better understanding of the acquired immunity and the possibility of re-infection. lateral flow immunoassays (lfia) for rapid detection of specific antibodies (igm and igg) against sars-cov- in different human specimens (whole blood, serum and plasma) have been developed in response to the pandemic [ , ] . this technique is quick and simple to perform and does not require special equipment. in addition, some studies have shown good concordance between the results obtained using whole blood samples (fingerstick blood) and serum or plasma samples so can be used as point-of-care (poc) immunodiagnostic tests, which makes it suitable for community surveillance [ , ] . the use of these poc immunodiagnostic tests for massive detection of antibody response to sars-cov- in the population would be an important step towards a better understanding of the outbreak's situation, which would help us to establish the right policies in the control of the covid- pandemic [ ]. however, the utility of these lfia assays is questionable due to the lack of official performance validation to evaluate the sensitivity and specificity of the tests, which are highly variable between the different commercially available serologic tests [ , ] .therefore, further studies are needed to assess the sensitivity and specificity of the serologic test launched to the international market. the aim of this study is to evaluate three immunocromathographic assays for igm and igg detection of covid- in serum samples. three different immunochromathographic (ict) assays for qualitative detection of igg and igm antibodies against in summary, for the three of them, μl of serum and drops of buffer solution were added to the corresponding loading area in the cassettes. after a short time (no longer than min), the interpretation of the results was done by two observers, based on the appearance of a coloured band. according to manufacturer's instructions, the appearance of a blurred band was considered as a positive result (weak positives). sienna® and prometheus® kits show igm and igg bands separately, while wondfo® detected total antibodies in one band. nasopharyngeal swabs were collected, and rna was extracted using an automated system and analysed using selected rt-qpcr commercial kits routinely used for diagnosis of covid- , which are addressed to detect the common targets of sars-cov- : nucleocapside (n), spike (s), envelope (e), orf ab and rdrp genes. rt-qpcr has been considered the reference for the evaluation of the ict strip assays. serum samples were collected between the th of march and the nd of april at hospital universitario la paz (madrid, spain), and were centrifuged at rpm during min. the evaluation was performed in two groups of patients. the first group was integrated by healthcare workers at hospital universitario la paz, who attended the occupational health consultation for the first time between the th march and the nd of april referring symptoms compatible with covid- . the second group included patients randomly selected who were admitted to the emergency department of the hospital with positive rt-qpcr or high clinical suspicion of covid- . in the first group of patients, serum samples and nasopharyngeal swabs were collected at the same time in patients, while in the other patients, the average time since the nasopharyngeal swab collection and the serum extraction was . days. regarding the in addition, extra serum samples of randomly selected patients from were tested as negative control with prometheus® and sienna® test. unfortunately, it was not possible to carry out this evaluation with wondfo® test due to lack of reagents. the statistical analysis was performed overall and divided in three different stages according to the time since the symptoms onset: early stage (first week), intermediate stage (second week) and late stage (third week). the statistical test parameters were calculated according to their definitions, with additional plots generated using graphpad prism (version ). the results from the negative controls were excluded from the overall statistical analysis. a total of serum samples from patients were collected: samples, which belonged to the group of healthcare workers, were tested with the three ict assays, samples ( from the first and from the second group of patients) were tested with sienna® and wondfo® and the other samples from the second group of patients were tested only with sienna. therefore, samples were tested with sienna®, with wondfo® and with prometheus®. demographic and clinical characteristics of the patients included in the study are collected in table . the overall results of sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) obtained were as follows: . %, %, . % and . % with sienna®; . %, . %, . % and . % with wondfo® and . %, . %, . % and . % with prometheus®. the accuracy of the test was . % with sienna®, % with wondfo® and . % with prometheus®, with a prevalence of covid- of . %, . % and . % respectively ( table ). the results of prevalence, accuracy, sensitivity, specificity, ppv and npv of prometheus® and sienna® for igg and igm detection separately are also available in table . detection rate of igm, igg and igm/igg antibodies against sars-cov- with the three ict strip assays in positive and negative rt-pcr patients along three periods of time since the onset of symptoms is shown in table . when the test was performed in patients with less than week since the symptoms onset, the results obtained with sienna® (n = ), wondfo® (n = ) and prometheus (n = ) respectively were as follows: sensitivity . %, in patients with more than days since the symptoms onset (n = ), sensitivity and npv reached %, with sienna® (n = ) and prometheus® (n = ), and . % and . % with wondfo® (n = ). specificity and ppv were . % and % with sienna®, . % and . % with wondfo® and % and % with prometheus®. (table ) . antibody detection rate based on the number of days after onset of symptoms of the three ict assays is determined and summarized in figs. , , and . detection rates of total antibodies (igm/igg) obtained with sienna® and wondfo® by the two groups of patients along the three stages since the symptoms onset are collected in table . the calculation of the different test parameters among the three stages was performed excluding three patients because the time since the onset of symptoms was unknown. the three of them were included in the global evaluation of sienna® and wondfo® and one was included in the evaluation of prometheus®. the serum samples from included as negative control of sienna® and prometheus® resulted in one positive sample with an igm band using sienna® while seven were positive ( igg and igm, igg and igm) with prometheus®. diagnosis of sars-cov- remains challenging in most fields. first, covid- patients present a range of unspecific clinical symptoms that are similar to those produced by common respiratory tract pathogens. second, laboratory diagnosis is currently based on rt-qpcr detection of viral rna from nasopharyngeal samples, which has limitations of sensitivity, especially in later stages of the disease. therefore, it is essential to optimize laboratory diagnosis, including serological tests as a complementary technique of rt-qpcr for sars-cov- diagnosis. in this study, we have investigated the diagnostic value of detection of sars-cov- igm and igg antibodies in different stages of the disease, using three ict strip assays, in comparison with rt-qpcr. it has been described that during the course of the disease, the sensitivity of the rt-qpcr technique tends to decrease in nasopharyngeal samples, due to a reduction of viral load in this location, while antibodies production raise and serological techniques show a remarkable increase of the sensibility [ ] . we found that the most sensitive test was prometheus®, whose sensitivity was significantly higher during the second and third week of the disease. sienna® and wondfo® presented a slightly lower sensitivity which also increased following the same pattern through the evolution of the disease. during the early stages of the disease (first week), the antibody detection rate with the three lfia test was low, as it has been observed in different published studies [ , , , ] . with prometheus® and sienna®, igm positive rate was higher than igg throughout the weeks. however, we have observed that igm presented a higher number of false positives, especially in the case of prometheus test. the most specific test was wondfo®, followed by sienna® and last prometheus®. both last showed higher specificity in igg detection. considering the high rate of false positives when using prometheus® test, an extra serum samples of randomly selected patients from were studied as negative controls and the results confirmed a high percentage of false positive with this assay ( %). the same was performed using sienna®, obtaining a much lower rate of false positives ( %). unfortunately, it was not possible to carry out this evaluation with wondfo® test due to lack of reagents. this indicated the need of further studies to evaluate the specificity and cross-reactions of serological test using negative controls. these results demonstrate that even though prometheus® presented a high sensitivity, the specificity was notably lower than the other two tests. sienna® showed the greatest contrast between sensitivity and specificity, achieving the best accuracy, followed by wondfo®. however, there are several limitations in the present study. the current pandemic situation has led to an interrupted availability of the tests, so it has not been possible to carry out the evaluation uniformly with the three kits in the same number of samples. moreover, the differences between the two groups of patients included in the study may be a potential bias. on one side, in the group of healthcare workers, probably more aware of disease symptoms than the general population, the time elapsed since the onset of symptoms until the attendance to the occupational consultation was considerably lower than in those patients who attended the emergency department, which were in more advanced stages of the disease with higher rates of antibodies production. furthermore, in most healthcare workers, nasopharyngeal swabs and serum samples were collected at the same time and the time between the onset of symptoms and serum sample collection was lower; hence, the sensitivity of serologic tests is reduced. on the other side, patients included in the second group, who were admitted to the emergency department, presented a previous positive rt-qpcr or a high clinical suspicion of covid- , and consequently the pre-test probability of antibodies production in this group of patients was higher. finally, the use of rt-qpcr as reference technique for the evaluation of serological test should be taken with care, since both techniques have different time windows for positivity during the course of the infection and rt-qpcr presented a substantial rate of false negatives. in contrast, the low rate of false positives using rt-qpcr enables a consisting evaluation of the sensitivity of serological tests in covid- patients with positive rt-qpcr. our study highlights the importance of performing the serological tests when the time since the onset of symptoms is larger than week, since the sensitivity of the three ict assays was notably higher during the intermediate and late stages of the disease. a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding detection of novel coronavirus ( -ncov) by real-time rt-pcr laboratory diagnosis of emerging human coronavirus infections -the state of the art the laboratory diagnosis of covid- infection: current issues and challenges world health organization.: world health organization ( ) laboratory testing for coronavirus disease ( covid- ) in suspected human cases: : interim guidance estimating false-negative detection rate of sars-cov- by rt-pcr correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases radiology, ) chest ct for typical -ncov pneumonia: relationship to negative rt-pcr testing one nosocomial cluster following with a familial cluster of covid- cases : the potential transmission risk in patients with negative swab tests antibody responses to sars-cov- in patients of novel coronavirus disease antibody responses to sars-cov- in covid- patients: the perspective application of serological tests in clinical practice kadkhoda: the role of antobody testing for sars-cov- : is there one? evaluations of serological test in the diagnosis of novel coronavirus (sars-cov- ) infections during the covid- outbreak molecular and serological investigation of -ncov infected patients: implication of multiple shedding routes development and clinical application of a rapid igm-igg combined antibody test for sars-cov- infection diagnosis lack of reinfection in rhesus macaques infected with sars-cov- reinfection could not occur in sars-cov- infected rhesus macaques new igm seroconversion and positive rt-pcr test after exposure to the virus in recovered covid- patient recurrence of positive sars -cov - in patients recovered from covid - positive rt-pcr test results in patients recovered from covid- stability issues of rt -pcr testing of sars -cov - for hospitalized patients clinically diagnosed with covid - false-negative of rt-pcr and prolonged nucleic acid conversion in covid- : rather than recurrence ) s e r o l o g i c a l immunochromatographic approach in diagnosis with sars-cov- infected covid- patients. medrxiv . ( ) advice on the use of point-of-care immunodiagnostic tests for covid- serology assays to manage covid- different longitudinal patterns of nucleic acid and serology testing results based on disease severity of covid- patients diagnostic value and dynamic variance of serum antibody in coronavirus disease publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -j wrtsxj authors: wagenvoort, j. h. t.; penders, r. j. r.; davies, b. i.; lütticken, r. title: similar environmental survival patterns of streptococcus pyogenes strains of different epidemiologic backgrounds and clinical severity date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: j wrtsxj nan group b included strains from less serious non-invasive soft tissue or wound infections, with subgroup (strains and ) being nosocomial and subgroup (strains and ) non-nosocomial. s. pyogenes strains , and were isolated from different patients during a hospital outbreak reported previously by davies et al. [ ] . in table of that report the respective patients were assigned the codes g, p and m . the influence of desiccation on the survival of the different s. pyogenes strains was evaluated and compared as described previously in detail for mrsa [ ] . suspensions containing approximately cfu/ml were prepared in sterile phosphate-buffered saline (pbs; ph . ). samples ( ml) of each suspension were transferred to -ml flat-bottomed glass bottles and allowed to dry. all bottles were plugged with cotton wool to allow free communication with the hospital environment through indirect northern light, ambient temperature and relative humidity. the fluid component of the suspensions had completely evaporated after days, and sampling was begun days later. remaining viable bacteria were recovered by adding ml of pbs to the bottle. after vigorous vortexing in the closed bottle, the suspension was flooded onto a blood agar plate and incubated for h at • c. for all strains, remaining colony forming units were measured at - -day intervals until extinction. the average relative humidity of the ambient air and temperature during the study period were % and • c, respectively. the survival rates of the different groups of s. pyogenes strains are shown in table . it can be seen that from an initial measurement of approximately cfu the strains died off rapidly, with the decline ranging from to -log cfu during the -day dry-out period to counts between and , cfu. after day , only more weeks passed until the last viable s. pyogenes strain was extinct. a gradual die-off pattern was noted for all strains within a range of up to circa -log cfu at the same measurement points. the last day on which a viable count was measured for each strain was between day and day . the nosocomial outbreak strains of subgroups and did not survive any longer than the non-outbreak strains in subgroups there was also no difference in the survival patterns exhibited by the virulent (group a) strains causing serious invasive infections (subgroups and ) and those of the less serious non-invasive (group b) strains (subgroups and ). in our approach the outcome was simple: no s. pyogenes isolate survived on glass for longer than month. the rapid decline of all s. pyogenes strains tested-even our own outbreak strain that had demonstrated mrsalike spread [ ] -contrasts sharply with the prolonged survival of around a year reported previously for epidemic mrsa strains [ ] . we did not find any survival characteristics that could clearly be correlated with a specific outbreak character. s. pyogenes strains thus seem to be disseminated in a fashion similar to s. aureus, with airborne spread playing a predominant role, supported by (intermediate) carriers via dispersal on skin scales from a carriage site or via direct transmission from hands or inanimate objects. environmental contamination was noted particularly in the outbreak related to strain no. , and mrsa-like spread was noted in the outbreak related to strain no. . the severity of disease caused by the various infecting strains did not correlate with any alternative or specific survival pattern. the potential danger of a contaminated environment has been recognized in earlier outbreaks [ , ] , and control measures aimed at removing dust and disinfecting surfaces were consequently implemented at our hospital during the outbreaks. although the -week survival period found for our s. pyogenes strains in the hospital environment is shorter than the period of months reported by lidwell and lowbury [ ] , it should be noted that their study measured survival in dust. since the influence of various dust mixtures can be surprisingly variable [ ] , we chose not to include dust samples in our investigational approach. our finding that s. pyogenes strains survive in the inanimate environment for up to month shows that contact transmission is facilitated in the short-term phase of an outbreak; however, long-term environmental survival cannot be considered an important factor in the dynamics of s. pyogenes transmission. the remarkable paucity of reports on the environmental survival of s. pyogenes strains could be related to the increasing interest in the behavior of other bacteria in the hospital environment, such as multiresistant pathogens, like mrsa [ , ] , vancomycin-resistant enterococci, clostridium difficile or acinetobacter baumannii [ ] , and the coronavirus causing severe acute respiratory syndrome. investigation of the last syndrome has identified the survival of the pathogen in fomites as a factor possibly related to transmission [ ] ; thus, multiple pathways must be considered for transmission of all pathogens, including s. pyogenes. an outbreak of streptococcal skin sepsis in a closed community group a streptococci in the s invasive group a streptococcus infection a streptococcus pyogenes outbreak caused by an unusual serotype of low virulence: the value of typing techniques in outbreak investigations a large outbreak of streptococcal pyoderma in a military training establishment dutch measures to control mrsa and the expanding european union better environmental survival of outbreak vs. sporadic mrsa isolates the survival of bacteria in dust the role of the hospital environment in the epidemiology of multi-resistant bacteria listening to sars: lessons for infection control key: cord- -q i fqu authors: dalhoff, a. title: antiviral, antifungal, and antiparasitic activities of fluoroquinolones optimized for treatment of bacterial infections: a puzzling paradox or a logical consequence of their mode of action? date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: q i fqu this review summarizes evidence that commercially available fluoroquinolones used for the treatment of bacterial infections are active against other non-bacterial infectious agents as well. any of these fluoroquinolones exerts, in parallel to its antibacterial action, antiviral, antifungal, and antiparasitic actions at clinically achievable concentrations. this broad range of anti-infective activities is due to one common mode of action, i.e., the inhibition of type ii topoisomerases or inhibition of viral helicases, thus maintaining the selective toxicity of fluoroquinolones inhibiting microbial topoisomerases at low concentrations but mammalian topoisomerases at much higher concentrations. evidence suggests that standard doses of the fluoroquinolones studied are clinically effective against viral and parasitic infections, whereas higher doses administered topically were active against candida spp. causing ophthalmological infections. well-designed clinical studies should be performed to substantiate these findings. the history of quinolones began in with the isolation of a byproduct of chloroquine synthesis by george yohe lesher and colleagues [ ] at the sterling-winthrop research institute in rensselaer, new york; this compound was found to be antibacterially active and was subsequently modified to yield nalidixic acid. nalidixic acid and chloroquine share structural features being essential for their antibacterial and antiparasitic activity, respectively. apart from its well-known antimalarial effects [ ] [ ] [ ] , chloroquine exerts direct antiviral [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , antifungal [ ] [ ] [ ] [ ] , and antibacterial effects [ , [ ] [ ] [ ] [ ] . furthermore, chloroquine exhibits immunomodulatory activity [ ] [ ] [ ] [ ] [ ] and was found to reverse p-glycoprotein (p-gp)-mediated multidrug resistance, thereby increasing the cytotoxicity of some antineoplastic agents [ ] [ ] [ ] [ ] [ ] . the antimalarial effects of chloroquine are due to its accumulation in acidic food vacuoles of intraerythrocytic trophozoites, thereby preventing hemoglobin degradation and inhibition of a haem polymerase enzyme [ , ] . the antiviral, antifungal, and antibacterial activities of chloroquine are ph-dependent [ , , , ]. this phenomenon is due to the fact that chloroquine is a weak base and, therefore, does not enter the cell if the extracellular fluid or the incubation medium is acidic. once chloroquine has entered cells, it intercalates into dna and prevents the introduction of topoisomerase ii-mediated dna breaks. the intercalation of chloroquine into dna protects cells against epipodophyllotoxins such as etoposide, acting as topoisomerase ii poison by hindering the dna cleavage reaction of this target enzyme [ , ] . the use of chloroquine in the treatment of some autoimmune diseases and its antiinflammatory properties may be due to the inhibition of mhc class ii antigen presentation; the inhibition of t-cell response may be due to a direct interaction of chloroquine with the cell membrane [ ] . furthermore, chloroquine was found to destabilize indirectly lysosomal and plasma membranes as a result of accumulation within the lysosome, followed by an increase in lysosomal volume; it also sequesters important cell membrane constituents in lysosomes [ ] . chloroquine was found to adsorb to the plasma membrane of yeasts, inhibit competitively the binding of immunoglobulin g to the cell surface, altered phospholipid turnover, and influenced directly but non-specifically the membrane integrity and permeability of renal brush border vesicles, mast cell membranes, and fibroblasts [ , [ ] [ ] [ ] . furthermore, chloroquine blocks the inward rectifier potassium channel kir . ; it is bound at the center of the cytoplasmic domain of the channel [ , ] . these data demonstrate that the congener of fluoroquinolones, i.e., chloroquine, exhibits, apart from its antimalarial activity, pleiotropic actions and interacts with multiple targets. as chloroquine and nalidixic acid share structural features being essential for their activity, it was not surprising that it has been recognized in the late s that nalidixic acid and oxolinic acid derivatives exert trypanocidal and antitumor activities [ ] ; in the early s, it was described that fluoroquinolones used for the treatment of bacterial infections exert not only an antibacterial but also an antiprotozoal activity [ ] and may find applications as antiparasitic, antifungal, or antiviral agents [ ] . furthermore, and in analogy to chloroquine, the activity of antibacterially active fluoroquinolones is ph-dependent [ ] , and they bind directly to bacterial dna, i.e., two molecules intercalate at the highly bent dna gate in the dna cleavage domain [ ] [ ] [ ] [ ] [ ] . despite these phenotypic and molecular homologies between chloroquine and fluoroquinolones, the pharmaceuticals industry invested financial and human resources into focused research programs on the application of developmental fluoroquinolones as antibacterials only and into pre-and postmarketing studies supporting the use of fluoroquinolones in the oncegranted indications. studies on the function of an antibacterial agent exerting pleiotropic anti-infective actions have never been performed systematically. surprisingly, the use of fluoroquinolones in indications other than bacterial infections has never been exploited, although not only nalidixic acid and its congener chloroquine exerts pleiotropic actions but, e.g., β-lactams and aminoglycosides are characterized by a broad range of biological activities too [ , ] , so that a multitude of antimicrobial effects would not have been unusual. this review summarizes the pleiotropic phenotypes of nonantibacterial actions of fluoroquinolones and addresses the question if the diversity of effects are due to one common mode of action of antibacterially active fluoroquinolones, i.e., inhibition of essential bacterial type ii topoisomerases, or if other mechanisms may mediate non-antibacterial activities. although the complexity and diversity of prokaryotic and eukaryotic topoisomerases is remarkable and little or no sequence homology of amino acids exists, type i and type ii topoisomerases share certain structural elements mediating identical functions like dna relaxation or dna transport in bacteria, dna viruses, yeasts, and parasites; the dna helicase coordinates the directionality of topoisomerase activity; rna helicases as present, e.g., in hepatitis c virus (hcv) directly interact with double-stranded dna or rna and assembles complexes with type ii topoisomerases [ ] [ ] [ ] [ ] [ ] . as dna topoisomerases are ubiquitous enzymes controlling dna topology, it is conceivable that antibacterially active quinolones may not only inhibit the growth of bacteria at clinically relevant concentrations, but that of other prokaryotic and even eukaryotic organisms as well. ciprofloxacin, ofloxacin, levofloxacin, and gatifloxacin were found to be clinically effective in the treatment of the singlestranded rna hcv and the non-enveloped, encapsulated dna polyomavirus bk [ ] [ ] [ ] [ ] [ ] [ ] [ ] . five and four patients with hcv-induced chronic hepatitis and compensated liver cirrhosis, respectively, were treated with to mg ofloxacin per day for one to eight weeks. in three patients with chronic hepatitis and one patient with compensated liver cirrhosis, hcv rna decreased at least by log titer [ ] . in another study, five patients with chronic hcv were treated with mg ciprofloxacin twice daily (b.i.d.) for days. serum hcv rna levels remained largely unchanged in these patients [ ] . the latter study indicates that the anti-hcvefficacy of quinolones may be limited in patients with advanced liver cirrhosis. ciprofloxacin decreased bk peak viral load after hematopoietic stem cell transplantation [ ] . a reduction of viremia was demonstrated two months after a -day course of gatifloxacin at mg/d in of transplant recipients with active bk virus replication [ ] . a retrospective analysis revealed that the use of either ciprofloxacin mg b.i.d. or levofloxacin mg once daily (q.d.) within the first month post transplantation and up to months after transplantation was associated with significantly lower one-year rates of bk viremia [ ] . a recent study in nine kidney transplant recipients with persistent bk infection revealed that, three months post ciprofloxacin treatment with mg b.i.d for days, the virus load was cleared completely in three patients and decreased by > % in another three patients [ ] ; patients were not treated with anti-infectives other than fluoroquinolones. fluoroquinolones inhibit bk viral replication in vitro. ofloxacin and levofloxacin inhibited polyomavirus bk replication in primary human kidney cells in a dose-dependent manner, yielding a~ % inhibition at μg/ml. bk virus genome replication was reduced by % at h post infection of the kidney cells. at h after inoculation of the kidney cells, the reduction in genome replication and protein expression was less pronounced. a dose-dependent cytostatic effect was noted. in infected cells, mg/l ofloxacin led to a % and % inhibition of cellular dna replication and total metabolic activity, respectively, while mg/l levofloxacin exhibited a slightly more marked cytostatic effect, particularly in uninfected cells [ ] . ciprofloxacin, moxifloxacin, levofloxacin, ofloxacin, gatifloxacin, and norfloxacin inhibited bk virus replication to % at concentrations ranging from . to . mg/l [ ] . ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin, and trovafloxacin inhibited viral replication of simian virus (sv ), another member of the polyomaviridae, in permissive monkey cells, as well as plaque formation, dna replication, and helicase activity. ciprofloxacin, levofloxacin, and ofloxacin inhibited "significantly" helicase activity at . , . , and . mm, whereas trovafloxacin inhibited helicase activity at μm [ , ] . recently, it was demonstrated that norfloxacin, ofloxacin, flumequine, enrofloxacin, cinoxacin, enoxacin, fleroxacin, lomefloxacin, balofloxacin, and difloxacin inhibited hcv replication, in particular, hepatoma huh- and huh- cell lines, and hcv ns helicase activity. the concentrations inhibiting hcv rna replication to % ranged from . to . μm and those inhibiting helicase activity ranged from . to . μm [ ] . the clinical studies reviewed above and one recent report of a successful treatment of a kidney retransplant patient with ciprofloxacin ( mg b.i.d. for days) who needed an overall increase of immunosuppression due to acute rejection [ ] suggest that fluoroquinolone treatment of polyomavirus bk infections in transplant patients may be beneficial. therefore, a study protocol for a randomized controlled clinical trial evaluating the prophylactic efficacy of fluoroquinolones has been designed and is registered at clinicaltrials.gov under nct ; levofloxacin at a dose of mg q.d. will be administered for months and will be compared to placebo [ ] . another clinical study on the use of ciprofloxacin ( mg q.d. for months as compared to placebo) for the prevention of bk infections is registered under nct [ ] . furthermore, it was demonstrated that ofloxacin [ ] and levofloxacin [ ] inhibited viral topoisomerase activity of vaccinia virus but not of herpes simplex virus and influenza virus [ ] . in agreement with this finding, it was reported that mg/l each of ciprofloxacin, lomefloxacin, ofloxacin, pefloxacin, and rufloxacin inhibited to % the cytopathic effect of herpes simplex virus type at concentrations being equivalent to the cytotoxic effect of the quinolones on the vero cells [ ] . fluoroquinolones inhibit not only enzymic activity of viral topoisomerases/helicases, but inhibit in vitro human immunodeficiency virus (hiv) reverse transcriptase as well; complete inhibition was observed at concentrations of ciprofloxacin and ofloxacin of μm and norfloxacin of μm, respectively [ ] [ ] [ ] . inhibition of rhinovirus (rv) infection by quinolones is due to the inhibition of cell functions required for viral replication. levofloxacin pretreatment of not yet infected human tracheal epithelial cells reduced the mrna level of intercellular adhesion molecule (icam- ), a receptor for rv, in the cells and the concentration of the soluble form of icam- in the supernatant, so that rv infection of the tracheal epithelial cells was significantly reduced. levofloxacin pretreatment also decreased the number of the acidic endosomes from which rv rna enters the cytoplasm. furthermore, levofloxacin pretreatment inhibited the activation of nuclear factor κb proteins. these data suggest that levofloxacin inhibits rv infections first by reducing icam- expression levels and the number of acidic endosomes, and second by modulating airway inflammation [ ] . fluoroquinolones other than levofloxacin have not been studied in this context. moxifloxacin and gatifloxacin inhibited, at a concentration of . % used for topical application in ophthalmology, candida spp. to > % [ ] . gatifloxacin and sparfloxacin showed activity in a qualitative paper disk diffusion test against trichophyton rubrum, fusarium solani, and candida albicans, but not against saccharomyces cerevisiae [ ] . ciprofloxacin, moxifloxacin, levofloxacin, trovafloxacin, and sitafloxacin enhanced the activities of antifungal agents against candida albicans and aspergillus fumigatus [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, ciprofloxacin showed synergism with azoles against histoplasma capsulatum and coccidioides posadasii [ ] , as well as in combination with amphotericin b against exophiala spinifera [ ] . several but still rare reports of clinical and microbiological cure of fungal keratitis by quinolones have been published; recently, five additional cases of fungal keratitis treated successfully with topical moxifloxacin monotherapy were published [ ] . the causative organisms curvularia spp., candida parapsilosis, paecilomyces lilacinum, and aspergillus fumigatus were treated with moxifloxacin . %, one drop every half-hour to every hour. all these cases of fungal keratitis were cured with topical moxifloxacin and the pathogens were eliminated [ ] . these data demonstrate that topical administration of quinolones, thus generating high target site concentrations, are clinically effective in the treatment of fungal ophthalmological infections. topoisomerase ii has been identified as the primary target for quinolones in yeast [ , ] , so that the antifungal activities of the fluoroquinolones tested are likely to be mediated by this enzyme. the dna topoisomerase ii isolated from candida albicans was more susceptible to quinolones than the calf thymus dna topoisomerase ii, despite the fact that both enzymes are of eukaryotic origin [ ] . yeast dna topoisomerase ii selected for resistance to quinolones are characterized by amino acid mutations which are homologous to mutations in gyra of escherichia coli [ ] [ ] [ ] . these differences between yeast and mammalian type ii topoisomerases may explain why fluoroquinolones exhibit an antifungal activity by maintaining in parallel a selective toxicity against prokaryotic topoisomerases. although antibacterially active fluoroquinolones were derived from the antimalaria agent chloroquine, the clinical efficacy of norfloxacin against plasmodium falciparum was discovered by chance when the agent was used for the treatment of typhoid fever in indian patients. norfloxacin was administered to nine hospitalized malaria patients orally with mg norfloxacin b.i.d. for three days; treatment led to disappearance of splenomegaly [ ] . later, another patients with uncomplicated malaria were treated with norfloxacin (ten with mg b.i.d. and five with mg b.i.d.) for three days [ ] . this study confirmed that norfloxacin is clinically effective in the treatment of falciparum malaria, but the efficacy of the lower dose was suboptimal. later, it was demonstrated that norfloxacin is inferior to chloroquine for falciparum malaria. a prospective, randomized trial revealed that the mean parasite clearance time as well as the mean defervescence time were shorter in the chloroquine group [ ] . fluoroquinolones like ciprofloxacin, amifloxacin, enoxacin, norfloxacin, ofloxacin, pefloxacin, grepafloxacin, trovafloxacin, and additional commercially available quinolones exhibit marked in vitro activity and in vivo efficacy against plasmodium spp. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nalidixic acid and several fluoroquinolones like ciprofloxacin, norfloxacin, enoxacin, ofloxacin, fleroxacin, clinafloxacin, pefloxacin, and sparfloxacin exerted an antitrypanosomal in vitro and in vivo effect at micromolar concentrations [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition, nalidixic acid, norfloxacin, ofloxacin, moxifloxacin, gatifloxacin, lomefloxacin, and some more fluoroquinolones inhibited growth of the microsporidia encephalitozoon intestinalis and vittaforma corneae to % at concentrations ranging from . to . μm [ ] . furthermore, ciprofloxacin caused a % growth inhibition of babesia microti, b. bigemina, b. caballi, b. equi, and b. bovis at concentrations of . to . μm [ ] . fluoroquinolones exerted antitoxoplasma activities as well. moxifloxacin, gatifloxacin, trovafloxacin, and grepafloxacin were the most active agents, inhibiting growth of t. gondii to % at concentrations ranging from . to . mg/l, while ciprofloxacin was poorly active, with a % inhibitory concentration value of . mg/l [ ] . the parasites of the phylum apicomplexa, i.e., plasmodium spp., toxoplasma spp., babesia spp., and leishmania spp. are characterized by the absence of organelles like mitochondria, but they have acquired a plastid by endosymbiosis of a green alga. the apicoplast is a nonphotosynthetic plastid in which several essential biosynthetic pathways are sequestered, so that interactions with these biosynthetic functions cause deleterious effects. elimination of the plastid or total inhibition of its function results in a "delayed death", i.e., the parasites grow and evade normally within and from the first host cell, but their replication is halted immediately after the invasion of a new host cell. the apicoplast harbors a circular dna and bacterial type dna gyrase. ciprofloxacin induced cleavage of apicoplast dna in p. falciparum, without targeting nuclear dna [ ] [ ] [ ] . exposure of toxoplasma gondii to ciprofloxacin resulted in a decrease of the apicoplast genome copy number during replication [ ] . although it was discussed that differences in the role of apicoplasts in toxoplasma and plasmodium may exist [ ] , the apicoplast dna gyrases isolated from both species were inhibited by almost identical concentrations; the apicoplast dna gyrase isolated from plasmodium falciparum is inhibited by ciprofloxacin concentrations ranging from to μm and trovafloxacin inhibits apicoplast dna gyrase activity isolated from toxoplasma gondii and plasmodium falciparum, respectively, at μm [ , [ ] [ ] [ ] [ ] [ ] . consequently, prokaryotic type ii dna topoisomerase of apicomplexan protozoa are effectively targeted by fluoroquinolones. it has been summarized previously that fluoroquinolones are active in preclinical infection models against quinoloneresistant bacteria as well as candida albicans infections [ , ] . furthermore, levofloxacin was active against rv infections [ ] . these phenomena were found to be directly correlated to the immunomodulatory activities of fluoroquinolones [ , ] . mechanisms underlying the various immunomodulatory effects of fluoroquinolones include an effect on intracellular cyclic adenosine- , -monophosphate and phosphodiesterases, as well as an effect on transcription factors and also a triggering effect on the eukaryotic equivalent of bacterial sos response with its ensuing intracellular events [ ] . fluoroquinolones are routinely prescribed for the treatment of coronavirus-associated severe acute respiratory syndrome (sars) or opportunistic bacterial infections in hiv-positive patients. upon elimination of the bacterial pathogen or exclusion of bacterial pathogens, antibiotic therapy can be withdrawn. however, patients may benefit from the immunomodulatory activities of fluoroquinolones, but their effect on the course of sars or acquired immune deficiency syndrome (aids) is undetermined. although it is well documented that nalidixic acid and fluoroquinolones modulate immune responses by the modulation of intracellular signaling cascades, it is unknown which mechanism(s) may trigger signal transduction. it has been d e m o n s t r a t e d t h at , i n a n a l og y t o c hl o r o q ui n e , fluoroquinolones bind to and insert into pro-and eukaryotic membranes, respectively, thereby altering their fluidity [ ] . changes in membrane fluidity may be sensed by the immunocompetent cells, so that gene expression may be controlled according to the signals triggered. furthermore, it can be hypothesized that fluoroquinolones exert direct antiinfective activities due to their physicochemical interactions with membranes, thus making the organisms leaky, followed by cell death. this latter aspect has never been addressed systematically. any fluoroquinolone used for the treatment of bacterial infections exerts, in parallel to its antibacterial action, antiviral, antifungal, and antiparasitic actions at clinically achievable concentrations. this broad range of anti-infective activities is due to one common mode of action, i.e., the inhibition of type ii topoisomerases, thus maintaining the selective toxicity of fluoroquinolones inhibiting microbial topoisomerases and eukaryotic topoisomerases of prokaryotic origin at low concentrations but mammalian topoisomerases at much higher concentrations. there is strong evidence that the broad range of anti-infective activities translates into the clinical arena. however, anti-infective activities other than antibacterial activities have never been evaluated systematically. this may be due to the strategy of both the pharmaceutical industry and regulatory authorities to develop an agent on the basis of its application, i.e., its use as an antibacterial agent. therefore, the antiviral or antifungal activities of fluoroquinolones have, so far, not been exploited systematically; two controlled studies evaluating the antiviral effects of fluoroquinolones have been initiated recently. the clinical evaluation of their antifungal and antiparasitic effects is justifiable and would be opportune. traditionally, clinical studies are designed on the basis of a monocausal microbe-outcome association, i.e., the presence of one bacterial species at the site of infection indicates pathogenicity. consequently, an anti-infective agent is considered to be effective if this single species is eradicated from the focus of infection. however, infections may be polymicrobial or chronically ill patients may suffer from opportunistic infections; hivpositive patients represent an extreme example for the acquisition of opportunistic infections caused in parallel by viruses, bacteria, and/or parasites. such patients could, in theory, benefit from treatment with agents which exert a broad range of anti-infective activities. a multifactorial analysis of the outcome of infectious diseases would be necessary. the corresponding outcome measures are quantifiable and can be linked to pharmacokinetics and overall clinical efficacy. in summary, based on one common mode of action, fluoroquinolones being commercially available as antibacterial agents are active against viruses, fungi, and parasites too, so this class of agents is probably representative of broad-spectrum anti-infectives in its true sense. brundage rp ( ) , -naphthyridine derivatives. a new class of chemotherapeutic agents inhibition by chloroquine of a novel haem polymerase enzyme activity in malaria trophozoites chloroquine: mechanism of drug action and resistance in plasmodium falciparum inhibition of glutathione-dependent degradation of heme by chloroquine and amodiaquine as a possible basis for their antimalarial mode of action the anti-hiv- activity of 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conflict of interest. key: cord- -nstfe a authors: cag, yasemin; icten, sacit; isik-goren, burcu; baysal, naciye betul; bektas, begum; selvi, ece; ergen, pinar; aydin, ozlem; ucisik, ayse canan; yilmaz-karadag, fatma; caskurlu, hulya; akarsu-ayazoglu, tulin; kocoglu, hasan; uzman, sinan; nural-pamukcu, muge; arslan, ferhat; bas, gurhan; kalcioglu, mahmut tayyar; vahaboglu, haluk title: a novel approach to managing covid- patients; results of lopinavir plus doxycycline cohort date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: nstfe a this manuscript aims to present a treatment algorithm we applied to manage covid- patients admitted to our hospital. during the study period, patients with suspected covid- were admitted to the emergency department. molecular tests indicated that of these patients tested positive for covid- . we administered hydroxychloroquine plus doxycycline to mild cases (isolated at home) for days and lopinavir plus doxycycline to moderate and severe cases (hospitalized) for days. the overall case fatality rate was . % ( / ). since the first report in december from wuhan, hubei province, china, the novel severe acute respiratory syndrome coronavirus (sars-cov- ) has spread quickly worldwide [ ] . available data indicate that the clinical course and outcome of sars-cov- are much milder than those of sars-cov and mers-cov [ ] . however, the socioeconomic consequences of the sars-cov- pandemic are enormous [ ] . the false news regarding the clinical course and fatality rates triggered a global panic epidemic which has spread even faster than the virus. social panic has the potential to accelerate the expected health burden of the disease [ ] . social panic causes excess inpatient capacity in hospitals as the number of individuals with mild nonspecific symptoms has been increasingly hospitalized. controlling adverse outcomes of the disease and the panic among the public and healthcare staff depends on running an effective triage and management algorithm. this manuscript aims to present a treatment algorithm we applied to manage covid- patients admitted to our hospital and describe the characteristics of covid- patients and the outcomes of the algorithm. this single-center, retrospective observational study was conducted in the istanbul medeniyet university goztepe ea hospital, a -bed affiliated hospital located in the anatolian side of istanbul. we obtained ethical approval from the institute ethics committee, and signed informed consent was waived ( / ). a case was defined as a patient with an epidemiologic risk factor who had body temperature of ≥ °c and/or respiratory system symptoms which cannot be fully explained by any other condition or disease (based on who approach). a mild case was defined to have no signs of respiratory dysfunctions, while a moderate case had any sign of respiratory dysfunction, and a severe case had acute respiratory failure (arf) and required icu support either via invasive or noninvasive means. noninvasive ventilation support was administered with high-flow masks. respiratory dysfunction was assessed in a patient having any of the following: (a) shortness of breath, (b) respiration rate of > breaths per minute, and (c) o saturation < in ambient air. hydroxychloroquine mg, lopinavir mg, and doxycycline mg were all orally administered twice daily as recommended. we managed covid- patients with a -step treatment approach in our institute. first, mild cases were isolated at home and prescribed with hydroxychloroquine plus doxycycline for days. second, moderate to severe cases were hospitalized and prescribed with a regimen of lopinavir plus doxycycline plus ceftriaxone for days. third, we used a salvage therapy for patients who did not respond to or whose conditions worsened under the lopinavir treatment. this therapy involved the oral administration of favipiravir mg twice daily after two loading doses. we performed all statistical analysis using the open-source r software (r foundation for statistical computing, vienna, austria) [ ] [ ] [ ] . from march to april , , patients were admitted to our emergency department, presenting symptoms compatible with those stated in our case definition. pcr was positive for nasopharyngeal samples of adult patients. we run a -step treatment algorithm, and our approach is displayed in fig. . we hospitalized moderate to severe cases and administered lopinavir combined with doxycycline and ceftriaxone to patients, among whom had positive pcr test results ( / , . %). unfortunately our lab ceased respiratory viral pcr panels and allocated all resources to sars-cov- pcr test during the study time. therefore, we could not identify other causes and diagnosed covid- pcr negative patients as viral respiratory tract infection of unknown etiology. we followed mild cases under the treatment with hydroxychloroquine plus doxycycline at home. besides, patients isolated at home were found to have positive pcr test results ( / , . %). pcr results were mostly available within h, and patients with positive pcr test results were further followed by filiation teams of the turkish health ministry. filiation teams provided them with a -day course of hydroxychloroquine. twenty-three of all patients treated at home were readmitted to the hospital because their initial symptoms worsened, and we administered lopinavir plus doxycycline. if these patients do not respond in h, we instituted favipiravir treatment. the overall case fatality rate was . % ( / ). two out of patients aged < years died ( . %), one of whom was under treatment for acute lymphoblastic leukemia. the other patient had an unidentified muscle disease affecting the respiratory muscles [ ] . there were three deaths among those aged - years ( / , %) and deaths among those aged > years ( / , . %). figure presents the daily incidence of pcr positive and negative patients. the burden of social panic is barely visible in this figure. most patients had subjective symptoms yet inquired attention; therefore, time, effort, and care should be taken to relieve these patients. in other words, if not correctly managed, this panic had the potential to consume hospital resources reserved for severe patients. table presents the baseline characteristics of pcr positive hospitalized patients. we administered our standard regimen, lopinavir plus doxycycline plus ceftriaxone, to these hospitalized patients. among cases, required icu support, and deceased during icu stay. however, of these patients were severe at admittance. of these nine patients immediately admitted to the icu, five of whom died. three other patients transferred to the icu on the second day of admittance to the hospital also died. of the hospitalized patients, acquired lopinavir for at least days before being admitted to the icu. only . % ( / ) required icu support with lopinavir treatment, two patients suddenly died, and patients recovered from the disease. only % ( / ) of patients had a fever (≥ °c). deceased patients were older, had a higher prevalence of hypertension, and had a higher neutrophil counts than the others, while their lymphocyte counts, platelet counts, and levels of oxygen saturation in ambient air were lower. no difference was observed between two genders. deceased patients had shorter elapsed time between the onset of symptoms and hospitalization. this study presents a -step treatment protocol to manage covid- patients. we administered hydroxychloroquine to mild cases isolated at home, lopinavir plus doxycycline to hospitalized moderate to severe cases, and favipiravir in the salvage treatment. we were able to run this approach smoothly. to our best knowledge, this study is the very first to report data from istanbul, turkey. more importantly, our data present the results of a unique combination of lopinavir and doxycycline. we, administered hydroxychloroquine to mild cases for only days because of its potential side effects on cardiac functions [ ] . the cardiac effects of hydroxychloroquine are demonstrated to depend on the accumulation of the drug and mostly start on the third day of the usage. these effects are more prominent among critically ill patients [ ] . we administered lopinavir to moderate to severe cases for days. clinical trials demonstrated its effectiveness in the treatment of patients with sars and mers [ ] . molecular analysis indicates that lopinavir has a potential role in inhibiting sars-cov- protease, thereby blocking viral replication [ ] . a recent study found a limited benefit of lopinavir compared with the standard of care treatment [ ] . however, this study had substantial methodologic limitations, which raises questions about its conclusions. we supplemented doxycycline to both lopinavir and hydroxychloroquine due to its immunomodulatory activity. recent findings revealed the adverse effect of dysregulated immunity on the outcome of covid- patients [ ] . doxycycline induces the suppressor of cytokine signaling (socs) proteins, a regulatory system on cytokine release [ ] . evidence accumulates that socs proteins, mainly socs- protein, prevent interleukin-and interferon-associated toxicity [ ] . notably, in the early stage of the disease, when there are enough healthy cells in the bronchi and alveoli, doxycycline might have some effect on preventing the upcoming cytokine storm. doxycycline had been successfully used in dengue hemorrhagic fever due to its immunomodulatory activity [ ] . however, we also consider covering other etiologies of community-acquired pneumonia. at admission, it is challenging to differentiate covid- from other etiologies of pneumoniae, such as mycoplasma infections [ ] . however, the study has several limitations which require to be addressed. the major limitation of this study lies in its retrospective and single-center nature which is a source of selection bias to evaluate the efficacy of a treatment. in our study, a considerable number of died patients were extremely severe at admittance and so directly allocated to icu care. a -step treatment algorithm ran smoothly in our hospital. we concluded that home isolation of mild cases is an effective means to manage the burden of disease, while lopinavir plus doxycycline is an alternative to current treatment regimens for covid- . however, in future epidemics, isolation of mild cases at new-settled fever clinics should be considered which might serve better to mitigate epidemics [ ] . conflicts of interest the authors declare that they have no conflicts of interest. a novel coronavirus from patients with pneumonia in china characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention coronavirus disease : the harms of exaggerated information and non-evidence-based measures wilder-smith a the pandemic of social media panic travels faster than the covid- outbreak regression modeling strategies building bivariate tables: the {comparegroups} package for {r} descriptive statistics guillain-barré syndrome associated with sars-cov- qtc interval prolongation in critically ill patients: prevalence, risk factors and associated medications the qt interval in patients with sars-cov- infection treated with hydroxychloroquine/azithromycin a systematic review of lopinavir therapy for sars coronavirus and mers coronavirus-a possible reference for coronavirus disease- treatment option lopinavir; a potent drug against coronavirus infection: insight from molecular docking study a trial of lopinavir-ritonavir in adults hospitalized with severe covid- lung pathology of fatal severe acute respiratory syndrome the suppressor of cytokine signaling (socs) and socs but not socs proteins inhibit interferonmediated antiviral and antiproliferative activities suppressor of cytokine signaling (socs- ) protects β-cells against interleukin- β-and interferon-γ-mediated toxicity dengue patients treated with doxycycline showed lower mortality associated to a reduction in il- and tnf levels ct imaging and differential diagnosis of covid- the role of fever clinic during the covid- pandemic: a case study of febrile patients publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations ethics approval the study protocol was approved by the clinical research ethics committee of istanbul medeniyet university goztepe training and research hospital, and signed informed consent was waived ( / ). key: cord- -qkkn authors: pegoraro, manuela; militello, valentina; salvagno, gian luca; gaino, stefania; bassi, antonella; caloi, cecilia; peretti, angelo; bizzego, silvia; poletto, laura; bovo, chiara; lippi, giuseppe; lo cascio, giuliana title: evaluation of three immunochromatographic tests in covid- serologic diagnosis and their clinical usefulness date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: qkkn results of three rapid immunochromatographic tests (icts) were compared with those obtained with two automated immunoassays for evaluation of their usefulness. one hundred fifty-nine patients and healthy volunteers were included. different assays demonstrate – % of diagnostic sensitivities and – % of specificities, with substantial agreement ( . – . %), but a high percentage of weak positive results ( – %) was observed with icts. icts performances were comparable to those of automated immunoassays. icts could have a role as screening approach due to their easy usability. subjective interpretation, significant rate of uncertain results, uncertainty on viral antigens source are undoubtedly drawbacks. as covid- pandemic is still ongoing [ ] , specific antibodies detection is of outmost importance. besides the availability of immunoenzymatic (elisa) and chemiluminescence (clia) anti sars-cov- antibodies immunoassays, many other rapid antibody-detecting tests based on immunochromatographic techniques have been recently commercialized. although the who does not clearly endorse their use for patient management, interest is raising around these devices, mainly supported by their easy usability [ , ] . the aim of this study was to compare diagnostic performances of three rapid immunochromatographic tests with those of an automated elisa and clia immunoassays, in order to evaluate their potential usefulness as diagnostic and/or epidemiological tools. study population consisted of patients ( males; females; median age ± years; range - years) admitted to the emergency room, medical, and intensive care units (icus) of the azienda ospedaliera universitaria integrata of verona with symptoms suggestive of sars-cov- infection, between the end of february and the beginning of april. control group consisted of healthy volunteer's ( males; females; median age ± years; range - years). for each patient, upper respiratory specimen and blood sample were collected for covid- molecular and serologic diagnosis, respectively. negative and inconclusive rt-pcr test results were investigated by systematically repeating the molecular test during some consecutive days. rt-pcr results were then matched with clinical and epidemiological data in order to identify confirmed and suspected covid- cases. in covid- confirmed cases (symptomatic patient with sars-cov- positive molecular detection), date of symptoms onset was used to timing infection at the moment of specimens' collection. three stages were identified: early ( - days from symptoms onset), intermediate ( - china), covid- igg/igm rapid test cassette (zhejiang orient gene biotech co., ltd huzhou, zhejiang, china), and prima professional (prima lab sa, balerbna, switzerland) are lateral flow immunochromatographic assays. they differ in sample (serum, plasma, or whole blood) quantity need ( - μl) and time of incubation, but test principle is identical. results are qualitative (i.e., positive/negative), interpreted by visual reading and are generated in - min. sars-cov- molecular detection was performed with a commercial real-time pcr method, seegene allplex tm -ncov (seegene, seoul, south korea). [ ] . for each assays, igg, igm, and iga positive and negative rates were calculated. sensitivities were assessed on confirmed covid- cases, combining igg and igm/iga positive results, while specificities were estimated on the group of healthy volunteer's. agreement with cohen's kappa test was used to compare icts vs elisa euroimmun and clia maglumi. the study has been cleared by the local ethical committee (university hospital of verona; sopav- ; protocol no. ) and was performed in agreement with the declaration of helsinki, under the terms of relevant local legislation. sars-cov- molecular detection was initially positive in ( %) patients, negative or inconclusive in ( %), and ( %) patients, respectively. nineteen out of patients with initial inconclusive rt-pcr, resulted positive when molecular assay was repeated in the following days. molecular results, matched with clinical and epidemiological data, allowed the identification of covid- and suspected cases (patients seeking medical care for respiratory symptoms with negative or inconclusive rt-pcr results, radiological findings not available). date of symptoms onset were available for out of ( %) covid- confirmed cases. in cases, blood samples were collected < days after symptoms onset (early stage), in patients between and days (intermediate stage), and in cases ≥ days (late stage). igg and igm positive rates varied between . - . % and . - . %, respectively (table ) . with icts, weak positivity rates varied from - % for igg ( % vivadiag; % covid- igg/igm rapid test cassette; % prima professional) to - % for igm ( % vivadiag; % covid- igg/igm rapid test cassette; % prima professional). with respect to the elisa assay, iga displayed an overall positive rate higher than that of igg ( % vs %). overall assays sensitivities were - % (table ) . according to disease stage, sensitivities increased from . - . % at early stage to - % at late stage ( since december , when sars-cov- pneumonia outbreak occurred in wuhan, many efforts have been made worldwide to quickly develop new diagnostic assays. molecular detection tests are considered the "gold standard" for diagnosing sars-cov- infection, though some reports have highlighted a high rate of false negative and/or inconsistent rt-pcr results [ ] . sensitive and specific serologic tests, able to detect neutralizing antibodies, are needed in order to establish seroprevalence and immunity. large variability among the positivity rates of anti-sars-cov- igg and igm has been observed: in the first / days after symptoms onset, assays sensitivities were lower than % even if igg/igm/iga positive results were combined [ ] . sensitivities reached - % after days of disease, but patients number included in this group was too low to be statistically significant. when assays performance has been analyzed according to disease stage, vivadiag was the best ict for igm detection, with a positivity rate increasing from to % from early to late stage. prima professional failed to detect any igm positive sample in early stage and identified only % of positive samples in the late phase. covid- igg/igm rapid test cassette displayed a diagnostic performance comparable to that of vivadiag, except for lower igm sensitivity in late stage. compared to clia-maglumi, ict tests displayed an overall better sensitivity, with the exception of prima professional whose performance was rather limited. with respect to igg, substantial agreement between elisa euroimmun and clia-maglumi has been already reported [ ] . among icts, prima professional showed the best performance with a positivity rate increasing from % (early phase) to % (intermediate) and % (late stage). compared with the automated immunoassays, the ability of icts to detect anti-sars-cov- igg was equivalent to that of clia-maglumi and better than elisa-euroimmun, whose igg positive rates ranged between and % at days after symptoms onset. in the early stage of disease, the positivity rates of igg/ igm/iga combined ranged between . and %. these data confirm previous reports which showed the emergence of igm together with igg, mostly during the second week of illness [ ] [ ] [ ] . igm was still detected in almost half of blood samples collected in the fourth week after symptoms' onset. this evidence reaffirms that the diagnostic value of serologic tests in the acute phase of sars-cov- infection is substantially limited and igm testing could be questioned. some important perspectives need to be considered. first, ict assays are plagued by subjective interpretation which may be difficult with weak positive bands: between and % of igg results were classified as "weak" in our study. second, none of the ict evaluated clearly declared the characteristics of the antigens used and/or the target viral protein(s), and this represents a foremost caveats in assessing the neutralizing activity of the specific anti-sars-cov- antibodies detected. this study has some limitations. first, patients were retrospectively identified, and thereby further prospective studies would be needed to validate our preliminary findings. then, about half of blood samples from covid- confirmed cases had been collected in the early stage, when antibodies are usually absent, thus partially impacting the diagnostic sensitivity. finally, assays' specificities were deduced from a limited number of healthy volunteers who may not represent specificities obtained using sera from patients with other diseases. in conclusion, despite heterogeneous diagnostic performances, some icts appear interesting as "first line" serologic tools since their good specificities would help ruling out the infection. the combined use of immunocromatographic and quantitative assays, able to detect neutralizing antibodies, within diagnostic algorithm could be seen as a valuable approach for the serologic diagnosis of covid- . funding information the manufacturer provided in-kind support in the form of equipment and consumables for the evaluation, but had no role in directing the study or influencing the study outcomes. conflict of interest the authors declare that they have no conflict of interest. time to use the p-word? coronavirus enter dangerous new phase sars-cov- specific antibody responses in covid- patients world health organization ( ) advice on the use of point-ofcare immunodiagnostic tests for covid- world health organization ( ) laboratory testing for coronavirus disease (covid- ) in suspected cases stability issues of rt-pcr testing of sars-cov- for hospitalized patients clinically diagnosed with covid- serological immunocromatographic approach in diagnosis with sars-cov- infected patients assessment of antibodies to sars-cov with the fully automated maglumi -ncov igg and igm chemiluminescence immunoassays. letter to editor antibody responses to sars-cov- in patients of novel coronavirus disease serologica assays for sars-cov- infectious disease: benefits, limitations andpperspectives performance of vivadiag covid- igm/igg rapid ttest is inadequate for diagnosis of covid- in acute patients referring to emergency room department. letter to editor publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -si t j authors: lu, q.-b.; wo, y.; wang, h.-y.; huang, d.-d.; zhao, j.; zhang, x.-a.; zhang, y.-y.; liu, e.-m.; liu, w.; cao, w.-c. title: epidemic and molecular evolution of human bocavirus in hospitalized children with acute respiratory tract infection date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: si t j human bocavirus (hbov) is a novel parvovirus, often associated with respiratory tract diseases in children. this study explored the epidemiological characteristics and molecular evolution of hbov- in southeastern china. nasopharyngeal aspirates were collected from children admitted to hospital with acute respiratory tract infections. hbov- was detected using real-time reverse transcription polymerase chain reaction and further characterized by complete genome sequences analysis. among the , recruited children, ( . %) were hbov- -positive and ( . %) had co-detection with other respiratory viruses. seasonal prevalence peaked in summer. hbov- presence was significantly associated with asthma attack [odds ratio = . ; % confidence interval: . , . ; p < . ]. similar results were obtained when either single detection or co-detection of hbov- was considered, demonstrating the minor impact of co-detection on the clinical characteristics or epidemic pattern. phylogenetic analysis based on the complete genome sequences showed that all the hbov- sequences clustered together and no branch was formed that was supported by bootstrap value ≥ . the overall evolutionary rate of the complete genome of hbov- was estimated at . × (− ) nucleotide substitutions per site per year (s/s/y) [ % highest probability density: ( . – . ) × (− ) s/s/y]. selective pressure analysis showed that all the ω-values were less than , suggesting that hbov- was under negative selective pressure. site-by-site analysis identified the codon site of the vp gene under positive selection. in conclusion, our study disclosed the epidemiological and genetic dynamics of hbov- epidemics in southeastern china in the most recent years, the information of which might help to further improve our understanding of hbov- infection and guide better surveillance and control strategies in the future. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. acute respiratory tract infections (arti) are a major cause of significant morbidity worldwide, especially in infants and children. viruses are leading pathogens of arti. although some viruses, such as influenza virus, respiratory syncytial virus (rsv), and human adenovirus (hadv), are responsible for most arti, etiological causes remain unknown in a proportion of arti [ ] . human bocavirus (hbov) is a novel parvovirus first described by allander et al. in [ ] . it has been classified in the family parvoviridae, subfamily parvovirinae, and genus bocavirus by genetic organization and sequence homology. other parvoviruses infecting humans included the adenoassociated viruses, the well-known pathogen parvovirus b , and the recently discovered human parvovirus [ ] . based on the phylogenetic analysis of viral genomes, four species of hbovs (hbov - ) have been identified [ ] , article note qing-bin lu and ying wo contributed equally to this work. electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. among which hbov- was mainly identified in respiratory specimens from children with acute respiratory disease [ ] [ ] [ ] . according to previous studies, hbov- has a global distribution, with prevalences varying considerably from . % to . % in arti patients [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, its role as a causative agent of respiratory tract diseases is frequently questioned due to its concurrent detection with other potential pathogens. possible interactions between hbov- and other viruses remained rarely investigated. in china, hbov- was detected in children with positive rates ranging from . % to . % [ , [ ] [ ] [ ] [ ] [ ] . however, hbov- was co-detected frequently with other viruses in patients with respiratory or enteric infections; therefore, the impact of hbov- on arti occurrence remains unclear. the seasonal patterns of hbov- infection in china, especially the surveillance data from the most recent years, are scarcely reported, probably owing to the lack of a systematic and prolonged surveillance. this study was aimed to explore the epidemiology pattern and clinical characteristics of hbov- infection in chinese children, as well as the molecular evolutionary pattern, for hbov- , by performing a -year laboratory surveillance of arti cases. the laboratory surveillance was performed to detect the viral infection in children with arti between june and may at the children's hospital of chongqing medical university, china. the arti was determined based on syndromes of cough, rhinorrhea and/or dyspnea, and/or fever of > . °c. fever caused by known chronic medical conditions was excluded. the pneumonia was defined by the presence of patchy alveolar opacities on chest radiographs, in addition to presenting symptoms of cough, dyspnea (lower chest wall indrawing), or tachypnea (in infants, > - breaths/min; in older children, > breaths/min). severe pneumonia was defined as pneumonia plus hypoxemia (maintained sao < % in air) or rising respiratory and pulse rates with clinical evidence of respiratory distress and exhaustion with or without raised paco . the definition of an attack was used as an episode of respiratory symptoms which prompts an urgent consultation with a doctor, is of sufficient severity to prevent the patient working/attending school/performing domestic duties/playing, and results in increased use of anti-asthma medication. the recruited patients had nasopharyngeal aspirates (npa) collected the day they were hospitalized. a standardized form was applied to extract the patients' information regarding demographic characteristics, underlying medical conditions, selected laboratory tests, radiographic findings, clinical manifestation, treatment course, and outcome. written informed consent was obtained from their parents. viral rna and dna were extracted from μl of the npa by using qiaamp® minelute virus spin kits (qiagen, hilden, germany). the cdna was synthesized by using the superscript®iii first-strand synthesis system for reverse transcription polymerase chain reaction (rt-pcr) (invitrogen, carlsbad, ca, usa). all samples were routinely screened for hbov- with the real-time pcr method [ ] . partial hbov- -positive samples were whole-genome sequenced following the method previously described [ ] . genomic sequences were assembled using lasergene's dna seqman software (version . . , dna star inc., madison, wi, usa). previously published genbank sequences of hbov- were used for comparison. all alignments and phylogenetic tree construction were performed by the neighborjoining (nj) method with , bootstrap replicates using clc genomic workbench . . similarities between strains were calculated by using bioedit version . (north carolina state university, raleigh, nc, usa; http://www.mbio.ncsu. edu/bioedit/bioedit.html). the substitution rates, the divergence over time, and the time to the most recent common ancestor (tmrca) of the current hbovs were estimated using a bayesian markov chain monte carlo (mcmc) approach, as implemented in the bayesian evolutionary analysis sampling trees (beast) package version . . . the value of ω and the individual site-specific selection pressure were measured by using the single likelihood ancestor counting (slac), fixed effects likelihood (fel), internal fixed effects likelihood (ifel), and fast unconstrained bayesian approximation (fubar) methods implemented in the hypothesis testing using phylogenies (hyphy) package performed at datamonkey online (http://www.datamonkey.org/). the sites were confirmed when they were selected by more than two methods. a single breakpoint recombination (sbp) test with both akaike information criterion (aic) and bayesian information criterion (bic) calculation was conducted. the overall ω-value and % confidence interval (ci) were estimated based on nj trees. selective pressure was defined as neutral evolution with ω= , purifying or negative selection with ω< , and positive selection with ω> . a p-value of less than . was considered as the threshold for strong evidence of selection in slac, fel, and ifel, and posterior probability ≥ . in fubar, respectively. statistical analysis was done by the chi-square test for categorical variables. logistic regression models were used to explore the factors associated with higher hbov- detection and to determine the association between clinical characteristics and hbov- infection, with clinical disease as the dependent variable and hbov- infection as the independent variable. odds ratios (ors) and their % cis were estimated using maximum likelihood methods. statistical significance was set at a p-value of less than . . all the tests were performed by using sas . (sas institute inc., cary, nc, usa). from june to may , , npa samples of hospitalized infants and children were collected. the median age was months (range months to years), and , ( . %) were boys. the median delay before hospital entrance was days (range - days). the hospitalization duration [mean ± standard deviation (sd)] was ± days. among the , children, ( . %) displayed severe pneumonia and ( . %) had severe asthma. in total, ( . %) children were positive for hbov- . the children aged between and months had higher detection rates of hbov- than children of the other age groups ( and severe asthma were identified in ( . %) and ( . %) hbov- -detected patients, among whom and four had single detection, respectively, which is comparable with the patients with co-detection. the monthly positive rates of hbov- displayed specific seasonal distribution, as plotted in fig. . one seasonal peak was observed in summer, demonstrated by remarkably higher infection rates during may-august. an exception was noticed in , when another low peak was also observed in the winter season from november to december. when the hbov- single infection was plotted, a similar seasonal pattern was derived, with a clearly increased activity of hbov demonstrated in summer. the phylogenetic tree based on , bp of the whole genome sequences showed that all the hbov- sequences, regardless of whether they were single detection or co-detection, clustered together and no other branch was formed that was supported by bootstrap value≥ (fig. ) . the identities for the hbov- complete genome were . - %. the phylogenetic trees were similarly established for the np , figure ) . the identities of hbov- were . - % for the np gene, . - % for ns , . - % for vp , and . - % for vp . all the hbov- viruses in the investigated localities were dominated by a single viral lineage, which had been dominant in most parts of china in recent years. neither a spatial nor a temporal specific branch was formed based on the whole-genome analysis. the overall evolutionary rate was estimated at . × − nucleotide substitutions per site per year (s/s/y) [ % highest probability density (hpd): ( . - . )× − s/s/y] based on the complete genome analysis. the highest evolutionary rate was derived from the np gene, while the lowest was from the ns gene sequences ( table ) . the sbp test showed no evidence of recombination for any of the four genes. selective pressure analysis showed that all the ω-values were less than ( table ), indicating that these hbov- genes were under negative selective pressure and the evolutionary process is shaped mainly by purifying selection. the np gene had the largest global rate ( . , % ci: . - . ) among the four genes (range . - . ), suggesting that it is subject to greater selective pressure than the other three genes. site-by-site analysis of the full data set consisting of hbov- coding sequences identified only one site on the vp gene (codon site ) undergoing significant positive selection and a number of negatively selected sites. a codon-based maximum likelihood reconstruction of the evolutionary history of codon site under positive selection was carried out (supplemental figure ) . the result disclosed that the substitution has occurred mostly in the strains from asia, especially in china, suggesting that hbov- strains from china undertake more selective pressure than those from other regions. in the current study, we have identified the clinical characteristics and temporal patterns of hbov- in southeastern china through a -year surveillance. no role of hbov- in severe pneumonia occurrence was observed, regardless of whether they were single detection or co-detection. in contrast, a significant association between hbov and asthma was determined, suggesting a possible causal relationship. based on the genetic analysis, hbov- in southeastern china have similar mean evolutionary rates as other species of the family parvoviridae and one positive selection site was also disclosed which differs from hbov of other origins. according to our results, the prevalence of hbov- in pediatric arti patients is higher than those from other hospital-based studies [ , , , ] , whereas it was lower than that previously detected in persistently wheezing children [ ] . hbov- was commonly co-detected with a median rate of . %, and the most frequent co-detected viruses included rhinovirus, enteroviruses, and influenza [ ] . we demonstrated a co-detection rate of . %, which is similar to the high proportion of co-detection as previously studied [ ] , yet significantly higher than those from other studies [ , ] . these discrepancies could be explained by the inherent differences in the population subsets, detection methods, as well as the epidemiological variability relating to geographic origin and climatic factors. seasonal circulations of hbov- vary, with most previous studies reporting hbov- peak attained during the winter season [ , , ] . choi et al. found a higher frequency of hbov- between may and july over a -year period among children with arti in korea [ ] , which, interestingly, is consistent with our results displaying a summer peak. although this temporal pattern coincided with the epidemic peaks of influenza circulation in southern china (http:// www.cnic.org.cn/chn/), the single detection of hbov also demonstrated a similar temporal pattern after excluding the the relative importance of hbov- as a causative pathogen for viral respiratory illnesses has not been determined, but it has been associated with respiratory diseases ranging from upper respiratory tract disease to bronchiolitis, and lower respiratory tract diseases, including pneumonia [ , , ] . vallet et al. found a high concordance between hbov- detection and exacerbation of asthma requiring eventual hospitalization [ ] . in our study, hbov- detection was not associated with pneumonia. instead, hbov- tended to cause more upper respiratory tract infections, and its infection was also related with asthma exacerbation. although a causal relationship between hbov- infection and asthma cannot be concluded based on the statistical results, their significant association suggest the need for further investigation. prospective longitudinal studies will hopefully increase our understanding of what, if any, role hbov plays in asthma. the epidemiology of the hbov- circulation in china has been previously studied; however, the viral gene sequence data acquired from these studies were rare and insufficient to reveal any temporal or spatial characteristics or any evolutionary dynamics. analysis of the genomic and epidemiological data of hbov- from the current study, as well as those available from different localities, revealed limited heterogeneity, and high conservation was demonstrated from both spatial and temporal perspectives. zehender et al. reported that the estimated mean evolutionary rate of the hbov- vp gene was . × − s/s/y using non-recombinant isolates [ ] , demonstrating that hbov- was characterized by a rapid evolution. the rate identified in the current study is slower, which might be partially caused by the sample size of analyzed hbov- sequences. a few of the previous studies explored the estimated evolutionary rates of parvoviruses in carnivore parvoviruses ( . × − - . × − s/s/y) [ ] and human parvovirus b ( . × − - . × − s/s/y) [ ] . the estimated mean evolutionary rates of the complete genome, vp , vp , and ns genes of hbov- were similar to those of carnivore parvoviruses and human parvovirus b . the ns gene was highly conservative among the four genes. surprisingly, the np gene showed a slightly higher substitution rate than the other genes. the icosahedral capsid consists of two structural proteins, vp and vp , which are identical except for a certain number of amino acids at the amino-terminal end of the vp protein, the so-called vp unique region (vp u). a conserved phospholipase a (pla ) motif, which resembles the catalytic motif of secreted pla (spla ), has been identified in the vp u of most parvoviruses [ ] . for hbov- , vp and vp have the same sequence of aa at the c termini, while vp has an additional aa peptide at the n-terminus. one study indicated that the vp u of hbov- also had spla -like enzymatic activity, and these residues are crucial for its spla -like activity [ ] . mutation of one of the amino acids ( pro, his, asp, or asp) in the vp u almost eliminated the spla activity of the hbov- vp u. interestingly enough, one positive selection site (codon ) was identified near codon and in the current study, which indicated a high probability of vp u undergoing selective pressure. in spite of this finding, the associations between the positive selection site and spla activity or amplification of dna need further investigation. in conclusion, our study, for the first time, disclosed the epidemiological characteristics and genetic dynamics of hbov- in southeastern china in recent years. 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activity-dependent stimulation of ca + entry by human parvovirus b capsid protein vp phospholipase a -like activity of human bocavirus vp unique region acknowledgments this study was supported by the china mega-project for infectious diseases grant ( zx - ) and the natural science foundation of china ( ). the authors do not have any commercial or other association that might pose a conflict of interest. all authors read and approved the final manuscript. key: cord- -sigw yxk authors: lampejo, temi title: influenza and antiviral resistance: an overview date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: sigw yxk influenza affects approximately billion individuals each year resulting in between , and , deaths. young children and immunocompromised individuals are at a particularly high risk of severe illness attributable to influenza and these are also the groups of individuals in which reduced susceptibility to neuraminidase inhibitors is most frequently seen. high levels of resistance emerged with previous adamantane therapy for influenza a and despite no longer being used to treat influenza and therefore lack of selection pressure, high levels of adamantane resistance continue to persist in currently circulating influenza a strains. resistance to neuraminidase inhibitors has remained at low levels to date and the majority of resistance is seen in influenza a h n pdm infected immunocompromised individuals receiving oseltamivir but is also seen less frequently with influenza a h n and b. rarely, resistance is also seen in the immunocompetent. there is evidence to suggest that these resistant strains (particularly h n pdm ) are able to maintain their replicative fitness and transmissibility, although there is no clear evidence that being infected with a resistant strain is associated with a worse clinical outcome. should neuraminidase inhibitor resistance become more problematic in the future, there are a small number of alternative novel agents within the anti-influenza armoury with different mechanisms of action to neuraminidase inhibitors and therefore potentially effective against neuraminidase inhibitor resistant strains. limited data from use of novel agents such as baloxavir marboxil and favipiravir, does however show that resistance variants can also emerge in the presence of these drugs. the world health organization estimates that annually there are approximately billion human influenza cases of which to million are considered severe (especially in children, the elderly and in the immunocompromised) and result in , to , deaths [ ] . influenza can be transmitted through the following routes: . respiratory droplets (> μm) generated e.g. by coughing and sneezing. these do not remain suspended in the air and settle to the ground within - m . contact transmission either through direct transfer of infectious particles from an infected to an uninfected individual or indirectly via contaminated surfaces or objects (i.e. fomites) and influenza can survive for hours on nonporous surfaces . possibly by airborne transmission via small aerosols (< μm) generated from breathing/talking (and can remain suspended in the air for minutes to hours) [ ] ; however, there is limited data to suggest that infectious particles can be transmitted over long distances (and special air handling and ventilation systems are not considered necessary to prevent spread) influenza belongs to the orthomyxovirus family and there are four influenza types a to d of which only influenza a, b and c can infect humans (influenza c is rare and usually causes a mild upper respiratory tract illness) [ ] . influenza a and b contain pieces of segmented single-stranded rna which encode various proteins including haemagglutinin (which facilitates attachment to the host cell) and neuraminidase (which facilitates release of new virus particles from the host cell). influenza a has the broadest host range of the influenza viruses and significant interspecies transmission occurs [ ] . eighteen haemagglutinin (h) and neuraminidase (n) subtypes have been described in influenza a (of which h and n subtypes have also been detected within avian species) [ ] . influenza b is far less genetically diverse than influenza a and has no distinct antigenic subtypes (mutates to times slower than influenza a and apart from humans, only seals and ferrets have demonstrated susceptibility) [ ] [ ] [ ] . influenza achieves antigenic diversity via two main mechanisms: . antigenic drift where mutations readily occur in ha and na resulting in new antigenic variants (thus avoiding preexisting host immunity); the error prone nature of the viral polymerase is a significant factor in this . antigenic shift due to reassortment of gene segments between two distinct influenza viruses within the same host giving rise to a novel strain the influenza a h n pandemic is thought to have arisen from reassortment between human and avian strains (based on sequencing of fixed, frozen lung tissue from victims) and similarly, the most recent 'swine flu' influenza a h n pandemic was thought to arise from a series of reassortment events between human influenza a h n , swine influenza a h n and avian influenza a h n [ , ] . lack of influenza b infection in several other species may explain why antigenic shift is not seen with influenza b [ ] . this potential for vast genetic variability within influenza viruses and their highly error-prone rna dependent rna polymerase does raise concerns regarding the possible emergence of treatment resistant strains and generates further questions regarding their viral fitness and transmissibility as well as which strategies to employ in rapidly identifying and effectively treating these resistance variants. this article discusses these issues including novel agents and experimental strategies that have been used in an attempt to treat as well as prevent the emergence of resistant influenza viruses in humans. the mechanism of action of the adamantanes is by blocking the m ion channel of influenza a thus preventing viral uncoating and the subsequent release of influenza a viral rna into the host cell. they have activity against influenza a but not influenza b (due to their lack of the m protein, influenza b has an alternative ion channel called bm ) [ ] . amantadine was approved for clinical use in and subsequently rimantadine in . both drugs were initially very effective in treating and preventing influenza a infection with efficacy rates of up to %. the resistance of influenza a to amantadine was first recognised during the influenza a epidemic [ ] . however, resistance to both drugs in seasonal influenza a subtypes was rare ( - % frequency) until after when there was a dramatic rise in rates of resistance. by , approximately % of all influenza a subtypes in circulation globally were resistant to the adamantanes (> % of h subtypes and % of h subtypes) [ ] . resistance (as a result of the s n mutation in the m protein) to the adamantanes occurs rapidly within - days of use and occurs in - % of both immunocompetent and immunocompromised patients [ , ] . due to such high levels of resistance, the adamantanes are no longer recommended for treatment of influenza a [ ] . neuraminidase inhibitors are currently first-line treatment for both influenza a and b in the united kingdom (uk) and most (if not all) parts of the world. they competitively inhibit neuraminidase on the surface of influenza a and b. they act by preventing cleavage of sialic acid residues on budding newly formed virus particles thus preventing release of new virus particles from infected host (ciliated epithelial) cells. resistance occurs much less readily in comparison with the adamantanes [ ] . the neuraminidase inhibitors, if given within h of onset, have been shown to reduce the duration of illness by % (with an approximately % reduction in illness severity) and, if given within h of symptom onset, even greater reductions in the duration of illness attributable to influenza (approximately % reduction) have been observed [ , ] . a decrease in the incidence of secondary complications, such as otitis media, sinusitis and pneumonia, with the use of neuraminidase inhibitors was also demonstrated [ ] . additionally, when used as prophylaxis (before or shortly after exposure), neuraminidase inhibitors can reduce the incidence of infection by approximately - % [ ] . in the uk, oseltamivir (oral agent; typically first line for influenza a and b treatment) and zanamivir (inhaled and also available as an aqueous solution which can be administered intravenously or via nebuliser) are licensed for the treatment of influenza a and b and also for prophylaxis. oseltamivir is licensed in the uk for use in all ages including neonates, whereas zanamivir is not licensed for children under the age of years. peramivir (a single-dose intravenous infusion) was licensed in the uk in but has not been marketed/launched (it is used in the usa, japan and south korea). laninamivir (an inhaled neuraminidase inhibitor) is licensed for use in japan. based on the data from animal models which demonstrated that oseltamivir-resistant viruses were unfit and poorly transmissible, resistance to neuraminidase inhibitors was not envisaged to become an important clinical issue [ ] . prior to , oseltamivir resistance was rarely seen in clinical practice (and low resistance rates of - % were reported in clinical trials) [ ] . human cases of oseltamivir-resistant influenza a h n began emerging during the - influenza season. many of the cases reported were individuals who had not taken oseltamivir demonstrating that resistant virus could be efficiently transmitted between humans [ ] . during the - influenza season, in some parts of the world, such as the usa, canada, the uk and australia, very high rates (> %) of oseltamivir-resistant seasonal influenza a h n strains were seen [ , ] . in , the pandemic (pdm ) influenza a h n strain emerged worldwide but in the majority of cases (< . % resistance) remained oseltamivir susceptible initially. oseltamivir resistant h n pdm cases did emerge but this was mostly immunocompromised patients that had received oseltamivir. subsequently, however, in the - influenza season in the uk (and other parts of the world), increasing numbers of oseltamivir resistant cases were identified with no previous oseltamivir exposure [ ] . resistance to oseltamivir was still seen with much greater frequency in immunocompromised individuals receiving oseltamivir (with very little cross-resistance seen with zanamivir). the neuraminidase mutation responsible for the oseltamivir resistance that emerged in the seasonal influenza a h n (a/brisbane/ / -like) strain in and then in the h n pdm and also subsequent h n strains is the h y mutation; a histidine to tyrosine substitution at amino acid of the influenza a n neuraminidase [ , ] . normally when oseltamivir binds to the neuraminidase on the influenza virion, the neuraminidase active site changes shape to accommodate oseltamivir. a neuraminidase mutation, such as h y, prevents this conformational change in the active site and therefore, oseltamivir is unable to bind. zanamivir, however, does not require this structural change in the neuraminidase active site in order to bind [ ] . the h y mutation reduces the susceptibility (ic ; the half maximal inhibitory concentration) of influenza a h n to oseltamivir by approximately -fold but not zanamivir [ , ] . peramivir binds to sialic acid residues in a similar manner to oseltamivir and is also affected by the h y mutation [ ] . this mutation has been shown to persist even after cessation of treatment, and strains harbouring this mutation are capable of causing outbreaks and significant morbidity and mortality in a similar fashion to their wild-type counterparts [ , ] . children and severely immunocompromised patients are at higher risk of developing resistance most likely due to higher viral loads and prolonged viral shedding [ ] . due to certain difference in the neuraminidase enzyme structure, neuraminidase resistance is less likely to occur in influenza a h n and influenza b compared with a h n pdm without causing significant loss of neuraminidase enzymatic function and reduced viral fitness [ , ] . in vitro and in vivo studies have demonstrated that influenza a h n and influenza b neuraminidase inhibitor resistant strains have a lower replicative capacity and less ability to transmit. in many of the case reports of influenza a h n and b resistant strains, resistance only occurs after prolonged treatment (> days but over month in many) and these resistant variants often disappear once treatment is ceased [ , ] . public health england (phe) publishes the most frequently observed influenza a and b mutations and their neuraminidase inhibitor resistance profiles in their 'surveillance and laboratory testing of influenza neuraminidase inhibitor resistance' reports [ ] . table summarises the centers for disease control and prevention (cdc) and phe resistance data for the usa and uk, respectively. the influenza resistance information study (iris) was a multicentre global observational study of neuraminidase inhibitor resistance and clinical outcomes in immunocompetent patients conducted from to [ ] . this study included patients over year of age presenting within hours of an influenza-like illness and/or had a positive rapid influenza test. nose throat swabs were collected on days , , and for influenza typing/subtyping, sequencing and neuraminidase inhibitor phenotypic susceptibility testing. there were influenza a and b reverse-transcription polymerase chain reaction (rt-pcr) positive patients in the study. except for patients with pre-treatment (i.e. transmitted) resistant influenza a h n strains, no resistance was detected in day samples. emergence of oseltamivir resistance after day was detected in / ( . %) of oseltamivir-treated influenza a positive patients; a higher frequency was seen in - year old ( . %) compared with those over years ( . %). all resistant h n viruses had the h y mutation and all resistant h n viruses had the r k mutation (conferring reduced susceptibility to both oseltamivir and zanamivir). virus clearance was a median of . days for treated patients with oseltamivir-resistant virus vs . days for untreated patients vs . days for treated patients with oseltamivir-resistant virus. time to alleviation of symptoms was day shorter in treated patients as compared with untreated patients. interestingly, the oseltamivir-resistant treated group exhibited the shortest duration of symptoms (symptoms resolved by day or earlier). dual resistance to oseltamivir and zanamivir is rare. a literature review by abed et al. identified published cases of human influenza a and b infections with mutations conferring reduced susceptibility to both oseltamivir and zanamivir [ ] . seven had influenza a h n pdm , had influenza a h n , had avian influenza a h n and had influenza b. the age range was months to years and out of the patients were immunocompromised. the other two patients had underlying chronic lung disease. thirteen out of patients had received neuraminidase inhibitor therapy before emergence resistance mutations ( oseltamivir alone, oseltamivir then zanamivir, zanamivir alone, zanamivir and oseltamivir simultaneously, oseltamivir then peramivir). there was a mean of . days (range - days) treatment before detection of the first mutation. mortality was high at % ( / patients). of the survivors, there was an immunocompetent asthmatic child with h n pdm who received no treatment, immunocompromised adult with h n pdm who received oseltamivir for days then inhaled zanamivir for days, immunocompromised adult with h n who had days of oseltamivir and immunocompromised child with h n who received oseltamivir for months and then inhaled zanamivir for days. in a recently published uk series of three cases of oseltamivir-resistant influenza a h n pdm that occurred in england in immunocompetent patients in the - influenza season, two of the patients (a -week old previously well boy and a -year old asthmatic woman) made a good recovery with days of oseltamivir despite whole genome sequencing revealing h y mutations in % and % of the virus population, respectively [ ] . the third case was a -month old girl with a developmental condition (najer syndrome) admitted with a -day history of respiratory illness who showed minimal clinical improvement with days of oseltamivir after which she was switched to intravenous zanamivir (as well as receiving antibiotics) and died soon after. phe sequencing data later revealed h y mutations in % of the virus population in a day nasopharyngeal aspirate specimen and this rapidly rose to % of the virus population harbouring h y mutations day later in a day nasopharyngeal aspirate sample. the treatment of influenza as recommended by phe is oseltamivir as first line for immunocompetent children and adults [ ] . for immunosuppressed patients, neuraminidase inhibitor choice is based on the dominant circulating strain in that particular season; oseltamivir is recommended when the dominant circulating strain is of lower risk for oseltamivir resistance (i.e. influenza a h n or b) and zanamivir is recommended when the dominant strain has a higher risk of oseltamivir resistance (e.g. h n pdm ). currently, available phe date for the - influenza season indicates that influenza a h n is the dominant circulating strain [ ] . in many laboratories, typing/subtyping results are becoming available early in the treatment course therefore guiding selection of treatment. neuraminidase inhibitor susceptibility testing should be considered particularly in young children and immunocompromised patients being treated with a neuraminidase inhibitor for influenza (especially h n pdm ), who are not responding to treatment and/or have persistently high viral loads (low cycle threshold values using rt-pcr) and/or exposed to a suspected or confirmed resistant case. given that resistance variants can emerge within only - days of treatment (as well as can be transmitted), resistance testing can be performed at any time prior to, during or after treatment. in england, phe offers both genotypic and phenotypic influenza a and b susceptibility testing which is summarised in table [ ] . relatively newer techniques also exist such as pyrosequencing, a high-throughput sequencing method that is able to type/subtype, screen for mutations and delineate the relative proportions of the various influenza variants [ , ] . another molecular technique, digital pcr (using a droplet-based system) has been shown to be highly accurate and precise in the identification and quantification of influenza sequence variants with the ability to detect rare single nucleotide polymorphisms present at levels as low as . % of the virus population [ ] [ ] [ ] . from a direct clinical perspective, identification of variants at such low proportions may not be relevant but these techniques may provide further insights into the viral dynamics in the emergence of resistant influenza viruses. baloxavir marboxil is a single dose oral agent for the treatment of influenza a and b (no data for it is used of prophylaxis). it was licensed in japan and usa in but is not currently licensed in the uk. it suppresses influenza replication by inhibition of cap-dependent endonuclease (an enzyme required for initiation of influenza mrna synthesis) and therefore, its mechanism of action is different to that of the neuraminidase inhibitors [ ] . there is limited data on resistance but a recent us/japan randomised controlled study of healthy adults/adolescents with influenza a and b treated with baloxavir marboxil found that . % ( / ) developed a specific mutation (pa/i x) - days after treatment and that the emergence of these pa/i x variants was associated with higher viral loads, prolonged detection of virus and a longer duration of symptoms compared with baloxavir marboxil treated individuals who did not develop the pa/i x mutation [ ] . favipiravir is an oral (and intravenous) antiviral which inhibits rna-dependent rna polymerases [ , ] . it has been approved for the treatment of influenza a and b in japan with very strict regulation for clinical use and is intended to be reserved for pandemics causes by novel/re-emerging influenza strains resistant to other antivirals. a recent study has demonstrated that a specific k r mutation in the pb subunit of the influenza virus polymerase results in reduced susceptibility to favipiravir in vitro and in cell culture. viral fitness, which was demonstrated to be impaired by this mutation, can be restored by a compensatory second (p l) mutation [ ] . the effects or these mutations in a clinical setting are yet to be determined. human clinical trials of dual therapy with oseltamivir plus zanamivir for h n have been investigated in various settings (including in ecmo patients) with varying outcomes and no clear benefit in terms of clinical outcome or prevention of drug resistance [ ] [ ] [ ] . studies in mice also failed to show a benefit [ ] . double dose oseltamivir did not reduce the risk of emergence of oseltamivir resistance in patients with influenza a h n pdm [ ] . this study was a small (n = ), randomised trial of patients treated in community. one patient in the single dose group and one in the double dose group developed oseltamivir resistance. there was no mention of any immunocompromised patients in the study. a triple-combination of amantadine, oseltamivir and ribavirin (tcad regimen) was shown in vitro and in the mouse model to have synergistic activity against sensitive and resistant influenza viruses (with greater synergy than any double antiviral regimen) [ ] . there was a phase i pilot study of tcad in to assess pharmacokinetics and safety in immunocompromised patients with influenza a (h n & h n ). five out of tolerated and completed the -day course of treatment ( patient had worsening respiratory failure and tcad was stopped) [ ] . a clinical response and a corresponding viral load reduction in the patients that completed treatment. no drug-drug interactions were seen and no haematological toxicity was seen with ribavirin. no new resistance mutations emerged on treatment. a study of tcad vs oseltamivir in critically ill mechanically-ventilated patients with pandemic h n showed that tcad was well tolerated but did not improve outcomes compared with oseltamivir alone [ ] . an in vitro study in of a zanamivir-oseltamivir hybrid inhibitor (ms- ) showed effectiveness against neuraminidase inhibitor-resistant influenza strains but there have been no further published studies to date [ ]. oseltamivir resistance is rare and zanamivir resistance is extremely rare. the presence of resistant virus does not necessarily mean a more severe infection and/or worse outcome, particularly in immunocompetent adults. in some instances, neuraminidase inhibitor resistant virus may actually be less fit (especially with h n and influenza b). there is an increased risk of resistance in the immunocompromised (but resistance does occur in the immunocompetent) and young children (< years); this is primarily with influenza ah n pdm but can occur less commonly with influenza a h n and b. neuraminidase inhibitor susceptibility testing should be considered primarily in h n infected immunocompromised patients and young children failing to respond to treatment, and clinicians should consider zanamivir in the intravenous form for patients that are critically ill/developing severe complications. there should potentially be a higher threshold for neuraminidase inhibitor susceptibility testing in influenza h n or b infected patients. this should generally be reserved for those that have had extended treatment (at least days) and not responded/deteriorated with persistent low ct values and no other identifiable cause. finally, there is no clearly proven benefit from combination (or double dose) antiviral therapy for influenza in terms of clinical outcome or emergence of resistance. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. global influenza strategy - infectious virus in exhaled breath of symptomatic seasonal influenza cases from a college community world health organization (who) viral determinants of influenza a virus host range mutations in influenza a virus neuraminidase and hemagglutinin confer resistance against a broadly neutralizing hemagglutinin stem antibody comparison of the mutation rates of human influenza a and b viruses influenza b virus in seals mouse adaptation of influenza b virus increases replication in the upper respiratory tract and results in droplet transmissibility in ferrets 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mutation in the influenza a/h n neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance. influenza other respir viruses permissive secondary mutations enable the evolution of influenza oseltamivir resistance infections with oseltamivir-resistant influenza a(h n ) virus in the united states world health organization (who). who. continued emergence and changing epidemiology of oseltamivir-resistant influenza a(h n ) virus, united kingdom, winter / a(h n ) influenza viruses in japan during the - season clinical implications of antiviral resistance in influenza oseltamivir resistance-disabling our influenza defenses global update on the susceptibility of human influenza viruses to neuraminidase inhibitors evaluation of intravenous peramivir for treatment of influenza in hospitalized patients systematic review of influenza resistance to the neuraminidase inhibitors structural and functional basis of resistance to neuraminidase inhibitors of influenza b viruses the potential impact of neuraminidase inhibitor resistant influenza mutations of neuraminidase implicated in neuraminidase inhibitors resistance recovery of drug-resistant influenza virus from immunocompromised patients: a case series surveillance and laboratory testing of influenza neuraminidase inhibitor five years of monitoring for the emergence of oseltamivir resistance in patients with influenza a infections in the influenza resistance information study. influenza other respir viruses a review of clinical influenza a and b infections with reduced susceptibility to both oseltamivir and zanamivir. open forum infect dis transmitted and acquired oseltamivir resistance during the - influenza season phe guidance on use of antiviral agents for the treatment and prophylaxis of seasonal influenza rapid detection and subtyping of human influenza a viruses and reassortants by pyrosequencing application of a seven-target pyrosequencing assay to improve the detection of neuraminidase inhibitor-resistant influenza a(h n ) viruses detection of rare drug resistance mutations by digital pcr in a human influenza a virus model system and clinical samples quantitative and sensitive detection of rare mutations using droplet-based microfluidics characterization of oseltamivir-resistant influenza virus populations in immunosuppressed patients using digital-droplet pcr: comparison with qpcr and next generation sequencing analysis baloxavir: first global approval treatment-emergent influenza variant viruses with reduced baloxavir susceptibility: impact on clinical and virologic outcomes in uncomplicated influenza mechanism of action of t- against influenza virus favipiravir (t- ), a novel viral rna polymerase inhibitor the mechanism of resistance to favipiravir in influenza failure of combination oral oseltamivir and inhaled zanamivir antiviral treatment in ventilator-and ecmo-treated critically ill patients with pandemic influenza a (h n )v efficacy of oseltamivir-zanamivir combination compared to each monotherapy for seasonal influenza: a randomized placebo-controlled trial oseltamivir-zanamivir combination therapy suppresses drug-resistant h n influenza a viruses in the hollow fiber infection model (hfim) system oseltamivirzanamivir combination therapy is not superior to zanamivir monotherapy in mice infected with influenza a(h n ) and a(h n )pdm viruses a randomized study of standard versus double dose oseltamivir for treating influenza in the community triple combination of amantadine, ribavirin, and oseltamivir is highly active and synergistic against drug resistant influenza virus strains in vitro combination therapy with amantadine, oseltamivir and ribavirin for influenza a infection: safety and pharmacokinetics triple-combination antiviral drug for pandemic h n influenza key: cord- -ooouqna authors: li, tao; wang, lianzi; wang, huihui; gao, yufeng; hu, xianwei; li, xuemei; zhang, shubing; xu, yuanhong; wei, wei title: characteristics of laboratory indexes in covid- patients with non-severe symptoms in hefei city, china: diagnostic value in organ injuries date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: ooouqna this study compared the laboratory indexes in non-severe covid- patients with those in healthy controls. in the peripheral blood system of non-severe symptom covid- patients, lymphocytes, eosinophils, basophils, total procollagen type amino-terminal propeptide, osteocalcin n-terminal, thyroid-stimulating hormone, growth hormone, and insulin-like growth factor–binding protein significantly decreased, and total protein, albumin, alanine transaminase, alkaline phosphatase, γ-glutamyl transferase, activated partial thromboplastin time, prothrombin time, fibrinogen, d-dimer, fibrinogen degradation products, human epididymal protein , serum ferritin, and c-reactive protein were elevated. sars-cov- infection can affect hematopoiesis, hemostasis, coagulation, fibrinolysis, bone metabolism, thyroid, parathyroid glands, the liver, and the reproductive system. since the unexplained viral pneumonia was reported in wuhan in december [ ] , local, national, and international transmission of severe acute respiratory syndrome coronavirus (sars-cov- ) [ ] has enabled it to spread rapidly to regions and countries around the world with more than million confirmed infections and , deaths [ ] . at present, there are many reports on the clinical characteristics of patients with severe disease and those who die [ ] [ ] [ ] ; however, there are few reports describing the clinical characteristics of non-severe patients. in this study, the clinical characteristics and laboratory indexes of patients with non-severe covid- treated in our hospital are described. additionally, we explored the characteristics and changing trends in these laboratory indexes. this was a cross-sectional study that recruited covid- patients from the first affiliated hospital of anhui medical university in hefei city, china, from january to march , diagnosed on the basis of results from the nucleic acid reverse transcription polymerase chain reaction (rt-pcr) test as well as the pathological changes observed in computed tomography (ct) images, and healthy individuals from the healthy examination center at the first affiliated hospital of anhui medical university over the same period, matched to the covid- patients based on age and gender. blood specimen collection two-milliliter blood samples were collected for all included participants after fasting for at least h. it was left standing for min and centrifuged for min at g within h. the serum was subsequently used to measure the different levels of biochemical markers, and abbreviations for the various biochemical markers are shown in table . furthermore, screening tests by serology for other pathogens were also conducted on the covid- patients. these tests were conducted to identify the hepatitis c virus antibody, the treponema pallidum antibody, the human immunodeficiency virus antibody, the hepatitis b surface antigen, the hepatitis b surface antibody, the hepatitis b e-antigen, the hepatitis b e-antibody, the hepatitis b core antibody, the serum igm antibody for q fever and rickettsia, mycoplasma pneumoniae, chlamydia pneumoniae, parainfluenza (including subtypes , , and ), respiratory syncytial virus, influenza a and b viruses, legionella pneumophila, and adenovirus. the covid- patients and controls were assessed at four different time points based on the number of hospital admission days: time , the st day of admission; time , the th day of admission; time , the th day of admission; time , the th day of admission. all patients received four serological examinations. spss (version ) was used for the statistical analyses of the data. two-sample t tests were used to analyze the differences in the continuous variables between two groups. one-way anova was to analyze the differences in the continuous variables between multiple groups; thereafter, the snk-q test was used to make comparisons between all of the groups. all continuous variables are expressed as means ± standard deviations (sd). chi-squared tests (χ ) were used for comparisons between categorical variables. p values less than . were considered statistically significant. statistical charts were drawn using graphpad prism . the lymph, eo, and bso counts in patients with covid- were significantly lower, compared with the controls on the st, th, th, and th days of admission, while the mono counts were significantly higher in covid- patients ( fig. a-d) . lymph, eo, and bso all presented upward trends. analyses of liver, skeletal muscle, and myocardial indexes tp and alb decreased significantly compared with the controls and showed a progressive downward trend, but glo was increased, which led to the decrease of a/g ( . ± . vs . ± . , p = . ) (fig. a-d) . overall, . % of the patients had a transient increase in alt and/or ast ( table ) ; eight of them had a simultaneous increase in ɑ-hbdh, my, ck, or ck-mb suggesting that these patients had liver injuries and some patients had skeletal muscle injuries ( table ) . there were abnormalities in cre, egfr, urea, and ca + in covid- patients (figs. a-c). in the process of continuous monitoring, the expression of cre in patients with covid- were significantly lower than those in the controls on the st, th, and th days of admission, and showed an overall downward trend (fig. a) . the expression of ca + in patients with covid- were significantly lower than those in the controls on the st, th, th and th days of admission, and showed an overall upward trend (fig. d ). *because the concentrations of il- β in patients and il- in controls are below the lower limit of the measurement range, we use the lower limit value of the measurement range as statistical data analyses of hemostasis, coagulation, and fibrinolysisrelated systems aptt and pt for covid- patients were significantly longer than those in controls, and fdp, d-d, fib, and pt-inr were higher compared with those in the controls (table and fig. a f). the expression of fib in patients with covid- were significantly higher as compared with those in the controls on the st, th, th, and th days of admission, and % of the patients' results were higher than the reference range ( table ) . analyses of thyroid axis and growth hormone axisrelated markers, the parathyroid gland, and bone metabolism-related markers the expression of pth was elevated in covid- patients on the th, th, and th days of admission (table and fig. a ). the expression of tsh, ft , p np, n-mid oc, (oh)d , gh, and igfbp- in covid- patients decreased compared with those in controls (fig. b-h) . the expression of he , sf, and crp significantly increased in covid- patients during the early stages of the disease (fig. b-d) . however, the expression of progrp, il- β, and il- significantly decreased in covid- patients compared with those in controls (fig. a , e, f). our study found that in the early stages of disease, lymph, eo, and bso counts were lower in covid- patients than in controls. in acute infection or inflammation, the number of circulating eos decreases rapidly and continuously [ ] . it is worth noting that two eosinophil granule proteins, eosinophil cationic protein (ecp) and eosinophil-derived neurotoxin (edn), neutralize most viruses [ ] . the decreasing eso in covid- patients may be caused by the sars-cov- virus attacking ecp and edn. besides, both bso and eo can produce il- , which is an important cytokine to stimulate the proliferation of activated b and t cells [ ] . therefore, the decrease of eo and bso counts in covid- patients may further lead to the decrease of lymph counts. impairment of liver function caused by sars-cov- has also been reported in many studies recently [ , ] . our results showed that the serum tp and alb of patients decreased significantly while the level of glo increased, leading to the abnormal frequency of a/g in the early stages of the disease. additionally, the discovery that eight covid- patients had a simultaneous increase in ɑ-hbdh, my, ck, or ck-mb suggested that we should pay attention to patients with different degrees of skeletal muscle injuries. it has been reported that the human kidneys are a specific target for sars-cov- infection [ ] . our results showed that there were abnormalities in cre, egfr, and urea in covid- patients, mainly in cre. in the process of continuous monitoring, we also found that the cre levels on the th, th, and th day of admission in covid- patients were our results also showed that the aptt of covid- patients were longer than that of controls, and the indexes of fdp, d-d, and fib were higher than those of the control, suggesting that sars-cov- has an important effect on the hematopoietic system and hemostasis. coagulation abnormalities such as prolonged pt and aptt, increased fibrin degradation products, and disseminated intravascular coagulation (dic) require continuous vigilance and timely interventions. we found that n-mid oc, pinp, and tsh were significantly decreased, while pth increased in covid- patients. some studies have already highlighted that sars-cov- can affect thyroid function [ , ] . therefore, thyroid damage caused by sars-cov- also needs further exploration. we found that gh and igfbp- significantly decreased in the serum of covid- patients, suggesting that sars-cov- may invade the central nervous system. baig et al. reported that the detection of sars-cov- in the cerebrospinal fluid of covid- patients suggests that the cns may be invaded by sars-cov- [ ] . inflammatory infection can promote the synthesis of ferritin and lead to an increase in ferritin. our study showed that sf and crp abnormally increased in covid- patients. an analysis of the data from covid- patients showed that the performance of sf in the patients significantly differed from that in the healthy control, which is consistent with our findings [ ] . sf needs to be further studied to assist in the diagnosis of sars-cov- infection or to be used to monitor disease activity, and response to treatment in sars-cov- patients. this study had some limitations. first, our sample size was small thus limiting the validity of our findings. second, the cross-sectional survey used in this study limited our ability to identify etiological factors associated with covid- patients. third, we were unable to obtain all the clinical and treatment information of the subjects enrolled in the study, which limited us to having an analysis combined with clinical feature and limited us in explaining the phenomenon fig. the expression of he , sf, crp, progrp, il- β, and il- in covid- patients (a-f) caused by sars-cov- . in further studies, we could focus on the specific biochemical marker difference in covid- patients, and try to figure out the mechanism. severe acute respiratory syndrome coronavirus (sars-cov- ) infection can affect hematopoiesis, hemostasis, coagulation, fibrinolysis, bone metabolism, thyroid and parathyroid glands, the liver, and the reproductive system. conflict of interest the authors declare that they have no conflicts of interest. ethics approval this retrospective chart review study involving human participants was in accordance with the ethical standards of the institutional and national research committee and with the helsinki declaration and its later amendments or comparable ethical standards. the ethics committee of the first affiliated hospital of anhui medical university approved this study and waived informed consent as a retrospective observational study. informed consent this study was a retrospective study, so formal consent was not required. china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia available online: https://gisanddata.maps.arcgis. c o m clinical features of covid- in elderly patients: a comparison with young and middle-aged patients clinical and immunologic features in severe and moderate coronavirus disease the clinical and chest ct features associated with severe and critical covid- pneumonia mechanisms of eosinophil-associated inflammation structure and chromosome localization of the human eosinophil-derived neurotoxin and eosinophil cationic protein genes: evidence for intronless coding sequences in the ribonuclease gene superfamily type and type cytokine dysregulation in human infectious, neoplastic, and inflammatory diseases a comparative study on the clinical features of covid- pneumonia to other pneumonias clinical characteristics of cases of corona virus disease (covid- ) in changsha human kidney is a target for novel severe acute respiratory syndrome coronavirus (sars-cov- ) infection. medrxiv neurological manifestations in covid- caused by sars-cov- a pathological report of three covid- cases by minimally invasive autopsies publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -f tzt jb authors: karolyi, m.; pawelka, e.; omid, s.; kelani, h.; mader, t.; baumgartner, s.; laferl, h.; traugott, m.; seitz, t.; zoufaly, a.; wenisch, c. title: late onset pulmonary embolism in young male otherwise healthy covid- patients date: - - journal: eur j clin microbiol infect dis doi: . /s - - -x sha: doc_id: cord_uid: f tzt jb sars-cov- infection is associated with increased risk of thrombosis in severely ill patients but little is known about the risk in outpatients with mild to moderate disease. our case series consists of four male otherwise healthy patients between and years of age. initial symptoms completely resolved but they developed new onset of dyspnea and thoracic pain at days to . ct scan revealed pulmonary embolism in all patients which led to hospitalization. standard anticoagulation practice needs to be re-evaluated and may be considered for certain outpatients with covid- . the sars-cov- pandemic led to more than , deaths worldwide, by may , (https://www.ecdc.europa.eu/ en/geographical-distribution- -ncov-cases). several risk factors for severe disease and mortality have been identified, e.g., advanced age, higher sofa score, elevated ferritin and ldh, number of comorbidities, decreased lymphocyte count, hypoalbuminemia, and elevated d-dimer levels [ , ] . abnormal coagulation parameters are a key feature of covid- especially in critically ill patients. one study showed that % of non-survivors fulfilled the laboratory criteria of dic [ ] . studies showed reduced mortality in hospitalized covid- patients treated vs not treated with anticoagulants in patients with a sepsis-induced-coagulopathy (sic) score ≥ or d-dimer > times upper limit of normal [ ] . thromboprophylaxis via low-weight-molecular heparin (lwmh) in hospitalized patients is commonly practiced but usually not considered necessary in outpatients. we might have to revaluate this practice in covid- patients and consider prophylactic anticoagulation even in mild cases treated on an outpatient basis. we describe a case series of four outpatients with proven sars-cov- infection who developed pulmonary embolism (pe) with a delay of - weeks after symptom onset with complete resolution of initial symptoms. four patients with suspected covid- who did not need initial hospitalization were visited by mobile sars-cov- sampling teams in vienna, austria. those trained professionals collected nasopharyngeal swabs and diagnosis of covid- was made via sars-cov- pcr in certified laboratories. after initial resolution of symptoms patients had to be admitted to the kaiser-franz-josef hospital because of new onset of symptoms. this hospital is the main treating facility for proven sars-cov- infected patients in vienna. collection of blood samples and ct scans took place there. data was collected retrospectively from patients' records. no statistical analysis was performed. patient's data are described descriptively. all patients were male and between and years of age. three of them had no underlying disease; one had bronchial asthma. none of them took medications regularly. two patients were overweight with a bmi of and , respectively. all patients initially complained of cough and fever and some felt weak or had throat pain as well. three patients were initially managed as outpatients in home isolation and did not receive any anticoagulation or further medication. initial symptoms completely resolved in these patients but they developed new onset of dyspnea and/or thoracic pain at days to and had to be admitted to the kaiser-franz-josef hospital. one patient (patient ) had to be admitted to the hospital due to covid- and received prophylactic anticoagulation (nadroparin . ml s.c. once daily) for days during his admission. he was discharged with complete resolution of symptoms but developed new onset of dyspnea the following day which lead to re-admission. inflammation markers were only slightly elevated in of patients on the day of pe diagnosis. no abnormalities in common coagulation tests were observed. two of the patients were already sars-cov- negative in nasopharyngeal swabs on the day of pe diagnosis. in of the patients, ecg showed sinus tachycardia ( - bpm), one patient had a right axis deviation. ct scans revealed large bilateral pe in two patients and smaller unilateral pe in the other two patients. d-dimer levels varied between . and . mg/l and where higher in patients with bilateral pe. none of the patients had signs nor history of deep vein thrombosis. after diagnosis of pulmonary embolism was established, patients were started on anticoagulation therapy with lwmh and direct oral anticoagulation (doac) and recovered. for further details see table . immobilization, dehydration, inflammation-induced hypercoagulability, and genetic thrombophilia are established risk factors for thrombosis associated with many bacterial and viral infections. our patients did not have any history of prior deep vein thrombosis, pe, or any other clotting disease. common clotting tests did not show any abnormalities beside elevated d-dimer levels. a chinese study found elevated d-dimer and decreased antithrombin levels even in non-severely ill patients [ ] . the prevalence of dvt in a small study of hospitalized covid- patients with a median age of years and % females who did not receive prophylactic anticoagulation was % [ ] . this unusual high rate of coagulopathies associated with covid- suggest additional causes and warrants further investigation. bmi body mass index, pe pulmonary embolism, pt prothrombin time, appt activated partial thromboplastin time, ecg electro cardiogram, bpm beats per minute, lwmh low weight molecular heparin, ()normal ranges sars-cov- enters human cells via the angiotensinconverting enzyme (ace ) receptor which may lead to a depletion of ace with the consequence of increased angiotensin ii (at ii) levels [ ] . studies showed that at ii stimulates tissue-factor (tf) expression in vascular endothelial cells [ ] and release of plasminogen activator inihibor- (pai- ) from adipocytes which leads to increased coagulation and impaired fibrinolysis [ ] . recently, a small chinese study showed an association between at ii levels, viral load, and severity of lung injury [ ] . this effect may be even more pronounced in overweight patients. pe was more severe in our overweight patients. furthermore, hypoxia is a common feature of covid- and might contribute to increased risk of thrombosis via hypoxemia-induced vasoconstriction which is a physiological reaction in the lung [ ] . none of our patient received oxygen supplementation before the onset of pe, and oxygen requirement was not monitored during their home isolation. therefore, we cannot rule out hypoxic states during this time. all patients showed a disease trajectory characterised by two peaks. this should raise suspicion of pe even in patients who received prophylactic anticoagulation as shown by patient . he received days of lwmh and did not show any clinical signs of dvt nor pe during hospitalisation but developed pe one day after discharge. a subclinical pe may have been unrecognised in patient during his hospital stay but is unlikely because he did not require any oxygen and felt completely well at the time of discharge. in summary, the late onset of pe after complete remission of initial covid- symptoms, which is shown here the first time, raises the question if standard anticoagulation practice needs to be revaluated and expanded to outpatients. further clinical research on timing, duration, and type of anticoagulation in outpatients with covid- is urgently needed. the characteristics of outpatients who are suitable for anticoagulation have to be determined.in conclusion, new onset of dyspnea and tachycardia after initial resolution of covid- symptoms ("disease trajectory characterised by two peaks") should raise suspicion of pe and a ct scan should be considered. risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease pneumonia inwuhan, china clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study. the lancet abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia anticoagulant treatment is associated with decreased mortality in severe coronavirus disease patients with coagulopathy prominent changes in blood coagulation of patients with sars-cov- infection prevalence of venous thromboembolism in patients with severe novel coronavirus pneumonia renin-angiotensin-aldosterone system inhibitors in patients with covid- angiotensin ii, tissue factor and the thrombotic paradox of hypertension angiotensin ii and its metabolites stimulate pai- protein release from human adipocytes in primary culture clinical and biochemical indexes from -ncov infected patients linked to viral loads and lung injury hypoxic pulmonary vasoconstriction publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that they have no conflicts of interest. key: cord- - f las y authors: wielders, c. c. h.; hackert, v. h.; schimmer, b.; hodemaekers, h. m.; de klerk, a.; hoebe, c. j. p. a.; schneeberger, p. m.; van duynhoven, y. t. h. p.; janssen, r. title: single nucleotide polymorphisms in immune response genes in acute q fever cases with differences in self-reported symptoms date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: f las y genes involved in human immune response are well recognized to influence the clinical course of infection. the association of host genetics with susceptibility to and severity of clinical symptoms in acute q fever was investigated. single nucleotide polymorphisms (snps) in the ifng (rs /rs ), stat (rs ), and vdr (rs ) genes were determined in patients from the dutch acute q fever outbreak, and a symptom score was calculated. ifng rs showed a significant association with the symptom score; ifng rs showed a similar trend. these snps were then used to reproduce results in a outbreak population (n = ). the median symptom score differed significantly in both populations: versus . the significant association of ifng rs with symptom score in the first population was not reproduced in the second population. we hypothesize that individuals in the second outbreak were exposed to a higher coxiella burnetii dose compared to the first, which overruled the protection conferred by the a-allele of ifng rs in the first population. from through , the netherlands faced the largest q fever outbreak recorded to date (> , notified cases) [ , ] . the causative agent of q fever is the intracellular bacterium coxiella burnetii. c. burnetii-infected individuals can remain asymptomatic or develop a flu-like illness, pneumonia, or hepatitis, known as acute q fever [ ] . approximately % of symptomatic acute q fever cases progress to chronic q fever [ ] . genes involved in human immune response, and also other genes, are well recognized to influence the clinical course of infection, especially in case of pathogens with cell dependency similar to that of c. burnetii. significant immunogenetic differences have been found comparing patients suffering from chronic sequelae due to q fever [q fever endocarditis or prolonged post-infection fatigue (i.e., q fever fatigue syndrome)] to patients who had an uncomplicated recovery from acute q fever or subjects from the general population [ , ] . polymorphic variations in individual "candidate genes" [single nucleotide polymorphisms (snps)] assumed to be of direct importance in pathogenesis may help clarify the relation between immune response genes and varying degrees of disease severity or susceptibility of subpopulations [ ] [ ] [ ] [ ] [ ] [ ] . the aim of this study was to assess whether snps in several immune response genes influence susceptibility to and severity of an acute c. burnetii infection, based on selfreported symptoms. we also compared the allelic frequencies observed in these individuals to frequencies in the general population. four snps in genes involved in innate and adaptive immunity were selected to study this association. the first notified cases in the dutch q fever outbreak ( ) ( ) ( ) ( ) were residents of a village in the southern parts of the netherlands (herpen), where a case-control study was performed for source identification and risk factor analysis in [ ] . this outbreak was likely caused by abortions on a single dairy goat farm in close vicinity to the village. acute q fever cases were selected from participants of this study [at least igm phase ii and/or igg phase ii antibodies against c. burnetii antigens ≥ : (indirect immunofluorescence assay, ifa; focus diagnostics, cypress, ca, usa)], based on available questionnaire data (self-reported symptoms), including no more than one person per household, and giving consent for the investigation of genetic differences. this study, including our analysis, was approved by the medical ethical committee of the university medical center utrecht (reference number: - ). an abortion wave on a dairy goat farm led to another singlepoint source outbreak in the southernmost part of the netherlands in (voerendaal). farm residents, employees, and visitors were serologically screened for c. burnetii and assessed for self-reported symptoms by means of a questionnaire almost identical to the one used in study population and administered immediately following laboratory notification [ ] . laboratory-confirmed acute q fever cases were defined as: a positive c. burnetii dna polymerase chain reaction (pcr) test and/or the presence of igm phase ii and/or igg phase ii antibodies [screening by enzyme-linked immunosorbent assay (elisa; serion elisa classic, institut virion\serion gmbh, würzburg, germany) and confirmation by ifa ≥ : (fuller laboratories, fullerton, ca, usa)] [ ] . in addition, laboratory-confirmed community cases of acute q fever subsequently notified to the regional public health service between march and april received a questionnaire with questions on age, sex, and specific symptoms within days following the laboratory diagnosis. in march , all farm and community cases who had earlier returned the questionnaire were selected for participation in our study, following approval by the medical ethical committee of the maastricht university medical centre (reference number: - - ). four snps in three candidate genes involved in innate and adaptive immunity to infection were selected based on previously published associations with various diseases: two snps in the ifng gene [interferon-γ (ifnγ); rs and rs ], one in stat (signal transducer and activator of transcription factor ; rs ) involved in the ifnγmediated signal transduction, and one in vdr (vitamin d receptor; rs ). the defense against intracellular c. burnetii mainly depends on cell-mediated immunity, including ifnγ-mediated macrophage activation [ ] [ ] [ ] . the ifnγ pathway is believed to be crucial for the host defense against this intracellular pathogen [ , ] , as well as other intracellular bacteria, like mycobacterium spp. [ , [ ] [ ] [ ] , but has also been associated with outcomes of, for example, severe acute respiratory syndrome (sars) [ ] , respiratory syncytial virus infection [ , ] , and gastroenteritis episodes [ ] . consequently, it is reasonable to assume that polymorphisms in the ifng gene and that of its receptor will modulate the ifnγ host pathogen (c. burnetii) interaction process [ ] . a previous study by vollmer-conna et al. showed a significant influence of functional polymorphisms in the ifng loci (rs ) on the severity and duration of illness after infection with several pathogens, including c. burnetii [ ] . vitamin d stimulates the innate immune response, but suppresses the adaptive immune response and is important in immunity to tuberculosis, diabetes, and respiratory syncytial virus [ ] [ ] [ ] . the association of these snps with other diseases increases the chance of selecting snps that are actually of functional importance. allelic frequencies of the four selected snps were investigated in study population . only those snps that showed a statistically significant association or a trend in association with disease severity were used for reproduction in population . the allelic frequencies for a dutch community control group were also available ("regenboog" study, a large dutch population health examination survey in including randomly selected individuals of all ages [ ] ). blood samples from population were collected and analyzed late and early for those patients that gave permission for the genetic analysis. subjects from population , selected for participation based on a returned questionnaire, received a self-administrable buccal swab kit accompanied by an information folder and an informed consent form in march . for both populations, dna was isolated as described by hoebee et al. [ ] using the qiaamp dna blood mini kit (qiagen nv, venlo, the netherlands). polymorphisms were genotyped using predesigned or custom taqman snp genotyping assays (life technologies, bleiswijk, the netherlands). for each sample, . μl of taqman fast universal pcr master mix (life technologies) and ng of genomic dna were used in a total volume of μl. primer and probe sequences and assay numbers are as described by doorduyn et al. [ ] . the protocol for amplification was s at °c and cycles of s at °c and s at °c. all genotyping assays were performed on a fast real-time pcr system (life technologies). questionnaire both questionnaires included demographic information and the following q fever-related symptoms: fever (> °c), malaise, headache, cough, severe fatigue, shortness of breath or respiratory difficulties, pain or pressure on the chest, diarrhea, joint pain, night sweating, loss of weight, itch, clinical diagnosis of jaundice/hepatitis, and clinical diagnosis of pneumonia. hospitalization at the time of the acute c. burnetii infection was also recorded. participants with missing self-reported symptom data were excluded. for each participant, a symptom score was calculated as the sum of all reported symptoms (range - ). the median score and corresponding interquartile range (iqr) were calculated for both study populations, and the mann-whitney u-test was used to assess statistical significance for the difference in the median score between both populations. multiple regression analysis was used to confirm independence of the symptom score from age and gender. the genotype data of all tested snps were used to estimate hardy-weinberg equilibrium by the comparison of genotype frequencies within population , population , and the community controls by a chi-square test [ ] . to study the association of the determined snps with the symptom score, allele frequencies of each snp in subjects who indicated no symptoms at all were compared with allele frequencies in subjects who reported at least one symptom ("symptomatic") by using cross-tabulations and chi-square tests. odds ratios (ors) and % confidence intervals ( % cis) were calculated as well. the same type of comparisons were performed for subjects with a symptom score up to and including the median number of symptoms ("symptomatic") compared with subjects with a symptom score higher than the median score ("severe symptomatic"). allele frequencies of the "symptomatic" and "severe symptomatic" groups were also compared with the allele frequencies of a dutch community control group. the distribution of the symptom score for the different genotypes was assessed as well. data were analyzed using ibm spss statistics version . . (spss inc.). all tested polymorphisms were in hardy-weinberg equilibrium (p> . ) in all three groups (population , population , and community controls). there was no substantial relationship between symptom score and the demographic factors age and sex. study population consisted of acute q fever cases after excluding three subjects with missing symptom data. the median age of the patients was years (iqr: - ) and ( %) were male. the self-reported symptom frequency ranged between % for hepatitis/jaundice and % for malaise, and % of the cases had been hospitalized ( table ) . the median symptom score was (iqr: - ). table presents the allele distribution of each snp by using the two different classifications of "symptomatic" (at least one symptom and "severe symptomatic", i.e., a symptom score above the median score). the dutch community control group consisted of , persons, of which % was male. a statistically significant difference was observed for both ifng snps when subjects who did not report any symptoms were compared to subjects who reported at least one symptom ( p= . ). a similar result was found for one of the ifng snps (rs ) when using the median symptom score as the cut-off (the a-allele was less frequently observed among the participants with the above median symptom score), while the other ifng snp (rs ) showed a trend in association in the same direction (the t-allele was less frequently observed among the participants with the above median symptom score), though it was no longer statistically significant. the gg genotype (rs ) and aa genotype (rs ) seemed to increase the symptom score (fig. ) , though the combination of both genotypes was not significantly associated with the two symptom classifications used. the other snps did not show an association with the symptom score. therefore, the two ifng snps were selected as the most likely candidates for reproduction of our results in study population . compared with the general dutch population, the a-allele of the ifng rs snp was more often present in participants with a low symptom score and less often in participants with a high symptom score; a borderline significant result was found for participants with a higher symptom score compared with the community controls (or: . ; % ci: . - . ; p= . ). from study population , individuals were invited, of whom ( %) submitted a buccal swab sample. three participants were excluded because only one member per household could be included and another three because of missing symptom data. in the remaining cases, the median age was years (iqr: - ) and ( %) were male; the self-reported symptom frequency ranged between % for hepatitis/jaundice and % for malaise; hospitalization was reported in % of the cases ( table ) . the median symptom score was significantly higher than in population : (iqr: - ; p< . ). non-responders from population did not differ from responders according to age, gender, and median symptom score (data not shown). figure shows the distribution of the symptom scores in both study populations. as there were only four patients who reported no symptoms in this second population, only the "severe symptomatic" classification with above median symptom score could be used for analysis (table ) . no statistically significant association between snp allele frequency and severity of symptoms was observed in this population. there is a less clear distinction between the genotypes and symptom score compared to population (fig. ) , i.e., all genotypes show moderate to high symptom scores. we also investigated whether fever in combination with other symptoms, hospitalization, pneumonia, or hepatitis was associated with the allele frequencies of the snps, but no clear associations were found. this study investigated the association of four snps in several immune response genes with susceptibility to and severity of self-reported symptoms in acute c. burnetii infection. both ifng snps seemed to be related to the symptom score in population . this association remained significant for the ifng rs snp irrespective of the cut-offs for symptom severity chosen (protection conferred by the a-allele, gallele is the risk allele), while a trend in association was found for the ifng rs snp (protection conferred by the tallele, a-allele is the risk allele). the other snps did not show an association with the symptom score. the associations found in the ifng snps in population , however, could not be reproduced in population . the a-allele of the ifng rs snp is associated with low ifnγ production [ , ] . this a-allele has been reported to significantly increase the susceptibility to develop tuberculosis [ , [ ] [ ] [ ] and sars [ ] . a dose-dependent association was found for this a-allele with susceptibility to sars [ ] . pacheco et al. suggested that the increased levels of ifnγ in the early events during infection could probably control the replication and spread of m. tuberculosis or other intracellular pathogens [ ] . with respect to disease severity, however, significant associations of the t-allele with a more severe acute sickness response to infection have been reported previously [ , ] . this suggests that high or low levels of ifnγ can have different effects on disease, depending on the outcome under investigation. fig. box plots of the ifng genotype and symptom score in study populations and : a ifng rs , b ifng rs . the numbers above the bars indicate the number of cases, the horizontal lines within the boxes represent the median symptom score, the lower and upper boundaries of the boxes represent the th and th percentiles, respectively, and the t-bars represent the . th and . th percentiles. outliers are indicated by the dots there are several explanations for our observation that findings from population could not be reproduced in population . the first observation is that, surprisingly, symptom severity appeared to be much higher in population compared to population , based on symptom scores and hospital admission rates ( vs. %). a possible explanation could be recall bias in study population , as participants were asked in september about their symptoms between may and july . nevertheless, the difference in symptom scores seems too large to be solely explained by recall bias in population . furthermore, self-reported hospital admission rates seem highly unlikely to be affected by recall bias. higher symptom severity in population could possibly be explained by higher environmental exposure dose, as reflected by the large numbers of abortions in goats in the outbreak and extremely high attack rates in farm contacts [ ] . a high c. burnetii dose in the environment is likely to lead to a higher probability of symptoms (i.e., a higher attack rate), even in less susceptible individuals. a dose-dependent attack rate has earlier been described for salmonella infections in humans [ , ] . a similar relationship for c. burnetii is suggested by experimental animal data [ ] , and a human dose-response model for c. burnetii was recently published as well [ ] . evidence from these studies suggests that higher doses may be likely to overrule the immune system of the exposed person, resulting in an increased probability of illness. this hypothesis, in the light of the findings from our study, leads us to presume that a high dose will not only cause symptoms in the genetically susceptible individuals, but also in those genetically less prone to symptoms. in other words, the effects of heterogeneity in host susceptibility are diminished or even extinguished. although we cannot exclude some influence of recall bias on our results of population , we presume that a higher c. burnetii dose was present in population in compared to population in . modeling studies investigating human exposure might give more insight in this hypothesis. an attempt to rule out dose effects in population by excluding subjects who we assumed had the highest degree of exposure [farm residents, employees, visitors, and people living close (< km) to the farm as a measure of exposure dose] still did not enable us to reproduce our results from population . this may be due to a loss in statistical power because of excluding a large number of subjects from our analysis. besides, the question remains as to whether the criteria we used for exclusion was a good measure for high exposure, even though it was the best available option in our study. symptoms associated with q fever are non-specific and are also common in other respiratory tract infections, such as influenza. it can, therefore, be questioned whether the reported symptoms can be ascribed to other respiratory tract infections. although the two outbreaks took place in different years, both occurred as seasonal peaks in spring (april-june), which corresponds to the main lambing season in goats [ , ] . influenza, however, mostly occurs in winter and the incidence in spring was low in both years in the netherlands [ ] . therefore, it is highly unlikely that other respiratory diseases played a major role in the two populations used for this study. in conclusion, a significant difference was found for the ifng rs snp between persons with a mild or more severe presentation of acute q fever, which was not confirmed in a second study population. such an effect could not be observed in the second outbreak due to the observed high rates of severe symptoms, possibly saturating the effects of host susceptibility factors. the - q fever epidemic in the netherlands: characteristics of notified acute q fever patients and the association with dairy goat farming shifting priorities in the aftermath of a q fever epidemic in to in the netherlands: from acute to chronic infection bell am ( ) q fever european centre for disease prevention and control (ecdc) ( ) risk assessment on q fever. ecdc immune response genes in the post-q-fever fatigue syndrome, q fever endocarditis and 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participants. key: cord- -prgj g authors: lehtoranta, l.; pitkäranta, a.; korpela, r. title: probiotics in respiratory virus infections date: - - journal: eur j clin microbiol infect dis doi: . /s - - -y sha: doc_id: cord_uid: prgj g viral respiratory infections are the most common diseases in humans. a large range of etiologic agents challenge the development of efficient therapies. research suggests that probiotics are able to decrease the risk or duration of respiratory infection symptoms. however, the antiviral mechanisms of probiotics are unclear. the purpose of this paper is to review the current knowledge on the effects of probiotics on respiratory virus infections and to provide insights on the possible antiviral mechanisms of probiotics. a pubmed and scopus database search was performed up to january using appropriate search terms on probiotic and respiratory virus infections in cell models, in animal models, and in humans, and reviewed for their relevance. altogether, thirty-three clinical trials were reviewed. the studies varied highly in study design, outcome measures, probiotics, dose, and matrices used. twenty-eight trials reported that probiotics had beneficial effects in the outcome of respiratory tract infections (rtis) and five showed no clear benefit. only eight studies reported investigating viral etiology from the respiratory tract, and one of these reported a significant decrease in viral load. based on experimental studies, probiotics may exert antiviral effects directly in probiotic–virus interaction or via stimulation of the immune system. although probiotics seem to be beneficial in respiratory illnesses, the role of probiotics on specific viruses has not been investigated sufficiently. due to the lack of confirmatory studies and varied data available, more randomized, double-blind, and placebo-controlled trials in different age populations investigating probiotic dose response, comparing probiotic strains/genera, and elucidating the antiviral effect mechanisms are necessary. respiratory tract infections (rtis) are a major cause of morbidity and mortality worldwide. viral pathogens are the most common etiological agents of acute respiratory disease. the social and economic impact of viral respiratory disease is substantial, due to hospitalizations, medical costs, missed work, and school and day care absences. for instance, estimates show that viral respiratory tract illnesses (mostly common colds) cost us$ billion annually in the united states alone [ ] . there are over different types of viruses which cause rtis in humans. human rhinoviruses (hrv) are the largest group of respiratory viruses, comprising over serotypes [ ] . in humans, the predominant illness caused by hrv is the acute upper rti, also known as the common cold. the second most common viruses infecting humans are the human enteroviruses (hev), which are associated with clinical manifestations ranging from mild respiratory symptoms to serious conditions [ ] . influenza viruses, respiratory syncytial virus (rsv), and adenoviruses are also major causative agents of both upper and lower rtis [ ] [ ] [ ] . in addition, many other viruses or virus groups cause rtis, e.g., parainfluenza viruses and coronaviruses can cause a broad spectrum of respiratory diseases, ranging from mild upper rtis to pneumonia [ ] . in recent years, with the rapid development of high-throughput molecular techniques, several new viruses associated with respiratory diseases, such as human bocavirus, human metapneumovirus, and the new coronaviruses hku and nl , have been identified as well [ ] . the prevention of viral respiratory infections is an important challenge to public health. currently, the only effective antivirals and vaccines for the prevention and treatment of respiratory virus infections are available against influenza viruses and adenoviruses. for the viruses causing common cold (hrv, hev), no effective therapies are available. large varieties of etiologic agents and increasing antibiotic and antiviral resistance challenge the development of efficient therapies. consequently, it is of importance to find alternative and safe ways to reduce the risk of these infections. even partially effective therapy in the treatment and prevention of viral rtis such as the common cold could have an impact on reducing morbidity and economic losses due to this illness. probiotics are defined as live microorganisms that confer a health benefit on the host [ ] . the most common types of microbes used as probiotics are lactobacilli and bifidobacteria, which are generally consumed as part of fermented foods, such as yoghurts or dietary supplements. criteria for probiotic bacteria include that the bacterial strain: ( ) must be able to survive in the gastrointestinal tract and to proliferate in the gut; ( ) should exert benefits to the host through growth and/or activity in the human body; ( ) should be non-pathogenic and non-toxic; ( ) provide protection against pathogenic microorganisms by means of multiple mechanisms; and ( ) should be lacking transferable antibiotic resistance [ ] . different bacterial strains of the same genus and species, verified also by genomic information, may exert completely different effects on the host. the most promising health effects of probiotics in human intervention studies include the amelioration of acute diarrhea in children, relief of children's milk allergy/atopic dermatitis, and relief of irritable bowel syndrome [ , ] . probiotics are likely to have an impact through gut mucosa by balancing the local microbiota by inhibiting the growth of pathogenic microorganisms [ ] , and by enhancing local and systemic immune responses [ ] . they may also influence the composition and activity of microbiota in the intestinal contents. considering the beneficial effects of probiotics in virus infections, specific probiotics have been suggested to be effective in alleviating the duration and severity of acute rotavirus gastroenteritis [ ] . in addition, increasing evidence shows that probiotics are beneficial in rtis [ ] , which, in most cases, are of viral origin. however, the mechanisms behind these effects are largely unknown. the aim of this review is to present the current knowledge of the health effects of probiotics on rtis in humans, with a focus on viral respiratory infections. in addition, possible antiviral mechanisms of probiotics are discussed in context with studies conducted in vitro and in animal models. a pubmed and scopus database search was performed up to january to review the relevant literature investigating the effects of probiotics on respiratory virus infections in cell culture, animal models, and clinical trials. the following search terms were used individually and in combination: ' probiotic' , ' lactobacillus' , ' bif idobacterium' , 'lactococcus', 'respiratory infection', 'respiratory virus', and 'influenza virus'. animal experiments provide insight on the clinical effects of probiotics against respiratory virus infections ( children altogether, five clinical trials have been conducted in children using l. rhamnosus gg as a probiotic [ ] [ ] [ ] [ ] [ ] . in healthy children attending day care, l. rhamnosus gg reduced the number of children experiencing rtis [ , ] , the number of upper and lower rtis [ ] , and the number of antibiotic treatments or absences from day care [ ] . in another study, no differences were reported between the l. rhamnosus gg and the control groups in the number of antibiotic treatments or respiratory symptom episodes [ ] . however, in a subgroup with l. rhamnosus gg identification in feces, l. rhamnosus gg usage reduced the duration of rtis. in hospitalized children, l. rhamnosus gg reduced the risk of rtis and duration of rti episodes [ ] . in preterm infants, l. rhamnosus gg reduced the incidence of rtis [ ] . in addition, a meta-analysis of four randomized controlled trials investigating the role of l. rhamnosus gg in the prevention of respiratory infections in children showed that l. rhamnosus gg has the potential to reduce the risk of upper rtis, incidence of acute otitis media, and antibiotic use. there were no significant differences between the l. rhamnosus gg and the control groups in the incidence of lower rtis [ ] . there are seven studies conducted with probiotic bacteria other than l. rhamnosus gg. l. casei rhamnosus in children reduced the number of rtis [ ] . also, l. casei dn reduced the incidence rate for upper rtis and decreased the duration (days) and incidence of only lower rtis, but not upper rtis [ ] . l. fermentum cect with prebiotics in infants, however, reduced the incidence of both upper and lower rtis [ ] . the use of b. animalis ssp. lactis bb in healthy newborns was able to reduce the number of rtis as well, but was ineffective in reducing the occurrence of acute otitis media (aom) or symptoms of otitis media [ ] . in healthy infants, treatment with l. reuteri sd , but not with b. animalis ssp. lactis bb , resulted in fewer days of absence from day care due to illness, lower number of days with fever, and clinical visits. both strains were ineffective in reducing the incidence or duration of rtis [ , ] . in healthy children, l. casei crl or l. reuteri dsm did not reduce the incidence, number, or duration of acute rtis or rti episodes [ ] . the effectiveness of several combinations of probiotics on rtis has been investigated in four clinical trials. a combination of l. rhamnosus gg, l. rhamnosus lc , b. breve bb , and p. freudenreichii ssp. shermanii js in otitis-prone children [ ] or a combination of l. rhamnosus gg and b. animalis ssp. lactis bb in healthy newborns [ ] both reduced the occurrence of recurrent rtis, but not the incidence of aom. a combination of l. acidophilus and b. bifidum in healthy children reduced the duration of acute rti symptoms, school absence, and the risk of upper rti symptoms as well [ ] . however, a combination of bacteria including species of lactobacillus, bifidobacterium, streptococcus, and enterococcus was not able to reduce the number of rtis [ ] . the viral etiologies of rtis were investigated in only five studies. in preterm infants, l. rhamnosus gg decreased the incidence of rhinovirus-induced episodes, but not rhinovirus load [ ] . in otitis-prone children, a combination of l. rhamnosus gg, l. rhamnosus lc , b. breve bb , and p. freudenreichii ssp. shermanii js reduced human bocavirus load in the nasopharynx [ ] , but not picornaviruses [ ] . in healthy children attending day care, l. rhamnosus gg was not able to decrease significantly respiratory viruses (hrv, hev, influenza viruses, parainfluenza viruses, rsv, adenovirus, and human bocavirus) in the upper respiratory tract [ ] . healthy children receiving l. casei rhamnosus had significantly lower odds of viral infection diagnosed by a doctor and a significant difference in doctor-diagnosed rti. however, specific viruses were not reported in that study [ ] . probiotics' effectiveness in rtis has been addressed in studies in healthy adults, in athletes, and in individuals under stressful conditions. in healthy adults, l. fermentum cetc reduced the number of rtis and increased antigen-specific iga formation after influenza virus vaccination [ ] . in addition, a combination of l. gasseri pa / , b. longum sp / , and b. bifidum mf / reduced the duration of rti symptoms [ ] , duration of rti episodes [ , ] , but not the severity of rti symptoms [ , ] . none of these trials reported the effects of combinations on respiratory virus load, although their viral etiology was studied. b. animalis ssp. lactis bl- reduced the risk of an upper rti episode [ ] . a combination of l. rhamnosus gg and b. animalis ssp. lactis bb reduced both the duration of upper rti and the severity of rti symptoms [ ] . altogether, seven trials have been conducted among athletes or stressed individuals, but they did not report studying the viral etiology. in male elite distance runners, l. fermentum vri reduced the duration of rti symptoms, but not the incidence of rtis or the severity of symptoms [ ] . in competitive cyclists, l. fermentum (pcc) had some decreasing effects on the symptoms of upper rti in males, but not in females [ ] . in rugby union players [ ] , a combination of l. gasseri, b. bifidum, and b. longum reduced the incidence of upper rtis, but not the severity of symptoms. however, in marathon runners, l. rhamnosus gg did not decrease the number of rti episodes or the severity or the duration of rti symptoms [ ] . in addition, in commando trainers, l. casei dn was ineffective in reducing the incidence of rtis or rti symptoms [ ] [ ] [ ] ] . similarly, l. salivarius did not lower the number of rti episodes or reduce the severity or the duration of rti symptoms in trainers [ ] . however, in shift workers, l. casei dn reduced the number of rtis and increased the function of immune cell activity [ ] . only five studies have investigated the effects of probiotics on rtis, but not on the occurrence of specific viruses, in the elderly. l. casei dn decreased the duration of rtis [ , ] , but had no effect on the incidence of rtis [ ] . l. casei shirota did not have an effect on the number of upper rtis or the severity of upper rti symptoms, but probiotics decreased the duration of upper rtis [ ] . however, in another study, l. casei shirota had no effect on the duration of rti symptoms [ ] . a combination of l. rhamnosus gg, l. rhamnosus lc , b. breve bb , and p. freudenreichii ssp. shermanii js was ineffective in lowering the number of rtis and reducing the duration of rti symptoms. however, the combination reduced the duration of rti episodes [ ] . the clinical trials in children, adults, and the elderly presented in this review are summarized in table . a variety of probiotic strains have been used in these clinical trials, most of them belonging to the genus lactobacillus. in addition, various combinations of probiotics have been used. of studies, altogether, studies reported that probiotics had beneficial effects in the outcome of rtis and five showed no clear benefit. only eight studies, however, reported investigating the viral etiology. of these, only one study showed a statistically significant reduction in the virus load in the probiotic group. a cochrane systematic review by hao et al. concluded that probiotics were better than placebo in terms of reducing the number of upper rti episodes, the incidence of acute upper rti episodes, and antibiotics used [ ] . although clinical trials show that the use of specific probiotics and probiotic combinations are beneficial in rtis, there are also studies that report no clear advantage. in addition, several viruses can cause respiratory illnesses, but only a few studies have investigated probiotics' effectiveness on viral agents. the lack of consistent evidence between probiotic strains/genera and even within strains may be due to variation in study designs and reported outcome measures, the length of intervention, study populations used (children vs. adults) or bacterial doses ( - cfu), and matrices (milk, yoghurt, capsule) used. in addition, in the elderly, decreased immunity due to aging may partly explain the conflicting results [ ] . clinical and animal studies have demonstrated that specific probiotics have antiviral effects, but the underlying mechanisms are unclear. additionally, the strain-to-strain variation may be relatively large concerning strain properties and efficacy. possible antiviral mechanisms of probiotics include: ( ) hindering the adsorption and ( ) cell internalization of the virus; ( ) production of metabolites and substances with a direct antiviral effect; and ) crosstalk (immunomodulation) with the cells in establishing the antiviral protection. the possible mechanisms of probiotics against respiratory viruses are presented in fig. . the respiratory tract is covered by mucosal epithelial surfaces, which are constantly exposed to numerous microorganisms and serve as primary ports of entry for respiratory viruses. virus attachment to a host cell is the first essential step in the disease process, and, therefore, interruption of this attachment could be beneficial to the host. probiotic bacteria may bind directly to the virus and inhibit virus attachment to the host cell receptor. for instance, there is evidence that specific strains of lactobacilli are able to bind and inactivate vesicular stomatitis virus (flu-like virus) in vitro [ ] . probiotics may also show direct antimicrobial activity against pathogens by producing antimicrobial substances such as organic acids, hydrogen peroxide, biosurfactants, and bacteriocins [ ] . in experimental studies in epithelial cells and macrophages, metabolic products of specific lactobacilli and bifidobacteria prevented vesicular stomatitis virus infection in a strain-specific manner [ ] . in addition, metabolites of bacteria in yoghurts showed antiviral activity, inhibiting influenza virus replication [ ] . the induction of low-level synthesis of nitric oxide may also be involved in the protective actions of probiotics against viruses in the respiratory cells, as shown in alveolar macrophages in vitro [ , , ] . however, it should be noted that respiratory viruses infect cells with different mechanisms by using various receptors and, also, the antiviral effects of probiotics are strain-specific. the induction of antiviral cytokines such as interferons (ifns), as well as proinflammatory cytokines and chemokines, upon antigen recognition in epithelial cells or underlying effector cells [macrophages, dendritic cells (dcs), neutrophils] play a key role in virus infections by initiating cell-mediated viral elimination and adaptive immune responses. probiotics may mediate their antiviral effects against respiratory viruses possibly by eliciting systemic immune responses via gut or enhancing cellular immunity in the airways with increased activity of natural killer cells and macrophages. in the gut epithelial cells and/or antigen-presenting cells, probiotics are recognized by toll-like receptors (tlrs) [ ] [ ] [ ] [ ] . probiotics may, therefore, modulate cytokine expression patterns through epithelial cells [ ] and through underlying professional antigen-presenting cells, such as macrophages and dendritic cells [ ] [ ] [ ] [ ] [ ] [ ] . many experimental studies in vitro and in animals show that specific strains of probiotics are capable of providing protection against virus infections by stimulating antiviral, cytokine, and chemokine responses in the respiratory and gastrointestinal epithelial cells or immune cells. in murine dcs, l. acidophilus ncfm and l. acidophilus x induced the expression of viral defense genes (ifn-β, il- , il- ) [ ] . in human macrophages, l. rhamnosus lc induced table summarizes the effects of probiotic bacteria on cell-mediated immunity upon respiratory virus challenge in animal models. data from animal studies indicate that strains of lactobacilli and bifidobacteria provide protection against respiratory virus infections also by inducing the synthesis of virus-specific immunoglobulins in the respiratory secretions and in serum [ , , , ] . in addition, studies in healthy human subjects suggest that specific probiotics may enhance the immunogenicity of viral vaccines. l. rhamnosus gg was effective in inducing protective immune response against the h n strain in influenza virus vaccine [ ] . moreover, l. fermentum cect ingestion in adults resulted in lower influenzalike illness, increased proportion of nk cells in blood, significantly higher tnf-α, and increased anti-influenza-specific iga and igm after influenza vaccination [ ] . the consumption of b. animalis ssp. lactis bb or l. paracasei ssp. paracasei l. casei also showed significantly greater increase in influenza virus vaccine-specific igg antibodies in plasma and secretory iga in saliva [ ] . in the elderly, the consumption of fermented yoghurt with l. casei dn- increased significantly influenza-specific antibody titers after influenza vaccination, especially against influenza b virus [ ] . these studies suggest that orally ingested lactobacilli and bifidobacteria have an adjuvant-like effect on the humoral responses. probiotics are frequently part of the normal gastrointestinal microbiota, and, therefore, probiotic therapy is generally considered as safe [ ] . however, probiotic therapy has raised potential safety concerns, including systemic infections, toxic or metabolic effects on the gastrointestinal tract, and the transfer of antibiotic resistance in the gastrointestinal microbiota [ ] . in rare cases, some studies have reported lactobacillus septicemia in children [ ] , in immunocompromised subjects [ ] , and detrimental effects in subjects with hepatitis [ ] . however, the european food safety authority (efsa) has concluded that there are no specific safety concerns regarding lactobacillus, bifidobacterium, or propionibacterium strains, as they have a long history of safe use in food [ ] . in addition, for instance in finland, increased consumption of probiotic products containing l. rhamnosus gg has not resulted in a significant increase in lactobacillus bacteremia [ ] and l. rhamnosus gg consumption is regarded as safe in immunocompromised human immunodeficiency virus (hiv)-infected patients [ ] . it should be taken into consideration that the safety of probiotics has not been as systematically investigated as in drugs, and the safety evaluation is partly based on long-term experience. the aim of this review was to summarize the current literature investigating the effects of probiotics in respiratory virus infections in cell models, in animal models, and in humans. in addition, possible antiviral mechanisms of probiotics in there are also contradictory data on probiotic use in the prevention of rtis. the variability in the outcomes between clinical trials studying probiotics' role in rtis may be explained by the use of different probiotic strains, bacterial dose, and matrices. in addition, it should be noted that the effects of probiotics are highly strain-specific and the adequate amount of bacteria transferred into the effector sites in the gut may be crucial. due to the lack of confirmatory studies and varied data available, more randomized, double-blind, and placebocontrolled clinical trials in different age populations investigating probiotic dose response, comparing probiotic strains, and elucidating the mechanisms of effects are necessary. as many animal studies show that probiotic administration through the nose is able to reduce viral titers and relieve clinical symptoms, nasal bacteriotherapy for viral rtis in humans could be worthy approach for consideration in the future. probiotics' ability to enhance local and systemic innate immunity during virus infection in animal experiments is a likely, yet unverified, effect mechanism behind beneficial effects, and an interesting area of future research. the inclusion of serological and immunological diagnostics, such as the identification of virus-specific immunoglobulins and cytokines, in clinical research would have clear benefits in providing valuable information on the effects of probiotics in respiratory virus infections. the economic burden of non-influenza-related viral respiratory tract infection in the united states picornavirus and enterovirus diversity with associated human diseases epidemiology and pathogenesis of influenza epidemiology and prevention of respiratory syncytial virus infections among infants and young children molecular evolution of human species d adenoviruses respiratory viruses other than influenza virus: impact and therapeutic advances new respiratory viral infections food and agriculture organization of the united nations/world health organization (fao/who) ( ) health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria guidance for substantiating the evidence for beneficial effects of probiotics: prevention and management of infections by probiotics probiotics and health: an evidence-based review antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens immunomodulatory effect of probiotic bacteria probiotics as prevention and treatment for diarrhea probiotics for preventing acute upper respiratory tract infections effect of intranasal administration of lactobacillus casei shirota on influenza virus infection of upper respiratory tract in mice reduction of influenza virus titer and protection against influenza virus infection in infant mice fed lactobacillus casei shirota oral administration of heat-killed lactobacillus plantarum l- enhances protection against influenza virus infection by stimulation of type i interferon production in mice efficacy of oral administration of heatkilled probiotics from mongolian dairy products against influenza infection in mice: alleviation of influenza infection by its immunomodulatory activity through intestinal immunity lactobacillus plantarum dk as a probiotic confers protection against influenza virus by modulating innate immunity oral administration of lactobacilli from human intestinal tract protects mice against influenza virus infection heat-killed lactobacillus gasseri tmc protects mice against influenza virus infection by stimulating gut and respiratory immune responses effects of oral administration of yogurt fermented with lactobacillus d e l b r u e c k i i s s p . b u l g a r i c u s o l l r - a n d i t s exopolysaccharides against influenza virus infection in mice oral administration of milk fermented with lactococcus lactis subsp. cremoris fc protects mice against influenza virus infection protection against influenza virus infection of mice fed bifidobacterium breve yit effect of long term consumption of probiotic milk on infections in children attending day care centres: double blind, randomised trial lactobacillus gg in the prevention of gastrointestinal and respiratory tract infections in children who attend day care centers: a randomized, double-blind, placebo-controlled trial lactobacillus gg in the prevention of nosocomial gastrointestinal and respiratory tract infections milk containing probiotic lactobacillus rhamnosus gg and respiratory illness in children: a randomized, double-blind, placebo-controlled trial prebiotic and probiotic supplementation prevents rhinovirus infections in preterm infants: a randomized, placebocontrolled trial the use of the probiotic lactobacillus rhamnosus gg and viral findings in the nasopharynx of children attending day care lactobacillus rhamnosus gg supplementation for preventing respiratory infections in children: a meta-analysis of randomized, placebo-controlled trials different effects of probiotic species/strains on infections in preschool children: a double-blind, randomized, controlled study effect of lactobacillus casei on the incidence of infectious conditions in children human milk probiotic lactobacillus fermentum cect reduces the incidence of gastrointestinal and upper respiratory tract infections in infants bifidobacterium animalis subsp. lactis bb- in reducing the risk of infections in infancy effect of a probiotic infant formula on infections in child care centers: comparison of two probiotic agents probiotics and otitis media in children randomized trial of probiotics and calcium on diarrhea and respiratory tract infections in indonesian children treatment of acute otitis media with probiotics in otitis-prone children-a double-blind, placebocontrolled randomised study specific probiotics in reducing the risk of acute infections in infancy-a randomised, double-blind, placebo-controlled study randomized controlled trial of probiotics to reduce common cold in schoolchildren human bocavirus in the nasopharynx of otitis-prone children probiotics in the prevention of clinical manifestations of common infectious diseases in children and in the elderly oral intake of lactobacillus fermentum cect enhances the effects of influenza vaccination effect of a dietary supplement containing probiotic bacteria plus vitamins and minerals on common cold infections and cellular immune parameters effect of lactobacillus gasseri pa / , bifidobacterium longum sp / , b. bifidum mf / on common cold episodes: a double blind, randomized, controlled trial probiotic bacteria reduced duration and severity but not the incidence of common cold episodes in a double blind, randomized, controlled trial probiotic supplementation for respiratory and gastrointestinal illness symptoms in healthy physically active individuals effect of lactobacillus rhamnosus lgg® and bifidobacterium animalis ssp. lactis bb- ® on health-related quality of life in college students affected by upper respiratory infections oral administration of the probiotic lactobacillus fermentum vri- and mucosal immunity in endurance athletes lactobacillus fermentum (pcc®) supplementation and gastrointestinal and respiratory-tract illness symptoms: a randomised control trial in athletes probiotic supplementation reduces the duration and incidence of infections but not severity in elite rugby union players the effect of probiotics on respiratory infections and gastrointestinal symptoms during training in marathon runners effect of a probiotics supplementation on respiratory infections and immune and hormonal parameters during intense military training effects of a lactobacillus salivarius probiotic intervention on infection, cold symptom duration and severity, and mucosal immunity in endurance athletes effects of consumption of a fermented dairy product containing the probiotic lactobacillus casei dn- on common respiratory and gastrointestinal infections in shift workers in a randomized controlled trial effect of fermented milk containing the probiotic lactobacillus casei dn- on winter infections in free-living elderly subjects: a randomised, controlled pilot study consumption of a fermented dairy product containing the probiotic lactobacillus casei dn- reduces the duration of respiratory infections in the elderly in a randomised controlled trial decreased duration of acute upper respiratory tract infections with daily intake of fermented milk: a multicenter, double-blinded, randomized comparative study in users of day care facilities for the elderly population efficacy of daily intake of lactobacillus casei shirota on respiratory symptoms and influenza vaccination immune response: a randomized, double-blind, placebo-controlled trial in healthy elderly nursing home residents use of a fermented dairy probiotic drink containing lactobacillus casei (dn- ) to decrease the rate of illness in kids: the drink study. a patient-oriented, double-blind, clusterrandomized, placebo-controlled, clinical trial probiotics and virus infections: the effects of lactobacillus rhamnosus gg on respiratory and gastrointestinal virus infections. dissertation a novel eukaryotic cell culture model to study antiviral activity of potential probiotic bacteria antiviral activities of cell-free supernatants of yogurts metabolites against some rna viruses interactions of macrophages with probiotic bacteria lead to increased antiviral response against vesicular stomatitis virus nitric oxide (no) production in mammalian nontumorigenic epithelial cells of the small intestine and macrophages induced by individual strains of lactobacilli and bifidobacteria lactobacilli and streptococci induce interleukin- (il- ), il- , and gamma interferon production in human peripheral blood mononuclear cells role of intestinal epithelial cells in immune effects mediated by gram-positive probiotic bacteria: involvement of toll-like receptors correlation between in vitro and in vivo immunomodulatory properties of lactic acid bacteria live lactobacillus rhamnosus and streptococcus pyogenes differentially regulate toll-like receptor (tlr) gene expression in human primary macrophages functional modulation of human intestinal epithelial cell responses by bifidobacterium infantis and lactobacillus salivarius potentially probiotic bacteria induce cytokine production and suppressor of cytokine signaling gene expression in human monocyte-derived macrophages lactobacilli and streptococci activate nf-kappa b and stat signaling pathways in human macrophages lactobacilli and streptococci induce inflammatory chemokine production in human macrophages that stimulates th cell chemotaxis streptococcus pyogenes and lactobacillus rhamnosus differentially induce maturation and production of th -type cytokines and chemokines in human monocyte-derived dendritic cells lactobacillus rhamnosus gg and streptococcus thermophilus induce suppressor of cytokine signalling (socs ) gene expression directly and indirectly via interleukin- in human primary macrophages lactobacillus acidophilus induces virus immune defence genes in murine dendritic cells by a toll-like receptor- -dependent mechanism lactobacilli and bifidobacteria induce differential interferon-beta profiles in dendritic cells nonpathogenic lactobacillus rhamnosus activates the inflammasome and antiviral responses in human macrophages augmentation of cellular immunity and reduction of influenza virus titer in aged mice fed lactobacillus casei strain shirota lactobacillus gg as an immune adjuvant for live-attenuated influenza vaccine in healthy adults: a randomized double-blind placebocontrolled trial evaluation of the immune benefits of two probiotic strains bifidobacterium animalis ssp. lactis, bb- ® and lactobacillus paracasei ssp. paracasei, l. casei ® in an influenza vaccination model: a randomised, double-blind, placebo-controlled study a probiotic fermented dairy drink improves antibody response to influenza vaccination in the elderly in two randomised controlled trials probiotic use in clinical practice: what are the risks? safety assessment of probiotics for human use lactobacillus sepsis associated with probiotic therapy lactobacillus rhamnosus infection in a child following bone marrow transplant probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial scientific opinion on the maintenance of the list of qps biological agents intentionally added to food and feed key: cord- -xd wxxf authors: monpoeho, s.; coste-burel, m.; costa-mattioli, m.; besse, b.; chomel, j.; billaudel, s.; ferré, v. title: application of a real-time polymerase chain reaction with internal positive control for detection and quantification of enterovirus in cerebrospinal fluid date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: xd wxxf a quantitative real-time reverse transcriptase-polymerase chain reaction (rt-pcr) method based on taqman technology was developed to determine the presence and amount of enterovirus rna. in order to prevent false-negative results, a one-step multiplex rt-pcr was optimized. it contains two dual-labelled fluorogenic probes to quantify the ′ noncoding region of enterovirus and detect an internal positive control. in the present study, cerebrospinal fluid samples collected during an outbreak of enteroviral meningitis were analyzed using this method. amplification of the internal positive control was effective in all but two specimens, confirming the absence of pcr inhibitors and allowing the results of amplification to be validated. the sensitivity of the rt-pcr was . %, while that of cell culture was . %. genomic viral loads found ranged between . and . log( ) copies per milliliter of cerebrospinal fluid (mean, . log( ) copies/ml). this fluorogenic enterovirus rt-pcr allows large numbers of samples to be screened rapidly. moreover, its sensitivity and reproducibility make it highly reliable. with these characteristics, the enterovirus rt-pcr can be a useful tool that may offer considerable benefit in the clinical management of patients with enteroviral infections. human enteroviruses (evs) of the picornaviridae family are the most common etiologic agents of aseptic meningitis. it is estimated that they cause - % of the cases of viral meningitis [ , ] . conventional laboratory diag-nosis of ev meningitis relies on isolation of the virus in cell culture with several cell lines. observation of cell cultures for the development of a cytopathic effect continues for about days and is then followed by neutralization typing. besides being time-consuming, this method is frequently unsuccessful because of the small volume of samples such as cerebrospinal fluid (csf) and the difficulty of propagating certain ev types in cell culture [ ] . as an alternative method, therefore, the use of molecular biology for the detection of ev genomes is of considerable interest. detection of evs by amplification of viral rna from csf using reverse transcriptase-polymerase chain reaction (rt-pcr) assay has already been reported [ , , ]. some studies have described the amplification of ev rna (by single or nested rt-pcr), followed by a variety of endpoint detection or quantification systems such as the colorimetric microwell hybridization assay [ , , , , ] . this last method is also time-consuming because of the post-pcr manipulations required, and its results are subject to error due to the plateauing effect [ ] . finally, substances present in the specimen can inhibit nucleic acid amplification and thus cause false-negative responses [ , ] . a new quantification method combining rt-pcr with a real-time detection system (abi prim ; pe applied biosystems, france) has recently been developed in our laboratory [ ] . up to now, false-negative responses have been averted by "spiking" all negative specimens with the rna standard for a second run, an impractical, laborious, and very expensive solution. in addition, the low amount of csf often available limits the ability to perform retesting. we therefore decided to improve our technique by incorporating an internal positive control (ipc) by which amplification and quantification results could be validated in a single run. the aim of the present study was to apply this technique by measuring the ev genomic viral load in csf collected during an outbreak of aseptic meningitis. between may and august , an outbreak of aseptic meningitis occurred in nantes, france. a total of csf specimens from young patients aged days to . years (median, . years) admitted to the hospital emergency department for neurological symptoms were collected in sterile containers and sent to our laboratory for diagnostic evaluation. csf samples were stored at - °c before being analyzed. cell culture and typing csf samples were cultured directly on two different cell lines (mrc- and hep- ). each type was inoculated with µl of specimen and then incubated at °c. cultures were observed daily for cytopathic effect characteristic of evs for days. cultures showing a cytopathic effect were subpassaged, and, if positive, the virus was typed by neutralization with the lim-benyesh-melnick equine antiserum pools [ ] . extraction of viral rna enterovirus rna was extracted from µl of csf using the qiamp viral rna kit (qiagen, france) according to the manufacturer's instructions. ev rna was eluted with µl of sterile water. primers and probes used for amplification of evs were as already published: ev [ ′-gattgtcaccataagcagc- ′], ev [ ′-cccctgaatgcggctaatc- ′] and ev probe [ ′-fam-cgga-accgactactttgggtgtccgt-tamra-phosphor- ′] [ ] . for absolute quantification, an ev rna standard representing the ′-noncoding region of ev rna was synthesized in vitro by plasmid cloning and in vitro transcription using complementary dna from mahoney type poliovirus and primers ev clon ′-tggccaatcgaattcgcttta- ′ and ev clon ′-ctacata-aggatcctccggcc- ′ [ ] . an exogenous ipc was introduced into each reaction well in order to prevent false-negative results. this was an rna template transcribed from the plasmid paw (paw rna), purchased from pe applied biosystems (france) and used as internal quality marker. we designed the primers and probe using the software primer express (pe applied biosystems) in order to use this rna in a pcr duplex format to monitor the quality of the amplification reaction. the software defined the primer paw [ ′-tccccag-gaacagttgaaaga- ′], primer paw [ ′-aacagggaac-ccaggctcc- ′] and paw probe [ ′-tet-cagtgcctgccc-attcggagga-tamra-phosphor- ′]. the compatibility of these primers and probe with those of ev amplification was tested using the software oligo (national biosciences, usa) in order to determine the multiplex conditions. the reaction mixture (final volume µl) was prepared in a single tube as follows: x taqman buffer a (pe applied biosystems), mm mgcl (pe applied biosystems), µm dntps (roche, france), nm each of the two primers ev and ev (genosys, uk), nm ev probe (eurogentec, belgium), nm each of the two primers paw and paw (genosys), nm paw probe (eurogentec), % glycerol (prolabo, france), . % pvp- (serva, france), µg t gene protein (amersham, france), . iu mulv (pe applied biosystems), iu amplitaq gold (pe applied biosystems), iu rnasin (promega, france), and µl ipc rna ( , copies/µl). twenty microliters of the reaction mixture was added to the pcr tubes containing µl rna extract from csf samples or ev rna standard in serial dilution. ev rna and the ipc rna were reverse transcribed into cdna ( min at . °c), and this was followed by the mulv/taq gold denaturation/activation step ( min at °c). the cdna fragments ( bp for ev rna and bp for ipc rna) were amplified by pcr ( s at °c and min at °c) for cycles on an abi prism (pe applied biosystems). real-time fluorescence measurements were taken and the threshold cycle (c t ) for each sample was calculated by determining the point at which fluorescence exceeded a threshold limit ( times the baseline standard deviation). a standard graph of the c t values obtained from serially diluted external ev rna standard was compiled. the correlation coefficient and the slope were calculated. c t values from the csf samples were plotted on the standard curve, and the number of copies was calculated automatically by sequence detector version . (pe applied biosystems). samples were defined as negative for evs if their c t values for evs (c t -evs) were cycles and their c t values for internal positive control (c t -ipc) < cycles. the optimal simultaneous amplification conditions were determined during the initial optimizing steps of the multiplex fluorogenic rt-pcr. the constructed ev rna standard was used at concentrations of - × copies/reaction and the ipc at , copies/reaction. the first results showed that the ipc was not amplified in the presence of ev rna standard at quantities over × copies/reaction, and that the ev rna detection limit was × copies/reaction. different concentrations of each primer ( - nm for evs and - nm for ipc) were thus tested systematically. final primer concentrations ( nm ev primer and nm ipc primer) enabled a constant amplification level for the ipc in the presence of ev rna standard at concentrations of - × copies/reaction. the detection limit, intra-assay variability, and interassay variability were determined in replicates of -fold dilutions of the ev rna standard from - × copies amplified in eight independent assays. intraassay coefficients of variation (cvs) varied between . and . % and interassay cvs between . and . %. the variability observed with copies of ev rna standard was acceptable, and the detection limit of the rt-pcr is therefore copies of ev rna standard per reaction. finally, the analytical sensitivity of our method, taking all extraction steps into account and using the ev rna standard serial dilutions, is copies/reaction, which is equivalent to , copies per milliliter of csf. smaller quantities, however, can be measured because the linearity of the standard curve allows values below the lower limit of the dynamic range to be interpolated [ ] . the fluorogenic rt-pcr was applied to detection of evs in the csf of patients presenting with signs of meningitis. the results of virus cell culture were analyzed in parallel. age of the patients as well as laboratory findings are summarized in table . mean biochemical parameters of csf such as chloride ions, protein, and glucose remained normal, while a relatively high cell count (mean, cells/mm ) was found in the majority of cases. the duration of hospitalization was short, ranging from to days, with a mean of only . days. validation of the results of the fluorogenic rt-pcr was performed by analyzing the multicomponent curves. any possibilities for the validation of the ev rt-pcr with the ipc are shown in fig. . no amplification took place in two cases, with c t -ev and c t -ipc of cycles, thus indicating the presence of pcr inhibitors. subse-quent amplification of a : dilution of these samples made it possible to conclude that they were negative, with c t -ev of cycles and c t -ipcs of . and . cycles. a comparison between the results of virus culture and those of the fluorogenic rt-pcr is summarized in table . of the csf samples, ( . %) were found to be negative by both rt-pcr and viral culture. twenty specimens were positive by both techniques. forty-three discrepant results were obtained: rt-pcr was positive in culture-negative samples, while culture-positive specimens were negative by rt-pcr. sixty-one of ( . %) specimens were positive in the rt-pcr, and ( . %) were positive in culture. of the ev strains isolated in culture, remained untypable, while were typed and yielded different ev serotypes (echovirus types , , and ). figure shows the distribution of the different serotypes isolated in the culture-positive csf samples. the most common serotype in this outbreak was echovirus type . for the two culture-positive specimens that were negative by rt-pcr, one remained untypable and the other was typed as echovirus type . using a "consensus positive" for ev infection, described by hadziyannis et al. [ ] , which was defined as either a culture-positive or rt-pcr-positive result and clinical evidence of enteroviral meningitis, the sensitivity and the negative predictive value of rt-pcr analysis were . % and . %, respectively, while these values for cell culture were . % and %. viral load in the rt-pcr-positive specimens was measured automatically in the abi prism software system and was found to range between . and . log, with a median of . log copies per milliliter of csf. mean viral loads calculated for the two groups rt-pcrpositive/culture-positive and rt-pcr-positive/culturenegative were . and . log, respectively. statistical comparison of these two means showed no significant difference (p> . ). we also compared mean cell counts, chloride ions, glucose, and protein in csf from patients with a viral load of < . copies/ml and those with a viral load of > . copies/ml, without finding any significant difference between the two groups. only duration of hospitalization showed a statistically significant decrease from . days for patients with negative pcr results to . days for patients with positive pcr results (p= . ). the diagnosis of aseptic meningitis by rt-pcr requires a sensitive technique with an internal control, in view of the often low levels of virus and the problems associated with pcr inhibitors. during the optimization of our previous fluorogenic rt-pcr [ ] , we overcame problems of sensitivity related to the secondary structures present in the ′ noncoding region of the ev genome and problems associated with pcr inhibitors by using pcr adjuvants such as t gene protein and polyvinylpyrrolidone [ ] . in order to detect pcr inhibitors without a second run, we decided to work on the incorporation of an internal control. real-time pcr assays allow the choice of internal controls totally different from those of the amplification target, as it is possible to evaluate competition between the two reactions with the amplification kinetic. the two amplification reactions must be independent. thus, our internal control is just an internal quality control of the amplification, and its amplification at the same level (ct= cycles) over a range of - × copies of evs rna standard improve its usability. the intra-and inter-assay variability values of . % and . % obtained with low numbers of ev rna standard copies agree with those found in work on other taqman rt-pcrs ( . % and . %) [ , ] . the presence of pcr inhibitors in of our ( . %) specimens confirms the importance of an internal control for rt-pcr assays in csf, as already demonstrated by other authors [ , ] who found amplification inhibitors in . % and . % of their csf specimens, respectively, using the competitive rt-pcr amplicor ev (roche molecular systems, usa). since the exact nature of these inhibitors is unknown, their effects cannot be excluded, even by using numerous amplification facilitators [ ] . in this study, rt-pcr was compared with ev isolation, which is routinely practiced in our laboratory to detect evs in young patients with suspected aseptic meningitis referred to the hospital during the period of an ev outbreak. our comparison confirms that rt-pcr is more sensitive than cell culture [ , ] . discrepant results (rt-pcr-negative/culture-positive) were obtained for two strains: one remained untypable, and for the second one, it is possible that problems occurred during rna extraction. retesting of the second strain, however, was prevented by the low quantity of csf. the nonsignificant difference found between mean viral load measured in patients with rt-pcr-positive /culture-positive and rt-pcr-positive/culture-negative results suggests that a negative culture result is not necessarily related to a low titre of virus and that other factors, related to the capacity of the virus to propagate in culture and, more particularly, to induce cytopathogenic effects, are more important. our method is relatively easy to perform, with results obtained in less time than that required for virus culture. thus, the entire process of rt-pcr analysis of multiple samples can be carried out easily in less than h, whereas virus culture of csf typically requires - days for a cpe to develop. furthermore, a real-time pcr assay eliminates the necessity for precautions that must be taken with amplified products to avoid contamination because the technique is performed in completely sealed wells. this is a great improvement over conventional pcr assays, which are associated with risks of carryover contamination and are more time-consuming. the other advantage of this method is that ev genome can be easily quantified using the standard curve. the ev viral loads detected in csf correspond with the infectious ev loads reported in the literature: log pfu per milliliter of csf was found for persistent echovirus type in a child with hypogammablobulinemia [ ] . persistent ev infections in agammaglobulinemic patients also have been demonstrated in csf by rt-pcr [ ] . our assay allowing quantification could be useful in detecting and monitoring the progression or eradication of ev infections in these patients, because cell cultures are often negative during antibody therapy, and monitoring virus eradication using cell culture is very difficult. if specific antiviral agents become available in the future, this method would be a powerful tool to evaluate their efficacy. the implementation of such a rapid, sensitive, and specific rt-pcr test with high negative predictive value ( . %) would have a considerable impact on patient care, especially during outbreaks [ ] . previous studies have shown that early diagnosis of enteroviral meningitis would provide potential cost savings by facilitating early patient discharge, discontinuation of inappropriate antibiotic therapy, and elimination of unnecessary investigation [ ] . this fluorogenic enterovirus rt-pcr allows a large number of samples to be screened rapidly during an epidemic, and its sensitivity, simplicity, and reproducibility make it a highly reliable and suitable tool in the clinical laboratory. quantitative pcr: theoretical considerations with practical implications evaluation of the roche amplicor enterovirus pcr assay in the diagnosis of enteroviral central nervous system infections amplicor enterovirus polymerase chain reaction in patients with aseptic meningitis: a sensitive test limited by amplification inhibitors quantification of enterovirus rna in sludge samples using single tube real-time rt-pcr typing of viruses by combinations of antiserum pools: application to typing of enteroviruses (coxsackie and echo) high-throughput real-time reverse transcription-pcr quantitation of hepatitis c virus rna one-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses effects of amplification facilitators on diagnostic pcr in the presence of blood, feces, and meat enzymatic rna amplification of the enteroviruses diagnosis of enteroviral central nervous system infection by polymerase chain reaction during a large community outbreak persistent meningo-encephalitis caused by echovirus type in a child with hypogammaglobulinemia (in french) persistent enterovirus infection in culture-negative meningoencephalitis: demonstration by enzymatic rna amplification circulation of enteroviruses and persistence of meningitis cases in the winter of potential cost savings through rapid diagnosis of enteroviral meningitis aseptic meningitis in infants < years of age: diagnosis and etiology viral meningitis role of the virology laboratory in diagnosis and management of patients with central nervous system disease comparison of use of cerebrospinal fluid, serum, and throat swab specimens in diagnosis of enteroviral acute neurological infection by a rapid rna detection pcr assay detection by pcr of enteroviruses in cerebrospinal fluid during a summer outbreak of aseptic meningitis in switzerland reverse-transcription polymerase chain reaction detection of the enteroviruses rapid detection of enterovirus in clinical specimens using pcr and microwell capture hybridization assay polymerase chain reaction for the laboratory diagnosis of aseptic meningitis and encephalitis diagnosis of enteroviral meningitis by using pcr with a colorimetric microwell detection assay a one-step rt-pcr assay using an enzyme-linked detection system for the diagnosis of enterovirus meningitis evaluation of a commercial dna enzyme immunoassay for detection of enterovirus reverse transcription-pcr products key: cord- -a q dq authors: pace, david; gauci, charmaine; barbara, christopher title: the epidemiology of invasive meningococcal disease and the utility of vaccination in malta date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: a q dq invasive meningococcal disease (imd) is a vaccine-preventable devastating infection that mainly affects infants, children and adolescents. we describe the population epidemiology of imd in malta in order to assess the potential utility of a meningococcal vaccination programme. all cases of microbiologically confirmed imd in the maltese population from to were analysed to quantify the overall and capsular-specific disease burden. mean overall crude and age-specific meningococcal incidence rates were calculated to identify the target age groups that would benefit from vaccination. over the -year study period, out of the eligible notified cases were confirmed microbiologically of which . % had septicaemia, . % had meningitis, and . % had both. the mean overall crude incidence rate was . / , population with an overall case fatality rate of . %. meningococcal capsular groups (men) b followed by c were the most prevalent with w and y appearing over the last years. infants had the highest meningococcal incidence rate of . / , followed by . / , in – year olds and . / , in – year old adolescents. the introduction of menacwy and menb vaccines on the national immunization schedule in malta would be expected to reduce the disease burden of meningococcal disease in children and adolescents in malta. invasive meningococcal disease (imd) manifests predominantly as meningitis and/or septicaemia with most affected individuals having a sudden presentation and rapid deterioration. although rare, imd affects all ages, and the brunt of the disease burden is highest in infants, children below years of age and adolescents [ , ] . compared with other age groups, there is also a relatively increased incidence of imd in the elderly who suffer the highest case fatality rates of all [ ] [ ] [ ] . despite advances in medical care, around % of individuals with imd die, and up to - % of survivors sustain permanent disabilities such as sensorineural hearing loss, neurodevelopmental problems, seizures and amputations [ ] [ ] [ ] [ ] . imd is a worldwide disease, but the epidemiology of the meningococcal capsular groups (men) is unpredictable and varies by geographical regions and over time. menb still causes the majority of imd within europe, followed by menc and more recently menw [ ] . in the usa, menb, menc and meny are each responsible for around one third of the imd cases [ ] . classically, mena was a major cause of epidemics within countries in the meningitis belt of sub-saharan africa [ ] , with recent emergence of menw, c and x disease [ , ] . nasopharyngeal carriage provides a continuous reservoir making eradication of the disease difficult. migration and international travel poses a risk of transmission of men capsular groups especially to family members within hosting countries [ ] . the introduction of meningococcal conjugate vaccines on national immunization programmes has resulted in significant reductions in the corresponding burden of imd. control of imd at a population level by meningococcal conjugate vaccines is not only a direct effect of protection of the vaccinated individuals but also is largely a result of their ability to interrupt transmission through reduction in meningococcal carriage rates, thus inducing a herd immune effect. this effect is only seen when adolescents, who are the main transmitters of the meningococcus, are included in meningococcal immunization strategies. drastic reductions in imd in all ages, including unvaccinated groups, have been observed with immunization programmes that included adolescents in catch-up or routine campaigns for menc in europe and salvador in brazil and mena in africa [ ] [ ] [ ] [ ] [ ] . in contrast, exclusive vaccination of at-risk children < years old against menw disease with a menacwy conjugate vaccine in chile and with a menc conjugate vaccine in bahia, brazil, without immunizing adolescents did not impact menw and menc disease in unvaccinated age groups, including the elderly, in the respective countries [ , ] . the recent introduction of proteinbased vaccines against menb in developed countries holds promise for control of the most prevalent meningococcal capsular group although up until now their effect is only envisaged to result from direct protection since impact on nasopharyngeal carriage has not been demonstrated [ , ] . the maltese archipelago is a small group of islands situated in the southern mediterranean area having a population of around , people, around . million tourists per year and a population density of persons per square kilometre, the highest in europe [ , ] . as yet, meningococcal vaccines have never been introduced on the national immunization schedule in malta although such vaccines are available privately for individuals wishing to protect themselves or their children against imd. we aimed to study the population epidemiology of imd in the maltese islands in order to assess the utility of a meningococcal vaccination programme. all microbiological confirmed cases of imd over an year period, from to , were collected from the bacteriology laboratory at mater dei hospital (mdh) which provides care for all patients suffering from imd. cases were included if they satisfied the european centre for disease prevention and control (ecdc) laboratory criteria for imd which consisted of (a) identification of the meningococcus through culture or molecular methods from usual sterile sites (blood, cerebrospinal fluid (csf), synovial fluid and any other usually sterile fluid) or from purpuric lesions or (b) detection of gram-negative diplococci from direct visualization of the csf or through a positive meningococcal rapid antigen screen (ras) [ ] . subsequently, isolates were classified according to the capsular group, if groupable. identification of the serogroups was performed by slide agglutination tests using specific capsular antisera (remel europe ltd., kent, uk). all isolates were sent to the public health england (phe) meningococcal reference unit (previously health protection agency meningococcal reference unit) in manchester, uk, where the capsular group was reconfirmed and phenotyping was performed by identification of the serotypes and serosubtypes with monoclonal antibodies as described by gray et al. [ ] . multilocus sequence typing (mlst) was not carried out. from to , a polymerase chain reaction assay (pcr) amplifying the specific capsular genes was performed at the phe laboratory on csf and/or blood of patients whose cultures were negative. in , a meningococcal screening pcr was introduced at the microbiological laboratory in mdh, and any positive samples were sent to the phe laboratory for capsular gene identification. meningococcal capsular gene detection was performed at mdh in . meningococcal isolates identified from non-sterile sites such as throat or nasopharyngeal swabs and sputum were excluded. notification of imd is statutory obligatory in malta. all notified cases of imd, identified through passive surveillance, were collected from the infectious disease prevention and control unit (idcu) in malta. the criteria for reporting adopted by the idcu were in accordance with the case definitions for confirmed imd set by the european union commission decisions in , and subsequently in [ , , ] which also included possible cases of imd as defined by the presence of one of the clinical criteria of meningitis, haemorrhagic rash, septic shock or septic arthritis or probable cases, as defined by the presence of clinical criteria with an epidemiological link to a case of imd, in the absence of laboratory confirmation of the infecting pathogen. cases were notified to the idcu by clinicians when a patient was clinically suspected to have imd and subsequently by the laboratory when the meningococcus was identified in clinical specimens. when microbiological results did not reveal an invasive pathogen, idcu contacted the clinicians to ascertain that the clinical picture, investigations and progress of the cases still satisfied the set case definitions of imd. these data were used to calculate the total number of imd cases per year in malta over the study period. the results of microbiological investigations of all notified cases were validated against electronic results and/or written case records. individuals with a foreign hospital number or whose laboratory results showed an alternative diagnosis were excluded from the analysis. population data were obtained from the national statistics office in malta. this study was approved by the faculty of medicine and surgery university o f m a l t a r e s e ar c h e t h i c s c om m i t t e e ( r e f n o : frecmds_ _ ). the primary objective of this study was to perform population-based descriptive statistics on imd in order to quantify the disease burden caused by the meningococcus in the maltese population. the median was used in preference to the mean to describe the age demographics as in view of the relative rarity of imd, it was expected to have a small number of cases with a wide age range. the proportion of laboratoryconfirmed imd cases was calculated per year from the total number of reported imd cases. the disease burden caused by the different meningococcal capsular groups was calculated for each year from the number of laboratory confirmed isolates. the distribution of cases was analysed according to prespecified age groups as follows: < , - , - , - , - , - , - , - and ≥ + years. a cut-off of < years was taken to indicate the paediatric population as determined by the hospital admission policy for children in mdh, malta. this enabled calculation of the age-specific incidence rates of meningococcal disease, and the corresponding % ci using a poisson regression analysis, in maltese children and identification of the target groups that would benefit from vaccination. fisher's exact test was used to analyse differences in the disease burden between the capsular groups. a p value < . for all analyses was taken as being statistically significant. the software stata was used for the analyses. a total of cases of meningococcal disease were identified over the -year study period: were notified, whilst additional cases had not been notified but were identified from the bacteriology lab from the results of their cultures. of these, individuals resident in malta were eligible to be included in the analysis, of whom . % were male ( / ). of the excluded cases, were tourists, and were found to have an alternative diagnosis on review of their electronic results or case records (either because csf was suggestive of viral meningitis in culture negative cases or a different pathogen was noted during validation of the electronic records). only . % ( / ) of cases were confirmed microbiologically, of which . % ( / ) were confirmed on culture ( fig. ). meningococcal pcr, introduced in , confirmed of the culture negative cases from to all of which were in children, although testing was performed at the request of the caring doctor rather than routinely on all suspected cases. the median age of the laboratoryconfirmed cases was . years (range, . - . years). out of the microbiologically confirmed cases of imd, . % ( / ) had confirmed septicaemia (confirmed from blood or petechial scrapings), . % ( / ) had confirmed meningitis, . % ( / ) had confirmed septicaemia and meningitis, . % had arthritis, and . % had pericarditis. *these cases were picked up by the microbiological laboratory but were not notified. pcr, polymerase chain reaction assay. ras, rapid antigen screen; men meningococcus. the variation of the annual total number of confirmed cases generally followed the variation of the total number of notified cases except for when only out of a total of notified cases ( . %) were confirmed microbiologically (fig. ) . the mean overall crude incidence rate of confirmed imd from to (table ) was . / , ( % ci, . to . ) population. although the mean crude incidence rate decreased from . / , ( % ci, . to . ) population in - to . / , ( % ci, . - . ) population from to , this was not statistically significant (difference . ; p = . ). the mean age-specific incidence rate of imd was significantly higher in infants ( . / , ; median age months; range, . - . months), - -year-old children ( . / , ; median age . years; range, . - . years) and - -yearold adolescents ( . / , , median age . years; range, . - . years) than the rest of the population (fig. ) with the highest burden being in infants. there were deaths caused by confirmed imd (table ) , of which ( . %) were in children < years old. the overall case fatality rate (cfr) was . % ( / ). in children < years of age, the highest age-specific cfr was in - -year-olds ( / ; . %), - -year-olds ( / ; . %) and - -year-olds ( / ; . %), whilst in adults, the highest risk of dying was in - -year-olds ( / ; . %) and in ≥ -year-olds ( / ; . %). the months with the highest total and similarly confirmed number of imd cases were january, february, march and august, whilst the lowest numbers were recorded in april and from october to december. no epidemics of meningococcal disease occurred during the study period. in view of the unexpected peak of imd in august, the cases of imd in tourists who became unwell whilst in malta were looked at (these had been excluded from the primary analysis). a peak in imd in tourists was also noted in august when / ( . %; median . years, range, . - . years) confirmed cases occurred. identification of the meningococcal capsule was successful in / cases, of which were isolated by culture (fig. ) . the most frequent isolated capsular groups were menb ( . %; / ) and menc ( . %; / ). capsular groups w and y and non-groupable meningococci collectively constituted a minor proportion of cases ( . %; / ), although capsular group y became prevalent from to ( . %; / cases) and capsular group w became more frequent from to ( . %; / cases) (fig. ) . menb disease was significantly more prevalent than other capsular groups in children < years of age. infants suffered the highest incidence of menb, menc and menw disease compared with all other age groups (fig. ) . meny disease was only observed in - year-olds, whilst menw affected teenagers and infants. the incidence rates of menb, c, w and y disease were not significantly different in elderly people ≥ years old. the overall mean crude incidence rate of menb in malta was . / , ( % ci, . to . ) population with an overall case fatality rate of . % ( / ). a statistically significant downward trend from . / , population, % ci . to . , over - , down to . / , population, % ci . to . , over - , (difference . ; p = . ) was observed (fig. ) . the highest disease burden of menb disease was in infants ( . / , ; % ci, . - . ) with a median age of . months, followed by - -year-olds ( . / , ; % ci: . - . ), median age . years, and - year-olds ( . / , ; % ci, . - . ), median age . years (fig. ) . only pora phenotyping was performed, with % ( / ) of the typed isolates having pora subtypes p . ,p . (table ) . % ci, . to . ), difference . , p = . (fig. ) . the mean age-specific incidence rate of menc disease was significantly higher in infants ( . / , infants; % ci, . - . ; median age . months, range, . - . months) compared with all other age groups (fig. ) . fatalities from menc disease were more common in children, with of the deaths occurring in children aged months, years and years. of the menc cases, three were confirmed by detection of the sia d gene by pcr, whilst were confirmed by culture. phenotyping of the cultured menc strains was performed on isolates, since of the meningococcal cultures did not remain viable in storage and could not be shipped to the phe laboratory in the uk for further identification ( table ) . out of the cases who succumbed to menc disease, were phenotyped, and all had the pora subtypes p . ,p . . the overall mean crude incidence rate of menw and y disease was . / , ( % ci, . - . ) each, with an associated cfr of % ( / ) and % ( / ), respectively. deaths occurred predominantly in > -year-olds although menw error bars represent % ci the average annual incidence of confirmed imd cases of . / , population ( % ci, . - . ) in malta remains significantly higher than the mean incidence reported overall from to in europe ( . / , ; % ci, . - . ; p = . ) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] and the usa ( . / , ; % ci, . - . ; p = . ) [ ] , where a significant decrease in overall imd has been observed over the last few years. the drop in menb disease observed in malta and similarly in the eu and the usa is very likely a result of natural variation in the epidemiological trends of menb. however, the reduction in menb disease has had no effect on the overall incidence of imd in malta since it was offset by the persistence of menc disease and the appearance of menw and men y disease since . in contrast, the incidence rates of menc imd in the eu were significantly reduced (from . / , , % ci, . - . in - down to . / , , % ci, . - . in - ; p = . ) as a result of the introduction of menc vaccination programmes in several european countries, many after catch-up campaigns targeting adolescents, and the introduction of routine adolescent vaccination [ , ] . in the usa, the incidence of menc and y disease was reduced through the introduction of routine adolescent menacwy vaccination since [ ] . in salvador, brazil, control of menc disease was similarly rapidly achieved within years following the introduction of a menc vaccination programme targeting children < years of age as well as - -year-old adolescents and young adults [ ] . similarly, control of a mena epidemic was achieved within years in mossala, chad, following the introduction of a mena conjugate vaccine targeting - -year-olds in [ ] . in contrast, a herd immune effect was not seen in the state of bahia, brazil (excluding salvador), when a different menc conjugate vaccination strategy that just targeted children from months to years of age was introduced [ ] . similarly, a strategy using a conjugate menacwy vaccine targeting -month to -year-old children to control menw disease in chile provided direct protection to the vaccinated group but did not result in a herd immune effect [ ] . these strategies emphasize that the success of meningococcal conjugate vaccines relies on their ability to interrupt meningococcal transmission through a decrease in meningococcal carriage, an effect that can only be achieved if adolescents are targeted in meningococcal vaccination programmes. control of menb disease is unlikely to be attained with a similar approach since menb protein-based vaccines have no effect on carriage [ ] ; however, as observed in the uk, direct protection of infants and young children can still be achieved with a modest vaccine effectiveness of . % years after an infant priming and boost schedule [ ] . discrepancies between malta and other countries could reflect geographical differences in the epidemiology of imd; however, the lack of a national meningococcal vaccination programme in malta is most likely contributing to the higher incidence rates of imd. the higher number of imd cases from january to march is similar to the seasonality of imd observed in europe and the usa [ , ] . in malta, these months of the year are the coldest months (mean - °c) with the highest values for relative humidity, which reaches around % [ ] , climatic factors that are known to be associated with a higher risk of imd [ ] . the peak in imd seen in august in malta is difficult to explain as this month is dry and is relatively less humid [ ] but is characterized by the highest number of inbound tourists, the majority of whom come from europe [ ] . inbound tourists reach a mean of , in august, reaching a proportion of % of the mean , individuals constituting the maltese population [ ] . intriguingly, a similar peak in the number of imd cases also occurs in august in tourists whose median age was . years compared with . years in maltese individuals with imd in the same month. nineteen per cent of tourists visiting malta in august are aged < years [ ] with adolescents and young adults, who have the highest rates of meningococcal carriage [ ] ; very likely to visit overcrowded places such as pubs, bars and discotheques; and human behaviour that is associated with a higher risk of meningococcal transmission [ ] [ ] [ ] [ ] . any causal association of climatic factors and imd is challenging to reach in view of the confounding effects of human behaviour and the seasonality of other infectious diseases caused by influenza and other respiratory viruses [ ] . in malta, infants suffer the highest overall rate of imd, followed by children aged - years and teenagers, similar to the age distribution of imd seen in europe and the usa [ , ] . the burden of capsular group b and c disease in malta is similarly disproportionately highest in infants but also affects - year-old children, adolescents and young adults. in european countries, the highest menb and c disease burden is similarly seen in infants (although the incidence rate reached . / , and . / , infants for menb and c, respectively, much less when compared with the mean incidence rate of . / , and . / , infants for the corresponding capsular groups in malta), with children less than years old and adolescents and young adults being more affected than other age groups [ , ] . decay of transplacentally acquired maternal menb and c antibody in infancy, subsequent lack of protective menb and c bactericidal antibody in children in the absence of a menb and c vaccination programme [ , ] and nasopharyngeal mucosal damage from concurrent respiratory tract viruses, which are more common in children < years old [ ] , possibly explain the higher risk of menb and c disease in these age groups. the increased risk among adolescents and young adults is likely a result of increased exposure from changes in social behaviour leading to enhanced transmission of the meningococcus and lack of immunity [ ] . the appearance of meny disease in adolescents and menw disease in infants reflects the recent rise of these capsular groups in europe and poses a risk of disease in both age groups [ , ] . natural immunity from asymptomatic menb and c carriage [ ] or cross-reactive immunity to other microorganisms, such as escherichia coli k which has a sialylated polysaccharide capsule that is structurally similar to that of menc [ ] and to neisseria lactamica which shares antigens found on meningococcal outer membrane proteins [ ] , could contribute to the decreased incidence of menb and c disease observed in the - -year-olds; however, there are no robust data to support this hypothesis. the rise in non-menb disease after years of age could plausibly be due to a decline in immunity from immunosenescence [ ] , but further studies are needed to ascertain this. the overall cfr of . % for cases of imd is similar to the - % reported in europe and the usa [ , ] . cfr from menb and c disease, which reached . % and . %, respectively, and which affected children predominantly, was also similar to the - % menb and c cfrs reported in the usa and europe [ , ] . although mlst typing was not done, the menb phenotype p . ,p . is known to belong to the sequence type clonal complex which is hyperendemic in europe [ ] , and the menc phenotypes c a:p . ,p . , c a:p . and c:nt:p . ,p . have all been previously identified as belonging to the hyperinvasive st- meningococcal clone in other european countries [ ] [ ] [ ] . currently, two protein-based menb vaccines are available: menb- c, (bexsero, glaxosmithkline biologicals, belgium) licenced from months of age, and a menb-fhbp vaccine (trumenba, pfizer ltd., new york) licenced from years of age [ , ] . introduction of a menb vaccine will address the most prevalent cause of imd in malta, although projections of vaccine effectiveness would require whole genome sequencing and vaccine antigen sequence typing of the menb isolates. in the past, a serotypespecific menb outer membrane vesicle vaccine, menzb, that matched the menb strain b: :p . - , causing a prolonged epidemic in new zealand (which was used in a dose schedule for mass vaccination of < year olds from to and subsequently for routine infant vaccination up to ), had an estimated effectiveness of % in children < years of age and an overall effectiveness of % [ , ] . a reduced two-dose infant schedule of the currently available menb- c vaccine at and months followed by a boost at months of age, as introduced in the uk, would be expected to result in a modest % vaccine effectiveness up to years following the last vaccination assuming high vaccine coverage and favourable vaccine sequence matching with invasive menb strains [ ] . in contrast to meningococcal conjugate vaccines, indirect protection in unvaccinated population groups from herd immunity is very unlikely to be observed since menb- c has not been shown to reduce menb carriage, or other pathogenic capsular types, in a large cohort of australian adolescents [ ] indicating that menb transmission will still occur despite vaccination. similarly, carriage data have also discouragingly not demonstrated a reduction in menb carriage following vaccination with a menb-fhbp vaccine during an outbreak [ ] . the lack of an impact on menb carriage makes the utility of catch-up vaccination campaigns against menb questionable [ , ] . protection against menb disease in infancy, early childhood and adolescence will have to rely on the direct protection offered by a menb vaccine. infant menb vaccine priming and boost schedules will still need boosting in adolescence as, similar to the antibody decline seen with conjugate menc vaccines, bactericidal antibody will not last into the teenage years [ , ] . introduction of menb vaccination at and months with a boost at months of age together with two-dose vaccination of adolescents starting from the age of years would be expected to have an impact on menb disease in malta. monovalent menc and quadrivalent menacwy vaccine formulations became available on the private market in malta in and , respectively, but were never introduced on the national schedule. a mean of conjugate vaccines against menc was given per year in children aged < years, amounting to vaccination of around % of the paediatric population per year (national immunization service, personal communication; pfizer malta, vivian corporation, personal communication). such practice of meningococcal conjugate vaccination on request results in individual protection, but considering the epidemiology of menc, w and y disease over the last years is evidently not enough to induce herd protection. control of infant menc and w disease in malta may be achieved with the introduction of a routine infant menacwy vaccination programme consisting of a single menacwy dose at months of age, followed by a month boost, a schedule which would induce protective menc bactericidal antibodies in infants and toddlers [ ] and with extrapolation also to menw. this would have potentially prevented . % ( / ) and % ( / ) of cases of invasive menc and menw disease, assuming % vaccine efficacy. an effective national menacwy conjugate vaccine strategy would also need to target adolescents, not only to target meny disease observed in - year olds in malta but also to contribute towards a desired herd immune effect and reduce the corresponding imd in non-vaccinated groups, especially in the elderly > years of age in whom . % ( / ) of imd was caused by menc, w and y. furthermore, adolescent vaccination would eventually serve the purpose of boosting those children who would have received the routine infant and toddler menacwy schedule since adequate protection against all of these capsular groups will not persist until adolescence [ , ] . alternatively, a single conjugate menacwy vaccine dose could be introduced at months of age concurrently with immunization of adolescents. such a schedule using a monovalent menc conjugate vaccine was introduced in ontario, canada, in / [ ] . although a reduction in invasive capsular group c disease was seen in vaccinated and unvaccinated age groups, the reduction in the incidence of menc disease in infants over the subsequent years was not statistically significant [ ] . in contrast, the introduction of routine mencc vaccination in in -month-old children following a catch-up vaccine campaign in -month to -year-olds in the netherlands resulted in control of invasive menc disease, even in infants [ ] . similarly, the introduction of routine infant menc conjugate vaccination in concurrently with a catch-up vaccine campaign in to -year-olds in the uk was also successful [ ] , although a menc boost was introduced at months of age in due to low vaccine efficacy rates just within years of infant vaccination [ ] . the importance of a catch-up campaign targeting adolescents and young adults to control menc disease was also observed in brazil where sole vaccination of children < years of age in the state of bahia did not result in a herd immune effect in contrast to salvador where the concurrent introduction of a catch-up campaign targeting - year-olds resulted in control of menc disease in all age groups [ ] . similarly, vaccination of -month to -yearold children with a conjugate menacwy vaccine without adolescent catch-up vaccination did not control menw disease on a population level in chile [ ] . considering such data, the introduction of a routine infant and toddler menacwy vaccination programme concurrently with the launch of a one-time menacwy vaccine catch-up campaign targeting children aged from to years old and adolescents and young adults aged from to years old in malta would not only induce direct protection of these highrisk age groups but also result in indirect protection of unvaccinated individuals from decreased menc, w and y transmission by adolescents. such induction of herd protection would translate in a reduction in imd in older unvaccinated individuals (as seen in countries introducing a menc catch-up vaccination programme), the disease burden in whom was substantial over the last years ( . %, / cases) in malta. herd immunity against menc, w and y could then be sustained through a routine menacwy vaccine dose at years of age. once the epidemiology of men disease shows that control of invasive menc and w disease in infants has been achieved (reflecting minimal menc and w transmission), which, as seen in the uk for menc, would be expected to be reached within years [ ] , then the infant menacwy dose may be dropped. this would leave a routine meningococcal conjugate vaccine schedule consisting of a single menacwy dose at months of age, which is important for priming, followed by a booster dose in adolescence. the induction of robust protection against invasive menacwy disease in adolescence would protect teenagers and young adults from possible transmission of these capsular groups from the large number of foreigners that visit malta and mix with the young population in summer. currently, the maltese national immunization schedule in early childhood consists of the following: diphtheria, tetanus, acellular pertussis, inactivated polio and hib (dtap-ipv-hib) vaccine at weeks, , and months; hepatitis b vaccine at , and months; and the measles, mumps and rubella vaccine at months and between and years of age. adolescents receive a tetanus, diphtheria and inactivated polio (td-ipv) vaccine at - years with girls being vaccinated with doses of the human papilloma virus vaccine at -< years of age. introduction of a conjugate menacwy vaccine at months and months of age with another routine dose at years would easily fit within this schedule even when a pneumococcal conjugate vaccine is eventually introduced. similarly, menb vaccination in a , infant priming and month boost in addition to two-dose vaccination starting from years of age can also be easily accommodated within the national vaccination programme. capsular groups b, c, w and y meningococcal disease are endemic in malta, a country with a relative high incidence of imd. the introduction of menacwy vaccination following a single dose in early infancy with a boost in the nd year of life and in adolescence would be expected to impact menc, w and y disease in malta. the concurrent launch of a one-time menacwy vaccine catch-up campaign in children aged between and years and adolescents and young adults aged - years would be expected to induce herd protection and help achieve a faster decline in menc, w and y disease. furthermore, a menb immunization programme consisting of a dose prime and boost menb infant vaccine schedule in addition to menb adolescent vaccination at years of age would also be projected to provide direct protection and reduce the incidence of menb, which is responsible for the highest meningococcal disease burden in malta. invasive meningococcal disease. annual epidemiological report for surveillance reports: neisseria meningitidis case fatality rates of invasive meningococcal disease by serogroup and age: a systematic review and meta-analysis investigators of the canadian immunization monitoring program outcomes of invasive meningococcal disease in adults and children in canada between and : a prospective cohort study cost impact of complications in meningococcal disease: evidence from a united states managed care population the clinical features and long-term sequelae of invasive meningococcal disease in children outcomes of invasive meningococcal serogroup b disease in children and adolescents (mosaic): a case-control study emergence and control of epidemic meningococcal meningitis in sub-saharan africa whole-genome characterization of epidemic neisseria meningitidis serogroup c and resurgence of serogroup w emergence of epidemic neisseria meningitidis serogroup x meningitis in togo and burkina faso the threat of meningococcal disease during the hajj and umrah mass gatherings: a comprehensive review effectiveness of meningococcal serogroup c conjugate vaccine years after introduction protection from routine vaccination at the age of months with meningococcal serogroup c conjugate vaccine in the netherlands impact and effectiveness of meningococcal c conjugate vaccine following its introduction in spain impact of meningococcal c conjugate vaccination programs with and without catch-up campaigns in adolescents: lessons learned from bahia impact of menafrivac in nine countries of the african meningitis belt, - 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the climate of malta: statistics, trends and analysis - environmental exposures and invasive meningococcal disease: an evaluation of effects on varying time scales the stonehouse survey: nasopharyngeal carriage of meningococci and neisseria lactamica united kingdom meningococcal carriage group ( ) social behavior and meningococcal carriage in british teenagers meningococcal carriage among a university student population -united states meningococcal carriage in dutch adolescents and young adults; a cross-sectional and longitudinal cohort study prevalence of meningococcal carriage in children and adolescents aged - years in chile in effectiveness and impact of a reduced infant schedule of cmenb vaccine against group b meningococcal disease in england: a national observational cohort study seroprevalence of meningococcal serogroup c bactericidal antibody in england and wales in the pre-vaccination era invasive pneumococcal and meningococcal disease: association with influenza virus and respiratory syncytial virus activity? meningococcal serogroup y disease in europe: continuation of high importance in some european regions in cross-antigenicity and immunogenicity between capsular polysaccharides of group c neisseria meningitidis and of escherichia coli k antigenic cross-reactivity between outer membrane proteins of neisseria meningitidis and commensal neisseria species b-cell responses to vaccination at the extremes of age changes in neisseria meningitidis disease epidemiology in the united states significance of meningococcal hyperinvasive clonal complexes and their influence on vaccines development genetic characterization of a new variant within the et- complex of neisseria meningitidis associated with outbreaks in various parts of the world serogroup and clonal characterization of czech invasive neisseria meningitidis strains isolated from to genotypic and phenotypic characterization of carriage and invasive disease isolates of neisseria meningitidis in finland summary of product characteristics: bexsero meningococcal group b vaccine for injection in prefilled syringe. available from www.medicines.org.uk summary of product characteristics: trumenba. available from www.medicines.org.uk effectiveness of a vaccination programme for an epidemic of meningococcal b in new zealand use of an observational cohort study to estimate the effectiveness of the new zealand group b meningococcal vaccine in children aged under years persistence of the immune response after cmenb vaccination, and the response to an additional booster dose in infants, children, adolescents, and young adults antibody persistence and immunological memory at age years after meningococcal group c conjugate vaccination in children in the united kingdom ) immunogenicity of reduced dose priming schedules of serogroup c meningococcal conjugate vaccine followed by booster at months in infants: open label randomised controlled trial persistence of the immune response at years of age following infant immunisation with investigational quadrivalent menacwy conjugate vaccine formulations antibody persistence up to years after vaccination of toddlers and children between months and years of age with a quadrivalent meningococcal acwy-tetanus toxoid conjugate vaccine epidemiology of serogroup c and y invasive meningococcal disease (imd) in ontario, - : vaccine program impact assessment is a single dose of meningococcal serogroup c conjugate vaccine sufficient for protection? experience from the netherlands meningococcal c conjugate vaccine: the experience in england and wales publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions all authors contributed to the study conception and design. material preparation, data collection and analysis were performed by david pace. the first draft of the manuscript was written by david pace, and all authors commented on previous versions of the manuscript. all authors read and approved the final manuscript. the authors declare that they have no conflict of interest. key: cord- -wu ygt w authors: tambyah, p. a. title: sars: responding to an unknown virus date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: wu ygt w the severe acute respiratory syndrome (sars) is an emerging infection caused by a novel coronavirus which first appeared in southern china at the end of . in early , through a single incident, it spread to hong kong, singapore, canada and vietnam. for busy clinicians in large public hospitals, the response to the virus was initially based on ensuring a high level of protection for staff. however, as the epidemic progressed and more information became available about the virus, procedures were rationalized and the virus is currently under control worldwide. there are, however, numerous unanswered questions concerning super-spreading events, the modes of transmission of the virus and, perhaps most importantly, the rapid detection of the virus early in the course of disease. these issues need to be addressed in case the virus becomes more widespread in the near future. the severe acute respiratory syndrome (sars) is a newly recognized coronavirus infection that emerged in southern china [ ] with subsequent global spread to countries [ ] [ ] [ ] [ ] . in countries where local transmission has occurred, hospitals have been the major foci of infection especially in singapore [ ] and canada [ ] . in february , reports began emerging on promed mail of an outbreak of atypical pneumonia in the guangdong province in southern china [ ] . it is now believed that the first cases of sars occurred in the city of foshan in the guangdong province [ ] . however, throughout november until the late part of february, sars was largely confined to the province of guangdong. the global dissemination of sars is believed to have begun on the ninth floor of hotel m in hong kong, where a physician from guangdong stayed for one night on february [ ] . at least individuals who were staying on the ninth floor of the hotel were subsequently infected with sars, although none of them reportedly had direct contact with the ill physician. the newly infected individuals traveled onward to their homes or next destinations in the usa, canada, singapore, hong kong and ireland sparking off epidemics of varying degrees of severity in each of those countries, mainly in hospitals but also in their respective communities. it is striking to realize that the entire global dissemination of this epidemic can probably be traced to this single event of one overnight hotel stay. it has almost become a cliché to report that the sars epidemic, the first emerging infectious disease of the st century, heralded an unprecedented collaboration between researchers across the globe. within weeks of the first cases, electronic publications reviewed the clinical syndromes [ , ] as well as the characteristics of the virus and methods for its detection by the polymerase chain reaction [ , ] . the genome of various strains of the virus were sequenced, which contributed tremendously to knowledge of its molecular epidemiology [ ] . there was an explosion of reports about the sars coronavirus, with more than a thousand publications available on pubmed by the beginning of march , the first anniversary of the global emergence of the virus. it is beyond the scope of this review to cover all of the virological and clinical information contained in these articles and i would refer the reader to the excellent review by peiris et al. [ ] . the present review focuses on the response to this emerging disease and its evolution in light of increasing information. outstanding issues that remain to be resolved are also highlighted. one of the first unusual aspects of this emerging infection was the recognition that healthcare workers (hcw) were uniquely susceptible to the then unknown etiologic agent. in hong kong, the first clue that a new infection had emerged was a cluster of ill hcws. the same was noted in vietnam and led to the world health organization (who) sending a team to vietnam under the leadership of dr carlo urbani, who later died from the virus he helped to define [ ] . as soon as sars was recognized as a nosocomial infection, guidelines were issued by various authorities including the who it is important to recognize that in the beginning of the sars outbreak there was no information about the agent responsible for the infection or its mode of transmission; hence, there was a tendency to "over-protect." as the epidemic evolved, so did the guidelines, which are constantly being updated and might indeed be out of date by the time this report is read. all guidelines are published on the internet [ - ], and the reader is encouraged to review the websites for the latest information. the physician from guangdong who became the source of the global epidemic through his stay at hotel m was admitted to a hospital in hong kong the day after his arrival in the city. he became progressively more ill and died days later. it is striking that although he was critically ill and intubated on a ventilator in the intensive care unit, only one hcw who attended to him in the emergency department became ill [ ] . the reason for this is likely that hong kong hospitals had been on the alert for highly pathogenic avian influenza. in february, there had been a small cluster of cases of avian influenza in a hong kong family that had traveled to mainland china [ ] . a directive had gone out from the hong kong department of health on february to maintain strict infection control with droplet precautions for all cases of "atypical" community-acquired pneumonia because of concerns that highly pathogenic avian influenza might be easily transmissible from person to person. one of the singaporean women who returned from hotel m was indeed isolated as a possible case of avian influenza in one of singapore's large general hospitals, and no secondary cases resulted from her. because of the concerns for possible avian influenza, or some unknown pathogen with an uncertain mode of transmission, most of the initial strategies devised for the prevention and control of sars were directed against a highly contagious airborne pathogen. in [ ] , previously healthy young people were infected and six died from highly pathogenic avian influenza in hong kong. this mortality rate ( %) is much higher than the normal mortality rate for influenza especially among young healthy individuals [ ] . in response to the outbreak, more than one million chickens were slaughtered and the disease was rapidly brought under control. seroepidemiologic studies of hcws done at the time [ ] demonstrated the efficacy of personal protective equipment (ppe) in preventing transmission and identified the risk associated with close personal contact in addition to the virus's lack of efficient humanto-human transmission capability [ ] . fears of a recurrence of a more virulent or easily transmissible form of avian influenza directed the initial efforts against sars. however, sars possessed an unusual quality in that it seemed to be transmitted in the healthcare setting far more efficiently than in households, where measles, varicella and other airborne viruses usually take rapid hold [ ] . this has yet to be explained completely, but it supports the argument that close contact is the major mode of transmission of the sars virus. fomites have been a cause for concern with the sars coronavirus since the initial global dissemination stemming from individuals in hotel m who had no direct contact with the index case but had stayed in the same corridor and probably had occasion to touch elevator buttons or railings that might have been contaminated with the sars virus. in the outbreak of sars in the amoy gardens apartments in hong kong [ ] , more than individuals who had no known direct contact were infected possibly through the aerosolisation of contaminated sewage. the implications of fomite transmission of sars are considerable and would mandate a much greater degree of environmental cleaning than is currently practiced. however, there are many unanswered questions in this arena. for example, why did the individuals staying on the same hotel floor as the index case in hotel m get infected but none of the staff? [ ] . sars has been convincingly demonstrated to be caused by a coronavirus [ ] . certain other characteristics have been ascertained from the previously known coronaviruses, e and oc , including their ability to survive after drying on inanimate surfaces in the hospital environment as well as differences in the viability of the virus at different conditions of temperature and humidity [ , ] . while the sars coronavirus has a certain amount of homology with the other pathogenic human coronaviruses [ ] , too little is known about its behavior under different environmental and atmospheric conditions to make a definitive statement about the role of the environment in nosocomial transmission. there have been reports of the sars coronavirus persisting for prolonged periods of up to days on environmental surfaces [ ] . survival in stool is reported to be even longer at up to days in alkaline diarrheal stools. this would certainly help explain such circumstances as the hotel m outbreak. the attack rates for sars have generally not been high. in singapore, for example, the index case for the national outbreak was nursed in a general ward by staff who were not wearing protective covering of any kind, and the attack rate was only / doctors, / nurses and / fellow patients in the same ward areas [ ] . again, the distribution of infections suggests that close contact is the most important factor leading to the transmission of this pathogen in hospitals. the widespread emphasis on ppe has been seen by some as placing an undue emphasis on hcw protection without adequately considering the protection of other at-risk individuals, such as other patients in the same area. the use of ppe is also not without its own adverse consequences [ ] as reactions to latex are common among hcws, some with serious consequences. it should also be noted that the use of respirators has been associated with fatal adverse events [ ] . costs are also an issue, and in resource-poor settings -ply cotton masks have been used, which have reportedly been effective in preventing the nosocomial transmission of the virus in at least one large public hospital [ ] . in a case-control study, seto et al. [ ] found that surgical masks were also effective in preventing transmission of sars to hcws, which is in line with our understanding of the epidemiology of the virus. in singapore, one of the successes of our approach to the control of sars was the widespread availability of full ppe for any staff member who requested it. this was even before the widespread dissemination of the virus led to the mandatory use of full ppe in all hospitals, and it provided staff the reassurance that their welfare was a high priority in the midst of an epidemic. there have been concerns that the use of n masks alone might not be adequate for preventing the nosocomial transmission of sars since cases have occurred among "fully protected" hcws [ , ] ; these cases possibly resulted from contact transmission. recognition of the role of contact transmission has led to the inclusion of recommendations for the use of gloves and gowns in all guidelines [ ] [ ] [ ] [ ] [ ] [ ] . while these garments have been shown to be effective in preventing the nosocomial transmission of other respiratory viruses [ ] , few data are available for their efficacy regarding sars. travel restrictions were among the more controversial aspects of the sars epidemic. the economies of most affected countries in east asia were devastated by these travel advisories. while other pathogens have been documented as being transmitted on airplanes, most notably influenza [ ] , the number of individuals infected with sars during air travel was remarkably low. according to the who [ ] , there have been flights carrying symptomatic probable sars patients and of those flights did not result in a single secondary infection. overall, cases of secondary infection resulted from symptomatic individuals. one flight alone, ca , which flew from hong kong to beijing on march, is now known to have accounted for of these cases. olson et al. [ ] reported that one flight with four symptomatic individuals with sars was associated with an attack rate (for confirmed sars) of zero while another flight with a single symptomatic individual was associated with an attack rate of %. the singapore experience [ ] was that three flights with symptomatic sars patients resulted in only one transmission. the overall attack rate for the flights into singapore was thus less than % despite one symptomatic individual being a so-called "superspreader" and another being critically ill at the time of the flight, requiring intubation soon after arrival. interestingly, olson et al. [ ] point out that fully % of the fellow passengers who became infected with sars had no direct contact, as defined by the who, with the index patient on their ill-fated flight. they do not offer any explanation for the differing attack rates, although a careful reader would realize that among the individuals allegedly infected on the flight, ten were traveling together as part of a tour group. also, the flight with the four symptomatic individuals was much shorter than the flight that was associated with widespread transmission. this is supported by icu data from canada [ ] that showed time of exposure to be a major risk factor. overall, however, what these reports demonstrate is that a much more detailed analysis is required in order to truly understand the epidemiology of this unusual virus. during the peak of the sars epidemic in china ( - april ), when the who had travel advisories and alerts in place, there were more than , visitors from china and hong kong [ ] who entered singapore and not a single case of transmission was recorded from any of these individuals. in taiwan, a strict -day quarantine [ ] was placed on all individuals returning from countries that were on the who list of sars-affected countries. a total of , individuals were quarantined, among whom had probable sars and only one of whom had laboratory-confirmed sars. thus, the detection rate was . % for probable sars and . % for laboratoryconfirmed sars. these figures have to be balanced against the costs and psychological impact of quarantine for more than , individuals who were perfectly well. thanks to excellent isolation and case finding with contact tracing, the number of people infected with sars with each successive generation of the outbreak was progressively reaching extinction levels [ ] in the sarsaffected countries. with the re-emergence of sars at the beginning of , drastic travel restrictions have fortunately not been instituted to date since there is no evidence yet of widespread dissemination of the virus across international air routes by travelers. it could also be argued that since we now have much better knowledge of the epidemiology of the virus, travel restrictions might not be necessary the next time around. all of the guidelines agree it would be ideal if patients with sars could be nursed in isolation rooms [ - ]. there are differences, however, in the recommendations for negative pressure with separate ventilation systems, and these perhaps reflect the differences in resources available for healthcare. one drawback of isolation rooms is that unless there are adequate nursing or medical resources, the degree of attention that the patient will receive in a single isolation room is obviously lower than in an open well-ventilated area. patients isolated for infection control purposes are known to be at risk for adverse events in hospital [ ] and this again has to be balanced against the benefits in terms of reduced nosocomial transmission. while all of the available evidence points to droplet and contact transmission, there is a possibility that the virus might be aerosolized during such procedures as high-flow oxygen therapy or possibly via the use of extractor fans, which were blamed for the aerosolisation of contaminated sewage during the amoy gardens outbreak [ ] . therefore, n respirators or higher should be used. this is a cause for concern as in many countries, including singapore, without adequate pre-prepared negative-pressure rooms, powerful extractor fans similar to the ones used in the bathrooms at the amoy gardens apartments are used to create a form of laminar uni-directional airflow. while these may serve to direct the flow of air away from areas of heavy traffic, it is possible that they might be hazardous by facilitating the aerosolisation of infectious droplets. in singapore [ ] and canada [ ] , transmission of the sars virus has been noted in crowded emergency rooms where patients routinely wait for hours for a hospital bed. in singapore, sars was documented as being transmitted to a patient's visitor who was waiting in a corridor during the patient's radiological procedure [ ] , again a common occurrence in many healthcare settings. in our own hospital, the national university hospital, the largest cluster of sars cases occurred in one of our eight-bed wards [ ] where patients are deliberately placed eight to a cubicle in order to support the philosophy of healthcare financing in singapore. the sars outbreak has clearly been a wake-up call for health authorities worldwide [ ] as they try to adjust health systems primarily designed to minimize costs into systems designed to protect staff and patients. the isolation and segregation of patients with suspect and probable sars has been credited with markedly reducing the transmission of the virus [ ] . lipsitch et al. [ ] reported a reduction in the time to isolation of patients with sars as the epidemic progressed with a corresponding decline in the number of secondary cases as knowledge of the virus increased. the majority of individuals with sars have not transmitted the virus to anyone [ ] . while it is tempting to ascribe this to infection control measures, many of these individuals were infected and hospitalized long before the institution of infection control methods. this has given rise to the concept of "super-spreaders." it is known that the presence of common viral upper respiratory tract infections can turn some hcws into so-called "cloud hcws" [ ] . these individuals have been linked with the airborne dispersal of agents that are normally only spread through contact, such as group a streptococci or staphylococcus aureus. the hypothesis is that the presence of upper respiratory tract infections transforms these individuals into efficient transmitters of pathogens through increased coughing, sneezing or nose rubbing. alternatively, airborne dispersal could result from the use of various respiratory therapies. the index patient for the singapore epidemic was not isolated, and hcws, visitors and fellow patients were infected [ ] . the second generation of cases associated with this cluster, before the institution of infection control practices or strict isolation, included only cases. the situation in canada was similar, with cases in the second generation occurring preisolation [ ] . it is quite clear, however, that non-isolated patients are hazardous to staff, visitors and other patients. single non-isolated patients have led to well-documented outbreaks in hospitals in singapore [ ] , taiwan [ ], canada [ ] and hong kong [ ] . the phenomenon of "super-spreaders" has been invoked to explain why so few transmissions resulted from the majority of non-isolated individuals while a few rare cases were associated with the vast majority of transmissions [ ] . the jury is still out as to whether these are indeed individuals who are for some reason more able to transmit infection or whether events, such as the use of high-flow oxygen therapy, are more responsible for what are probably more accurately described as "super-spreading events." until we have more virologic information, we have to assume that all individuals with sars are "super-spreaders" until proven otherwise and we have to take all the necessary precautions. again, because of the concern that an undiagnosed patient might turn out to be a "super-spreader," the threshold to isolation has become progressively lower. initially, hospitals and clinics were using the who case definitions of suspect and probable sars cases to determine which cases to isolate. as we, and others, have pointed out, atypical presentations [ ] are the achilles heel of such a strategy and these have been associated with significant nosocomial transmission. in a very important report from a sars screening clinic, rainer et al. [ ] pointed out that the who criteria, which were actually designed for epidemiologic purposes, while relatively specific, have a sensitivity of only about % in predicting which individuals will turn out to have sars. the implications are that a large number of individuals will need to be isolated and monitored very closely until their clinical course becomes evident. in practice in singapore, this resulted in the conversion of large numbers of hospital wards to isolation facilities, cancellation of elective surgeries and an overall paralysis of the healthcare system. we used a regime of -hourly temperature monitoring without any use at all of antipyretics together with daily serial chest radiographs and blood counts and comprehensive chemistry work ups. with this regime, we found a sensitivity of %, specificity of %, positive predictive value of % and negative predictive value of % for the who criteria at patient presentation [ ] . it is clear that an accurate rapid diagnostic test is urgently needed to allow us to filter out individuals who are at low risk of sars or, better still, at lower risk of transmitting the virus should they not be isolated. current diagnostic tests [ ] , which are based on either molecular methods or serological diagnosis, are severely limited not predominantly by sensitivity or specificity but by the fact that they take awhile to become positive, during which time widespread dissemination of the virus could have occurred from a single non-isolated "super-spreader." fever screening is widely practiced as a sars-prevention measure. there was even a period when the who called for fever screening at airports to prevent the global spread of sars. unfortunately, fever is a non-specific and insensitive screening tool for sars [ ] . atypical presentations of sars without fever have been reported especially in older and immunocompromised patients [ ] . one case is particularly illustrative [ ] . a -year-old man cleared a fever triage area in an emergency room as he was afebrile; he was then admitted to a general ward (not a "fever ward") as a case of heart failure, and he remained afebrile until he developed a lowgrade temperature after being transferred to the medical intensive care unit for progressive shortness of breath. two other patients, one visitor and one nurse who had been in the same emergency department area as this patient were infected with sars. the visitor, a previously healthy -year-old woman, died and her husband and son were subsequently infected. in the brief period the index patient was in the general ward, two other patients and the entire shift of nurses working in the ward at the time, who were only wearing n masks, were infected. by the time the patient became febrile in the intensive care unit, staff were wearing full ppe and no further infections resulted. thus, a single patient who "passed" two strict fever screens managed to be the source of at least nine infections in less than h. this patient was critically ill and died days later; thus, he may have had a very high viral load. this case illustrates the limitations of "cookbook screening" by using fever protocols without paying attention to a careful clinical history and physical examination. in this case, an alert cardiology team who re-did the patient's history and examination and performed a bedside echocardiograph to prove that he was suffering from pneumonia not heart failure made the diagnosis. during the sars outbreak, the inter-hospital transfer of patients in canada [ ] , taiwan [ ] and singapore [ ] was a very efficient means of dissemination of the sars virus. in singapore, on march, the decision was made to close one hospital to new admissions and to concentrate all sars patients there [ ] . this unfortunately led to patients recently discharged from this hospital being shunted to other hospitals and starting off epidemics there. now, in singapore, once a cluster of cases with even a low degree of suspicion is identified, the unit is "locked down" with no admissions, transfers or discharges in order to prevent a recurrence of the former situation. this strategy was also used successfully in vietnam to contain the sars virus, which led to vietnam being the first country to be declared free of local transmission of sars [ ] . while we have learned a tremendous amount in the brief period since sars first emerged in november , there are still a number of unresolved issues for practicing clinicians. the cases of sars in early have quashed hopes that the virus was "put back in the box," and in the formerly affected countries many clinicians are deeply worried about dealing with a devastating resurgence of the virus. i have a personal "wish list" of questions that i would like answered before too long. these include: what are the conditions required for the airborne transmission of the sars coronavirus? when can we be sure that transmission does not occur? when can we get a good rapid diagnostic test that is positive early in the course of the illness? what makes a super-spreading event? is quarantine really necessary? i can only hope that the answers to these and numerous other questions raised by infection control practitioners, hospital epidemiologists, infectious disease clinicians and researchers can be answered before we face the next sars outbreak or something worse! even as this is being written, avian influenza rampages across east asia affecting primarily birds, but also claiming the lives of more than individuals. if this becomes a pandemic form of influenza, sars will pale in comparison. epidemiology and cause of severe acute respiratory syndrome (sars) in people's republic of china a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada 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procedures to prevent nosocomial infection with respiratory syncytial virus an outbreak of influenza aboard a commercial airliner global surveillance for severe acute respiratory syndrome transmission of the severe acute respiratory syndrome on aircraft low risk of transmission of severe acute respiratory syndrome on airplanes: the singapore experience accessed use of quarantine to prevent transmission of severe acute respiratory syndrome-taiwan transmission dynamics and control of severe acute respiratory syndrome safety of patients isolated for infection control preventing local transmission of sars: lessons from singapore identification and containment of an outbreak of sars in a community hospital update: severe acute respiratory syndrome-singapore the sars outbreak: how many reminders do we need? cloud" healthcare workers update: severe acute respiratory syndrome-taiwan update: severe acute respiratory syndrome-toronto, canada sars experience at prince of wales hospital, hong kong atypical presentations of sars evaluation of who criteria for identifying patients with severe acute respiratory syndrome out of hospital accuracy of who criteria for sars was similar in a non-sars hospital in singapore crouching tiger, hidden dragon: the laboratory diagnosis of severe acute respiratory syndrome atypical sars in a geriatric patient vietnam-sars free key: cord- -cfv ta authors: patel, kishan p.; vunnam, srinivas r.; patel, puja a.; krill, kaleigh l.; korbitz, parker m.; gallagher, john p.; suh, jane e.; vunnam, rama r. title: transmission of sars-cov- : an update of current literature date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: cfv ta severe acute respiratory syndrome coronavirus (sars-cov- ), the etiologic agent for the coronavirus disease (covid- ) pandemic, has caused a public health emergency. the need for additional research in viral pathogenesis is essential as the number of cases and deaths rise. understanding the virus and its ability to cause disease has been the main focus of current literature; however, there is much unknown. studies have revealed new findings related to the full transmission potential of sars-cov- and its subsequent ability to cause infection by different means. the virus is hypothesized to be of increased virulence compared with previous coronavirus that caused epidemics, in part due to its overall structural integrity and resilience to inactivation. to date, many studies have discussed that the rationale behind its transmission potential is that viral rna has unexpectedly been detected in multiple bodily fluids, with some samples having remained positive for extended periods of time. additionally, the receptor by which the virus gains cellular entry, ace , has been found to be expressed in different human body systems, thereby potentiating its infection in those locations. in this evidence-based comprehensive review, we discuss various potential routes of transmission of sars-cov- —respiratory/droplet, indirect, fecal-oral, vertical, sexual, and ocular. understanding these different routes is important as they pertain to clinical practice, especially in taking preventative measures to mitigate the spread of sars-cov- . sars-cov- , previously known as the novel coronavirus, is an enveloped non-segmented positive-sense rna virus responsible for the coronavirus disease (covid- ) pandemic. it is classified as a beta coronavirus, rendering mammalian hosts susceptible to infection. six other coronaviruses have been identified to infect human hosts, resulting in epidemics, including severe acute respiratory syndrome-related coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) [ ] . as of may , , the global count has reached , , confirmed cases with , covid- -related deaths [ ] , causing growing public health concern. r t , the effective reproduction number, approximates the potential for a virus to spread given a specific measure of time in relation to control measures in place. as suggested by inglesby in an article in jama, without measures such as social distancing in place, the r t is estimated to be similar to that of its value in january ranging between and [ ] . the transmission potential of the virus may be due in part to its structure and overall tenacity. sars-cov- was found to have one of the hardest outer protective shells among all coronaviruses. this is believed to result in more stable viral particles, resulting in its greater resilience in bodily fluid [ ] . in a nejm study, sars-cov- was found to remain viable, in aerosolized form for up to h, and stable on plastic and stainless steel surfaces for up to h [ ] . the virus was found to be more stable on smooth surfaces [ ] . however, it should be noted that the findings presented are in the absence of in vivo data and based solely on a limited number of studies, potentially creating a gap in our understanding. studies have demonstrated that the mechanism by which sars-cov- gains cellular entry in the respiratory tract is through the angiotensin-converting enzyme (ace ) receptor [ ] . the first step involves viral attachment to ace to enable host cell entry; the second step involves activation of the virus internalization process by proteases, particularly tmprss [ ] . ace has been noted to be expressed in various tissues and organ systems throughout the body, including the central nervous system, gastrointestinal system, heart, lung, testes, and kidney [ ] [ ] [ ] . it is hypothesized that this ace -mediated mechanism impacts the tropism of sars-cov- and its ability to invade different organ systems and cause damage [ , ] . studies have shown the detection of viral rna in various bodily fluid samples. wang [ ] . a preprint study reported a covid- patient with no urinary symptoms having tested positive for sars-cov- in the urine [ ] . one case report depicted a patient with meningitis/encephalitis associated with covid- , whereby sars-cov- rna was detected in cerebrospinal fluid (csf) [ ] , whereas another study of pediatric covid- patients showed that four patients with neurological symptoms had negative csf samples for viral rna [ ] . a prospective study by sun j et al. examining covid- patients in china noted that persistent prolonged viral rna shedding has been detected in body fluids, particularly that of nasopharyngeal and fecal samples [ ] . additionally, while viral shedding has been widely noted, a study by xiao et al. had successfully isolated infectious sars-cov- from fecal matter [ ] . it is important to note that although quantitative viral rna has been detected from various body sources, the ability to recover viable virus is of critical importance to understand transmission routes. as some of the studies included in this review lack this data, it may be very difficult to draw reasonable conclusions. in this review, the latest literature pertaining to the potential for various routes of transmission of sars-cov- will be discussed. this narrative review serves to provide information on the latest literature available pertaining to the transmission potential of sars-cov- . this paper was not systematically conducted, and the data presented was made available according to the authors' preference and may be subject to bias or missing data. as the covid- pandemic is evolving, research becomes progressively available, keeping in mind that some of the studies collected thus far may consist of case reports or series with lowquality methodology as they were obtained when this review was written. therefore, the conclusions of this review may be limited in evidence as further research becomes readily available. similar to sars-cov and mers-cov, sars-cov- is predominantly characterized as a respiratory tract infection. the virus commonly presents with nonspecific lower respiratory tract infection symptoms, such as fever, cough, and shortness of breath [ ] . the first cases of covid- took place in wuhan, china, where a group of individuals were evaluated for pneumonia of unknown etiology [ ] . following the outbreak, it was suspected that the virus propagated by means of community and intrafamilial spread [ ] . the transmission is thought to be predominately person to person, either by direct contact or through droplets originating from infected individuals [ ] . droplets can be formed through coughing, sneezing, singing, breathing, and speaking [ ] . droplet transmission involves exposing an entry point, such as mucosa (nose and mouth) or conjunctiva, to potentially infective respiratory droplets (typically > - μm in diameter) produced by someone having respiratory symptoms within a m proximity [ ] . the inhalation of small, exhaled respiratory droplets containing infectious virions is thought to occur; there is a growing concern for long-range human-to-human infection should these droplets remain airborne and viable [ ] . another medium to consider is airborne dust, as inhalation of virus-laden fine particles may transport the virus in deeper bronchial and alveolar regions [ ] . a study analyzing covid- patients from three hospitals in china demonstrated higher positive rates of viral rna in bronchoalveolar lavage fluid, sputum, and nasal swabs [ ] . furthermore, in a study evaluating environmental contamination in symptomatic patients, one of the three subjects had % of his tested room sites, including air outlets, return positive for viral rna despite only having mild upper respiratory tract involvement. environmental contamination through airflow may perpetuate viral transmission through infectious droplets [ ] . compared with direct human-to-human spread, the role of indirect transmission is less understood. airborne transmission occurs when larger respiratory droplets, which have evaporated, or dust particles harbor microbes in the droplet nuclei with a size < μm in diameter. this allows microbes to remain in the air for longer periods of time and travel greater than -m distance. airborne transmission becomes a threat when aerosolization of particles takes place, especially in essential procedures such as endotracheal intubation, bronchoscopy, and cardiopulmonary resuscitation [ ] . liu et al. investigated the aerodynamic nature of the virus based on findings from hospitals in china. the concentration of viral rna in aerosols detected in isolation wards and ventilated patient rooms was very low but elevated in toilet areas. additionally, airborne viral rna levels in the majority of public areas were undetectable with the exception of a few areas prone to crowding [ ] . in addition to aerosolization of microbe particles, infection secondary to contact with contaminated objects or exposure to fomites may occur. a study from the nejm characterized the viability of the virus in multiple surfaces of inoculated samples, with their results indicating that indirect transmission by means of fomite and aerosolization was plausible [ ] . in an early study by cai et al., covid- patients were tracked to identify their environmental exposures within a shopping mall. the findings suggested that viral spread was unlikely due to respiratory droplet transmission alone. direct contact with other subjects, asymptomatic individuals, or exposure to contaminated environments accounted for transmission in some subjects [ ] . moriarty et al. highlighted the transmission potential of the virus on cruise ships totaling more than confirmed cases; viral rna was discovered on multiple surfaces of cabins in symptomatic and asymptomatic individuals despite quarantining efforts, questioning the role of transmission by fomites [ ] . additionally, a preprint study conducted by santarpia et al. highlights environmental viral shedding observed in quarantined individuals in the usa; of surface and aerosol samples collected, ( . %) tested positive by reverse transcriptase-polymerase chain reaction (rt-pcr) for sars-cov- . furthermore, . % of all room surface samples tested positive for the presence of viral rna, supporting significant indirect contamination and likely airborne transmission [ ] . it should be noted that to the best of our knowledge, no viable virus has been isolated from fomite samples indicating limited data on its transmissibility. thus far, there have been many studies analyzing the possibility of fecal-oral transmission of covid- . potential sars-cov- infection in the gastrointestinal tract has been discussed in regard to the expression of ace and tmprss in the epithelium [ , ] . additionally, studies have noted that its fecal-oral transmission potential may lie in the fact that prolonged viral shedding can occur in fecal matter-one case reported an asymptomatic covid- patient experiencing viral detection in the stool for up to days, while nasopharyngeal sampling was negative [ ] . prolonged viral detection in stool samples has also been detected in pediatric patients following the recovery of covid- pneumonia [ ] . in another study involving pediatric covid- patients postdischarge by zhang et al., all subjects had positive stool rt-pcr results after days, without positive throat swabs, clinical symptoms, or imaging findings [ ] . similar findings were demonstrated in adults, whereby patients had positive rt-pcr results in anal swabs after recovery, yet still met criteria for hospital discharge due to negative nasopharyngeal testing [ ] . while prolonged viral shedding in the stool has been noted, whether or not these particles are infectious and have the potential of being spread fecal-orally is questionable. supporting its potentiation, one study by zhang et al. has characterized the presence of live virus in stool; they were able to successfully culture sars-cov- in vero cells which was isolated from a stool specimen of a patient with severe covid- pneumonia [ ] . additionally, a study published in jama by ong et al. supported the possibility of this transmission by demonstrating extensive environmental contamination from a symptomatic covid- patient. samples were collected from the room of a patient whose fecal matter tested positive for sars-cov- by rt-pcr prior to routine cleaning, including from the surface of the toilet bowl, inside of the bowl, and door handle-all of which tested positive. however, samples obtained post-cleaning were negative, implying that current decontamination measures are effective. these findings suggested that viral shedding in the stool could be contributing to a possible route of transmission [ ] . yeo et al. discussed the clinical implications that fecal-oral transmission of covid- may have in infection control, especially in areas with poor sanitation [ ] . with new findings, it was recommended that when handling stool of covid- patients, strict precautions be practiced [ ] . in fact, the detection of sars-cov- in untreated wastewater was confirmed in australia [ ] . yeo et al. also discussed the need for hospital-directed recommendations regarding proper management and disinfection of sewage due to growing concerns for the existence of fecal-oral transmission [ ] . although vertical transmission of covid- has been studied, there still remains a need for further conclusive evidence. certain studies have suggested evidence for vertical transmission on the basis that some neonates born to covid- positive mothers had elevated igm antibodies following birth [ ] [ ] [ ] . in a study by dong et al., a neonate born to a covid- -positive mother was found to have elevated igm antibodies h after birth but tested negative for covid- on nasopharyngeal specimens. typically, igm antibodies do not appear until - days after infection in part due to its molecular structure, but this elevation was present soon after birth in the setting of negative maternal vaginal secretions for sars-cov- [ , ] . another study conducted by zeng et al. examined pregnant covid- patients and highlighted that two infants had elevated igm levels [ ] . in a study by parazzini et al., covid- mothers with both vaginal and cesarean deliveries were assessed ( and , respectively). two neonates tested positive for sars-cov- on rt-pcr testing; three neonates had elevated sars-cov- igg and igm levels but tested negative on rt-pcr. it was concluded that the rate of vertical or peripartum transmission of covid- is low to none for cesarean delivery, but no data was available for vaginal delivery [ ] . additionally, a study involving covid- pregnant mothers reported no vertical transmission in their neonates or placentas [ ] . zamaniyan et al. reported a pregnant woman with severe covid- pneumonia having given birth to a preterm baby at weeks gestation with no evidence of sars-cov- infection. however, testing for covid- by rt-pcr was positive in both an amniotic sample and a second nasal and throat test the neonate underwent h after birth via cesarean delivery; testing was negative in the vaginal secretion sample, umbilical cord blood, and first neonate test. since the amniotic fluid and the neonate tested positive, it may suggest that the newborn was affected intrauterine by sars-cov- [ ] . to oppose, in a retrospective review of nine covid- pregnant mothers who underwent cesarean section, six patients had samples of amniotic fluid, cord blood, neonatal throat swab, and breastmilk samples tested for sars-cov- , and all were negative [ ] . furthermore, there has been a case of neonate in china who tested positive for covid- via rt-pcr of pharyngeal swabs h after birth; however, vertical transmission in the case was not confirmed [ ] . the impact that covid- pregnancies can have on neonates is largely unknown. in a clinical analysis of neonates born from nine covid- mothers by zhu et al., it was found that perinatal infection of sars-cov- may lead to adverse effects in newborns, as some of the neonates experienced fetal distress, premature labor, respiratory distress, thrombocytopenia, abnormal liver function tests, and even death [ ] . a recent study in jama by baud et al. depicted a case of miscarriage during the second trimester in a covid- female related to placental infection with sars-cov- . the fetal surface of the placenta tested positive for sars-cov- ; histopathology revealed mixed inflammatory infiltrates and funisitis. although vertical transmission was not proven, no other etiology of fetal demise was identified [ ] . nevertheless, further research is needed to determine whether sars-cov- crosses the placental barrier. in a cross-sectional study examining adult males recovering from covid- , sars-cov- was not detected in any of the subjects' semen approximately month after initial disease confirmation. however, the findings were unable to definitively rule out the presence of sars-cov- in seminal fluid due to a lack of testing during the acute phase of infection [ ] . however, a recent cohort study by li et al. demonstrated that semen testing for sars-cov- resulted positive for six of the male covid- patients, four of which were in the acute stage of infection and two being in the recovery phase [ ] . the study by pan et al. also characterized that there was low overlapping gene expression of ace and tmprss in human testes, suggesting that the sars-cov- would not likely be able to gain testicular cellular entry via this mechanism [ ] . however, as stated by sama et al., ace has been noted to be highly expressed in the testis [ ] . to et al. noted the detection of sars-cov- in selfcollected saliva specimens in . % ( / ) of covid- patients [ ] . the need for safe sexual intercourse behavior may be heightened due to the fact that the virus has been detected in high concentrations in saliva and nasal mucosa and may have potential receptor binding in rectal and anal epithelial cells. lastly, the potential for an ocular route of transmission has recently been explored. certain studies have noted the detection of viral rna in conjunctival specimens, while others demonstrated limited evidence of its presence. a case study of an ocular manifestation of covid- presenting as bilateral conjunctivitis days after onset of illness suggested that viral shedding occurs in the eyes; rt-pcr of conjunctival samples on days and following the onset of symptoms tested positive. the conjunctiva was confirmed to be a site of viral replication when he tested positive via rt-pcr of consecutive conjunctival swabs. on day , the conjunctival swab specimen resulted negative [ ] . moreover, covid- patients at a hospital center in china were retrospectively reviewed for ocular manifestations; the study concluded that about one-third of patients with covid- had ocular abnormalities, which frequently occurred in patients with more severe disease [ ] . in a case series of covid- patients published in jama ophthalmology, . % ( % ci, . - . ) of subjects had ocular manifestations consistent with conjunctivitis such as conjunctival hyperemia, chemosis, or increased secretions. similar to the prior study, this more commonly occurred in patients with severe systemic manifestations or abnormal laboratory tests. there was a low prevalence of viral nucleotides in conjunctival specimens of . % ( % ci, . - . ) [ ] . a recent case report of the first patient in italy to be diagnosed with covid- depicted bilateral conjunctivitis, in addition to fever, respiratory symptoms, nausea, and vomiting. sars-cov- rna was detected via conjunctival swab on day of hospitalization while still having persistent conjunctivitis-it remained positive until day and again on day (conjunctivitis resolved on day ). in order to demonstrate that the ocular samples contained infectious virus, an rna-positive ocular sample was inoculated in vero e cells, with a cytopathic effect being observed days later. viral replication was confirmed via rt-pcr from purified rna in the cell growth medium. implications of these findings are that ocular fluid of covid- patients may contain infectious virus and serve as a possible source of infection [ , ] . however, a few studies have demonstrated limited evidence for an ocular route of transmission. in a study of covid- patients, tears were sampled and all samples resulted negative for the presence of sars-cov- , while nasopharyngeal samples were positive. likewise, patients with upper respiratory tract infections did not demonstrate any evidence of viral shedding in tears, suggesting that transmission through tears is likely low [ ] . a couple of preprint studies have arrived at similar conclusions. in a study by sun et al. involving patients with laboratory-confirmed covid- , only two patients had conjunctivitis, but sars-cov- rna fragments were found in the ocular discharge of one of the two [ ] . another preprint study which was a retrospective cohort study enrolled confirmed and suspected covid- patients, of which were laboratory confirmed. of the laboratory-confirmed patients, conjunctival specimens from one patient were positive on pcr testing and two patients had probable positive results-none of these three had ocular symptoms [ ] . the outbreak of covid- and evolving pandemic warrant the need for better infection control by means of identifying viral sources. emerging studies have detected the presence of viral rna in multiple regions of the human body, raising concern for its ease of transmissibility. thus far, covid- was originally thought to be spread by respiratory droplets; however, newer studies have highlighted the potential for alternative routes. detection of viral rna shedding in multiple bodily fluids/samples suggests the potential for additional modes of transmission, such as bloodborne, urinary, and fecal-respiratory. transmission by such means remains controversial given the limited supporting data for each mode. clinicians should be aware that the detection of viral rna in numerous bodily fluids can potentiate new routes of transmission as more data on sars-cov- emerges becoming more readily available. these findings emphasize the importance of control measures as they could impact daily practices, from precautions taken by the general public to prevent asymptomatic spread of disease to the isolation precautions practiced in healthcare facilities. limitations to consider when interpreting certain studies discussing transmission routes are availability of research, sample size of patients, and testing capacity in relation to diagnostic supply shortages. the role of whole genome sequencing in characterizing the presence of sars-cov- has been discussed as a comprehensive detection method. the limitations with this method include cost, time, and complications; however, it may play a part in elucidating further understanding of viral transmission pathways. as covid- is not completely understood, the need for further research confirming and/or refuting these potential routes of transmission remains a priority. the origin, 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single-center cross-sectional study. medrxiv ophthalmologic evidence against the interpersonal transmission of novel coronavirus through conjunctiva. medrxiv publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions the authors contributed to and reviewed all aspects of the article. competing interests the authors declare that they have no competing interests. key: cord- -ivu j authors: hernes, s. s.; quarsten, h.; hagen, e.; lyngroth, a. l.; pripp, a. h.; bjorvatn, b.; bakke, p. s. title: swabbing for respiratory viral infections in older patients: a comparison of rayon and nylon flocked swabs date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: ivu j the purpose of this study was to compare the sampling efficacy of rayon swabs and nylon flocked swabs, and of oropharyngeal and nasopharyngeal specimens for the detection of respiratory viruses in elderly patients. samples were obtained from patients years of age or above who were newly admitted to sorlandet hospital arendal, norway. the patients were interviewed for current symptoms of a respiratory tract infection. using rayon swabs and nylon flocked swabs, comparable sets of mucosal samples were harvested from the nasopharynx and the oropharynx. the samples were analysed using real-time polymerase chain reaction (pcr) methods. a total of patients (mean age . years, standard deviation [sd] . years) were swabbed and a virus was recovered from % of the symptomatic patients. regardless of the sampling site, a calculated . times higher viral load ( % confidence interval [ci] . – , p = . ) was obtained using the nylon flocked swabs as compared to the rayon swabs. also, regardless of the type of swab, a calculated times higher viral load was found in the samples from the nasopharynx as compared to the oropharynx ( % ci . – . , p < . ). when swabbing for respiratory viruses in elderly patients, nasopharyngeal rather than oropharyngeal samples should be obtained. nylon flocked swabs appear to be more efficient than rayon swabs. in recent years, respiratory viruses have been established as significant causes of mortality and morbidity in the older population [ ] [ ] [ ] [ ] [ ] . however, as respiratory pathogens may cause similar clinical pictures, the aetiological diagnosis depends on laboratory confirmation [ ] . as demonstrated with respiratory syncytial virus (rsv), viral detection is more demanding in the elderly than in young individuals, as older people tend to shed less virus and have shorter viral-shedding periods [ ] . hence, rapid viral antigen tests that are useful in children may fail in the elderly [ ] . due to its high sensitivity and specificity, realtime polymerase chain reaction (pcr) on respiratory viral samples represents an important diagnostic opportunity also in the older age groups. samples for the diagnosis of a respiratory viral infection can be obtained by swabbing the oropharynx, the nasal cavity, the nasopharynx or alternatively, by nasopharyngeal aspiration (npa) or nasopharyngeal washings (npw). carefully conducted npa and npw usually provide sufficient diagnostic material in children and young adults. however, in frail elderly, npa and npw are less well tolerated [ , ] . for this reason, nasopharyngeal swabbing (nps) is the preferred sampling method in older adults [ ] , although slightly fewer epithelial cells are recovered by nps as compared to npa and npw [ ] . two structurally different swabs are available for microbial sampling in the upper airways: rayon swabs and nylon flocked swabs. a few studies comparing the respective efficacies of these two swabs in providing diagnostic specimens have been performed in children, but, to our knowledge, no such studies have been conducted in the elderly. the aim of this study was to compare the respective efficacies of rayon swabs and nylon flocked swabs in providing material for direct respiratory virus detection by real-time pcr in adults above years of age. we also compared the diagnostic yield of samples recovered from the oropharynx and the nasopharynx. the study design was approved by the norwegian regional committee of research ethics. the study took place from february until february at the department of internal medicine, sorlandet hospital arendal, norway. twice a week, all patients born in or earlier and admitted to the hospital the previous day were interviewed by two team members for symptoms as described in table . during the first and the last four weeks of the study, patients were asked to take part regardless of symptoms, whereas during the rest of the study period, only patients with symptoms were asked to participate. if a patient was unable to supply the information needed, the next of kin and/or the concerned hospital staff were interviewed. if a patient was not able to give informed consent, the next of kin was asked for permission. rayon swabs virocult swabs mw and mw (virocult, medical wire & equipment, corsham, uk) were used in the oropharynx and nasopharynx, respectively. the rayon swabs were inserted into the virocult medium immediately after sampling and the tip of the tube was squeezed as instructed by the manufacturer. nylon flocked swabs nylon flocked swabs cs regular and cs nasopharyngeal (copan italia, brescia, italy) were used in the oropharynx and nasopharynx respectively. the nylon flocked swabs were inserted into universal transport medium (utm) tubes ( . ml medium, no beads, copan italia) and the tubes were shaken for s. swabbing in the oropharynx the sample was taken from the tonsils or posterior oropharyngeal area. the nylon flocked swab was used on one side and the rayon swab on the other. a tongue depressor was used during the procedure and care was taken to avoid touching the tongue with the swabs. swabbing in the nasopharynx the swab was inserted through the nostril, pushed back as far as possible, then rotated, and withdrawn. the nylon flocked swab was used in one nostril and the rayon swab in the other. swabbing was conducted by two experienced team members only. the patients underwent mucosa swabbing of the oropharynx and nasopharynx the day after admittance to the inclusion criteria patients born in or earlier and at least one of the following current symptoms with debut less than three weeks ago: • nasal congestion or runny nose • throat pain • fever • malaise • muscle pain • self-diagnosis of "the common cold" • diarrhoea or eye infection combined with laboratory values supporting an infection table inclusion criteria hospital. after four weeks, all patients testing positive for a respiratory virus were retested with oropharyngeal and nasopharyngeal swabs. the swabbing was performed in the following order: right oropharyngeal area, left oropharyngeal area, left nasopharynx and right nasopharynx. the order of the two types of swabs used was decided by block randomisation. only one swab was used per transport tube, regardless of the type of swab or the location swabbed. the tubes were stored at room temperature and transported to the microbiological laboratory within h. specimens were kept at room temperature until nucleic acid extraction, which usually took place within h and always within h. pcr analyses were performed within the next h or else the nucleic acid eluates were stored at − °c until testing. using monoplex (rsv and human metapneumovirus) or multiplex (influenza a/b, adenovirus/internal control and parainfluenza virus - ) pcr methods, the specimens were examined for, in total, nine different respiratory viruses (table ) . to achieve a more complete diagnostic coverage of respiratory pathogens, pcr assays were added for the detection of mycoplasma pneumonia, chlamydophila pneumonia and bordetella pertussis. extraction of total nucleic acids the rayon swabs were incubated for min in a tris-edta (te) buffer. after thorough stirring, μl of each sample was subjected to nucleic acid extraction in a magnapure lc instrument (roche diagnostics, mannheim, germany) using the magna total nucleic acid isolation kit (roche ca. no. ). the samples were eluted in μl buffer. to be able to detect inhibition of the pcr assay by specimen-specific inhibitors, a final concentration of , λ phage dna copies/ml (tib molbiol cat. no. - - ) were added to the te buffer (not done with the flocked swabs). a control sample with only λ phage dna was included in all extraction runs, in order to ensure that negative results were not due to poor processing. cdna synthesis cdna synthesis was performed with the qscript cdna synthesis kit (quanta biosciences, gaithersburg, md, usa; cat. no. ) following the manufacturer's protocol. the reaction was incubated for min at °c and for min at °c, and then inactivated by min at °c. the cdna samples were subjected to amplification immediately. real-time pcr assay adenovirus real-time pcr assays were performed using lightmix® kit tib molbiol (cat. no. - - ) according to the manufacturer's protocol. the other real-time pcr was performed using μl of (c) dna in a -μl reaction mixture consisting of mm mgcl , units uracil-dna glycosylase (eurogentec s. a., seraing, belgium), lightcycler faststart dna master mix (roche cat. no. ), . - . μm of the primers and . - . μm of each of the probes. all primers and probes are listed in table . the pcr cycling conditions were initiated with min at °c, followed by min at °c and cycles of s at °c, s at °c and s at °c. during pcr amplification, the amount of dna is doubled in each cycle until the components in the reaction reaches a critical level. there is an exponential relationship between the initial amount of template dna and the cycle threshold (ct) values. a high ct value represents a low microbial load in the specimen. samples that differ by a factor of two in the original dna concentration would be expected to be one cycle apart in the run, whereas samples that differ by a factor of ten would be approximately . cycles apart. the differences in ct values obtained from the same experimental run demonstrate a relative difference in the concentration between samples, and is used for calculating differences in viral load. the amplification efficiency for all of the pcr assays used in this study was measured to be above %. descriptive statistics were used when describing the population. a linear mixed model was applied to the data when comparing the two types of swabs or the origin of the samples with a random effect of patient. the statistical analysis was based on ct values. a ct value of was designated as the cut-off value for positive results. thus, a lower ct value in a sample corresponds to a higher microbial load in the sample. the ct values were analysed as continuous data. all statistical analyses were performed using spss . (spss inc., chicago, il, usa). a total of patients were interviewed for current symptoms of a respiratory tract infection (table ) . of these, ( %) patients reported at least one such symptom and agreed to be tested according to the study protocol. twenty-six patients refused to participate ( male, female, mean age . years, standard deviation [sd] . years). during the first and last four weeks of the study, patients without symptoms agreed to be swabbed as well. altogether, patients ( males, females, mean age . years, sd . years) were swabbed and swabs were used. a total of patients were swabbed with all four swabs, patients with three, five patients with two, and one patient with one swab only. a virus was recovered from at least one swab in ( %) of the symptomatic patients (table ), all of whom had reported symptoms of respiratory tract infection. no patients tested positive for more than one virus and no virus was found when reswabbing previously positive patients after four weeks. one patient tested positive for bordetella pertussis and none for chlamydophila pneumonia and mycoplasma pneumonia. as bacterial infections were not the focus of the current study, these results were excluded from the following analysis. in general, nasopharyngeal swabs were positive at a lower ct value compared to oropharyngeal swabs (mean difference ct . , % ci . - . , p < . ), regardless of the sample type (fig. ) . the calculated viral load was times higher ( % ci . - . ) than in the oropharynx. influenza a virus-positive samples showed a lower ct value in the nasopharynx than in the oropharynx, with a mean difference in ct value of . ( % ci . - . , p < . ), which represents a calculated times higher ( % ci - , ) viral load in the nasopharynx. to our knowledge, no comparison between nylon flocked swabs and rayon swabs has been conducted in the elderly population. the present study favours nasopharyngeal sampling with nylon flocked swabs: a calculated . times higher viral dna concentration was found on the nylon flocked swabs, regardless of the sample origin. nylon flocked swabs seem to adhere more epithelial cells than rayon swabs [ ] and this might, at least in part, explain the lower ct values obtained by using flocked swabs. nasopharyngeal aspirate, which has been considered as a gold standard material for the diagnosis of respiratory viruses in children, has a sensitivity at the level of, or slightly above, nasopharyngeal nylon flocked swabs [ , ] . a comparison between nylon flocked swabs and nasopharyngeal aspirate in the elderly population has not been published. in terms of ct, we found nasopharyngeal samples to be superior to oropharyngeal samples, yielding a calculated times higher concentration of viral nucleic acids. this corresponds to the results found in a study by lieberman et al. [ ] , with a much broader variation in patient age. our subgroup analysis of influenza a virus reveals a calculated times higher viral load in samples from the nasopharynx. as shown in table , several samples were negative for influenza a virus in the oropharynx and positive in the nasopharynx. nasopharyngeal swabs are easy to use after proper instruction, but swabbing for microbiological agents is often left with the least trained medical staff. in such cases, suboptimal swabbing may be relatively common. the sample collection in our study was performed by two experienced team members, thus, minimising the risk of suboptimal sampling. whereas usually, considerable efforts are made to optimise the diagnostic procedures in the laboratory, less emphasis tends to be placed on the preceding procedures of sample collection. we believe that there is a lot to be gained from an increased awareness of proper sampling techniques and sampling tools. for semi-quantitative assessments of respiratory pathogens in children, immunofluorescent assays or viral culture have been widely applied [ , [ ] [ ] [ ] , whereas in the current study, ct values were obtained for this purpose. most studies applying real-time pcr for the detection of respiratory agents simply determine whether there is an infection or not. however, within each pcr experiment, there is a close relationship between the achieved ct value and the initial amount of specific nucleic acids in the sample. in this study, only samples taken at the same time from the same patient are compared and the ct values used for the calculations are collected from the same experiment. the calculations of differences in viral load relied upon the use of pcr assays with high amplification efficiencies. factors other than the ones we have been examining are then minimised. in this manner, the method provides valid relative quantitative data when comparing distinct sample materials. we found respiratory viruses in % of our study population. corresponding numbers reported by other authors range from to % [ , ] , depending on which viruses were included in the pcr analysis. we chose to include only microbial agents responsible for significant illness, thus, excluding frequently occurring causes of upper respiratory tract infections, such as rhinovirus and coronavirus. the number of patients diagnosed with influenza a and rsv infections were lower in our study than those reported by others, probably due to annual variations. we did not find any respiratory viruses in non-symptomatic patients. during the two periods when all patients regardless of symptoms were swabbed, respiratory viruses were found in . % ( patients) of the total study population. some authors have concluded that the combination of oropharyngeal sampling, nasopharyngeal sampling and npa represents the ideal sampling method for the diagnosis of respiratory tract viruses [ ] . however, in a busy clinical setting, it may be hard enough to ensure one properly collected sample from each of the concerned patients. our conclusion is that, when a respiratory tract infection requires aetiological clarification, a rational approach to the virological part of the investigation is to obtain a nasopharyngeal sample, preferably collected by trained personnel and, at least where costs are comparable, using nylon flocked swabs rather than rayon swabs. an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility respiratory syncytial virus infection in elderly and highrisk adults respiratory syncytial virus pneumonia among the elderly: an assessment of disease burden a summer outbreak of human metapneumovirus infection in a long-term-care facility mortality associated with influenza and respiratory syncytial virus in the united states is clinical recognition of respiratory syncytial virus infection in hospitalized elderly and high-risk adults possible? evaluation of four methods for the 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nasopharyngeal versus oropharyngeal sampling comparison of nasopharyngeal aspirate and nasopharyngeal swab specimens for respiratory syncytial virus diagnosis by cell culture, indirect immunofluorescence assay, and enzymelinked immunosorbent assay comparison between pernasal flocked swabs and nasopharyngeal aspirates for detection of common respiratory viruses in samples from children nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses surveillance of respiratory virus infections in adult hospital admissions using rapid methods development of a genomics-based pcr assay for detection of mycoplasma pneumoniae in a large outbreak in new york state evaluation of real-time quantitative pcr for identification and quantification of chlamydia pneumoniae by comparison with immunohistochemistry the use of taqman pcr assay for detection of bordetella pertussis infection from clinical specimens real-time quantitative pcr assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients clinical diagnosis of influenza virus infection: evaluation of diagnostic tools in general practice increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time pcr in samples from patients with respiratory symptoms comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses , , , and real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages acknowledgements this project was financed with the aid of extra funds from the norwegian foundation for health and rehabilitation in association with the norwegian heart and lung patient organisation, from the south-eastern norway regional health authority and from sorlandet hospital hf. the nylon flocked swabs and utm transport tubes were donated by copan italia, brescia, italy, whereas the rayon swabs and transport medium were donated by chemi-teknik a/s, oslo, norway. both suppliers delivered the swabs free of charge. the suppliers had no role in the planning, running, evaluation or reporting of this trial. the authors declare that they have no conflict of interest related to this report.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord- -fiqx tll authors: mäkelä, m. j.; halminen, m.; ruuskanen, o.; puhakka, t.; pirhonen, j.; julkunen, i.; ilonen, j. title: lack of induction by rhinoviruses of systemic type i interferon production or enhanced mxa protein expression during the common cold date: journal: eur j clin microbiol infect dis doi: . /s sha: doc_id: cord_uid: fiqx tll to study whether mxa protein expression is systemically upregulated during rhinovirus infection, blood specimens were collected from patients with common cold and mxa expression in mononuclear cells analyzed by flow cytometry. none of the patients with a confirmed rhinovirus infection (n= ) or with an infection of unknown etiology (n= ) had elevated expression of the mxa protein (median fluorescence intensity, and , respectively) when compared to healthy controls (n= , median ). patients with influenza infections had significantly elevated values (n= , median ), and interferon could be detected only in serum samples from influenza patients. in conclusion, expression of mxa in blood lymphocytes and an apparently systemic type i interferon response is not induced during rhinovirus infection or during most other cases of common cold in young adult patients. interferons (ifns) form the first line of defence against viral infections. type i interferons (ifn-a/b) are often produced by many types of cells, and ifn-a can be detected in serum. measurement of ifn-a in serum has been used as a marker to differentiate viral and bacterial infections [ ] [ ] [ ] . the sensitivity of these assays for indicating viral infection ranges from to %. therefore, other assays based on the expression of ifn-inducible genes have been developed. one of these ifn-a/b-inducible genes, mxa, encodes for a cytoplasmic gtpase. this protein has antiviral activity against many viruses [ , ] and is expressed in lymphocytes and in some tissues under strict regulation by type i ifns [ ] . previously, we showed via flow cytometry that nearly all febrile children with a documented viral infection have elevated mxa levels [ ] . recent studies suggest that rhinovirus is the single most important respiratory virus because of its role in the common cold and in the pathogenesis of otitis media, sinusitis and exacerbations of asthma [ ] . little is known about the significance and function of the ifn system in rhinovirus infections. in the present study, the induction of a systemic ifn response as reflected by the expression of mxa protein in blood lymphocytes of patients with rhinovirus infection was examined. a total of subjects were recruited for the study. blood was drawn from patients with common cold within h of the onset of symptoms. the median age of the patients was . years (range . - . years). diagnosis of viral infection was performed as described previously [ ] . briefly, antigen detection or culture from nasopharyngeal aspirate and serological tests were used for detection of adenovirus, respiratory syncytial virus, influenza viruses a and b and parainfluenza virus types , and . rhinoviruses were detected both by culture and by polymerase chain reaction (pcr). based on the viral diagnosis, the patients were divided into three groups. there were rhinovirus-positive subjects ( culture positive, of whom were also pcr positive; serum type i ifn levels were also analyzed by two different assays as described below. since we were not able to detect elevated mxa levels in the rhinovirus-positive patients in the first phase of the study, we wanted to study whether these patients would have type i interferon in serum. for this purpose, two assays were used as described below. there was no serum left of the influenza virus-positive patients analyzed in the mxa assay, and, therefore, the serum specimens from influenza virus-positive patients whose lymphocytes were used for mxa expression analysis were not available. therefore, in our analysis, we used paired serum samples from patients with a marked rise in serum antibodies against influenza a. these frozen serum samples were obtained from a collection of serum samples stored at the department of virology, university of turku (j.i.). mxa protein was analyzed from peripheral blood mononuclear cells by indirect immunofluorescence and flow cytometry as described previously [ ] . the lymphocyte population was identified by the typical pattern of side scatter (intracellular structure) and forward scatter (size) parameters in flow cytometry. the mxa level was indicated by the median channel of logarithmic fluorescence counts from the cytometer. the nonparametric mann-whitney u test was used for comparisons between the groups. serum ifn-a levels were determined using a commercial assay kit (human ifn alpha elisa; endogen, usa) according to the manufacturer's instructions. the sensitivity of the assay was theoretically pg/ml, which corresponds to approximately . - iu/ml of ifn-a. only two of the influenza virus patients were positive by this assay, suggesting inadequate sensitivity. therefore, serum specimens were also analyzed by a biological ifn assay. the presence of biologically active ifn-a/b in sera was measured in hep- cells by a vesicular stomatitis virus plaque reduction assay as described previously [ ] . the results are expressed as international units (iu/ml), using an international control ifn-a prepared as a laboratory standard. the ethical committee of the turku university hospital approved the study. informed consent was obtained from all patients. the median intensity values of mxa-specific fluorescence in peripheral blood mononuclear cells of different patient groups are shown in figure . based on earlier data from healthy children and patients with bacterial infections, the cut-off value was set at , which is two standard deviations above the mean of healthy children [ ] . in the present study, the median fluorescence intensity value for the healthy adults was ( table ) . none of the patients with rhinovirus infection (np ) had values above , whereas all influenza virus-positive patients (np ) had values above this limit, with an upper range of . the median value in the rhinovirus-negative group (np ) was , which does not differ significantly from the median value in the rhinovirus-positive group (pp . ) but is significantly lower than the value in the influenza group (pp . ). to study whether the capacity of lymphocytes to produce mxa protein was blocked by some repressors or other inhibitory factor, we also cultured the cells for h in the presence of exogenous ifn-a ( iu/ml). in all groups the induced values were significantly higher than the baseline values (table ) . ifn-a/b levels were measured in serum, since mxa expression is regulated by type i ifns. none of the patients with rhinovirus infection had detectable ifn-a/b in serum as measured by either enzyme immunoassay or by a biological assay. a similar observation was made in the rhinovirus-negative patients and in the control group. ifn-a/b was detectable in the serum of seven of the . in six of the seven patients, ifn was detected in the acute-phase serum but not in the convalescent serum, and in one patient, ifn was detected only in the convalescent serum sample. low mxa values in patients with common cold were not expected, particularly in light of our earlier findings in children with viral infections in whom mxa was induced in practically all patients with influenza, adenovirus, respiratory syncytial virus and rotavirus infection [ ] . moreover, rhinovirus has been shown to induce ifn in vitro [ ] , and ifn has been found in the nasal secretions of volunteers infected with rhinovirus [ ] . stimulation with exogenous ifn-a, on the other hand, resulted in significant induction, suggesting that there is no functional defect associated with the rhinovirus infection per se. it is known that viruses and, probably, virus strains differ in their ability to induce ifn production, and there is also significant individual variation [ ] . ifn synthesis is often short-lived, but mxa is a stable protein with a half-life of several days [ ] . since all patients were examined within h after the onset of symptoms, it is unlikely that potential mxa upregulation would have been missed due to delayed analysis [ , ] . the mxa gene is under the strict control of type i ifns, and thus expression of the mxa protein indirectly reflects the presence of ifn in the host. serum ifn-a/b levels were therefore measured as well. the findings were in accordance with the study of parry and parry [ ] , who found interferon in the sera of nine of their patients with influenza but in none of the patients with rhinovirus infection. our data suggests that type i ifn production in serum is not comparable to that seen in most other respiratory viral infections in vivo, since the rhinovirus-positive patients had only low or no expression of the mxa protein and no ifn-a/b was detectable in serum samples. there is still uncertainty about the presence of rhinovirus in different parts of the airways, but according to recent studies with experimental infections in humans, rhinoviruses seem to infect epithelial cells both in the upper and lower airways [ ] . it is possible that, in most cases, the rhinovirus infection is limited mainly to the respiratory epithelium without major systemic influence. in conclusion, this study suggests that the systemic type i ifn response and subsequent upregulation of ifninducible mxa protein expression is poor in rhinovirus infection. mxa protein expression may, therefore, not be a suitable marker of viral infection in the common cold. it is still unknown whether the lack of a systemic ifn response plays a significant pathophysiological role during rhinovirus infection. although we did not find ifn or clearly induced mxa protein expression in the peripheral blood of any subject with rhinovirus infection, we cannot rule out the possibly significant role of ifn in local epithelial response against the virus. interferon assay as a viral diagnostic test serum alpha-interferon in lower respiratory tract infections of children detection of serum interpheron-alpha by dissociation-enhanced lanthanide fluoroimmunoassay. studies of patients with acute viral and bacterial infections inhibition of puumala and tula hantaviruses in vero cells by mxa protein resistance to influenza virus and vesicular stomatitis virus conferred by expression of human mxa protein control of ifninducible mxa gene expression in human cells expression of mxa protein in blood lymphocytes discriminates between viral and bacterial infections in febrile children what's new with common colds? complications and management viruses and bacteria in the etiology of the common cold rapid production of interferon-gamma in uninduced human leukocyte suspensions induction of interferon in human leukocyte cultures by natural pathogenic respiratory viruses interferon and resistance to upper respiratory virus illness mxa protein in infants and children with respiratory tract infection interferon assay as a diagnostic test detection of rhinovirus rna in lower airway cells during experimentally induced infection acknowledgements this work was supported by the finnish cancer foundation, the maud kuistila memorial foundation and the academy of finland. dr. david smith is acknowledged for revising the language of the manuscript. key: cord- -iz l i d authors: o’gorman, c.; mchenry, e.; coyle, p. v. title: human metapneumovirus in adults: a short case series date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: iz l i d this study was carried out to further the available information on adult cases of human metapneumovirus (hmpv), a recently described cause of respiratory infection. among a cohort of symptomatic patients tested since , the virus was diagnosed in six adults using reverse transcriptase polymerase chain reaction. of the six, two were from the community, two were hospital inpatients with chronic obstructive pulmonary disease and two were immunocompromised patients, both of whom required ventilation and later died. this report discusses the clinical features, epidemiology and diagnosis of hmpv, highlighting that this infection may be associated with death in high-risk adults. for adults presenting with respiratory symptoms and a background of pre-existing respiratory disease or who are immunocompromised, nucleic acid-based techniques are a cost-effective means of making the viral diagnosis in a clinically relevant time frame. acute respiratory tract infections are a major cause of morbidity and mortality worldwide, and they account for a high percentage of hospital admissions. however, up to % of cases of community-acquired pneumonia in adults and - % of cases of bronchiolitis and pneumonia in children are of uncertain aetiology [ ] [ ] [ ] . it is clear that respiratory viruses are the principal pathogens in a proportion of such cases [ ] . hmpv is a recently described respiratory pathogen of the subfamily pneumovirinae, which is divided into the pneumovirus genus, containing respiratory syncytial virus, and the metapneumovirus genus, containing hmpv. in young children and elderly patients hmpv is most commonly associated with a clinical diagnosis of bronchiolitis or bronchitis, respectively, whereas in middle-aged adults, it may produce an influenza-like illness, which can be complicated by pneumonitis in the presence of immunocompromising factors [ ] . hmpv has been detected as the sole pathogen in the nasopharyngeal aspirate of a haematopoietic stem cell transplant recipient who subsequently died of respiratory failure following an upper respiratory prodrome [ ] . the regional virus laboratory in belfast carries out viral diagnostics for northern ireland, a region with a population of , , . it began routine use of a multiplex reverse-transcriptase polymerase chain reaction (rt-pcr) method for virus detection from respiratory samples in july of [ ] . the samples were from three sources: adult hospital inpatients ( . % of total processed), paediatric hospital inpatients ( . %) and general practitioners participating in a sentinel influenza surveillance program ( . %). the laboratory tested any specimens of respiratory tract material including bronchoalveolar lavage, nasopharyngeal and tracheal aspirates, and sputum, nose and throat swabs. this retrospective observational study reviewed all cases of hmpv detected in patients over years of age, from the time the rt-pcr method was adopted in july through to january . we selected adult cases for review as there is less data in the literature pertaining to hmpv infection in this age group. the routine assay used to detect respiratory viruses in the regional virus laboratory is a multiplex, nested rt-pcr for common respiratory pathogens: influenza a (h and h ) and b; parainfluenza virus types , and ; respiratory syncytial virus a and b; adenovirus; human rhinovirus; coronavirus e; and hmpv. the pcr target for hmpv was the fusion gene. sensitivity was determined at × copies per ml using an hmpv amplicon cloned from a clinical specimen during the preliminary development of the c. o'gorman (*) . e. mchenry . p. v. coyle regional virus laboratory, royal group of hospitals trust, belfast bt ba, northern ireland e-mail: ciaran.ogorman@bll.n-i.nhs.uk tel.: + - - fax: + - - molecular strip assay. direct comparison of the assay with an alternative was not possible because of the lack of specimens containing hmpvor alternative tests; virus identification was confirmed by selected amplicon sequencing [ ] . following ethics committee approval, we obtained written consent from the living patients in whom hmpv was detected. we reviewed their clinical case notes, noting presenting symptoms, significant medical and drug histories, results of other infectious investigations, chest radiograph findings, and clinical outcomes. a total of respiratory samples from adults were tested over the winters of / and / . six were positive for hmpv, accounting for a period prevalence in the population assayed of . %. in all cases, hmpv was the only virus detected. unless otherwise stated, bacterial culture and serology for atypical respiratory pathogens were negative. the clinical characteristics of these six patients are summarised in table . of the six cases, five were female and one male. their ages ranged from to years (mean . years). two cases presented to sentinel general practitioners with selflimiting influenza-like illness (cases and ). neither had a background respiratory disease or any other significant aspect of their medical history. two cases were diagnosed while admitted for acute exacerbations of chronic obstructive pulmonary disease (cases and ). they presented with dyspnoea, decreased exercise tolerance, cough and lowgrade pyrexia with a background of respiratory disease requiring home oxygen and nebulised corticosteroids. both had been smokers, had received prednisolone as inpatients, and were discharged on maintenance oral steroids. sputum culture of case revealed serratia liquefaciens, which was treated with intravenous ertapenem; for case , hmpv was the sole pathogen isolated. two cases ( and ) were fatal. the fatalities occurred in individuals receiving immunosuppressive therapy for rheumatological conditions. case had rheumatoid arthritis for which she received prednisolone and methotrexate. she presented in a collapsed and unresponsive condition following a -week history of cough, nausea and increasing dyspnoea. within h of admission she was intubated, ventilated and admitted to the intensive care unit (icu). hmpv was the sole pathogen isolated. she died with multiorgan failure days after admission. case six was admitted in an acute confusional state attributed to the oral steroids she was taking for polymyalgia rheumatica. she had been an inpatient for days when she became acutely dyspnoeic with hypoxia, hypothermia and hypotension requiring intubation and admission to the icu. she was found to have bacteremia caused by escherichia coli and was treated with piperacillintazobactam, fluconazole and pulsed methylprednisolone. on the first day of her icu stay, having been an inpatient for days, she was found to have hmpv. this patient made no significant progress while receiving maximal inotropic and ventilatory support and she died after weeks in the icu. whilst our detection of hmpv in symptomatic patients does not establish causation, it is possible that the virus may be associated with serious sequelae in high-risk adults as reported previously [ ] and supported by the fact that two of our six cases required icu admission and ventilation. we cannot be sure that case represented nosocomial acquisition as our assay may detect hmpv up to weeks after clinical infection. the clinical syndrome associated with this virus is probably too indistinct to be of diagnostic value. treatment is principally supportive; at present there are no antiviral agents active against hmpv, nor any available vaccine. effective diagnosis of viral respiratory pathogens is necessary to elucidate their epidemiology and assess their clinical impact. moreover, such data is crucial for effective infection control in hospitals and may ultimately allow more rational prescribing of antibiotics and corticosteroids [ ] . defining the assay sensitivity by comparison with a gold-standard detection system is a growing problem for viruses such as hmpvor human rhinovirus, for which pcr is becoming adopted as the diagnostic test of choice [ ] . we recommend the routine use of rt-pcr or other nucleic acid-based techniques for adults presenting with respiratory symptoms and a background of pre-existing respiratory disease or immunocompromise. the tucson children's respiratory study ii. lower respiratory tract illness in the first year of life etiology of community-acquired pneumonia: impact of age, comorbidity, and severity prospective comparative study of viral, bacterial and atypical organisms identified in pneumonia and bronchiolitis in hospitalized canadian infants virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratorytract infections in all age groups a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus in a haematopoietic stem cell transplant recipient with fatal lower respiratory tract disease a touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients acknowledgements the royal group of hospitals trust (royal research office, grosvenor road, belfast) has agreed to act as sponsor for this study. ethical approval for this study was obtained on january from the office for research ethics committees northern ireland (orecni); rec reference number: /nir / . written consent was obtained from case patients as required by the research ethics committee. key: cord- -rb n wc authors: poole, stephen; townsend, jennifer; wertheim, heiman; kidd, stephen p.; welte, tobias; schuetz, philipp; luyt, charles-edouard; beishuizen, albertus; jensen, jens-ulrik stæhr; del castillo, juan gonzález; plebani, mario; saeed, kordo title: how are rapid diagnostic tests for infectious diseases used in clinical practice: a global survey by the international society of antimicrobial chemotherapy (isac) date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: rb n wc novel rapid diagnostic tests (rdts) offer huge potential to optimise clinical care and improve patient outcomes. in this study, we aim to assess the current patterns of use around the world, identify issues for successful implementation and suggest best practice advice on how to introduce new tests. an electronic survey was devised by the international society of antimicrobial chemotherapy (isac) rapid diagnostics and biomarkers working group focussing on the availability, structure and impact of rdts around the world. it was circulated to isac members in december . results were collated according to the un human development index (hdi). responses were gathered from different countries. % of institutions reported the availability of any test / . in more developed countries, this was more for respiratory viruses, whereas in high and medium/low developed countries, it was for hiv and viral hepatitis. only % of those carrying out rapid tests measured the impact. there is no ‘one-size fits all’ solution to rdts: the requirements must be tailored to the healthcare setting in which they are deployed and there are many factors that should be considered prior to this. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. rapid diagnostic tests (rdts) are increasingly used in clinical practice to provide actionable information for patient care in a timely manner, ideally at the time and location of the patient's interaction with health care systems. rdts (often referred to as point-of-care tests (poct) when deployed near-patient) are often simple to use and therefore can offer diagnostic support in resource-limited settings or away from more sophisticated diagnostic laboratory support, for example in primary care. the treatment of many infectious diseases is time-critical. a test that facilitates early-directed therapy increases the chance of good patient outcomes and promotes good antimicrobial stewardship. furthermore, the early identification of highly transmissible illnesses allows healthcare services in high-income countries to rapidly isolate patients and limit the spread of disease: a benefit which has been particularly highlighted with the emergence of sars-cov- . the last decade has seen a boom in rapid diagnostic products, with many developed and approved by healthcare authorities around the world [ ] for a variety of different infections including gastroenteritis [ ] , bloodstream infections [ ] , pneumonia [ ] and respiratory viruses [ ] . formats of these tests include lateral flow assays and polymerase chain reaction (pcr). there are potential pitfalls around the implementation of rdts. many are expensive, and robust evidence for tangible clinical benefit to justify this outlay can be lacking. for some, sensitivity and specificity may be lower than established laboratory tests and therefore require that these can only be applicable to specific situations (e.g. when the pre-test probability is high). governance, quality control and assurance can be challenging, particularly when rdts are not sited within a traditional laboratory setup. these challenges differ around the world depending on local health diagnostic regulations, availability of resource, local epidemiology and patient expectation. the international society of antimicrobial chemotherapy (isac)'s rapid diagnostics and biomarkers working group conducted this international survey aiming to identify and highlight some key issues related to rdts and their impacts in clinical practice and provide a number of key points to consider while adopting a rdt. a questionnaire (survey monkey®) was devised and approved by the working group (supplementary materials). the survey included questions about the experience of rdts: the questionnaire was circulated to isac members via isac secretaries and respondents were given weeks to respond during december and january with at least two reminders. everyone surveyed was either in a position to request or deliver tests. among the questions, there was an optional question asking responders to provide their specific role and institution. the location of institutions was linked to a united nations (un) human development index (hdi) ranking (very high, high, medium or low) [ ] . this is a widely used, blunt representation of a nation's development which considers life expectancy, income per capita and education. responses were received from isac members representing countries (fig. ) . this represented % of those initially surveyed. six respondents did not disclose their nationality. % of those who did disclose their nationality were received from countries classified as very highly developed on the un hdi, % were from highly developed nations and the remaining % from countries classified as having medium or low levels of development. / ( %) respondents reported no available rdts. the proportion of those who have these available is reported in fig. . the main barrier reported for not adopting rdts was financial ( %), and other reasons were a lack of expertise ( %) or lack of applicability to their clinical setting ( %). % cited a lack of interest in the tests. only % of those with rdts reported measuring the impacts of their tests in any way (fig. ) . % of those with rdts reported the laboratory carrying out the test. % reported the emergency department performing them. other clinical settings rarely carried out the tests ( % in clinics and % inwards). the governance structure for rdts is presented in fig. . the most common way for reporting was in the electronic patient file ( %); fewer institutions generate the report in real time ( %). % of institutions directly phone the result to the requesting clinician. one respondent reported still generating paper reports, one reporting by email, one by sms and one not generating any specific laboratory reports as the test is done in the assessment area. the survey has given us an insight into what is happening globally with rdts. many respondents reported / availability of tests. very high-income countries had higher proportional availability of rapid influenza and respiratory virus tests. in lower-income countries, however, a lower proportion of respondents reported the availability of these tests, but hiv and hepatitis testing were available in greater proportions. the explanation for this pattern is likely multifactorial. in general, the epidemiology of chronic viral hepatitis and hiv is such that they are more prevalent in developing countries where public health interventions are less likely to identify and treat patients early in the course of illness [ ] . the priorities for treatment are also different: influenza other resp multiplex, other respiratory multiplex; hep, hepatitis; gi, gastrointestinal tests management in secondary care is a less pressing need in resource-restricted settings where patient isolation facilities are less readily available. furthermore, the clinical impact relative to the cost of identifying a case of influenza is less than hiv or viral hepatitis where early identification and treatment make a greater difference [ , ] . the relative cost of each test is likely to also be a factor in the difference of availability, with multiplexed assays generally being considerably more expensive and requiring more complex logistical support. methods for reducing the costs of many rdts are lacking, which limit their availability in low-income settings. there are still major gaps in capturing the impact of rdts on decision making in a systematic manner. only % of users measure impact. % of those surveyed reported that lack of money was the major barrier to bringing in rdts in their institution. developing robust impact recording systems, such as regular audit cycles, coupled with cost-effectiveness analyses are crucial to support business cases for new rdts. the current setup of rdts appears to be more laboratory centred: governance and quality control are the responsibility of laboratories in the vast majority of those surveyed. % of those who responded to the survey said tests were carried out in their institution by laboratory staff. simpler tests lend themselves more towards near-patient deployment and a clia waiver is often a good indicator of this. while there are a number of existing international regulatory processes for drugs and medications, providing safeguards for their safety and efficacy, they are often lacking for rdts [ , ] . as a result, diagnostic tests are often sold and used in the developing world without any evidence of effectiveness. for example, mak et al. [ ] reported the sensitivity of an rdt for sars-cov- of . - . % when the manufacturer had claimed it was %. the benefit of rdts can be lost if not coupled with rapid pre-and post-analytical phases. the survey identified that less than half of the results are communicated to the requester directly, and only % of reports are generated in real-time on computers. this means delays are introduced as clinicians look up results. interestingly in some institutions, results are sent out by sms or email to requesting clinicians which would optimise the reporting process. identification of certain infectious organisms may have wider public health implications, for example, legionella; therefore we advocate real-time connection for these results to systems that allow rapid reporting to responsible public health authorities. a limitation to the method we should consider is the selection bias towards isac members who would be motivated to respond to the survey: potentially those who have the greatest interest in rdts or who are highly critical of them. there is also a bias towards respondents with greater resources suggested by the fact that at least % of tests had a laboratory involvement. furthermore, the survey size is relatively small and certain world regions (especially southeast asian nations and sub-saharan african nations) are poorly represented. the main aims of rdts are to improve patient care most efficiently within well-managed healthcare systems. we therefore suggest a number of best practices for implementation of rdts (table ) . for rdts there is no 'one-size-fits-all' model; modelling of tests and costs are wildly different for different healthcare systems. our survey highlights the availability of these tests in different resource settings, as well as the current models for governance, quality control and reporting. point-of-care testing for infectious diseases: past, present, and future multiplex molecular panels for diagnosis of gastrointestinal infection: performance, result interpretation, and cost-effectiveness a review of novel technologies and techniques associated with identification of bloodstream infection etiologies and rapid antimicrobial genotypic and quantitative phenotypic determination rapid syndromic molecular testing in pneumonia: the current landscape and future potential routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial human development index (hdi) (n.d.) human development reports hepatitis b virus burden in developing countries cost-benefit analysis of real-time influenza testing for patients in german emergency rooms the economic burden of late entry into medical care for patients with hiv infection a guide for diagnostic evaluations point-of-care tests for diagnosing infections in the developing world evaluation of rapid antigen test for detection of sars-cov- virus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments the authors would like to thank the isac secretariat, key: cord- -wqlp ogb authors: liu, wen-kuan; chen, de-hui; tan, wei-ping; qiu, shu-yan; xu, duo; zhang, li; gu, shu-jun; zhou, rong; liu, qian title: paramyxoviruses respiratory syncytial virus, parainfluenza virus, and human metapneumovirus infection in pediatric hospitalized patients and climate correlation in a subtropical region of southern china: a -year survey date: - - journal: eur j clin microbiol infect dis doi: . /s - - -x sha: doc_id: cord_uid: wqlp ogb to investigate the features of paramyxovirus respiratory syncytial virus (rsv), parainfluenza virus (piv), and human metapneumovirus (hmpv) infection and determine the effect of meteorological conditions in guangzhou, a subtropical region of southern china. we collected , respiratory samples from hospitalized pediatric patients with acute respiratory illness between july and june in guangzhou. the samples were tested simultaneously for respiratory pathogens using real-time pcr. local meteorological data were also collected for correlation analysis. of , patients tested, ( . %) patients tested positive for one or more pathogens; rsv, piv, and hmpv were the first, sixth, and ninth most frequently detected pathogens, in ( . %), ( . %), and ( . %) patients, respectively. a total . % ( / ) of patients with positive results had coinfection with other pathogens. significant differences were found in the prevalence of rsv, piv, and hmpv among all age groups (p < . ). rsv and hmpv had similar seasonal patterns, with two prevalence peaks every year. piv appeared alternatively with rsv and hmpv. multiple linear regression models were established for rsv, piv, and hmpv prevalence and meteorological factors (p < . ). rsv and piv incidence was negatively correlated with monthly mean relative humidity; rsv and hmpv incidence was negatively correlated with sunshine duration; piv incidence was positively correlated with mean temperature. we described the features of paramyxovirus infection in a subtropical region of china and highlighted the correlation with meteorological factors. these findings will assist public health authorities and clinicians in improving strategies for controlling paramyxovirus infection. respiratory syncytial virus (rsv), parainfluenza virus (piv), and human metapneumovirus (hmpv) are enveloped, nonsegmented, negative-sense, singlestranded rna viruses belonging to the paramyxoviridae family. these viruses are a significant cause of morbidity and mortality globally, especially among children in developing countries [ ] [ ] [ ] [ ] [ ] [ ] [ ] . rsv is the most important pathogenic infection of childhood worldwide, causing a variety of manifestations from mild upper respiratory tract illnesses or otitis media to severe and potentially lifethreatening lower respiratory tract illnesses [ , [ ] [ ] [ ] [ ] . four piv types (piv - ) have been identified [ , ] . piv and piv are best known as the cause of croup whereas piv is a common cause of bronchiolitis and pneumonia [ ] . piv infection has low prevalence [ ] . hmpv was first discovered in patients with acute respiratory illness (ari) in [ ] . since then, hmpv has been associated with ari in children as well as elderly and immunocompromised adults [ ] [ ] [ ] . rsv, piv, and hmpv are also important causes of nosocomial infection, which might be life-threatening in certain individuals, such as transplant or immunocompromised patients [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . until now, no effective vaccine for rsv has been available. the rsv-specific monoclonal antibody palivizumab has been advocated for use as prophylaxis in high-risk patients against rsv infection [ ] [ ] [ ] . however, there is no available vaccine or specific antiviral treatment for piv and hmpv infection. consequently, it is imperative to conduct further research, especially in low-and middle-income countries, to understand the epidemiological features of these pathogens in different areas and populations. in general, the prevalence of viruses can vary because of factors such as geographical location, climatic conditions, population, and social activity [ ] . guangzhou, which is located on the subtropical coast of china, has a maritime subtropical monsoon climate. guangzhou is china's first gateway hub to southeast asia and oceania. the city is densely populated and frequent exchanges of domestic and international personnel and materials take place in the area. guangzhou has been a hotbed of activity for various respiratory pathogens. investigation of respiratory pathogen epidemics in the region is critical. in this study, we analyzed paramyxovirus infection among children hospitalized with ari over a -year period in guangzhou, and we collected local meteorological data for climate correlation analysis. these data will be helpful for the prevention and control of these viruses. we performed a cross-sectional study in three tertiary hospitals between july and june in guangzhou, southern china. pediatric patients (n = , ) hospitalized with ari were enrolled in this study. the detail inclusion criteria were pediatric patients (≤ years old) who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, or dyspnea during the previous week. patients who were diagnosed with pneumonia by chest radiography during the previous week were also included in the study, even if they did not show the clinical features described above. some patients who had been cured and discharged some time ago but were then readmitted because of a new episode of ari were included as new cases if met the recruitment criteria; otherwise, they were excluded. chest radiography was conducted according to the clinical situation of the patient. respiratory samples, including throat swab, sputum, or bronchoalveolar lavage fluid, were collected from the enrolled patients for routine screening of respiratory viruses, mycoplasma pneumoniae (mp), and chlamydophila pneumoniae (cp), according to established clinical protocols [ ] . the samples were refrigerated at - °c in viral transport medium, transported on ice, and analyzed immediately or stored at − °c before analysis, as described previously [ ] . we also collected meteorological data of guangzhou (longitude e ° ′ to e ° ′, latitude n ° ′ to n ° ′), including the monthly mean temperature (°c), mean relative humidity (%), rainfall (mm), sunshine duration (h), mean wind speed (m/s), mean air pressure (hpa), and mean vapor pressure (hpa) from the china meteorological administration between july and june . real-time pcr for detection of rsv, piv, hmpv, and common respiratory pathogens taqman real-time pcr was conducted to detect rsv, piv - , hmpv, and other common respiratory pathogens, including influenza a virus (infa), influenza b virus (infb), human rhinovirus (hrv), enterovirus (ev), four types of coronaviruses (hcov- e, -oc , -nl , and -hku ), adenovirus (adv), human bocavirus (hbov), mp, and cp, as previously reported [ ] . briefly, real-time-pcr and rna/ dna extraction kits were purchased from guangzhou huyansuo medical technology co., ltd. rna/dna was extracted from -μl samples, according to the manufacturer's protocol. the cycling conditions were °c for min, °c for min, and then cycles of °c for s and °c for s. the amplified products were detected using the applied biosystems real-time pcr system (life technologies, singapore). the sensitivity of the detection kits was copies/ml and copies/ml for the target dna and rna, respectively. statistical analysis was performed using spss . (spss inc., chicago, il, usa). numerical data were presented as mean ± standard deviation for continuous variables of meteorological data, percentage for normal discrete variables, or median (interquartile range, iqr) for age distribution. categorical data were compared with the chi-squared test. multiple linear regression analysis was performed with rsv, piv, and hmpv prevalence as dependent variables and meteorological factors as independent variables. linear correlations of meteorological independent variables were analyzed to exclude any effect on the final multiple linear regression analysis. the independent variable mean temperature drop month (mean temperature in the preceding month) was also included as an independent variable in the multiple linear regression analysis because of its delay effect. a p value < . (two-tailed) was considered statistically significant. over a -year period, a total of , patients were enrolled in the study and screened for rsv, piv, hmpv, and respiratory pathogens ( table ). the median age of patients was . years (iqr, . - . ), and the sex ratio was . : . of the , patients tested, ( . %) had positive results for one or more of the pathogens of interest, and . % ( / ) of positive patients were found to have coinfection with two or more pathogens. the median age of pathogen-positive patients was . years (iqr, . - . ), and positive patients had a higher sex ratio ( . : ) than patients who tested negative for all pathogens ( . : ) (p = . ). rsv, piv, and hmpv were the first, sixth, and ninth most frequently detected pathogens, with prevalence of . % ( / ), . % ( / ), and . % ( / ), respectively ( fig. , table ). the median age of patients who tested positive for these paramyxoviruses was . years (iqr, . - . ), . years (iqr, . - . ), and . years (iqr, . - . ), respectively. the sex ratio of patients positive for these three viruses was . : , . : , and : , respectively. age distribution of patients with rsv, piv, or hmpv infection patients were divided into seven age groups to clarify the age distributions for these three paramyxoviruses, as follows: age - months, - months, - months, - years, - years, - years, and - years. significant differences were found in the prevalence of rsv, piv, and hmpv among all age groups (p < . ), and prevalence declined with age for rsv. peak prevalence was found in patients aged - months ( . %, / ) for piv; high hmpv prevalence was found in patients months to years old, . % ( / ) to . % ( / ) (fig. ). overall, in the -year period study, rsv and hmpv had similar seasonal patterns as well as two clear prevalence peaks each year. the larger peak of rsv and hmpv prevalence appeared during the change of season from winter to spring, mainly occurring in february to april every year. the smaller peak was mainly observed in august to october each year, during the shift from summer to autumn (fig. ) . piv prevalence increased as autumn turned to winter and summer turned to autumn, and appeared between peaks of rsv and hmpv prevalence (fig. ) . to explore the correlation of paramyxovirus prevalence with climate conditions in guangzhou, we collected meteorological data for the -year period. between july and june , the mean temperature was . ± . °c, mean relative humidity was . ± . %, sunshine duration was . ± . h, mean wind speed was . ± . m/s, rainfall was . ± . mm, mean air pressure was . ± . hpa, and mean vapor pressure was . ± . hpa. multiple linear regression analysis was conducted to explore the correlation between meteorological conditions and paramyxovirus prevalence. we first analyzed linear correlations among meteorological independent variables. we excluded the independent variables mean air pressure (adjusted r = . , p < . ) and mean vapor pressure (adjusted r = . , p < . ), which were linearly associated with mean temperature, and we excluded rainfall (adjusted r = . , p < . ), which was strongly correlated with mean relative humidity. thus, the independent variables included in the final multiple linear regression analysis were mean temperature, mean relative humidity, sunshine duration, and mean wind speed. multiple linear regression models were established for rsv, piv, and hmpv prevalence and meteorological data (p < . ) ( table ) . rsv prevalence was negatively correlated with relative humidity and sunshine duration (coefficient = − . and − . , respectively) (p < . ). piv prevalence was negatively correlated with relative humidity (coefficient = − . ) and positively correlated with temperature (coefficient = . ) (p < . ). hmpv prevalence was negatively correlated with sunshine duration (coefficient = − . ) (p < . ) ( table , fig. ). mean temperature drop month (mean temperature in the preceding month) was also included as an independent variable in the analysis; however, no effective regression model was established (p > . ). ari is one of the most common human diseases resulting in high mortality and mobility, and it is predominantly caused by respiratory viruses [ , ] . paramyxoviruses, including rsv, piv, and hmpv, are the most important respiratory viruses in patients with ari all over the world, especially among [ , ] . these viruses are also an important cause of nosocomial infection [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . currently, no effective vaccines or drugs have been developed against these viruses, except palivizumab for rsv prophylaxis [ ] . thus, additional research must be carried out. in the present study, we sought to analyze the features of paramyxovirus infection and correlation with meteorological conditions in a subtropical region of southern china, by collecting respiratory samples from pediatric patients (≤ years old) hospitalized with ari in guangzhou and testing for rsv, piv, hmpv, and other common respiratory pathogens. meteorological data were also collected between july and june , for further correlation analysis. the goal of the study was to reveal information that could be useful in the prevention and control of these viruses. the median age of the , enrolled pediatric patients was . years (iqr, . - . ), indicating the high public health burden among infants and young children, as previously reported [ , ] . nearly half of all patients had positive test results for one or more of the pathogens of interest; moreover, all pathogens in the panel were detected, indicating the complexity and diversity of ari etiology (table ) . higher pathogen prevalence was found among male patients than female ones (p = . ), similar to previous reports [ ] . rsv, infa, mp, adv, hrv, and piv were the six most frequently detected pathogens in this study (fig. ) ; these results are consistent with previous reports from china as well as international studies [ , , , ] . rsv, piv, and hmpv are the most important respiratory viruses, causing lower respiratory illnesses among children worldwide [ ] [ ] [ ] ] . in this study, rsv, piv, and hmpv were the first ( . %), sixth ( . %), and ninth ( . %) most frequently detected pathogens (table , fig. ). in children with positive results for these three paramyxoviruses, the median age was highest in children who were hmpv positive and lowest for those positive for rsv. rsv mostly affected children under years old, and the prevalence decreased with age (p < . ). piv showed peak prevalence among patients aged - months (p < . ), and there was high hmpv prevalence among patients aged months to years (p < . ) (fig. ) . the different age distributions of these three viruses may be helpful in determining appropriate pediatric care and disease diagnosis; however, laboratory testing is still necessary because of the complex diversity and similarities in clinical manifestations of respiratory pathogens [ , , ] . in general, the epidemiology of the main respiratory viruses in patients with ari has been closely monitored in developed countries; however, these data are less available in developing countries, mostly because of the high cost of these studies. rsv is known to occur in well-defined, recurrent epidemics during the cold season in temperate climates [ ] , with peaks occurring more often during the rainy season in tropical and subtropical areas; locations close to the equator have less consistent patterns, with some showing nearly continuous rsv activity and varying seasonal peaks [ ] . in this study, we found that rsv occurred during the change of seasons from winter to spring and from summer to autumn. this pattern is similar to those in previous reports [ ] . hmpv had a similar epidemic pattern to rsv, consistent with previous reports from other subtropical areas [ , ] , and differed from the pattern in temperate climates, which peaks at the end of winter or in early spring [ , ] . in previous reports from the usa, piv is second only to rsv as a cause of hospitalization for ari ( - %) among children aged younger than years [ , , ] . seasonal peaks of pivare mostly driven by piv- and piv- , whereas there are a small number of piv and especially piv infections [ , ] . in this study, piv was isolated throughout the year and appeared to alternate with peaks in rsvand hmpv infection, increasing as autumn turned to winter and summer turned to autumn (fig. ) . these results differ from previous reports of biennial piv epidemics [ , ] . the different geographic location might lead to the different seasonal distribution of piv observed in the present study. in addition to pathogenic characteristics, pathogenic epidemics are closely related to geographic environment, local climate, social development level, population structure, ethnic characteristics, social interaction, and so forth. guangzhou has a maritime subtropical monsoon climate, with high temperatures (mean temperature . ± . °c) and high relative humidity ( . ± . %). investigation of respiratory pathogen epidemics in the region is of great importance. in this study, we analyzed the correlation between the prevalence of paramyxoviruses among pediatric hospitalized patients and multiple linear regression analysis was performed for monthly prevalence of three paramyxoviruses as the dependent variable, and monthly mean temperature, mean relative humidity, sunshine duration, and mean wind speed as the independent variables data in italics are significant meteorological conditions in guangzhou. multiple linear regression analysis was performed with monthly rsv, piv, and hmpv prevalence as the dependent variable and current mean temperature (or mean temperature in the preceding month), mean relative humidity, mean wind speed, and sunshine duration as the independent variables. regression models were established for rsv, piv, and hmpv using the current monthly temperature model (p < . ) ( table ) . however, regression models using mean temperature in the preceding month model were not established. in general, the trend of associations between climate factors and respiratory pathogen activity varies with geographic location [ , [ ] [ ] [ ] [ ] [ ] [ ] . in this study, rsv and hmpv had similar seasonal distribution patterns, and both were negatively correlated with sunshine duration ( fig. ); this might be owing to the sensitivity of rsv and hmpv to ultraviolet light. in subtropical and temperate regions, rsv prevalence is more consistently positively correlated with lower temperatures and higher relative humidity [ ] . however, we found a negative correlation between rsv prevalence and relative humidity (p < . ) ( table , fig. ) , which might be owing to the high relative humidity in the guangzhou region (monthly mean relative humidity . ± . %). piv had an alternating seasonal distribution pattern with rsv and hmpv in this study. piv incidence was negatively correlated with relative humidity, similar to rsv; however, piv incidence was positively correlated with mean temperature, and the absolute value of the correlation coefficient of relative humidity (|− . |) was smaller rsv prevalence was negativelycorrelated with relative humidityand sunshine duration (p< . ). piv prevalence was negatively correlated with relative humidityand positivelycorrelated with temperature (p< . ). hmpvprevalencewas negativelycorrelated withsunshine duration (p< . ). than the absolute value of the correlation coefficient of temperature (| . |) ( table ) , which means that the effect of temperature on the distribution of piv was greater than the relative humidity. this might explain the different distribution patterns between rsv and piv. overall, the established models were found to be of value for understanding the epidemic patterns of rsv, piv, and hmpv (fig. ) . some limitations of this study should be noted. first, because our study was mainly focused on the circulation of paramyxoviruses among hospitalized patients with ari, paramyxoviruses in outpatients and the asymptomatic population were not included. second, many factors can affect virus epidemics; meteorological data analysis alone may be insufficient to reach a final conclusive interpretation. third, the current study was only conducted in three hospitals and may not be representative of the overall population. in conclusion, this study provided a better understanding of paramyxoviruses infection and highlighted the correlation with climate factors, revealing the potential for modeling and risk assessment. the findings of this work will help public health authorities and clinicians to improve strategies for controlling paramyxoviruses infection. viral pneumonia aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis paramyxovirus infection: mortality and morbidity in a pediatric intensive care unit epidemiological and clinical features of 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cross-sectional study human metapnuemovirus infections in hospitalized children and comparison with other respiratory viruses. - prospective study children with respiratory disease associated with metapneumovirus in hong kong human metapneumovirus associated with respiratory tract infections in a -year study of nasal swabs from infants in italy prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children bronchiolitis-associated hospitalizations among us children seasonal trends of human parainfluenza viral infections: united states the isolation of parainfluenza subtypes a and b in england and serological studies of their prevalence seasonal pattern in childhood viral lower respiratory tract infections in melbourne correlations between climate factors and incidence-a contributor to rsv seasonality climatic factors and lower respiratory tract infection due to respiratory syncytial virus in hospitalised infants in northern spain a four year seasonal survey of the relationship between outdoor climate and epidemiology of viral respiratory tract infections in a temperate climate seasonal distribution and epidemiological characteristics of human metapneumovirus infections in pediatric inpatients in southeast china seasonal patterns of respiratory syncytial virus infection in hong kong: a preliminary report incidence of common respiratory viral infections related to climate factors in hospitalized children in hong kong acknowledgments we thank the study volunteers for their generous participation. we thank huang-xi liang, mei-xin chen, chi li, and zhengshi lin for their technical assistance. conflict of interest the authors declare that they have no conflicts of interest.ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the first affiliated hospital of guangzhou medical university ethics committee and with the helsinki declaration and its later amendments or comparable ethical standards.informed consent this study is a retrospective study, and a formal consent is not required.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - y n ea authors: el kholy, a. a.; mostafa, n. a.; ali, a. a.; soliman, m. m. s.; el-sherbini, s. a.; ismail, r. i.; el basha, n.; magdy, r. i.; el rifai, n.; hamed, d. h. title: the use of multiplex pcr for the diagnosis of viral severe acute respiratory infection in children: a high rate of co-detection during the winter season date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: y n ea respiratory tract infection is a major cause of hospitalization in children. although most such infections are viral in origin, it is difficult to differentiate bacterial and viral infections, as the clinical symptoms are similar. multiplex polymerase chain reaction (pcr) methods allow testing for multiple pathogens simultaneously and are, therefore, gaining interest. this prospective case-control study was conducted from october to february . nasopharyngeal (np) and oropharyngeal (throat) swabs were obtained from children admitted with severe acute respiratory infection (sari) at a tertiary hospital. a control group of asymptomatic children was included. testing for viruses was done by real-time multiplex pcr. multiplex pcr detected a viral pathogen in / ( . %) patients admitted with sari. there was a high rate of co-infection ( . %). dual detections were observed in ( . %), triple detections in ( . %), and quadruple detections in ( . %) of samples. seventy-eight patients required intensive care unit (icu) admission, of whom ( . %) had co-infection with multiple viruses. adv, hbov, hrv, hev, and hcov-oc were also detected among asymptomatic children. this study confirms the high rate of detection of viral nucleic acids by multiplex pcr among hospitalized children admitted with sari, as well as the high rate of co-detection of multiple viruses. adv, hbov, hrv, hev, and hcov-oc were also detected in asymptomatic children, resulting in challenges in clinical interpretation. studies are required to provide quantitative conclusions that will facilitate clinical interpretation and application of the results in the clinical setting. respiratory tract infection is a major cause of hospitalization in children. most such infections are of viral origin, but viral infection is often hard to distinguish from bacterial infection [ ] . pathogen-specific clinical symptoms are often lacking [ ] . specific diagnosis, therefore, relies almost entirely on laboratory investigation [ ] . treatment with antibiotics induces the development of antibiotic resistance in bacteria [ ] and has a negligible effect on most acute respiratory infections (aris), which are generally of viral origin [ ] . nevertheless, antibiotics are frequently prescribed due to a lack of clinically valid diagnostic tests verifying a viral etiology [ ] . sensitive methods, such as quantitative real-time polymerase chain reaction (qpcr) analyses on nasopharyngeal samples, for a number of viruses have been introduced in hospitals as a sensitive diagnostic tool among children with respiratory tract infection [ ] . investigations using specific pcr for individual viruses (bmonoplex pcr^) are too time consuming and elaborate for the laboratory, and are usually used to detect influenza and respiratory syncytial virus (rsv). when the viruses targeted by monoplex pcr are negative, no specific etiology is identified to help clinical management of the patient and epidemiological monitoring of infections. multiplex pcr methods, on the other hand, enable testing for many pathogens in parallel in a single analysis and are, therefore, increasingly gaining importance [ ] . this prospective case-control study was conducted during the period from october to february (winter season) at cairo university pediatric hospitals ( beds) and included all children who were hospitalized in intensive care units (icus) and hospital wards with severe acute respiratory infections (sari) according to the world health organization (who) case definition for sari [ ] . a standardized study protocol was developed to record the demographic characteristics and medical history of the patients, clinical features including symptoms and signs, outcome of the illness, hospital course, and laboratory and radiological findings. forty healthy age-matched asymptomatic children with no history of a recent respiratory tract infection during the previous weeks, who were not admitted to the hospital, and who do not have any chronic underlying illness were included as a control group. informed verbal consent was obtained from the parents of all the patients and the controls. the study design conformed to the revised helsinki declaration of bioethics and was approved by the scientific ethics committee of the department of pediatrics, faculty of medicine of cairo university. sampling and sample processing: nasopharyngeal (np) and oropharyngeal (throat) swabs were obtained, transported, and preserved on viral transport media. the swabs inside the -ml tube were agitated vigorously for s using a vortex mixer to free cells from the swab tip. viral testing was done by real-time multiplex pcr using anyplex™ ii rv detection (v . ) (cat. no. rv g y) supplied by seegene, operated on a cfx ™ real-time pcr detection system (bio-rad), which simultaneously detects respiratory viruses [adenovirus (adv), influenza a virus (flu a), influenza b virus (flu b), parainfluenza viruses - (piv- , - , - , - ), rhinovirus (hrv), respiratory syncytial virus (rsv), bocavirus (hbov), metapneumovirus (mpv), coronavirus e (cov e), coronavirus nl (cov nl ), coronavirus oc (cov oc ), and enterovirus (hev)]. nucleic acid extraction was done automatically using seeprep ™ viral (#spn ) supplied by nordiag, using the extraction seeprep machine (seegene), as indicated as a nucleic acid extraction option by the manufacturer. protocol viral na was operated using μl from the sample to result in an eluted volume of μl. reverse transcription was done using the cdna synthesis kit for manual set up cdna synthesis premix (sgrt ) from seegene. interpretation of the results was done according to the manufacturer's instructions, in addition to automatic analysis using the seegene viewer software after exporting the run data to it [ ] . data were coded and entered using the statistical package spss version (ibm corp., armonk, ny, usa). data were summarized using mean [standard deviation (sd)] or median (range) for quantitative variables, and number and percentage for qualitative variables. comparison between groups was done using the chi-square test for qualitative variables, and the mann-whitney u-test was used for quantitative variables that were not normally distributed. a p-value < . was considered significant. the turnaround time for multiplex pcr was h, but the test was done in batches three times per week. nucleic acids of respiratory viruses causing sari were detected in / ( . %) patients enrolled in the study. the demographic and clinical characteristics of the patients are shown in table , and the frequency and monthly distribution of pathogens among the patients are shown in table and fig. . the median interval between the onset of symptoms and testing in our study was days. the nucleic acids of more than one virus were detected in patients ( . %). dual detections were observed in ( . %), triple detections in ( . %), and quadruple detections in ( . %) of samples. the frequency of co-detection with each of the studied viruses among the patients is shown in table . important combinations included rsv with other viruses in patients. rhinovirus was mostly detected in association with other viruses ( out of patients positive for hrv). bocavirus was detected in patients. it was the only virus detected in three of them, while it was detected in association with another virus in patients. a large proportion of the patients had received antibiotic therapy prior to hospital admission ( patients, . %), and almost all the patients received antibiotic therapy during hospital admission ( patients, . %). seventy-eight patients required icu admission, of whom ( . %) had co-infection with multiple viruses as follows; had co-infection with two viruses, three had co-infection with three viruses, and one patient had co-infection with four viruses. twenty-one of the patients who were admitted to the icu had rsv in association with other viruses, of whom had co-infection of rsv and rhinovirus. among the healthy asymptomatic children who were included as a control group, no virus was detected in of them, while a viral pathogen was detected in controls. the only viruses that were detected among the control group were adv, hbov, hev, hcov-oc , and hrv, either as a single detection or as a co-detection (table ) . a dual detection of these viruses was present in six controls, while triple detections of these viruses were present in four controls. the frequency of detection of these viruses among the patients and the controls is shown in fig. . the following viruses were not detected at all among the control group, either alone or as a co-detection with other viruses: rsv, hmpv, flu a, flu b, hpiv- - - - , hcov-nl , hcov- e. we used multiplex pcr for the detection of respiratory viruses among patients admitted with sari. we also assessed the presence of viral nucleic acids by pcr in healthy asymptomatic children in order to better understand the etiologic role of the tested viruses in respiratory disease. viral nucleic acids were detected in . % of patients admitted with severe lower respiratory tract infections. this is in keeping with a previous study that reported the detection of a viral pathogen by pcr in . % of children aged ≤ years [ ] , and with bierbaum et al., who reported viral pathogen detection in children in excess of %, compared to only % in adults presenting with acute respiratory illness [ ] . similar to previous reports [ , ] , the most frequently detected virus in our study was rsv ( %). flu a was detected in . % of the samples and flu b was detected in . % of the samples. these results are similar to those of lam et al., who reported a positive rate of . % for flu a by pcr and a positive rate of . % for flu b [ ] . some - % of all children use healthcare services every year because of influenza [ ] . additionally, many cases of infection with influenza virus are not diagnosed as such [ ] . early confirmation of influenza viruses is helpful because treatment with neuraminidase inhibitors then becomes an option [ ] . hpivs are important causes of upper respiratory tract illness and lower respiratory tract illness, especially in children [ , ] . collectively, the four types of hpivs were detected in % of the patients with sari in the current study. similar to previous studies, multiplex pcr allowed the detection of hrv, hcov-oc , and hmpv, which were not detectable by conventional cell culture isolation or direct detection using immunofluorescence assay. the improvement in the diagnostic yield by adding hrv was confirmed in our study, and also reported previously by gruteke et al. [ ] . [ ] . these findings are comparable to previous reports [ , ] . in view of the fact that respiratory tract infections in children occur in close succession during the winter season, it can be assumed that infections actually overlap. this may provide an explanation for the fact that viral nucleic acids of different viruses in one specimen are found virtually only in childrenfor example, in % of all specimens analyzed by bierbaum et al. [ ] and . % of the samples in our study. co-detections were reported more commonly than single infections in children than older adults (≥ years; p = . ) [ ] . co-infection of rsv with other respiratory viruses is common and can increase the severity of the disease [ ] . in the current study, co-infection with rsv was present in . % of the patients who tested positive for rsv. this may have increased the severity of the disease, resulting in hospital admission. moreover, . % of the patients who were admitted to the icu had rsv in association with other viruses. for a long time, it was assumed that symptomatic infection with respiratory tract viruses was regularly shown by the confirmation of viral nucleic acids in respiratory secretions. however, in recent years, there have been increasing indications that this is not necessarily the case [ ] . data are available from only a few case-control studies that examined to what extent specific respiratory viruses cause disease [ , [ ] [ ] [ ] [ ] . in the current study, adv, hbov, hrv, hev, and hcov-oc were not only detected in patients admitted with sari, but were also detected in asymptomatic children. the clinical importance of bocaviruses remains the subject of controversy. bocavirus dna can be confirmed in the respiratory tract of asymptomatic children; a high concentration of these viruses in respiratory specimens seems to be associated with symptoms [ ] . only quantitative monoplex pcr or multiplex pcr can provide information about virus concentration. bocavirus was detected by pcr in nasopharyngeal swabs samples of children suffering from acute respiratory tract infection in saudi arabia [ ] . in our study, hbov was detected among symptomatic children admitted with sari, as well as in asymptomatic children. six different human pathogenic coronavirus species (hcov) are known to date [ ] . as a rule, they cause benign disorders of the upper respiratory tract [ ] . however, after the sars (severe acute respiratory syndrome) epidemic in with the sars cov, hcov-nl was described in and hcovhku in [ ] . the middle eastern respiratory syndrome (mers) is caused by mers-cov [ ] . human corona viruses (hcov-nl , hcov-oc , hcov- e) were detected in seven patients admitted with sari. however, hcov-oc was also detected in six asymptomatic children. adenovirus was the third most frequently detected virus in our study population. adenovirus dna is also often found in the respiratory secretions of asymptomatic children [ ] , and was detected among patients as well as asymptomatic children in our study. hrvs are frequently found in asymptomatic children [ ] , but are also detected in patients with symptoms ranging from mild common colds [ ] to serious lower respiratory tract disease [ ] . since the development of molecular assays for the detection of hrvs, the detection rate of hrv in patients with respiratory infections has increased to up to % [ ] . hrv was only second to rsv in frequency of detection by multiplex pcr in swab samples of children admitted with sari in our study. however, hrv was also detected in / controls. hrvs are frequently detected in asymptomatic children, making it difficult to interpret the clinical significance of a positive pcr finding [ ] . moreover, hrvs could be detected until - weeks after the onset of symptoms [ ] . in spite of these facts, rhedin et al. [ ] reported that their data indicate that % [ % confidence interval (ci): to ] of acute respiratory infections in their population could be attributed to hrv. the potentially important rate of excreters of viral nucleic acids of specific pathogens that are not directly associated with the acute illness presents a problem. to date, quantitative conclusions are not reliably established, neither for monoplex pcr nor for multiplex pcr [ ] . rhedin et al. showed that the nucleic acids of rsv, hmpv, and parainfluenza virus can be confirmed almost exclusively in symptomatic children and very rarely in asymptomatic children. their results indicate that a qpcr finding of rsv, hmpv, or piv is likely to be causative of disease in children with acute respiratory infection, and that detection of several other viruses, such as hbov, hrv, adv, hcov, and hev, must be interpreted with caution, due to the high detection rates among healthy children [ ] . the detection of adv, hbov, hrv, hcov-oc , and hev among asymptomatic children in the current study and the absence of rsv, hmpv, flu a, flu b, hpiv- - - - , hcov-nl , and hcov- e in these children support these findings. whereas the detection of some viruses, such as influenza virus and respiratory syncytial virus (rsv), is clearly predictive for respiratory disease [ ] [ ] [ ] , the clinical significance upon the detection of several other viruses needs further investigation. the interpretation of a viral detection is complicated by the fact that infections with multiple viruses are common in children with acute respiratory infection and that many viruses have lately been reported to be found also in asymptomatic children [ ] . most respiratory tract infections in children are of viral origin, especially during the winter season, which, in egypt, is from november to february. almost all the patients in our study received antibiotic therapy during hospital admission. the identification of specific viral pathogens will limit the unnecessary use of antibiotics and will facilitate giving specific antiviral therapy when indicated (e.g., neuraminidase inhibitors in confirmed influenza virus infection). better understanding of how to interpret viral findings by the use of new technologies is important to improve management decisions, which, in turn, will ameliorate patient outcomes and reduce unnecessary use of antibiotics. this study confirms the high rate of detection of viral nucleic acids by multiplex pcr) among hospitalized children admitted with severe acute respiratory infection, as well as the high rate of detection of multiple viruses. adenovirus, hbov, hrv, hev, and hcov-oc were also detected in asymptomatic controls, resulting in challenges in clinical interpretation. further studies are required to provide quantitative conclusions that will facilitate clinical interpretation and application of the results in the clinical setting. funding source none. the authors declare that they have no conflict of interest. ethical approval informed verbal consent was obtained from the parents of all the patients and the controls. the study design conformed to the revised helsinki declaration of bioethics and was approved by the scientific ethics committee of the department of pediatrics, faculty of medicine of cairo university. the role of multiplex pcr in respiratory tract infections in children rapid multiplex nested pcr for detection of respiratory viruses effect of azithromycin and clarithromycin therapy on pharyngeal carriage of macrolide-resistant streptococci in healthy volunteers: a randomised, double-blind, placebo-controlled study antibiotics for bronchiolitis in children the effect of rapid respiratory viral diagnostic testing on antibiotic use in a children's hospital new molecular virus detection methods and their clinical value in lower respiratory tract infections in children who regional office for europe guidance for sentinel influenza surveillance in humans comparison of 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predict subsequent childhood wheezing role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study highly frequent infections with human rhinovirus in healthy young children: a longitudinal cohort study the burden of respiratory syncytial virus infection in young children influenza a and b virus infections in children viral etiology of severe pneumonia among kenyan infants and children key: cord- -s nsiano authors: muller, m. p.; richardson, s. e.; mcgeer, a.; dresser, l.; raboud, j.; mazzulli, t.; loeb, m.; louie, m. title: early diagnosis of sars: lessons from the toronto sars outbreak date: - - journal: eur j clin microbiol infect dis doi: . /s - - -x sha: doc_id: cord_uid: s nsiano the clinical presentation of sars is nonspecific and diagnostic tests do not provide accurate results early in the disease course. initial diagnosis remains reliant on clinical assessment. to identify features of the clinical assessment that are useful in sars diagnosis, the exposure status and the prevalence and timing of symptoms, signs, laboratory and radiographic findings were determined for all adult patients admitted with suspected sars during the toronto sars outbreak. findings were compared between patients with laboratory-confirmed sars and those in whom sars was excluded by laboratory or public health investigation. of cases, ( %) had confirmed sars, ( %) were excluded, and ( %) remained indeterminate. among confirmed cases, exposure occurred in the healthcare environment ( %) or in the households of affected patients ( %); community or travel-related cases were rare (< %). fever occurred in % of patients by the time of admission. respiratory findings including cough, dyspnea and pulmonary infiltrates evolved later and were present in only , and % of patients, respectively, at admission. direct exposure, fever on the first day of illness, and elevated temperature, pulmonary infiltrates, lymphopenia and thrombocytopenia at admission were associated with confirmed cases. rhinorrhea, sore throat, and an elevated neutrophil count at admission were associated with excluded cases. in the absence of fever or significant exposure, sars is unlikely. other clinical, laboratory and radiographic findings further raise or lower the likelihood of sars and provide a rational basis for estimating the likelihood of sars and directing initial management. since the end of the global sars outbreak, the world health organization has continued to recommend surveillance for the re-emergence of sars, while acknowledging that prompt diagnosis remains a challenge due to the nonspecific nature of the clinical illness and the inability of diagnostic tests to provide sensitive and specific results early in the disease course [ ] . cases of sars due to laboratory or animal exposure have been reported since the global outbreak ended, and these events highlight the importance of prompt recognition of cases in preventing larger outbreaks from occurring [ ] [ ] [ ] . toronto was the site of the largest sars outbreak outside of asia, but previous clinical descriptions of the toronto outbreak did not include all patients and did not use microbiological methods to classify cases [ ] [ ] [ ] [ ] [ ] . we conducted a retrospective study of the entire toronto sars outbreak, using microbiological methods for case classification, to identify epidemiological and clinical findings that may be useful in early diagnosis of sars. the charts of all adult (age ≥ years) patients initially diagnosed with suspect or probable sars based on world health organization or health canada criteria and admitted to a toronto area hospital between february and june were reviewed. patients were classified as confirmed sars, indeterminate sars or excluded sars based on their exposure history, clinical illness and the results of microbiological testing. patients were classified as confirmed sars if they had a compatible clinical illness (fever or nonproductive cough or dyspnea), an exposure to sars (direct contact with a known sars case or travel to a sars-endemic area or time spent at an institution where sars transmission was occurring, within days of symptom onset), and a positive microbiological test (positive acute or convalescent serology, or positive pcr from clinical or pathological specimens). sars was excluded in patients that had negative convalescent serology, or when retrospective investigations ruled out an exposure to sars (e.g. a case where the patient's only exposure to sars was direct contact with a suspected sars patient who later was determined to be seronegative). patients were classified as indeterminate if they had a documented exposure and compatible clinical history, but did not have definitive results from microbiological testing (i.e. convalescent serology was not performed, acute serology was not performed or was negative, and pcr testing was not performed or was negative). a description of the specimen collection protocol and the methods used for serologic and diagnostic testing has been published previously [ ] . serologic testing was performed at the national microbiology laboratory, winnipeg, manitoba and at mount sinai hospital, toronto, ontario using an enzyme-linked immunosorbent assay and an immunofluorescent assay to detect sars-cov antibodies (euroimmun, lubeck, germany). serological testing was considered negative if a sample drawn days or more after illness onset was negative and positive if igm or igg antibodies were detected at predefined dilutions [ ] . rt-pcr was performed at the national microbiology laboratory, the ontario central public health laboratory and at mount sinai hospital. the hospital charts of all identified patients were reviewed by trained research nurses, and data was recorded on a standardized form. ten percent of charts were randomly selected for duplicate abstraction. data was double-entered into a computerized database. the study was approved by the research ethics boards at all participating hospitals and at mcmaster university, hamilton, ontario. analysis was conducted using sas version (sas institute, cary, nc, usa). variables were compared between groups using the chi-square or fisher's exact test for categorical variables and the wilcoxon rank sum test for continuous variables. to account for differences in illness duration at hospital admission, cases were stratified into quartile groups based on the time from symptom onset to admission. the cochrane-armitage trend test and spearman's correlation coefficient were used to test for trends in symptom incidence between quartile groups for categorical and continuous variables. kaplan-meier survival curves were used to plot time-to-event data. for adult patients admitted to hospital with possible sars, charts were available for review. sars was excluded in ( %) patients due to lack of a significant exposure ( ), negative convalescent serology ( ) or both ( ) . sars was confirmed in ( %) patients, based on positive serology ( ), positive histopathology and pcr ( ) or positive pcr alone ( ) . the remaining ( %) cases were classified as indeterminate. a review of serological results from all inpatients tested during the outbreak did not identify any additional cases. for the confirmed cases, the median age was years (interquartile range, - years), % were female and % had no comorbidities (table ) . exposure occurred in the healthcare environment in % of cases; in hospitals ( %), physician's offices ( %) or during patient transport ( %). transmission outside the healthcare environment occurred among the household contacts of cases ( %, n= ); travel-related disease was confirmed in two cases (excluding the index case), and community transmission resulted in eight confirmed cases, six of whom were exposed at the same community event. because the onset of illness and the initial symptoms were difficult to discern in patients who developed sars while hospitalized for another illness, these cases (n= ) were excluded from the analysis of clinical presentation. results are for the confirmed cases, unless otherwise specified. patients were admitted to hospital a median of days (interquartile range, - days) after illness onset. the initial symptoms of sars among the confirmed cases, and the symptoms present at admission to hospital, are shown in fig. . initial symptoms included fever in %, nonspecific influenza-like symptoms such as myalgia, malaise or headache in %, respiratory symptoms in % and gastrointestinal symptoms such as diarrhea or nausea in %. at admission to hospital, fever was present in % of patients, influenza-like symptoms in %, respiratory symptoms in % (cough in %, dyspnea in %) and gastrointestinal symptoms in %. following admission to hospital, fever resolved in a median of days (interquartile range, - days), while respiratory symptoms continued to evolve. by the end of the first week in hospital, % of patients had respiratory symptoms ( % cough, % dyspnea). fever was the most common symptom, both initially and at admission. of the % (n= ) of patients without fever at admission, eight had had fever that resolved prior to admission; thus, % of all patients had symptomatic fever at, or prior to, admission. of the remaining nine patients, eight developed fever while in hospital, and one never had fever. sars patients without fever by the time of admission were older ( vs. years, p= . ), had more comorbid illness ( vs. %, p< . ), and were more likely to die from sars ( vs. %, p= . ) than patients with fever. although symptomatic fever was present in % of patients on the day of admission, a documented temperature of ≥ . °c was seen at admission in only % of cases. initial radiographic investigations identified pulmonary infiltrates in % ( / ) of patients, of whom % ( / ) had bilateral infiltrates. infiltrates continued to evolve following admission and were present in % of patients by the end of the first week in hospital. common laboratory abnormalities included leukopenia (leukocyte count < . × /l) in %, lymphopenia (absolute lymphocyte count < . × /l) in %, neutrophilia (absolute neutrophil count > . × /l) in %, thrombocytopenia (platelet count < × /l) in %, elevated creatine kinase (ck > iu/l) in % and elevated lactate dehydrogenase (ldh > iu/l) in %. the incidence of the clinical, radiographic and laboratory findings of sars varied depending on the duration of illness at the time of presentation (table ) . while fever and influenza-like symptoms were common throughout the first week of illness, respiratory symptoms and radiographic findings were significantly more common in patients a age is presented in years as median (interquartile range) b cases with more than one possible site of exposure c n= for exposure status of excluded cases since cases initially felt to have been exposed did not have any true exposure upon detailed review fig. symptom frequency on the first day of illness (white bar) and at the time of admission (black bar) for confirmed cases in the toronto sars outbreak. x-axis=% presenting towards the end of the first week of illness: for example, the incidence of nonproductive cough, dyspnea or pulmonary infiltrates in patients presenting within days of onset was , and %, respectively; after days of illness; these figures rose to , and %, respectively. the time of onset of fever, dyspnea, nonproductive cough and pulmonary infiltrates relative to symptom onset are shown in fig. . these results confirm that fever is an early symptom in most patients, while cough, dyspnea and infiltrates develop significantly later. comparison of the clinical and epidemiological features of patients with confirmed sars and patients with excluded sars identified several features that discriminate between these groups (table ) . findings associated with a confirmed diagnosis included direct exposure to a known case (or, . ; %ci, . - . ), symptomatic fever as an initial symptom (or, . ; %ci, . - . ), a documented temperature of . °c on admission to hospital (or, . ; %ci, . - . ), and the presence of a pulmonary infiltrate by the time of admission (or, . ; %ci, . - . ). findings associated with the absence of sars included rhinorrhea initially (or . ; % ci, . - . ) or at admission (or . ; % ci, . - . ), sore throat initially (or . ; % ci, . - . ), or at admission (or . ; % ci, . - . ), vomiting at admission (or . ; % ci, . - . ), or productive cough initially (or . ; % ci, . - . ) or at admission (or . ; % ci, . - . ). when laboratory results were classified as normal or abnormal, a low leukocyte count (or, . ; %ci, . - ), low absolute lymphocyte count (or, . ; %ci, . - . ), and low platelet count (or, . ; %ci, . - . ) were predictive of confirmed sars, while a high absolute neutrophil count (or, . ; %ci, . - . ) made confirmed sars less likely. although the median ck was higher in patients with confirmed sars ( vs. iu/l, p= . ), the proportion of patients with ck above the normal range was not statistically significantly different between groups ( vs. %, p= . ). for ldh there was no difference in either the proportion of patients with elevated ldh between groups ( vs. %, p= . ) or in the median ldh ( vs. iu/l, p= . ). this study provides the first description of the clinical presentation and early clinical course of sars in all laboratory-confirmed cases admitted to hospital during the toronto sars outbreak. because evidence suggests that findings indicate percentage of laboratory results that were classified as above (anc, ck, ldh) or below (wbc, alc, plt) the normal range the sensitivity and specificity of the case definitions used for sars diagnosis may be limited [ , ] and because inclusion of even small numbers of non-sars cases within a cohort of sars patients can limit the ability to recognize features unique to sars, we focused our analysis on the confirmed patients. although it was not possible to determine the incidence of sars in the group of indeterminate patients, the incidence of clinical findings in this group tended to be intermediate between those with confirmed and those with excluded sars, further suggesting that this was a mixed group of sars and non-sars cases. transmission of sars in the toronto outbreak was nosocomial in % of cases; an additional % of patients were exposed at home. thus, % of cases had exposure either to the healthcare system or to an ill contact within the household (often a healthcare worker). transmission due to travel or exposure within the community was distinctly uncommon. the tendency of sars to transmit primarily within the nosocomial setting has been described in the majority of other sars outbreaks [ ] [ ] [ ] [ ] [ ] . the clinical features of sars in confirmed patients demonstrated that sars begins as a febrile illness, either with fever alone or with fever and a combination of nonspecific influenza-like symptoms such as myalgia and headache. fever, although nonspecific, is the central symptom of sars; it was often the initial symptom of illness, was present at or prior to admission in % of cases, and often persisted into the second week of illness. respiratory symptoms, although a common feature of sars frequently used in diagnostic algorithms, are often absent initially, rise in incidence over time, and are present in the majority of patients only late in the first week or early in the second week of illness, often after patients have been diagnosed and admitted to hospital. the incidence of pulmonary infiltrates seen on chest radiography parallels the evolution of respiratory symptoms, and many patients did not have documented infiltrates at the time of admission to hospital. although our comparative analysis of confirmed and excluded sars cases did not identify any individual finding diagnostic of sars, a number of features were identified that can increase or lower the probability of sars. an approach that combines these findings with an understanding of the incidence and timing of the clinical, laboratory and radiographic findings of sars will provide the best possible estimate of the likelihood of sars in individual patients. our results suggest that, during an evolving sars outbreak, and in the absence of evidence suggesting widespread transmission within the community, the absence of any direct or indirect exposure to the healthcare system makes a diagnosis of sars unlikely, while direct exposure to a known case significantly raises the risk of sars. although the presence of fever is a nonspecific finding, the absence of a history of fever in a patient presenting to hospital several days after the onset of illness suggests that sars is unlikely, given that % of sars patients have fever by day of illness, and % have fever by admission. on the other hand, the absence of respiratory symptoms or pulmonary infiltrates early in disease does not reduce the likelihood of sars, as these may be late-onset findings. in some cases, careful follow-up with repeated clinical, laboratory and radiographic assessment can aid diagnosis, and this approach has been demonstrated to be useful [ , ] . the presence of symptoms common in other infectious illnesses, but uncommon in sars, is useful in lowering the probability of sars, i.e., rhinorrhea, age is reported as median years and the odds ratio is per years of age c direct exposure is defined as close contact with a known case of sars within days of symptom onset; indirect contact is defined as travel to a sars-endemic area or time spent at an institution where sars transmission was occurring, within days of symptom onset d laboratory results are presented as the percentage above the normal range for anc, ck and ldh and as the percentage below the normal range for wbc, alc and plt sore throat, productive cough and an elevated absolute neutrophil count are more common in patients without sars. features more common in confirmed sars than in excluded cases included the presence of fever on the first day of illness, documented fever at admission, pulmonary infiltrates at admission, and leukopenia, lymphopenia or thrombocytopenia at admission. the presence of these findings may raise concerns about sars, but they may be less useful diagnostically as they occur frequently in other conditions that may mimic sars, such as communityacquired pneumonia [ ] . conversely, their absence lowers the likelihood of sars, but none as much as the absence of fever at admission, given that the incidence of the other findings in confirmed sars cases is considerably lower than the incidence of fever at admission. our study has several limitations. data collection was retrospective and based on chart review. biased or incomplete recording of clinical information could thus affect the accuracy of our results. patients not admitted to the hospital were not considered. patients that developed sars following hospital admission were excluded from the analysis of clinical presentation. finally, the number of patients in the excluded group was small, limiting our power to detect differences between the confirmed and excluded groups. despite these limitations, we believe the results of our study are both valid and important. the unexpected emergence of sars prevented large-scale prospective evaluations from being undertaken in most areas; we are therefore dependent on retrospective data to improve our understanding of sars, despite the inherent limitations of this approach. because asymptomatic sars is rare, transmission has rarely been documented from mildly symptomatic cases, and > % of seropositive patients identified in toronto were admitted to hospital, we believe our results are widely applicable [ ] [ ] [ ] . because the patients that developed sars during hospitalization were excluded, extrapolation of our data to such patients should be made cautiously. finally, although the small size of our excluded group suggests that the results of the between group comparisons should be interpreted cautiously, our results are consistent with those of other studies [ ] . ongoing surveillance for, and prompt diagnosis of, future sars cases remains the most important strategy to prevent or limit future sars outbreaks. due to the limitations of current microbiological tests the best approach to diagnosis remains an assessment of exposure risk and clinical presentation. our results identify a number of clinical, laboratory and radiographic findings that are useful in establishing the likelihood of sars and demonstrate the importance of considering these factors in the context of disease duration. taken together with the results of diagnostic testing for other pathogens and current global sars epidemiology, these findings provide the best information upon which to establish a "pre-test" probability of sars that can guide initial decisions pertaining to patient isolation, patient management and the ordering and interpretation of specific microbiological tests for sars. who guidelines for the global surveillance of severe acute respiratory syndrome laboratory-acquired severe acute respiratory syndrome laboratory diagnosis of four recent sporadic cases of community-acquired sars identification of severe acute respiratory syndrome in canada clinical features and shortterm outcomes of patients with sars in the greater toronto area clinical course and management of sars in health care workers in toronto: a case series identification and containment of an outbreak of sars in a community hospital critically ill patients with severe acute respiratory syndrome interpretation of diagnostic laboratory tests for severe acute respiratory syndrome: the toronto experience updated interim surveillance case definition for severe acute respiratory syndrome (sars)-united states sars surveillance during emergency public health response evaluation of who criteria for identifying patients with severe acute respiratory syndrome out of hospital: prospective observational study epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong a major outbreak of severe acute respiratory syndrome in hong kong clinical description of a completed outbreak of sars in vietnam clinical manifestations, laboratory findings, and treatment outcomes of sars patients acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome the spectrum of severe acute respiratory syndrome-associated coronavirus infection can routine laboratory tests discriminate between severe acute respiratory syndrome and other causes of community-acquired pneumonia? anti-sars-cov immunoglobulin g in healthcare workers seroprevalence of antibody to severe acute respiratory syndrome (sars)-associated coronavirus among health care workers in sars and non-sars medical wards asymptomatic severe acute respiratory syndrome-associated coronavirus infection a clinical prediction rule for diagnosing severe acute respiratory syndrome in the emergency department acknowledgements the authors would like to thank and acknowledge l. moss, m. keogh, j. pound, m. mione, j. dejager and c. harltonstezov and all others who assisted with data collection, data entry and data management. thanks also to k. austin for helpful comments on the manuscript. finally, we would like to thank all toronto area clinicians who assisted with case identification and who dedicated themselves to the care of these patients.members key: cord- -qldawc m authors: edouard, s.; colson, p.; melenotte, c.; di pinto, f.; thomas, l.; la scola, b.; million, m.; tissot-dupont, h.; gautret, p.; stein, a.; brouqui, p.; parola, p.; lagier, j.-c.; raoult, d.; drancourt, michel title: evaluating the serological status of covid- patients using an indirect immunofluorescent assay, france date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: qldawc m an indirect in-house immunofluorescent assay was developed in order to assess the serological status of covid- patients in marseille, france. performance of ifa was compared to a commercial elisa igg kit. we tested rt-qpcr-confirmed covid- patients ( serum samples) and controls including sera collected before the pandemic, sera known to be associated with nonspecific serological interference, sera from non-coronavirus pneumonia and sera from patient with other common coronavirus to elicit false-positive serology. incorporating an inactivated clinical sars-cov- isolate as the antigen, the specificity of the assay was measured as % for iga titre ≥ : , . % for igm titre ≥ : and . % for igg titre ≥ : after testing a series of negative controls. ifa presented substantial agreement ( %) with elisa euroimmun sars-cov- igg kit (cohen’s kappa = . ). the presence of antibodies was then measured at % before a -day evolution up to % after more than days of evolution. we observed that the rates of seropositivity as well as the titre of specific antibodies were both significantly higher in patients with a poor clinical outcome than in patients with a favourable evolution. these data, which have to be integrated into the ongoing understanding of the immunological phase of the infection, suggest that detection anti-sars-cov- antibodies is useful as a marker associated with covid- severity. the ifa assay reported here is useful for monitoring sars-cov- exposure at the individual and population levels. supplementary information: the online version contains supplementary material available at . /s - - - . the sars-cov- is a coronavirus belonging to the genus betacoronavirus that emerged in humans in december [ ] . it was first described in china before spreading and being classified as a pandemic [ ] . it causes a respiratory disease known as covid- that is usually mild but can result in a severe and even life-threatening pneumonia, particularly in elderly people [ ] [ ] [ ] . on september , , , sars-cov- infections and , associated deaths had been reported worldwide (https://coronavirus.jhu.edu/map.html). to date, the virological diagnosis of infections by sars-cov- has been essentially based on real-time reverse transcription pcr [ ] . this virus has been shown to elicit specific antibodies during the course of infection [ , ] . this serological response has mainly been analysed using enzyme-linked (elisa) or chemiluminescence immunoassays among exposed populations in china and neighbouring countries. previous studies showed that specific igg, igm and iga were produced in response to the infection [ ] . the kinetics of these three classes of antibodies have been described, yet correlations with the clinical outcome of the patients has been poorly reported [ ] . in this study, we are reporting our experience to develop an indirect immunofluorescent assay (ifa) for the detection of anti-sars-cov- antibodies that we implemented before any other serological test was available in france. performance of ifa was compared to a commercial elisa anti-sars-cov- igg kit when they became available. we found significant differences in the rates of seropositivity and antibody titres between groups of patients depending on their clinical outcome. study design an in-house ifa was developed to assess the serological status of a cohort of patients with confirmed sars-cov- infection at the institut hospitalo-universitaire (ihu) méditerranée infection in marseille, france [ ] . all patients presenting symptoms compatible with covid- and contacts of suspected and confirmed covid- cases were tested using a sars-cov- -specific rt-qpcr assay [ , ] . treatment with hydroxychloroquine (hcq) associated with azithromycin (az) was proposed to all rt-qpcr-positive patients who enrolled on a voluntary basis if they did not present contraindications [ ] . patients were followed up on an out-patient basis at our day care hospital or were hospitalised in the infectious disease units of the ihu, in intensive care units or in other medical departments of the assistance publique-hôpitaux de marseille, depending on the severity of the disease. we included in the present study all patients from the previous study by million et al. for whom ≥ serum sample was available for serological testing as part of the routine care of these patients. meanwhile, the sera from controls randomly selected were tested to evaluate specificity of ifa. also, we further compared the specificity and sensitivity of ifa to a sars-cov- igg elisa which became commercially available months after we set up ifa. all the sera were tested retrospectively. the time of serum collection was determined relative to the date of the onset of symptoms. the non-interventional nature of this study was approved by the ethical committee of the ihu méditerranée infection under no. - . case definition sars-cov- infection was defined by clinical, radiological, and microbiological criteria as previously reported [ , ] . briefly, the national early warning score (news) for covid- was used for the classification of clinical presentation of patients. this score was based on physiological parameters including respiratory rate, oxygen saturations, temperature, systolic blood pressure, heart rate and level of consciousness to predict deterioration risk in acute ill patients. three risk categories were defined for clinical deterioration: low score (news - ), medium score (news - ), and high score (news ≥ ). virological evidence of the infection was based on a positive rt-qpcr on a nasopharyngeal sample or another respiratory sample. pulmonary involvement was evaluated by chest low-dose computed tomography for all patients. five groups of patients were constituted according to the following criteria [ ] : ( ) patients with mild disease and good clinical and virological outcome (go; n = ); ( ) patients with poor virological outcome defined by persistence at day or more of viral rna load in respiratory samples (pviro; n = ); ( ) patients who received hcq + az treatment for more than days, with poor clinical outcome requiring prolonged hospitalisation for days or more despite days or more of hcq + az treatment (pclino ; n = ); ( ) patients who received hcq + az treatment less than days, with poor clinical outcome requiring prolonged hospitalisation for days or more (pclino ; n = ); ( ) patients with poor clinical outcome requiring prolonged hospitalisation for days or more leading to death (pclino ; n = ) including who received hcq + az treatment. main characteristics of the patients in each group are summarised in table . indirect immunofluorescence assay anti-sars-cov- antibodies were detected using an in-house indirect immunofluorescence assay (ifa), as previously described [ ] . vero e cells (atcc crl- , rockville, md, usa) infected with the sars-cov strain ihu-mi (full genome sequence of this strain was deposited under the european molecular biology laboratory embl project accession no. prjeb ) [ ] were used as the antigen. infected cells were harvested between and h post-inoculation when cytopathic effect began to be observed, before massive cell lysis. infected cells were washed with sterile phosphate buffered saline (pbs) (oxoid, dardilly, france) and inactivated using % paraformaldehyde. the antigen ( nl) was spotted on each well of a -well microscope glass slide using echo liquid handler instruments (labcytes, cannock, uk) that uses acoustic energy to transfer liquid from a -well plate containing the antigen to slides. fifty nanolitres of uninfected vero cells were also spotted on each well as a negative control and a clinical isolate of staphylococcus aureus (identified by matrix-assisted laser desorption ionisation time of flight mass spectrometry) [ ] was spotted on each well in order to ensure further serum deposition, as previously described [ ] . each slide was air dried, fixed in acetone for min and conserved at °c in the dark. in a first step, each serum sample was screened for the presence of anti-sars-cov- antibodies using the ifa, as previously described [ ] . serum samples were heatdecomplemented for min at °c, diluted in % pbsmilk and μl of a : dilution and a : dilution were pipetted onto a -spot slide then incubated for min at °c in the dark to be screened for the detection of total immunoglobulin (igt). after washing thrice, the slides with sterile pbs for min, μl of total fitc-conjugated igt anti-human immunoglobulin (bio-rad, marnes-la-coquette, france) with . % evans blue (bio-rad) were incubated for min at °c. after washing, slides were examined under a fluorescence microscope (axioskop , zeiss, marly le roi, france). in a second step, all the serum samples screened positive at a : dilution were quantified for igg, igm and iga as reported above, except that serum samples were diluted up to : for iga and igm and : for igg; and anti-igg, anti-igm and anti-iga conjugates were used (bio-rad). serum samples exhibiting positivity at : were further tested up to : , . serum samples exhibiting positivity at : were further tested up to : , . antibody titre was determined by the last dilution with detected fluorescence. reading of slide was performed in duplicate by two experienced laboratory technicians or medical microbiologists. in for the studied of seropositivity rate, we considered only the sera with the higher igg titre or with the higher igm or iga titre or only one negative sera when several sera were available for a same patients in the same studied period time case of discrepancy, a third operator read the slide. a serum sample collected from one patient who was positive by sars-cov- rt-qpcr and exhibiting igg, igm and iga titres of : , : and : , respectively, was anonymised and used as a positive control. it was used on each slide for screening and on each run for antibody quantification allowing to daily endure the robustness of the technique and validate any new lot of antigen. a negative serum collected in december from a patient and pbs-milk % were used as negative controls on each slide screened. in order to interpret the ifa, any serum sample exhibiting igg ≥ : with or without igm and/or iga ≥ : was considered as positive, as well as any serum sample exhibiting isolated igm or iga : (fig. ) . chlamydia pneumoniae infections, in order to assess for potential cross-reactivity. we evaluated repeatability of ifa by testing sera in triplicate by a same operator and reproducibility testing sera by independent operators. elisa to compare our ifa with commercial elisa igg, we randomly selected sera with possible cross-reactivity (including sera with possible nonspecific serological interference and sera from patients diagnosed with common others human coronavirus), sera collected before the pandemic and sera from our cohort of sars-cov- -infected patients among all the sera that we tested by ifa. positive rt-qpcr was considered as definite criteria for sars-cov- infection and the serological techniques were compared in these groups of patients. euroimmun® sars-cov- igg elisa (euroimmun france®, bussy saint-martin, france) using recombinant s protein as antigen was performed using elispeed duo system (euroimmun france®) according to the manufacturer's recommendations. the ratio (auc sample/auc calibrator) was interpreted according to the manufacturer's recommendations: < . negative; ≥ . to < . undetermined (grey zone); ≥ . positive. we considered results in grey zone as negative for statistical analyses. statistical analysis to avoid bias in data analysis, we studied the serological response according to the time of sampling of the sera related to the date of the onset of symptoms. we represented the kinetics of the antibodies in our cohort of sera collected before d (n = ) in fig. . an earlier and higher increase of igg, igm and iga was found in patients with poor outcome compared to patients with good outcome and with virological persistence. detailing the results for each group of patients, the median time of serum sampling was , , , and days after the onset of symptoms for dead patients (pclino ), poor outcome patients with hcq+az < days (pclino ), poor outcome patients with hcq+az ≥ days (pclino ), with persistent viral shedding (pviro) and with good outcome (go), respectively. rates of positive serology by group were % ( / ) in pclino (dead), % ( / ) in pclino (hcq+az < days), % ( / ) in pclino (hcq+az ≥ days), % ( / ) in pviro (virological persistence) and % ( / ) in patients with good outcome (go). higher rates of seropositivity were observed in group of patients with poor clinical outcome (pclino , pclino , pclino ) compared to patients with virological persistent shedding and patients with clinical good outcome (fig. ) for each period time but significant results were observed after days. higher rates of seropositivity were found in pclino ( %), pclino ( %) and pclino ( %) compared to patients with good clinical outcome (go) ( %), p = . , p = . and p = . , respectively (fig. d) . in particular, the five dead patients had exhibited positive serology after day . no significant difference was observed between patients with persistent viral shedding (pviro) and patient with good outcome (go). we observed lower rate of seropositivity among the asymptomatic patients ( / , %) compared to symptomatic patients with low news score ( / , %) but this difference was not significant, p = . . we did not observe significant difference in the time of occurrence of the different classes of antibodies from the onset of symptoms in our cohort and we noted presence of igg in most of patients with positive serology (n = ). the median of occurrence of igg with titre ≥ : was days (range - ) and were detected in sera from patients ( %) (table s ). iga seems to have better sensitivity than igm at the acute phase of the disease. igm with titre ≥ : were detected in patients ( %) with a median of days (range - ), and iga with titre ≥ : were detected in patients ( %) with a median of days (range - ). three patients presented isolated iga ≥ : and patients presented isolated igm ≥ : in early sera. all the other patients presented concomitant igg ≥ : . we did not detail data for igg because they were very similar to data of seropositivity including all classes of antibodies as described above. however, we observed significant higher seropositivity of iga in patients with poor clinical outcome ( / , %) compared to patients with persistent viral shedding (pviro) ( / , %) and patients with good outcome (go) ( / , %) (p = . and p < . , respectively). for igm, we found a significant higher prevalence in all patients with poor clinical outcome ( / , %) and with virological persistence ( / , %) compared to patients with good outcome ( / , %) (p = . and p = . , respectively). we also compared igg titre between the five groups of patients but we included only sera collected at least days after the onset of symptoms (n = ). we found significant higher igg titre in patients with a poor clinical outcome (died pclino , pclino , pclino ) compared to patients with good outcome (go) (p = . ) (fig. ) . the median of igg titre was : for patients with poor clinical outcome and : for both patients with viral persistent shedding and patients with good outcome. we did not observed significant difference of igm and iga titre between the different groups of patients. to date, many methods exist for both rapid (lateral flow assays) and semi/quantitative (clia, elisa) measurement of sars-cov- antibodies [ ] . however, at the beginning of the pandemic, most of currently commercially available serological tests were not available for several months. in this context, we developed an in-house indirect immunofluorescence assay for the detection of igg, igm and iga anti-sars-cov- antibodies using sars-cov- antigen produced fig. comparison of rates of seropositivity among the five groups of patients (a) between days and , (b) between days and (c) between days and and (d) after day directly in our biosafety level laboratory. while this technique was time-consuming and could lack of standardisation, it presented the advantage to be performed only with our materials and human resources without dependence of any suppliers. we used it to assess the serological status of hundreds of covid- patients and controls; as such, an assay has been only reported on a very small group of patients [ , ] . in order to avoid false-negative results, the assay incorporated s. aureus as a control of deposition of tested sera, as s. aureus protein a and protein m bind non-specifically to any serum antibody [ ] . the assay also incorporated noninfected vero cells on which the viral antigen has been produced, in order to identify false-positive reactivities. reading of both controls was incorporated into the interpretation algorithm. accordingly, the specificity of the assay was measured at % for iga, . % for igm and . % for igg. substantial agreement was found between commercial elisa igg kit and our ifa technique which attests to the reliability and robustness of our in-house ifa assay compared to a standardised commercial test. using this assay, we observed low rates of seropositivity, at % in rt-qpcr-confirmed covid- patients, ranging precisely from % before days' evolution to % after days' evolution. however, seroconversions of specific igm and igg antibodies were observed as early as day four after the onset of symptoms, as previously described [ ] . this low rate of seropositivity is here observed in a population of treated patients with a favourable clinical evolution and outcome in most of these patients. a limit of our study is that collection of sera was not yet protocolised according to time interval after the onset of symptoms at the beginning of our study, therefore is not homogeneous time interval between the patients, and sera were analysed only at the time for which we received the sera and we did not have a kinetic for most of the patients. in contrast, we identified that patients with severe disease developed a serological response in most cases (and all patients who died) that was characterised by high levels of igg as was also observed for sars-cov infection [ ] and in agreement with others reports about sars-cov- infection. most study reported higher antibody levels after a severe and critical infection than after a mild infection [ ] [ ] [ ] [ ] especially on sera taken - days after the onset of symptoms. these findings are confirmed for different subtype of antibodies directed against s , rbd and s protein [ ] as well as for neutralising antibody [ , , ] . only minority of study did not find this correlation [ , ] . most of them include few numbers of sera or did not analysed data according to collection sample time after symptoms onset. in fact, no significant difference was mostly found on early sera [ , ] . the discrepancies between the studies can also be explained by the nature and the target of the antigens used. some other studies also reported an earlier serological response in severe compared to mild sars-cov- infection [ , , ] that is consistent with the earlier seroconversion that we found in patients with poor clinical outcome (pclino). on the other hand, an analysis of patients with mild symptoms of covid- showed that sars-cov- can persist in patients who developed specific igg antibodies for a very long period of time, up to days, whereas only one patient who did not develop an igg response cleared the virus after days [ ] . our study included a few number of asymptomatic patients. we observed lower rates of seropositivity compared to symptomatic patients but this difference was not significant; these results need to be confirmed on a larger sample size. however, most of other studies reported production of neutralising antibodies even in asymptomatic subjects but with lower antibody titre and/or delayed response than in symptomatic patients [ , , , ] . detecting anti-sars-cov- antibodies is useful as a marker associated with covid- severity. serology also assesses exposure to the virus, at the individual level for middle long-term medical monitoring of the patients; and at the population level for monitoring the circulation of the virus, as it is one of the markers contributing to assessing the effectiveness of countermeasures. fig. comparison of median of igg titre detected at least days after the onset of symptoms between the different groups of patients infected with sars-cov- (only the sera with higher igg titre were considered for this analysis when multiple sera were available for a same patient) supplementary information the online version contains supplementary material available at https://doi.org/ . /s - - - . china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china antibody detection and dynamic 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nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements the authors acknowledge the contribution of the technical staff of the ihu méditerranée infection laboratory. this work was supported by ihu méditerranée infection, marseille, france. key: cord- -qdq authors: reckziegel, maria; weber-osel, claudia; egerer, renate; gruhn, bernd; kubek, florian; walther, mario; wilhelm, stefanie; zell, roland; krumbholz, andi title: viruses and atypical bacteria in the respiratory tract of immunocompromised and immunocompetent patients with airway infection date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: qdq respiratory tract infections (rti) can take a serious course under immunosuppression. data on the impact of the underlying pathogens are still controversial. samples from the upper (n = ) and lower rt (n = ) were collected from children and adults; among them have been immunocompromised patients. exclusion criteria were presence of relevant cultivable microorganisms, c-reactive protein > mg/dl, or procalcitonin > . ng/ml. samples were tested by pcr for the presence of herpesviruses (hsv- /- ; vzv; cmv; hhv ; ebv), adenoviruses, bocaviruses, entero-/rhinoviruses (hrv), parechoviruses, coronaviruses, influenza viruses (iv), parainfluenza viruses as well as for pneumoviruses (hmpv and rsv), and atypical bacteria (mycoplasma pneumoniae, m.p.; chlamydia pneumoniae, c.p.). viral/bacterial genome equivalents were detected in more than two-thirds of specimens. under immunosuppression, herpesviruses (ebv . %/ . %, p < . ; cmv . %/ . %, p < . ; hsv- : . %/ . %, p = . ) were frequently observed, mainly through their reactivation in adults. immunocompromised adults tended to present a higher rsv prevalence ( . %/ . %, p = . ). immunocompetent patients were more frequently tested positive for iv ( . %/ . %, p = . ) and m.p. ( . %/ . %, p < . ), probably biased due to the influenza pandemic of and an m.p. epidemic in . about . % of samples were positive for a single pathogen, and among them ebv ( . %) was most prevalent followed by hrv ( . %) and iv ( . %). hsv- and c.p. were not found. marked seasonal effects were observed for hrv, iv, and rsv. differences in pathogen prevalence were demonstrated between immunocompetent and immunocompromised patients. the exact contribution of some herpesviruses to the development of rti remains unclear. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. infections of the upper respiratory tract (urti) are among the most frequent infections worldwide. these are mainly caused by rna viruses. among them, influenza viruses (iv), pneumoviruses (respiratory syncytial virus, rsv; human metapneumovirus, hmpv), but also parainfluenza viruses (piv), coronaviruses (cov), and rhinoviruses (hrv) are considered by world health organization a global health burden [ ] . serious rti through respiratory viruses are frequently observed under immunosuppression, for example, in solid organ transplant recipients [ ] . the current breakthroughs of immunomodulating therapies in medicine contribute to the continuous increase of patients being under iatrogenic immunosuppression and being at risk for pulmonary infections [ ] . in general, suppression of t cell function is associated with a higher susceptibility for infection or reactivation of various viruses [ , ] . impairment of th cell activity but also of humoral immunity, both, facilitates the development of viral rti. in immunocompromised patients, higher morbidity and sometimes also mortality rates through infections, for example, with adenoviruses (adv), iv, piv, rsv, hmpv, but also of secondary complications like bacterial pneumonia have been observed [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, particularly transplant patients are at risk for reactivation of diverse herpesviruses (herpes simplex virus- /- , hsv- /- ; varicella zoster virus, vzv; cytomegalovirus, cmv; human herpesvirus , hhv- ; epstein-barr virus, ebv) [ , , [ ] [ ] [ ] [ ] . multiple viral infections/reactivations can occur [ ] as well as indirect interactions of viruses with bacteria [ ] [ ] [ ] . these aspects may challenge interpretation of diagnostic findings. the frequency of viral infections/reactivations is also influenced by factors like the underlying disease, therapeutic regimes, as well as the type of transplant and hla mismatches [ , ] . thus, fast and efficient diagnostic methods that cover a broad spectrum of viruses, bacteria, but also fungi and parasites are in urgent need to deal with the aforementioned challenges. the availability of such methods is of particular importance in stem cell transplant recipients where clinical symptoms of rti are variable or may be mimicked by graft-versus-host disease [ , ] . early diagnosis enables limited antiviral interventions [ , ] and may prevent further cross-transmission [ ] . however, detection of viral genome equivalents does not necessarily mean a causative role of this virus, and particularly immunocompromised patients can shed viruses over a prolonged time period [ , , ] . furthermore, there is increasing evidence of the existence of a respiratory virome which is defined by the presence of common viral pathogens, rare viruses, and viruses of unknown pathogenicity [ ] . thus, the exact contribution of a single virus to the development of rti is still controversial. we tried to consider most of these aspects by performing an observational study addressing the spectrum and impact of respiratory viruses but also of herpesviruses and the atypical bacteria mycoplasma pneumoniae (m.p.) and chlamydia pneumoniae (c.p.) in patients with respiratory symptoms. for this, an underlying infection through relevant cultivable microorganisms was largely ruled out. data were analyzed with respect to patients' immune status and age. this study included samples from the upper (nasal or throat swabs and washings), and samples from the lower (broncheoalveolar/tracheal washings, induced sputum) respiratory tract (urt/lrt) collected over a period of months beginning in september to december from healthy and immunocompromised patients with symptoms of a rti (i.e., common cold, cough with/without sputum, dyspnea, and fever). this setting included samples from patients with respiratory symptoms under neutropenia or lungtransplant recipients with a recently observed decrease in forced expiratory volume in one second (fev ). a compromised immune status was defined (i) for solid organ or stem cell transplant recipients under iatrogenic immunosuppression, (ii) in patients with autoimmune disorders under immunosuppressing therapy but also (iii) in cancer patients under chemotherapy/radiation, and (iv) in patients with primary or secondary causes of immunodeficiency including hiv infection. samples found to contain relevant cultivable bacteria (streptococcus pneumoniae, staphylococcus aureus, haemophilus influenzae, various enterobacterales and nonfermenting gram-negative bacteria, mycobacterium tuberculosis) and fungi were excluded. furthermore, specimens obtained from patients with a present bacteremia/ sepsis were excluded as well as samples from patients with rti through pneumocystis jirovecii or legionella pneumophila. the oropharyngeal and tracheopulmonal flora was considered if data were available from routine diagnostics. in addition, samples obtained from the lrt of patients with more than , colonies/ml of oropharyngeal or tracheopulmonal flora, or more than , colonies/ml of enterococcus spp. or candida spp., were also excluded since such high concentrations may represent an infection rather than colonization. in addition, the presence of procalcitonin (pct) > ng/ml and/or of c-reactive protein (crp) > mg/ dl in serum leads to exclusion of the rt sample. the remaining samples were obtained from immunocompetent (median age . years; males/ females) and immunocompromised (median age . years; males/ females) children and adolescents as well as immunocompetent (median age . years; males/ females) and (median age: . years; males/ females) immunocompromised adults. among the immunocompromised cohort, most samples were obtained from patients with hemato-oncological malignancies ( . %), followed by samples from patients after organ ( . %) and stem cell ( . %) transplantation or with autoimmune disorders ( . %). about . % of samples were included from patients with other conditions of immunosuppression or from solid tumor patients ( . %). specimens were immediately deep frozen (− °c) until nucleic acid extraction. the extraction was done manually with the qiaamp minelute virus spin kit or automatically with the ez virus mini kit (both qiagen, hilden, germany). the nucleic acids were stored at − °c and used for synthesis of copy dna (cdna) applying the revertaid h minus first strand cdna synthesis kit (thermofisher scientific, langen, germany) with random hexamers. integrity of cdna was demonstrated by amplification of ß-actin or glyceraldehyde -phosphate dehydrogenase (gapdh) dna [ , ] with dreamtaq dna polymerase (thermofisher scientific). presence of herpesviral dna (hsv- /- , vzv, cmv, and hhv- ) was demonstrated by applying conventional pcr [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] together with the hotstartaq dna polymerase and qsolution (qiagen). quantitative detection of ebv dna in samples from the lrt was done in accordance with krumbholz et al. ( ) [ ] , but sybr-green (quantitect sybr green pcr kit; qiagen) was used instead of hybridization probes. these diagnostic pcrs were continuously approved by successful participation in the external quality assurance service (eqas) program of instand e.v. (düsseldorf, germany). for detection of diverse respiratory viruses, all cdnas were tested with the seeplex rv ace (covers iv-a; iv-b; rsv-a/-b; adv; piv - ; bocavirus, bov; hmpv; hrv-a/ b; cov e/nl /oc /hku ) and rv (covers the same spectrum as rv , but is not able to detect bov) ace detection kits. the seeplex rv ace detection kit (includes also piv- , hrv-c, and enterovirus detection but is not able to detect cov hku ) was used from november , since the distribution of rv and rv versions was abandoned (all kits seegene, eschborn, germany). all three multiplex assays have been established in the laboratory using defined eqas samples from instand e.v. before testing of study samples. the rv kit is a screening kit and neither allows discrimination between adv, piv, and bov nor between hmpv, hrv, and cov. to overcome this problem, amplicons were purified after agarose gel-electrophoresis applying the qiaquick gel extraction kit (qiagen). purified dna was ligated into the pdrive cloning vector included in the qiagen pcr cloning kit, and used for transformation of competent escherichia coli cells. then, colonies were screened for inserts by pcr applying the dreamtaq dna polymerase and oligonucleotides specific for pdrive. amplicons with inserts were purified and sequenced using the dtcs quick start master mix. sequence analysis was done on a beckman ceq genetic analyzer (all beckman coulter, krefeld, germany). for detection of human parechovirus (hpev) genome equivalents, a semiquantitative real-time pcr was established using cdna and oligonucleotides [ ] together with the quantitect sybr green pcr kit (qiagen) on a lightcycler . (roche, mannheim, germany). positive controls for hpev-pcr were kindly provided by dr. corinna pietsch and prof. dr. uwe gerd liebert (institute of virology, university of leipzig, germany). since enteroviruses were not covered by rv and rv assays, nearly all samples were screened by a nested pcr protocol detecting a conserved sequence of the ′nontranslated region ( ′-ntr) [ ] . then, rough-typing was done by sequence analysis of purified pcr products. detection of hrv was performed by nested amplification of the vp / -encoding region [ ] . parallel testing for m.p. and c.p. was done by applying the diagenode mycoplasma pneumoniae and chlamydophila pneumoniae kit (r-diamcpn, diagenode s. a., liège, belgium) on an abi real-time pcr system (thermofisher scientific). sequence data were analyzed using mega . [ ] . all other data were analyzed applying the two-sided fisher's exact test implemented in ibm® spss® statistics . a p value < . was considered statistically significant. this study included samples from immunocompromised or immunocompetent patients with symptoms of rti collected over a period of months. all samples tested positive for the presence of gapdh and/or ß-actin. thus, quality of sampling and nucleic acid extraction was demonstrated (data not shown). among the overall study population, genome equivalents of ebv were most frequently detected ( . %, / ), followed by hhv- ( . %, / ), hrv ( . %, / ), cmv ( . %, / ), rsv ( . %, / ), and iv ( . %, / ). genome equivalents of hsv- and c.p. were generally not detected (table ) . with respect to patients' immune status, dna of ebv ( . % vs. . %), cmv ( . % vs. . %), and hsv- ( . % vs. . %) was significantly more prevalent in immunocompromised patients while genome equivalents of iv ( . % vs. . %) or m.p. ( . % vs. . %) were more frequently observed in their immunocompetent counterpart. the higher prevalence of cmv and ebv was only observed (supplementary fig. ). in . % ( / ) of samples found to be pathogen-positive, multiple agents were detected. among them were samples with two ( . %), three ( . %), four ( . %), or even five ( . %) different viruses/bacteria (supplementary fig. ). the combination of two herpesviruses (hhv- /ebv . %, / ; cmv/ebv . %, / ; cmv/hsv- . %, / ) but also of ebv and iv ( . %, / ) or m.p. ( . %, / ) as well as of rsv and hrv ( . %, / ) was frequently observed. bocaviral dna was found together with other viruses/m.p. supplementary fig. ). significant seasonal effects were recorded in the immunocompetent group for hrv with a high prevalence in autumn and for iv with an increased prevalence in winter. seasonal effect was significant in both patient groups for rsv with increased prevalence in winter and spring (supplementary table ). interestingly, slight seasonality was also observed for hhv- in the immunocompetent group. in addition, some interannual variation was found for m.p. and iv (data not shown), which was most likely associated to the influenza pandemic and an m.p. epidemic in , respectively. in this monocentric study, genome equivalents of viruses and m.p. were frequently detected in immunocompromised ( . %) and immunocompetent ( . %) patients with respiratory symptoms (table ) . since a contribution of relevant cultivable microorganisms to patient symptoms was largely excluded, a causative role of the pathogens detected in this study has to be considered. previously, a comparable approach was used to identify viral causes of severe rti in children [ ] . the stringent exclusion criteria may account for the low number of patient samples included in this study and may have neglected possible additive or synergistic effects between bacteria, fungi, and viruses. in particular, we found a high prevalence of herpesviruses in immunocompromised adults with respiratory infections. nearly one-third of them was tested positive for ebv and every fourth patient presented cmv in his respiratory tract. in children, herpesviral dna was rarely detected which reflects the generally increasing infestation rate observed over life-time [ ] [ ] [ ] [ ] [ ] and indicates viral reactivation as a major cause for pathogen detection. the higher prevalence of ebv, cmv, and hsv- in the airways of adults was associated with the state of immunosuppression. this is in line with the fact that herpesviral reactivation is facilitated by the impaired immune system [ ] . it is still controversial whether this reactivation contributes to respiratory pathology or just represents an indicator of excessive immunosuppression. for cmv, however, there is no doubt that this betaherpesvirus is responsible for lrti in immunocompromised patients [ , ] . cmv pneumonia is considered as likely when viral dna has been detected in bal of symptomatic patients [ ] . thus, in our study, a remarkable proportion of immunocompromised adults revealed signs of suspected cmv pneumonia (table ) and may benefit from antiviral prophylaxis or therapy. while hsv- dna was slightly more prevalent in the urt of immunocompromised patients, we found nearly comparable detection rates in the lrt of both patients groups (table ). this is in line with a previous study [ ] . interestingly, the same authors found that higher hsv- concentrations were associated with a poor patient outcome [ ] . as for cmv, definitive diagnosis of hsv- pneumonitis depends on the presence of viral antigen within the lrt tissues [ ] . in contrast to a recent report [ ] , we found two-times higher ebv dna prevalence in immunocompromised patients compared to their immunocompetent counterparts (table ) . previously, ebv dna was frequently detected in patients with pneumonia, respiratory insufficiency, and other bronchopneumopathies, but its presence was not associated with increased -day mortality [ ] . in addition, the same authors reported no difference in ebv concentration between immunocompromised and immunocompetent patients [ ] , which is in contrast to our findings (fig. , supplementary table ). nevertheless, the contribution of ebv to the development of respiratory symptoms is still controversially discussed in the literature and remains unclear so far [ ] [ ] [ ] . there is, however, some evidence that ebv reactivation-like that of other herpesviruses-may trigger fig. comparison of ebv-dna copies/ml in respiratory specimens from immunocompromised and immunocompetent patients. data are presented in a logarithmic scale. the median ebv concentration is significantly higher in immunocompromised patients (p = . , mann-whitney u test) inflammation which is associated to transplant rejection or interstitial lung disease [ , [ ] [ ] [ ] . hhv- dna was found at similar high frequencies of ca. % in both patient collectives. interpretation of our results, however, is limited since our pcr protocol may have also detected chromosomally integrated viral dna [ ] and cannot differentiate between hhv- a and hhv- b. the latter variant is more commonly implicated in human disease [ ] . moreover, hhv- was frequently observed in combination with other herpesviruses ( table ) as also seen by others [ ] . thus, the contribution of hhv- to rti remains unclear. other herpesviruses (vzv, hsv- ) were found to be negligible in this study (table ) which is in line with the literature [ , , , ] . most of our results were obtained by end-point pcr. the consideration of viral concentration-as it is exemplarily shown here for ebv-may be useful in order to better unravel the contribution of herpesviruses to the development of lung pathology [ ] . the observed frequencies of respiratory viruses were comparable to data from the german laboratory network (https://clinical-virology.net/en/charts/chart/ctype/ count/network/resp/section/viruses) and to another study from germany [ ] . genome equivalents of rsv and hrv were prevalent in children while hrv and iv were frequent in adults. interestingly, immunocompromised adults tended to have a higher prevalence of rsv (table ) . this supports previous data on the contribution of rsv to morbidity and mortality in this patient group [ ] . there were various examples of single detections, which are probably indicative for infection, but also of co-presence of two or more pathogens ( table , supplementary fig. ). bocaviral dna, for instance, was frequently found in combination together with further viral genomes as also reported by others [ ] . in children of years and younger, however, this parvovirus was detected solely. previously, isolated bov infection was shown to be a likely cause of severe acute rti in children [ ] . in a german study, bov dna was demonstrated in . % of nasal swabs obtained from children with respiratory symptoms [ ] . this prevalence is largely comparable to our results. same authors indicated a mean age of . years table detection of multiple pathogens in the respiratory tract of the overall study population (a) as well as of immunocompromised (b) and immunocompetent (c) patients. the gray boxes indicate frequent co-infections. note that due to multiple detection (i.e., more than two pathogens), the sum of the frequencies given in these boxes may be higher the total frequency given in the black box. see also suppl. figure a) overall study populaƟon adv bov cov ev hmpv hpev hrv iv-a iv-b piv rsv-a rsv-b cmv ebv hhv- hsv- for bov detection. in . %, bocaviral dna was detected together with other pathogens [ ] . interestingly, analysis of the ev ′-ntr sequences gave some evidence for the presence of ev-d in the airways of three adults and one toddler. ev-d infection is associated with the development of acute flaccid myelitis and severe respiratory illness [ ] . parechoviral rna was found only in a -year-old immunocompetent child with ia-v infection. human parechoviruses can cause mild gastrointestinal and respiratory disease but also sepsis-like illness and meningitis in infants [ ] . the general low prevalence of hpev in this study is in line with a previous report [ ] . the possible etiology of rti was not clarified in . % of immunocompromised children (table ) . under these conditions, application of broad diagnostic technologies like nextgeneration sequencing could be useful in identification of the underlying pathogen [ ] . moreover, cmv detection rate in samples from the urt of immunocompetent children was surprisingly high (table ) . seasonal effects were evident for several respiratory viruses (supplementary table ). slight seasonality was also observed for hhv- in immunocompetent patients. interpretation of this finding, however, is unclear. the high prevalence of m.p. in may be explained by an epidemic observed in germany [ ] . in line with this, the pandemic caused by a/h n pdm may account for a further study bias. dna of c.p. was generally not detected in our study. this is in line with the low prevalence of . % reported recently [ ] , but also with data from the respiratory viruses network (https://clinical-virology.net/en/charts/chart/ctype/count/ network/resp/section/bacteria). it is hypothesized that the prevalence of c.p. was overestimated in previous reports, most likely due to usage of nested-pcr methods or inclusion of serological data. previously, both c.p. and m.p. were found to be not relevant in critically ill patients with 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posttransplant lymphoproliferative disease after hematopoietic stem cell transplantation the role of infection in interstitial lung diseases: a review epstein-barr virus-associated pneumonia and bronchiolitis obliterans syndrome in a lung transplant recipient detection of epstein-barr virus dna in peripheral blood is associated with the development of bronchiolitis obliterans syndrome after lung transplantation chromosomally integrated human herpesvirus : questions and answers human herpesvirus- infections in kidney, liver, lung, and heart transplantation: review varicella pneumonia in adults detection of respiratory viruses using a multiplex real-time pcr assay in germany respiratory syncytial virus infection-associated hospitalization in adults: a retrospective cohort study human bocaviruses: possible etiologic role in respiratory infection human bocavirus infection as a cause of severe acute respiratory tract infection in children frequent detection of bocavirus dna in german children with respiratory tract infections current understanding of human enterovirus d enterovirus and parechovirus infection in children: a brief overview a method to identify respiratory virus infections in clinical samples using next-generation sequencing capnetz study g ( ) mycoplasma pneumoniae and chlamydia spp. infection in community-acquired pneumonia low yield by molecular detection of chlamydophila pneumoniae in respiratory samples in belgium questioning its etiological role in respiratory tract infections m. pneumoniae and c. pneumoniae are no relevant pathogens in critically ill patients with hospital-acquired respiratory tract infections publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments the authors would like to thank all patients and their families for study support. furthermore, the great contribution of attended physicians is kindly acknowledged. the authors thank fabian cundano maltez, rosemarie carius, and martina müller for their excellent technical service. in addition, we are grateful for the continuous support given authors' contributions ak and rz conceived the study, helped with interpretation of data, and wrote the manuscript together with mw who performed statistical analyses. mr and cwo tested all respiratory tract samples by pcr and analyzed available patient data in frame of their m.d. theses. testing of nucleic acids for the presence of ev, hrv, and hpev was partially done by fk and sw as part of their bachelor theses. bg and re continuously supported the collection and analysis of samples. all authors read and approved the final version of this manuscript. the study was approved by the ethics committee of the jena university hospital ( - / ). the authors declare that they have no conflict of interest. key: cord- -s wjg ar authors: cobrado, l.; silva-dias, a.; azevedo, m. m.; rodrigues, a. g. title: high-touch surfaces: microbial neighbours at hand date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: s wjg ar despite considerable efforts, healthcare-associated infections (hais) continue to be globally responsible for serious morbidity, increased costs and prolonged length of stay. among potentially preventable sources of microbial pathogens causing hais, patient care items and environmental surfaces frequently touched play an important role in the chain of transmission. microorganisms contaminating such high-touch surfaces include gram-positive and gram-negative bacteria, viruses, yeasts and parasites, with improved cleaning and disinfection effectively decreasing the rate of hais. manual and automated surface cleaning strategies used in the control of infectious outbreaks are discussed and current trends concerning the prevention of contamination by the use of antimicrobial surfaces are taken into consideration in this manuscript. in spite of the growing global commitment towards an effective reduction of healthcare-associated infections (hais), it is unfortunately certain that such infections will continue to be responsible for very high morbidity, increased costs and length of stay (los) for the coming decades [ , ] . among potential sources of pathogens causing hais, the most frequent are the patient's microbiota and the hands of healthcare personnel [ ] . additionally, evidence that hightouch surfaces (hts) will work as an extra source of microbial pathogens accumulated over the years, e.g., several microorganisms can survive on medical equipment for hours to months, improved cleaning and disinfection of surfaces decrease the rate of hai, and hospital environmental screening results and the study of clonal outbreaks, all have given support to the role of contaminated hts in the transmission of pathogens between patients and healthcare personnel [ ] . from surfaces, microbial transmission may occur either through direct patient contact or, indirectly, through healthcare personnel hands or gloves [ ] . therefore, upon potentially preventable sources of microorganisms, contaminated hts deserve strong consideration. microbial pathogens most frequently involved in the contamination of hospital environmental surfaces are (methicillinresistant) staphylococcus aureus (mrsa), vancomycinresistant enterococci (vre), clostridium difficile, multidrug resistant gramme-negative bacilli (such as pseudomonas, acinetobacter and enterobacteriaceae), norovirus, coronavirus and candida species [ ] [ ] [ ] [ ] [ ] . strategies for cleaning contaminated hts may include manual and automated techniques. wipes and cloths with application of detergents or disinfectants are examples of manual techniques, while automated methods may involve uv light, hydrogen peroxide, steam vapour, ozone and hins (high-intensity narrow-spectrum light). on the other side, in order to prevent contamination of hts, antimicrobial surfaces are being developed. the inhibition of microbial adhesion with repellent films is a possible strategy, as it is the surface treatment with antimicrobial coatings of silver, copper, polycations, triclosan, bacteriophages or, even, light-activated biotoxic radicals. the aim of this manuscript is to review the role of hightouch surfaces in healthcare-associated infections, from the aetiology to strategies for surface cleaning and addressing preventive trends. as early as , spaulding proposed a classification of inanimate surfaces into three general categories based on the risk of infection if the surfaces were contaminated at the time of use [ ] . these categories can be applied to devices or instruments as follows: critical (exposed to normally sterile areas of the body; require sterilization), semi-critical (touch mucous membranes; may be sterilized or disinfected), and noncritical (touch skin or come into contact with people only indirectly; can be either cleaned and then disinfected with an intermediate-level disinfectant, sanitized with a low-level disinfectant or, simply, cleaned with water and soap). in , the cdc proposed environmental surfaces (floors, walls and other bhousekeeping surfaces^that do not make direct contact with a person's skin) as an additional category [ ] . more recently, the cdc's and healthcare infection control practices advisory committee's guidelines for environmental infection control in healthcare facilities [ ] divided surfaces into patient care items and environmental surfaces. environmental surfaces were further divided into medical equipment and patient room surfaces (table ) . over the years, research has been done in order to better target room disinfection practices. following recommendations made by the cdc to clean and disinfect hts more frequently than minimal-touch surfaces, data published in by huslage et al., based on the real frequency of contact, defined the top five most touched surfaces in hospitals: bed rails, bed surface, supply cart, over-bed table and intravenous pump [ ] . hts may be classified as non-critical items (the contact occurs with intact skin that effectively acts as a barrier to most pathogens, but not with mucous membranes) and must be subject to cleaning and disinfection procedures as recommended, but with no absolute need for sterilization [ ] . many pathogens may thrive on healthcare-associated equipment and environmental surfaces. among such organisms, mrsa, vre, c. difficile, p. aeruginosa, a. baumannii, enterobacteriaceae, stenotrophomonas maltophilia, burkholderia cepacia, norovirus, coronavirus and candida spp. may persist and contribute to the infection risk to which patients are systematically exposed. several studies have demonstrated that basic cleaning leads to mrsa elimination from environmental surfaces and enhanced cleaning may terminate outbreaks in intensive care units, with cost savings of $ , up to $ , per year [ ] [ ] [ ] [ ] . recently, two studies reported positively about pulsed xenon uv and hydrogen peroxide methods to boost the decontamination of patient rooms, contributing towards a reduction of mrsa bioburden [ , ] . its inherent ability to resist certain antimicrobial agents (such as cephalosporins and aminoglycosides) allied to a great capacity to acquire determinants of antibiotic resistance (like gene clusters of vancomycin resistance) turn enterococci into a versatile nosocomial multidrug-resistant pathogen. the number of vre infections has been increasing worldwide, most frequently afflicting patients with serious comorbidities or undergoing prolonged hospitalization [ ] . vre are known to survive for a long time in the hospital environment. viability on surfaces may range from days to months [ ] . moreover, enterococci are tolerant to heat, chlorine and some alcohol preparations, making them very resilient to conventional cleaning practices, thus becoming easily disseminated among healthcare facilities [ ] . therefore, besides thorough environmental cleaning several times a day with disinfectants, vre management protocols should include strict adoption of contact precautions and implementation of comprehensive educational programs for staff [ , ] . spores of c. difficile can hold on to a healthcare environment for more than months [ , ] . fortunately, the use of chlorine-releasing disinfectants reduces the amount of spores in the environment, with some evidence suggesting that it may reduce the risk of recurrence and transmission of c. difficileassociated infections [ ] . transmission of p. aeruginosa may easily occur from contaminated sinks to hands of healthcare personnel during washing, since this organism may thrive in biofilms that are adherent to sink traps, pipes, water lines and hospital drains [ ] , turning these fashion-organized bacteria more prone to resist to disinfectants [ ] . additionally, p. aeruginosa can resist hours to months on dry inanimate surfaces [ ] . programs to control transmission should include, therefore, repeated cleaning with chlorine-based disinfectants, physical removal of persistent biofilm, replacement of components whenever feasible and regular inspection [ , ] . the increase in the number of hais caused by a. baumannii might be explained not only by its ability to persist from days to more than months in undisturbed surfaces of healthcare equipment [ ] , but also by its high resilience to cleaning with conventional detergent and alcohol disinfectants [ ] . hence, outbreaks in hospital or other healthcare settings are difficult to contain because of the easy environmental contamination by this pathogen [ ] [ ] [ ] . targeted infection control measures may be needed, including intensive cleaning with sodium hypochlorite and subsequent measurement of cleanliness, hand hygiene training, adoption of barrier precautions and contact isolation, as well as patient surveillance [ , ] . there has been a growing concern about klebsiella pneumoniae infections, mainly because of its extensive β-lactamase resistance. k. pneumoniae are usual colonizers of the human gastrointestinal tract, pharynx and skin that may cause wound infections, pneumonia and sepsis, particularly in immunocompromised patients [ ] . more recently, given its wide dissemination and selective advantage to resist to carbapenem antibiotics, k. pneumoniae have been showing a propensity to cause outbreaks in healthcare institutions [ ] . it is known that k. pneumoniae may survive for more than months in the healthcare environment [ ] and that the origin of some outbreaks has been related to sinks and related pipes [ , ] . another member of the enterobacteriaceae family, serratia marcescens, are known to cause pneumonia, meningitis, urinary tract and bloodstream infections. mdr isolates, including colistin resistant [ ] , have been responsible for serious outbreaks among intensive care units and critically ill neonates [ ] [ ] [ ] . s. marcescens are known to survive up to months on dry inanimate surfaces [ ] and have frequently been recovered from water pipes and hospital disinfectants [ ] . because of the easy transmission and environmental persistence of enterobacteriaceae in healthcare facilities, adequate solutions aiming its eradication should ensure comprehensive educational interventions, hand hygiene training, chlorine-based cleaning and even the replacement of sinks and pipes [ , , ] . similarly to other bacteria, it can persist in biofilms that may turn cleaning products and disinfectants more ineffective [ ] . long-term control of s. maltophilia will be dependent upon the integration of an efficient cleaning strategy into a targeted healthcare facilities maintenance program [ ] . it is widely distributed in soil and water habitats and recent healthcare-associated outbreaks have been linked to b. cepacia persistence in disinfectants, drugs, medical devices (e.g., respiratory nebulizers), sinks and contiguous aerator filters [ ] [ ] [ ] . strict and repeated cleaning and replacement of aerators with flow straighteners may be required to stop outbreaks [ , ] . the origin of norovirus outbreaks in healthcare facilities has been traced not only to sites near bathroom showers and toilets but also to sites near patients, including clinical equipment (e.g., blood pressure and pulse oximeter monitors), thermometers, trolleys and soap and alcohol gel containers [ ] . after suspected or confirmed case contact, use of soap and running water is recommended [ ] , probably with a superior efficacy than ethanol-based sanitizers [ ] . however, detergent-based cleaning may be insufficient to eliminate norovirus from the environment and, therefore, hypochlorite solutions of at least ppm for an appropriate contact time represent a better strategy for cleaning [ , ] . human coronavirus, usually responsible for acute respiratory syndromes, have been causing increased concern due to contact transmission during healthcare-associated outbreaks. viral persistence on doorknobs and surgical boom shelves has already been identified, with a presumed viability of h; scrupulous environmental cleaning is certainly highly advisable in reducing the spread [ , ] . moreover, biocidal surfaces based on copper alloys are very effective in inactivating coronavirus and could be employed in high touch surfaces in order to prevent the transmission of this respiratory virus [ ] . although candida spp. are more resistant to germicidal chemicals than most vegetative bacteria, there are no specific recommendations other than general healthcare surface decontamination with disinfectants. nevertheless, in order to control a recent outbreak by a mdr c. auris, measures implemented included isolation of cases and contacts, protective clothing, screening of all other ward patients, skin decontamination with chlorhexidine, environmental cleaning with chlorine-based disinfectants and hydrogen peroxide vapour [ ] . a clinical alert issued in june by the cdc on the global emergence of invasive infections caused by the mdr c. auris recommended thorough daily and terminal cleaning and disinfection of patient rooms using an epa-registered hospital grade disinfectant with a fungal claim. preventing the environmental surface transmission of healthcare-associated pathogens general strategies based on patterns of microbial resistance to physical and chemical germicidal agents and on the instrument/surface classification, spaulding has proposed three levels of disinfection [ ] : high-level disinfection, that inactivates all vegetative bacteria, mycobacteria, viruses, fungi and some bacterial spores by the action of chemicals such as glutaraldehyde, peracetic acid and hydrogen peroxide; intermediate-level disinfection, which is effective against vegetative bacteria, some spores, mycobacteria, fungi, lipid and medium size viruses, but not against all nonlipid and small size viruses (e.g., sodium hypochlorite, alcohols, some phenolics and some iodophors); and low-level disinfection, that inactivates vegetative bacteria, fungi, enveloped viruses and some non-enveloped viruses (e.g., adenoviruses) by the action of quaternary ammonium compounds, some phenolics and some iodophors [ ] . in order to prevent the persistence of microbial pathogens on medical equipment and environmental surfaces, education of healthcare staff, checklists and assessment of the adequacy of cleaning (by direct observation, use of fluorescent markers, of atp bioluminescence systems, swab cultures or agar slide cultures) with feedback to the staff are general interventions that need to be implemented to improve the frequency of adequate cleaning [ ] [ ] [ ] . as general principles, all patient care items should be cleaned and/or decontaminated before and after use, for all patients [ , ] ; whenever these items come into contact with blood or other body fluids, stringent cleaning and disinfection is warranted before and after use [ ] . manufacturers of medical equipment usually provide care and maintenance instructions regarding servicing decontamination, compatibility with germicidal agents and water-resistance. in the absence of such instructions, the cdc and the healthcare infection control practices advisory committee (hicpac) recommend non-critical medical equipment (e.g., stethoscopes, blood pressure cuffs, equipment knobs and controls) to be subject to low or intermediate-level disinfection after cleansing, depending on the nature and degree of contamination. for instance, ethyl or isopropyl alcohol ( - % v/v) may be used to disinfect small surfaces (e.g., rubber stoppers of multiple-dose medication vials and thermometers) and surfaces of healthcare equipment (e.g., stethoscopes and ventilators) [ ] , while for large surfaces it may be impractical due to the rapid evaporation of alcohol and absence of the adequate contact time [ ] . as a whole, frequently touched environmental surfaces benefit from enhanced cleaning. routine decontamination and disinfection are practices normally included within institutional cleaning policies. nevertheless, evidence has been built in order to favour the use of less toxic detergents over disinfectants in non-outbreak situations, without losing cleaning efficacy or adding costs [ ] . detergents are less likely to contribute to the accumulation or dispersal of tolerance or resistance genes among healthcare-associated microbial isolates [ , ] . according to the cdc, for medical equipment (particularly in the case of monitor touch screens, controls and cables), a disposable plastic barrier protection can be useful whenever these surfaces, touched frequently by gloved hands, may become contaminated with body fluids or present difficulties to cleaning. manual cleaning the physical removal of soil is a very important step in the cleaning process since its presence will impede the microbicidal activity of disinfectants, if needed. in order to control the bioburden on regular wards, daily cleaning with neutral detergent wipes is usually sufficient. however, more attention is essential on high-risk intensive care units because of the easiness of microbial recontamination [ ] . moreover, patients colonized or infected with specific pathogens may demand cleaning regimens with disinfectants with registered label claims [ ] . after patient discharge, terminal or deep cleaning is usually performed by removal of all detachable objects from the room and systematically wiping all surfaces downward to the floor level, with detergent cloths or disinfectant wipes. new liquid disinfectants are under development and include: improved hydrogen peroxide disinfectants, effective in reducing bacterial levels on surfaces [ , ] , related to fewer hais [ ] and able to reduce contamination by mdr pathogens on soft surfaces such as bedside curtains [ ] ; peracetic acid and hydrogen peroxide disinfectants, a sporicidal combination that was shown to lower bacterial levels on surfaces and to reduce the contamination by mrsa, vre and c. difficile as effectively as sodium hypochlorite [ ] ; electrolyzed water (hypochlorous acid) disinfectant, which may reduce bacterial levels on surfaces near patients in a higher degree than quaternary ammonium disinfectants [ ] ; further promising, electrolyzed water has been sprayed onto medical equipment (with a short contact time and without the need for wiping because no toxic residue remains on surfaces) with a reduction of aerobic bacteria and c. difficile spores [ ] ; coldair atmospheric pressure plasma systems, which generate reactive oxygen species (ros) with bactericidal activity and have potential use as surface disinfectants [ , ] ; nebulized polymeric guanidine, under investigation for its antimicrobial activity against several healthcare-associated pathogens [ ] . together with disinfectants, novel materials for liquid application such as microfiber cloths or mops and ultramicrofiber cloths are under development. when used according to manufacturers' instructions, an increased cleaning efficacy is to be expected as compared to standard cotton cloths or mops [ ] . automated cleaning on the pathway to improve quality and ease of cleaning environmental surfaces, considerable efforts have been dedicated towards the development of automated devices. however, because of yet unsolved safety risks, mainly for patients, automated solutions are invariably targeting terminal cleaning. in most instances, these solutions do not preclude preliminary manual cleaning of surfaces to remove residual debris and reduce the bioburden. the microbicidal effect of uv light has been in use for disinfection of environmental surfaces, instruments and air. by damaging the molecular bonds in dna, a reduction in contamination by mrsa, vre and c. difficile on high-touch surfaces has been achieved [ ] . automated mobile uv light devices are easy to use, with minimal need for special staff training. nonetheless, several issues have been raised that may hinder its efficacy, namely, the time and intensity of light exposure and potential barriers that may exist between the lamp and its target surface. as such, uv light is regarded as an effective adjunct, but not a stand-alone strategy for disinfection [ ] . by producing free radicals that lead to oxidation of dna, proteins and membrane lipids [ ] , vapour and aerosol hydrogen peroxide systems have already been shown to be effective against mrsa, vre, mdr gramme-negative bacilli, c. difficile, viruses and fungi [ ] [ ] [ ] [ ] [ ] [ ] . this excellent wide spectrum antimicrobial activity is not without drawbacks, such as toxicity after accidental exposure, minor erosion of environmental polymers and damage of electronic equipment. in addition, there is the need for trained operators, long cycle times for disinfection and the cost is high [ ] . experiments suggest that vapour-phase hydrogen peroxide is a more potent oxidizer of protein than liquid-phase hydrogen peroxide [ ] and, when supplementing other strategies, microcondensation hydrogen peroxide vapour systems may have contributed to control outbreaks by mrsa, mdr grammenegative bacteria and c. difficile in intensive care units, surgical wards and long-term care facilities [ , [ ] [ ] [ ] [ ] [ ] . a novel silver-stabilized hydrogen peroxide is under investigation for its enhanced biocidal activity towards gramme positive and negative bacteria capable of producing catalase, both in planktonic and biofilm cultures. silver probably helps to stabilize and target hydrogen peroxide to the bacterial cell surface acting, therefore, synergistically [ ] . in fact, a previous report on the effect of a dry-mist system using a mixture of hydrogen peroxide ( %) and silver cations (< ppm) was effective in decontaminating burn patient rooms, as well as a fungal research laboratory: a reduction in growth of at least two log was observed for tested bacteria, mycobacteria and fungi [ ] . steam cleaning is a non-toxic and rapid method that may reduce the total bioburden from environmental surfaces by more than % [ ] , with effectiveness against mrsa, vre and gramme-negative bacilli [ ] . concerns about security when steam is applied to electrical items such as switches and buttons and risk of burns and scalds when cleaning a crowded ward are the reasons precluding its widespread use in healthcare facilities [ ] . the oxidizing capacity of ozone justifies its previous evaluation as a gaseous decontaminant for controlling c. difficile on environmental surfaces and e.coli in hospital laundries [ , ] . while it seems highly effective against vegetative bacterial cells, a smaller impact has been found in case of bacterial spores and fungi [ ] . moreover, corrosiveness and toxicity issues may restrain further the use of ozone in healthcare settings [ ] . by targeting intracellular porphyrins that absorb the light and produce ros with bactericidal activity [ ] , highintensity narrow-spectrum (hins) light stands as another light-based method with possible application for decontamination of high-touch surfaces, although its efficacy is lower than uv light. as clear advantages, hins light is safe for patients, allowing continuous decontamination of the clinical environment [ ] and it exhibits a wide-range microbicidal activity that includes mrsa, p. aeruginosa and a. baumannii [ , ] . however, hins light has yet to prove its effectiveness in clinical settings and benefits upon hai rates, given the small range of published studies [ , , ] . antimicrobial surfaces instead of focusing on the reduction of the bioburden on surfaces solely by cleaning, there are solutions designed to prevent surfaces from working as a microbial reservoir and that may be used as an adjunct to other strategies in reducing hais. antiadhesive surfaces target microbial adhesion usually by the interaction of antagonist physicochemical properties. easy-clean surfaces that are hydrophobic repel bacteria better than glass-coated controls [ ] , while hydrophilic surfaces favour water sheeting and subsequent cleaning. similarly, polyethylene glycol coated surfaces promote a hydrophilic interaction against bacteria, preventing attachment [ ] . the use of diamond-like carbon films has already been tried for medical implanted devices such as joint prostheses and stents in order to repel microbial adhesion [ ] . despite being non-toxic and appealing, the lack of biocidal properties may turn discouraging a more generalized implementation of such easy-clean technologies. currently, there are available antimicrobial coatings that can produce a microbicidal effect and could lead to an effective reduction of high-touch surface bioburden. for instance, inorganic metals have been investigated for a long time and it is known that silver binds with disulphide and sulfhydryl groups present in proteins of microbial cell wall leading to death [ ] , inhibiting not only environmental contamination but also colonization of medical implanted devices [ , ] ; copper and copper alloys may form reactive oxygen radicals that damage nucleic acid and proteins [ ] and have already demonstrated a potent antimicrobial effect when applied to surfaces, reducing the rate of healthcare-associated infections [ , ] . polycationic surfaces, such as those coated with polyethyleneimines, hydrophobically attract and kill bacteria by physically damaging the cell wall [ ] . triclosan has been in use for more than years in detergents, soaps and cosmetics. at lower concentrations, it is bacteriostatic by inhibiting an enzyme involved in fatty acid synthesis and, at higher concentrations, it is bacteric i d a l b y d e s t a b i l i z i n g m i c r o b i a l m e m b r a n e s . compatibilization of triclosan with polymers may extend the duration of its wide-spectrum antimicrobial activity [ ] and could prove effective in reducing environmental surface load of pathogens. bacteriophages applied to surfaces and targeting specific microorganisms have been attempted and mixtures of phages have been further suggested in order to effectively reduce the environmental bioburden. particularly interesting in healthcare settings is the fact that mdr pathogens keep vulnerable to the lytic action of phages [ , ] . light-activated antimicrobial surfaces, such as those coated with titanium dioxide and activated by uv light [ ] , generate reactive oxygen radicals with nonselective toxicity towards both bacteria and yeasts [ ] . similarly, photosensitized surfaces could reduce the healthcare bioburden without promoting microbial drug resistance mechanisms. although antimicrobial coatings may seem very promising, especially as an adjunct measure to more traditional and proven cleaning strategies, some concerns keep hindering its wider use in healthcare settings. robust cost-effectiveness studies are still lacking since reliable information about antimicrobial coatings durability, resistance and possible toxicity is yet somewhat insufficient [ , ] . given the high morbidity and costs associated with hais, improved strategies are urgently needed to reduce effectively the rate of infection. certainly, one good step forward would be the blockade of transmission from environmental hightouch surfaces. at the moment, manual and automated techniques for cleaning surfaces exhibit variable success. concerns over durability, resistance and toxicity may be precluding a much wider application of the novel antimicrobial coatings. admitting an albeit limited performance of the traditional cleaning methods, the supplementation with newer technology should be indicated. hence, more randomized controlled trials and cost-effectiveness studies are needed and further investigation on antimicrobial surfaces is welcomed in order to face the challenge imposed by the global advance of antimicrobial drug resistance and the pressure to reduce bed turnover times with shortages in nursing personnel, housekeeping 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antimicrobial polymers potential of bacteriophage phi ab as an environmental biocontrol agent for the control of multidrug-resistant acinetobacter baumannii antimicrobial surfaces and their potential in reducing the role of the inanimate environment in the incidence of hospital-acquired infections comparison of infectious agents susceptibility to photocatalytic effects of nanosized titanium and zinc oxides: a practical approach light-activated antimicrobial coating for the continuous disinfection of surfaces key: cord- -xbpb nfi authors: ge, huipeng; wang, xiufen; yuan, xiangning; xiao, gong; wang, chengzhi; deng, tianci; yuan, qiongjing; xiao, xiangcheng title: the epidemiology and clinical information about covid- date: - - journal: eur j clin microbiol infect dis doi: . /s - - -z sha: doc_id: cord_uid: xbpb nfi in december , pneumonia of unknown cause occurred in wuhan, hubei province, china. on january , a novel coronavirus, named as severe acute respiratory syndrome coronavirus- (sars-cov- ), was identified in the throat swab sample of one patient. the world health organization (who) announced the epidemic disease caused by sars-cov- as coronavirus disease (covid- ). currently, covid- has spread widely around the world, affecting more than seventy countries. china, with a huge burden of this disease, has taken strong measures to control the spread and improve the curative rate of covid- . in this review, we summarized the epidemiological characteristics, clinical features, diagnosis, treatment, and prognosis of covid- . a comprehensive understanding will help to control the disease. at the end of , several cases of pneumonia with unknown etiology emerged in wuhan, hubei province, china [ ] [ ] [ ] . the pneumonia spread quickly to other provinces of china and overseas. at early stage, it was reported that most patients had the contact history with huanan seafood market [ ] [ ] [ ] . after that, more and more patients had fever and cough symptoms. on january , a novel coronavirus was identified in the throat swab sample of one patient by the chinese center for disease control and prevention (cdc), and was subsequently named as ncov by world health organization (who) [ , ] . as the situation got worse, the who declared the outbreak as the public health emergency of international concern (pheic) [ ] . on february , the international committee on taxonomy of viruses renamed the virus as severe acute respiratory syndrome coronaviruse- (sars-cov- ) [ ] . and who announced the epidemic disease caused by sars-cov- as coronavirus disease (covid- ) [ ] . this is the third coronavirus pneumonia in the past years around the world. in november , a novel betacoronavirus called severe acute respiratory syndrome coronavirus (sars-cov) emerged in guangdong, china, and resulted in more than infections and deaths in countries. in , middle east respiratory syndrome coronavirus (mers-cov), which was first detected in saudi arabia, affected individuals and caused fatalities [ , ] . after the outbreak of covid- , the chinese government has initiated a level- public health response to prevent the spread of the disease on january [ ] . as of : on march (beijing time), the sars-cov- has resulted in , laboratory and clinical confirmed cases in the mainland of china, and patient deaths [ ] . currently, there are many studies of sars-cov- and covid- . this review makes a comprehensive introduction about this disease, including the genome structure and receptor of sars-cov- , epidemiology, clinical features, diagnosis, treatment, and prognosis of covid- . we hope our work can provide more information in understanding this disease, and more research findings are needed to help to limit spread of the disease and to invent vaccine and specific drugs. huipeng ge and xiufen wang contributed equally to this work. about two thirds of the overall genome, encoding nonstructural proteins (nsps), while the remaining one-third encodes accessory proteins and structural proteins [ , ] . as shown in fig. , there are some slight differences in the reported genome structure, mainly in accessory proteins [ , , , [ ] [ ] [ ] . for example, the significant difference of two accessory proteins (orf b and orf ) on the gene sequence between sars-cov- and sars-cov was reported by several studies [ , ] . using the different genome sequences as comparison can partly account for the results. as for the novel proteins of sars-cov- , whether involving in pathogenesis of the virus or not is unclear. inspired by the pathogenesis of sars-cov, the sars-cov- was presumed to infect the human cells by spike glycoprotein binding to its cellular receptor, angiotensinconverting enzyme (ace ). in fact, current evidence supports this idea. as for the spike protein of sars-cov- , it contains two regions, s subunit and s subunit, which consists of amino acids [ ] . the amino acid identity of spike protein between sars-cov- and sars-cov was about % [ , ] . generally, s domain is linked to receptor binding; s domain is linked to cell membrane fusion. similar to sars-cov, s contains the n-terminal domain (ntd) and a receptor-binding domain (rbd) which contains core domain and external subdomain (esd). s contains three functional domains, fusion peptide (fp), and heptad repeat (hr) and (fig. ) . whether sars-cov- can combine with host cells or not is determined by the affinity between the viral rbd and ace of human cells [ ] . once rbd binds to the receptor, the s changes conformation to facilitate the membrane fusion by three functional domains. although sars-cov is not the closest to sars-cov- at the wholegenome level, the rbd of sars-cov- is closer to that of [ , , , [ ] [ ] [ ] . orf = open reading frame (orange). structural proteins including s, e, m, n (blue) (s = spike, e = envelope, m = membrane, n = nucleocapsid). accessory proteins including , a, b, , , , a, b, b, , (purple). sp = signal peptide. s = subunit . s = subunit . ntd = n-terminal domain. rbd = receptor binding domain. esd = external subdomain. fp = fusion peptide. hr = heptad repeat . hr = heptad repeat . tm = transmembrane domain. cp = cytoplasmic domain. the length of genes is not drawn in scale sars-cov, and - . % amino acid sequences of rbd in both are identical [ , , ] . several critical residues in sars-cov- rbd have good interactions with human ace . most residues of rbd interacting with ace are fully conserved [ , ] . as for function domains of s , there is no difference between sars-cov- and sars-cov except some non-critical amino acids in hr region [ ] . another strong piece of evidence supporting ace as a receptor of cells is that hr and hr domain of sars-cov- can fuse with each other to form -hb following sars-cov's fusion mechanism [ ] . despite this, several studies speculated sars-cov- has less affinity with ace than sars-cov [ , , ] . on the contrary, chen et al. [ ] reported sars-cov- provided a stronger interaction with ace than sars-cov by structure analysis of the receptor binding of sars-cov- . by elucidating cryo-em structure of sars-cov- spike protein, wrapp et al. [ ] also found that compared with sars-cov, sars-cov- bound to ace with -to fold higher affinity. in order to identify the suppose of entering cellular by ace , peng et al. [ ] found that sars-cov- was able to use ace protein of many kinds of cells, including human cells, as an entry receptor in the ace -expressing cells, but not cells without ace . it also could not use aminopeptidase n and dipeptidyl peptidase which are other coronaviruses' receptors. in most recent studies, the results provided direct evidence to support ace as the receptor. yan et al. [ ] reported the ace -b at (a neutral amino acid transporter) complex can combine with two spike proteins by structural modeling in a structure analysis of fulllength human ace , and the extracellular peptidase domain (pd) of ace has the direct interaction with polar residues of rbd [ , ] . overall, there is sufficient evidence to support that sars-cov- infects cells by using the human ace . as the cryo-em structure of spike protein and human ace were revealed successfully, we have more opportunity to clarify the detailed process of entering cells [ , , ] . in early studies, - % patients had the contact history of huanan seafood market, where various kinds of living wild animals were on sale, including poultry, bats, and marmots [ , , ] . it is currently speculated that the outbreak of covid- in wuhan is associated with wild animals. according to who, the environmental samples taken from huanan seafood market were tested positive for sars-cov- [ ] , but the specific animals associated with the virus have not been identified. based on previous evidence, the bats, the host of more than coronaviruses [ ] , may be the origin of covid- . the bats are the natural reservoir of sars-cov and mers-cov, and spread to human through the palm civets and dromedary camels, respectively [ ] . the ratg , which is a short rna-dependent rna polymerase (rdrp) region from a bat coronavirus, was closest to sars-cov- with . - . % identity in whole-genome sequence [ , , , ] . the other two bat-derived sars-like coronaviruses, bat-sl-covzxc and bat-sl-covzc , were closer to sars-cov- than sars-covand mers-cov, which have approximately % nucleotide identity [ , [ ] [ ] [ ] [ ] . as for the intermediate hosts of sars-cov- , recent studies suggested pangolins were the most probable animal. two sub-lineages of sars-cov- were found in organs of pangolins obtained from anti-smuggling activities in guangdong and guangxi province of china by metagenomic sequence [ ] . xiao et al. [ ] reported the sars-cov- was derived from the reorganization of pangolin-cov-like virus and a bat-cov-ratg -like virus. however, the pangolin may not be the only intermediate reservoir, because sars-cov- was not originated from pangolin-cov-like virus directly, which was demonstrated by the molecular and phylogenetic analyses in liu's study [ ] . to sum up, the bats are the most probable original reservoir based on the current evidence. however, it is notable that wuhan huanan seafood market may not be the only source of sars-cov- spreading globally. cohen pointed out wuhan huanan seafood market was not the only origin of sars-cov- by analyzing the epidemiology of cases in the earliest study [ ] . pangolins may act as one of intermediate hosts. more work is needed to provide more precise information about original reservoir and intermediate hosts of sars-cov- . table lists some information of studies about the genome sequence identity with sars-cov- . transmissibility is an important factor of an epidemic [ ] . data as reported by am cet on march , sars-cov- has been responsible for , confirmed cases with ( . %) deaths around the world [ ] . the reported median age of patients was ranged from to years [ , , ] . male made up the majority of patients with the proportion of - % [ ] [ ] [ ] ] . due to different data sources, the infection rate of medical staff has a huge difference, with . - % [ , ] . approximately . - . % sars-cov- infected patients had one or more underlying diseases (table ) , including hypertension, diabetes, chronic obstructive pulmonary disease, cardiovascular disease, and malignancy [ - , , ] . the covid- 's median incubation period from exposure to illness onset was . days in a -case cohort and . days in a -case cohort [ , ] . it was also reported the longest incubation period was days [ ] . the percentage of patients exposed to huanan seafood market varies between . % and % [ ] [ ] [ ] ] . patients with no contact history of huanan seafood market and medical staff infection [ , , ] indicated human-to-human transmission mainly via droplets from coughing or sneezing or direct contact [ , [ ] [ ] [ ] [ ] . in addition, several studies reported fecal-mouth pathway may also be a potential way for the transmission of sars-cov- [ , ] , and the sars-cov- was also isolated from urine of a patient in a recent new [ ] . but it is unclear whether humanto-human transmission can be implemented by the above routes. the basic reproduction number r , the important property of transmission, is commonly used to estimate the average number of secondary cases generated by an infectious case in a fully susceptible population during the early phase of the outbreak [ , ] . if the r is more than , human-tohuman transmission may persist. a range from . to . of r was estimated by various methods [ , [ ] [ ] [ ] . the different values of estimated r can attribute to different data and different modeling methods [ ] . liu et al. [ ] came to conclusion that the average of estimate r was . with a median of . by an analysis of studies. so, around - is indeed a reliable range, suggesting the potential of sustained human-to-human transmission [ , ] . additionally, so many factors can affect the value of r , including estimation period, utilized models, and datasets [ ] . as various aspects of measures have taken effect, the estimated r is mutable. as david said, it is impossible to know what will happen so early in this sars-cov- epidemic [ ] . the initial r estimation for sars-cov was more than . , but the predicted large outbreak did not occur [ , ] . it is also notable that asymptomatic patients can also be a source of infection [ , ] . the super communicator is worthy of notice, who can infect more than individuals [ ] . the immediate priority is to clarify all potential routes of transmission. after all, superspreading has a huge impact on the epidemic. anyway, it is necessary for us to take effective measures and keep alert to control the epidemic. fortunately, the number of confirmed cases maintains at a lower level in mainland of china with confirmed cases on march [ ] . at this critical period, it is apparently wise to continue taking steps to control the outbreak. the clinical manifestations of sars-cov- -infected patients ranged from mild non-specific symptoms to severe pneumonia with organ function damage. the common symptoms were fever ( . - . %), cough ( . - . %), fatigue ( . - . %), dyspnea ( . - . %), myalgia ( . - . %), sputum production ( . - . %), and headache ( . - . %) [ - , , ] (table ). sore throat, rhinorrhea, chest pain, hemoptysis, conjunctival congestion, diarrhea, nausea, and vomiting were less common [ - , , ] . but one study showed . % of confirmed covid- patients had gastrointestinal symptoms [ ] , and . % patients presented with gastrointestinal discomfort at onset in wang's study [ ] . patients did not necessarily have fever at onset, some patients developed after hospitalization [ ] , and some severe patients even did not have fever. sars-cov- , sars-cov, and mers-cov infections share many similar clinical symptoms [ ] , including fever, cough, myalgia, and dyspnea. however, patients with sars and mers have more gastrointestinal involvement (about one-third) than covid- patients [ ] . and mers has a high incidence of renal failure, which is a typical characteristic not often found in other human coronavirus infections [ , ] . of confirmed patients, . - . %, . - . %, and . - . % had lymphopenia, thrombocytopenia, and leukopenia, respectively [ - , , ] ( table ) . c-reactive protein (crp), erythrocyte sedimentation rate (esr), serum ferritin, and interleukin- (il ) elevated prominently in chen's research [ ] . many patients also had increased levels of d-dimer, lactate dehydrogenase (ldh), creatine kinase (ck), prolonged prothrombin time, alanine aminotransferase (alt), and aspartate aminotransferase (ast) [ - , , , ] . however, elevated levels of procalcitonin, troponin i, and creatinine were uncommon [ , , ] . the typical imaging features of chest computed tomography (ct) for novel coronavirus pneumonia (ncp) included ground-glass opacity, bilateral patchy shadows, and subsegmental areas of consolidation, sometimes with a rounded morphology and a peripheral lung distribution [ - , , , ] . changes in the disease were accompanied by changes in ct imaging, reflecting the severity of the disease [ ] . pan et al. [ ] analyzed ncp patients from initial diagnosis until recovery (without severe respiratory distress during a hospital stay), and they found chest ct scan showed that the lung abnormalities were the most severe about days after the initial symptoms onset. overall, the ct manifestations of ncp were diverse and fast-changing [ ] . however, a normal chest ct image cannot exclude the diagnosis of sars-cov- infection [ ] . by obtaining biopsy samples at autopsy of one covid- patient [ ] , the lung biopsy specimens showed bilateral diffuse alveolar damage with cellular fibromyxoid exudates. bilateral lung tissue indicated pulmonary edema with hyaline membrane formation, reflecting ards. meanwhile, flow cytometric analysis of peripheral blood suggested the reduced counts of peripheral cd and cd t cells but a hyperactivated status. a report on systemic anatomy at autopsy from the other patient [ ] indicated gray white patchy lesions in lungs, gray white viscous fluid overflow in the lung section, and pulmonary fiber bands, reflecting that covid- caused an inflammatory response characterized by deep airway and alveolar damage. the pathological results of the first one resembled those seen in sars-cov and mers-cov infection [ ] . however, the latter found pulmonary fibrosis and consolidation were less severe than sars, but exudation was more obvious [ ] . these findings, with more pathological ards, acute respiratory distress syndrome; crp, c-reactive protein; ldh, lactose dehydrogenase; ck, creatinine kinase; alt, alanine aminotransferase; ast, aspartate aminotransferase; esr, erythrocyte sedimentation rate research, will make a great importance in understanding the pathogenesis and making therapeutic strategy for covid- . the covid- patients around the world were diagnosed based on world health organization interim guidance [ ] , and china updated the novel coronavirus pneumonia diagnosis and treatment program (trial version) (in chinese) according to epidemic situation and improved awareness of disease. a laboratory confirmed case with sars-cov- infection was defined as a positive result to high-throughput sequencing or real-time reverse-transcriptase polymerase-chain-reaction (rt-pcr) assay for sars-cov- [ , ] . chest ct, as a diagnostic method for covid- with a high sensitivity, is being given more important value for diagnosis [ ] . in the fifth trial version of novel coronavirus pneumonia diagnosis and treatment program (in chinese) [ ] , clinical diagnosis was proposed for cases in hubei province, who had epidemiology history, the above clinical features along with typical chest ct imaging. and more than , patients for clinical diagnosis got early treatment [ ] . the sixth trial version (in chinese) removed the clinical diagnosis, for reduced suspected cases and improved nucleic acid detection capability [ ] . in several clinical studies of confirmed cases, strategies for covid- patients included antiviral treatment, empirical antibiotic treatment, corticosteroid, intravenous immunoglobulin therapy, oxygen support (nasal cannula, mask oxygen inhalation, non-invasive ventilation, invasive mechanical ventilation), continuous renal replacement therapy (crrt), and extracorporeal membrane oxygenation (ecmo) [ - , , ] . due to the absence of clinical evidence, there were no approved drugs for antiviral therapy against sars-cov- . of three clinical cohort studies, oseltamivir was used for antiviral therapy in . % ( / ) patients, . % ( / ) patients, and . % ( / ) patients, respectively [ , , ] . another research [ ] included covid- patients, in which . % patients received antiviral treatment, including oseltamivir, ganciclovir, and lopinavir and ritonavir tablets, and the duration of antiviral treatment was - days. though oseltamivir had high application rate in the early cohort studies, its drug efficacy on covid- was not obvious, and the sixth trial version of novel coronavirus pneumonia diagnosis and treatment program (in chinese) did not recommend it [ ] . case report implied that lopinavir and ritonavir therapy may be beneficial for covid- cases [ , ] . remdesivir, a nucleotide analogue rna polymerase inhibitor with broad-spectrum antiviral activity, was demonstrated that it could be against ebola virus in rhesus monkeys [ ] . wang et al. [ ] found that remdesivir and chloroquine were highly effective in the control of sars-cov- infection in vitro. what's more, the first reported covid- case of the usa was cured by intravenous remdesivir [ ] and other supportive care. more researches from cells, animals, and clinical need to explore the effect of remdesivir on sars-cov- . other methods of antiviral drug administration may also have some certain function. it was reported that different combinations of interferon alpha inhalation, lopinavir/ritonavir, and arbidol may have some effects [ ] . furthermore, combined chinese and western medicine treatment, including lopinavir/ ritonavir, arbidol, and shufeng jiedu capsule (a traditional chinese medicine) may also be beneficial to treatment which deserved further study [ ] . as for the corticosteroid therapy for sars-cov- , current interim guidance from who on clinical management of severe acute respiratory infection, when sars-cov- infection is suspected (released jan , ), suggests not routinely using systemic corticosteroids unless indicated for another reason [ ] , and there were contradictory opinions from professors [ , ] . corticosteroid therapy was used in approximately - . % covid- patients [ - , , ] . compared with non-severe patients, severe patients got more corticosteroid therapy ( . % vs. . %) with the median of maximal daily dose up to . ( . - . ) (mg/kg) [ ] . according to pathological findings of one covid- patient, proper use of corticosteroid together with other support care should be considered for the severe patients to prevent ards development [ ] . there are more than running or pending clinical trials on potential treatments for covid- in china [ ] . studies of recombinant human angiotensin-converting enzyme (rhace ), mesenchymal stem cell, pd- blocking antibody, bevacizumab injection, and immunoglobulin of cured patients are registered in the website of clinical trial [ ] , and some are recruiting patients. safe and effective clinical trials will find more therapeutic possibilities for covid- patients. many patients infected withsars-cov- , especially for severe patients, had complications (table ) , including ards, shock, acute renal injury, acute cardiac injury, and secondary infection [ ] [ ] [ ] ] . the mortality rate of covid- ranged from to . % [ - , , , ] . however, yang et al. [ ] reported of ( . %) critically ill adult icu patients had died at days. it is not hard to know the disease severity is an independent predictor of poor prognosis [ , ] . compared with non-icu patients, the icu patients were older with a greater number of comorbid conditions and had more common symptoms of dyspnea, abdominal pain, and anorexia [ ] . meanwhile, it was reported that icu patients had higher plasma cytokine and chemokine levels of il , il , il , gscf, ip , mcp , mip a, and tnfα [ ] . non-survivors had more severe lymphopenia and higher blood cell counts, neutrophil counts, d-dimer and fibrin degradation product than survivors [ , ] . wang et al. [ ] analyzed the first deaths up to january , announced by the china national health commission found that the median days from first symptom to death were . (range - ) days, and seemed to be shorter among old people [ ] . in yang's study [ ] , the survival time of the non-survivors is likely to be within - weeks after icu admission. in summary, severe patients or icu patients have a relatively higher mortality [ , ] . age, comorbidity, some symptoms (dyspnea, abdominal pain, etc.), and more prominent laboratory abnormalities (lymphopenia, elevated d-dimer, etc.) may be risk factors for poor outcome [ , , , ] . according to the latest data released at : on march (beijing time), the fatality is . % and . % for the mainland of china and wuhan, hubei province, respectively [ ] . overall, sars-cov- has a relatively lower mortality rate than sars-cov and mers-cov ( . % and . %, respectively) [ ] . covid- has spread to countries, territories, or areas around the world, and is responsible for , patients as of am cet on march . the virus may be related to bats, but wuhan huanan seafood market may not be its sole origin. whatever the case, banning of wildlife sales and removing them from wet markets are beneficial to control the epidemic. except several genes of accessory proteins, sars-cov- is almost identical to sars-cov in genome organization. hence, we obtain the fact that ace is the receptor of sars-cov- entering cells. human to human transmission can be realized mainly by droplets from coughing or sneezing or direct contact. fever and cough are the main symptoms. chest ct examination is an important tool for diagnosis, and confirmed cases are diagnosed by detecting sars-cov- of specimens taken from the upper respiratory tract and lower respiratory tract. as for the treatment, there are no specific drugs for the infection, and many therapies, with preliminary good clinical response, are being tested in clinical trials. we hope that increased awareness of the virus and ongoing clinical trials can help to find effective treatment against sars-cov- . though the fatality of sars-cov- is lower than sars-cov and mers-cov, the overall mortality of sars-cov- remains to be established in the future, because a large number of confirmed and suspected cases are still in hospital; even worse, the confirmed cases are increasing in other countries, like korea and japan. further study is necessary for us to control this epidemic disease. authors' contribution huipeng ge, xiufen wang: manuscript writing, bibliographic retrieval, making tables and figure. xiangning yuan, gong xiao, chengzhi wang, tianci deng: manuscript writing. qiongjing yuan, xiangcheng xiao: manuscript editing and critical review. conflict of interest all authors declared that they had no conflicts of interest. clinical features of patients infected with novel coronavirus in wuhan epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan what next for the coronavirus response notice of 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covid- infected pneumonia monitored by quantitative rt-pcr therapeutic efficacy of the small molecule gs- against ebola virus in rhesus monkeys remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro clinical characteristics and therapeutic procedure for four cases with novel coronavirus pneumonia receiving combined chinese and western medicine treatment on the use of corticosteroids for -ncov pneumonia clinical evidence does not support corticosteroid treatment for -ncov lung injury more than clinical trials launch to test coronavirus treatments accessed abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia updated understanding of the outbreak of novel coronavirus ( -ncov) in wuhan publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -v eh rtk authors: boattini, matteo; almeida, andré; christaki, eirini; cruz, lourenço; antão, diogo; moreira, maria inês; bianco, gabriele; iannaccone, marco; tsiolakkis, georgios; khattab, elina; kasapi, diamanto; charrier, lorena; tosatto, valentina; marques, torcato moreira; cavallo, rossana; costa, cristina title: influenza and respiratory syncytial virus infections in the oldest-old continent date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: v eh rtk sars-cov- dramatically revealed the sudden impact of respiratory viruses in our lives. influenza and respiratory syncytial virus (rsv) infections are associated with high rates of morbidity, mortality, and an important burden on healthcare systems worldwide, especially in elderly patients. the aim of this study was to identify severity predictors in the oldest-old admitted with influenza and/or rsv infections. this is a multicenter, retrospective study of all oldest-old patients (≥ years old) admitted for laboratory-confirmed influenza and/or rsv infection in three tertiary hospitals in portugal, italy, and cyprus over two consecutive winter seasons. the outcomes included the following: pneumonia on infection presentation, use of non-invasive ventilation (niv), and in-hospital death (ihd). the association with possible predictors, including clinical features and type of virus infection, was assessed using uni- and multivariable analyses. a total of oldest-old patients were included in the study. pneumonia was evident in . % (n = ). niv was implemented in . % (n = ), and ihd occurred in . % (n = ). multivariable analyses revealed that chronic obstructive pulmonary disease (copd) or asthma was associated with pneumonia (or . ; % ci . – . ; p = . ). copd or asthma (or . ; % ci . – . ; p = . ), rsv (or . ; % ci . – . ; p = . ), and influenza b infections (or . ; % ci . – . ; p = . ) were associated with niv use, respectively, while chronic kidney disease was associated with ihd (or . ; % ci . – . ; p = . ). among the oldest-old, chronic organ failure, such as copd or asthma, and ckd predicted pneumonia and ihd, respectively, beyond the importance of viral virulence itself. these findings could impact on public health policies, such as fostering influenza immunization campaigns, home-based care programs, and end-of-life care. filling knowledge gaps is crucial to set priorities and advise on transition model of care that best fits the oldest-old. introduction sars-cov- dramatically revealed the sudden impact and the utmost importance of respiratory viruses in our lives. influenza and respiratory syncytial virus (rsv) infections are associated with high rate of morbidity, mortality, and an important burden on healthcare systems worldwide, especially among elderly patients [ ] [ ] [ ] [ ] [ ] [ ] . oldest-old is a term meant to include people aged years and older that represent a growing population in the old european continent. despite being a non-homogeneous group, they are professionally retired individuals that usually experience multimorbidity and disability and may face a limited life expectancy. recent evidence suggests that there is a high research interest towards addressing their needs and establishing the best standard of care [ , ] . especially in epidemiological research, oldest-old patients are considered to be a part of the wider age group of the elderly (≥ years old), and there is limited published evidence about predictors of severity of illness and mortality in viral infections, such as caused by influenza and rsv. the aim of this study was to describe the clinical features of an oldest-old population admitted with influenza and/or rsv infections in three southern european hospitals over two consecutive winter seasons and identify predictors of pneumonia, non-invasive ventilation (niv), and in-hospital death (ihd). such knowledge might provide insight to assist healthcare policymakers managing chronic conditions, improving patient satisfaction, and reducing hospital utilization. this is a multicenter, retrospective study of all oldest-old patients (≥ years old) who were either admitted to the hospital for laboratory-confirmed influenza and/or rsv infection or developed it during the course of admission for other causes, from october to april and from october to april in three tertiary hospitals in portugal, italy and cyprus. the laboratory confirmation was based on a positive xpert flu/rsv pcr (cepheid diagnostics, sunnyvale, ca, usa) and/or allplex respiratory panel (allplex, seegene, republic of korea) on naso/ oropharyngeal swabs obtained from patients with signs or symptoms of viral infection. for patients with more than one positive pcr in a seasonal period, the first episode was considered for study purposes. the infection was characterized as hospital-acquired if symptoms pertaining to viral infection began after h from admission. variables assessed included demographics, smoking status, co-morbidities, virus type, nosocomial acquisition, pulmonary infiltrate on chest x-ray taken when symptoms were observed, neuraminidase inhibitor use, length of stay (from admission to discharge), niv, mechanical ventilation, and ihd. this study was conducted in accordance with the declaration of helsinki. formal ethical approval was obtained by the institutional review board of the coordinating center (central lisbon hospital center, no. _ ). informed consent was not deemed required for the purposes of this study. descriptive data are shown as absolute (n) and relative (%) frequencies for categorical data and as mean ± standard deviation (sd) and median and interquartile range (iqr), as appropriate, for continuous variables. on univariate analysis, chi-square test for categorical variables and student's t test or wilcoxon rank-sum test, as appropriate, for continuous variables were carried out to identify factors associated with pneumonia, niv, and ihd. odds ratios (or) and their % confidence intervals ( % ci) were also calculated to estimate the strength of those associations. multivariable analysis models were then fitted to investigate the independent effects of type of virus infection and clinical variables that turned out to be significantly associated with the outcomes at univariate analysis, adjusting for possible confounders like age and gender. for all tests, a p value ≤ . was considered significant. all analyses were performed with stata . a total of patients aged ≥ years old were admitted for influenza a/b and/or rsv infections during the study period in the three centers. oldest-old patients were ( . %), of which ( %) had hospital-acquired influenza a/b and/or rsv infections. clinical features of oldest-old patients included in the study were reported in table . mean age was . ± . (range, to ) years, ( . %) were men, and . % was current active smoker. the co-morbidities mainly observed were diabetes ( . %), copd or asthma ( . %), chf ( %), and ckd ( . %). the viral agents identified were influenza a ( . %), influenza b ( . %), rsv ( . %), influenza a + influenza b ( . %), and influenza a + rsv ( . %) co-infections. among influenza a infections, h n was the most common ( . %) followed by h n ( %), . % not having been subtyped. radiological signs of pneumonia were present on the chest x-ray exams of . % (n = ) following laboratory diagnosis of viral infection; . % (n = ) were submitted to niv and only one patient ( . %) was invasively mechanically ventilated. a total of patients ( . %) did not survive admission. among patients submitted to niv, . % (n = ) survived admission. antiviral treatment with a neuraminidase inhibitor was started in . % of patients. mean length of stay of patients with community-and hospitalacquired infections was ± . (median , iqr - ) and . ± . (median , iqr - ) days, respectively (p < . ). overall, ihd was . %, being % and . % for community-and hospital-acquired infections, respectively, with no significant difference. results of univariate and multivariable analyses were shown in table . at univariate analysis, copd or asthma was significantly associated with pneumonia (or . ; % ci . - . ) and use of niv (or . ; % ci . - . ); rsv infection turned out to be another significant factor associated with niv use (or . ; % ci . - . ), while ckd was the only clinical feature significantly associated with ihd (or . ; % ci . - . ). finally, we considered three logistic regression models, where, for each outcome, age, gender, copd or asthma (for pneumonia and use of niv models), ckd (for ihd model), and type of virus infection were the independent variables. among all patients, multivariable analyses revealed that copd or asthma was significantly associated with radiologically confirmed pneumonia (or . ; % ci . - . ; p = . ); copd or asthma (or . ; % ci . - . ; p = . ), influenza b (or . ; % ci . - . ; p = . ), and rsv infections (or . ; % ci . - . ; p = . ) were associated with niv use; ckd turned out to be the only predictor significantly associated with ihd (or . ; % ci . - . ; p = . ). sars-cov- pandemic and its unsustainable burden supplanted every hierarchy of interest in medical research but highlighted how viral infections' knowledge is crucial in clinical practice. among the aged population, influenza and rsv infections are important causes of hospital admission during autumn and winter months. the highlights of this study are the following findings: ( ) the proportion of oldest-old patients among total hospitalizations was remarkable; ( ) radiological pneumonia, use of niv, and ihd were considerable; ( ) influenza a h n infection was the most prevalent; influenza b and rsv infection were significantly associated with niv use; ( ) copd or asthma was associated with both pneumonia and niv use; ( ) ckd was a predictor of ihd. to the best of our knowledge, our line of research is quite novel and barely comparable to previous reports given the higher mean age of patients involved. overall, the number of hospitalizations over the study period was remarkable comparing to recent reports [ ] , showing that over two years one out of five admissions with influenza and/or rsv infections involved oldest-old. moreover, in our study, hospital-acquired influenza and/or rsv infections were not identified as predictors of pneumonia, use of niv, and ihd for patients aged years and older, moving away from evidence available so far [ , ] . pneumonia on infection presentation was very frequent, in line with more recent reports [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , revealing how an important proportion of patients showed a direct viral injury in lung parenchyma and/or bacterial co-infection. however, despite being a life-threatening condition, it was neither predictive of niv use nor of ihd. the use of niv was considerable when comparing with available evidence in a cohort of younger patients [ ] . this finding might be related to both the type of respiratory failure on infection presentation and a remarkable rate of diagnosed or likely under-diagnosed chronic obstructive lung disease [ ] . similarly, in our population, niv seemed to be effective since data about its use in respiratory viral infections are limited and uncertain, especially in the presence of pneumonia, hypoxemic respiratory failure, and sofa ≥ , and no copd and/or cardiogenic pulmonary edema [ ] . on the other hand, invasive mechanical ventilation rate was performed only once, probably according to ethical and prognostic considerations, such as coexistence of frailty and patient end-of-life preferences. the discrepancy between the use of these two modalities of ventilation could be presumably due to accept niv as a ceiling of therapeutic effort. ihd was also remarkable. studies suggest mortality rate ranging from . to . % [ - , - , ] and up to % in icu patients [ ] . nevertheless, these studies were performed on elderly people with lower median age while our data should be comprehensible for an oldest-old cohort of patients. infection due to influenza a h n was the most prevalent, rsv infection was also very frequent, confirming that epidemic viral subtypes and their affinity for the lower respiratory tract differ according to the study period [ ] . severity of illness and ihd due to rsv infection were similar as compared to influenza viruses, but according to literature, these can vary from season to season [ , ] . therefore, the role of virus type in morbidity and mortality remains controversial. our findings might suggest that rsv and influenza b probably caused infections with a clinical scenario that benefited from application of niv [ ] more than influenza a. copd or asthma was independently associated with pneumonia on infection presentation and niv use, suggesting the importance of the aged lung [ ] and the attempt to overcome respiratory failure through this widespread and generally welltolerated ventilation technique. ckd was the only significant predictor ihd, and it could represent an interesting clinical tool. indeed, previous studies included acute kidney injury and renal disease as predictors of mortality and disease severity, respectively [ , ] , but no prognostic factors have been identified for the oldest-old. our study had limitations. a -h period might have led to misclassify the community-vs. hospital-acquisition of the viral infection since evidence suggests longer incubation periods [ ] . several factors contributing to disease severity and mortality, including nursing home residency, frailty scores, bedridden status, immunization status, malnutrition, sarcopenia, presence of mixed viral and bacterial pneumonia, respiratory failure, occurrence of systemic complications, and physicians' attitude towards more intensive care, were not assessed. moreover, our study lacks an assessment of postdischarge disability and follow-up. in conclusion, this study provided one of the largest assessments available so far of clinical features and factors contributing to severity of illness in the oldest-old admitted with influenza and/or rsv infections in southern europe. chronic organ failure, such as copd or asthma and ckd, predicted pneumonia and ihd, respectively, surpassing the importance of viral virulence. these findings could impact on public health policies, such as fostering influenza immunization campaigns, home-based care programs [ ] , and endof-life care. filling knowledge gaps is crucial to set priorities and advise on transition model of care that best fits the oldestold. european all-cause excess and influenza-attributable mortality in the / season: should the burden of influenza b be reconsidered? estimates of global seasonal influenzaassociated respiratory mortality: a modelling study respiratory syncytial virus infection in adults respiratory syncytial virus infection in older adults: an under-recognized problem respiratory syncytial virus infection in elderly adults caring for critically ill oldest old patients: a clinical review a bibliometric study of research pertaining to the oldest-old (age eighty-five and older) analysis of acute respiratory infections due to influenza virus a, b and rsv during an influenza epidemic predictors of mortality of influenza virus infections in a swiss hospital during four influenza seasons: role of quick sequential organ failure assessment postpandemic influenza a/h n pdm is associated with more severe outcomes than a/h n and other respiratory viruses in adult hospitalisations clinical characteristics of influenzaassociated pneumonia of adults: clinical features and factors contributing to severity and mortality clinical and radiographic comparison of influenza virusassociated pneumonia among three viral subtypes intensive care admissions and associated severity of influenza b versus a during influenza b vaccinemismatched seasons comparative outcomes of adults hospitalized with seasonal influenza a or b virus infection: application of the -category ordinal scale outcomes and prognostic features of patients with influenza requiring hospitalization and receiving early antiviral therapy: a prospective multicenter cohort study high morbidity and mortality in adults hospitalized for respiratory syncytial virus infections the aging lung critical care management of adults with community-acquired severe respiratory viral infection rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults seasonal influenza activity during the sars-cov- outbreak in japan risk of mortality associated with respiratory syncytial virus and influenza infection in adults official ers/ats clinical practice guidelines: noninvasive ventilation for acute respiratory failure incubation periods of acute respiratory viral infections: a systematic review avoiding hospital admission through provision of hospital care at home: a systematic review and meta-analysis of individual patient data publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments the authors thank dr. miguel toscano rico for supporting the project and sharing his knowledge and expertise. key: cord- -u dhbmht authors: keske, Şiran; ergönül, Önder; tutucu, faik; karaaslan, doruk; palaoğlu, erhan; can, füsun title: the rapid diagnosis of viral respiratory tract infections and its impact on antimicrobial stewardship programs date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: u dhbmht we aimed to describe the potential benefit of new rapid molecular respiratory tests (mrt) in decreasing inappropriate antibiotic use among the inpatients presenting with influenza-like illness (ili). we included patients from inpatient and outpatient departments who had ili and performed mrt between january and december in a -bed private hospital in istanbul. at the end of , we implemented antimicrobial stewardship including systematic use of mrt. then, we compared our observations between the year and the year . we designed the study according to the strengthening the reporting of observational studies in epidemiology (strobe) tool. a u.s. food and drug administration (fda)-cleared multiplexed polymerase chain reaction (pcr) system (biofire filmarray, idaho technology, salt lake city, ut) which detects viruses and three bacteria was used for diagnosis. in total, patients were included; ( %) were inpatients and ( %) were older than years of age. at least one virus was detected in ( %) patients. rhinovirus/enterovirus, influenza virus, and adenovirus were the most commonly detected. among hospitalized patients, in children, a significant decrease in antibiotic use ( . % in and . % in , p = . ) was observed, but in adults, the decrease was not statistically significant ( % in and % in , p = . ). the duration of antibiotic use after the detection of virus was significantly decreased in both children and adults (p < . and p = . , respectively). by using mrt, inappropriate antibiotic use and, also, duration of inappropriate antibiotic use after the detection of virus was significantly decreased. it is time to increase the awareness about the viral etiology in respiratory tract infections (rtis) and implement mrt in clinical practice. respiratory tract infections (rtis) are the most common reasons for admission to healthcare facilities and one of the leading causes of hospitalization [ , ] . the viral pathogens in the etiology of rtis became more detectable along with the improvement in molecular diagnostic methods in recent years. in adults, influenza virus, rhinovirus, adenovirus, respiratory syncytial virus (rsv), human coronavirus, and parainfluenza virus cause infections with considerable morbidity and mortality [ , ] , and in infants, rsv is the most common reason for rtis among hospitalized patients [ ] . the antibiotic prescription rate was reported to be more than % despite the high proportion of viral etiology [ ] [ ] [ ] [ ] . unnecessary antibiotic administration in viral infections and antimicrobial resistance prompted the implementation of antimicrobial stewardship (ams) programs. the bimplementing an antibiotic stewardship program^guideline by the infectious diseases society of america (idsa) suggested rapid viral testing for respiratory pathogens to decrease inappropriate antibiotic use [ ] . in this study, we aimed to describe the viral etiology in influenza-like illness (ili) in children and adults and to show the benefit of new rapid molecular respiratory tests (mrt) in decreasing inappropriate antibiotic use. we designed the study according to the strengthening the reporting of observational studies in epidemiology (strobe) tool [ ] . we included consequent patients from inpatient and outpatient departments, who had moderate to severe ili, and, therefore, needed further diagnostic evaluation between january and december in a -bed private hospital in istanbul. only the patients whom mrt was implemented for diagnosis were included in this study. mrt was introduced in our hospital in , and the demand for mrt increased over the years. at the end of , we enhanced our training on antimicrobial stewardship (ams). the intervention consists of training physicians to increase awareness about mrt, highlighting antimicrobial resistance because of unnecessary antibiotic consumption at ad hoc meetings in each related department. then, we compared our observations between the year and the year . this was a retrospective study performed by reviewing the patient charts. clinical features of the patients, demographic characteristics, chronic diseases including malignant disorders, diabetes mellitus, chronic kidney and liver diseases, and detailed information about antibiotic and antiviral drugs during hospitalization were collected by the chart review. complete blood count, c-reactive protein, and liver and kidney function tests were studied among all patients. bacterial cultures (throat, sputum, blood, and urine) were obtained, and procalcitonin (pct) was studied in all patients with suspicion of bacterial infection and/or in critical patients. chest x-ray and lung computed tomography was done if lower respiratory tract infection (lrti) was suspected. ili was defined according to the world health organization (who) and explained as an acute respiratory infection with fever of ≥ °c and cough and with onset within the last days [ ] . inappropriate antibiotic use was defined as antibiotic use despite detection of virus without documented and/or suspicious bacterial infection. antibiotic use duration was calculated as the duration of antibiotic use after the detection of virus in patients with inappropriate antibiotic use. for molecular detection and identification of respiratory viral pathogens, a u.s. food and drug administration (fda)cleared multiplexed respiratory polymerase chain reaction (pcr) system (mrt), biofire filmarray (idaho technology, salt lake city, ut) which detects viral respiratory pathogens (adenovirus, coronavirus hku , coronavirus nl , coronavirus e, coronavirus oc , hmpv, rhinovirus/enterovirus, influenza a, influenza a/h , influenza a/h - , influenza a/h , influenza b, parainfluenza , parainfluenza , parainfluenza , parainfluenza , and rsv) and three bacteria (bordetella pertussis, chlamydophila pneumoniae, and mycoplasma pneumoniae) was used according to the manufacturer's instructions. mrt yielded results within a few hours and the results were immediately reported to the physician of the patients by laboratory staff. in the statistical analysis, the t-test for continuous variables and the chi-square test for comparison of categorical variables were used. in the analysis, stata (statacorp, college station, tx) was used and a p-value < . was set as being in total, patients whom mrt was implemented were included; ( %) were inpatients and ( %) were older than years of age. at least one virus was detected in ( %) patients and ( %) of them were inpatients (fig. (fig. ) . in patients out of ( . %), multiple viral pathogens were detected. the overall rate of lrtis was % among inpatients. among inpatients infected with rsv, the rate of lrti was % in adults and % in children. similarly, in hmpv infections, the rate of lrti was % in adults and % in children (table ) . in total, among hospitalized patients, antibiotics were continued inappropriately in % ( table ). in children, the antibiotic continuation rate was . % in and decreased to . % in (p = . ); in adults, there was a decrease towards but this was not significant ( table ). the mean duration of inappropriate antibiotic use after the detection of virus decreased in both adults and children in when compared with . the most commonly inappropriately used antibiotics were third-generation cephalosporins, quinolones, and second-generation cephalosporins. in our study, rhinovirus/enterovirus, influenza, and adenovirus were the most commonly detected viruses in the etiology of ili. in a recent study by jain et al. [ ] , a population-based surveillance for community-acquired pneumonia among adults aged years and older was conducted. human rhinovirus, influenza a and b virus, and hmpv were the most commonly detected viruses in etiology. influenza virus and rhinovirus were reported to be the most commonly detected viruses in both adults and children in turkey [ ] . among inpatients older than years of age, % had lrti, and influenza a, rhinovirus, rsv, and hmpv were the most common agents. among children, % had lrti, and rhinovirus, rsv, and hmpv were the most common agents ( fig. ; fig. the most commonly detected viruses among all patients in whom at least one virus was detected. rsv: respiratory syncytial virus; hmpv: human metapneumovirus table ). any effort targeted to reduce the unnecessary use of antibiotics in viral pneumonia could be useful in this era of antibiotic resistance. because of the high rate of inappropriate antibiotic prescription despite the availability of mrt, we decided to improve training sessions about the diagnosis and management of respiratory system infections at the end of . then, we performed an observational comparison between the practice in and . there was a decrease in antibiotic use towards among all inpatients. however when analyzed separately, a significant reduction in children but not in adults was detected ( table ). the significant reduction of antibiotic use in children but not in adults could be explained by differences in physicians' experience in mrt. despite the fact that there was a reduction in antibiotic use in children after the detection of virus, antibiotic prescription rates were still high. in a recent study, the impact of mrt on antibiotic use was investigated and it was seen that antibiotics were still continued in about % of the patients, despite the detection of virus. this finding was explained by being a new test [ ] . in another study, afzal et al. [ ] reported that a positive result for mrt decreased antibiotic use duration and prescription rate but the decrease in antibiotic prescription was not statistically significant. timbrook et al. [ ] studied mrt in combination with pct and concluded that a lower level of pct and detection of virus by mrt were not associated with decreased use of antibiotics in rtis, but in another study, a lower level of pct in combination with detection of virus was found to be associated with shortened duration of antibiotic usage [ ] . in a randomized controlled study, the effect of both mrt and pct on antibiotic use was evaluated and the duration of antibiotic use was found to be decreased in cases of viral infection with lower levels of pct [ ] . one of the strongest parts of our study was having the opportunity of ordering mrt whenever needed in routine clinical practice, so we reached a large sample size. we had the opportunity of getting the results of c-reactive protein, pct, and mrt within a few hours, so we discriminated bacterial or viral etiology quickly. one of the limitations of this study was being conducted in a single center, but our center was unique for implementing this test in clinical practice. we are unique because mrt are still expensive to perform in routine clinical practice in other hospitals. future studies for rapid point-of-care testing for respiratory viruses might improve clinical care by reducing unnecessary antibiotic use [ ] . future cost-effective studies for the implementation of mrt will be useful. by using molecular rapid tests (mrt) in our hospital, inappropriate antibiotic use and also duration of inappropriate antibiotic use after the detection of virus was significantly decreased among inpatients. the treatment paradigm for respiratory tract infections (rtis) is changing, but molecular viral diagnostic tests are in their infancy. it is time to increase the awareness of the viral etiology in rtis and implement mrt in clinical practice. funding no funding of any kind has been received. the data were generated as part of routine work. conflict of interest none to declare. ethical approval koç university irb approved the study. informed consent not applicable. mean duration of inappropriate antibiotic use (days) . (sd . ) . (sd . ) < . . (sd . ) . (sd . ) . rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults ecil- ): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus community-acquired pneumonia requiring hospitalization among u.s. adults role of influenza and other respiratory viruses in admissions of adults to canadian hospitals antibiotic use for viral acute respiratory tract infections remains common ambulatory antibiotic prescribing for acute bronchitis and cough and hospital admissions for respiratory infections: time trends analysis antibiotic prescription rates for acute respiratory tract infections in us ambulatory settings prevalence of inappropriate antibiotic prescriptions among us ambulatory care visits implementing an antibiotic stewardship program: guidelines by the infectious diseases society of america and the society for healthcare epidemiology of america the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies world health organization (who) ( ) who surveillance case definitions for ili and sari. case definitions for influenza surveillance prevalence and seasonal distribution of respiratory viruses during the - season in istanbul evaluating the impact of the multiplex respiratory virus panel polymerase chain reaction test on the clinical management of suspected respiratory viral infections in adult patients in a hospital setting clinical diagnosis, viral pcr, and antibiotic utilization in community-acquired pneumonia antibiotic discontinuation rates associated with positive respiratory viral panel and low procalcitonin results in proven or suspected respiratory infections the clinical impact of the detection of potential etiologic pathogens of community-acquired pneumonia serum procalcitonin measurement and viral testing to guide antibiotic use for respiratory infections in hospitalized adults: a randomized controlled trial routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial key: cord- -y zi iz authors: liu, wei; ren, xiaojuan; wang, qian; zhang, yan; du, junfeng title: pharmacological inhibition of poly (adp-ribose) polymerase by olaparib ameliorates influenza-virus-induced pneumonia in mice date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: y zi iz treatments against influenza a viruses (iav) have to be updated regularly due to antigenic drift and drug resistance. poly (adp-ribose) polymerases (parps) are considered effective therapeutic targets of acute lung inflammatory injury. this study aimed to explore the effects of parp- inhibitor olaparib on iav-induced lung injury and the underlying mechanisms. male wild-type c bl/ mice were intranasally infected with iav strain h n to mimic pneumonia experimentally. olaparib at different doses was intraperitoneally injected days before and consecutive days after virus stimulation. on day post-infection, lung tissues as well as bronchoalveolar lavage fluid (balf) were sampled for histological and biochemical analyses. olaparib increased the survival rate of iav mice dose-dependently. olaparib remarkably reduced iav mrna expression, myeloperoxidase (mpo) level, and inflammatory cell infiltration in iav lungs. moreover, olaparib significantly reduced the level of interleukin (il)- β, tumor necrosis factor (tnf)-α, interferon (ifn)-γ, il- , and il- and increased il- in iav lungs. also, olaparib efficiently reduced il- , monocyte chemotactic protein (mcp)- , granulocyte colony-stimulating factor (g-csf), tnf-α, chemokine (c–x–c motif) ligand (cxcl) , cxcl , chemokine (c–c motif) ligand (ccl) , and regulated on activation, normal t cell expressed and secreted (rantes) release in iav balf. olaparib decreased parylated protein content and p , iκbα phosphorylation in iav lung tissues. this study successfully constructed the pneumonia murine model using iav. olaparib decreased iav-induced mortality in mice, lung injury, and cytokine production possibly via modulation of parp- /nf-κb axis. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. influenza is an acute infectious disease affecting respiratory tracts accompanied with different clinical manifestations ranging from wild to lethal. influenza causes seasonal, unpredictable epidemics and it is now one of the major public health concerns worldwide [ , ] . according to the report of the world health organization (who) posted online in , only influenza a virus (iav) has caused pandemics up to now, and most human influenza cases are due to the infection of two iav strains, h n and h n . iav have laid heavy burdens on global population and economy these years [ ] . although vaccine inoculation and antiviral drug administration have been proved to be effective ways to control influenza, epidemics occur sometimes as a result of antigenic drift, which urges the development of novel anti-influenza drugs [ ] . poly (adp-ribose) polymerases (parps) family is composed of members which are involved in the bioprocesses, including dna repair, cell cycle regulation, transcription, and so on [ , ] . abnormal expression of parps is correlated with necrotic cell death, cancer, and some inflammatory disorders [ ] . parp- activation has been regarded as one of the critic mechanisms underlying lung inflammation in the context of lipopolysaccharide (lps) and elastase stimulations experimentally [ , ] . evidences indicated the abnormal increased expression of parp- in non-pulmonary cells, alveolar epithelial cells, and lung tissue after iav infection [ , ] , suggesting its potential as the target for the treatment of iav infection. one of the parp inhibitors, olaparib, is reported to ameliorate acute lung injury induced by elastase and lps [ , ] . yet the role of olaparib on iav-induced lung injury has rarely been reported. this study aimed to explore the effects of parp inhibition by olaparib on iav infection. animals specific-pathogen-free -to -week-old male wild-type (wt) c bl/ mice, weighing to g, were purchased from the nanjing model animal center and kept at °c in a -h light/ dark cycle, with free access to food and water. animals were allowed to acclimate to the housing environment for week before experimental procedures. all the animal-involved experiments were performed in accordance with the animal care and use committee of cangzhou central hospital. the a/font monmouth/ (h n , fm ) mouse-adapted influenza virus (chinese center for disease control and prevention) was first plaque purified in the madin-darby canine kidney mdck cells, followed by replication in -day-old chicken embryos. the virus pool was pretitrated in mice before further studies in order to determine a suitable challenge dose. the murine model of viral pneumonia was constructed by intranasal infection with h n according to previously described [ ] . briefly, ketamine ( mg/kg weight) and pentobarbital ( mg/kg weight) were intraperitoneally injected to induce the mild anesthesia in mice. next, a -μl influenza virus in ice-cooled pbs, containing fifteen % mouse lethal challenge doses (mld ), was infected intranasally. mice were kept on a °c thermal insulation blanket for min to recover. the day of virus infection was defined as experimental day and -days post-infection. two grouping methods were applied in this study. for the part of survival rate analysis, mice were randomly divided into groups, that is, the influenza a virus (iav) group (h n virus + normal saline), the ola groups (h n virus + . , , mg/kg olaparib), and the positive control group (h n virus + mg/kg oseltamivir). for the part of biochemical detection, mice were divided into groups, the control group (normal saline), the iav group (h n virus + normal saline), and the iav + ola group (h n virus + mg/kg olaparib). olaparib (selleck chemicals, houston, tx, usa) at different doses was intraperitoneally injected days before and consecutive days after h n virus challenge. the day of virus challenge was defined as experimental day . oseltamivir (hoffmann-la roche, basel, switzerland) was administered in a similar manner to olaparib. after anesthetization, mice were infected intranasally with -μl influenza virus in ice-cooled pbs, containing five mld , at experimental day ( days post-infection). then mice in different groups were followed for consecutive days post-infection with the number of deaths being monitored daily. the severity of pulmonary edema induced by h n virus challenge was quantified by lung indexing. five days after virus infection, the body weight of mice was measured using an electronic analytical balance. then lungs were dissected, washed in pre-cooled pbs, and weighed. the lung index was calculated by the following formula: lung weight/body weight × %. at day post-infection, lungs were dissected and fixed in % paraformaldehyde for further histopathological analysis. paraffin sections in -μm thickness were stained with hematoxylin and eosin (h&e) and examined using a light microscope. the severity of the lung injury was assessed by scoring the h n -induced lung histopathological changes using the scoring system previously described [ ] . in brief, the grading was conducted in a blinded manner with the grader unaware of the concrete group or mice being reviewed. the scores of to were defined to represent normal, mild, severe, and very severe lung injury, respectively. in concrete, was valued for normal lung, for lower than %, for - %, for - %, and for higher than % lung involvement, respectively. at day post-infection, lung homogenates and balf were sampled from different groups of mice. for lung homogenates, the lungs were dissected and homogenized in ice-cold ripa lysis (sigma) supplemented with protease inhibitor (sigma) on ice for h. after °c centrifugations at g for min, supernatants were collected and stored at − °c for further biochemical analysis, including myeloperoxidase (mpo), cytokines, and targeted protein levels. total protein content of lung homogenates was determined using a bca assay kit (beyotime, shanghai, china). for balf sampling, bronchoalveolar lavage was conducted according to previously described by yashiro m et al. [ ] . briefly, after anesthesia, the left main bronchus was ligated and the right lung was quickly lavaged twice by ml of cold sterile pbs. then the collected balf was centrifuged at rpm for min at °c, and the supernatant was stored at − °c for measurement of cytokine levels. mpo activity was assessed by , ′, , '-tetramethylbenzidine (tmb, sigma-aldrich, st. louis, mo, usa) method according to previously described [ ] . in brief, equal amount of sample ( μl) was sequentially mixed with . -mm hydrogen peroxide ( μl), . -mm tmb dissolved in . % dimethyl sulfoxide ( μl), and -mm sodium phosphate buffer (ph . ) in a -well plate for -min incubation at °c. then -m sulfuric acid (sigma) was added to stop the reaction and absorbance at nm was measured to evaluate mpo activity in each sample. the mpo levels were expressed as pmol mpo per milligram of lung tissue (pmol/mg protein). cytokines and chemokines in lung homogenate and balf samples were measured using commercial assay kits according to the recommended protocols. interleukin- β (il- β), interleukin- (il- ), interleukin- (il- ), interleukin- (il- ), interferon-γ (ifn-γ), and tumor necrosis factor-α (tnf-α) were measured by enzyme-linked immunosorbent assay (elisa) kits (r&d system, minneapolis, mn, usa). monocyte chemotactic protein- (mcp- ), granulocyte colony-stimulating factor (g-csf), chemokine (c-x-c motif) ligand (cxcl ), chemokine (c-x-c motif) ligand (cxcl ), chemokine (c-c motif) ligand (ccl ), and regulated on activation, normal t cell expressed and secreted (rantes) were measured using a mouse multi-cytokine/chemokine magnetic bead panel (millipore, billerica, ma, usa) and analyzed on a luminex system (luminex, austin, tx, usa). the level of target factors was expressed as pg/mg protein for lung homogenate detection and pg/ml for balf detection. real-time quantitative polymerase chain reaction (rt-qpcr) viral load was determined by rt-qpcr according to previously described by li y et al. [ ] with slight modifications. the total rna of isolated lung tissues at day post-infection was extracted using trizol method (life technologies, carlsbad, ca, usa) according to the manufacturer's instructions. then equal amount of rna for each sample was transcribed into complementary dna (cdna) using the first strand cdna synthesis kit (takara, dalian, china) following the instructions. quantitative pcr (qpcr) was performed using a sybr green suit (takara) to determine the mrna expression levels of target genes, iav m gene, and gapdh, in an abi real-time pcr system (applied biosystems, new york, ny, usa), with the following amplification procedures: -min preincubation at °c, cycles of °c for s, °c for s, and °c for s. each sample was performed in triplicate in one single technical repetition. the primer sequences used in this study were as follows: iav m, '-aatggtgcaggcgatagag- ′ (forward) and '-tacttgcggcaacaacgagag- ′ (reverse); gapdh, '-tgaggtcaatgaaggggtcg- ′ (forward) and '-tgaggtcaatgaaggggtcg- ′ (reverse). the relative quantitation of iav was calculated using the comparative −ΔΔct method. gapdh was used as the inner control. the parylated protein content in the lung tissues was analyzed by western blot. briefly, − °c stored lung homogenate samples were thawed on ice. for each sample, -μg protein was separated through % sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (millipore), followed by -h block in % nonfat milk at room temperature. then the membranes were incubated at °c overnight with primary antibodies against par ( : , abcam, cambridge, uk), phospho-p (p-p , : , abcam), p ( : , abcam), phospho-iκbα (p-iκbα, : , abcam), iκbα ( : , abcam), or β-actin ( : , santa cruz, ca, usa), respectively. after the -h incubation with peroxidase-conjugated secondary antibodies (abcam) at room temperature, the enhanced chemiluminescence kit (millipore) was used to detect the proteins of interest in the uvp biospectrum imaging system (biospectrum, ca, usa). βactin served as the inner control. each experiment was performed for at least three times. data were expressed as mean ± sd for each assay. statistical analysis was conducted by a one-way analysis of variance (anova) test using the spss software (chicago, il, usa). p < . was considered to be statistically significant. for the body weight change analysis, the two-way anova followed by tukey's multiple comparison test was used. this study aimed to investigate whether olaparib (chemical structure in fig. a ) possessed protective effects against influenza virus challenge. from day post-infection, the iavinfected mice began to exhibit clinical symptoms, and on day post-infection, the symptoms got worse, which included but not limited to the following, such as weight loss (fig. s ) , inactivity, rapid shallow breathing, and poor appetite, indicating the successful construction of viral pneumonia model experimentally. figure b showed that the iav-only mice began to die on day post-infection until day post-infection. the positive control, oseltamivir, improved the survival rate of infected mice significantly compared with those in the iavonly group. to the expectations, mice in the olaparib group showed higher survival rate compared with that in the iav group in a dose-dependent manner, indicating that olaparib could powerfully protect against influenza virus challenge in by h&e staining and the quantitative analysis of histological changes in the lung tissues (e) (n = for each group). data were presented as mean ± sd. ## p < . and ### p < . compared with the control. * p < . and ** p < . compared with iav mice. additionally, olaparib-treated iav mice exhibited lower weight loss compared with untreated ones, suggesting it might possess lighter side effects (fig. s ) . also, the dose of mg/kg olaparib was chosen for further investigation as a result of the highest survival rate among all olaparib groups. as olaparib treatment evidently reduced the virus-induced mortality, pathological changes were further explored to assess the influences of olaparib on the lung injury severity. in comparison with the control group, the lung index of mice in the iav group was significantly higher than that in the control group, and olaparib treatment remarkably reduced the lung index, suggesting that olaparib could alleviate the pulmonary edema induced by iva infection (fig. a) . as shown in fig. b , higher mrna expression of iav was detected in iav lungs, while no virus was detected in the control lung tissues, indicating the direct relationship between iav infection and pathological manifestations of mice model. unsurprisingly, less iav was detected in the lungs of olaparib-treated iav group, indicating the antiviral effect of this drug functions in the iav model mice. virus infection always correlated with leukocyte filtration to the target organs. we next determined the mpo levels and the marker of leucocytes, in lung tissues. figure c showed that iav infection elevated the mpo levels compared with that in the control group, whereas olaparib evidently reduced mpo levels, illustrating that olaparib reduced leucocyte infiltration to the lungs of iav mice. morphologic analysis was performed using h&e staining of lung tissues. as shown in fig. d , extensive inflammatory cell infiltration, especially around bronchioles, was presented in iav lungs, signifying lung edema might lead to the breathing difficulty in infected mice. and olaparib treatment attenuated the pathological abnormalities in iav lungs. lung histopathological grading numerically pointed that olaparib rectified the lung injury caused by iav infection (fig. e) . virus-infected pneumonia always comes along with abnormal release of inflammatory cytokines. next the level of inflammatory cytokines in lung homogenate was detected to investigate whether olaparib could influence the release of inflammatory cytokines in lung tissue. as shown in fig. a , b, c, d, e, compared with those in the control group, pro-inflammatory cytokines, il- β, tnf-α, ifn-γ, il- , and il- , were significantly elevated, and anti-inflammatory cytokine, il- , was remarkably descended in the iav group, while olaparib treatment obviously rectified the abnormal release of the above inflammatory cytokines, meaning that olaparib might possess anti-inflammatory effect in murine lung tissue under the iav context. the detection of cytokine/chemokine in balf samples at day post-infection showed that il- , mcp- , g-csf, tnf-α, cxcl , cxcl , ccl , and rantes were remarkably increased in the iav group compared with those in the control group, while olaparib treatment significantly reduced the abnormal increased levels of the above cytokine/chemokines, which was similar with the results obtained from lung tissue (fig. a-h) . to explore the mechanisms underlying the protective effect of olaparib against iav-induced injury to murine lungs, western blot was performed to detect the parps, the marker of apoptosis. iav infection increased the parylated protein content in lung homogenate samples compared with the control group, while parp inhibitor olaparib significantly reduced the parylated protein content in iav-infected samples (fig. a and c) . nf-κb genes are reported to be the targets of olaparib in the context of lps stimulation to the lungs [ ] . as shown in fig. b , d, and e, iav remarkably elevated the p-p and p-iκbα protein expression in lung homogenates compared with those of the control group, while olaparib obviously decreased the abnormal increase of the two proteins, suggesting that olaparib inhibits the activation of nf-κb signaling pathway in the condition of iav infection. and there were no significant differences of the total expressions of p and ikba among different groups (fig. b) . the highly transmissible human pathogen iav mainly attacks human respiratory epithelium with its hemagglutinin binding with sialic acid to initiate endocytosis [ ] . severe symptoms after iav infection are commonly found in the infant, elderly, pregnant, as well as the immunocompromised populations [ ] [ ] [ ] [ ] . iav can cause serious pandemics in a short time, with shocking and heart-wrenching facts in history, e.g., the great influenza killed almost / of the global population only in . years. also iav is capable of generating new strains adapted to human beings. that is the very reason that influenza vaccines have to be updated regularly. also antiviral drugs have to be replaced as a result of the increasing drug resistance developed by viable iav subtypes. thus, it is urgent to explore novel anti-iav drugs to enrich the available antiviral drug bank. this study investigated for the first time the possible role of olaparib on iav infection in a murine model and found that parp- inhibitor olaparib remarkably relieved iav-induced lung injury and lung inflammation possibly via inhibition of parp, as well as p , and iκbα phosphorylation. parp has been indicated to be the target of treatment strategy against cancers and inflammatory disorders [ , ] . the pro-inflammatory effect of parp- has been highlighted in a variety of non-pulmonary and pulmonary inflammatory diseases, including arthritis, allergic encephalomyelitis, asthma, acute lung injury (ali), and so on [ ] [ ] [ ] . parp- is activated and involved in the lungs of allergen-induced asthma animal models via modulating immune cell recruitment, airway modeling, as well as cytokine production, mainly th cytokines [ ] [ ] [ ] . parp- is reported to play a critical role in lps-induced and mechanical ventilation-induced ali in mice [ , ] . hence parp- might be a convincing target for the prevention and treatment of lung inflammatory injury. genetic as well as pharmacological measures modulating parp- activity were proved to be effective to alleviate the lung inflammation and injury experimentally from the aspects of reducing neutrophil infiltration and macrophage accumulation [ ] , blocking the activation of nf-κb and ap- [ ] . additionally, parp- inhibition can relieve the secondary kidney injury induced by ali [ ] . as a potent parp inhibitor, olaparib has already been approved to be used in cancer patients for clinical trials with acceptable toxicity. olaparib downregulates nf-κb-related genes including tnf-α and il- β, decreases neutrophil, and alleviates edema of lpsstimulated murine lungs [ ] . our study found that olaparib possesses protective effects against iav-induced lung inflammation, suggesting that this drug might exert non-specific anti-inflammatory roles. as the outbreak of severe coronavirus infection since december , it is urgent to explore the mechanisms of virus-induced damage to lung tissues or other organs, as well as develop more effective antiviral drugs or therapies to prevent related damages to patients. our study western blot analysis of parylated protein content (a) and p-p , p , p-iκbα, and iκbα (b) in lung samples from each experimental group. the relative expressions were normalized to control (c, d, e). data are presented as mean ± sd. ## p < . and ### p < . compared with the control. ** p < . and *** p < . compared with iav explored the protective effects of olaparib in experimental virus-induced pneumonia, suggesting its possible application in viral pneumonia treatment in the future. there are some shortcomings in our study. we did not perform toxicity analysis about the influence of olaparib on normal physiology of mice. the relationship between parp- /nf-κb and iav-induced lung inflammation as well as the concrete mechanisms was not well illustrated. related experiments would be performed in the future study. the present study successfully constructed the pneumonia murine model using iav. olaparib decreased iav-induced mortality in mice, lung injury, and cytokine production, possibly via modulation of parp- /nf-κb axis. this study indicates the non-specific anti-inflammatory effect of olaparib in lung disorders and would shine light on the development for treatment against pneumonia infected by viable iav subtypes. conflict of interest the authors declare that they have no competing interests. ethical approval all the animal-involved experiments were performed in accordance with the animal care and use committee of cangzhou central hospital. informed consent not applicable. the genomic and epidemiological dynamics of human influenza a virus global circulation patterns of seasonal influenza viruses vary with antigenic drift rationale and opportunities in estimating the economic burden of seasonal influenza across countries using a standardized who tool and manual variable influenza vaccine effectiveness by subtype: a systematic review and meta-analysis of test-negative design studies poly (adp-ribose) polymerases in double-strand break repair: focus on parp , parp and parp role of poly (adp-ribose) polymerase in cell-cycle checkpoint 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crosstalk induced by intratracheal lipopolysaccharide instillation in rats publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - nhade s authors: seleem, noura m.; abd el latif, hemat k.; shaldam, moataz a.; el-ganiny, amira title: drugs with new lease of life as quorum sensing inhibitors: for combating mdr acinetobacter baumannii infections date: - - journal: eur j clin microbiol infect dis doi: . /s - - -z sha: doc_id: cord_uid: nhade s the emergence of multidrug-resistant (mdr) strains is a major health problem worldwide. there is an urgent need for novel strategies to combat bacterial infections caused by mdr strains like pseudomonas aeruginosa and acinetobacter baumannii. quorum sensing (qs) is a critical communication system in bacterial community controlling survival and virulence. the awareness of the importance of qs in bacterial infections has stimulated research to identify qs inhibitors (qsis) to defeat microbes. in this study, four fda-approved drugs (besides azithromycin as positive qsi) were tested for potential qs inhibition against clinical a. baumannii isolates and p. aeruginosa (pao ) standard strain. the inhibitory effect of these drugs on virulence factors of both microbes has been investigated. the studied virulence factors include biofilm formation, twitching and swarming motilities, proteolytic enzyme production, and resistance to oxidative stress. the four tested drugs (erythromycin, levamisole, chloroquine, and propranolol) inhibited qs in chromobacterium violaceum by , , . , and . %, respectively. they also significantly inhibited virulence factors in both pao and a. baumannii at sub-inhibitory concentrations. these findings were confirmed by qrt-pcr and mice mortality test, where tested drugs highly repressed the expression of abai gene and showed significantly improved mice survival rates. in addition, molecular docking studies against abai and abar proteins of qs system in a. baumannii revealed the potential inhibition of qs by tested drugs. beside their known activities, the tested drugs could be given new life as qsis to combat a. baumannii nosocomial infections (alone or in combination with antimicrobials). electronic supplementary material: the online version of this article ( . /s - - -z) contains supplementary material, which is available to authorized users. acinetobacter baumannii and pseudomonas aeruginosa are two of the main superbug bacteria responsible for nosocomial infections in severely ill patients. they cause a wide spectrum of infections from skin and wound infections to septicemia and hospital outbreaks worldwide; both organisms are resistant to several classes of antibiotics making their treatment very difficult [ ] . the mdr pattern can be transferred to the other organisms that initially do not show such resistance [ ] . in addition, biofilm production in a. baumannii and p. aeruginosa encourages increased colonization and persistence in hospital environments leading to higher rates of device-related infections [ ] . to solve the problem of limited therapeutic options, it is very vital to find new therapeutic strategy to combat a. baumannii infections. it is well known now that quorum sensing (qs), a cell-tocell communication system, regulates the expression of several virulence and survival genes in many organisms at high cell densities using n-acyl-homoserine lactone (ahl) signaling molecules; thus, qs plays key role in the establishment of microbial infections [ ] . therefore, interfering with qs is considered as a promising strategy to combat bacterial infections by inhibiting bacterial virulence hence affecting the ability of pathogens to cause diseases rather than affecting their growth which does not impose a selection pressure and helps to avoid emergence of resistance [ ] . consequently, qsi would increase the susceptibility of the pathogen to host defense and clearance by the host immune system [ ] . twenty qs-positive clinical a. baumannii isolates were selected for this study. they were obtained from different intensive care units (icus) of zagazig university hospitals, and the international medical center, egypt. all strains have been identified and confirmed to be qs-positive mdr a. baumannii. the isolates were of clinical sources, from tracheal aspirate, from blood, and from wound (supplementary table ). they were maintained routinely on luria-bertani (lb) agar. two reference strains were used in this study: pseudomonas aeruginosa o (pao ) as a qs-positive strain and chromobacterium violaceum (cv ), a biosensor for qs which is violacein-negative strain, and its violacein purple pigment is induced by ahls (acyl-homoserine lactones). chloroquine (cq), erythromycin (e), levamisole (lev), and propranolol (ppl) were obtained from the national organization of drug control and research (nodcar) cairo, egypt. the antibiotic azithromycin (az) was used as qsi-positive control in all experiments. all drugs were dissolved in water except erythromycin and azithromycin which were dissolved in dimethylsulfoxide (dmso). overnight-grown c. violaceum cv cells ( ml) were added into ml of . % molten lb agar (oxoid, hampshire, england) that has been supplemented with nhexanoyl homoserine lactone (c -hsl, . μg/ml) purchased from sigma-aldrich chemie gmbh (steinheim, germany). cv agar suspension was poured into petri dishes and allowed to solidify; wells were made using sterile pipette tips. the tested drug solutions ( μl of mg/ml) were placed in each well and the solvent served as negative control. the plates were incubated at °c for h. halo formation on a purple background suggested that the tested drugs exhibited anti-qs activity [ ] . first, minimum inhibitory concentration (mic) of the potential qsis was determined by broth microdilution according to clsi methodology [ ] . briefly, four morphologically similar colonies from cv were touched with a sterile loop and transferred to müller hinton broth (mhb), oxoid (hampshire, england), and broth was incubated with shaking at - °c until the visible turbidity was equivalent to . mcfarland. the bacterial suspension was diluted : in mhb, so the final concentration of bacteria will be approximated × cfu/ml. twofold serial dilutions of each drug were prepared directly in -well microtiter plates (double strength of the required concentrations) in a final volume of μl per well. one well was left without drug to serve as a positive growth control. each well was inoculated with μl of inoculum and incubated at °c for to h. the microtiter plates were examined for growth; mic was considered as the lowest concentration of qsi at which there was no visible growth of the organism. the inhibition of violacein pigment production using sub-mic of tested drugs was quantified spectrophotometrically [ ] . briefly, ml of lb broth supplemented with c -hsl ( . μg/ml) either in the absence or presence of and ¼ mic of the drugs were inoculated with ml of overnight culture of cv (diluted to cfu/ml) and incubated at °c for h. one milliliter of culture from each sample was centrifuged at , rpm for min. then, culture supernatant was discarded, and the pellet was solubilized in ml of dmso, vortexed until the violacein was extracted, and centrifuged at , rpm for min. optical density (od) of each violacein-containing supernatant was measured at nm using micro-titer plate reader (biotek spectrofluorimeter, biotek, usa); results were expressed as percentage of violacein production inhibition with respect to negative controls. the control sample was cv inoculated in lb medium containing c -hsl without qsis. measurement of p. aeruginosa and a. baumannii virulence factors in presence of the mics of four qsis (erythromycin, chloroquine, levamisole, propranolol) against pao and clinical qs positive a. baumannii isolates were first determined by broth microdilution method in addition to azithromycin as positive qsi control. the virulence factors were detected in absence and presence of mic of potential qsis. assay of biofilm formation, twitching motility, sensitivity to oxidative stress and protease, and gelatinase activity were performed for both p. aeruginosa (pao ) and a. baumannii isolates. pyocyanin production, swarming motility, and rhamnolipid production were done only for p. aeruginosa, while surface-associated motility, esterase, and phospholipase c production were performed only for a. baumannii clinical isolates. the modified method of stepanovic et al. [ ] was used to test biofilm formation. overnight culture of pao and a. baumannii either with and without mic of qsis were prepared, diluted with mhb, and adjusted to a cell density of × cfu/ml. aliquots of μl of adjusted bacterial suspension were inoculated to the wells of sterile -well polystyrene micro-titer plates. after incubation for h at °c, the contents of the wells were gently aspirated, and the wells were washed three times with sterile phosphate buffered saline (pbs, ph . ). the adherent cells were fixed with μl of % methanol for min and then stained with μl crystal violet ( %) for min. the excess dye was then removed under running distilled water, and then the plates were left to air-dry. the bound dye was extracted by the addition of μl of % glacial acetic acid, and the optical densities were read at nm using micro-titer plate reader. the experiment was performed in triplicate, and the mean optical densities were calculated, wells using media only was used as negative control. the cut-off od (odc) was defined as three times standard deviations above the mean od of the negative control. according to the criteria of stepanovic et al. [ ] , the test isolates were categorized into four groups: non-biofilm forming (od ≤ odc), weak biofilm forming (od > odc, but ≤ × odc), moderate biofilm forming (od > × odc, but ≤ × odc), and strong biofilm forming (od > × odc). semisolid lb media (lb broth with % agar) either with or without qsis were used for the motility assay [ ] . plates were made by pouring ml medium into a -mm petri dish, and allowing the medium to air-dry with the lid off for min in a laminar flow hood, plates were prepared on the same day as the inoculation. a μl of freshly grown cultures, adjusted to . mcfarland, were stab-inoculated into semisolid agar to enable spread of bacteria on the interphase between the bottom of the petri dish and the lb medium. after inoculation, plates were sealed with parafilm and incubated at °c for h. to visualize the bacteria at the interface, agar was removed from each plate, and the plates were air-dried and stained with % cv. for each isolate, assays were performed at least three times. results interpreted according to the criteria of vijayakumar et al. [ ] . the average diameter of zone of twitching was determined; isolates are classified as non-motile (nm, < mm), intermediately motile (im, - mm), and highly motile (hm, > mm). first, sterile supernatant was prepared according to gupta et al. [ ] . briefly, . ml of . macfarland overnight cultures was inoculated in -ml lb broth with and without / mic of qsis and incubated at °c for h. the supernatants were separated by centrifugation at g for min and were filtered using . -μm syringe filter. this sterile supernatant was used in testing protease, gelatinase, and rhamnolipid production. protease activity was tested using skimmed milk agar media [ ] . the media consists of % skimmed milk in . % lb agar. skimmed milk agar plates were prepared, and μl of sterile supernatants were added to the wells made in the plates using a sterile pipette tip. the plates were incubated overnight at °c for h, and the clear zones surrounding the wells were measured which indicates positive proteolytic activity in presence and absence of qsis. the experiment was performed in triplicate. the method of su et al. [ ] was used to detect gelatinase production. briefly, . % lb agar supplemented with % gelatin with a final ph . was prepared, and isolate culture was spot inoculated on the surface of the plates and incubated for h at °c. the plates were observed for growth and subsequently flooded with ml of frazier's solution ( . g mercuric chloride, ml hydrochloric acid ( % v/v), and distilled water to ml) to precipitate the unhydrolyzed gelatin. the plates which showed area of opaque layer with zone of clearance (transparent halo) around the colonies were taken as positive for gelatin hydrolysis. the experiment was performed in triplicate. az was used as control qsi. the results were recorded as gz values by calculating the ratio between the colony diameter and the gelatin hydrolysis zone diameter, and the isolates were classified according to the method of sanchez et al. [ ] , where gz of . was evaluated as negative (−), . - . as low (+), . - . as moderate activity (++), and < . as high (++++) activity. sensitivity to oxidative stress was carried out by the modified disk assay [ ] . briefly, -μl aliquots from lb broth cultures in mid-log or stationary phases of growth were uniformly spread on the surface of mha plates containing % agar with and without sub-mic of qsis. sterile -mm diameter filter paper disks were placed on the surface, and the disks were spotted with μl of . % h o . the diameter of the zone of growth inhibition around each disk was measured after h of incubation at °c. the experiment was performed in triplicate. the increase in the diameter of the inhibition zone indicates the ability of potential qsis to inhibit resistance or tolerance to oxidative stress (i.e., augment susceptibility to oxidative stress). pyocyanin production by untreated and qsi-treated pao was assayed as described previously [ ] . pao overnight culture in lb broth was prepared and diluted to od of . . luria-bertani broth ( ml) with and without qsis was inoculated with μl of diluted culture suspension, and the cultures were incubated at °c for h. to remove the cells and obtain the supernatants, the tubes were centrifuged at , rpm for min, and the pyocyanin was quantified in the supernatants by measuring the absorbance at nm by biotek spectrofluorimeter (biotek, usa). the experiment was performed in triplicate. swarming motility was detected according to rashid and kornberg [ ] . lb medium used for assay and contained . % agar, tryptone g/l, nacl . g/l, and yeast extract g/l with or without potential qsis. swarm plates were typically allowed to dry at room temperature before being used. the swarm lb agar plates were spot inoculated by μl of . mcfarland solution of pao overnight culture, and incubated for h at °c. this assay was performed three times, and the average of the diameter of swarm colony was recorded in mm. the oil spreading technique was carried out as described by youssef et al. [ ] . briefly, -ml distilled water was added to -mm petri dish followed by addition of -μl crude oil to the surface of water. then, -μl pao supernatant with and without sub-mic of qsis was dropped onto the crude oil surface. the diameter of the clear zone on the oil surface was measured. the experiment was performed in triplicate, and the average was calculated. surface motility plates were prepared according to vijayakumar et al. [ ] with modification (tryptone g/l, nacl . g/l, yeast extract g/l, . % triphenyl tetrazolium chloride (ttc), . % agar). in order to reduce variation between plates, plates were prepared on the same day, by pouring ml of surface motility medium in each plate in a laminar flow hood with the lids off and allowing it to dry for min and then quickly used for motility assays. two microliters of a bacterial suspension normalized to . mcfarland standard were surface inoculated. after inoculation, the plates were sealed with parafilm and incubated for h at °c. surface-associated motility assays were conducted on at least three separate occasions. swarming positive isolates were defined as those strains that showed a zone of > mm around the site of inoculation. the esterase production assay was performed according to slifkin [ ] . two microliters of overnight cultures of a. baumannii isolates grown on lb broth and adjusted to . mcfarland turbidity were transferred to tween medium (containing . % tween ) with or without sub-mic of qsis. the inoculated agar plates were incubated at °c for days. the tests were performed in triplicate. the presence of an opaque halo around an inoculated site on the medium, viewed with transmitted light, indicated a positive result. esterase activity (ez) was calculated as the ratio of the diameter of the colony to the diameter of the colony plus that of the opaque zone, which correlates with hydrolysis of tween . the isolates were classified according to the method of sanchez et al. [ ] , where ez of . was evaluated as negative (−), . - . as low (+), . to . as moderate activity (++), and < . as high (++++) activity. phospholipase c (lecithinase) activity was done by inoculating μl of overnight cultures (on lb) equivalent to . mcfarland turbidity of each isolate into egg yolk agar ( . % lb agar containing . % egg yolk and . % calcium chloride) with or without sub-mic of qsis [ ] . inoculated plates were incubated at °c for h. the experiment was performed in triplicate. phospholipase c positive colonies were clearly marked by dense white zone of precipitation extending from the edge of the colony. colony diameter and colony diameter plus precipitation zone were measured for each isolate, and the zone of phospholipase activity (pz value) was calculated. pz value was calculated as the ratio of the diameter of the colony to the diameter of the colony plus that of the precipitation zone. the isolates were classified according to the method of sanchez et al. [ ] , where a pz of . was evaluated as negative (−), . - . as low (+), . to . as moderate activity (++), and < . as high (++++) activity. real-time pcr was performed in absence of the potential qsis on the qs positive isolates while qrt-pcr was performed in presence of / mics of potential qsis for a. baumannii isolate number (ab ). az was used as control. total rna was extracted using genejet rna extraction kit (thermo fisher scientific inc., germany) according to the manufacturer's instructions. briefly, -ml lb broth of a. baumannii isolates was inoculated with . -ml culture equivalent to . macfarland and incubated at °for h. cdna was generated by reverse transcription of μg of total rna using oligo dt primers and . μl reverse transcriptase in a total reaction volume of μl. primers for abai (abaif ′ ccgccttcctctagcagtca ′ abair ′aaaacccg cagcacgtaataa ′) and srrna (forward ′ actcctacgggaggcagcagt ′ and reverse ′ tattaccg cggctgctggc ′) were adapted from selasi et al. [ ] and clifford et al. [ ] , respectively. the rt-pcr master mix was prepared in -μl final volume comprised of the following: μl of × sensifast™ sybr (bioline, london, uk), . μl of each primer ( μ m), . μ l reverse tra nscriptase, . μ l ribosafernase inhibitor, water up to μl, and μl of rna template. the reaction mix was applied in rt-pcr (stepone applied biosystem, foster city, usa). three-step cycling was used as follows: cycle reverse transcription at °c for min, then pcr consists of initial activation cycle of °c at min followed by cycles of °c for s, °c for s, and °c for min. fold changes in gene expression were calculated using the comparative ct method ( −ΔΔct ), and samples were normalized to s rrna expression [ ] . a murine model of peritoneal sepsis by a. baumannii was established by intra-peritoneal inoculation of bacteria in mice [ ] . an overnight culture of a. baumannii ab was inoculated in lb medium with and without the qsis (at / -mic concentration) at °c for h. ten milliliters of the culture (adjusted to . mcfarland) was harvested by centrifugation at , g at °c for min. the pellet was resuspended in . ml pbs by vortexing. a μl of the suspended cells, equivalent to × cells, was injected into the abdominal cavity of four to -week-old-specific pathogen-free swiss albino ( - g weight) female mice. mortality was observed during days period for mice for each treatment. dmso, dmf, and pbs were injected into mice as negative control groups (n = /group). experimental design and animal handling procedures were performed according to the guidelines for animal use of the ethical committee of the faculty of pharmacy, zagazig university. the structure of autoinducer synthase abai protein model (code: b fln ) was retrieved from swiss-model (b fln (abai_aciba) acinetobacter baumannii, acylhomoserine-lactone synthase. https://swissmodel.expasy.org/ repository/uniprot/b fln ). the active site was identified through protein structure alignment tool in the molegro virtual docker (mvd version . ) with the template structure (pdb code: p h) [ ] , and the ligand s-adenosyl methionine (sam) was transferred to the model active site. this study was carried out on erythromycin, propranolol, chloroquine, and levamisole along with sam. the studied compounds were drawn into marvin sketch (v . . ). the most energetically favored conformer was saved as (*.mol ) file format for docking. the optimal geometry of the ligand was determined during the docking process. moldock optimizer algorithm in molegro virtual docker software (mvd) was chosen to perform docking process with runs per ligand, population size, max iteration, and poses for each ligand. moldock docking engine using the optimized ligands was executed [ ] . finally, the top-returned poses were chosen for analysis. the structure of abar receptor model (code: a a c tfv ) was retrieved from swiss-model (a a c tfv (a a c tfv _aciba) acinetobacter baumannii, https:// swissmodel.expasy.org/repository/uniprot/a a c tfv ). the docking study was carried out on erythromycin, propranolol, chloroquine, and levamisole along with natural ligand n-( -hydroxydodecanoyl)-l-homoserine lactone oh-dhl) with the same docking procedure as previously mentioned. in presence of c -hsl (cognate signal), cv produces violacein purple pigment, which inhibited in presence of qsi that can affect the signal production or signal reception. among the tested drugs, erythromycin, levamisole, propranolol, and chloroquine showed the highest inhibition of quorum sensing in cv as they produced a large clear zone around each well on a purple background (fig. ) ; hence, these four drugs were tested in the further experiments. azithromycin was used as a positive qsi. it was noticed that drugs had antibacterial activity that appeared as growth inhibition zone beside the anti-qs activity. erythromycin and azithromycin also produce darker ring of purple color after the anti-qs zone. the mics of the four qsis and azithromycin against cv were determined by broth microdilution method. the mics were . , , , , and , μg/ml for azithromycin, erythromycin, propranolol, levamisole, and chloroquine, respectively. the inhibition of violacein pigment production in cv in presence of ( / , / mic of qsis) was quantified spectrophotometrically. od values at nm were used to calculate the percentage of inhibition of pigment production ( table ). the results revealed that all tested drugs had high percentage of qsi; the most effective drug was erythromycin ( % at / mic, . % at / mic) followed by azithromycin, levamisole, chloroquine, and propranolol. all tested drugs inhibited pao growth at different values, . μg/ml for azithromycin, μg/ml for propranolol, μg/ml for erythromycin, μg/ml for levamisole, and . mg/ml for chloroquine. to evaluate the anti-qs and antivirulence activity of tested drugs, / mic of each drug was used. the obtained results revealed that pao biofilm (strong) was markedly inhibited in presence of / mic of tested drugs (> %); the highest inhibition was observed with erythromycin ( . %) as shown in table and fig. a . the effect of the tested drugs on twitching and swarming of pao was determined. swarming motility was more inhibited than twitching; the highest inhibition of twitching and swarming motility was reported in presence of erythromycin ( . % and . %, respectively), and the lowest inhibition in both motilities was observed with propranolol ( table and fig. b, c) . pao was found to be strong gelatinase producer as gz value was . . gelatinase production was highly inhibited in presence of erythromycin and chloroquine ( . % and . %, respectively), while protease activity was moderately inhibited in presence of qsis. the lowest inhibition in protease activity was recorded in presence of levamisole and propranolol (table and fig. d, e) . the effect of h o was highly augmented in presence of erythromycin as tolerance to oxidative stress was reduced by . % (table and fig. f) ; lower augmentation was observed with azithromycin and levamisole ( . % for both) and propranolol and chloroquine ( . % for both). the pyocyanin production was significantly inhibited in presence of erythromycin, azithromycin, levamisole, and chloroquine with percentages of . , . , . , and . %, respectively. lower inhibition ( %) was observed with propranolol ( table and fig. g) . the rhamnolipid production was highly inhibited in presence of all qsis (table and fig. h ) as spreading of oil on water surface was decreased. azithromycin and erythromycin showed very significant inhibition ( . %) followed by chloroquine ( %) and levamisole ( . %), while propranolol showed the lowest inhibition ( . %). the mics of qsis against qs-positive mdr a. baumannii isolates were determined by broth micro-dilution method (supplementary table ). mic was calculated; azithromycin had the lowest mic ( μg/ml) while chloroquine had the highest ( μg/ml). to evaluate the anti-qs and anti-virulence activity of tested drugs, / mic of each drug was used (see supplementary tables s -s for detailed results of virulence inhibition and supplementary fig. s for representative examples of virulence inhibition in a. baumannii). quantification of biofilm in presence of / mic of qsis was performed. more than % inhibition of biofilm formation appeared in of a. baumannii isolates with all tested drugs (table and fig. ). chloroquine showed the highest range of percentage inhibition of biofilm ( . - . %). twitching motility was tested in a. baumannii isolates, % of isolates were intermediately motile, and % were highly motile. all qsis inhibited the twitching motility in all isolates with varying degrees more than % inhibition in twitching motility noticed in isolates. the highest inhibition was recorded with levamisole. in addition, ≥ % inhibition was detected in isolates in presence of erythromycin and one isolate in presence of propranolol and levamisole (table and fig. ). the results revealed that % of the a. baumannii isolates were positive for surface-associated motility. all qsis inhibited surface motility; the inhibition was ranged from . to % in presence of azithromycin, erythromycin, and (table and fig. ). all a. baumannii isolates strongly produced esterase enzyme, except one isolate. the highest inhibition range was recorded in presence of erythromycin; esterase was inhibited in a range of . - . %, with isolates being inhibited by ≥ % (table and fig. ) . the effect of hydrogen peroxide was highly augmented in presence of sub-mic of erythromycin and levamisole. the augmentation ranged from to % in case erythromycin and from . to % in case of levamisole (table and fig. ) . only isolates ( %) were phospholipase producers, two isolates were strong producers (pz = . ), and the other isolates were low producers (pz = . - . ). the highest (table and fig. ). protease and gelatinase activities were not detected in any of the tested isolates. relative expression of qs regulatory gene abai to the reference gene s rrna was tested. all tested isolates were positive for the expression of the abai gene. in addition, qrt-pcr was performed on isolate no. (ab ) either treated with the four qsis or untreated culture as negative control. az was used as positive control. relative expression levels of abai gene was calculated using the ^− ΔΔct method according to livak and schmittgen ( ) . at sub-inhibitory concentrations, erythromycin, azithromycin, levamisole, chloroquine, and propranolol highly repressed the expression of abai gene by , . , . , , and %, respectively (fig. ) . the fold decrease in the expression of abai gene was . , . , . , , and times in presence of erythromycin, azithromycin, levamisole, chloroquine, and propranolol, respectively. in mice mortality test, the mice injected with ab alone started to die after h of injection, and all of them were dead after h. the control mice groups which were injected with solvents and saline were % alive throughout the experiment period. mice groups injected with ab treated with sub-mic of potential qsis showed significant improvement in survival rates; % survival was recorded in the group injected with culture grown with erythromycin (fig. ) . fig. a -e. the moldock score for sam was − . , and the interacting residues were shown in supplementary table s . from the docking scores, the erythromycin showed higher binding affinity than sam and the other studied compounds. the molecular docking study ranked the activity of compounds (moldock score) as inhibitors for abar receptor as supplementary table s . from the docking scores, chloroquine showed the highest binding affinity among the studied compounds. it is well known that qs controls virulence and pathogenicity of microbes; hence, it is a good target to design novel antimicrobial therapeutics by developing qsis that target virulence through either inhibiting qs signal production, signal reception, or degrading ahls [ ] . in the present study, it was found that the tested drugs have inhibited qs in cv biosensor as indicated by inhibition of violacein pigment production. cv responds to c -hsl and produces violacein, but in presence of qsis, no pigment is produced. it is noticed that azithromycin and erythromycin formed an intense violet ring around the inhibition zone. this may be attributed to a potentiating effect for violacein production with a certain concentration of these inhibitors. this finding was in accordance with that of liu et al. [ ] . the highest inhibition of violacein pigment was recorded for erythromycin and azithromycin followed by levamisole, chloroquine, and propranolol; erythromycin has the highest qs inhibitory activity. this was consistent with several reports which reported that antibiotics interfere with qs by blocking c -hsl production in p. aeruginosa [ , ] . pseudomonas aeruginosa has three major qs systems lasi/r, rhli/r, and pseudomonas quinolone system (pqs) [ ] which work together to control cell survival, biofilm formation, and virulence [ , ] . it is very critical to inhibit biofilm as it is responsible for high antibiotic resistance rates [ ] , and it participates in serious infections [ ] . the tested drugs significantly reduced the ability of pao to form biofilm as they had very strong anti-biofilm activities, with erythromycin being the most powerful qs inhibitor. many reports stated the high inhibition of biofilm in pao by other qsis [ ] [ ] [ ] . in the current study, sub-mic of tested drugs highly inhibited both types of motility in pao . the swarming motility was significantly inhibited in a range of . - . %; erythromycin had the strongest inhibitory activity. consistent with our results, other studies reported high [ , ] . twitching motility was inhibited in a range of . - . %, and the highest inhibition was also in presence of erythromycin, which was comparable with previous work [ ] . it was reported that swarming motility is affected by qs, rhamnolipid production, type iv pili, and flagella [ ] . this may explain the higher inhibition in swarming motility than twitching that was observed in the current study. in the present study, the effect of qsis on hydrolytic enzymes like protease and gelatinase was investigated. these enzymes are very crucial for degrading host tissue components which aid in bacterial colonization [ ] . the protease production by pao was inhibited by . % in presence of tested drugs. similarly, other qsis reduced pao proteolytic activity [ , ] . moreover, gelatinase production by pao was much more inhibited than protease enzyme by a range of - . %. in accordance with our results, macrolides inhibited gelatinase in p. aeruginosa [ ] . pyocyanin is responsible for tissue damage due to hydroxyl radical formation and also it suppresses the acute inflammatory response [ , ] . the tested drugs inhibited pyocyanin production in pao by a range of - . %. erythromycin and levamisole were the most effective inhibitors. consistent to our findings, several reports showed reduction in pyocyanin production by other drugs [ , ] . rhamnolipid (biosurfactant) is a key virulence determinant regulated by qs. it has antimicrobial activity against different bacteria and fungi [ , ] . in the present study, rhamnolipid production was markedly inhibited in a range of . - . %. similar to our findings, baicalin demonstrated significant suppression in pao rhamnolipid production [ ] . pao resistance to oxidative stress is under the control of qs [ ] . in the current study, qsis showed moderate increase in the sensitivity of pao to h o ; the highest reduction in tolerance to oxidative stress was obtained with erythromycin ( . %). similar to our findings, metformin and glyceryl trinitrate caused augmentation in the effect of h o [ , ] . it is well known that azithromycin and erythromycin (macrolide antibiotics) act mainly on gram-positive bacteria. so, their inhibitory effect in p. aeruginosa and a. baumannii is not due to their antimicrobial activity, but due to their inhibitory activity on qs system [ ] . in this study, several virulence factors in a. baumannii clinical isolates were investigated. biofilm and twitching motility were detected in all isolates ( %), followed by esterase (n = ) then surfaceassociated motility (n = ). the least detected virulence was phospholipase (n = ), while none of the isolates produced proteolytic enzymes. five isolates produced all these factors including isolate (no. ) which has the highest virulence expression; hence, it was used in animal study and rt-pcr experiments. biofilm formation is considered one of the main virulence factors in a. baumannii [ ] . in the present study, qsis markedly reduced biofilm formation in a. baumannii clinical isolates as % inhibition was observed in % of a. baumannii isolates. chloroquine showed the highest range of inhibition ( . - . % ). this finding was consistent with many reports that linked qs to biofilm formation in a. baumannii [ , ] . also, chloroquine sensitizes biofilm of candida to azoles [ ] . twitching motility allows a. baumannii to spread rapidly on semisolid surfaces; twitching is mediated by type iv pili (tfp) system [ , ] . all of our isolates showed twitching motility, while vijaykumar et al. [ ] found that . % of isolates had twitching motility. twitching motility is regulated by several environmental factors [ , , ] . the tested qsis inhibited twitching motility in all a. baumannii isolates with varying degrees. the highest inhibition was recorded in presence of levamisole ( . - . %). the difference in results between this work and other studies may be related to different factors including source and nature of isolates, and experimental conditions. many strains of a. baumannii have been found to exhibit unique surface-associated motility on semisolid surface that resembles swarming motility of p. aeruginosa; however, swarming motility is flagellum-coordinated movement [ ] . a. baumannii surface-associated motility is a flagellum-independent, complex process that is dependent on many factors including qs [ , , ] . it has been speculated that tfp might be involved in the surface-associated motility of a. baumannii [ ] . in the present study, % of a. baumannii isolates were positive in surface-associated motility. in agreement with our results, eraç et al. [ ] reported that % of a. baumannii isolates were motile. at the contrary, vijaykumar et al. [ ] reported that only % of isolates swarm, while skiebe et al. [ ] reported nearly all isolates had swarming-like motility on . % agarose-containing media; however, we used . % agar. any variation in the physical factors could have effect on motility; we also found that using low salt concentration in the media gives better motility. all the tested qsis inhibited surface motility; ≥ % inhibition was observed in presence of azithromycin, erythromycin, and levamisole. in addition, ≥ % inhibition was recorded in isolates in presence of erythromycin. at the contrary, % of a. baumannii isolates underwent a decrease in surface-associated motility in presence of virstatin [ ] . in the present study, % of a. baumannii isolates were strong esterase producers. valli and gopinath [ ] found that % of isolates were esterase positive which agreed with our results to some extent. in the current study, the highest inhibition range of esterase production was recorded in presence of erythromycin ( . - . %) with the highest number of isolates being inhibited by ≥ %, followed by chloroquine and levamisole. pathogens are exposed to killing by host via various reactive oxygen species (ros). pathogen is able to resist ros and persist under oxidative stress by production of superoxide dismutase (sod) and catalase enzymes. sod was found to play vital role in resistance to oxidative stress in a. baumannii [ ] . autoinducer synthase mutant was found to produce small amount of antioxidant enzymes indicating the importance of qs in response to oxidative stress [ ] . in the current study, the effect of h o was highly augmented (tolerance to oxidative stress was reduced) in presence of sub-mic of erythromycin ( - %) and levamisole ( . - %). a recent study showed that curcumin enhances the therapeutic efficacy of antibiotics by increasing ros and compromising oxidative stress defense [ ] . in the current study, results revealed that only % of a. baumannii were phospholipase producers. in agreement with our results, two previous studies found similar percentage of plc production [ , ] . regarding the inhibition of plc, our results revealed that at least one isolate had % inhibition in phospholipase production in presence of some tested drugs. the highest number of isolates inhibited by ≥ % was recorded with levamisole, propranolol, and azithromycin. in our study, both protease and gelatinase activities were not detected in a. baumannii, in accordance with abdulla et al. [ ] . however, other studies reported proteolytic activity in a. baumannii [ , ] . the qrt-pcr was used to study the relative expression of qs regulatory gene abai in a. baumannii isolates, and to calculate the relative expression of abai gene before and after treatment with sub-mic of qsis. it was found that erythromycin, azithromycin, levamisole, chloroquine, and propranolol highly repressed expression of abai by , . , . , , and %, respectively. these results confirm the qs inhibitory activity of the drugs at molecular level. this finding provides a basis to explain the remarkable decrease in the production of qs-regulated virulence factors. similarly, significant downregulation of qs regulator systems of pao treated with sub-inhibitory concentration of qsis was reported [ , ] . it is worth mentioning that the concentrations of the drugs used as qsis do not always meet the pharmacological limits for human use. levamisole and propranolol therapeutic plasma levels . - . μg/ml and . μg/ ml, respectively [ , ] , are much lower than their concentrations as qsis in this study. on the contrary, erythromycin and azithromycin therapeutic plasma levels are . μg/ml and . μg/ml, respectively [ ] , and are higher than the concentrations used as qsis in this study. the in vitro results must not be considered as final applicable conclusions in clinical situations. moreover, the behavior of bacteria in lab differs from that in human body, and characteristics of drugs in lab also differ from those in human body where the drug may undergo degradation, conformational changes, re-arrangement, and plasma protein binding. therefore, all in vitro studies are considered as a beginning step and must be fulfilled with in vivo studies. to compensate this shortage in reaching the drug therapeutic plasma level with some drugs, it could be possible to use qsis with high mic values in topical formulations like dressings or ointments for skin and soft-tissue infections and aerosols for respiratory infections. also, qsis with high mic can be tested in future study in combination with antibiotics using low concentration of these qsis that not exceed their accepted therapeutic levels. to evaluate the effect of the qsis in vivo, the mice mortality test was performed. first, mice were injected with μl of a. baumannii (ab ) grown without qsis, which cause % mortality within h. several studies have reported other numbers of bacteria injected in the mice, some agree with our number, and others differ [ , , ] . this was strain-dependent, and many reports account this to the probable negative correlation between mdr phenotype and virulence [ , ] . the obtained results revealed that mice which were injected with ab grown with sub-mic of qsis showed significant improvement in survival rates, which reached % in case of erythromycin followed by % in presence of propranolol. a more confirmation of qs inhibitory mechanism of the tested drugs was done using the molecular docking study. it was revealed that erythromycin showed higher binding affinity than sam (the natural ligand of abai) while levamisole had the lowest binding affinity. moreover, all drugs bound with the active site of the ligand-binding domain of abai by hydrophobic interactions and h-bonding. in addition, molecular docking study ranked the activity of the compounds as inhibitors for abar receptor with chloroquine being the most active, followed by propranolol with a score similar to the natural ligand. the binding interactions with the receptor were by hydrophobic interactions and h-bonding except levamisole, which had no h-bonding. erythromycin exhibited the lowest inhibition for abar receptor. therefore, the potential mechanism of qs inhibition by the tested drugs may be due to either binding to the abai autoinducer synthase preventing ahl synthesis, or binding to abar receptor preventing ahl signal reception. finally, erythromycin m a r k e d l y i n h i b i t e d q s i n v i v o a n d i n v i t r o i n a. baumannii isolates. this may be due to inhibiting the synthesis of ahl signals by erythromycin more than inhibiting signal reception. in addition, chloroquine inhibited qs in a. baumannii by inhibiting ahl signal reception more than ahl signal synthesis. several studies performed the docking of qsis against either lasr or rhlr of pao , and a significant binding potential to the active site of these proteins was revealed confirming the qs inhibitory activity [ , , ] . the study demonstrated that qs could control the virulence, as most of virulence factors of both pao and a. baumannii clinical isolates were highly inhibited by qsis. the most effective drug as qsis was erythromycin. to the best of our knowledge, this study showed that antibiotics like erythromycin can inhibit qs beside their antimicrobial activity; the other tested drugs (i.e., chloroquine, levamisole, and propranolol) were studied for the first time as inhibitors for qs in a. baumannii. results obtained 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effect on virulence and bacterial fitness interplay between antibiotic resistance and virulence during disease promoted by multidrug-resistant bacteria publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements we would like to thank islam mohsen and fatma yehia (assistant lectures in microbiology & immunology department, zagazig university) for providing acinetobacter baumannii isolates, and the department of microbiology and immunology, ain shams university, for providing the cvo strain. authors' contributions nms, hka, and ae conceived and designed the experiments. nms performed the experiments except the molecular docking studies which were done by mas. ae and nms analyzed and interpreted the data. nms wrote the manuscript draft. hka and ae revised and edited the manuscript. all the authors approved the final manuscript. ethics approval experimental design and animal handling procedures were performed according to the guidelines of the ethical committee of for animal use in the faculty of pharmacy, zagazig university.competing interests the authors declare that they have no competing interests. key: cord- -z frgm s authors: gidari, anna; nofri, marco; saccarelli, luca; bastianelli, sabrina; sabbatini, samuele; bozza, silvia; camilloni, barbara; fusco-moffa, igino; monari, claudia; de robertis, edoardo; mencacci, antonella; francisci, daniela title: is recurrence possible in coronavirus disease (covid- )? case series and systematic review of literature date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: z frgm s can a patient diagnosed with severe acute respiratory syndrome coronavirus- (sars-cov- ) be infected again? this question is still unsolved. we tried to analyze local and literature cases with a positive respiratory swab after recovery. we collected data from symptomatic patients diagnosed with sars-cov- infection in the italian umbria region that, after recovery, were again positive for sars-cov- in respiratory tract specimens. samples were also assessed for infectivity in vitro. a systematic review of similar cases reported in the literature was performed. the study population was composed of patients during a -month study period. among the new positive samples, six were inoculated in vero-e cells and showed no growth and negative molecular test in culture supernatants. all patients were positive for igg against sars-cov- nucleoprotein and/or s protein. conducting a review of the literature, similar cases have been found. the presumptive reactivation occurred in . days on average (standard deviation, sd, . days) after covid- onset, when the . % of patients presented fever and the . % symptoms. the outcome was favorable in . % of patients, while the . % of them were still hospitalized at the time of data collection and the . % died. several hypotheses have been formulated to explain new positive respiratory samples after confirmed negativity. according to this study, the phenomenon seems to be due to the prolonged detection of sars-cov- rna traces in respiratory samples of recovered patients. the failure of the virus to replicate in vitro suggests its inability to replicate in vivo. severe acute respiratory syndrome coronavirus- (sars-cov- ) is the etiologic agent of coronavirus disease (covid- ) that has been declared a global pandemic by the world health organization (who) in march . sars-cov- was discovered in december , in wuhan city (the capital of hubei province), china [ ] . the origin of the virus remains unknown. however, newly diagnosed cases were initially linked to the huanan seafood wholesale market where people can buy animals that are butchered in loco. the virus was identified as a novel enveloped rna beta-coronavirus that has been named sars-cov- [ ] . as of september , the total worldwide confirmed cases are , , [ ] . albeit our knowledge of this virus has improved, it is still a challenging matter whether patients with sars-cov- anna gidari and marco nofri contributed equally to this work. infection will reactivate the illness, and which risk factors predict eventual recurrence. unraveling this topic is important to better understand the immune response to the virus and contain disease transmission. after a prolonged scientific debate, the characteristics of a patient that can be discharged from the hospital have been standardized, in particular, improvement of symptoms (normal temperature lasting longer than days and no more respiratory symptoms) and two negative swabs collected at least h apart [ ] . however, from june , who simplified criteria for discharge: for symptomatic patients, days after symptom onset, plus at least additional days, without symptoms (including no fever and respiratory symptoms) is enough to discharge patients from isolation [ ] . however, several authors observed that in some cases patients presented a new positive respiratory sample after discharge. it is not clear yet what this means in terms of clinical course and contagiousness. here, we report the clinical and virologic findings of nine patients with a positive swab that had the criteria for discharge, in the italian umbria region, from march to september. we also systematically revised all the similar cases reported in the literature so far, evaluating the possibility that covid- may recur after recovery. criteria for patients' selection were diagnosis of sars-cov- infection [ ] ; the subsequent meeting of criteria for hospital discharge (improvement of symptoms and two negative swabs collected at least h apart) [ ] ; and a positive respiratory sample collected after discharge. all subjects provided informed oral consent to clinical data collection. the study was approved by the ethics committee of the umbria region (protocol number / /ov). respiratory samples (nasopharyngeal swabs, sputum, tracheal aspirate, or bronchoalveolar lavage, bal, fluid) were tested for sars-cov- rna by a commercial reverse transcriptase real-time pcr assay (rt-pcr assay, allplex™ -ncov assay, seegene, seoul) and/or with the xpert® xpress sars-cov- (cepheid, sunnyvale, ca, usa). in particular, allplex™ -ncov assay was performed according to the manufacturer's instructions, using μl of respiratory samples and μl of the provided internal control (ic). the envelope (e) gene (specific of the subgenus sarbecovirus), the nucleocapsid gene (n), and the rna-dependent-rnapolymerase (rdrp) genes (both specifics of the sars-cov- ) were the targeted genes of the rt-pcr. the assay was considered valid if the cycle threshold (ct) value of the ic was ≤ . samples with or viral targets (ct ≤ ) were considered positive. samples with only target (whatever ct) or with or targeted genes > ct were considered indeterminate, requiring a new specimen for retesting. samples were considered negative in the absence of any targeted gene. the fully automated xpert xpress® sars-cov- assay was performed on the genexpert® platform (cepheid) according to the manufacturer's instructions, loading μl of the sample into a single-use disposable cartridge. targets were e and n genes. after results were automatically passed, they were interpreted as positive if both e and n targets were detected. if a gene alone was detected, the sample was considered indeterminate, and a new sample was retested. the virus isolation was conducted in a biosafety level- facility. samples tested positive for the sars-cov- in real-time rt-pcr analyses were mixed with a : nystatin ( , u/ml) and penicillin-streptomycin ( , u/ml) mixture in a : ratio, and left to react at °c for h. the samples were then centrifuged at ×g for min, and the supernatant was used as the inoculant. for the cell inoculation, vero e (atcc® - ) were maintained in eagle's minimum essential medium (mem) supplemented with % fetal bovine serum, and % penicillin-streptomycin at °c in the presence of % co . on the day prior to inoculation, the cells were seeded at . × cells/cm into a t flask. the inoculated cells were cultured for days as above described. to evaluate the viral replication process, the cytopathic effect was observed at different exposure times, and rna from the cell culture supernatant was extracted and assessed for the presence of sars-cov- using real-time rt-pcr [ , ] . the titer of igg against the sars-cov- s /s subunit of spike protein was measured with clia liaison® sars-cov- s / s igg (diasorin). following the preferred reporting items for systematic reviews and meta-analyses (prisma) statement protocol [ ] , a systematic review has been performed concerning the patients with a diagnosis of covid- that, after clinical and virological recovery, presented a new positive respiratory sample (swab, sputum, saliva, tracheal aspirate, or bal). a systematic search on pubmed and google scholar was performed from january to september , . we used the following searching strategy: every search included the terms "sars-cov- " or "covid- " and "reactivation" or "recurrence" or "relapse." furthermore, we performed the following research: "(covid- or sars-cov- ) and positive and (recovered or discharged)." each article was analyzed to establish eligibility criteria and pertinency to our research. the inclusion criteria were as follows: english, italian, or spanish languages; pertinence on the question; articles that reported sufficient patients' information such as age, sex, and clinical and radiological presentation; treatment; and outcome. the exclusion criteria were as follows: different languages from those mentioned above and abstract and posters from conference proceedings since they did not go through peer review. data were summarized using descriptive analysis where possible. we included our presented cases in the analysis. statistical analysis was performed with prism graphpad software. the figure was created using microsoft powerpoint . from march to september , , patients meeting the inclusion criteria were included in the study. patient characteristics and laboratory findings are summarized in table . a -year-old man with a previous diagnosis of hbv infection developed fever on february , , and cough on february , . he was hospitalized on march , after being diagnosed with sars-cov- infection with a nasopharyngeal swab (allplex™ -ncov assay). on admission, he presented a severe respiratory failure. although antiviral and antimicrobial therapies were started, blood gas exchanges rapidly worsened, and he was intubated and transferred to the intensive care unit (icu), where he remained for days. the clinical course in icu was complicated with two episodes of ventilator-associated pneumonia (vap) and a central venous catheter (cvc)-related bloodstream infection due to a carbapenem-resistant klebsiella pneumoniae. two consecutive endotracheal samples resulted negative for sars-cov- on march and march . he underwent further endotracheal samples (march , april , and april ), two of which came indeterminate and the other two negatives (allplex™ -ncov assay). between april and april , qualitative serological tests (screen® test covid- , screenitalia) resulted positive for both igg and igm. a nasopharyngeal swab on april tested positive. another sample collected on april resulted negative while another one collected on april was positive (nasopharyngeal swab; xpert® xpress sars-cov- , cepheid). on april and , two additional nasopharyngeal swabs tested negative again (fig. a) . his sars-cov- serology, collected on may , was compatible with a previous infection (igg au/ml, liason® sars-cov- s /s igg, diasorin). as the clinical table severity of symptoms, laboratory exams, serology for severe acute respiratory syndrome coronavirus (sars-cov- ), and sars-cov- culture at the first admission (where available) and at the presumptive recurrence of coronavirus disease (covid- ) symptoms abbreviations: wbc white blood cells, crp c-reactive protein, pct procalcitonin, ast aspartate-aminotransferase, alt alanine aminotransferase, cpk creatine-phosphokinase, ldh lactate dehydrogenase, na not available, +++ severe symptoms, + mild symptoms, − absence of symptoms conditions were good, and the criteria for discharge were met, the patient was transferred to a covid-dedicated rehabilitative ward. the swab sample collected on april (resulted positive) was stored, and culture-based virus isolation in vero e cells was performed as previously described to evaluate the potential infectivity of the clinical specimen [ ] . no virus was isolated, and viral rna on supernatant was not detected (xpert® xpress sars-cov- , cepheid). a -year-old man with a history of hypertension, tobacco use, and dyslipidemia developed fever until . °c with no other apparent symptoms on march , . he underwent a nasopharyngeal swab which came positive for sars-cov- with rt-pcr (allplex™ -ncov assay). he was not hospitalized, as he did not complain of dyspnea and gradually became afebrile. he performed a follow-up nasopharyngeal swab, which came negative on april (allplex™ -ncov assay). afterwards, he went to the hospital for persistent retrosternal sense of weight with efforts. he was hospitalized once he underwent a screening swab test (xpert® xpress sars-cov- , cepheid) in the emergency room that tested positive on april . on april , his serology was compatible with a previous sars-cov- infection (igg . au/ ml). he denied contact during quarantine with confirmed or possible covid- cases. in the following days, the pain disappeared, and his clinical features resulted normal. the patient was discharged in good clinical conditions with indication to repeat quarantine and swab tests that came negative for sars-cov- (allplex™ -ncov assay) on april and (fig. b) . a -year-old woman with a history of iatrogenic hypothyroidism developed fever until °c with arthro-myalgia, cough, headache, and conjunctival hyperemia from march , . she also complained of dyspnea and desaturation from march . she was not in contact with a known case of sars-cov- . she underwent a nasopharyngeal swab, which tested positive (allplex™ -ncov assay). she was not hospitalized. fever and other symptoms disappeared after weeks, while arthro-myalgia and headache persisted. she performed two follow-up nasopharyngeal swabs on april and , which tested negative (allplex™ -ncov assay) and a serologic test that was positive for igg (screen® test covid- , screenitalia). while on quarantine, she referred a sense of chest weight and underwent computed tomography (ct) of the thorax that showed bilateral ground-glass opacities and interlobular septal thickening. on april , she was hospitalized after a positive nasopharyngeal swab test; rna gene "n" of sars-cov- was detected with a ct value of (xpert® xpress sars-cov- , cepheid). at the same time, her sars-cov- serology was positive for igg au/ml. during the hospitalization, pain progressively disappeared, and her clinical features resulted normal. no complications occurred. the patient was discharged in good clinical condition after two subsequent negative nasopharyngeal swabs (may and , respectively; allplex™ -ncov assay) (fig. c) . a -year-old man with no history of previous diseases developed fever until °c with anosmia, dysgeusia, and lack of appetite on march , . he denied dyspnea. his probable covid- contact was his wife, so he underwent a nasopharyngeal swab, which came positive on march (allplex™ -ncov assay). considering his mild symptoms, he was not hospitalized. he performed two follow-up pharyngeal swabs on april and , which came negative (allplex™ -ncov assay). he also underwent a sars-cov- qualitative serologic test that was positive for igg (screen® test covid- , screenitalia). during the following quarantine, he was always asymptomatic, but his ct of the chest showed bilateral interlobular septal thickening without interstitial pneumonia signs. basing on this radiological sign, he underwent a nasopharyngeal swab test for sars-cov- that resulted positive on may with detection of rna genes "e" and "n" at ct values of and , respectively (xpert® xpress sars-cov- , cepheid). the patient presented two subsequent negative nasopharyngeal swabs on may and , both performed with rt-pcr assay and xpert® xpress sars-cov- (cepheid) (fig. d) . at the same time, his sars-cov- serology was positive for igg ( . au/ml). a -year-old woman with no history of previous diseases developed cough, mild dyspnea, sore throat, anosmia, and dysgeusia on march , . she denied fever. she underwent a nasopharyngeal swab that tested positive on march (allplex™ -ncov assay). considering her mild symptoms, she was not hospitalized. during the quarantine period, she was always afebrile while the other symptoms gradually improved and completely disappeared on april . she performed two follow-up nasopharyngeal swabs on april and , which came negative (allplex™ -ncov assay). she also underwent a sars-cov- qualitative serologic test that was positive for igg (screen® test covid- , screenitalia). on may , because of chest pain and arthro-myalgia, the patient was hospitalized. she underwent a nasopharyngeal swab test for sars-cov- that resulted positive with detection of genes "e" and "n" at ct values of and , respectively (xpert® xpress sars-cov- , cepheid). she was discharged h later. the patient presented two subsequent negative nasopharyngeal swabs on may and , respectively (allplex™ -ncov assay) (fig. e) . as in case , the swab sample of may was cultured with vero e cells. no viral replication was observed, and no viral rna on the supernatant was detected. a -year-old woman with chronic respiratory disease and cardiomyopathy had presumably a close contact with a positive case. she underwent nasopharyngeal swab on march that came positive (allplex™ -ncov assay). the day after, she started complaining of generalized arthro-myopathy but no respiratory symptoms. she was considered healed when her nasopharyngeal swabs for sars-cov- came negative (may and , allplex™ -ncov assay). she tested positive in an ulterior test, performed on may before a cardiological follow-up visit with detection of genes "e" and "n" at ct values of and , respectively (nasopharyngeal swab; xpert® xpress sars-cov- , cepheid). the following swab was performed on june and came negative (rt-pcr assay) (fig. f) . at the same time, sars-cov- serology was compatible with a previous infection (igg . au/ml). the sample of may was inoculated onto vero e cells, without any cytopathic effect development, and viral rna on the supernatant was not found. a -year-old physician working in a hospital in northern italy probably came in contact with a positive case during his professional activity. on march , , he developed headache, arthro-myalgia, ageusia, anosmia, and dyspnea on exertion. he underwent a nasopharyngeal swab that tested positive on march (rt-pcr assay). considering his mild symptoms, he was not hospitalized. during the quarantine period, his clinical conditions were fine, but he kept on having mild cough and complaining of dyspnea. he performed follow-up nasopharyngeal swabs on april , , and , which came negative (rt-pcr assay), and a highresolution ct that showed two millimetric ground-glass opacities. on may , , he repeated the nasopharyngeal swab and serology test (method not available) that resulted negative. on june , he was admitted to perugia hospital for a bone marrow donation and performed a new nasopharyngeal swab test for sars-cov- that resulted positive with detection of genes "e" and "n" at ct values of and , respectively (xpert® xpress sars-cov- , cepheid). the patient presented two subsequent negative pharyngeal swabs on june and , respectively (rt-pcr assay) (fig. g) . his sars-cov- serology was positive for igg ( . au/ml). the swab sample of june , , was inoculated in vero e cell culture. no virus was isolated, and no viral rna on the supernatant was detected. a diabetic man, aged years old, was hospitalized on march for dyspnea after being diagnosed with covid- with a nasopharyngeal swab (allplex™ -ncov assay). during the hospitalization, his respiratory exchanges gradually worsened, needing noninvasive ventilation. t o c i l i z u m a b , w i d e -r a n g e a n t i b i o t i c t r e a t m e n t , hydroxychloroquine, and corticosteroids were also administered. his infection was classified as overcome once he collected two negative nasopharyngeal swabs (allplex™ -ncov assay) (fig. h) . he underwent a follow-up ct scan that showed the ground-glass aspect of parenchyma and "crazy paving" phenomena in the inferior and superior right lobes. he was discharged on april . the patient went to the hospital on june for the persistence of headache, arthro-myalgias, asthenia, and insomnia. he was hospitalized again as a sample performed in the emergency room resulted positive (nasopharyngeal swab; xpert® xpress sars-cov- , cepheid). at the same time, sars-cov- serology showed igg = au/ml. subsequently, he was rapidly discharged after two negative pharyngeal swabs and negative sars-cov- culture in vero e cells. a -year-old man with a history of cardiomyopathy, atrial fibrillation, diabetes mellitus type , alcoholic cirrhosis, myelodysplastic syndrome, and hepatic neoformation developed fever with no other apparent symptoms on april , . he was hospitalized after performing a nasopharyngeal swab with rt-pcr assay, which came positive (allplex™ -ncov assay). as he became afebrile and his parameters were in range, he was discharged after a follow-up pharyngeal swab that came negative on may (allplex™ -ncov assay). during quarantine, he was asymptomatic. on july , he went to the emergency room after seeing blood in his stools. he underwent a screening nasopharyngeal swab (xpert® xpress sars-cov- , cepheid) that tested positive with detection of genes "e" and "n" at ct values of and , respectively. his serology was compatible with a previous infection with sars-cov- (igg = . au/ml). he denied contact during quarantine with confirmed or possible covid- cases. the patient was discharged in good clinical conditions after two further swab tests for sars-cov- (allplex™ -ncov assay), which came negative on july and (fig. i) . the swab sample of june , , was inoculated in vero-e cell culture. no virus was isolated, and no viral rna on the supernatant was detected. a total of papers matched the eligibility criteria . no clinical or observational trials were found. among those, there were case reports and case series. furthermore, review/minireview articles have been found [ , , [ ] [ ] [ ] . applying clinical criteria based on literature sources [ ] , we identified cases of covid- that presented new positive respiratory samples after recovery. we analyzed data also including our nine presented cases, with a total of . the characteristics of the patients are summarized in table . recurrence indistinctly occurred in the same rate in male and in female patients with a small predominance of female cases (male . %, number of patients, n ). the patients were admitted to the hospital in a mean of . days (standard deviation, sd, . days; n ) after symptoms onset. only four patients were not hospitalized (cases , , , and ). recurrence was not associated with a specific age (mean age, . years old; sd, . ; range, - years old; n ) or comorbidity ( . % of patients had at least one comorbidity, n ). the most common symptoms at infection onset included fever ( . %, n ), cough ( . %, n ), dyspnea ( . %, n ), nausea or diarrhea ( . %, n ), and arthro-myalgia ( . %, n ). as shown in table , poor data are available about laboratory findings, but coherent to literature data concerning patients with sars-cov- infection [ ] . many cases also had ct scans or chest x-rays of patients which revealed the typical radiological sign of sars-cov- lung involvement ( . %, n ). most patients underwent some antiviral treatment ( . %, n ) . only six patients (included case of our case series) required icu admission ( . %, n ). in particular, case and other cases have been in icu before the presumptive reactivation of sars-cov- infection, and only two patients needed icu admission at the second episode [ , ] . the first positive respiratory sample was collected in mean after . days (sd . , n ) from symptom onset. in that phase, . % (n ) of patients presented symptoms related to covid- , and . % (n ) of patients had fever. in table , we reported the first negative respiratory sample only if followed by a second negative sample. it occurred in mean after . days (sd . days, n ). in this phase, . % (n ) of patients presented persisting but improving symptoms. only in one case ( . %, n ) did fever persist because of a klebsiella pneumoniae bloodstream infection, documented at the same time. these patients underwent follow-up respiratory sample collection after being declared recovered. new positive respiratory samples were found in mean . days (sd . days, n ) after symptoms onset. some patients presented fever ( . %, n ) and/or were still symptomatic ( . %, n ) . most of them reported mild cough and respiratory symptoms. at that time, . % of patients already had igg against sars-cov- . data concerning the following negative respiratory samples are available for patients; none presented fever and % of the patients were mildly symptomatic. the outcome was available for patients. among these, . % recovered, . % improved, . % were still hospitalized at the time of data collection, and . % died. sars-cov- is a new coronavirus that since its discovery has been spreading all over the world, causing an impressive amount of deaths [ ] . it is believed that patients infected with sars-cov- carry protective antibodies after recovery [ ] . in literature, nonetheless, a series of cases of recurrences is reported, which means positivity for sars-cov- after two negative respiratory samples [ ] . we need further data to determine risk factors, timing, and mechanisms that can cause it. recurrence might probably be related to host factors (virologic status, underlying medical conditions, and therapy administered), characteristics of the virus itself, sample collection, and processing [ ] . currently, no reliable predictive marker of reactivation was found. sars-cov- recurrence will be a vexing and persistent problem. considering numerous patients infected or previously exposed to the virus, such a possibility poses a major public health burden [ ] . the aim of the present study was to explore if the cases of suspected recurrence reported in the literature and in our clinic may be caused by a flawed sample or an incomplete clearance of the virus, or if effectively recovered people may be infected anew. according to the presented data, the hypothesis of a recurrence seems to be improbable. indeed, most of the patients were afebrile ( . % of patients had fever), and symptoms were mild. these data depose for slow viral clearance and disease resolution instead of reactivation. almost % of patients subsequently improved or recovered. among cases, four patients died after presumptive recurrence, but it is not clear if the episode could be due to recurrence of covid- [ ] . interestingly, lafaye et al. described a geriatric case (case ) with probable covid- recurrence as he had clinical and radiological worsening, absence of neutralizing antibody, and positive cell culture during the second episode [ ] . a real case of re-infection was described by wang to et al. in this case, epidemiological, clinical, serological (igg seroconversion), and genomic analyses confirmed that the patient had re-infection by a different strain of sars-cov- [ ] . osman et al. performed a review of re-positive cases after discharge from hospitals in china. they concluded that the repositivization might be attributed to false-negative laboratory results and prolonged viral shedding, rather than re-infection [ ] . however, the authors, in line with another review performed by han et al., suggested that health authorities need to consider the importance of maintaining social distancing, even after the patients' recovery [ , ] . cento et al. retrospectively analyzed data of recovered covid- . negative-to-positive rt-pcr fluctuations occurred in / patients, while none of them has ever shown a recurring of covid- symptoms, regardless of rt-pcr results [ ] . kang et al. analyzed clinical and epidemiological information of re-positive cases from the south korea centers for disease control and prevention (kcdc). patients were asymptomatic or complained about minor symptoms. the authors suggested that rna of the "dead virus" remaining in the recovered patient's body is amplified during the rt-pcr process. furthermore, as sars-cov- does not cause chronic infection (seen as a latent stage), its reactivation is not virologically possible. they also concluded that comprehending the above evidence, re-positive cases are not contagious [ ] . concerning our nine cases, none presented severe disease or complications at the time of presumed recurrence. all tested patients (n ) had antibody against the s and s subunit of sars-cov- spike protein with sufficient titer immediately before or during the presumptive recurrence. these data are in contrast with the hypothesis of recurrence of covid- . another important issue was to establish if these patients could be contagious again. to this purpose, swab samples of the supposed "recurrence" in our patient cohort were stored, and the cultivability of sars-cov- in vero e cells was assessed as previously described [ ] . in all these cases, after h of culture on vero e cells, no virus replication has been observed, and viral rna on supernatant was not detected. since the first sars-cov- virus was isolated by our virology laboratory, many other swab samples have been cultured to isolate as much different virus strains as possible. we found that symptomatic patients with positive rt-pcr tests with ct values < easily provided cultivable sars-cov- viruses from their specimens. as previously reported, the results of rt-pcr from swab samples of re-positive patients of our cohort always showed ct values above , so the uncultivability of these samples should be considered easily predictable [ ] . this also strengthens the hypothesis of a "dead virus" that remained in the recovered respiratory tracts instead of a recurrence of infection. furthermore, our results confirmed what previously declared by kcdc that tested negative viral cell culture of re-positive cases [ ] . although these data should be confirmed in a larger number of cases, they strongly suggest that patients previously considered recovered and with a subsequent new sample positive for sars-cov- should not be considered contagious. limits of our study are the small number of cases; the incompleteness of some literature data, such as the detailed description of criteria for positivity and negativity; and the heterogeneity of the entire study cohort. strengths are the analysis of all available literature about the recurrence of covid- and the univocal answers to the issues. in conclusion, our results suggest that in most cases the presumptive recurrence is indeed a prolonged, not contagious, viral rna persistence in the respiratory tract, probably due to a slow disease resolution. further studies are necessary to definitively understand if a covid- recurrence is possible and whether it could be considered as a real threat. code availability available on reasonable request. author contributions daniela francisci, anna gidari, marco nofri, and luca saccarelli contributed to the study conception and design. material preparation and data collection and analysis were performed by anna gidari and marco nofri. the first draft of the manuscript was written by anna gidari, marco nofri, and luca saccarelli, and all authors commented on previous versions of the manuscript. all authors read and approved the final manuscript. funding open access funding provided by università degli studi di perugia within the crui-care agreement. data availability available on reasonable request. conflict of interest the authors declare that they have no competing interests. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not 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