key: cord-0008890-qnl24rrf
authors: Pan, Tingting; Tan, Ruoming; Qu, Hongping; Weng, Xing; Liu, Zhaojun; Li, Meiling; Liu, Jialin
title: Next-generation sequencing of the BALF in the diagnosis of community-acquired pneumonia in immunocompromised patients
date: 2018-11-23
journal: J Infect
DOI: 10.1016/j.jinf.2018.11.005
sha: ea94d5fd7507f67e9b3ffbe3c8d5c2d115fa8833
doc_id: 8890
cord_uid: qnl24rrf

nan

. Isoelectric focusing for VZV IgG. Isoelectric focusing of paired CSF and serum ( A and B ) stained for IgG in CSF, showing prominent oligoclonal bands in CSF, but absent from serum (consistent with local or intrathecal synthesis). The patterns have been aligned against the prominent band in the centre of the pattern on CSF to allow easy comparison. Although the distribution of bands appears slightly different between CSF samples this just reflects inter-assay variability in the analytical system (each sample pair was run on a different gel). The anode ( + ) electrode is on the left. The paired CSF and serum in images A and B are from samples taken approximately two months apart during acute illness episodes. temporaneous serum VZV DNA PCR was mostly negative except once when it was positive at a very low level (63 copies/ml of VZV DNA). Repeat isoelectric focusing and immune-blotting revealed an intrathecal oligoclonal IgG response, together with a VZV-specific monoclonal IgG response ( Fig. 2 ).

Further treatment with oral valacyclovir (10 0 0 mg TDS for 7 days) followed by a suppressive dose (valacyclovir, 500 mg BD) for three months was then tried. However, the patient continued to experience meningitis symptoms on this medication. Treatment was continued with oral valacyclovir (10 0 0 mg BD), but now in combination with monthly VZV hyper-immune globulin (VZIG, IM, 400 units). Although there was some improvement, the meningitis symptoms recurred every 3 weeks.

Several years later, we received an update from the patient. He had been on holiday in South Africa, during which he had mistakenly received 3 doses of the paediatric varicella vaccine instead of the regular VZIG. His symptoms improved for 4-5 weeks. On returning to The Netherlands, on this basis, his neurologist suggested zoster vaccine (which contains at least 19,400 PFU -plaque forming units -of VZV, per dose) for longer-term symptom relief. As this was not yet available, he was given 18 doses of paediatric varicella vaccine (which contains at least 1350 PFU, per dose) instead. This resolved his symptoms. He then received a depot corticosteroid injection for shoulder pain, after which his meningitis symptoms returned 3 weeks later. After a further 18-dose course of paediatric varicella vaccine, he has remained symptom-free.

Varicella-zoster virus remains latent in the neurons of cranial nerves and dorsal root ganglia for the entire life of the host after primary infection (chickenpox). 3 Herpes zoster or shingles is the most common form of VZV reactivation and is characterised by a dermatomal rash and radicular pain. Rarely, VZV spreads to the spinal cord and brain. When this occurs, CNS complications may develop without concomitant zoster rash. 4 . This pattern is also just visible in the serum samples. The ladderpattern is characteristic of a monoclonal antibody. Due to the strong staining, the anodic (left-most) bands have merged. The absence of any bands in the control immunoblot (using a cell-line without VZV, B ) shows that the IgG in the patient's CSF and serum is highly VZV-specific.

to VZV is seen in both immunocompetent as well as immunocompromised patients. 6 The diagnosis is usually confirmed by a positive CSF VZV DNA PCR result, or less commonly, by the intrathecal detection of VZV-specific IgG. 7 The repeated failure to detect CSF VZV DNA in this case during acute episodes may be due to the chronic course of the disease. A similar decrease in the percentage of CSF HSV PCR-positive cases can be seen within 2 weeks for HSV encephalitis, 8 which may also be true for VZV. The onset of symptoms in this patient was 2 years before the first CSF VZV PCR was performed. His symptoms were also limited to viral meningitis rather evolving into encephalitis, which may have also explained the negative VZV PCR results. 9 However, the extensive intrathecal VZV IgG production confirmed the diagnosis of VZV aseptic viral meningitis.

The finding of a distinctly monoclonal intrathecal VZV-specific IgG is unusual and atypical ( Fig. 2 ) . Normally, an oligoclonal response is required to clear the virus efficiently. Therefore, the patient may have a defect in his VZV-specific antibody affinity maturation, i.e. a partial 'immunological scotoma' to VZV, explaining his inability to clear (i.e. 'neutralise') the virus efficiently, allowing recurrent episodes of meningitis. Note that this case does not meet Bruyn's diagnostic criteria for Mollaret's meningitis, 10 as the patient was not completely symptom-free between episodes.

To our knowledge, such chronic-relapsing varicella zoster viral meningitis of such periodicity has not been previously described. The remarkable response to multiple doses of paediatric varicella vaccine was both fortunate and serendipitous. This patient likely suffers from a VZV-specific antibody maturation deficit that appears to have been overcome by the massive immune stimulation provided by multiple doses of paediatric varicella vaccine. Whilst such cases are rare, this report may assist in their management.

To the editors , We would like to take the opportunity to comment on the systematic review by Alene et al. 1 The authors pooled data of prevalence in patients with multidrug-resistant tuberculosis (MDR-TB). However, we found that prevalence differed extremely among a Only the total number of psychosis and depression was reported ( n = 33). b Only baseline information before treatment initiation was reported. c For diverse tools were applied in prevalence estimate of depression and diagnosis standard was not uniformed, we did not extract some event numbers in this Table. studies, ranged from 3% to 79%, leading to a high statistical heterogeneity ( I 2 = 98%).

After checking, we surprisingly found that only four studies reported the prevalence of depression after MDR-TB treatment, one study reported the prevalence at baseline, while the others reported the incidence of depression as an adverse effect of MDR-TB treatment ( Table 1 ). The authors may not carefully distinguish the conception of prevalence and incidence. In addition, for those studies adopting different scales and diagnosis criteria, the prevalence rate may change according to the criteria. It is not recommended to pool these data into meta-analysis. We hope future studies can adopt a unified standard of depression in the research, to better estimate the prevalence of depression after MDR-TB treatment.

The we conducted a meta-analysis by STATA 15.1, to calculate the incidence of depression as an adverse effect of MDR-TB treatment. The results showed an incidence rate of 11% (95% Confidence Intervals: 8-14%, I 2 = 77.5%) ( Fig. 1 ) . We consider that this result may provide more reference to clinical doctors.

The authors have discussed that the cause of psychosis may be cycloserine. 1 In addition, several studies mentioned that depression might also be an adverse effect of cycloserine. 2, 3 To summarize, we appreciate the authors' hard work, but there were some mistakes in data pooling. We estimated the incidence rate of depression as an adverse effect of MDR-TB treatment by performing a meta-analysis. Future studies are needed to estimate the prevalence.

None. 

Dear editor , We previously reported in this Journal that, 1 in early 2016, the public authority of China enforced the detection of imported zika cases in international airports regarding to the continuing global transmission of Zika virus (ZIKV). During February to September, 2016, 28 imported ZIKV infection cases were detected in China, of which 15 were imported from Venezuela, Suriname and Guatemala to Enping County, Guangdong, China ( Fig. 1 ) where is the hometown of more than 450,0 0 0 Guangdong residents work and live in South America and South Pacifica countries. Most of these residents maintain their permanent home and travel frequently between Enping County and ZIKV endemic countries in South America or South Pacifica. Therefore, Enping was considered as the most threaten area by ZIKV in Guangdong, China due to high incidence of importation of zika cases. Additionally, Guangdong Province is a hyper-endemic areas of arboviruses located in Southern China, where both Aedes albopictus and aegypti mosquitoes are encountered simultaneously. Dengue occurred annually. Chikungunya were also occasionally imported and caused an local outbreak in 2010. 2 , 3 However, whether there were previously importation of ZIKV within returned ex-patriot Chinese in Enping, Guangdong, China and result in unware local transmission before 2016 in naïve population is still remained unknown.

During February to October 2016, a community based serological surveillance study of returned ex-patriot Chinese and naïve population was conducted in Enping County, Guangdong Province, China to address whether those populations have been exposed to ZIKV before the importation in 2016. The 227 ex-patriot Chinese were randomly selected from 1161 Guangdong residents work and live in South America and South Pacifica countries returned to Enping County. The information of returned ex-patriot Chinese (travelers in total, 227; male, 129; female, 98; range 1-76 years old) were informed by entry-exit inspection and quarantine bureau of Guangdong Province. All these travelers showed no symptoms of ZIKV infection, either fever ( > 38 °C), rash, arthralgia or conjunctivitis. Local Center for Disease Control and Prevention (CDC) staff contacted these travelers at the time of their arrival in Enping County by phone or household visits. Blood sample were taken at the local hospital upon their arrival, and transferred to the Guangdong Provincial CDC for serum separation and ZIKV RNA testing. The naïve population were choosed from the communities in Enping County where the 15 imported zika cases detected, and in a neighbors Heshan County (fifty kilometers away from Enping) where most of the oversea Chinese live in non ZIKV endemic areas of Southeast Asia ( Fig. 1 ) . The naïve population was recruit from the local residents who have no travel history to ZIKV endemic areas of South America and South Pacifica countries, and without any contact history with imported zika cases. Finally, a total of 665 naïve residents (male, 278; female, 387; range 1-95 years old) were recruit, of which 330 and 335 came from Enping and Heshan County, respectively. The blood samples of these 665 residents were taken from each of them, and transferred to the local CDC. A total of 892 serum samples were collected and transferred to Guangdong Provincial CDC for further ZIKV RNA and neutrality antibody detection.

The quantity of ZIKV RNA Envelope gene was determined by quantitative real-time reverse transcription PCR (qRT-PCR) as we previously reported. 4 No ZIKV RNA were detected among all the 892 serum samples which indicated no acute stage infection of ZIKV. Although cross-reactive binding antibodies are commonly detected in flavivirus-immune serum, neutralization assays are more specific and can distinguish the antibodies response to various flaviviruses exposure previously, 5 thus, serum antibodies against to ZIKV in both 227 returned and 665 naïve residents were determined to make sure whether those persons exposed to ZIKV or not by a Vero cell based micro-neutralization assay against to ZIKV, CHIKV, DENV 1, and DENV 2. The results indicated that ZIKV could be neutralized by serum samples of 32 (No. 1-32) (14.1%, 32/227) returned persons (IC 50 range from 1:8 to 1:1024) who had travel history in South America and South Pacific areas ( Table 1 ) . Twenty one of these 32 ZIKV antibody positive serum samples showed across-neutrality effects against to DENV-2 but not DENV-1. Four of these 32 samples showed minor cross-neutrality effects against to CHIKV (IC 50 range from 1:8 to 1:32). Moreover, 18 serum samples from returned persons showed minor cross-neutralization against to CHIKV (No. 33-50, IC 50 range from 1:8 to 1:32) but not ZIKV or DENV. Surprisingly, one serum samples (No. 51) from one naïve resident in Enping County showed significantly neutrality ability against to ZIKV (IC 50 = 1:1024) and CHIKV (IC 50 = 1:1024), while the remained 664 of 665 naïve residents samples showed no any neutrality effects against to ZIKV, CHIKV, as well as DENV.

Transmission of infection through international travel is a growing health issue, and the frequency of imported infection is increasing in China. The recently studies report the profile of travel-related infections among arriving travelers in mainland China, and highlight to increase public awareness of the potential risk of imported infections. 6 Indeed, the global transmission of ZIKV has been suspected through international travelers between endemic areas and local countries, and addressed by recently phylodynamic analysis. 7, 8 In Guangdong, China, the airport active screening system found 18 imported zika cases which indicate Guangdong is under a high risk of zika transmission due to wide distribution of competent vector of ZIKV, Aedes albopictus and aegypti . Although the present community based follow-up serological survey found 14.10% (32/227) of ex-patriot Chinese returned from ZIKV endemic countries were ever exposed to ZIKV, none of them were ZIKV cases at acute stage according to the results of ZIKV RNA detection. In contrast, we found most of naïve residents were not exposure to ZIKV. In addition, ZIKV antibody positive serum samples from returned population showed minor crossneutrality effects against to DENV-2 and CHIKV but not DENV-1.

It is not surprising because of continuously circulation of DENV-2 and CHIKV in South American countries, 9, 10 therefore, those travelers may have been infected by DENV-2 or CHIKV before they returned to China. However, we did not observed any crossneutrality effects against to ZIKV, CHIKV and DENV among the 665 naïve residents except for the unique suspect asymptomatic ZIKV and CHIKV case who do not want declare his travel history.

In conclusion, this report indicated that although numbers of zika case had been imported to a hyper endemic area of arboviruses in Guangdong, China, the community based surveillance of naïve populations showed ZIKV was only tentatively imported to Guangdong, whereas no local transmission has been established in Guangdong, China before 2016. It highlight the control strategies taken by public health authorities in Guangdong 2016 probably prevent the onward transmission of ZIKV in these areas. In recent years, pathogenicity and transmissibility as well as increasing potential human infection ability of H5N6 avian influenza viruses (AIVs) have been documented in this journal. 1,2 Since 2003, H5N1 highly pathogenic avian influenza viruses (HPAIVs) have become enzootic in many countries and pose a serious challenge to the poultry industry and public health. 3 As of 2013, H5N6 HPAIVs have emerged and circulated in poultry, causing sporadic human infections in China. 4 Since 2014, H5N8 HPAIVs have been circulating in Asia, North America, and Europe. 5 Wild birds are considered a natural reservoir for AIVs and play an important role in viral transmission. 6 In this study, we have presented a brief characterization of three H5 HPAIVs with different NA subtypes (H5N1, H5N6, and H5N8) isolated from wild birds.

During AIV surveillance in wild birds in 2015-2016, we isolated three strains of H5 HPAIVs, designated A/Anser cygnoides/Jiangxi/ P126/2015(H5N1) (P126/H5N1), A/Anser fabalis/Jiangxi/P560/2015 (H5N6) (P560/H5N6), and A/swan/Shanxi/ST/2016(H5N8) (ST/ H5N8), from Poyang Lake of Jiangxi Province and Shengtian Lake of Shanxi Province. To clarify their genetic characteristics, we sequenced all gene segments of the three viruses and compared them with influenza virus sequences in GenBank. The pathogenicities of the three isolates were additionally determined in chickens, ducks and mice.

For virus isolation, feces samples were resuspended in PBS solution with antibiotics and subsequently inoculated into specific pathogen-free (SPF) embryonated chicken eggs as described previously. 7 RNA was extracted using the Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The full genomes of the viruses were sequenced and deposited in GenBank under accession nos. MK6160 6 6-MK616089.

To characterize the three isolates, we determined clade distribution based on the HA gene sequences. P126/H5N1, P560/H5N6, and ST/H5N8 isolates belonged to clade 2.3.4.4 ( Fig. 1 ) . Sequence analyses further showed high nucleotide identities of HA genes of the three H5 viruses with A/chicken/Tong Hai/ 302/2014(H5N1), A/oriental magpie robin/HK/6154/2015(H5N6), and A/chicken/Uganda/17RS115-15/2017(H5N8), which are currently circulating in Asia ( Fig. 1 ) .

To assess the risk of likelihood of transmission from avian to mammalian species, genotypic markers were evaluated based on their genome sequences. The receptor binding sites in the HA gene of three H5 HPAIVs contained Q226 and G228 (H3 numbering), suggesting binding preference for avian-like receptors. 8 The H5 viruses shared four to six amino acid changes within the receptor binding site (RBS) of HA, specifically, S133L (ST/H5N8), and S137A in the 130-loop, D187N, K193N , and Q196K in the 190-helix, and S227R (ST/H5N8) in the 220-loop (Table S1) . Remarkably, HA genes of the three H5 isolates lacked an N-linked glycosylation site at position 158, whose absence is considered to promote receptor binding affinity in guinea pig transmission. 9 Mutations within the RBS and loss of the glycosylation site at residue 158 suggested that these H5 isolates confer receptor preference changes.

Mutations related to pathogenicity in mammals, 10 such as E627K and D701N in PB2, were not detected in the three avian isolates. However, substitutions in internal genes were identified, including nine in PB2 (T63I, L89V, G309D, T339K, Q368R, H447Q, R477G, I495V, and A676T), eight in PB1 (A3V, L13P, R207K, K328N, S375N, H436Y, L473V, and M677T), two in PA (H266R and S/A515T), three in MP (V15I, N30D, and T215A), and six in NS (A/P42S, T/D92E, L98F, I101M, V149A, and N200S), that potentially enhanced polymerase activity and increased virulence in mammals. These mutations appeared to confer not only increased receptor binding properties but also greater virulence of H5 HPAIVs, thus posing an increased threat to public health.

To evaluate the pathogenicity of the three H5 isolates in chickens, groups of ten 6-week-old SPF chickens were intravenously inoculated with 0.1 mL (1/10 dilution) of the allantoic fluid of each virus and mortality observed over a 10-day period. The characteristics of P126/H5N1, P560/H5N6, and ST/H5N8 viruses, which were highly pathogenic in chickens, are presented in Table S2 .

To evaluate the pathogenicity of the H5 isolates in ducks, we inoculated groups of 10 3-week-old SPF ducks intranasally with 10 6 EID 50 of each virus and observed mortality over a 10-day period. The characteristics of P126/H5N1, P560/H5N6, and ST/H5N8 viruses are shown in Table S2 . Our data clearly indicated high pathogenicity of these viruses in ducks.

To determine the pathogenicity of the viruses in mammalian hosts, we inoculated BALB/c mice intranasally with 10 6 EID 50 of each virus in a total volume of 50 μL. Five inoculated mice were euthanized on day 5 post-inoculation (p.i.) for virus titration in the lungs, nasal turbinate, liver and brain. The remaining 10 mice were monitored daily for weight loss and mortality for a total of 2 weeks. After intranasal administration of P126/H5N1 and P560/H5N6, high virus titers were detected in lung, nasal turbinate and liver, but not brain, while the virus was identified in lung and nasal turbinate, but not liver or brain of the ST/H5N8 group. Mice showed signs of illness. Over the 14-day observation period, all mice in groups inoculated with P126/H5N1 and P560/H5N6 isolates died ( Fig. 2 ) . However, no mortality was observed in the ST/H5N8 group ( Fig. 2 ) , suggesting that P126/H5N1 and P560/H5N6, but not the ST/H5N8 strain, were highly pathogenic in mice.

In summary, we characterized three H5 HPAI viruses with different NA subtypes isolated from apparently healthy wild birds in 2015-2016. All three isolates were highly pathogenic in chickens and ducks while the H5N1 and H5N6 subtypes showed high pathogenicity in mice. Many species of wild birds have longdistance dispersal and migration capacity. This behavior increases the likelihood of HPAIV spread, which presents a serious threat to poultry and human health. 

There are no potential conflicts of interest. The NA gene of F352 showed the highest similarity to A/duck/Zhejiang/727042/2014 (H6N2) between nucleotide positions 101 and 583 and 700 and 1301, except for 583 −700, which was most similar to A/duck/Hunan/S2046/2011 (H4N2) and A/muscovy_duck/Vietnam/LBM201/2012 (H3N2). These findings indicated that the NA gene of F352 was mainly derived from the N2 segments of the major parent A/duck/Zhejiang/ 727042/2014 (H6N2), but underwent reassortment with A/duck/ Hunan/S2046/2011 (H4N2) and A/muscovy_duck/Vietnam/LBM201/ 2012 (H3N2) ( Fig. 2 D) .

To estimate the rates of nucleotide or codon substitutions, we gathered sequences of currently available H3N2 subtype AIVs from NCBI and GISAID from January 2015 to March 2018 and divided them into two groups (isolates of 2015-2016 and 2017-018) for analysis by the uncorrelated relax-clock Bayesian Markov chain Monte Carlo method using BEAST v1.8.4. 8 BEAST analysis showed that the mean rates of HA and NA gene nucleotide substitutions during 2017-2018 were decreased when compared with 2015-2016 ( Table 1 ). In addition, the mutation rates of second nucleotide of each codon within the open reading frame (ORF) of the HA gene was 0.188 during 2015-2016, but the rates were 0.454 during 2017-2018, which confirmed that the mutation rates had doubled ( Table 1 ) . Interestingly, most of the H3N2 subtypes of AIVs belonged to the Eurasion lineage and contained six potential N-glycosylation sites, including positions 24 NST 26 , 38 NGT 40 , 2.393 2.507 * HPD: highest probability density. * Position: the three nucleotides of each codon. E: scientific notation. 54 NAT 56 , 181 NVT 183 , 301 NGS 303 , and 499 NGT 501 in the HA protein; however, mutation of amino acids at the position 24N → S resulted in 24 NST 26 N-glycosylation sites of some strains disappearing and a new 22 NDS 24 site appearing in the HA1 protein since 2010. Strikingly, the codon of amino acids at position 24 had a one nucleotide (AAC) difference from the nucleotide (AGC) that leads to the mutation of 24N → S. This might have been related to the mutation rates of second nucleotide of each codon doubling with time, thereby changing the N-glycosylation sites of F138 and F352, which indicated viral replication and adaptation of H3N2 subtypes AIVs might have evolved substantially in the hosts.

In conclusion, AIVs with the H3N2 subtype will be ignored because of their low pathogenic and isolation rates. Our findings revealed that the HA and NA gene of F138 and F352 reassortant with the H3N2, H3N3, H4N2, H6N2 subtypes AIVs and the mean rates of second nucleotide of each codon of the HA gene had doubled. However, although the mean rates of the HA and NA gene nucleotide substitutions decreased, the reassortant process might have been affected by the frequent changes in antigenicity of other prevalent AIV strains so that better conditions are needed for the reassortant process in H3N2 subtypes of AIVs to proceed. In addition, consistent with Gao' research, 9 the binding affinity for the α(2-3)-linked sialic acid receptor of H7N9 subtype AIVs in 2013 was decreased, mainly due to the lack of N-glycosylation sites at amino acid position 150 in the HA protein. The H3N2 influenza virus has a wide host range and new N-glycosylation sites of F138 and F352 have been found, indicating that the H3N2 subtype AIVs might have attempted to adapt to different hosts. Importantly, H5N1 and H7N9 subtype AIVs have adapted to mammals and now pose a public health concern to mammals and humans. Hence, ongoing surveillance of H3N2 subtypes of AIVs in birds is warranted.

The author declare no competing financial interests.

We read with great interest the recent publication by Xie et al., addressing the issue of next-generation sequencing (NGS) for diagnosis of severe pneumonia in China. 1 They suggested that NGS might lead to more rapid and accurate diagnosis with better clinical prognosis than conventional detection methods in severe pneumonia in ICU. NGS is a novel method to DNA/RNA sequencing. Due to its high-throughput capacity and fast turnover time, NGS technology has been widely applied to the field of medical microbiology in recent years. NGS has been reported in the application for pathogen detection from blood, cerebrospinal fluid, tissue and intraoperative samples. [2] [3] [4] However, no studies have been reported from bronchoalveolar lavage fluid (BALF) samples in community-acquired pneumonia (CAP) patients with immuno- suppression. Compared with the immunocompetent host, CAP can be potentially fatal and present significant challenges in immunocompromised host (ICH). In addition to common pathogens, some unusual organisms can cause severe infection in ICH. Besides, multiple concurrent infections are also common in ICH. 5 These characteristics pose a severe challenge to establish a specific diagnosis to guide therapy. A diagnostic tool should be pursued aggressively for favorable outcomes in this population. We recently explored the utility of NGS of bronchoalveolar lavage on the diagnosis of pneumonia in immunocompromised patients. Thirteen immunocompromised patients with a diagnosis of communty-acquired pneumonia (CAP) were recruited from critical care department of Shanghai Ruijin Hospital. The diagnosis of CAP is based on the presence of select clinical features (e.g., cough, fever, sputum production, and pleuritic chest pain) and is supported by imaging of the lung, usually by chest radiography. Immunosuppression, defined as chemotherapy or neutropenia < 10 0 0 μL during the past 28 days; treatment ≥ 20 mg corticosteroids daily for ≥ 14 days; human immunodeficiency virus infection; immunosuppressive therapy after organ or bone marrow transplantation; and active tuberculosis. Fiberoptic bronchoscopy was performed under local anesthesia with topical lidocaine for patients. BALF specimens using three aliquots of 20 mL of 0.9% saline were obtained. The first was discarded as recommended. The others were pooled and separated into two aliquots. One aliquot was sent to the microbiology lab for direct examination with Gram stain and culture. The other one was sent for DNA extraction and sequencing.

Standard procedures (SP) of BALF microbiologic testing were performed in all patients, which included quantitative cultures for bacteria, mycobacteria and fungi. In addition, multiplex PCR for influenza A/B, parainfluenza 1/2/3/4, coronavirus OC43, coronavirus 229E/NL63, respiratory syncytial virus A/B, adenovirus, human metapneumovirus, human rhinovirus, enterovirus and Human bocavirus was performed on BALF and nasopharyngeal. CMV, EBV, HSV-I, and HSV-II serology for IgG and IgM antibodies were measured.

Clinical characteristics including sex, age, APACHE II score at the time of diagnosis of CAP were demonstrated in Table 1 . All patients were considered to be immunocompromised due to corticosteroids therapy or other immunosuppressive drugs. 6 SP identified pathogens in 6 patients (6/13), four of which were bacteria, one fungi and one virus detected by clinical respiratory viral PCR panels ( Table 2 ) . It is worth noting that two samples positive for Acinetobacter baumannii by SP were considered co-infected Table 2 Next-generation sequencing of BALF for the patients with pneumonia.

with PJP or CMV respectively by NGS. NGS detected pathogens in 12 patients (12/13) and one sample with non-infectious etiologies was confirmed by NGS ( Table 2 ) . Importantly, five PJP infections and one Aspergillus fumigatus infection were recognized by NGS. Moreover, among the six fungi-positive samples, CMV was also identified in three of them (two PJP and one Aspergillus fumigatus ). Being relatively uncommon pathogens among the immunocompromised, PJP were identified in 5 cases and CMV in 4 cases by NGS. However, CMV serology for IgM were negative in these patients. No methenamine silver staining was performed in these cases, for the detecting item has not been carried out in our hospital. Overall, four bacteria, one fungus and one virus were detected by SP. By comparison, four bacteria, seven fungi and five viruses were identified by NGS ( Table 2 ).

In terms of bacteria detection, NGS showed no significant advantages in this study. It is noteworthy that our study demonstrated NGS has a distinct advantage in the field of fungi and viral testing, especially PJP and CMV detection.

The definitive PJP diagnosis usually requires demonstration of fungus in tissue, BALF, or induced sputum samples. Methenamine silver staining and PCR in BALF samples have good sensitivity and specificity and is an emerging modality for diagnosis in the correct clinical setting. 7, 8 There are a few laboratory tests to detect CMV infection. 7 Histopathologic analysis or viral culture from tissue, blood, urine, or respiratory specimens is less utilized because of poor sensitivity and longer time-consuming. CMV serology for IgG and IgM antibodies is another diagnostic tool but may be negative and inconclusive in immunocompromised hosts. The other methods such as PCR and pp65 antigenemia assays have not been carried out widely. 7 , 9 NGS technology showed its remarkable advantages in terms of opportunistic pathogens in patients with immunocompromised states.

In recent years, clinicians have been increasingly aware of the problem of mixed infection in immunocompromised hosts. 10 However, to diagnosis the co-occurrence of two commonly seen opportunistic infections is difficult in immunocompromised patients with lung infiltrates. It is worth noting the distinct advantage of NGS in concurrent detection of bacteria, fungi and viruses. In our study, standard procedures performed poorly in detecting mixed infections, whereas five cases with coinfection of bacteria, fungi or viruses were identified by NGS. This advantage may help clinicians more comprehensive evaluation of patients and make effective treatment.

In conclusion, our study explored the application of NGS in the microbiologic diagnosis in CAP patients with immunosuppression. NGS technology showed its remarkable advantages in detecting opportunistic pathogens and mixed infection in those patients.

The study protocol was approved by the Ruijin Hospital Ethics Committee, Shanghai Jiaotong University School of Medicine, China. Formal consent was obtained from the patient or the next of kin.

This study was supported by the National Key R&D Program of China ( 2017YFC1309700 , 2017YFC1309705 ), National Natural Science Foundation of China ( 81772040 , 81770 0 05 , 81801885 ) and Shanghai Sailing Program ( 18YF1413800 ).

The authors declare no conflicts of interest.

Varicella-zoster virus (VZV) DNA in serum of patients with VZV central nervous system infections

Adults with suspected central nervous system infection: a prospective study of diagnostic accuracy

Neurologic complications of the reactivation of varicella-zoster virus

Varicella-Zoster virus infections of the nervous system: clinical and pathologic correlates

Fatal varicella-zoster virus meningoradiculitis without skin involvement

The neurotropic herpes viruses: herpes simplex and varicella-zoster

Etiology of aseptic meningitis and encephalitis in an adult population

Rapid diagnosis of herpes simplex encephalitis by nested polymerase chain reaction assay of cerebrospinal fluid

The value of cerebrospinal fluid antiviral antibody in the diagnosis of neurologic disease produced by varicella zoster virus

Herpes simplex virus type 2 (Mollaret's) meningitis: a case report

Mental health disorders, social stressors, and health-related quality of life in patients with multidrug-resistant tuberculosis: a systematic review and meta-analysis

The treatment results of patients with multidrug resistant tuberculosis and factors affecting treatment outcome

Adverse events in the treatment of multidrug-resistant tuberculosis: results from the DOTS-Plus initiative

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The epidemiological characteristics and genetic diversity of dengue virus during the third largest historical outbreak of dengue in Guangdong

Presence of Zika virus in conjunctival fluid

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Genomic epidemiology reveals multiple introductions of Zika virus into the United States

Establishment and cryptic transmission of Zika virus in Brazil and the Americas

Emergence and potential for spread of Chikungunya virus in Brazil

Dengue in latin America: systematic review of molecular epidemiological trends

Increasing the potential ability of human infections in H5N6 avian influenza A viruses

Pathogenicity and transmissibility of three avian influenza A (H5N6) viruses isolated from wild birds

H5N1 highly pathogenic avian influenza in Southeast Asia

Novel Reassortant Avian Influenza A(H5N6) Viruses in Humans

Outbreaks among Wild Birds and Domestic Poultry Caused by Re

Is the gene pool of influenza viruses in shorebirds and gulls different from that in wild ducks?

Novel variants of clade 2.3.2.1 H5N1 highly pathogenic avian influenza virus in migratory waterfowl of Hongze Lake

Structural determinants for naturally evolving H5N1 hemagglutinin to switch its receptor specificity

Identification of amino acids in HA and PB2 critical for the transmission of H5N1 avian influenza viruses in a mammalian host

Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses

Moreover, the HA gene of F138 showed the highest similarity to A/duck/Hubei/ZYSYF2/2015(H3N6) between nucleotide positions 101 and 1621, except around position 823, where it was similar to A/duck/Hubei/ZYSYF2/2015(H3N6) and A/duck/Fujian/SD063/2017(H3N3) based on Sim ilarity Plot ting ( Simplot) ( Fig. 2 A). These findings suggested the HA gene of F138 was mainly derived from the A/duck/Hubei/ZYSYF2/2015(H3N6) but reassorted with A/duck/Fujian/SD063/2017(H3N3). F352 showed the highest similarity to A/chicken/Guangxi/165C7/2014 (H3N2) between nucleotides 101 and 1621

Simplot analyses showed that the NA gene of F138 was most similar to A/duck/Zhejiang/ 727042/2014(H6N2), A/duck/Hunan/S2046/2011(H4N2) and A/ muscovy_duck/Vietnam/LBM201/2012(H3N2) between nucleotides 101 and 262, 262 and 382, and 382 and 502, respectively. The greatest similarity was with A/duck/Hunan/S2046/2011 (H4N2) between nucleotides 502 and 1063; however, positions 1063 −1301 showed the highest similarity with A/duck/Zhejiang/ 727042/2014(H6N2) ( Fig. 2 C). These findings indicate the NA gene of F138 mainly originated from the N2 segments of A/duck

Human infection with an avian-origin influenza A (H7N4) virus in Jiangsu: a potential threat to China

Origin and progress of the 1968-69 Hong Kong influenza epidemic

Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts

Evolution and ecology of influenza A viruses

Transmission of avian influenza virus (H3N2) to dogs. Emerg Infect Dis

Characterization of a novel H3N2 influenza virus isolated from domestic ducks in China

jModelTest 2: more models, new heuristics and parallel computing

Bayesian phylogenetics with BEAUti and the BEAST 1.7

Human infection with a novel avian-origin influenza A (H7N9) Virus

Next generation sequencing for diagnosis of severe pneumonia: China, 2010-2018

Detection of virus in CSF from the cases with meningoencephalitis by next-generation sequencing

Diagnosis of sepsis with cell-free DNA by next-generation sequencing technology in ICU patients

Diagnosis of local hepatic tuberculosis through nextgeneration sequencing: smarter, faster and better

Respiratory tract infections in the immunocompromised

Undiagnosed diabetes mellitus in community-acquired pneumonia: a prospective cohort study

Opportunistic pulmonary infections in immunocompromised hosts

Guidelines for prevention and treatment of opportunistic infections in HIV-infected adults and adolescents: recommendations from CDC, the National Institutes of Health, and the HIV medicine association of the infectious diseases society of America

Infection in solid-organ transplant recipients

Epidemiology, co-infections, and outcomes of viral pneumonia in adults: an observational cohort study

We thank the patient for his cooperation in the reporting of this case, as well as other members of the NHNN team that have been involved in his management over the years, particularly Geoff Keir who produced the images shown in Figs. 1 and 2 .

We thank the laboratory and administrative personnel at Guangdong Provincial Center for Disease Control and Prevention and the Jiangmen City Center for Disease Control and Prevention for their contribution to the follow-up investigation. This project was supported by grants from the Guangdong Provincial Science and Technology Program ( 2015A020213004 ), the Na- 

Xuanjiang Jin National 

We thank all the staff for their valuable contribution to the study.

None of the authors have any conflicts of interest to declare.

No conflict of interest were reported.

Supplementary material associated with this article can be found, in the online version, at doi: 10 .1016/j.jinf.2019.03.011 .

Recently, we read with interest a letter in the Journal of Infection , which confirmed avian influenza viruses has posed a new challenge for public health. 1 The H3N2 subtype influenza virus has resulted in global pandemics since 1968, 2 and H3 has a wide host range from birds to various mammalian species, including humans, pigs, dogs, and horses. 3 , 4 Previous studies have shown that avian influenza viruses (AIVs) such as H5N1 and H7N9 could adapt to mammals and gain the ability to infect humans. Song reported cross-species transmission of an intact avian influenza virus (H3N2) to dogs in South Korea that caused acute respiratory disease. 5 In addition, seasonal influenza viruses of strain H3N2 have circulated in the human population every year, and sequence analysis has shown that the novel H3N2 contains genes from H7N3 and H7N7 that viruses are closely related to the H7N9 virus, suggesting that reassortment occurred between H3N2 and other influenza virus subtypes. 6 Thus, evolution of H3N2 AIVs was demonstrated in this study.Two influenza A(H3N2) virus-positive isolates, A/duck/ Guangdong/F138/2017(H3N2) and A/duck/Guangdong/F352/2018 (H3N2) (hereafter F138, F352), were collected from live poultry markets in Guangdong province during November 2017-March 2018. Molecular characterization of the two H3N2 isolates confirmed an amino acid HA-226Q, HA-228 G (H3 numbering), NA-292R (N2 numbering) and motif PEKQTR ↓ GL at its HA-cleavage site, which was the characteristic of low-pathogenic AIVs. Moreover, these strains were susceptible for NA inhibitors (oseltamivir and zanamivir). The detailed information of two H3N2 isolates were shown in Supplementary Table 1 .Available reference sequences, including those of avianorigin, canine-origin, swine and human-origin of the H3N2 subtype used for phylogenetic analysis, were acquired from GenBank ( http://ncbi.nlm.nih.gov/genbank/ ) and GISAID ( http://platform.gisaid.org/ ), and phylogenetic trees of all sequences were prepared by the Bayesian method using MrBayes version 3.2.6. Preliminary analyses were conducted to estimate the best-fit nucleotide substitution model using MrModeltest version 2.3. 7 Phylogenetic analysis confirmed that the HA and NA genes of F138 and F352 belonged to the avian-Eurasion lineage and were not related to canine, swine or human influenza viruses ( Fig. 1 A and B) . The HA segments of F138 and F352 showed 

Supplementary material associated with this article can be found, in the online version, at doi: 10