key: cord-0006304-jrvh9q3c
authors: nan
title: 9th Congress of Infectious Diseases and Tropical Medicine: This issue is available online at www.springerlink.com/content/1439-0973
date: 2008
journal: Infection
DOI: 10.1007/s15010-008-1001-9
sha: 7971a7173d046b139df174f571b2e96cc8ca3ab8
doc_id: 6304
cord_uid: jrvh9q3c

nan

Implant-associated Infections: Basics, Diagnostics, Therapy Implantat-assoziierte Infektionen: Grundlagen, Diagnostik, Therapie Treatment of Antibiotic-associated Diarrhea Behandlung der Antibiotika-assoziierten Diarrhoe Infection Abstracts KIT2008

Basics, Diagnostics, Therapy

Pathogenesis of implant-associated infections caused by staphylococci D. Mack (Swansea, UK) Staphylococcus epidermidis is the prototype organism involved in medical biofilm disease resulting in infected implants like intravascular catheters or joint prostheses yearly affecting millions of patients worldwide. These infections persist despite antimicrobial treatment due to organization of S. epidermidis in surface adherent biofilms regularly requiring device removal. Biofilms are formed in two phases: initial attachment of bacteria is followed by accumulation of bacteria in multiple layers. Attachment is a multifactorial process involving a variety of specific protein and polysaccharide factors depending on surface properties. We identified polysaccharide intercellular adhesin (PIA) as the central functional factor in biofilm accumulation. PIA is a homoglycan of -1,6-linked N-acetylglucosamine residues of which 15-20% are deacetylated. PIA is synthesised by the gene products of the icaADBC locus. Epidemiological studies defined PIA as the main functional molecule involved in biofilm accumulation in S. epidermidis. Using isogenic biofilm-negative icaA-insertion mutants expression of PIA and biofilm formation were defined as essential virulence factors of S. epidermidis in foreign body infection models. Study of isogenic biofilm-negative mutants with regulatory defects in transcription of icaADBC, allelic gene replacement analysis of regulatory gene loci, and transcriptional analysis of icaADBC expression defined a complex regulatory network controlling expression of icaADBC and PIA synthesis involving the alternative sigma factor B, the transcriptional repressor IcaR, sarA, the new regulatory locus barAB, a glucose-induced protein, and at least one other uncharacterised locus. The expression level of methicillin resistance is influenced in parallel by some of the regulatory mechanisms controlling expression of PIA. Study of the molecular epidemiology of S. epidermidis strains revealed that almost all isolates from port-catheter infections were icaADBCpositive and proficient for PIA-synthesis while the strains from prosthetic joint infections produced biofilms frequently in an icaADBCand PIA-independent manner. Consequently we discovered a second mechanism of biofilm accumulation, which was completely polysaccharide-independent. In this case the highly prevalent accumulation associated protein (Aap) is activated by proteolytic processing by staphylococcal or host proteases generating an intercellular adhesin mediating biofilm accumulation. Apparently, S. epidermidis can use factors of innate immunity to generate phagocytosis resistant bacterial aggregates and biofilm leading to immune escape and persistence.

Microbiological methods for the diagnosis of implant-related infections E. Presterl (Vienna, AT) In the face of an aging population on one side and an increasing group of active people exercising risk sports there is an increasing demand for surgery (arthroplasty, osteosynthesis) to repair loss of function either due to fracture or arthrosis. It is estimated that these infections occur in about 5 percent after trauma surgery and in about 2 percent after planned joint repair. Implant related infections are associated with the formation of microbial biofilms on the surface of the foreign body. The biofilm consists of a structured community of bacterial cells enclosed in a self-produced polymeric matrix Early diagnosis is pivotal for the establishment of appropriate antimicrobial treatment in order to eradicate bacteria and the associated biofilm and to improve the clinical outcome of such patients. and adherent to an inert or living surface. Within biofilms bacteria are less susceptible to antimicrobial agents. The basic principle for routine microbiological testing for bone and joint infections is that sampling time, site and diagnostic procedure to obtain material for the microbiological examination are pivotal. Superficial swabs from wounds or fistulas are not diagnostic. "Open" procedures (open surgery, arthroscopy, CT-steered biopsy) are preferred to blind biopsy or aspiration. According to recommendations in the literature any antimicrobial treatment should be delayed to after the diagnostic procedure (debridement, biopsy, aspiration). Swabs, drainage fluids or any superficial material are not diagnostic for the presence of bone, joint or implant infections. If any antimicrobial treatment is already administered, it should be stopped and the diagnostic procedure should be performed only 10-14 later. Perioperative prophylaxis (30 minutes before the incision) should only administered if a prosthesis is implanted. No antimicrobial prophylaxis should be administered to avoid false negative culture results if a pathogen has been not been yet recovered from blood cultures or joint aspiration and the patient is clinically stable. For removed implants a newly published method using sonography to disrupt the biofilms of the surface is more effective to culture the causative pathogens. Treating biofilm-associated infections with antibiotics is often inefficient. Bacteria in biofilms resist killing by antibiotics and disinfectants in comparison to free-floating cells. Because of the resistance mounted to antimicrobial agents in biofims, existing methods for measuring bacterial killing which use planktonic cell cultures are probably not ideal to predict the treatment response. There is, however, not yet a generally accepted method for susceptibility testing within biofilms.

Therapy of implant-associated infections A. Trampuz (Basel, CH) Orthopedic devices are increasingly used to alleviate pain (joint prostheses) and to fixate bone fractures (intramedullary nails, externalfixation pins, plates and screws). Infections associated with orthopedic implants occur rare, but cause significant morbidity and consume substantial healthcare expenditures. Implant-associated infections are typically caused by microorganisms attached to a surface and embedded in an extracellular matrix. This structure is called microbial biofilm, organized as complex communities with structural and functional heterogeneity resembling multicellular organisms. Existence of adhering stationary-phase (biofilm) microorganisms makes implant-associated infections difficult to treat. The antimicrobial susceptibility of biofilm organisms to cell-wall active antibiotics (such as beta-lactams or glycopeptides) may be decreased up to 1000-times compared to the planktonic counterparts. Prosthetic joint infection is defined by the presence >1 of the following criteria: (i) microbial growth from synovial fluid or intraoperative tissue (>2 positive cultures are required for low-virulent organisms), (ii) visible purulence, (ii) acute inflammation on histopathological examination or (iii) presence of a sinus tract. Infections are classified according to the onset of clinical symptoms after implantation into early (<3 months), delayed (3-24 months) or late (>24 months) infections. Early and delayed infections are usually acquired during implant surgery (perioperatively), whereas late infections are predominantly acquired by hematogenous seeding. Successful treatment of implant-associated infection requires adequate surgical procedure combined with long-term antimicrobial therapy (3-6 months), ideally with an agent acting on adhering stationary-phase (biofilm) microorganisms. Rifampin has an excellent activity on biofilm staphylococci, but must always be combined with another drug (e.g. quinolones, beta-lactams, glycopeptides, lipopeptides, oxazolidinones) to prevent emergence of resistance. Surgical modalities include debridement with retention of the prosthesis, one-stage or two-stage exchange, resection arthroplasty, arthrodesis and amputation. Debridement with retention is a reasonable option, if the duration of clinical signs and symptoms <3 weeks, the implant is stable, the

soft tissue is in good condition, and an agent with activity against biofilm microorganisms is available. In the presentation, current treatment guidelines and new developments will be reviewed.

Genomics in sepsis -update 2008 J. Lohmeyer (Giessen, DE) Morbidity and mortality to infection including sepsis has been found to be strongly influenced by the genetic background in animals and human populations, but the polygenic traits underlying disease susceptibility are complex and poorly understood. Polymorphisms found to be associated with sepsis development and outcome have been inconsistent with frequent failures to replicate positive findings. For polygenic diseases, like sepsis and critical illness, unbiased genomic approaches like genomewide genetic analyses and expression profiling were expected to overcome the limitations of conventional reductionist approaches focused on the contribution of individual genes and proteins. Therefore, a broad range of functional genomic approaches for host and pathogens including bioinformatically predictive and comparative genome analysis are currently applied to identify new host and microbial molecules that mediate pathogenicity in both systemic and local infection. Indeed, transcriptome analyses have been used successfully to identify new candidate gene involved in the host inflammatory response. In clinical sepsis settings progress was slower as expected due to a number of both theoretical and technical hurdles. However, with the standardization of target cell collection, nucleic acid isolation and microarray analysis and the development of new statistical approaches gene expression profiling seems now ready to be re-evaluated in complex clinical settings like sepsis. Adequately powered studies in well documented clinical sepsis cohorts are needed to identify surrogate genetic markers or gene expression profiles suitable as risk factors for clinical use in septic patients. It remains to be found out whether whole blood approaches, blood cell subset gene profiles or other tissue samples are most informative and how many genes have to be included in a ´sepchip´ approach. Signatures based on mRNA profiles will have to compete with the dynamic development of proteomic approaches as sepsis biomarkers. All these approaches aim at adapting sepsis therapy according to the patient´s genetic background and inflammatory status to overcome the strategic shortcomings of current clinical sepsis care.

Fokus HCV: Immunologie

Virus-spezifische T-Zellen spielen eine entscheidende Rolle im natürlichen Verlauf (Viruselimination bzw. -persistenz) und der Pathogenese der Hepatitis-C-Virus (HCV)-Infektion. Die akute, selbst-limitierte HCV-Infektion ist mit einer multispezifischen und starken T-Zellantwort assoziiert, die gegen verschiedene virale Proteine gerichtet ist. Die Mechanismen, die zum Versagen der T-Zellantwort und somit zur häufigen Viruspersistenz beitragen, sind bisher erst wenig charakterisiert. Diskutiert werden z. B. ein primäres T-Zellversagen oder T-Zellerschöpfung, virale Escapemutationen sowie T-Zell-Dysfunktionen. T-zell Dysfunktionen können u.a. durch eine fehlende Hilfe von CD4+ Zellen oder durch direkte suppressive Wirkungen von regulatorischen T-Zellen oder des Interleukins 10 bedingt sein. Auch die genetische Restriktion der Immunantwort spielt eine wichtige Rolle im Ausgang der Infektion, so ist z.B. HLA-B27 häufig mit einer spontanen Elimination des Virus assoziiert. Bedeutenderweise haben die Erkenntnisse über die entscheidende Rolle von Virus-spezifischen T-Zellen bereits zu ersten immunprophylaktischen und therapeutischen Ansätzen in der Klinik geführt. Ein besseres Verständnis der immunologischen Mechanismen von Viruselimination bzw. -persistenz ist Grundvoraussetzung für die Weiterentwicklung dieser prophylaktischen und therapeutischen Immunstrategien, bevor sie Eingang in den klinischen Alltag finden können.

T.F. Baumert (Strasbourg, FR) With an estimated 170 million infected individuals, hepatitis C virus (HCV) has a major impact on public health. A vaccine protecting against HCV infection is not available and current antiviral therapies are characterized by limited efficacy, high costs and substantial side effects. Binding of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between virus and the target cell that is required for the initiation of infection. Since this step represents a critical determinant of tissue tropism and pathogenesis, it is a major target for host cell responses such as antibody-mediated virus-neutralization -and a promising target for a new antiviral therapy. The recent development of novel tissue culture model systems for the study of the first steps of HCV infection has allowed rapid progress in the understanding of the molecular mechanisms of HCV binding and entry. Several host cell molecules have been identified as HCV co-entry factors: these include heparan sulfate, CD81, scavenger receptor BI and claudin-1. This presentation summarizes the impact of recently identified viral and host cell factors for HCV attachment and entry. Furthermore, clinical implications of this important process for the pathogenesis of HCV infection and novel therapeutic interventions are discussed.

Parvovirus B19 infection during pregnancya complex of problems S. Modrow (Regensburg, DE) Introduction: Parvovirus B19 (B19V) is the causative agent of erythrema infectiosum, a generally benign and self-limiting rash disease associated with anemia. In addition some patients develop severe long-lasting symptoms as arthritis and arthalgia. Hepatitis, encephalitis, meningitis and myocarditis are rarely described complications. In seronegative pregnant women acute B19V-infections might result in fetal death and/or hydrops fetalis. Since the majority of infections occur during childhood an increased risk is anticipated for the development of complications in seronegative pregnant women working in occupational fields with frequent contacts to children, particularly in nursery and elementary school teachers as well as in children's medical units. In consequence seronegative women with occupational contacts to children are banned from employment during pregnancy. Methods: To obtain an overview of the epidemiological situation and to identify groups with high risk for B19V-infection we analyzed the Infection Abstracts KIT2008

seroprevalence of B19V using the collection of 6,583 sera of the German National Health Survey that is representative for the population in Germany and of 649 sera from healthy Thuringian children and adolescents. Results: In adults the overall seroprevalence of B19V was 72.1%, rising from 20.4% in children (1-3 years) and 66.9% in adolescents (18-19 years) to 79.1% in the elderly (65-69 years). Significant differences were observed between females (73.3%) and males (70.9%) and inhabitants of small (74.8%) and big cities (69.0%). For women during child-bearing age (18-49 years) highest values were observed for those living in joint households together with 2 or more children (81.6%) and in women with occupational contact to children below 6 years (88.9%). In contrast to these groups seroprevalence was significantly lower in age-matched female singles (64.8%) and in women with occupational contact to children above 6 years and adolescents (63.8%). Conclusion: High risk for B19V-infection is evident in women working in kindergarden or nursery schools with frequent contact to children younger than 6 years and in women in families with two or more children. In health care workers and in school teachers (elementary and secondary school) seroprevalence to the one observed in the agematched female population in Germany. The study might be used as a basis for the consideration of problems associated with B19V-infections in maternity protection and public health.

Prenatal and perinatal enterovirus infections: a significant risk for mother and child?

A. Heim (Hannover, DE) Perinatal and neonatal enterovirus infections are significant causes of morbidity (e. g. myocarditis, hepatitis, meningo-encephalitis and virus-"sepsis") and mortality of newborns, whereas disease manifestation of the mother is usually mild or subclinical. Mother to child transmissions and nosocomial transmissions on neonatal wards are facilitated by shedding of enterovirus with feces and by its resistance to detergents and many non-virucidal disinfectants. By contrast, the clinical significance of prenatal enterovirus infection has not yet been completely established. Therefore, the association of prenatal enterovirus infections with malformations and intrauterine fetal death will be reviewed in more detail.

Recently, a few studies demonstrated enterovirus infection of the placenta and the fetus during the second and third trimester in cases of intrauterine fetal death by PCR, in situ hybridization, in situ PCR or immunohistochemistry, whereas control placenta specimens were enterovirus negative (Satosar 2004; Nuovo 2005; Petersson 2004 ). Enterovirus was also found frequently by PCR (7-12%) and virus isolation (1.2%) in amniotic fluid samples of fetuses with abnormalities detected by prenatal sonography, e.g. non immune hydrops (Veyver, 1998; Reddy 2005 , Petrikovsky, 2003 . However, amniotic fluid samples of healthy fetuses were also occasionally positive (2 of 44 samples) for enterovirus RNA by PCR suggesting either asymptomatic infections or contaminations (Baschat, 2003) . Moreover, a clear association between enterovirus detection and a specific malformation syndrome has not yet been established. Association of enterovirus infections of the placenta with fetal mortality and severe respiratory distress of the newborn was suggested (Satosar, 2004; Euscher, 2001; Garcia 1991) .

In addition, several case reports described typical symptoms of postnatal enterovirus infections (e.g. meningo-encephalitis, skin lesions, hepatitis) in newborns presumably infected in the third trimester.

As the incidence of enterovirus infections is high in the average population (and also in pregnant women), the individual risk of placental transmission and fetal enterovirus disease seems to be only low in cases of enterovirus infections of pregnant women (Amstey, 1988) . However, the high incidence of enterovirus RNA detection in studies on intrauterine fetal death, and to a lower extent also in studies on fetuses with abnormalities, urgently requires future studies on the topic of prenatal enterovirus infections.

Varicella during pregnancy and in newborn infants P. Wutzler (Jena, DE) Varicella is rare in pregnancy. Only 3-7% of women of child-bearing age in Europe are susceptible to primary infection with the varicellazoster virus (VZV). However, varicella during pregnancy can be associated with severe illnesses for both the mother and her neonate.

The consequences for the in-fant depend on the time of maternal disease. They range from asymptomatic infection to fetal loss especially in case of severe maternal disease. The varicella pneumonia is the most common serious maternal complication in pregnancy and seems to occur more often in the third trimester. The disease must be regarded as medical emergency since pregnant women are at risk of life-threatening ventilatory compromise and death. After maternal chickenpox in the first and second trimesters, the congenital varicella syndrome may occur in nearly 2%. The characteristic symptoms consist of skin lesions in dermatomal distribution, neurological defects, eye diseases, and skeletal anomalies. Nearly 30% of infants die during the first months of life. If the mother develops the varicella rash between day 4 (5) ante partum and day 2 post partum, generalized neonatal varicella leading to death in about 20% of the cases has to be expected. Normal zoster has not been shown to be associated with maternal pneumonia, birth defects or problems in the perinatal period. Intrauterine VZV infection can be evidenced by virological and serological methods. The causal relationship between maternal varicella infection and congenital abnormalities is verified most convincingly by detection of the viral DNA in the infant. An effective prophylaxis of varicella in pregnant women is only possible by active immunization of seronegative women before pregnancy. Passive immunization of the mother has been recommended for prevention of severe maternal varicella, but there is no evidence that this prevents fetal disease. At first signs of varicella pneumonia or other disseminated infections, an antiviral treatment with aciclovir has immediately to be introduced. Passive immunization of the newborn is indicated to prevent severe neonatal chickenpox. If varicella occurs, aciclovir treatment has to be administered promptly.

A. Peschel (Tübingen, DE) Defensin and bacteriocin-like endogenous cationic antimicrobial peptides (CAMPs) belong to the most ancient and efficient components of host defense. It appears enigmatic that bacteria have not been able to develop highly effective CAMP resistance mechanisms such as those that thwart most therapeutic antibiotics. We propose that CAMPs and CAMP resistance mechanisms have co-evolved leading to a transient host/pathogen balance that shapes the existing CAMP repertoires. In particular, CAMPs appear to exhibit unique modes of action and target specificities that have been particularly difficult for bacteria to overcome. Elucidating and harnessing the underlying principles of CAMP-mediated host defense could be of pivotal importance in the development of more sustainable antibiotics. In fact, several CAMP-like molecules have yielded promising results in clinical trials or, in the case of daptomycin, are already successfully used in antimicrobial therapy. Weidenmaier C, Kristian SA, Peschel A (2003) Curr Drug Targets 4:643-649 Peschel A, Sahl HG (2006) Nat Rev Microbiol 4: 529-36 Infection Abstracts KIT2008

Common variable Immunodeficiency -a clinical update on the most common primary immunodeficiency K. Warnatz (Freiburg, DE) The unusual frequency or unusual nature of infections in one individual is the clinical hallmark of immunodeficiency. Common variable immunodeficiency (CVID) represents the most common primary immunodeficiency. While most cases of CVID are sporadic about 20% show a familial trait. Four underlying genetic defects have been identified within recent years (TACI, ICOS, BAFF-R, CD19) accounting for about 10% of CVID. CVID is defined as a heterogeneous antibody deficiency syndrome characterized by recurrent upper and lower respiratory (ca. 90% of patients) as well as gastrointestinal bacterial infections (ca. 40% of patients). Especially infections due to encapsulated bacteria are frequent. Susceptibility to viral infections is not a general trait, but seems to define a subgroup of CVID patients. Recently, HHV8 infection was identified in CVID patients with granulomatous and interstitial lung disease. It remains open whether the viral infection is cause or result of the underlying immunodeficiency. Similarly, some patients suffer from recurrent CMV reactivation. Fungal infections are not common among patients with antibody deficiency. Among parasitic infections giardiasis and enteritis due to cryptosporidia or microsporidia need to be excluded in CVID patients presenting with diarrhoea. The sometimes difficult clinical care for these patients requires a close interaction between immunologists and infectious disease specialists for the prevention and treatment of infection.

The immune reconstitution inflammatory syndrome in HIV-therapy G. Behrens (Hannover, DE) Antiretroviral combination therapy results in suppression of viral replication leading to effective immune reconstitution of previously immuno-compromised HIV-patients. Depending on the cohort studied, up to 25% of HIV-patients with advance cellular immunodeficiency present signs and symptoms of a pathological immune reaction against infectious or self antigens weeks or months after initiation of HIVtherapy. This abnormal antigen-specific immune reaction is frequently termed "immune reconstitution inflammatory syndrome" (IRIS) and is associated with atypical clinical manifestations. Among various bacterial and viral infectious diseases, cryptococcal meningitis and mycobacterial infections are most frequently associated with the development of an IRIS. Patients with IRIS due to M. tuberculosis present often develop new pulmonary infiltrations or focal and mediastinal lymphadenitis. IRIS against MAC are characterised by focal lymphadenitis or granulomas mainly in the abdomen together with fever. Observation data suggest that it might be beneficial to first treat mycobacterial or cryptococcal infections before initiating HIV-therapy, if the opportunistic infections are diagnosed. In cases of IRIS, treatment with corticosteroids or antiphlogistic drugs has been described as effective although no controlled trails have been performed. Interruption of HIV-therapy is rarely required and should be avoided. The etiology of IRIS remains obscure but significantly enhanced antigenspecific CD4+ T-lymphocyte responses have been described in patients at time of IRIS. Additional genetic risk factors have been proposed.

Alte und neue Immunsuppressiva K. Resch (Hannover, DE) Immunsuppressiva greifen vor allen an den zentralen Regulatorzellen adaptiver Immunreaktionen, die T-Helfer-Lymphozyten (CD4-positive Zellen), an. Antimetabolite, wie die älteren Pharmaka Azathioprin oder Methotrexat, oder auch neue Immunsuppressiva, wie Leflunomid oder Mycophenolat, hemmen als Zytostatika die Proliferation von Lymphozyten. Glucocorticoide, z.B. Prednisolon, wirken auf andere Weise immunsuppressiv: Sie verhindern in hohen Konzentrationen die Aktivierung von T-Lymphozyten. Mit Einführung von Ciclosporin wurde dieser Mechanismus als alleiniges immunsuppressives Therapieprinzip verfügbar, später gefolgt von dem sehr ähnlich wirkenden Tacrolimus. Beide hemmen die für die Weiterleitung des Aktivierungssignals in T-Lymphozyten wichtige Phosphatase Calcineurin und dadurch vor allem die Synthese des Wachstumsfaktors Interleukin-2. Sirolimus (Rapamycin) und sein Derivat Everolimus hemmen die Proteinkinase mTOR ("mammalian target of rapamycin"), die in den Signalweg eingeschaltet ist, durch den Interleukin-2 die Zellteilung in T-Lymphozyten vorantreibt. Hemmung der IL-2-Synthese wie Hemmung der IL-2-Wirklung verhindern gleichermaßen -und synergistisch -die Vermehrung der gegen ein Antigen gerichteten (T-) Lymphozyten ("klonale Expansion"). T-Helfer-Lymphozyten reagieren nur auf Antigen-Peptide, die von Zellen (Dendritischen Zellen, B-Lymphozyten) präsentiert werden. Zur Aktivierung benötigen sie zusätzliche kostimulatorische Signale. Abatacept -ein Konstrukt aus der extrazellulären Domäne des negativen (= inhibitorischen) Kostimulator-Rezeptors CTLA4 und IgG -blockiert wirksam die Kostimulation. Rituximab, ein Antikörper gegen CD20, zerstört selektiv B-Lymphozyten. Neben der Verminderung der Synthese von Antikörpern sind auch T-Lymphozyten durch Verminderung der Antigen-Präsentation betroffen. Immunologische Immunsuppressiva -Antikörper gegen Aktivierungsrezeptoren von T-Lymphozyten -verhindern ebenfalls selektiv die Aktivierung und klonale Expansion. Muromonab CD3 regelt den Antigen-Rezeptor herab; Basiliximab und Daclizumab blockieren den Rezeptor für Interleukin-2. Eine größere Zahl weiterer monoklonaler Antikörper befinden sich in klinischer Prüfung, z.B. Keliximab, das gegen das CD4-Molekül der T-Helfer-Lymphozyten gerichtet ist. Die Aktivierung von T-Lymphozyten erfolgt in den regionalen Lymphknoten. Von dort müssen sie über das Blut in die betroffenen Organe einwandern. Efalizumab, ein humanisierter monoklonaler Antikörper gegen eine Untereinheit des Leukozytenfunktionsantigens 1 (LFA-1), verhindert die Bindung an den Bindungspartner von Gefäßendothelzellen und damit das Auswandern ins Gewebe. Natalizumab hemmt ein anderes Integrin mit ähnlichen Folgen. Auch hier befinden sich mehrere Pharmaka in klinischer Prüfung, darunter Derivate von FTY720, einem Analogon von Sphingosin-1-Phosphat, das das Auswandern aktivierter T-Lymphozyten aus den Lymphknoten verhindert.

Immunantwort im Rahmen der HPV-Impfung A. Kaufmann (Berlin, DE) Vaccination strategies against Human Papillomavirus (HPV) induce immune responses that prevent primary infection (prophylaxis) or treat an ongoing infection, or premalignant or malignant disease (therapy) with the aim to prevent cervical cancer. For prophylaxis virusneutralizing antibodies have to be induced that are secreted into epithelia and mucus. They bind to virus particles and prevent initial binding and infection of target cells. Prophylactic HPV vaccines induce Infection Abstracts KIT2008 specific serum IgG in extremely high concentrations. These antibodies correlate with protection. All clinical studies with the virus-like-particle (VLP)-based HPV vaccines measured antibody titers that were 100fold higher than after natural infection. After initial decline until month 18 post vaccination course, titers stabilized above/around natural titers. These concentrations were detectable until 5 years. Seroconversion rate and effectivity of the vaccination was up to 100%. This is true for both vaccines on the market and for HPV16 and 18 as well as HPV6 and 11 infections, depending on the vaccine. Duration of long term protection has to be demonstrated. Vaccination of children 9-15 years of age resulted in higher titers as compared to young women. This may translate into a longer lasting protection until age of highest risk. Vaccination of women until age of 55 showed lower but comparable titers. Cross protection to non-vaccine HPV types could be demonstrated e.g. to closely related HPV31 and 45. Importantly immunologic memory was demonstrated by a booster vaccination after 5 years reaching initial antibody titers. Few studies have been conducted measuring T cell responses to prophylactic vaccines. Strong proliferative and cytokine responses can be demonstrated in vaccinees. Therapeutic vaccines were tested in about 30 published clinical trials.

Immunogenicity and safety was regularly demonstrated. In in vitro experiments and animal tumor regression models effector responses were demonstrated. In patients convincing results are lacking although tumor-infiltrating cytotoxic T cells can be found. Immunoevasion and immunosuppression by tumor cells hinder effectivity of this strategy. Therefore, HPV prophylaxis is the most promising measure to date until therapeutic vaccines are available.

Leitlinie HPV-Impfung G.E. Gross (Rostock, DE) Die Kernaussagen der im Rahmen des Konsensusverfahrens entstandenen AWMF-S 3-Leitlinie zu "Impfprävention HPV assoziierter Neoplasien" sind: Sowohl die HPV 6, 11, 16, 18-Virus-like-Particle (VLP)-Impfung (Quadrivalente HPV-Impfung) als auch die HPV16, 18-VLP-Impfung (Bivalente HPV-Impfung) werden für Mädchen zwischen 9 und 17 Jahren empfohlen. Die drei notwendigen Impfdosen sollten möglichst vor dem ersten Geschlechtsverkehr vollständig verabreicht worden sein. Bei Frauen über 17 Jahren sollte in einem individuellen Gespräch die Nutzen-Risiko-Kosten-Abwägung diskutiert werden und eine Entscheidungsfindung ermöglicht werden. Eine eindeutige und allgemeine Empfehlung kann für diese Altersgruppe nicht gegeben werden. Die Wirksamkeit der Impfung hinsichtlich der Verhinderung der HPV16/18 -positiven Vorstufen von Gebärmutterhalskrebs (cervikale intraepitheliale Neoplasie, CIN) ist für HPV16/18-DNA-negative Frauen (9 Studien mit Evidenzgrad A2 -EN1) überzeugend nachgewiesen worden. Der Impfstoff schützt auch vor den impftypassoziierten vulvären und vaginalen intraepithelialen Neoplasien (VIN, VAIN). Die Impfung von männlichen Jugendlichen mit dem Ziel die CIN zu verhindern, wird in Deutschland nur dann als erforderlich angesehen, falls durch die Vakzinierung von weiblichen Jugendlichen keine hohe Durchimpfungsrate erreicht werden kann. Für die quadrivalente Impfung wurde die Wirksamkeit bezüglich des Schutzes vor den zu über 90% durch HPV6-und HPV11-verursachten Genitalwarzen (Condylomata acuminata) bei HPV 6-und HPV11negativen Frauen eindeutig gezeigt (3 Studien mit Evidenzgrad A2-EN1). Zuverlässige Daten hinsichtlich Wirksamkeit und Sicherheit des Impfstoffes wurden über Studien an 15-25-jährigen Frauen gewonnen. Die Impfung mit dem gegen HPV6-und HPV11 gerichteten quadrivalenten HPV 6, 11, 16, 18 Impfstoff sollte ebenfalls bereits zwischen dem 9. und 17. Lebensjahr erfolgen und möglichst vor dem ersten Geschlechtsverkehr abgeschlossen sein. Für männliche Jugendliche wurde bisher die Immunogenität des quadrivalenten Impfstoffes gezeigt. Ergebnisse aus klinischen Unter-

Abstracts KIT2008

Proteolytic destruction of complement as an immune evasion mechanism in cerebral aspergillosis G. Rambach, D. Dum, I. Mohsenipour, M. Dierich, C. Speth (Innsbruck, AT) Background: Spreading of the fungus into the central nervous system (CNS) is a frequent and lethal form of invasive aspergillosis. The complement system is a central component of the local immune defence in the CNS. To clarify the reason for the inefficiency of complement to eliminate the invading fungus we studied Aspergillus-induced complement degradation as a putative evasion strategy. Furthermore we tried to develop strategies to interfere with this evasion mechanism. Methods: Aspergillus was grown in Sabouraud medium or cerebrospinal fluid (CSF), with or without supplements. Complement degradation was investigated by Western Blot. Hyphal opsonization was examined by immunofluorescence. Cellular expression of complement receptors was quantified by FACS. Results: Aspergillus grown in cerebrospinal fluid secretes proteases in a time-dependent manner. These proteases were able to degrade various soluble complement factors, thus interfering with all three complement activation pathways. Furthermore the expression of cell membrane-anchored complement proteins on immune cells decreased after incubation with the fungal supernatants. The amount of the released proteolytic potency was dependent on the Aspergillus species with A. fumigatus producing the highest concentrations of the proteases. We examined two possible strategies to interfere with the Aspergillusinduced complement degradation. First we tested several protease inhibitors and found a significant decrease of complement degradation using the serine protease inhibitor PMSF; other cocktails of inhibitors did not show any effect. As the growth in Sabouraud medium completely abolished the proteolytic activity, we investigated the dependence of protease secretion on the presence of several nutrients. Supplementation of CSF with various nitrogen sources diminished the production of complement-degrading proteases in a dose-dependent manner. Glutamine, a naturally occurring amino acid, was one of the most potent nitrogen compounds.

The production of complement-degrading enzymes and subsequent destruction of soluble and membrane-bound complement factors are likely to constitute an important evasion mechanism and thus to contribute to the high lethality of cerebral aspergillosis. This offers new possibilities for supportive antifungal therapy. Protease inhibitors could prevent complement demolition, and the supply of nitrogen or other nutrients might abolish the secretion of proteases.

Mycotoxins in cerebral aspergillosis: virulence factors and therapeutic targets C. Speth, C. Kupfahl, M. Hagleitner, M.C. Deutinger, G. Rambach, I. Mohsenipour, M.P. Dierich (Innsbruck, AT; Heidelberg, DE) Objective: The high lethality of cerebral aspergillosis urgently asks for a deeper insight into the pathogenic mechanisms of this disease and for new therapeutic approaches which help the local immunity in the brain to cope with the infiltrating fungus. Therefore we studied whether secreted mycotoxins contribute to neural damage and inefficient immune response in cerebral aspergillosis and thus provide a putative target for a supportive antifungal therapy. Methods: Different Aspergillus-derived mycotoxins were tested for their effect on the viability and proliferation of astrocytes, neurons and microglia using MTS assays. The secretion of gliotoxin was measured using HPLC and tandem mass spectrometry. Phagocytic activity of cells in the presence of mycotoxins was quantified with fluorescent latex beads and subsequent microscopy; oxidative burst was studied by FACS. Results: Pathogenic Aspergillus species secrete a variety of mycotoxins including patulin, gliotoxin, verruculogen, fumitremorgin C and citrinin. We measured a direct toxic effect of these substances on astrocytes, neurons and microglia, and could show that all mycotoxins affect their viability. Different brain cells differed in their susceptibility towards the fungal substances with neurons and microglia being the most sensitive cell types. In addition we demonstrated that Aspergillus fumigatus is able to produce and secrete gliotoxin when cultivated in cerebrospinal fluid; the produced amounts were sufficient to induce a significant toxic effect on all brain cell types. Furthermore, we used subtoxic concentrations of the mycotoxins to study a putative modulation of the immunological activity of local and infiltrating immune cell types; their dysfunction induced by the fungal compounds might contribute to the failure in antimicrobial defence and consequently to the high lethality. First experiments showed that infiltrating peripheral immune cells revealed a diminished capacity of antigen presentation and phagocytosis after incubation with the mycotoxins.

A number of neuroprotective substances is already used routinely in different neurodegenerative diseases, or is at least tested successfully in animal models. We performed some tests with these substances in order to protect the cells from mycotoxin-induced damage. Carnitin proved to be the most promising compound and could at least partly inhibit the mycotoxins-induced decrease in cell viability.

These data indicate an essential contribution of mycotoxins in the pathogenesis of cerebral aspergillosis. As a therapeutic approach diverse neuroprotective substances known to be generally effective in neurodegenerative diseases might be used to neutralize the toxic or immune-inhibitory activity of the mycotoxins.

Changes in the epidemiology of invasive pulmonary aspergillosis after introduction of posaconazole prophylaxis in AML/MDS patients J. Vehreschild, R. Sims, A. Stollorz, S. Prasse, M. Rüping, F. Farowski, P. Frommolt, M. Hallek, O. Cornely (Cologne, DE) Introduction: Long-term neutropenic patients are at high risk of contracting invasive fungal infections (IFI). Large randomized controlled trials have shown significant reductions of morbidity and mortality by antifungal prophylaxis with posaconazole. However, the value of prophylaxis has been questioned in centers with preemptive treatment strategies and a low basic incidence of IFI. We conducted a prospective cohort study to document the impact of posaconazole prophylaxis in our centre. Methods: Neutropenic episodes of patients with de novo or relapsed acute myelogenous leukemia (AML) and myelodysplastic syndrome Infection Abstracts KIT2008

(MDS) admitted for remission induction chemotherapy between January 2003 and April 2007 were included. Posaconazole prophylaxis was introduced in January 2006. We acquired descriptive data to assess group homogeneity as well as efficacy parameters including occurrence and duration of fever, imaging results as well as serological and microbiological findings. No major changes of diagnostic and treatment standards other than the introduction of posaconazole prophylaxis were introduced during the course of the trial. Results: Overall, 98 patients were included, 58 received no systemic prophylaxis while 40 received posaconazole prophylaxis. Both groups were well matched for gender, age, hematopoietic growth factors, duration of neutropenia, length of stay, and stage of disease. Lung infiltrates occurred in 39 (67.2 %) and 11 (27.5 %) patients, respectively (P< 0.001, Chi-square test), 21 (36.2 %) and 3 (7.5 %) of these showed typical radiological signs of invasive pulmonary aspergillosis (IPA) (P= 0.001, Chi-square test). Incidence of probable IPA according to EORTC/MSG criteria was 9 (15.5 %) and 1 (2.5 %), respectively (P= 0.034, Fisher's exact test). Kaplan-Meier estimate of one year survival was 58.6 % and 70.1 %, respectively (NS). Conclusion: Even a small cohort of patients sufficed to demonstrate a significant reduction of patient morbidity following introduction of posaconazole prophylaxis. Incidences of lung infiltrates and IFI were substantially improved in patients receiving posaconazole oral suspension. There is a trend towards an improved one year survival following the intervention. Posaconazole prophylaxis should be routinely administered during AML/MDS induction chemotherapy.

Prolonged Posaconazole Exposure and Emergence of multiple-triazole-resistant Candida glabrata S. Weiler, C. Lass-Flörl, J. Auberger, M. Stein, M. Joannidis, R. Bellmann (Innsbruck, AT) Introduction: Posaconazole is a new triazole with an extended spectrum of antifungal activity, which is increasingly used for the prophylactic administration in high risk patients. Patients and Methods: Two cases of multiple-triazole resistance in hematologic patients, who have been enduringly exposed to posaconazole, are reported. Patient 1 is a 20-year-old woman with acute myelogenous leukemia. During aplasia the patient received amphotericin B colloidal dispersion, followed by posaconazole for antifungal prophylaxis. Later the patient was readmitted to the hospital and transferred to the ICU because of severe sepsis. Empiric antibiotic treatment was started with broad-spectrum antibiotics and posaconazole prophylaxis was continued. After an initial improvement, the patient's condition worsened again. At this time, multiple-triazole resistant Candida glabrata was cultivated from blood and gastric juice. Therefore, posaconazole was substituted by amphotericin B colloidal dispersion and caspofungin was added. Patient 2 is a 39 year old male suffering from acute myeloid leukaemia. A pulmonary mycosis was successfully treated with posaconazole. During episodes of aplasia the patient received posaconazole again for prophylaxis. After ten months, the patient was readmitted to the hospital because of a relapse of his underlying disease. Due to a blood stream infection broad-spectrum antibiotics were initiated together with amphotericin B colloidal dispersion. Three weeks later posaconazole was added. The patient was transferred to the ICU as he developed ARDS. Two positive blood cultures revealed multiple-triazole resistant Candida glabrata. The central venous catheter was removed. The dosage of amphotericin B colloidal dispersion was increased and therapy with caspofungin was started. Administration of posaconazole was stopped. Results: After prolonged Posaconazole exposure Candida glabrata isolates from both patients were resistant to posaconazole, fluconazole, itraconazole and sensitive to amphotericin B, caspofungin and voriconazole. Antifungal treatment was adjusted to susceptibility patterns of the clinical isolates. Patient 1 was treated successfully. Patient 2 developed a prolonged septic shock and died due to multiorgan failure.

Multiple-resistant fungal infections following broad-spectrum azole exposure may occur along with the intensified antifungal application.

Fungiscope -the Global Rare Fungal Infection Registry M.J. Rueping, J.J. Vehreschild, C. Beisel, U. Auerbach, C. Mueller, C. Wickenhauser, O. Cornely (Cologne, DE) Introduction: The incidence of invasive fungal infections is increasing worldwide and rare fungi -neither belonging to the genera Aspergillus, Candida, Pneumocystis or Cryptococcus, nor being endemic or regional, such as Histoplasma spp. 

Pathogens: across borders, without frontiers T. Grein (Genf, CH) The world has changed dramatically since 1951, when WHO issued its first set of legally binding regulations aimed at preventing the international spread of disease. At that time, the disease situation was relatively stable. Concern focused on six "quarantinable" diseases: cholera, plague, relapsing fever, smallpox, typhus and yellow fever. New diseases were rare, and miracle drugs had revolutionized the care of many well-known infections. People travelled internationally by ship, and news travelled by telegram.

Since then, profound changes have occurred in the way humanity inhabits the planet. Population growth, incursion into previously uninhabited areas, rapid urbanization, intensive farming practices, envi-

ronmental degradation, and the misuse of antimicrobials have disrupted the equilibrium of the microbial world. Mainstay antimicrobials are failing at a rate that outpaces the development of replacement drugs. New diseases are emerging at the historically unprecedented rate of one per year. Airlines now carry more than 2 billion passengers annually, vastly increasing opportunities for the rapid international spread of infectious agents and their vectors. Industrialization of food production and processing, and globalization of marketing and distribution mean that a single tainted ingredient can lead to the contamination of tons of food items in scores of countries. Dependence on chemicals has increased, as has awareness of the potential hazards for health and the environment. These threats have become a much larger menace in a world characterized by high mobility, economic interdependence and electronic interconnectedness. Traditional defences at national borders cannot protect against the invasion of a disease or vector. Real time news allows panic to spread with equal ease. Shocks to health reverberate as shocks to economies and business continuity in areas well beyond the affected site.

To address these issues, global public health security is required -the reduced vulnerability of populations to such and other acute threats to health. One of the pillars of global health security is the revised International Health Regulations (2005). These Regulations are an international legal instrument designed to achieve maximum security against the international spread of diseases. They also aim to reduce the international impact of public health emergencies. The IHR (2005) expand the focus of collective defence from just a few "quarantinable" diseases to include any emergency with international repercussions for health, including outbreaks of emerging and epidemic-prone diseases, outbreaks of foodborne disease, natural disasters, and chemical or radionuclear events, whether accidental or caused deliberately. In a significant departure from the past, IHR (2005) move away from a focus on passive barriers at borders, airports and seaports to a strategy of proactive risk management. This strategy aims to detect an event early and stop it at its source -before it has a chance to become an international threat. Today, the public health security of all countries depends on the capacity of each to act effectively and contribute to the security of all. Therefore, the first steps required are to develop core detection and response capacities in all countries, and to maintain new levels of cooperation between countries to improve risk management. This entails countries strengthening their health systems and ensuring they have the capacity to prevent and control epidemics that can quickly spread across borders and even across continents. Where countries are unable to achieve prevention and control by themselves, it means providing rapid, expert international disease surveillance and response networks to assist them -and making sure these mesh together into an efficient safety net. (WHO, 2002) . Although both forms of trypanosomosis (the term "trypanosomiasis" has to be considered as obsolete and should be replaced by trypanosomosis) belong to the most serious parasitic diseases of man, they are attributed to the "negligible infectious diseases" nowadays, which is presumably due to the fact that patients suffering from sleeping sickness or from Chagas disease are very rarely diagnosed in (Central) European countries. (In Austria no case of sleeping sickness has been documented (at least) during the last 29 years.) Thus, this paper is dedicated to these two trypanosomoses and will give a synoptic overview on the epidemiology, nosology, diagnosis, therapy and prophylaxis of these serious parasitic diseases. References World Health Organization (2002) 

For debate: antibacterial and antifungal chemoprophylaxis in cancer patients -pro antibiotics W.V. Kern (Freiburg, DE) Antibacterial chemoprophylaxis in cancer patients with neutropenia has already been studied in the late 60s. It was not until the availability of cotrimoxazole and, later, fluoroquinolones that benefits in terms of morbidity (less fever episodes and/or less defined infections) have been convincingly shown in studies powered to do so. Major drawbacks have been and still are the development of resistance. The trade-off between benefits and this adverse effect, however, has often been discussed based on emotions rather than based on data. We updated a recent metaanalysis of antibacterial chemoprophylaxis in cancer patients by adding the results of two large trials (Cullen et al. NEJM 2006 , Bucaneve et al. NEJM 2006 . The updated meta-analysis (Leibovici et al. Cancer 2006) shows that antibiotic prophylaxis in neutropenic patients does not only reduce febrile episodes and bacterial infections, but can also reduce mortality. For high-risk patients, prophylaxis with fluoroquinolones diminished the risk of death from any cause by 33% (95% CI 2% -54%). Thus, 55 patients with acute leukemia or stem cell transplantation need to be given prophylaxis to prevent one death. One of the two recent large trials was conducted in a population with nearly 20% resistance in gram-negative isolates, and yet, showed benefits. Similar beneficial effects were shown in before-and-after studies conducted in Germany -at a similar level of fluoroquinolone resistance in the community and paients admitted. Now, studies should attempt to define the threshold of resistance at which these beneficial effects by prophylaxis will disappear.

O. Cornely (Cologne, DE) Deferring treatment until proof of diagnosis increases morbidity and decreases survival in immunocompromised hosts. On the opposite, the scientific community achieved major advances by initiating treatment for opportunistic infections as early as possible. Early treatment, i.e.

empiric treatment or prophylaxis were proven beneficial regarding solid trial endpoints, i.e. established diagnosis and death. All these successes have been accomplished primarily in the field of fungal infection, and less so with some viral infections, namely CMV diseases. It is a reasonable step to aim for the same successes in the much more frequent bacterial infections as well. Many attempts have been made in this direction. In times of evidencebased medicine we may constantly ask ourselves: What has been achieved in terms of solid endpoint clinical trials? Historically, trimethoprim/sulfamethoxazole has been used for prophylaxis as well as ß-lactam antibiotics. Today cotrimoxazole use is primarily driven by the risk of Pneumocystis jirovecii pneumonia, e.g. in patients receiving corticosteroids as part of the chemotherapy regimen or in neoplasms coming with a T cell defect. Only few clinical trial groups evaluated ß-lactam prophylaxis, which has been left soon after. Reasons are manifold and include the difficulty to treat breakthrough infections without using ß-lactams. The use of fluoroquinolone prophylaxis has attracted more interest. The largest and most well-designed clinical trials on antibacterial prophylaxis are two placebo controlled levofloxacin trials (Bucaneve NEJM 2005 , Cullen NEJM 2005 . Levofloxacin reduced the incidence of fever, but had no significant effect on mortality. Prophylaxis always means over-treatment of those who would never develop the infection under investigation. Other arguments against prophylaxis include, but are not limited to increases in fungal (Viscoli CID 2001) and Clostridium difficile disease (Biller ICHE 2007) , and increasing bacterial resistance (Kern JAC 2005) . Unfortunately, the shortcomings of antibacterial prophylaxis have not been outweighed by a decrease in mortality, which has not been achieved in any clinical trial so far. Why is antibacterial prophylaxis less successful than antifungal prophylaxis? Bacterial infection is more readily diagnosed than fungal infection, i.e. blood culture versus a complex EORTC/MSG score; empiric treatment is broad spectrum and thus includes >95% of the causative bacteria, and finally, mortality attributable to the infections causing the first fever (early mortality) is very low. At this time the risk benefit ratio does not support the wide spread use of antibacterial prophylaxis.

Hepatitis und Lebertransplantation U. Spengler (Bonn, DE) Terminal cirrhosis and liver cancer in chronic hepatitis B (infection with HBV) and C (infection with HCV) have become major indications for liver transplantation. Both infections tend to regularly reinfect the graft leading to graft loss and the need for re-transplantation in a considerable proportion of patients. Recurrent fibrocholestatic hepatitis under immunosuppressive therapy can complicate transplantation in both types of viral hepatitis and carries a grave prognosis. The recent development of antiviral drugs inhibiting HBV polymerase has decisively improved prognosis in hepatitis B. To minimize the risk of subsequent graft infection treatment should be started when patients enter the waiting list. In selected patients antiviral therapy may result in marked improvement of liver function even in advanced liver disease so that transplantation can be avoided. To prevent infection of the graft after transplantation, re-infection prophylaxis has been established as an effective strategy. Prophylaxis comprising anti-HBs hyperimmunoglobulins and nucleoside analogues must be instituted during transplantation and maintained life-long.

In contrast to hepatitis B, re-infection is still universal in hepatitis C but the clinical course may vary considerably. On average, 20% and 50% of patients will develop cirrhosis in the graft after 5 and 10 years, respectively. Approximately 10% of patients will eventually require re-transplantation. HCV genotype and viral load as well as host-related factors affect the clinical course in recurrent hepatitis C but cannot reliably predict the individual risk of disease progression. Antiviral therapy with interferon and ribavirin markedly improves the prognosis of recurrent hepatitis C but is effective in only 80% and 20% of patients infected with genotypes 2 or 3 versus 1, respectively. In addition, optimal timing and duration of antiviral therapy have not yet been established in patients with liver transplantation. Type and severity of immune suppression further determine the prognosis in recurrent hepatitis C. Thus, prolonged treatment and high-dose pulses with corticosteroids as well as monoclonal antibodies against IL2 receptor or CD3 should be avoided in HCV re-infected patients. Nevertheless liver transplantation is an effective and beneficial treatment option in patients with hepatitis C, but graft re-infection and posttransplant care should best be managed by a physician with special expertise in this field.

Skin, Soft Tissue, Bones

J.-M. Schröder (Kiel, DE) Human skin is covered with microorganisms, but usually not infected. This is achieved by the physical skin barrier, which includes the stratum corneum and lipid layers. In addition, skin is equipped with a "chemical barrier", consisting mainly in antimicrobial peptides (AMPs). Some of the AMPs are constitutively produced, among them the principal healthy skin AMPs lysozyme, RNase-7 and psoriasin. Psoriasin (S100A7) is a very lipophilic protein, which is secreted from keratinocytes and which covers, partly dissolved in lipids, the skin surface. It preferentially kills E. coli, thus protecting us from gramnegative gut bacterial infection. RNase7 is another major skin-AMP, which represents a preferentially Enterococci killing broad spectrum-peptide antibiotic that may control grampositive gut bacterial growth at the skin surface. It is believed, that these AMPs play a major role in the protection of healthy skin. When microbes come into contact with the living epidermis e.g. by a defective physical barrier, these will induce additional AMPs like ß-defensins. Human ß-defensin-2 (hBD-2) is the most abundant inducible human peptide antibiotic, expressed in uppermost epidermal layers upon inflammation and infection. It is a rather gramnegative bacteria-selective peptide antibiotic. Apart from gramnegative bacteria-derived "pathogen-associated molecules", proin-flammatory mediators like IL-1ß and TNF-? or IL-17 and IL-22 have a high capacity to induce hBD-2. Human ß-defensin-3 (hBD-3), which is a broadspectrum AMP, is selectively upregulated by growth factors and important in wound healing. Another inducible AMP is the cathelicidinfragment LL-37, a major AMP in neutrophils, which is also produced by keratinocytes during infection and inflammation. Recent evidence suggests that LL-37 has proinflammatory properties, linking innate and adaptive immunity by formation of stable complexes with DNA, which induce in plasmacytoid dendritic cells IFN-?. As yet, a few skin diseases have been proven to be associated with alterations of AMP-production or activity. These include Psoriasis and Rosacea with increased production of AMPs and Atopic Dermatitis with decreased AMP production, which is possibly important for increased S. aureus infection. Future studies will show, whether in cases of reduced AMP-production an artificial induction of AMPs will Risk and manifestation of Plasmodium falciparum malaria are subject to diverse factors including, among others, transmission intensity, access to treatment, and genetic background of the population. Malaria is the strongest known force of evolutionary selection in the recent history of the human genome, and susceptibility to malaria and disease severity are associated with a wide range of human genetic polymorphisms. In fact, rougly a quarter of the variation in the incidence of malaria in children in sub-Saharan Africa is attributed to host genetics. Polymorphisms protecting against malaria involve genes regulating erythrocyte structure and function, cytoadherence and immune response. Genetic predispostion has also been observed, e.g. in conditions of deficient innate responses to the parasite. For several of these traits, their high frequency in sub-Saharan Africa cannot easily be explained by Haldane's malaria hypothesis, i.e. selection of a genetic trait due to protection from malaria. Respective examples are presented. Potential factors involved in the seemingly paradoxical selection of polymoprphisms disposing to malaria comprise multiple, partially counterbalancing, selective pressures as well as interactions of these with each other and with environmental and parasite variables.

Research on genetic disposition in malaria has the potential to reduce the global burden of disease due to P. falciparum by elucidating mechanisms of disease, susceptibility, and resistance, thereby conveying the development of anti-malarial interventions. However, with only a few exceptions, this has not been achieved so far.

Plasmodial GPI recognition and TLRs in malaria J.P. Cramer (Hamburg, DE) Plasmodium falciparum malaria is confined to blood stage parasites. Host immune response to parasite antigens is essential for parasite clearance but also contributes to disease manifestation. Plasmodial glycosylphosphatidylinositol (GPI) contributes to malaria pathology by induction of excessive cytokine release via Toll-like receptor (TLR) 2. Since GPI is a promising anti-disease vaccine candidate, identification of structural requirements for TLR-activation and understanding the mechanisms underlying GPI-induced parasite clearance is important. Applying synthetic P. falciparum GPI-glycan analogues we identified GPI moieties required for immune activation. On RAW264.7 macrophages, GPI substructures lacking the diacylglycerol moiety were still stimulatory. However, at least four mannose residues were required for TNF-alpha induction. Integration of lipidated GPI into erythrocyte membranes induced increased TNF-alpha and IL-12 compared to pure compound. Assessing the role of TLRs in parasite clearance, MyD88-/-mice infected with non-lethal P. yoelii were unable to control parasites while parasitemia did not differ in TLR2/4/9-/-and wildtype mice. These findings correlated with reduced IL-12p40-and IFNgamma production in MyD88-/-but not in TLR-/-mice. We conclude that the glycan moiety is the immune-stimulatory moiety while the acyl chains confer optimal presentation of the molecule on cell surfaces, and that GPI-vaccination may not interfere with host parasite clearance.

Abstracts KIT2008

Malaria Therapy and Drug Resistance H. Noedl (Vienna, AT) Throughout the last decades malaria therapy has become ever more challenging. Despite considerable efforts to eradicate malaria in the 20th century, malaria remains one of the most prevalent and most devastating tropical diseases. In spite of the promising early results of malaria control, using chloroquine and insecticides, the incidence of malaria increased in many countries largely due to the development and spread of strains of Plasmodium falciparum resistant to chloroquine and subsequently to other antimalarials. Ever since the discovery of the first cases of chloroquine resistance along the Thai-Cambodian border in the late 1950s, Southeast Asia has played a critical role as a focus for the development of antimalarial drug resistance. The onset of chloroquine resistance marked the beginning of a new chapter in the history of malaria in Southeast Asia. In the early sixties spreading chloroquine resistance lead to a significant increase in mortality and by 1973 chloroquine finally had to be replaced by the combination of sulphadoxine and pyrimethamine as first-line drug for the treatment of falciparum malaria in Thailand. This resulted in a short-lived decrease in the incidence of malaria. In the mid 1980s, eventually SP was replaced by mefloquine. The rapid development of resistance to this new drug lead to the introduction of artemisinin as a combination therapy in the mid 1990s. Since then artemisinin-based combination therapies (ACTs) have become an integral part of the treatment of multidrug resistant isolates of P. falciparum throughout the world. Antimalarial drugs in the pipeline include numerous combination partners for ACTs, however, currently there is no alternative to the artemisinins. Data from our recent studies in Cambodia indicate the emergence of the first cases of genuine clinical artemisinin resistance. Although these data suggest that resistance is still confined to a relatively limited area, further studies are needed to determine the rate of increase, geographical extent and potential genetic markers of this emerging problem so that strategies for containment can be determined. While being the most potent and rapidly acting antimalarial drugs, the widespread use of artemisinins in the treatment of P. falciparum malaria makes malaria control more vulnerable to the consequences of drug resistance. In the current situation the spread of artemisinin resistance could very well be the most devastating event in the history of malaria control in the 21st century and could severely impact the ability of many countries to treat falciparum malaria.

Antibiotic resistance of Neisseria gonorrhoeae in Slovenia M. Potocznik (Ljubliana, SI) Gonorrhoea is the second most common bacterial sexually transmitted infection in the EU and in Slovenia. In 2006 35 cases of gonorrhoea were reported, the incidence rate was 1,7/100,000 inhabitants, what is indubitably underreported. 33 of them were males, M:F ratio was 16:1. In the past five years gonorrhoea in Slovenia is decreasing, but it is increasing in MSM, especially in the group having sexual contacts in the other countries of the EU. Sexual contacts abroad significantly increase the chance of infection with a resistant strain. Because of high specifity and sensitivity a Gram stain of male urethral specimen can be considered diagnostic for gonococcal infection, but not for endocervical, pharyngeal, or rectal specimens. The ability of Neisseria gonorrhoeae to become resistant to cheap and effective antibiotics is well recognised and has significantly compromised both individual and public health management of gonorrhoea. The number of strains of gonococci that are resistant to antibiotics is increasing all over the world and also in Slovenia. In all patients with gonococcal infection diagnosed in the Department of Dermatovenereology in Ljubljana culture and antimicrobial susceptibility is performed in the Institute Microbiology and Immunology. Recent data have shown antibiotic resistance to different antibiotics including to quinolones. Early and successful treatment of gonococcal infection is important not only for the individual patient but is also a significant factor in control of disease and the prevention of complications. The increasing antimicrobial resistance of N. gonorrhoeae emphasizes the need for the performance of gonococcal culture. To prevent treament failures, continuous surveillance of trends in gonococcal resistance to antimicrobials is an imperative. National treatment gudelines should be revised according to the resistance proof.

Chlamydia trachomatis infection during pregnancy T. Meyer, A. Clad (Hamburg, DE) Besides transfer to newborns some other adverse pregnancy outcomes resulting from genital Chlamydia trachomatis (CT) infections during pregnancy have been described, including post-abortal PID, secondary sterility (one-child-infertility) due to post-partum infections, and ectopic pregnancies. In addition, CT may be associated with early and late spontaneous abortion, premature delivery, and stillbirth, although evidence for these complications is rather low. In Germany, Chlamydia testing was taken up into the prevention program of maternity guidelines in 1995, primarily to prevent infections of newborns and post-partum infertility. Efficiency of pregnancy screening is unclear so far, since no data about frequency of CT infections of newborns are available in Germany.

To estimate efficiency of CT screening we compared prevalence of CT infection of pregnant women tested routinely within the prevention program (n=7249) and of women immediately after delivery (n=2943). CT infections were analyzed by NAAT (nucleic acid amplification: PCR and SDA) using swabs and urine samples. Women analyzed after delivery were also tested for CT during pregnancy, which, however, was performed usually by antigen tests and not by NAAT, according to maternity guidelines. Women of both groups were 31 years old on an average. Also, mean age of CT positive individuals was similar (26 years of pregnant women compared to 27 years of those after delivery). CT-infections were detected in 149 (2.1%) of 7249 pregnant women and 44 (1.5%) of 2943 women after delivery. Prevalence of CT infection was age dependent, with the highest frequency found in women of age 20 (12.6%). In women of age 30 and over CT prevalence was only 0.9%. With respect to reduction of infections at delivery efficiency of CT screening of pregnant women is limited, as indicated by only about 30% decrease of frequency of CT infections. This mainly depends on using tests with insufficient sensitivity, since maternity guidelines consider antigen tests and hybridization tests only for CT testing in pregnancy until recently. Because the mean age at first delivery in Germany is 30 years now, pregnancy screening will miss the majority of infections, which were shown to occur mainly in younger women. These women may wish to have children later, but are at risk for CT-associated complications including infertility. Thus, a general screening of women up to 25 years has started in 2008 in Germany.

Microbial patterns signaling via Toll-like receptors 2 and 5 contribute to epithelial repair, growth and survival R. Shaykhiev, R. Bals (Marburg, DE) Introduction: Epithelial cells (ECs) continuously interact with microorganisms and detect their presence via different pattern-recognition receptors (PRRs) including Toll-like receptors (TLRs). Ligation of epithelial TLRs by pathogens is usually associated with the induction of pro-inflammatory mediators and antimicrobial factors.

In this study, using human airway ECs as a model, we found that detection of microbial patterns via epithelial TLRs directly regulates tissue homeostasis. Staphylococcus aureus (S. aureus) and microbial patterns signaling via TLR2 and TLR5 induce a set of non-immune epithelial responses including cell migration, wound repair, proliferation, and survival of primary and cancerous ECs. Using small interfering RNA (siRNA) gene targeting, receptor-tyrosine kinase microarray and inhibition studies, we determined that TLR and the epidermal growth factor receptor (EGFR) mediate the stimulating effect of microbial patterns on epithelial repair. Conclusions: Microbial patterns signaling via Toll-like receptors 2 and 5 contribute to epithelial repair, growth and survival. This effect is independent of hematopoietic and other cells as well as inflammatory cytokines suggesting that epithelia are able to regulate their integrity in an autonomous non-inflammatory manner by sensing microbes directly via TLRs.

Polyomavirus JC with rearranged noncoding control region are found in cerebrospinal fluid, but not in urine and increase viral early gene expression R. Gosert, A. Egli, N. Khanna, H. Hirsch (Basel, CH) Introduction: JCV, with a seroprevalence of ~70%, persists in the renourinary tract with urinary shedding in 20-40% of individuals. The viral genome can be divided in the noncoding control region (NCCR) with the origin of replication and promoter/enhancer functions driving early (large and small T-antigen) and late (agno, capsid proteins VP1-3) gene expression. In immunocompromised patients, JCV can cause progressive multifocal leukoencephalopathy (PML), a rare, usually fatal disease of the CNS. In PML tissue, rearranged (rr)-NCCR have been detected and proposed to cause PML but the rr-NCCR impact is unclear.

The NCCR was amplified from urine, plasma and cerebrospinal fluid (CSF) of JCV-positive patients, sequenced and cloned into a dual-GFP-reporter vector. After transfection into cells, the transcription efficiency of the early (RFP, red) and late (GFP, green) region was evaluated by fluorescence microscopy. Results: We investigated 2 HIV/AIDS patients with JCV loads in CSF, plasma and urine of 12.8E7(3.3E4), 553(1.0E4), 6.4E5 Geq/mL. Sequencing of NCCRs isolated from urine demonstrated archetype (ww) architecture. However, we found rr-NCCRs isolated from plasma and CSF. The NCCR rearrangements changed transcription factor binding sites and enhancer elements, but no common feature was apparent. The dual reporter assay revealed that ww-NCCRs were mainly associated with late gene expression. In contrast, rr-NCCRs were characterized by increased early gene expression but decreased late gene expression. JCV-specific cellular immune responses were not detectable, but antibodies against JC VLP were found. Conclusions: Naturally occurring NCCRs show in an in vivo assay significant differences in their potential to transcribe the early versus the late region. PML corresponds to secondary JCV replication in patients without detectable JCV immune responses favoring increased early gene expression in brain tissues.

TREM-1 improves the inflammatory response and outcome to Streptococcus pneumoniae infection by reducing levels of negative regulators in the lung H. Lagler, O. Sharif, U. Matt, I. Haslinger, K. Stich, T. Furtner, K. Schmidt, S. Knapp (Vienna, AT) Background: Triggering receptor expressed on myeloid cells (TREM-1) is a cell surface receptor present on both monocytes/macrophages and neutrophils that has been shown to amplify cytokine and chemokine production in response to bacterial Toll like receptor (TLR) ligands. Blocking studies revealed TREM-1 as a valuable target to prevent overwhelming inflammation during sepsis. At the same time, TREM-1 is highly expressed within the pulmonary compartment with soluble TREM-1 being a valuable marker indicating the presence of pneumonia in humans. The biological role of TREM-1 during community acquired pneumonia, such as pneumococcal pneumonia is not known. The aim of the present study was to determine the function of TREM-1 in the inflammatory response to Streptococcus pneumoniae infection in vivo, the mechanism where by this occurred, and the outcome of this on infection. Methods: C57BL/6 mice were intranasally infected with S. pneumoniae followed by i.p. treatment with PBS, isotype Ab or agonistic TREM-1 mAb. Bacterial counts, lung histology, neutrophil influx, chemokine/cytokine responses and signaling mediators as well as survival were evaluated in vivo in a time dependent manner. In vitro, immortalized lung alveolar macrophages (MHS) and respiratory epithelial cells were utilized to study the response to S. pneumoniae treatment.

Results: Mice pre-treated with agonistic TREM-1 displayed a significantly amplified cytokine and chemokine responses (TNF, MIP-2 and IL-6) as well as neutrophil influx to the lungs 6h after induction of pneumonia. This enhanced early inflammation was associated with lower numbers of bacteria in the lungs and reduced pulmonary inflammation at 48hr, resulting in improved survival. In vitro studies corroborated these results at the early time point. A reduction of negative regulators of TLR signaling in lungs was observed in mice pre-treated with TREM-1 in vivo. Conclusion: TREM-1 boosts the early inflammatory response during S. pneumoniae infection in vivo through lowering levels of negative regulators in the lung, which results in accelerated bacterial clearance and ultimately improved survival.

The antimicrobial peptide cathelicidin protects from sepsis K. Kändler, O. Pinkenburg, S. Al Alwani, R. Gallo, R. Bals (Marburg, DE; San Diego, US) Introduction: Cathelicidin is an antimicrobial peptide that protects the body against infection. Recent findings indicated that the peptide plays a larger role in innate immunity as an endogenous antibiotic.

To study the role of the murine cathelicidin CRAMP in vivo, we analyzed the response of CRAMP-deficient mice to endotoxin shock and infection with Gram-negative bacteria. CRAMP -/-animals showed higher mortality in a model of peritoneal LPS application. A model of pneumogenic sepsis applying Escherichia coli resulted in increased mortality of the cathelicidin deficient animals. Pulmonary infection with Pseudomonas aeruginosa results in increased inflammation in CRAMP-deficient animals and a reduced bacterial load. This is explained by the observation that CRAMP inhibits the activation of macrophages and neutrophils by LPS.

Conclusions: These are the first results from a knockout model that substantiates the role of cathelicidin peptides in the regulation of the innate immune response.

Encephalitozoon cuniculi (Microsporidia) Modulates Apoptosis in Infected Cells C. Franzen, B. Salzberger (Regensburg, DE) Introduction: Protozoan parasites which reside inside a host cell avoid direct destruction by the immune system of the host. Programmed cell death (apoptosis) is an important regulator of the host's response during infection with a variety of intracellular protozoan parasites. Apoptotic cells are recognised and phagocytosed by macrophages and the parasite is potentially eliminated together with the infected cell. Different parasites have evolved diverse strategies to inhibit host-cell apoptosis, thereby faciliating intracellular survival. Microsporidia are intracellular parasites that infect a variety of cells. Amongst others they replicate in short living cells such as gastrointestinal enterocytes. We investigated if Encephalitozoon cuniculi modulates host cell apoptosis in HCT-8 and HT-29 cells.

Methods: Cells were infected with E. cuniculi and after one, three or five days apoptosis was induced by adding of camphotericin B, TNF, cycloheximide, or aktinomycin D to nearly confluent monolayers for 24 hours. Apoptosis was measured by FACS after annexin V/PI staining. Caspase-3, activated caspase, bcl-2, bax, and p53 levels were measured by Western blot and ELISA. DNA fragmentation was determined by fluorescent microscopy after staining with Hoechst 33258. Results: Infected cells (see figure down right) showed significantly lower rate of apoptosis after induction of apoptosis by a variety of inducers, as measured by Annexin V staining (FACS analysis). We have started to study different apoptotic mediators during microsporidial infection by Western blot. Caspase-3 cleavage was clearly inhibited at different times of cell infection with E. cuniculi. Significant changes in the expression of bcl-2, bax, or p53 levels, as it has been reported for other intracellular parasites, were not observed, but the relative ratio of bcl-2/bax was elevated in infected cells.

Conclusions: Microsporidia modulates apoptosis in infected host cells. The mechanisms are not clear, and further studies are needed to elucidate the regulation induced by microsporidia infection in the host cell.

Antigen presentation by dendritic cells for CTL immunity against HSV-1 A.C. Jirmo, C.H. Nagel, B. Sodeik, G.M.N. Behrens (Hannover, DE) Dendritic cells (DC) sample pathogens which they have acquired either by infection or as a result of scavenging from other infected cells. Following antigen processing, peptides are presented via MHC class I molecules for CTL immunity via direct or cross-presentation. We and others have shown that induction CTL against cellular or viral antigens (HSV-1) is restricted to CD8+ DC in mice. Using HSV-1 entry receptor specific antibodies, novel HSV-1 constructs and mutants, HSV-1-specific TCR-transgenic mice and in vitro and in vivo antigen presentation assays we studied the requirements for antigen acquisition and presentation by splenic DC subsets. Studies with HSV-1 mutants deficient of relevant glycoproteins (gH, gB) for attachment, entry and immunogenicity, revealed that DC can acquire HSV-1 by multiple pathways including direct infection, endocytosis and phagocytosis of infected cells. We provide evidence that CD8+ DC mainly cross-present antigens in viral immunity and that these DC do not present antigens due to endogenous synthesis upon phagocytosis of infected cells. Finally, CD8+DC have the unique ability to cross-present incoming viral surface antigen after inoculation. We conclude that although infection amplifies the amount of antigen, cross-presentation of HSV-1-derived antigens is a dominating an effective route for generation of an effective CTL-response.

P. Kern, B. Grüner, G. Härter, W. Kratzer, K. Buttenschön, P. Kern, T. Romig (Ulm, DE) The term echinococcosis describes two diseases which markedly differ in their presentation, behaviour and management: Alveolar echinococcosis (AE) is caused by the larva of Echinococcus multilocularis and cystic echinococcosis (CE) by E. granulosus, respectively. The larval growth of the two bugs is very different and separates the «malignant» AE from the «benign» CE. Thus, current concepts and treatment guidelines are discussed separately. AE: The European project EchinoRisk discovered a continuous increase and spread of the parasite in the final host (fox). In parallel, human cases were observed in regions outside the known endemic areas, such as in the Baltic States, in Hungary and Slowakia. New foci have been discovered in China/Tibet with a prevalence rate of up to 4% of the affected population. Risk factors for humans are farming and a long-standing, close contact to dogs. The initially asymptomatic liver disease is still rare in Central Europe, and diagnosis is often made at a late stage of the disease. An anatomical classification system has been proposed by the WHO expert group (PNM classification adapted from the TNM system of liver tumours). It describes the extent of the lesion in the liver, in the neighbouring and/or distant organs. This classification allows a comparison of the disorder at the time of first diagnosis in different clinical settings. Imaging techniques, such as Ultrasound, CT, MRI and PET/CT contributed to a much better description of the lesions. However, the expertise of the radiologist is often not available due to the low incidence of AE. Serology is helpful, but may be misleading due to non-standardized tests. Molecular tools have been developed, and help histopathologists to clearly separate the two bugs by microscopy. Due to the potential «malignant» features of the disease palliative surgery and liver transplantation are 

Moderne Diagnostik der Pneumocystis-Infektion

The diagnosis of pneumocystis pneumonia requires microscopical examination in order to identify pneumocystis from a clinically relevant source such as specimens of induced sputum, bronchoalveolar fluid, or lung tissue, because pneumocystis cannot be cultured. Trophic forms can be detected with modified Papanicolaou, Wright-Giemsa, or Gram-Weigert stains. Cysts can be stained with Gomori methenamine silver, cresyl echt violet, toluidine blue O, or calcofluor white. Monoclonal antibodies for detecting pneumocystis have a higher sensitivity and specificity in induced-sputum samples than conventional stains. An advantage of monoclonal antibodies is their ability to stain both trophic forms and cysts, which is important because the trophic forms are generally more abundant during pneumocystis pneumonia. The use of PCR to detect pneumocystis nucleic acids has been an active area of research. PCR has been shown to have greater sensitivity and specificity for the diagnosis of pneumocystis pneumonia from specimens of induced sputum and bronchoalveolar-lavage fluid than conventional staining when using PCR primers for the gene for mitochondrial large-subunit ribosomal RNA. Although an elevated serum lactate dehydrogenase level has been noted in patients with pneumocystis pneumonia, it is likely to be a reflection of the underlying lung inflammation and injury rather than a specific marker for the disease. Lower levels of plasma S-adenosylmethionine were observed in seven patients with confirmed pneumocystis pneumonia than in patients with other pulmonary infections, but the usefulness of this test will need to be confirmed in a larger cohort of patients. The role antibody detection and ß-d-glucan (cell-wall-component) Staphylococcus aureus remains one of the most frequent causes of both hospital and community-acquired infections with vast impact on healthcare and economics. Colonizing distinct ecological niches, this pathogen is involved in a broad spectrum of infections resulting in substantial morbidity and mortality. While it was shown that the human anterior nares are the endogenous reservoirs for subsequent, often severe infections due to S. aureus, strategies to reduce infections by eradication of nasal carriage were only in part successful. One of the reasons for the success of this pathogen seems to be its versatility. New, highly virulent and transmissible S. aureus clones, e.g. strains carrying the Panton-Valentine leukocidin as well as multi-resistant clones as response to the selective pressure of antibiotics emerged worldwide. In addition, the ability to form a biofilm on inserted or implanted foreign bodies demonstrates the versatility and the capacity of S. aureus to adapt to various conditions and thus, to be a successful pathogen. In the past decade, interest in Staphylococcus aureus smallcolony variants (SCVs) emerged, a phenotypic subpopulation of the species typically associated with chronic and persistent infections. These variants, defined by mostly non-pigmented and non-haemolytic colonies about 10 times smaller than the parent strain, seem to be specifically adapted for an intracellular life style. The intracellular location may shield the SCVs from host defences and antibiotics, thus providing one explanation for the difficulty in clearing S. aureus SCVs from host tissues. This subpopulation potentially uses the up-regulated arginine-deiminase pathway to produce ATP or through ammonia production to counteract the acidic environment that prevails intracellularly. Due to their changed characteristics, these variants present a challenge to clinical microbiologists with regard to recovery, identification and susceptibility testing, probably leading to an underestimation of their prevalence. In conclusion, the often not successful treatment of infections due to S. aureus seems to be associated with the remarkable versatility of this pathogen.

Aktive und passive Vakzine: the way to go?

The prevalence of Staphylococcus aureus as an unabated and even emergent pathogen in community and nosocomial infection requires novel strategies for combat and prevention. Coagulase-negative staphylococci have a devastating potential in very low birth weight children. Therefore, staphylococcal vaccine research is of great importance and has a long tradition, however, it has hitherto not translated into a universal vaccine strategy. On the other hand, detailed insight in pathogenic mechanisms as well as delineation of immunodominant pathogen surface structures, the advent of potent conjugative vaccine techniques and availability of advanced antibody techniques have all accumulated to a number of promising novel vaccine approaches and candidates. In this presentation, an overview on staphylococcal components which have been used in the recent past for experimental vaccine approaches as well as on the current or recently performed clinical vaccine candidates and vaccine studies will be presented. This will include discussion of the potential for toxins, 'MSCRAMM' type of adhesive wall components, secreted 'SERAM' type of adhesins as well as the role of expolysaccharides and defined staphylococcal regulators to be used as vaccines or inflammatory modulators. Among these staphylococcal factors, a number of vaccine and antibody preparations are currently in phase I -III clinical trials. These include active vaccines directed against cell wall components, capsular polysac-

charides, or toxins, either alone or in combination, as well as passive antibody developments including mono-and polyvalent antibodies against surface components, capsular polysaccharides, lipoteichoic acids, and metabolic factors. The presentation will further discuss potential reasons for the recent failures of large phase III clinical trials and will try to indicate a perspective for future developments. Doubtlessly, despite these backlashes in clinical vaccine development, the enormous burden of clinical staphylococcal disease will continue to foster vaccine development for defined risk group of patients, and it is hoped that lessons from previous studies will now translate into a safe, immunogenic, and protective active or passive vaccine.

Detection of viral nucleic acids by the innate immune system V. Hornung (Worcester, UK) The rapid production of type I interferon (IFN) following viral infection is critical for the control of viral replication. In recent years, the effects of IFN on immune function have received considerable attention. We now have a detailed understanding of how IFNs signal and control anti-viral immune responses. Much less is known, however, concerning the complex recognition machinery responsible for the virus-triggered induction of the IFN genes themselves. The acute IFN response is regulated by multiple germline-encoded innate immune receptors, often referred to as pathogen recognition receptors (PRRs). These PRRs include membrane bound toll-like receptors (TLRs) and cytoplasmic RNA helicases, including RIG-I (retinoic acid inducible gene-I) and MDA5 (melanoma differentiation antigen-5). All PRRs that have been shown to play an important role in antiviral defense detect viruses via their nucleic acid genome. The fact that nucleic acids are an essential constituent of every living organism demands the existence of a mechanism in our immune system that enables the discrimination between self and non-self to recognize DNA or RNA as foreign. Two major concepts seem to be in place to distinguish nonself from self via this protein receptor-based recognition system: (1) Detection of a certain molecular pattern that is specific for the pathogen and does not occur in the host system and (2) recognition based on pathogen-specific compartimentalization. For detection of pathogen-derived nucleic acids both mechanisms apply. In the case of the cytoplasmic RNA helicases, structural signatures exist that allow these immune receptors to determine the origin of RNA. We have recently characterized the first natural ligand for RIG-I -5'-triphosphate RNA. In mammalian cells, under physiological conditions, 5'-triphosphate RNA is absent, yet can be generated by many viral polymerases during replication and transcription. While the Picornavirus EMCV (Encephalomyocarditis virus) has been identified as a natural stimulus for MDA5, the exact nature of the ligand recognized by MDA5 is still elusive. The nucleic acid sensing TLR system on the other hand, rather relies on the localization of its cognate ligand than on its structural features. Given their expression in the endosome, this family of TLRs is primarily activated by RNA ligands that are targeted to this compartment. Both pathways form a non-redundant, highly efficient system mastering the detection of foreign nucleic acids. Results: Overall, the sera of 20/289 children (6.9%) contained detectable amounts of HBoV-DNA (quantities ranging between 1x103-4.6x106 geq/ml). 150/298 children (51.9%) were admitted due to diseases of the respiratory tract, HBoV genomes could be detected in 16 of these patients (10.7%). Highest prevalences of 14.3% (7/49) and 13.0% (6/46) were observed in patient subgroups with symptoms of the lower respiratory tract and pneumonia, respectively. In contrast, in children with symptoms of the upper respiratory tract HBoV genomes were detected less frequently (3/55, 5.5%). Furthermore HBoV viremia could be shown in 3/48 patients (6.3%) presenting with infectious gastroenteritis and in one child with chronic inflammatory bowel disease (1/9). Whereas the mean age of all patients tested was 53.5 months, acute HBoV infection was observed preferentially in small children (mean age: 24.7 months; range 3-44 months). HBoV-specific IgM was observed in 12/24 of the DNA-positive patients, 10 of these were tested positive for IgG in addition. None of the children of the control group was found to be viremic or tested positive for HBoVspecific IgM. Seronegative children were observed preferentially in the age-groups between 7-23 months indicating that seroconversion takes place rather early in childhood.

The study presents a comprehensive overview on the frequency and impact of acute HBoV-infection in hospitalised children and adolescents. It could be shown that HBoV-infection mainly takes place in children younger than 48 months and is associated with diseases of the respiratory tract, but also with gastroenteritis and diarrhea.

2007: year of the hantaviruses in Germany? 

Abstracts KIT2008 more than1400 clinically apparent human hantavirus infections have been reported until October, implying a 3 up to 10 fold increase of the number of infections in comparison to the previous years.

The objective of our study was to identify and characterise hantavirus strains involved in this increased occurrence of Nephropathia epidemica (NE) in humans in Germany including also military training areas in South-West Germany. The serological investigations were based on a novel recombinant nuucleocapsid protein of a PUUV strain originating from Lower Bavaria. Results: In all investigated regions in Baden-Wuerttemberg, North Rhine Westphalia, Lower Saxony and Bavaria a high PUUV prevalence of up to 76% was observed in trapped bank voles. RT-PCR amplification and subsequent phylogenetic analysis of S-and M-genome sequences demonstrated significant differences between PUUV strainsoriginating from the different regions in Germany. A nucleotide-sequence divergence of about 16% was observed in S-and M-segments between the PUUV strains. Conclusions: Our investigations demonstrated that different PUUV strainsare presently circulating in bank voles in Germany. Embedded in the German-wide network "Rodent-borne pathogens" future rodent trapping activities are dedicated to monitor the population density of rodents as well as the prevalence and molecular variation of PUUV in these rodent populations. These studies will allow conclusions on the evolution of PUUV and changes in their distribution which might result in a risk assessment for human infections in the investigated regions.

Viral load predicts outcome of hepatitis C after liver transplantation I. Graziadei, K. Nachbaur, A. Schloegl, R. Margreiter, W. Vogel (Innsbruck, AT) Hepatitis C virus (HCV) -associated liver failure is the most common indication for liver transplantation (LT) worldwide. Recurrent HCV infection is almost ubiquitous after LT and leads to graft loss and reLT in about 10-20% of LT recipients. Risk factors associated with HCV recurrence include donor, recipient and vial parameters. Conflicting data have been reported regarding viral load and severity of recurrent HCV disease. The aim of this study was to analyse the impact of viral load on severity of recurrent HCV infection. Between 1980 and 2006 185 HCV patients were transplanted at our institution. However, only patients who survived more than 6 months and with histological assessment of recurrent HCV infection were included in this study. The overall study group consisted of 130 patients (mean age: 56.8±8.9 years; 31 females, 99 males). Viral loads were measured at wk 2, ms. 3, 6 and 12 post LT, using the bDNA HCV RNA 3.0 assay (Bayer Diagnostics). The patients were divided into group A -patients with severe recurrent hepatitis C (cholestatic type and/or liver cirrhosis) and B -recurrent hepatitis with fibrosis score 0-3. The overall follow-up was 6.1 ± 3.6 years. There was no difference between both groups regarding major patients' characteristics and follow-up period. The actuarial patient survival of the overall group at 1-, 2-, 5-and 10 years were 96%, 90%, 77% and 63%, respectively. Patients in group A had significantly decreased survival rates at 1-, 5-and 10 years compared to group B (90%, 65%, 45%% vs. 98%, 81%, 71%; p <0.01 This data show that a serum viral concentration >3.2x106 IU/mL, measured between ms. 3 and 12 after LT, is predictive of a severe course and/or high mortality due to HCV recurrence. These results indicate that antiviral therapy should be considered in these patients in the first year following LT.

Detection of Herpesviruses and Adenovirus Co-Infections in Patients Undergoing Allogeneic Stem Cell Transplantation with a Diagnostic DNA-Microarray A. Ditzen, A. Rutzka, M. Stichling, R. Müller, R. Ehricht, T. Illmer, E. Jacobs, J. Rohayem (Dresden, Jena, DE) Introduction: Herpesviruses and adenovirus are an important cause of end-organ disease (EOD) and are associated with graft versus host disease (GVHD) in patients undergoing allogeneic stem cell transplantation (SCT). Detection and monitoring of those viruses is routinely performed by simplex quantitative real-time PCR. Since more than one herpes-or adenovirus are reported to be present shortly after transplantation, detection of all possible (sub-)clinical viral infections is still time-consuming and cost-intense.

We therefore designed a DNA-microarray (VINAray) for the simultaneous detection of herpes-simplex-virus-1 and -2 (HSV-1/2), cytomegalovirus (CMV), varicella-zoster-virus (VZV), Epstein-Barr-virus (EBV), human herpesvirus-6 (HHV-6) and adenovirus. The VINAray was validated in comparison to simplex quantitative PCR routinely used in our diagnostic laboratory. We retrospectively screened 615 samples of 35 patients undergoing allogeneic SCT on a regular basis until day 100 post-transplantation. The detection limit of the VINAray is 10 genome equivalents (GE)/ml.

We detected one or more viruses in 357 samples (58,1%). In the positive samples (n=357), 444 viruses were detected. Among those, CMV was detected most frequently in 228 samples (51,4%), followed by EBV in 94 (21,2%), HHV-6 A/B in 73 (16,4%), adenovirus in 29 (6,5%), HSV-1/2 in 14 (3,2%) and VZV in 6 samples (1,4%), respectively. Of the samples containing two viruses (n=61), we detected CMV/HHV-6 A/B in 23 (37,7%), CMV/EBV in 13 (21,3%), and EBV/ HHV-6 A/B in 7 (11,5%) samples, respectively. CMV/adenovirus/ HHV-6 A/B was the most frequent combination (n=4, 36,4%) detected in the samples containing 3 viruses (n=11). The assay displayed a high diagnostic accuracy for the detection of the different viruses within the 95% analytical sensitivity limit of the PCR used.

We conclude that the VINAray is a highly sensitive and specific method for the simultaneous detection of up to six viruses in a single sample. By screening and monitoring SCT-patients for viruses known to be associated with EOD or GVHD we herewith introduce an innovative molecular technique that can simultaneously detect multiple viral infections on a large scale. The assay is currently used to determine the impact of multi-virus detection on clinical end-points like GVHD and infectious complications in patients undergoing allogeneic stem cell transplantation.

Polyomavirus BK (BKV) and JC (JCV) replication in plasma and urine in healthy blood donors A. Egli, L. Infanti, C. Stebler, J. Samaridis, S. Bodaghi, R. Gosert, H. Hirsch (Basel, CH) Introduction: Polyomaviruses have high impact in immuno-suppressed patients: In kidney transplant patients, BKV replication is detected in blood and urine samples of 12% and 30%, and serves as a risk-marker for polyomavirus-associated nephropathy. In stem cell transplantation BKV is associated with hemorrhagic cystitis and in HIV patients JCV causes progressive multifocal leukoencephalopathy. In healthy individuals, BK and JC viruria and viremia is not well characterized. Viral transmission is unresolved. We are interested in the

rates of BKV and JCV in urine and blood of healthy blood donors and their potential role in transmitting the viruses. Methods: We enrolled adult healthy blood donors from Basel blood donation center (51.1% male, median age: 45.5y range: 18-67). We quantified BKV and JCV DNA in plasma and urine using two standard real-time PCR protocols with a detection limit of 1000 geq/mL. We used ELISA assay to determine IgM and IgG immune responses against BKV and JCV virus-like particles (VLP, positive: >0.11 optical density, OD). Results: Seroprevalence: We found 12/264 plasma samples being positive for BKV-VLP IgM (4.5%; median 0.137, range 0.1104-0.2386OD) and 222/264 for IgG (84.1%; median 0.5485OD, range 0.1117-2.7071OD). 14/264 were positive for JCV-VLP IgM (5.3%; median 0.1305OD, range 0.1129-0.1397OD) and 161/264 for IgG (61%; median 0.3902OD, range 0.1116-2.0711OD). A significant increase of JCV seroprevalence with increasing age was observed, whereas BKV IgG-titers significantly drop with age. Viral shedding: We detected BKV in urine of 26/264 (9.9%; median 2975c/mL, range 1000-64900c/mL), and JCV in 54/264 urine samples (20.5%; median 44037c/mL, range 1000-100x10^6/mL). JCV loads in urine were significantly higher than BKV loads. In older age groups, JCV shedding was more frequent and higher, which was not apparent for BKV. Both viruses were more often detected in males, but in females viral loads were higher. We could not detect BKV and JCV in plasma.

Our results showed that seroprevalence for BKV was 84.1% and 61% for JCV. BKV can be found in 9.9% of urine samples and JCV in 20.5%. BKV and JCV were not found in plasma. Blood transfusion seems to be a minority routine of transmission. Nevertheless, due to high shedding in urine, active replication in healthy donors can be suggested and because of potential high impact in immuno-suppressed patients, we would like to encourage PCR routine screening of blood products in future.

A. Dumoulin, H. Marti, C. Hatz, M. Panning, H. H. Hirsch (Basel, Hamburg, CH) Introduction: Dengue viruses (DENV) infections are increasingly diagnosed in travellers returning from tropical zones. The clinical pattern ranges from flu-like illness, to life-threatening hemorrhagic fever and shock syndrome. DENV are immunologically grouped in four serotypes and secondary infection by a different serotype is presumably favouring the hemorrhagic presentation. Routine diagnostics rely on the detection of DENV-specific immunglobulins. However, the low antibody titres in the acute phase may produce false-negative results. RT-PCR allows detection of the virus in this early phase. The objective of this study was to improve DENV-diagnostics at the "Zentrum für Infektionsdiagnostik" (ZID: IMM and STI) by realtime RT-PCR for early detection and serotyping.

Methods: Serological testing of patient samples was performed by using a rapid commercial assay detecting DENV-specific antibodies. In parallel, a quantitative, serotype-independent pan-dengue real-time RT-PCR (PAND-PCR) and a serotype-specific multiplex real-time RT-PCR were used. Overall, 188 serum samples from 175 travellers were analysed with both methods: subset A was a retrospective panel of 25 IgM-positive serum samples from the STI; subset B consisted of 163 serum samples (43 IgM-positive, 120 IgM-negative) collected prospectively at the ZID. Results: Of 67 IgM-positive samples, 26 (39%) were positive by PAND-PCR (subset A: 4 from 25, 16% / subset B: 22 from 42, 52%). Of 121 IgM-negative samples (all subset B), 10 were positive by PAND-PCR (8%). The detected viral load ranged from 255 copies/ml to 7.13e7 copies/ml (median 1.33e5 copies/ml). Of 24 samples tested with the serotype specific RT-PCR, 15 (63%) could be attributed to a subtype: 9 to subtype I, 2 to subtype II, 2 to subtype III, and 2 to subtype IV. For 54 Dengue-positive samples, information on the time between beginning of the symptoms and sampling was available. Mea-sured viral loads were significantly higher in samples taken earlier in the course of infection.

Our results show that a combination of serology and RT-PCR are required for rapid (<24h) and reliable DENV diagnostics at all stages of infection.

Clostridium difficile C. Wenisch (Vienna, AT) Clostridium difficile is an anaerobic gram positive, spore-forming bacterium which is responsible for 15-25% of antibiotic-associated diarrhea and for more than 95% of pseudomembranous colitis (PMC). Clostridium difficile-associated diarrhea usually occurs as a complication of antibiotic treatment. Recent data shows an increase in incidence rate of CDAD and higher rates of morbidity, colectomy and death. Since 2003, outbreaks of severe C. difficile-associated diarrhea (CDAD) have been increasingly reported. The management of CDAD involves discontinuing the inciting antibiotic agent and treatment with metronidazole or vancomycin. The reduced response rates and higher recurrence rates with metronidazole treatment reported in recent studies raise the question of the effectiveness of metronidazole therapy. After each recurrence, the risks for further relapses grow even bigger (after two recurrences, the risk being greater than 50%) and the management of recurrent CDAD becomes a challenge. Even after a careful review of available data on various drugs and having the experience of managing many cases of CDAD, one might find difficult to present with a successful "recipe" for treating severe CDAD. Every case is different and different management plans can lead to full recovery. First episode are metronidazole. If there is no improvement in three days or white blood cell count is more than 12,000 or creatinine level is high, metronidazole should be discontinued and vancomycin should be started. The latest trend of CDAD with more severe cases and increasing morbidity and mortality may be an incentive for using vancomycin as first line in some ases for RCDAD. Adding S boulardii to vancomycin or metronidazole from the first or second relapse and using pulse/tapering vancomycin therapy have been beneficial in decreasing the relapse rate. For patients with RCDAD, vancomycin therapy followed by rifaximin for two weeks looks promising. New therapies with, nitazoxanide, tinidazole, tiacumicin, rifaximin and ramoplanin are being evaluated and future reports and trials will show their efficacy. Immune therapy is also a promising option treatment in evaluation, showing seroconversion and protective antibody levels in initial tests in healthy volunteer. Passive immunization is also considered but for all these new therapy options, further randomized studies are needed. Prevention is also very important in controlling this disease: first by limiting the use of broad spectrum antibiotics and secondly by controlling the environmental spreading through gloves, handwashing and disposable thermometers.

Treatment and Prevention of Antibiotic-Associated Diarrhea: Role of Probiotics J. Hübner (Freiburg, DE) Diarrhea associated with antibiotic use is a serious problem that often complicates the treatment of infectious diseases. Between 5% and 30% of patients treated with antibiotics will develop antibiotic-associ-

Abstracts KIT2008 ated diarrhea, with higher rates observed for broad-spectrum antibiotics and antibiotics that display activity against anaerobes. Clostridium difficile-associated diarrhea can range from mild diarrhea to lifethreatening disease, and is associated with a recurrence rate of 10-40% despite appropriate treatment. Clostridium difficile has emerged as a major nosocomial pathogen and several outbreaks with increased severity, high mortality, and high relapse rates have been associated with a hypervirulent C. difficile strain (ribotype 027). There is growing evidence that probiotics may be useful in a number of gastrointestinal diseases, although there is only limited information on the possible mechanisms of these effects. Several large trials and meta-analyses have shown that probiotics are able to prevent antibiotic-associated diarrhea and treat C. difficile disease in children or adults. However, studies have also confirmed that only specific probiotic strains are active, while others seem to have no effect. While probiotics are generally considered to be safe, there are also several reports of patients who have developed bacteremia and sepsis due to lactobacilli or saccharomyces strains used as probiotics. In addition, there are studies indicating that a number of commercial products contain bacterial species other than those designated on the product label. While growing evidence and an increasing number of randomized and controlled studies support use of specific preparations, it is also important to be aware of the risks associated with probiotic treatments.

J.C. Wasmuth (Bonn, DE) Background: In recent years outbreaks of sexually acquired acute HCV-infections have been observed within HIV-infected men who have sex with men. Reasons are unclear as both viruses have been known for more than a decade and sexual transmission of hepatitis C has been rare. Recent findings: For effective sexual transmission of hepatitis C several factors comprising HIV-infection, concomitant sexually transmitted infections and sexual practices with a risk for mucosal damage appear to be important. However, sexual transmissions have been described in HIV-negative persons as well underlining that the degree of mucosal damage, facilitating blood to blood transmission, is the key risk factor. After acquisition of acute hepatitis C a spontaneous resolution is observed in 25% of HIV-infected individuals. Early treatment with pegylated interferon and ribavirin results in sustained virological response rated in 60 -75% of HIV co-infected patients. Summary: Sexual transmission of hepatitis C occurs more frequently in recent years within a highly selected population. A surge of classic sexually transmitted infections as well as behavioral changes appear to fuel this epidemic.

Vector-borne diseases: climate change and socio-economic factors S. Randolph (Oxford, UK) Objectives: To explain the extreme spatio-temporal heterogeneity in the emergence of tick-borne encephalitis (TBE) in Europe. Methods: Time series data on the changing TBE incidence, abiotic and biotic environmental factors (including climate) and socio-economic conditions have been analyzed at national and regional scales. Results: Changes in climate, particularly increased temperatures in the spring in many parts of Europe, are such as could enhance the transmission of TBE virus between co-feeding nymphal and larval ticks. These change, however, are too uniform across wide areas to account for the variable timing and degree of upsurge of TBE. Instead, a multi-factorial synergistic system of changes, including land cover, host availability and human behaviour related to soco-economic conditions, can better explain the variation in TBE upsurge. Decreases in incidence have also occurred, apparently due to human responses to perceived risk. In Austria this is linearly related to increased vaccination coverage, but in the Baltics decreased TBE incidence far exceeds vaccination and is more likely to be due to reduction in high-risk behaviour. Conclusions: Climate change is unlikely to be the sole, perhaps not even the most important, cause of the recent emergence of a range of vector-borne diseases. Other factors that underpin changes in both infection risk and human exposure to that risk must be recognised so that they can be dealt with more effectively and rapidly than can climate change.

Hantaviruses in Europe: Molecular epidemiology and virulence D.H. Krüger (Berlin, DE) Hantaviruses, family Bunyaviridae, are emerging viruses that cause two life-threatening human zoonoses: hemorrhagic fever with renal syndrome (HFRS) in Asia and Europe and hantavirus cardiopulmonary syndrome in the Americas. Kidney failure is a prominent symptom of HFRS. In contrast to other members of the Bunyaviridae, hantaviruses are not transmitted by arthropods but are spread by aerosolized excreta of rodents and shrews. In Europe, a widespread hantavirus is the Puumala virus (PUUV). The virus is harbored by the bank vole (Myodes glareolus) as reservoir host. In 2007, a large PUUV outbreak has occurred in the Southern part of Germany leading to about 1,600 HFRS cases. Moreover, two other hantaviruses, Dobrava-Belgrade virus (DOBV) and Tula virus (TULV), were shown to be of relevance in Europe. DOBV was originally found in the yellow-necked mouse, Apodemus flavicollis, in Southeast Europe where it causes moderate and severe HFRS. However, members of DOBV are also harbored by at least two other Apodemus species, the striped field mouse (A. agrarius) and the Caucasian wood mouse (A. ponticus). DOBV-Aa is regularly found in HFRS patients in the Northern part of Germany and causes large HFRS outbreaks in the Central region of European Russia. Infections by DOBV-Ap were demonstrated in the Southern region of European Russia. Both viruses, DOBV-Aa and DOBV-Ap, were isolated on cell culture, genetically characterized and used for typing of neutralizing antibodies from the HFRS patients under BSL-3 conditions. The clinical courses of DOBV-Aa infections resemble those of PUUV infections (rather mild to moderate) while DOBV-Ap infections cause moderate to severe courses more frequently. TULV genetic material was amplified from Microtus arvalis animals in the surroundings of a first HFRS patient in Germany. However,

TULV infections seem to play a minor role in human morbidity whereas DOBV and PUUV are the major HFRS-inducing pathogens in Europe. Since the main target cells involved in HFRS pathogenesis -endothelial cells -are not cytolytically destroyed by the infecting virus, it is assumed that the virus infection leads to a destruction of the microvascular endothelium by immunopathological mechanisms. The most useful assays for the diagnostics of hantavirus infections are IgM and IgG ELISAs (which can be combined with confirmatory assays as immunoblot or IFA). Hantavirus typing could be performed by determination of neutralizing antibodies or (even more valuable) by PCR amplification, sequencing and phylogenetic analysis of virus RNA from clinical materials.

Minireview: Jüngste Resistenztrends bei Gramnegativen Bakterien

Antimicrobial resistance in Germany and the central European region is monitored by several surveillance studies like the European Antimicrobial Resistance Surveillance System (EARSS), the German Network for Antimicrobial Resistance Surveillance (GENARS), the Antimicrobial Resistance Surveillance Programme of the Paul-Ehrlich-Society for Chemotherapy (PEG), and the Surveillance of Antimicrobial Use and Antimicrobial Resistance in Intensive Care Units (SARI). EARSS provides data on the spread of resistance among major invasive bacteria, while GENARS, PEG and SARI monitor the distribution of resistance among hospital isolates from all clinical sources. The increase of resistance of Escherichia coli to relevant drug classes is of great concern. Aminopenicillins and cotrimoxazole, with resistance rates of >50% and >30%, can no longer be recommended for empiric treatment of UTI. Regarding the fluoroquinolones (FQ), resistance markedly increased over the past ten years, as in most other European countries. According to data from EARSS, FQ resistance rose from ~10% in 2001 to >20% in 2006. During the same period, the proportion of ESBL producing strains increased from <1% to 4-7%. The proportion of ESBL producing strains among Klebsiella isolates also increased. In contrast, resistance to carbapenems remained rare among Enterobacteriaceae isolates. Overall rates of resistance in Pseudomonas aeruginosa for antipseudomonal ß-lactams and FQ range between 10% and 20%. However, resistance is more frequently encountered among intensive care isolates than non-intensive care isolates and the proportion of multi-resistant strains increased over time. Multiple resistance in Acinetobacter baumannii and Stenotrophomonas maltophilia is also worrying. With <5% resistant strains, the carbapenems have been considered the drugs of choice for the treatment of A. baumannii infections. The majority (~80%) of S. maltophilia isolates displayed susceptibility to cotrimoxazole. On the one hand, the observed increase in the prevalence of antimicrobial resistance can be explained by the nosocomial, clonal spread of resistant bacteria. On the other hand, the selection pressure caused by an inadequate use of antibiotics may also have contributed to the development of resistance.

The most frequent cause of resistance against extended spectrum cephalosporins like cefotaxime, ceftriaxone, ceftazidime, and cefepime is constitutive expression of chromosomal ampC ß-lactamases (bla). However, plasmid encoded extended spectrum ß-lactamases (ESBL) generated by point mutations in genes of common bla types TEM, SHV, and CTX-M are causing increasing problems. Presently more than 200 different ESBL have been described (www.lahey.org/ studies/webt.htm). ESBL mediate decreased susceptibility or gross resistance against oxyimino cephalosporins and monobactams. Epidemiology of ESBL-carrying Enterobacteriaceae has been changing in recent years from predominantly hospital acquired disease caused by organisms carrying predominantly TEM-and SHV-type ESBLs to CTX-M-type ESBLs occurring in E. coli isolated from hospital acquired as well as genuine community acquired infection. Outbreaks caused by single clones of CTX-M-15 carrying E. coli were observed all over the United Kingdom including many community acquired cases. Carbapenems are the therapy of choice in severe infection. Immediate institution of infection control policies is mandatory to prevent uncontrolled spread of these multi-resistant problem organisms. Many different resistance phenotypes occur, which make detection of an ESBL challenging, especially as some of the compounds may appear susceptible. ESBL occur with increasing frequency predominantly in Escherichia coli und Klebsiella spp., but have been detected in other Enterobacteriaceae like Proteus spp. and Enterobacter spp.. ESBL are detected by a positive screening test using one or several of the oxyimino cephalosporins. Presence of an ESBL is confirmed by an additional phenotypic assay using the typical inhibitory activity of clavulanic acid against ESBL. MIC or disk-diffusion synergy assays are recommended by the NCCLS. Variations of these assays are the E-Test ESBL strips and double-disk approximation assays. Semi-automatic systems for identification and susceptibility testing like VI-TEK2 (bioMerieux), Phoenix (Becton-Dickinson), or MicroScan Walkaway (Dade-Behring) together with their expert systems are also helpful in detection and confirmation of ESBL. Special problems are encountered in organisms with overlap of ampC and ESBL mediated resistance. In ESBL-positive organisms all cephalosporins and monobactams are reported resistant.

The opportunistic pathogen Pseudomonas aeruginosa is endowed with numerous intrinisic mechanisms of antibiotic resistance. Its flexible genome rapidly acquires resistance by mutation in intrinsic chromosomal genes or by horizontal gene transfer of new resistance determinants. Active efflux causes concerning multidrug resistant (MDR) phenotypes in P. aeruginosa. Twelve genetically distinct systems have been identified in the P. aeruginosa genome. Each system is composed of three proteins. A cytoplasmic membrane protein acts as an energy dependent pump with broad substrate specificity. A second protein is located in the outer membrane. A third protein is located in the periplasmic space and makes a link between the other proteins. Only the MexAB-MexOprM system is constitutively expressed. The four biochemically characterized pumps display different resistance phenotypes. MexAB-OprM confers resistance to most ß-lactams except imipenem, as well as to quinolones, chloramphenicol, trimethoprimsulphonamides, and tetracycline. MexCD-OprJ extrudes fourth generation cephalosporins, quinolones, macrolides, chloramphenicol and trimethoprim. MexEF-OPrN has the narrowest substrate profile, affecting chloramphenicol, quinolones and trimethoprim-sulphonamides. MexXY-OprM confers resistance to aminoglycosides, erythromycin, and tetracycline. Overexpression and mutation of elements Infection Abstracts KIT2008 of the Amp operon (ampD, ampR, ampG) are further clinically relevant MDR mechanisms. Of the transferable elements, the class B acquired carbapenemases that can efficiently inactivate all penicillins, cephalosporins and carbapenems, deserve particular attention. The genes encoding carbapenemase genes are typically located together with aminoglycoside modifying enzymes in integrons and thus confer a MDR phenotype. During chronic infections hypermutable strains often emerge that genetically diversify and will rapidly adapt to their host habitat. Moreover, a global metabolic control of resistance is operating in P. aeruginosa that modifies the bacterial fitness to evade the action of antimicrobials. This intrinsic resistome consists of about 2 % of all chromosomal genes. We have developed a binary multilocus microarray as a point-of-care tool for the surveillance of hospital infections with P. aeruginosa

Mononuclear phagocytes -alveolar recruitment and response to lung injury J. Lohmeyer (Giessen, DE) Mononuclear phagocytes are long living cells with broad differentiation potential entering lung tissue by two pathways: (i) constitutively to regenerate resident alveolar macrophage and lung dendritic cell pools and (ii) inflammation-driven to foster host defense. A role of alveolar resident and recruited mononuclear phagocytes in initiating inflammation in lung infection has been convincingly demonstrated. Our group has shown a key role of the CC-chemokine ligand 2 (CCL2, MCP-1) and its receptor CCR2 for inflammatory mononuclear phagocyte recruitment into lung tissue, expanding and replacing the resident alveolar macrophage pool. CCL2/CCR2 driven transendo-/epithelial monocyte migration was found to activate distinct genetic programs, leading to further differentiation into alveolar macrophages or dendritic cells. Using a model of Flt3-Ligand elicited expansion of lung DCs, we could further show that in response to both bacterial toxins and gram-negative bacteria, lung DCs contribute critically to cytokine production, especially IL-12, and to the control of the lung barrier integrity. In addition, recent reports suggest that mononuclear phagocytes, either lung resident or recruited during inflammation, also contribute to resolution and repair processes in acute lung inflammation that are essential for return to homeostasis. We found that mononuclear phagocytes can modulate alveolar epithelial cell (AEC) function during influenza virus infection inducing both inflammatory and repair processes. Re-epithelialisation following injury of the alveolar compartment is considered a critical step to restore normal gas exchange conditions in the lung. This involves AEC proliferation, migration and polar differentiation with restoration of junctional structures. Using a co-culture model of primary murine resident alveolar macrophages and AEC, we identified macrophage-epithelial communication molecules that significantly contributed to AEC type II proliferation and transdifferentiation into AEC type I as a prerequisite to replace injured AEC type I. Mononuclear phagocyte subsets/differentiation stages and their signal pathways/mediator molecules that start inflammation, initiate exudate resolution and promote tissue repair are candidates for selective therapeutic targeting.

Innate defence against L. pneumophila in human host cells

Legionella pneumophila is a Gram-negative, intracellular bacterium. While in most mice strains powerful innate immune mechanisms mediate resistance to Legionella infection, humans are susceptible to this bacterium and can develop severe pneumonia (Legionnaires´ disease). This talk will focus on different innate defence mechanisms against L. pneumophila in human cells, which are dependent e.g. on NOD-like receptors, Toll-like receptors, as well as type I IFNs, and discuss similarities and potential differences in human and murine innate immunity which might influence differential susceptibility in mice and men.

Tuberculosis -paleomicrobiological aspects

Paleomicrobiology studies ancient infectious diseases by detection of specific pathogen in ancient materials like mummies and skeletons, by elucidation of the epidemiology of past infections and of the genetic evolution of the respective microorganism. Progress in this field is tightly bound to research of ancient mycobacterial DNA since mycobacteria are -for several reasons -ideal microorganisms to examine antique DNA. Detection of mycobacterial ancient DNA has first been published in 1993 when mycobacterial DNA was amplified from pre-European contact Borneo, a spine from Byzantine Turkey, and a lumbar-sacral spine from 17th century Scotland. Up to now mycobacterial ancient DNA has been detected (among others) in northern-amercian bisons of the Pleistocene (17500 b.C.) and in Peruvian mummies dating back to the pre-Columbian era. Also detection of mycobacterial DNA in Egyptian mummies shows the high prevalence of tuberculosis in ancient Egypt. Spolygotyping of ancient mycobacterial DNA allowing species-identifcation proved the existence of M. africanum in Egypt 2500 b.C.. The low prevalence of M. bovis-DNA in ancient bones -M. bovis has only recently been identified in sibirian bones dating from 1000 to 400 b.C. -agrees with the assumption that M. bovis has emerged from a common M. tuberculosis ancestor, as suggested by the genomic comparison of region of differences in the M. tuberculosis complex. A large spolgyotype database of more than 40.000 actually prevalent strains of the M. tuberculosis complex has recently been established representing more than 1900 different spolygotypes from 122 countries worldwide. The geographic distribution of these spolygotypes shows co-migration of human and mycobacteria during the global distribution of humans. The construction of a phylogenetic tree of these spolygotypes shows that the main eastafrican indian linage is evolutionary the oldest supporting the hypothesis that tuberculosis is as old as humanity and originated in East Africa. Paleomycobacteriology provides a fascinating insight into the co-evolution of humans and mycobacteria.

Tuberkulose-Pathogenese

Inhalierte Tuberkulose Bakterien werden in der Lunge durch Alveolarmakrophagen und dendritische Zellen phagozytiert. Der weitere Verlauf der Primärinfektion hängt von dem Immunstatus des Wirtes 

C. Lange (Borstel, DE) Tuberculosis (TB) is one of the leading causes of morbidity and mortality worldwide. Symptoms of active TB like fever, nightsweats and weight loss are unspecific and do not distinguish TB from other causes of chronic inflammatory or neoplastic diseases. The diagnosis of TB is based upon a thorough medical history and clinical examination as well as on the results of radiological, microbiological, immunological, molecular-biological and histological examinations. Cultural confirmation of the growth of Mycobacterium tuberculosis (MTB) from a biological specimen is the gold-standard for the diagnosis of TB. However, results from mycobacterial cultures are not readily available and up to 20 % of cases with active TB remain undetected by MTB-culture. Therefore other diagnostic tests are welcome which enable clinicians to develop rapidly a diagnosis or exclusion of active tuberculosis in clinical practice. When acid-fast-bacilli (AFB) are visible in biological specimen, the diagnosis of TB or infection with non-tuberculous mycobacteria can readily be made by mycobacterial species-specific nucleic acid amplification (NAT) with almost 100 % test sensitivity. The sensitivity of all tests, including NAT, decreases, when AFBs are not visible in biological probes. Under these circumstances further diagnostic procedures are necessary (e.g. bronchoscopy in case of suspected pulmonary TB) to establish a diagnosis by the examination on cells and biological tissue with cytological/histological and microbiological methods. The tuberculin-skin-test (TST) has little value in the diagnosis of acute tuberculosis and is being replaced by T-cell interferon-gamma release assays (TIGRA). A negative test result in a TIGRA can almost rule out MTB-infection; however TIGRAs may lack sensitivity in immunocompromised hosts. Neither a TST nor a TIGRA test result can allow a distinction between active TB and latent MTB infection (LTBI), when the TIGRA is performed on peripheral blood cells only. When a MTB-specific ELISPOT is performed on blood mononuclear cells and cells from the site of the infection (e.g. from a bronchoalveolar lavage or on pleural fluid) in parallel, recruitment of antigen specific T-lymphocytes to the site of the infection is seen in active TB. Very recently additional diagnostic methods have been explored to distinguish between active TB and LTBI by the analysis of blood only.

These tests include the quantification of heparin-binding hemagglutinine, the chemokine CXCL10/IP-10 and of immune responses against selective Region of Difference-1 specific oligopeptides. The immunological differentiation of separate stages of MTB-infection may also be possible by immunophenotyping of cell surface markers or the cytokine profiles of MTB-specific T-cells alone.

MDR/XDR-Tuberkulose: Einblicke -Perspektiven M.P. Grobusch (Johannesburg, ZA) Multiresistente Mycobacterium tuberculosis-Stämme sind in den letzten Jahren weltweit identifiziert und charakterisiert worden; schon im Jahre 2004 

A. Kola, F. Schwab, S. Bärwolff, T. Eckmanns, K. Weist, E. Dinger, I. Klare, W. Witte, H. Rüden, P. Gastmeier (Hannover, Berlin, Wernigerode, DE) Introduction: Nosocomial infections (NIs) contribute significantly to morbidity and mortality in intensive care units (ICUs). An important instrument of infection control is the surveillance of nosocomial infections. Though surveillance data of device associated infection rates are not able to distinguish between the endogenous or exogenous origin of infections, they are increasingly used for public reporting and interhospital comparisons of infection control management. The utility of NI rates for benchmarking was revised by analysing the association between NI rates and bacterial cross-transmissions. Methods: Over a 2 years period, primary isolates of 6 bacterial indicator organisms (A. baumanii, E. faecium, E. faecalis, K. pneumoniae, P. aeruginosa, S. aureus) cultured from clinical samples of ICU-patients were genotyped by PFGE (pulsed field gel electrophoresis). Genetically indistinguishable isolates from different patients were considered as cross-transmissions if the patients were hospitalized on the same ICU in a temporal proximity of at least 9 days. Surveillance of NIs was performed following the protocol of the German Nosocomial Infection Surveillance System KISS (which is based on NNIS System methodology). Results: During 100 781 patient days, 100 829 microbiological specimens from 24 362 patients were sampled (average investigation density: 1.0 samples per patient * day) and 3 419 indicator organisms were Infection Abstracts KIT2008 cultured. Altogether, 462 cross-transmissions (incidence density of 4.6 transmissions per 1 000 patient days, ranging between 1.4 -14.7 per 1000 patient days depending on the respective ICU) and 1 216 nosocomial infections (incidence density of 12.1 per 1 000 patient days, range: 6.2 -16.6) were discerned. Correlation analysis was unable to reveal any association between the incidence of cross-transmissions and nosocomial infections, duration of hospitalisation or device utilisation. Conclusion: The differences in nosocomial infection rates between study ICUs are not only explained by differences in infection control practices. In particular, they are not due to bacterial cross-transmissions. Additional factors like the severity of the patients underlying diseases, the patients endogenous flora or invasive procedures have to be taken into account. This should be kept in mind when nosocomial infection rates are used for external benchmarking.

Antibiotic Treatment of Respiratory Infections of Non-Hospitalized patients contributes to increased numbers of C. difficile associated diseases in South-eastern Germany S. Borgmann, T. Jakobiak, E. Scholz, H. Gruber, K. Pohl, M. Kist, C. von Eichel-Streiber, B. Schulte (Weiden, Mainz, Tübingen, Freiburg, DE) Introduction: In our lab we perform microbiological analyses for about 3500 facilities comprising more than 40 hospitals in South-eastern Germany allowing examination of C. difficile prevalence in the local region (Northern Bavaria). In winter and early spring 2006 increased numbers of C. difficile associated diseases (CDAD) were observed. However, infection control analyses performed in affected hospitals revealed no obvious hygienic failures. Methods: Numbers of patients exhibiting either C. difficile toxin positive (Tcd) stool samples or H. influenzae in respiratory secretions between 2001 and 2006 were systematically determined by hybase analyses. C. difficile isolated from Tcd positive stool samples were analyzed by pulsed-field gel-electrophoresis (PFGE) and ribotyping. Resistance to chinolones was examined by E-test procedure. Antibiotic subscriptions to outpatients were requested from the WIDO institute. Results: From 2001 to 2006 the number CDAD affected patients per year increased from 154 to 811. The most prominent peak appeared in January 2006 due to a five-fold increase when compared with November 2005. The number of affected patients treated at the three largest hospitals showed very similar temporally courses suggesting that community factors might have contributed to CDAD increase. While in 2000/1 the number of CDAD patients was negatively correlated with the number of H. influenzae bearing patients, a positive correlation was observed in 2005/6 (R = 0.68) indicating that antibiotic treatment of respiratory infections has contributed to increased CDAD numbers in the late observation period. Ribotyping and PFGE of 21 strains revealed identical results: Ten C. difficile isolates were ribotype 001 while each of other ribotypes was observed only once. Whereas all C. difficile strains were resistant to ciprofloxacin, only ribotype 001 C. difficile were resistant to moxifloxacin. Since the year 2000 a continuous increase of moxifloxacin for treatment of respiratory infections was indicated by subscription data. Conclusion: One C. difficile strain seems to be prevalent in Southeastern Germany. The high number of CDAD cases in winter 2006 was probably promoted by common events, e.g. antibiotic treatment of respiratory infects in winter. As only the prevalent strain exhibited resistance to moxifloxacin increased treatment of respiratory infections with broad-spectrum quinolones might has facilitated increased numbers of CDAD affected patients.

Impact on antibiotic use and resistance: enhanced antibiotic stewardship and reduced length of antibiotic phrophylaxis E. Meyer, F. Schwab, A. Pollitt, M. Trautmann (Freiburg, Berlin, Stuttgart, DE) Objective: To evaluate the impact of a reduced length of antibiotic prophylaxis for cerebrospinal shunts on total antibiotic use and key resistant pathogens. Methods: In 1/2004 antibiotic prophylaxis was reduced to a single shot dose. Before intervention prophylaxis with 2nd generation cephalosporins was applied during the whole duration of external liquor drainage. The effect on the antibiotic use density (AD: DDD = defined daily doses per 000 patient days) was calculated before (1/2002-12/2003) and after the intervention (1/2004-12/2006 ) by segmented regression analysis of interrupted time series. Resistance proportions (RP) and resistance densities (RD) defined as resistant pathogen/1000 patient days (pd) were compared by Fisher's exact test before and after the intervention. Results: Total antibiotic use significantly decreased in level (-147 .3) after the intervention when pre-operative prophylaxis for shunt catheters was changed into single shot prophylaxis. This was mainly due to a reduction of cefuroxime which was used for prophylaxis. The reduction of total antibiotic consumption was sustainable because it did not increase again over the next 36 months. RR and RD of 3rd generation resistant E. coli increased after 1/2004 whereas the percentage of MRSA significantly decreased. Conclusion: Change to single shot prophylaxis along with an ongoing antibiotic stewardship program resulted in a cut down in as much as 15% of total antibiotic use. Therefore, targeting reduction of antibiotic prophylaxis in surgical ICUs might be most worthwhile.

Is Fluoroquinolone Prophylaxis in Hematology-Oncology patients still useful? A before and after ecological and individual data analysis Pritzkow, G. Peyerl-Hofmann, K. de With, H. von Baum, C. Schneider, M. Dettenkofer, U. Frank, H. Bertz (Freiburg, Ulm, DE) Background: Fluoroquinolone prophylaxis in patients (pts) with cancer and neutropenia has been shown to reduce morbidity and mortality, but these beneficial effects may be lost with increasing rates of resistance in target bacteria, i.e. primarily E. coli. We assessed the impact of a recent introduction of fluoroquinolone prophylaxis as a standard of care for pts with neutropenia on the incidence density of bloodstream infection and rates of in vitro fluoroquinolone resistance (FQR). Methods: We planned to introduce in 4/2005 a new antibiotic policy of fluoroquinolone prophylaxis in pts with neutropenia. In a before and after ecological analysis, unit-wide antibiotic consumption, bloodstream infection and numbers of in vitro FQR bloodstream isolates were evaluated during four 3-month periods before and after introduction of FQ prophylaxis with levofloxacin (500 mg single daily dose). Individual patient data before and after the new policy were also evaluated. Results: Baseline FQR of E. coli bloodstream isolates was 15% in the hematology-oncology unit, and 7% in other medical services, respectively. The unit-wide incidence density of gram-negative bacteremia (per 100 patient-days) fell from 0.07-0.11 before to =< 0.04 after the introduction of the policy, respectively, with a number of 2 (before) and 1 (after) FQR E. coli bacteremias unit-wide, respectively. FQR rates in E. coli bloodstream isolates changed from 15% to 25% in the hematology-oncology unit compared with a change from 7% to 12% in other medical services, respectively. Individual patient data analysis (253 pts without FQ prophylaxis before and 103 pts with FQ prophy-

laxis after the intervention) confirmed a significantly decreased incidence of gram-negative bacteremia (p=0.06, adjusted for acute leukemia, and duration of neutropenia), and a tendency for fewer deaths (3/103 vs. 15/254, not significant) in pts given prophylaxis. The "cost" was an increase in total antibiotic exposure by 25%: 28 versus 35 DDD/patient (individual patient data), or 133 vs 166 DDD/100 occupied bed days (unit-wide before/after data). The incidence of C. difficile infection remained unchanged. Conclusion: At a given background of 7-15% FQREC rates, FQ prophylaxis in pts with cancer and neutropenia still appears to provide beneficial effects at least on morbidity and at little cost. Ecological analysis may be useful to monitor this trend but needs to include infection incidence rates rather than resistance rates.

Acquisition of antibiotic-resistant Enterococcus faecium strains during long-term hospitalization and fast adaptation of enterococcal flora to antibiotic treatment S. Borgmann, C. Wolz, U. Schumacher, P. Heeg, B. Schulte (Weiden, Tübingen, DE) Introduction: Recently, a university hospital in South-western Germany was affected by a large outbreak of vancomycin-resistant E. faecium (VRE). As shown by pulsed-field gel-electrophoresis (PFGE) typing outbreak was caused by two strains. On the other hand, other groups had suspected that long durations of hospitalization might be a possible risk factor to get colonized by multiple VRE strains. Therefore, we examined if VRE affected patients were colonized by multiple VRE strains. Methods: Usually only those VRE were analysed by PFGE typing that had been isolated from a certain patient for the first time. However, when VRE exhibiting altered antibiotic susceptibilities were observed also subsequently found VRE were typed by PFGE. Antibiotic susceptibility was analysed by E-test procedure and carriage of enterococcal surface protein gene (esp) was examined by PCR. PurK allele was determined by PCR and sequencing of a 492 base pair fragment. Antibiotic treatment of VRE positive patients was reconstructed from patients´ histories. Results: Four patients exhibited at least two VRE strains. Moreover, one patient who stayed for four months at the hospital exhibited at least four different E. faecium strains. Usually all of these enterococci were resistant to antibiotics that had been applied previously to enterococcal isolation. All of the examined VRE were esp gene positive and belonged to purK strain E0158 suggesting that the bacteria were hospital adapted clonal complex 17 VRE. Conclusion: These findings indicate that due to long hospital stay patients might be colonized with multiple Enterococcus faecium strains and that the enterococcal flora quickly adapts due to antibiotic exposure. As it seems not to be possible to eradicate VRE from colonized patients antibiotic treatment should be restricted to patients suffering from enterococci infections.

Helicobacter pylori -Antimicrobial Resistance and Treatment

Actual standard treatment of Helicobacter pylori (Hp) infections in Europe is based on first line antimicrobial schemes including clar-ithromycin or metronidazole combined with amoxicillin and standard doses of a proton pump inhibitor (PPI) (Maastricht Consensus 2005) . Quadruple therapy consisting of PPI, bismuthsubcitrate, tetracycline and metronidazole is equally effective but not often used due to problems with compliance. Nevertheless, antimicrobial treatment of Helicobacter pylori (Hp) infections is jeopardized if H. pylori developes resistance against anti-infectives in use. Development of resistance in H. pylori, in contrast to resistance in campylobacter, which is a multifactor process depending on prevalence of resistant strains in the environment, in food of animal origin, as well as on development of resistance under treatment, depends nearly exclusively on the management of the individual patient. This particular characteristic of H. pylori antimicrobial resistance allows identification of risk factors for resistance development in H. pylori much easier than in more complex situations given with rather ubiquitously distributed pathogens, and in addition better allows control of resistance development by individual patient adapted clinical and therapeutical management strategies. To investigate risk factors for the development of antimicrobial resistance in H. pylori the nationwide study ResiNet was launched by the National Reference Centre (NRZ) for Hp in 2001. For this purpose the NRZ involved 16 microbiological centres (MC), each collaborating with 3 to 7 gastroenterologists (GE) in clinical practice. During "study weeks" GE, without pre-selection, enrol consecutive patients into the study, sending gastric biopsies for microbiological investigation and completing a questionnaire. All MC use identical culture media lots and standardized operation procedures during each study week. At end of December 2007 a total of 1525 patients were investigated, and from a total of 983 individuals data were complete for evaluation. Overall 41% and 25 % of isolates were resistant against metronidazole (MTZ) or clarithromycin (CLA) respectively, and 17% showed double-resistance against both drugs. The frequencies of primary resistance (N=622) were 28 % (MTZ), 6.0% (CLA), 3 % (MTZ and CLA), and 15% (CIP) compared to 52 % (MTZ), 57 % (CLA), 31 % (MTZ and CLA), and 15.1% (CIP) in patients pre-treated once (N=124). Repeated pre-treatment (N=133) was associated with increase of resistances to MTZ (83 %), to CLA (77 %), and of double-resistances up to 67 % to MTZ and CLA, clearly indicating that a significant increase in resistance to MTZ and CLA already occurs after the first treatment failure, reaching dramatically high resistances after repeated empirical eradication trials. In conclusion, these results underline the hypothesis that development of resistance in Hp in contrast to other pathogens is nearly exclusively dependent on treatment strategies in the individual patient. Further, the data clearly demonstrate that culture and sensitivity testing of Hp from gastric biopsies should be mandatory already after the first treatment failure. The latter should be recognized in upcoming guidelines.

The threat of avian influenza A (H5N1): Molecular clues to pathogenicity and pathology S. Ludwig (Münster, DE) Influenza is currently considered one of the most important threats to human health and animal welfare. Besides yearly influenza epidemics these viruses have the potential to cause pandemic outbreaks that have resulted in millions of death in the past. The ongoing infections of humans with highly pathogenic H5N1 influenza viruses (HPAIV) should be taken as a warning sign that this can happen again any time. HPAIV of the H5N1 subtype have become endemic in poultry populations throughout Southeast Asia and continue to infect humans with Infection Abstracts KIT2008 a greater than 50% case fatality rate. So far, human-to-human transmission of these viruses has been limited. The special molecular features of H5N1 influenza viruses that might affect their pathogenicity will be discussed, especially in the light of the current lack of efficient human-to-human transmission. Another focus of the presentation will be the development of new treatment options that may prevent generation of resistant virus variants.

Effects of socio-economic factors and intermittent preventive antimalaria treatment (IPT) on development of young African children B. Kreuels, R. Kobbe, S. Adjei, S. Ehrhardt, C. Kreuzberg, C. von Reden, O. Adjei, J. May (Hamburg, DE; Kumasi, GH) Introduction: Intermittent preventive antimalarial treatment in infants (IPTi) with sulfadoxine-pyrimethamine (SP) is a promising new tool in the global campaign to reduce mortality of Plasmodium falciparum malaria. While its effect on malaria and anaemia has been evaluated in a number of studies, little is known about the effects on child health and development. Methods: A randomized, double-blinded, placebo-controlled trial with administration of sulfadoxine-pyrimethamine based IPTi was conducted with 1070 children in an area in Ghana where malaria is holoendemic. Participants were monitored for 21 months until the age of two years. Children's length and weight were measured at 3 monthly intervals while mid-length arm circumference was measured at 3, 9 and 15 months of age. Anthropometric z-scores for weight-for-age (indicating underweight), length-for-age (indicating stunting), and weight-for-height (indicating wasting) were calculated according to "WHO Child Growth Standards". The dependency of scores on socioeconomic factors, treatment arm and their time-dependent development were assessed by Generalized Linear Regression models. Results: At time of recruitment (month 3 of age) 5.6% of the children were underweight, 3.9% were stunted, and 8.0% were wasted. These proportions increased over time and at one year of age 20.1% of the children were underweight, 10.7% were stunted, and 20.9% were wasted. A number of fixed cofactors influenced the development of anthropometric z-scores during the first two years of life. Stratification of anthropometric data for the treatment arm showed higher scores indicating underweight, wasting for children having received IPTi. however the statistical significance of this influence was inconsistent. Conclusion: In our study population different factors had an influence on children's development and health condition during the first two years of life. The specific impact of IPTi on anthropometric data was inconsistent.

Ototoxicity, tolerability and efficacy of Artemether/ Lumefantrine, Quinine and Atovaquone/Proguanil in the treatment of Falciparum Malaria R. Gürkov, I. Barreto Miranda, T. Eshetu, N. Berens-Riha, Y. Mamo, T. Girma, E. Krause, M. Schmidt, J. Hempel, T. Löscher (Munich, DE) Background: Due to increasing drug resistance, artemisinin-based combination chemotherapy (ACT) has become the first-line treatment of falciparum malaria in many endemic countries. However, recent reports suggested irreversible ototoxicity as a serious limitation in the use of ACT. Our aim was to compare ototoxicity, tolerability, and efficacy of ACT with that of quinine and atovaquone/proguanil in the treatment of uncomplicated falciparum malaria. Methods: 97 patients in south west Ethiopia with slide-confirmed malaria were randomly assigned to receive either artemether/lumefantrine or quinine or atovaquone/proguanil and followed-up for 90 days. Comprehensive audiovestibular testing by pure tone audiometry (PTA), transitory evoked (TE) and distortion product (DP) otoacoustic emissions (OAE), and brain stem evoked response audiometry (BERA) was done before enrolment and after 7, 28 and 90 days. Results: After correcting for reinfections by genotyping, parasitological failure rates on day 28 were 0, 9 and 6% for artemether/lumefantrine, quinine, and atovaquone/proguanil respectively. PTA and OAE levels revealed transient significant cochlear hearing loss in patients treated with quinine but not in those treated with artemether/lumefantrine or atovaquone/proguanil. There was no evidence of drug-induced brain stem lesions by BERA measurements. Conclusions: We could not detect any detrimental effect of a standard oral regimen of artemether/lumefantrineon peripheral hearing or brainstem auditory pathways in patients with uncomplicated falciparum malaria. In contrast, transient hearing loss is common after quinine therapy and due to temporary outer hair cell dysfunction. Adjei, B. Fleischer, T. Löscher, G. Bretzel (Munich, DE; Agogo, Dunkwa, GH; Hamburg, DE; Kumasi, GH) Introduction: Buruli Ulcer disease (BUD)has been recognized to be an emerging public health problem in many tropical countries, especially in the West African Region. In Ghana prevalence rates of up to 150 per 100.000 individuals were reported from most affected districts, making BUD the most prevalent mycobacterial disease in these areas. In the context of improving the quality of clinical management, the need for follow-up studies to monitor the treatment outcome has been emphasised. Methods: A follow-up study of 129 laboratory confirmed BUD patients who received surgical treatment in two treatment centres in highly endemic areas of Ghana was conducted. The aim was to assess the frequency of recurrences and functional limitations as late sequelae of BUD in association to clinical and treatment specific factors as well as psychosocial aspects in the follow-up sample. Results: Out of a total of 129 laboratory confirmed BU-patients, 79 (61%) were retrieved for follow-up. In the follow-up sample 7(9%) had a recurrence of BUD. In the analysis of factors influencing the probability of recurrence no significant association was found between recurrence and size/type of the primary lesion, wound closure technique, use of antimycobacterial treatment, duration of disease before treatment and place of treatment. 27% (n=21) of the followed-up patients showed a functional limitation caused by a reduced range of motion of one or more joints as a sequel to the BU-infection. There was no significant correlation concerning type and size of primary lesion; however, no functional limitation was caused by a nodule as primary lesion. Regarding the wound closure technique a significant association was noted. In 90% (n=19) of the patients presenting with functional limitations at follow-up, skin grafts were applied for wound closure. In patients over 15 years of age (n=55) the acceptance of the outcome after clinical interventions was with 73% (n=40) quite high. 29% (n=16) in this group reported that due to functional impairments they were not able to fulfil all tasks of daily life.

M. Schunk, W. Thompson, E. Klutse, J. Nitschke, K. Asamoah- Opare, R. Thompson, E. Fleischmann, V. Siegmund, O.

The results show a low recurrence rate in our study group, which is attributed to an effective clinical management of BUD in the study area. However, functional limitations were relatively frequent. This emphasises the importance of the improvement of preand post-operative wound care to avoid functional sequelae and the urgent need for the provision of rehabilitation programmes.

O143 Matrix Metalloproteinases and their Tissue Inhibitors in Plasmodium falciparum Malaria: Serum TIMP-1 levels are associated with Severity A. Dietmann, R. Helbok, P. Lackner, S. Issifou, B. Lell, P.B. Matsiegui, M. Reindl, E. Schmutzhard, P.G. Kremsner (Innsbruck, AT; Lambaréné, GA; Tübingen, DE) Background: Each year 350 and 500 million cases of malaria occur worldwide, and over one million people die, most of them young children in sub-Saharan Africa. Molecular mechanisms involved in the pathogenesis of severe malaria (SM), caused by Plasmodium falciparum, are not yet fully understood. Matrix metalloproteinases (MMP) are enzymes, proteolytically degrading both extracellular matrix (ECM) and non-matrix substances with various functions in the inflammatory response. Their key inhibitors are the tissue inhibitors of metalloproteinases (TIMP). Methods: We studied sequential serum levels of MMP-8, MMP-9, TIMP-1 and TIMP-2, using enzyme-linked-immunosorbent-assay (ELISA) technique, from Gabonese children with severe malaria (SM, n = 50) and compared them to those with uncomplicated malaria (UM, n = 43) and healthy controls (HC, n = 27). Levels were than correlated with clinical and laboratory parameters. Results: Serum MMP-8 and TIMP-1 levels were significantly higher in SM and UM compared to HC (p < 0.001) and TIMP-1 was significantly higher in SM compared to UM (p < 0.001). High TIMP-1 levels were significantly associated with the simplified Multi-Organ-Dysfunction-Score (sMODS; r = 0.55; p < 0.001), reflecting disease severity. TIMP-2 levels were significantly higher in HC compared to both malaria groups (p < 0.001). MMP-9 were not significantly different between groups. Conclusions: TIMP-1 is associated with signs and symptoms of severe malaria. Furthermore, MMP-8 is elevated in P. falciparum malaria. Our findings of high TIMP-1 and low TIMP-2 levels in SM support the concept, that TIMP-1 is more important in acute inflammatory conditions, whereas chronic inflammatory diseases present a rather TIMP-2 predominant pattern. Both TIMP-1 and MMP-8 can be seen as markers of acute inflammation, relevant in the pathogenesis of severe malaria.

Epilepsy and Neurocysticercosis in Northern Tanzania J. Blocher, E. Schmutzhard, T. Gotwald, H. Auer, W. Matuja, A. Winkler (Innsbruck, Vienna, AT; Dar es Salaam, TZ; Ulm, DE) Neurocysticercosis (NCC) is a common cause of epilepsy in developing countries. Tanzania is a country, where Taenia solium is endemic. Veterinary studies showed a high prevalence of cysticercosis among pigs in the Mbulu district, northern Tanzania [1]. However, to date studies on NCC and epilepsy are missing. We interviewed 212 people with epilepsy (PWE) and performed cerebral CT (cCT) scans with i.v. contrast medium administration at Haydom Lutheran Hospital. As controls served 198 consecutive cCTs of individuals without epilepsy. Sera of PWE and lesions highly suggestive of NCC (n = 20), PWE without lesions (n = 20), controls without epilepsy (n = 20) and CSF samples of PWE and lesions highly suggestive of NCC (n = 11) were investigated for anti-cysticercal antibodies using the enzyme linked immunosorbant assay (ELISA) and western blot techniques. Based on the Del Brutto criteria [2], cCTs in PWE showed definite NCC lesions in 5 (2.4%), highly suggestive lesions of NCC in 24 (11.3%) and lesions compatible with NCC in 9 PWE (4.2%). This compares to the control group where 2 people (1.0%) had definite NCC lesions, 2 (1.0) had lesions classified as highly suggestive and 6 individuals (2.9%) showed lesions compatible with NCC. Significantly more NCC lesions were found in PWE than in controls (p < 0.001 ). With cCT scan, serum and CSF analysis, we diagnosed 22 (10.4%) cases of probable and seven cases (3.3%) of definite NCC. In the CT control group, we found two cases (1.0%) with definite NCC and two cases (1.0%) with lesions highly suggestive of NCC. With regard to predisposing factors, we found people, who consumed pork, to have significantly (p = 0.023) more NCC than people who didn't eat pork, close contact with pigs, the absence of latrines and number of people in one household, however, did not show any significant influence on the prevalence of NCC. Our data clearly demonstrates that NCC is a major cause of epilepsy in northern Tanzania 

Pseudomonas aeruginosa: new insights into transmission pathways between hospital water and patients

Pseudomonas aeruginosa is a well known nosocomial pathogen causing a variety of infections such as ventilator-associated pneumonia, urinary tract infection and septicemia. During the last two decades, it has been recognized that P. aeruginosa resides in various moist habitats on hospital wards. Typical locations are sink drains, tap water outlets, therapy pools and drinking fountains. However, before the advent of DNA-based typing techniques, it was doubted that P. aeruginosa strains residing in water habitats are the same that cause nosocomial infections in patients. By molecular typing, we and others have shown that a significant proportion of infections on the intensive care unit are caused by strains previously found in water outlets. In a pilot study carried out on a 16 bed intensive care unit, it was found that during a 6-month period, 5/17 (29 %) invasive Pseudomonas infections ware caused by strains previously isolated from water. In a subsequent study, we demonstrated that 39 % of strains causing invasive infections in patients were present in water outlets in the same room. The opposite direction of transmission could also be proven: 5 patients transmitted their strains to tap water outlets in their individual rooms. Thus, tap water outlets play a pivotal role as a reservoir of transmission for P. aeruginosa strains in the hospital. In another study, we were able to drastically reduce nosocomial infections on a surgical intensive care unit by mounting water filters to all patient-related tap water outlets. Recent studies by authors from other countries (Spain, France, Switzerland, Italy) confirmed these results.

Nosocomial Infections caused by vancomycin-resistant enterococci (VRE)

The incidence of vancomycin-resistant Enterococci (VRE) has increased significantly in German hospitals, mostly in consequence of VRE outbreaks (VR-E. faecium) in several hospitals between the years 2004 and 2006. The German National Nosocomial-Infection-Surveillance-System (KISS) has also noted a sharp increase in the incidence of nosocomial VRE-infections per 10,000 patients from 0.5 in 2003 to 11.0 in 2005. This was accompanied by a rise in VRE-associated mortality. According to data provided by EARSS, ,vancomycin resistance has increased significantly in Germany, Greece, Ireland, Israel and Slovenia [www.rivm.nl/earss] over the past five years. The

Abstracts KIT2008 rapid expansion of VR-E. faecium in these countries is typically the result of nosocomial outbreaks and does not therefore represent the situation in hospitals that have remained unaffected. A decrease in vancomycin resistance has been observed in Austria. Since VRE may cause severe nosocomial infections, especially in high risk patients (immuno-compromised, undergoing cardiac surgery or liver transplant), spread from patient to patient must be strictly prevented. One major constraint is that in the majority of cases only colonisation with VRE occurs and that without systematic microbiological screening a hidden problem is likely to be detected late. In Germany, guidelines have been published by two working groups (B.-W. and Deutsche Gesellschaft for Hygiene und Mikrobiologie, DGHM) for the prevention of VRE transmission in both the endemic and the epidemic setting. The following topics are addressed: Indication for VRE screening, microbiological diagnostics, general infection control measures (isolation precautions and use of protective clothing), and special hygiene measures in the case of nosocomial VRE outbreaks.

German hand hygiene campaign: "Aktion saubere Hände" P. Gastmeier (Hannover/Berlin, DE) Hand hygiene is a core element of patient safety for the prevention of healthcare-associated infections and the spread of antimicrobial resistance. Its promotion represents a challenge that requires a multimodal strategy using a clear, robust and simple conceptual framework. The World Health Organization has started an international hand hygiene campaign: First Global Patient Safety Challenge ‹Clean Care is Safer Care›. Over 70 member states have signed a national pledge to tackle health care-associated infection. One of the pledges made by those countries relates to national campaigning, and health ministries commit to work to reduce health care-associated infection through actions such as hand hygiene campaigns. The German national hand hygiene campaign started in January 2008. The title of the campaign is «Aktion saubere Hände". All German hospitals are invited to participated, and a lot have already declared an interest to participate. The campaign is providing educational materials and posters and further support in organizing local campaigns in the individual hospitals. It is also a platform to collect examples from various hospitals and to exchange experience. In order to measure success a further KISS-component will be created to compare the amount of alcoholic hand hygiene compounds per 1000 patient days used for hand disinfection in the individual departments and hospitals over time. National campaigns are a powerful symbol, indicating that a country is serious about infection control, hand hygiene and therefore, patient safety.

Chemoprophylaxis against Malaria versus stand by treatment: PRO

Chemoprophylaxis is a well established tool to protect the traveler against malaria. The permanent intake of an antimalarial to prevent a life-threatening disease has been proven to be effective and safe. This kind of chemoprophylaxis is out of doubt for many so called "high risk areas" where highly resistant P.falciparum malaria is predominant. However, in geografic regions with a considerably lower risk of infection there is a longstandig discussion if a stand by treatment against malaria should be favorized for travelers to these regions instead of the permanent intake of a drug with potential side effects. In my part of the debate I will raise arguments which favor a permanent prophylaxis instead of stand by treatment: -Our knowledge on malaria risk for the individual traveler in some areas is not satisfying and therefore risk assessment is often not possible -Permanent prophylaxis is safe and effective, side effects are calculable and not severe -Intake is simple -Permanent intake avoids emergency situations for the traveler -The traveler himself does not decide whether to take an antimalarial or not -Some antimalarials are badly tolerated if taken as a treatment

Infectiology -Part I S158

The 

The existing Division of Infectious Diseases and Clinical Immunology of the University Hospital will be integrated in, and thus, upgraded to a Comprehensive Infectious Diseases Center (CIDC). The new structural organisation includes the establishment of interdisciplinary In-

fectious-Diseases (ID)-Boards. The ID-Boards guarantees sustainability by the development of treatment algorithms and guidelines. The research unit of the CIDC Ulm focuses on the immune reconstitution after allogeneic stem cell transplantation, after starting antiretroviral therapy in HIV/AIDS, and after starting treatment with TNF-? inhibitors or other biologicals. The patients will be systematically and prospectively recorded in view of development of opportunistic, mycobacterial or viral diseases. In cooperating projects the account of certain HIV-or HCMV variants for reactivation of infectious diseases will be analysed. The identification of immunological deficiencies and genetic factors may allow a risk stratification and detection of predictive factors for development of bacterial or viral infections. Additionally a vaccination with CMV-pp65 peptide will be tested in a phase I/II study in patients after allogeneic stem cell transplantation.

Risk factors for indeterminate results of a Tuberculosis-Specific Gamma interferon release assay in immunocompromised patients B. Lange, M. Vavra, W.V. Kern, D. Wagner (Freiburg, DE) Introduction: Tuberculosis (TB)-specific interferon-gamma release assays (TIGRA), either as ELISA (QuantiFERON TB Gold in tube (QFT-IT), Cellestis) or as ELISPOT (T-SPOT.TB, Oxfordimmunotec), have been introduced as an alternative to tuberculin skin test (TST) into routine for the diagnosis of latent TB in the last few years. Both tests use stimulation of T-cells with phythemagglutinin as positive control, which -in contrast to TST -allows to identify false negatives. Little data exist on reliability of these tests in immunocompromised (IC) patients. In this study we prospectively tested the QFT-IT in patients with diverse conditions of immunosuppression. The primary objective was to determine the rate of and identify risk factors for indeterminate results (IR). Methods: 208 patients, 128 (62%) male and 80 (39%) female, with a mean age of 51,7 years were included in the study. 65% (136/208) of the study population had either kidney or liver transplants, 11% (23/208) were patients with HIV infection, 14% (30/208) had rheumatic/autoimmune diseases under immunosuppressive therapy, and 9% (19/208) had primary immunodeficiency. Medical students and healthcare workers served as control. Data collection included demographic data, medical history, medication, and laboratory values. For statistical analysis the Chi Quadrat based measure Cramér V was used to determine relationship of categorical variables and tested for significance in SPSS. Results: Only few controls (1/77, 1%) had IR, significantly less than patients in the IC cohort (17/208; 8%; p<0.05). IR rates were higher in female patients (13/80; 16%) than in male patients (4/128; 4%; p<0,001), and a low percentage of IR was found in male patients across all disease groups. Patients older than 55 years had a higher risk of an IR than younger patients (13 vs 4%, p<0.05). IR rates varied significantly dependant on disease groups. Patients with rheumatic/autoimmune diseases had a higher percentage of IR as organ-transplant patients (p<0.05). Conclusion: Although IR rates are higher in IC patients than in healthy subjects, QFT-IT still produces reliable results in >90% of this patient group. Patients with organ transplantation had the smallest rate of IR among disease groups. We confirm that old age is associated with a higher rate of IR. The study shows a gender difference of IR results using QFT-IT in IC patients that has previously not been reported and needs confirmation in future studies.

Striking enrichment of Mycobacterium tuberculosis specific CD4+ T cells to the site of infection in active tuberculosis J. Nemeth, M. Ramharter, H.-M. Winkler, R. Zwick, R. Rumetshofer, P. Schenk, O. Burghuber, W. Graninger, S. Winkler (Vienna, AT) Introduction: Accurate and early diagnosis of active tuberculosis (TB) is problematic, as current diagnostic methods show low sensitivity (acid-fast bacilli smears), are time-consuming (culture of biological samples) or show variable results (Mycobacterium tuberculosis [MTB] specific PCR). In the course of infection MTB specific T cells become concentrated at the site of infection and are likely to play an important role in the local cell mediated immune response. Therefore, we hypothesized that the assessment of MTB specific T cells at the site of disease may be used as diagnostic marker for active TB. Methods: Using flow cytometry we studied the frequency of MTB specific, Interferon(IFN)-gamma (g) expressing CD4+ T cells obtained from peripheral blood and the site of disease in 25 patients with suspected tuberculosis (n = 11 bronchoalveolar lavage, n = 6 pleural fluid, n = 1 ascites, n = 1 joint fluid, n = 5 cerebrospinalfluid). Results: 15 patients showed proven active Tb infection. Amongst them, a striking increase of MTB specific T cells was detected at the site of infection compared to peripheral blood in (median increase: 28.5-fold, range: 7.25 -531-fold; median of IFN-g-producing CD4+ T cells from blood: 0.02 %, range: 0 -0.52 %; median of IFN-g-producing CD4+ T cells from the site of infection: 1.81 %, range: 0.29 -6.55 %, p <0.001). No enrichment of MTB-specific T cells was found for 12 patients with non-tuberculous disease. In 6 TB patients, ESAT-6 reactive T cells were found only at the site of disease but not in peripheral blood.

The assessment of MTB specific T cells at the site of infection may prove as useful diagnostic marker for an accurate and early diagnosis of active TB.

Procalcitonin predicts patients at low risk of death from community-acquired pneumonia across all CRB-65 groups at risk S. Krüger, S. Ewig, R. Marre, J. Papassotiriou, K. Richter, H. von Baum, N. Suttorp, T. Welte (Aachen, Bochum, Ulm, Hennigsdorf, Berlin, Hannover, DE) Background: Aim of this CAPNETZ substudy was to investigate the prognostic value of procalcitonin (PCT) compared to the established inflammatory markers C-reactive protein (CRP) and leukocyte count (WBC) alone and in combination with the CRB-65 score in CAP patients. Methods: We enrolled 1671 consecutive patients (61 +/-18 y, 55 % m) with proven CAP. Extensive microbiological workup was performed. In all patients PCT, CRP, WBC and CRB-65 score were determined on admission. Patients were followed-up for 28 days for survival. Results: In contrast to CRP and WBC, PCT levels markedly increased with the severity of CAP as measured by the CRB-65 score (p < 0.0001). In 70 patients who died during follow-up, PCT levels on admission were significantly higher compared to levels in survivors (mean (IQ) 0.88 (0.32 to 3.38) vs. 0.13 (0.08 to 0.38) ng/mL, p < 0.0001). In receiver operating characteristic (ROC) analysis for survival, the area under the ROC curve (AUC) for PCT and CRB-65 was comparable (0.80, 95% CI 0.75 to 0.84 vs. 0.79, 95% CI 0.74 to 0.84, n.s.), which was significantly higher compared to CRP (0.62, 95% CI 0.54 to 0.68, p < 0.01) and WBC (0.61, 95% CI 0.54 to 0.68, p < 0.01). The prognostic accuracy of the combination of CRB-65 with PCT (0.83, 95% CI 0.77 to 0.88, p < 0.01) was superior compared to CRB-65 alone. When patients were grouped according to three groups at risk (CRB-65 0, CRB-65 1-2, and CRB-65 3-4) mortality was significantly different according to PCT with a threshold <0.228 ng/mL in all three risk groups.

Conclusion: PCT levels on admission predict the severity and outcome of CAP with a similar prognostic accuracy as the CRB-65 score and a higher prognostic accuracy compared to CRP and WBC. The prognostic accuracy is improved by the combination of PCT and CRB-65 score compared to CRB-65 alone.

Value of serum procalcitonin, neopterin and C-reactive protein in differentiating bacterial from viral etiologies in patients presenting with lower respiratory tract infections M. Ip, T. Rainer, N. Lee, C. Chan, S. Chau, W. Leung, M. Leung, T. Tam, G. Antonio, G. Lui, T. Lau, D. Hui, D. Fuchs, R. Renneberg, P. Chan (Shatin, Hong Kong, HK; Innsbruck, AT) Introduction: Differentiating viral from bacterial causes of lower respiratory tract infections (LRTI) is important so that, first, optimal treatment is started, second, inappropriate antibiotic treatment and subsequent resistance is prevented, and third, patients with mixed infection are not cohorted together. This study evaluated the values of procalcitonin (PCT), neopterin, and C-reactive protein (CRP) alone and in combination to differentiate bacterial from viral etiology in patients with lower respiratory tract infections (LRTIs). Methods: Sera obtained on the day of hospitalization for LRTI (139 patients with confirmed bacterial etiology and 128 with viral etiology, age was similar; female/male = 1/1.15) were examined. A further 146 sera from healthy Chinese subjects with no infection were included as controls. Results: PCT (medians: 0.18 vs. 0.08 ug/L, P <0.005) and CRP (91.7 vs. 58.1 mg/L, P <0.0001) were higher in bacterial than in viral etiology, and higher than in healthy subjects (PCT: 0.03 ug/L, P <0.001; CRP: 1.5 mg/L, P <0.0001). Neopterin was higher in patients with viral than with bacterial infections (30.5 vs. 18.7 nmol/L, P <0.05), and higher than in healthy subjects (5.8 nmol/L, P = 0.001). The area under the receiver operating characteristic curve (ROC-AUC) for distinguishing bacterial from viral infections was 0.838 for CRP, 0.770 for PCT and 0.832 for neopterin (all P <0.05). When the markers were used in combination, ROC-AUC increased to 0.857 (CRP/neopterin) and 0.856 (CRP x PCT)/neopterin was 0.856. Conclusions: The application of a single marker such as PCT alone may not be discriminatory enough to indicate an infection of bacterial etiology or excludes viral etiology. However, the use of 2 or more of these markers may improve the diagnostic accuracy for selection of patients for antibiotic treatment. In this study, the combination of all 3 markers (CRP x PCT/neopterin) gave the highest ROC-AUC in our cohort of patients with LRTI of confirmed bacterial and viral etiology. This suggests that in a strategy for intervention with antibiotics in this group, the combination of 2 or all 3 of the markers should be more discriminatory and effective at targeting appropriate treatment in the patients with LRTI of bacterial etiology.

Pro-atrial natriuretic peptide and pro-vasopressin to predict severity and prognosis in communityacquired pneumonia S. Krüger, J. Papassotiriou , R. Marre, K. Richter, N. Suttorp, T. Welte (Aachen, Hennigsdorf, Ulm, Berlin, Hannover, DE) Background: Community acquired pneumonia (CAP) is the most important clinical infection. Therefore, CAP competence network CAP-NETZ was instituted in Germany. Aim of this substudy was to evaluate the value of pro-atrial natriuretic peptide (MR-proANP) and pro-vasopressin (CT-proAVP) for severity assessment and outcome prediction in CAP.

We enrolled 589 consecutive patients (61 ± 18 y, 46% f) with proven CAP. MR-proANP, CT-proAVP, C-reactive protein (CRP) and CRB-65 score were determined on admission. Results: MR-proANP and CT-proAVP levels but not CRP, increased with increasing severity of CAP, classified according to the CRB-65 score. In patients who died during 28-days follow-up, median MR-proANP and CT-proAVP levels (237.0 vs. 93.5 pmol/L, and 44.2 vs. 12.4 pmol/L, each p < 0.0001) were significantly higher compared to levels in survivors. In receiver operating characteristic (ROC) analysis for survival, the area under the curve (AUC) for CT-proAVP (0.86, 95% CI 0.83 to 0.89) and MR-proANP (0.76, 95% CI 0.72 to 0.80) was similar to the AUC of CRB-65 (0.73, 95% CI 0.70 to 0.77). In multivariable Cox proportional-hazards regression analyses including MR-proANP/CT-proAVP, coexisting illnesses and CRB-65, increased MR-proANP and CT-proAVP concentrations were the strongest predictors of mortality. Conclusions: MR-proANP and CT-proAVP are useful new biomarkers for the risk stratification of CAP-patients. They are significantly lower in CAP survivors and correlate with the severity of the disease measured by CRB-65 score.

H. Wisplinghoff, A. Kaasch, K. Achilles, A. Langhorst, G. Peyerl-Hoffmann, A. Wöhrmann, G. Fätkenheuer, B. Salzberger, W. Kern, H. Seifert (Cologne, Freiburg, Regensburg, DE) Introduction: Nosocomial bloodstream infections (BSIs) are important causes of morbidity and S. aureus is one of the most important nosocomial pathogens. Methods: INSTINCT was established to prospectively monitor clinical features of S. aureus BSI (SAB) in Germany using a web-based database. Data are collected by infectious diseases physicians aided by specially trained medical personnel to monitor risk factors, predisposing conditions, portal of entry, clinical course, diagnostic and therapeutic procedures as well as outcome up to one year after SAB. Results: Between January 2005 and September 2007, 468 cases of SAB were detected in the 3 participating university hospitals. Patients had a mean age of 61 years and 33% were female. Length of stay averaged 33 days. SAB were classified as nosocomial in 54% of patients, as healthcare-associated in 26%, and as community-acquired in 20%. Primary SAB was seen in 61% of cases, of which 28% were catheterrelated. Among secondary SAB (39% of cases), skin and soft tissue infections (7%), surgical wound infections (5%) and respiratory tract infections (4.5%) were the most frequent portals of entry. Complicated SAB was reported in 14% of patients. 18% of S. aureus isolates were methicillin-resistant. The mean duration of S. aureus bacteremia was 3.5 days. Severe Sepsis and septic shock at onset of SAB were seen in 4.5% and 2.5% of patients, respectively. Adherence to ID consult recommendations such as TEE (recommended in 50%, followed in 34%) and follow-up blood-cultures (61%; 48%) varied significantly between centers. Patients received a mean of 16±13 days of appropriate anti-staphylococcal therapy. The crude 7-day-mortality was 15%, in-hospital mortality was 23%. Conclusion: INSTINCT currently represents one of the most detailed SAB studies. Data from the first years underscore the importance of SAB and confirm the feasibility of this very detailed prospective monitoring design.

16S rDNA PCR and sequence analysis in the diagnosis of bacterial infections C. Schabereiter-Gurtner, M. Nehr, P. Apfalter, A. Makristathis, M.L. Rotter, A.M. Hirschl (Vienna, AT) In routine laboratory praxis, culture may be negative despite a bacterial infection, while the application of molecular techniques may lead to diagnosis. At our department, a highly universal and sensitive Taq-Man-based broad-range real-time PCR assay has been designed. This assay targets to the 16S rRNA gene and facilitates the unselective and culture-independent detection of bacterial DNA in clinical specimens. Species are identified by sequencing of the generated PCR amplicon and comparative phylogenetic sequence analysis in official databases (BLAST analysis). For clinical evaluation, 136 specimens were analyzed. Specimens were taken from normally sterile body-sites and included pleural fluid (n = 38), synovial fluid (n = 35), cerebrospinal fluid (n = 16), ascitic fluid (n = 11), amniotic fluid (n = 2), drain fluid (n = 2), aspirates (n = 7), tissue biopsies (n = 13), EDTA-blood (n = 5), dialysates (n = 4), periosteum (n = 1), and others (n = 2). Materials were cultured by means of standard cultivation methods also. Out of the 136 clinical samples, 24.3% were considered as positive upon a positive culture or PCR-result; 16.2% were both culture-and PCR-positive, 1.5% were culture-positive but PCR-negative, and 6.6% were culture-negative but PCRpositive. The remaining 75.7% were both culture-and PCR-negative. BLAST analysis revealed members of Acinetobacter, Escherichia, Haemophilus, Klebsiella, Leuconostoc/Enterococcus, Mycobacterium, Mycoplasma, Neisseria, Propionibacterium, Pseudomonas, Staphylococcus, Streptococcus, Treponema, and Ureaplasma. Due to its good clinical applicability, the in house assay is now applied in our daily routine diagnosis since two years. In 2007, in 14 out of 86 culture negative cases infections with Staphylococcus aureus (n = 3), Propionibacterium acnes (n = 2), Abiotrophia detectiva (n = 1), Corynebacterium sp. (n = 1), Treponema pallidum (n = 1), Enterococcus faecalis (n = 1), Escherichia coli (n = 1), Haemophilus influenzae (n = 1), Pseudomonas aeruginosa (n = 1), Staphylococcus sp. (n = 1), and Fusobacterium necrophorum (n = 1) were detected. Concluding, the introduced approach certainly does not displace culture in clinical routine, but offers an improvement in the diagnosis of bacterial infections. It is suitable for use in laboratories with specialized staff and can be used in the clinical routine allowing for diagnosis when antimicrobial therapy has already been initiated or when culture remains negative due to infections caused by bacteria with unusual growth requirements. Diagnosis can be obtained within two days.

Diagnosing fungal infections remains a problem, particularly in the immunocompromised patient. Symptoms are mostly non-specific and colonization is difficult to distinguish from invasive disease. Existing diagnostic tools often lack sensitivity. Thus, the combination of various diagnostic tools is mandatory to allow earlier diagnosis of systemic fungal infections. Microscopy, culture based methods, antigen detection, and molecular techniques such as PCR may help to facilitate and accelerate the diagnosis. Sensitive and specific PCR assays to detect fungal DNA are an important part of the diagnostic approach. But extensive validation and standardization is strongly needed, before PCR assays can be used in a routine laboratory.

Primary and secondary resistance is an essential problem in the increasing number of systemic fungal infections as well as the unknown susceptibility of emerging pathogens. The Clinical and Laboratory Standards Institute (CLSI) established standardized methods for antifungal susceptibility testing of Candida spp. and Cryptococcus neoformans and molds including interpretative breakpoints for a selection of various antifungals. Also, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) established a standard method. However, both standards are known to be time consuming, labour intensive and difficult to use, thus preventing their application as routine techniques. Alternative approaches are still needed. Agar diffusion methods and commercial microdilution methods have also been developed and are still being evaluated.

Pathogen-specific DNA concentration step combined with a novel multiplex PCR assay improve molecular diagnosis of sepsis S. Sachse (Jena, DE) Sepsis is defined as complex of infection caused by micro-organisms and the host systemic defence against invasive microbial pathogens. The progressive development within the continuum of sepsis (sepsis to severe sepsis to septic shock) demands a rapid and direct identification of causing pathogens to initiate an appropriate antibiotic therapy. Presently, the diagnosis of the micro-organisms bases on culture-dependent methods, e.g. blood culture systems. However, these methods fail in blood samples of antibiotic pre-treated patients or in fastidious micro-organisms. An additional aggravating issue is the long time period up to 2 to 6 days which is required for microbial findings, automation of some processes notwithstanding. Consequently, the decisions for initial therapeutics rely only on clinical situation of patients without knowledge about pathogen findings. Against this background of insufficient time consuming techniques, the obligations for development of rapid, high sensitive culture independent analytical systems like nucleic acids amplification techniques (NAT) draw the focus of research in recent years. However, the sensitivity of NAT is often hampered by factors like haemin or immunoglobulins and mainly by unpropitious ratio between microbial DNA and high amount of human DNA in patient samples. Therefore an appropriate sample preparation method becomes indispensable. Our key is an innovative pre-analytical tool (LOOXSTER ® ) based on a recombinant human protein with preferential binding to microbial DNA in combination with a novel multiplex PCR (VYOO ® ) designed for the fast detection of 45 microbial sepsis pathogens and common resistances. We performed clinical pilot studies with the aim to evaluate the combination of the pre-analytical tool and multiplex-PCR for microbial diagnostic especially for septic patients. We compared the sensitivity and specificities of standard procedures (e.g. blood culture systems) with our novel sample preparation and detection systems. The results confirm noteworthy a higher sensitivity and specificity for NAT in contrast to culture-based methods in less time. Obviously, the combination of both sample preparation tool and multiplex-PCR could represent the basics for physicians to start a pathogen directed antibiotic regime within the first 24 hour which results in better outcomes for the patients.

Novel approaches to diagnose invasive fungal infections D. Buchheidt (Mannheim, DE) Over the recent years, novel methods have been developed to improve the diagnosis of invasive fungal infections in patients at high risk, especially patients with malignant haematological diseases undergoing intensive chemotherapy. Early diagnosis and treatment are crucial in the management of invasive fungal infections. However, diagnosis often remains difficult as most of the diagnostic tools in clinical use at present either lack specificity or acceptable sensitivity in the early phase of the infection. The clinical value, advantages and problems of novel approaches to diagnose invasive fungal infections are reviewed.

Adoptive Immunotherapy with Anti-Aspergillus T-Cells in Patients Undergoing Allogeneic Stem Cell Transplantation?

T. Lehrnbecher (Frankfurt, DE) Invasive aspergillosis is still a life-threatening complication in patients undergoing allogeneic stem cell transplantation (alloSCT). Since CD4+ T-cells play a major role in the secondary host defense against Aspergillus fumigatus, the most frequent cause of invasive aspergillosis, adoptive therapy with ex vivo generated human anti-Aspergillus T-cells may be a promising strategy in the prophylactic or therapeutic setting in hematopoietic stem cell recipients. In preliminary experiments, we were able to select and to expand anti-Aspergillus T-cells in healthy individuals. T-cells were first activated with a cellular extract of Aspergillus fumigatus, then isolated using the interferon (IFN)-gamma secretion assay and expanded for up to 28 days. Phenotypic characterization revealed a memory, activated T-helper cell population (positive for CD3, CD4, CD45RO and HLA-DR). In addition, the cells secreted IFN-gamma and TNF-alpha, but no IL-4 or IL-10, indicating that the generated cells were activated TH-1 cells. The isolated and expanded cells proliferated upon restimulation and showed a reduced alloreactivity compared to unselected CD4+ cells. The generated T-cells responded upon activation with antigen extracts of A.fumigatus, A.flavus, A.niger and P. chrysogenum, whereas no significant response was observed upon activation with antigen extracts of A. alternata and C. albicans. In addition, the generated Tcells alone induced damage to A.fumigatus hyphae and also significantly increased hyphal damage induced by human neutrophils. Based on these results, we developed a method for the clinical-scale generation of functionally active anti-Aspergillus T-cells according to good manufacturing practice (GMP) conditions. After stimulation of a total of 1.1x109 white blood cells derived from a leukapheresis product with Aspergillus antigens, cells were selected by the IFN-gamma secretion assay and then expanded over a period of 12 days. In three experiments, a median number of 2x107 CD3+CD4+cells (range, 0.9-3.2x107) was obtained. The cultured cells exhibited a memory activated T-helper cell phenotype. In line with the preliminary results, the generated T-cells produced IFN-gamma, but no interleukin IL-4 or IL-10, proliferated upon restimulation, and showed reduced alloreactivity compared to unselected CD4+ cells. Further studies have to identify those hematopoietic transplant recipients who will ultimately benefit from adoptive immunotherapy with anti-Aspergillus T-cells and to define the optimal time point(s) for application.

Etiology of acute gastroenteritis in three sentinel general practices, Austria 2007 F. Allerberger (Vienna, AT) We studied the etiology of acute gastroenteritis in a village with a total population of approximately 6000. This is the first study in Austria that has investigated a broad range of pathogens recovered from an unselected population of patients who had consulted general practitioners because of gastroenteritis. In 2007, all patients who vistited one of the three local general practitioners for acute gastroenteritis were invited to provide stool specimens to be tested for salmonella, shigella, campylobacter, enterohemorrhagic E. coli (EHEC) (mTSB-enrichement [R-Biopharm] followed by toxin-ELISA plus culture), enteropathogenic E. coli (EPEC), yersinia, Vibrio cholerae, Clostridium difficile (toxin plus culture), rotavirus plus adenovirus (RIDA ® Quick Rotavirus/Adenovirus Combi), Giardia lamblia plus Cryptosporidium parvum (RIDA® Quick Cryptosporidium/Giardia Combi), astrovirus (ELISA), and norovirus (RT-PCR). Stoolspecimens were provided by 283 patients (150 female) with acute diarrhoe. Patients' age ranged from 1 to 89 years (mean: 37, median: 36). A pathogen could be detected in 24% of patients, with incidence peaks in February and in June. Norovirus accounted for 36.8% of positive results, Clostridium difficile for 19.1%, rotavirus 16.2%, campylobacter 8.8%, salmonella 5%, adenovirus 5.9%, Giardia lamblia and Cryptospordium parvum for 2.9% each, and Yersinia enterocolitica for 1.4%. No case of shigellosis or infection with EHEC, EPEC and astrovirus was diagnosed. Viruses accounted for 58.8% of the 68 positive results, bacteria for 35.3%, and parasites for 5.9%. Our findings compare well to those presented in the 2006 annual Community Summary Report, with campylobacter, salmonella and yersinia infections being the most commonly reported zoonotic disesase in humans in the EU. Our study underlines a dominant role of norovirus and toxigenic Clostridium difficile as etiologic agents for acute gastroenteritis in general practice. The high prevalence of individuals infected with Clostridium difficile in the community is noteworthy. In Austria, both agents are not in the scope of a routine laboratory's stool test panel reimbursed by sickness funds.

Mikrosporidiose -Update C. Franzen (Regensburg, DE) Microsporidiosis is an opportunistic infection associated with a wide range of clinical syndromes in humans. These organisms are small single-celled, obligate intracellular parasites that were considered to be early eukaryotic protozoa but were recently reclassified with the fungi. The reduced and compact microsporidian genome has generated much interest for better understanding the evolution of these parasites, and comparative molecular phylogenetic studies continue to support a relationship between the microsporidia and fungi. Through increased awareness and improved diagnostics, microsporidiosis has been identified in a broader range of human populations that, in addition to persons with HIV infection, includes travellers, children, organ transplant recipients, and the elderly. Of the 14 species of microsporidia currently known to infect humans, Enterocytozoon bieneusi and Encephalitozoon intestinalis are the most common causes of human infections and are associated with diarrhoea and systemic disease. Species of microsporidia infecting humans have been identified in water sources as well as in wild, domestic, and food-producing farm animals, raising concerns for waterborne, foodborne, and zoonotic transmission. Current therapies for microsporidiosis include albendazole which is a benzimidazole that inhibits microtubule assembly and is effective against several microsporidia, including the Encephalitozoon spp., but is less effective against E. bieneusi. Fumagillin, an antibiotic and anti-angio-

genic compound produced by Aspergillus fumigatus, is more broadly effective against Encephalitozoon spp. and E. bieneusi but is toxic when administered systemically to mammals. Gene target studies have focused on methionine aminopeptidase 2 for characterizing the mechanism of action and for identifying more effective, less toxic fumagillin-related drugs. Polyamine analogues have shown promise in demonstrating anti-microsporidial activity in culture and in animal models, and a gene encoding topoisomerase IV was identified in Vittaforma corneae, raising prospects for studies on fluoroquinolone efficacy against microsporidia. Effective commercial therapies for E. bieneusi, the most common microsporidian species identified in humans, are still lacking, making the need to develop tissue culture and small animal models increasingly urgent. Questions still exist about whether microsporidia infections remain persistent in asymptomatic immune-competent individuals, reactivate during conditions of immune compromise, or may be transmitted to others at risk, such as during pregnancy or through organ donation. Reliable serological diagnostic methods are needed to supplement polymerase chain reaction or histochemistry when spore shedding may be sporadic.

Giessen -Overview: Pneumonia -molecular signatures of compartimentalized and barrier penetrating alveolar infection J. Lohmeyer (Giessen, DE) Infections of the distal respiratory tract are high incidence diseases in Germany with significant mortality and high impact on health economics. Novel therapeutic approaches beyond antibiotics are urgently needed and depend essentially on a fundamental understanding of pathogen host cell interactions the lung. The clinical research unit in clinical infectiology therefore aims at elucidating the clinically relevant molecular interplay between infectious agents and the host related to lung specific organ features and to develop novel tailored strategies for the diagnosis and treatment of pneumonia. In tissue/organ models, whole animals and humans the contribution of microbes and their products to the course of pneumonia will be systematically evaluated to identify the molecular checkpoints in the microbial host interplay that discriminate protective host defence from injurious inflammation with loss of alveolo-capillary barrier function. The clinical research unit is structured as interdiscipinary network of the infectious disease core unit in the department of internal medicine with theoretical (institute of biochemistry) and theoretical clinical (institutes of medical microbiology, medical virology, clinical immunology, pathology) institutions long term active in the research field of inflammation and infections. In 5 subprojects the contribution of microbial agents and their components to pneumonia pathogenesis will be systematically evaluated in tissue and organ models in vitro and in animal infection/inflammation models as well as in humans in vivo. In addition, the host molecular signatures of lung parenchymal infections will be defined and evaluated for their potential as biomarkers for diagnostics and clinical course. Long term diagnostic algorithms and intervention strategies based on these results will be evaluated in clinical settings. To this end, prospective asservation of thoroughly characterized patient material has been already started in the associated biomaterial bank project Z. This interdisciplinary research approach aims at rapidly translating basic science results into clinical studies and medical care in the field of infectious diseases currently underdeveloped in Germany.

Diagnosis of fungal infections imported to Central Europe

Mycoses, which are imported to Central Europe, are caused by a broad spectrum of fungi. This includes primary pathogens such as Histoplasma capsulatum and Coccidioides species as well as opportunistic fungi like the infectious agents of eumycetoma, chromoblastomycosis and dermatomycosis. The clinical manifestation of fungal infections is rarely characteristic. Diagnosis of an imported fungal infection starts with the knowledge of the clinical spectrum of mycoses, long term anamnesis including the country of residence and travels abroad to certain endemic areas and the Entnahme of adequate clinical samples. The optimal diagnostic strategy for fungal infections depends on the immunological status, the clinical manifestation of the suspected fungal disease and the geographical location, where the infection might have been aquired. Histology and detection of fungal cells by microscopy of biopsies, BAL etc. have a high diagnostic value for a preliminary diagnosis, identification of the fungal agent is rarely possible. Cultural detection of the suspected fungus should still be the aim of laboratory diagnostics. Many of the fungal agents are slow growing organisms, therefore molecular methods -although still under evaluation -can be highly beneficial for detection of imported fungal infections, especially for diagnosis of histoplasmosis and other systemic infections by dimorphic fungi. Nevertheless the results of PCR and sequencing have always to be checked for plausibility. Antibody detection against Histoplasma capsulatum and other primary pathogens also has a high diagnostic value in non-endemic areas, because patients are often immunocompetent and able to produce specific antibodies. Tests are available for serodiagnosis of histoplasmosis, coccidoidomycosis and paracoccidioidomycosis. Other serological assays are routinely not available. Infections due to dimorphic fungi like H. capsulatum, Coccidioides spec. or Penicillium marneffei, pathogens of chromoblastomycosis, eumycetoma and others will be presented by case reports, adequate diagnostic tools are suggested.

A novel enzymatic process for the synthesis of new antifungals V. Hahn, A. Mikolasch, F. Schauer (Greifswald, DE) Introduction: Candida species are today the most important cause of opportunistic mycoses in the world (Cihlar, 2007) . Therefore, we developed a new technology to synthesize novel antifungal substances using fungal laccases as catalyst. Laccases [E.C. 1.10.3.2] are polyphenoloxidases, which oxidize aromatic phenols to reactive radicals, which can undergo non-enzymatic coupling reactions. The heteromolecular coupling reactions mediated by laccases can be applied for an environmentally friendly synthesis of a variety of chemical substances and bioactive compounds (Mikolasch et al., 2007) . According to these advantages we used this reaction for the derivatization of basic structures of antifungal compounds like azoles or morpholines to increase antifungal activities, to improve bioavailability or to reduce side effects. Methods: The laccases were obtained from the ligninolytic fungi Pycnoporus cinnabarinus (Pcl) and Myceliophthora thermophila (Mtl). The reaction mixtures were analyzed using an HPLC system with DAD and a RP 18 column. For mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy the products were isolated via solid phase extraction and dried by lyophilization. The antifungal susceptibility testing was performed with a modified method of E.Dis. 7.1 (Rodriguez-Tudela et al., 2003) . Results: In course of our investigations concerning the modifications of azoles and morpholines, Pcl and Mtl has been found to be able to catalyze the synthesis of novel antifungal compounds. During the laccase-catalyzed transformation of para-hydroxylated substrates into quinones a nucleophilic attack of azoles or morpholines occurs, resulting in C-N-coupled heteromolecular products. Depending on the substituents mono-or diaminated products were formed. Yields of 25-74% showed the high efficiency. The MS and NMR analyses confirmed the supposed structures of the newly synthesized compounds. Among the isolated products we found substances with antifungal activity, although the substrates were not or less active. Conclusions: Through the developed enzymatic process we could synthesize different potential antifungal substances. This process of combinatorial biochemistry may be suitable for the production and improvement of properties of many other biological active substances. Literature Cihlar, R.: Candida albicans (2007) 

Enhanced expression of RND efflux pumps in pairs of isogenic clinical strains of Pseudomonas aeruginosa with acquired multidrug resistance E. Fähnrich, A. Huth, A. Sigge, M. Vavra, H. von Baum, W.V. Kern (Freiburg, Ulm, DE) Background: P. aeruginosa (PA) is a nosocomial pathogen which often shows or develops multidrug resistance (MDR). Mechanisms of MDR in PA can be diverse and may include decreased intracellular penetration of antibiotics associated with overexpression of efflux pumps. Detailed information about the risks of efflux-associated MDR in PA is limited since pairs of isogenic isolates are infrequently available for further study.

Methods: Seven pairs of clinical PA isolates with identical genotypes (as assessed by pulsed-field electrophoresis PFGE) were isolated from adult patients hospitalized at an intensive care unit of Ulm University Hospital. Susceptibilities to a panel of different antibiotics were studied by microbroth dilution in the presence or absence of the putative efflux pump PAßN. The quinolone-resistance-determining regions (QRDRs) of gyrA and parC were sequenced. Intracellular dye (pyronin Y) and PAßN accumulation was measured by fluorometry. Quantitative RT-PCR (qRT-PCR) was performed to determine the expression of different RND efflux pumps (mexA, mexD, mexE and mexX) and a porin (oprD). The expression of two independent housekeeping genes was used as a relative standard (rpsL and rpo). Wildtype strain PAO1, efflux-pump knockout strain PA1425 and mexAB/oprM overexpressing strain PA1426 were used as controls. Results: MIC tests confirmed decreased susceptibility of the second isolate to ß-lactams, fluoroquinolones, tetracycline and/or other antibiotics compared to the parental isolate. A change in the QRDR of gyrA was documented in one pair (83 Thr ->Ile) although in 4 of the 7 cases, a fluoroquinolone had been given between isolation of the two strains. Interestingly, this was the only strain with increased intracellular dye and/or PAßN accumulation compared to the presumed parenteral strain. qRT-PCR revealed overexpression of mexD or mexA in the second compared to the initial isolate in 3 cases. PAßN and pyronin Y accumulation was not consistently correlated with efflux pump gene expression. PAßN accumulation, however, correlated well with the intrinsic antibacterial activity of PAßN. Conclusions: Efflux mechanisms play an important role in the development of MDR resistance during therapy with antimicrobial substances. In 6 out of 7 pairs decreased dye or PAßN accumulation was observed, and in 3 an enhanced activity of efflux pumps was detected and likely responsible for reduced susceptibilty to different classes of antibiotics.

Analysis of the beta-Tubulin Gene from Vittaforma corneae Suggests Benzimidazole Resistance C. Franzen, B. Salzberger (Regensburg, DE) Introduction: Vittaforma corneae is a microsporidium causing corneal and disseminated infections in humans. Benzimidazoles are widely used as antihelmintic drugs in veterinary and human medicine and as antifungal agents in agriculture, and albendazole is one of the most commonly used drugs for treating microsporidiosis in humans. However, clinical data show poor response of Vittaforma infections to albendazole. Methods: We amplified, cloned, and sequenced the beta-tubulin gene of V. corneae and benzimidazoles were assayed for antimicrosporidial activity using an in vitro assay with V. corneae and Encephalitozoon cuniculi (as control). Microsporidian spores and drugs were added to monolayers of MRC-5 cells on day 0, and the numbers of organisms were measured on day 10. Results: The sequence showed 72% similarity with the beta-tubulin of Enterocytozoon bieneusi and 72 to 74% similarity with beta-tubulins of Encephalitozoon spp. Analysis of the obtained sequence provides an explanation for the observed clinical resistance of V. corneae to albendazole. The beta-tubulin gene encoded by the sequence has a substitution at Glu198 (with glutamine), which is one of six amino acids reported to be associated with benzimidazole sensitivity. Albendazole expressed significant higher MIC50s for V. corneae than for E. cuniculi. Conclusions: Molecular data explain the clinicaly observed resistance of V. corneae to benzimidazoles, that could be shown also in vitro. Infections with this microsporidium should not be treated with benzimidazoles and other treatment options should be used.

Influence of different efflux pump inhibitors on antibiotic resistance in multi-resistant bacteria and control strains J. Nemeth, A. Abrahim, M. Zeitlinger (Vienna, AT) Introduction: It is widely recognized that expression of efflux pumps is largely responsible for the phenomenon of both intrinsic and acquired antibiotic resistance. Thus, inhibition of bacterial efflux mechanisms appears to be a promising target in order to achieve or restore drug susceptibility. Methods: Two previously described efflux pump inhibitors (EPI), 1-(1-naphthylmethyl)-piperazine (NMP) and phenyl-arginine-betanaphthylamide (PAßN), were compared with two novel, specific pglycoprotein inhibitors, tariquidar and elacridar, in terms of effects on in vitro antibacterial activity. Antimicrobial susceptibility to ciprofloxacin, clarithromycin and clindamycin in the absence and presence of NMP, PAßN, tariquidar and elacridar were tested in the following strains: Staphylococcus aureus ATCC 29213 (SA), ciprofloxacin resistant S. aureus SA-1199B (rSA), Pseudomonas aeruginosa ATCC 27853 (PS) and Stenotrophomonas maltofilia ATCC BAA-85 (SM).

Results: Inhibition of p-glycoprotein mediated drug efflux by tariquidar and elacridar increased susceptibility of rSA towards ciprofloxacin in a dose depended manner, whereas this effect was absent in SA and gram-negative strains. Unspecific inhibition of drug efflux by MNP and PAßN increased susceptibility towards ciprofloxacin, clarithromycin and clindamycin in all resistant strains tested. Unspecific efflux inhibition resulted in bacteriostatic effects for clarithromycin and clindamycin for in intrinsic resistant PS and SM. Conclusions: Our findings suggest that acquired antibiotic resistance is associated with p-glycoprotein mediated efflux in particular strains. Moreover, our data underline the assumption that unspecific efflux mechanisms are the main cause of non-susceptibility to antibiotics in PS and SM.

Prevalence of Plasmodium falciparum crt(K76T) and resistance to chloroquine and amodiaquine in Ghana S. Ehrhardt, T.A. Eggelte, G.D. Burchard, S.D. Anemana, U. Bienzle, F.P. Mockenhaupt (Hamburg, DE; Amsterdam, NL; Takoradi, GH; Berlin, DE) Introduction: Chloroquine (CQ) treatment has been the mainstay of malaria control in sub-Saharan Africa but now fails because of intensifying drug resistance. Artemisinin-based combination therapy (ACT), e.g., amodiaquine-artesunate (AQ-AS), is being implemented as policy in Ghana as well as in many other African countries. CQ resistance is associated with a mutation in the Plasmodium falciparum CQ resistance transporter gene [Pfcrt(K76T)]. Pfcrt(K76T) is associated with AQ resistance, possibly to a lesser degree than with CQ resistance. We examined geographical patterns of Pfcrt(K76T) and blood CQ concentrations in > 4000 Ghanaian children. Methods: In 30 settlements in Tamale (urban) and surrounding districts (rural), 70 children aged 0.5 to 9 years were randomly recruited.

Sampling was done twice, from January to April (dry season) and from July to October (rainy season). Anaemia was defined as Hb < 11 g/dl. Microscopy of Giemsa-stained thick blood films was performed. Plasmodium infection was confirmed by PCR and Pfcrt(K76T) by PCR-restriction fragment length polymorphism analysis. CQ blood concentrations were measured by enzyme-linked immunosorbent assay. Continuous variables were compared by the Mann-Whitney U test and proportions by chi-squared tests. Factors independently associated with the presence of Pfcrt(K76T) were identified by logistic regression analysis. Results: Regardless of the season, some three-quarters of the children were infected with P. falciparum. Nearly 80% of infected children harbored parasites with the resistant Pfcrt(K76T) genotype. Blood CQ levels were observed in 22% of children during the dry season and in 34% during the rainy season. In both the dry and rainy seasons, the prevalences of CQ in blood differed between the 30 communities surveyed (P was < 0.0001 for each). Factors associated with the prevalence of Pfcrt(K76T) were CQ in blood, low parasitemia, asymptomatic infection, and the dry season. No geographical pattern predicted Pfcrt(K76T). A trend for a higher prevalence of Pfcrt(K76T) with increasing district population size was observed. The same trend was seen for the prevalence of CQ in blood. Conclusions: Residual CQ concentrations and Pfcrt(K76T) are common in a representative sample of children in one area in sub-Saharan Africa. If the Pfcrt(K76T) mutation is confirmed to translate into impaired AQ efficacy in this part of Africa, the useful life span of AQ-AS may be shortened considerably.

Efflux-mediated enhanced macrolide resistance by AcrAB-TolC from Escherichia coli versus MexAB-OprM from Pseudomonas aeruginosa: a single amino acid makes the difference C. Wehmeier, S. Schuster, E. Fähnrich, W.V. Kern (Freiburg, DE) Introduction: Tripartite, so-called resistance nodulation cell division (RND) protein complexes from gram-negative bacteria provide resistance to a wide variety of different antimicrobial agents. AcrAB-TolC is the major RND efflux pump in E.coli, whereas MexAB-OprM is one of several active RND efflux pumps in P.aeruginosa. The amino acid identity of the periplasmic space spanning pump component AcrB from E. coli and the homologous MexB protein from P.aeruginosa is 70%. We wondered whether amino acid differences in the region 610 to 628, a region likely to be critical for substrate recognition by AcrB based on crystal structure studies, might explain substrate differences between acrB and MexB. Methods: We initially engineered a chimeric AcrB efflux protein, in which the amino acid region 615 up to 628 was exchanged with the corresponding amino acids of MexB. Chromosomal mutagenesis in an AcrAB overexpressing E.coli strain (3AG100) was done by homologous recombination, using the Red/ET counter-selection modification method. Susceptibility to several antimicrobial agents and dyes was tested by broth microdilution. Effects of the 615-628 AcrB/MexB substitution were also investigated by exchange of single amino-acids within the AcrB/MexB complex and within AcrB as control experiment. Results: The exchange of region 615-628 from AcrB with the corresponding region from MexB resulted in four amino-acid substitutions: G616 was altered to N, N623 and T624 to S and I626 to M. Compared with the wildtype, the chimeric mutant showed 4-8 fold increased susceptibility to the macrolides, whereas the susceptibility to several other antibiotics and dyes was less affected or unchanged. Single substitution of amino acid 616, 623, 624 and 626 in AcrB/615-628MexB back to the original amino acid as well as single substitutions within wildtype AcrB to the corresponding amino acids of MexB gave evidence for the G616 to N exchange as specific for macrolide susceptibility. Conclusion: The MexB efflux protein from P.aeruginosa provides much less to macrolide resistance than the homologous AcrB efflux protein from E.coli. We confirmed involvement of the AcrB aminoacid region 610-628 in encoding substrate specificity by investigating chimeric AcrB/MexB chromosomal mutants. We found that a single amino acid, G616, plays a major role for elevated macrolide resistance mediated by AcrB.

Differential effects of the Val-612 to Phe mutation in the E. coli AcrB efflux pump protein on resistance to linezolid, fluoroquinolones and chloramphenicol versus other AcrAB efflux pump substrates E. Fähnrich, J. Bohnert, S. Schuster, W.V. Kern (Freiburg, DE; Berkeley, US) Introduction: AcrAB is the major resistance nodulation cell division (RND) efflux pump in E. coli and provides multidrug resistance (MDR) including resistance to fluoroquinolones (FQ), macrolides (ML) and linezolid (LZ), but the E. coli genome encodes other RND pumps, such as AcrEF and MdtEF (formerly YhiUV), which also provide multidrug resistance (MDR) when overexpressed in an AcrABdeficient background. We have previously shown that the spectrum of substrates of MdtEF does not include LZ but that a specific periplasmic loop mutation in MdtF (Val-610 to Phe) restores LZ resistance, increases FQ resistance but decreases ML resistance of E. coli. In this study we introduced this mutation in AcrB and evaluated its effects on the susceptibility of a variety of drugs. Methods: The Val-610 to Phe mutation in MdtF corresponds to the Val-612 to Phe mutation in AcrB. We introduced this mutation in an AcrAB overexpressing E. coli laboratory strain by a phage lambdabased homologous recombination method (Red/ET system) and tested the changes in pump substrate susceptibility by microbroth dilution. Results: The AcrB Val-612 to Phe mutation was confirmed by nucleotide sequencing, and the growth rate of the mutant strain was comparable to that of a Phe-628 to Phe (TTC to TTT) pseudomutant. The Val-612 to Phe provided a substantial loss of resistance to almost all AcrAB substrates including ML (to near pump knockout levels) but not FQ, chloramphenicol and LZ. Western blot experiments (polyclonal AcrB antibody) initially failed to show expression of the mutant protein presumably due to conformational changes and antibody epitope masking but experiments with high-expression plasmids yielded results similar to wildtype. Also, AcrA protein expression and acrB gene expression were similar to wildtype. Conclusion: Similar to the mutation in MdtF, the Val-612 to Phe mutation in AcrB differentially affects resistance to LZ and FQ versus ML and others indicating that this amino acid is involved in substratespecific pump function. We speculate that this site has a role in substrate-specific binding and transport, and that the mutation is associated with reduced binding and enhanced transport of a given substrate and increased binding and reduced transport of another one. This data indirectly confirms crystal structure information that proposes Val-612 as one among other residues that participate in forming an AcrB substrate binding pocket.

Ampicillin/Sulbactam (AS) for the treatment of aspiration pneumonia and primary lung abscess H. Lode (Berlin, DE) Aspiration pneumonia (AP) and primary lung abscess (PA) are important complications following aspiration of infectious material from the oropha-rynx or stomach;fatality rates of up to 20% have been reported(1).The principal therapeutic strategy is a prolonged course of antibiotic therapy after exclusion of bronchial obstruction.-In two prospective studies between 1995 and 2005 a total of 234 patients with these infections were investigated (2,3).AS was compared to Infection Abstracts KIT2008 clindamycin(CL) plus a cephalosporin in the first study and to moxifloxacin(MX) in the second study.-166 patients (mean age 60,5 years,72% males)were treated according to the protocol;85 pat. received AS in an i.v. starting dose of 3,0g three times daily followed by twice a day orally 750 mg.Leading pathogens collected by bronchoscopy were S.aureus, Klebs. pneum. and anaerobes.Overall clinical response rate at end of therapy was 69,8% for AS, 66,7% for CL and 66,7% for MX.Mean duration of treatment was between 22+/-23 days for AS and 26+/-29 days for MOX.All three antibiotic regimes were well tolerated. Conclusion: Ampicillin/Sulbactam proved equally ef-fective and safe as comparators (clindamycin plus cephalosporin or moxifloxacin)in the treatment of aspiration pneumonia and lung abscess. 

Levofloxacin 500 mg once daily oral in the treatment of chronic bacterial prostatitis (CBP)

Introduction: Levofloxacin 500 mg is a fluoroquinolone given once daily (OD) active against a wide range of gram-negative and grampositive bacteria which has demonstrated its efficacy in the treatment of complicated urinary tract infections. It is also as effective as ciprofloxacin 500mg twice daily in the treatment of chronic bacterial prostatitis (CBP). The study was aimed at further confirming in Europe the efficacy and safety of levofloxacin in chronic bacterial prostatitis. Methods: Men with a history of CBP (category II), clinical signs and symptoms and laboratory evidence of prostatitis (Meares-Stamey "four glass" procedure (MS)) were enrolled in a prospective, multinational (7 countries) open-label study to receive levofloxacin 500mg OD per os for 28 days. They were followed for 6 months. Clinical signs and symptoms were evaluated 5-12 days and then at 1, 3 and 6 months post-treatment. Microbiological (by MS) eradication rate was determined at 1 and 6 months post-treatment. A medical expert team (MEA) validated clinical and microbiological end-points. Spontaneously reported adverse events were collected. The statistical analysis consists of point-estimates for the proportion of subjects and corresponding 95%-confidence intervals calculated using Pearson-Clopper values. Results: A total of 117 patients were treated (age 45.0 years, duration of CBP 48,0 months, median values). A Gram-negative bacteria was identified in 55 patients (mainly E.coli, n=36) and a Gram-positive bacteria in 50 cases (mainly E.faecalis, n=13 and S.epidermidis, n=11), Of the 100 men evaluable for clinical outcome (ITT population), the clinical success rate (cured and improved patients) was 92% [84.8, 96 .5], 77.4%[68.2, 84.9], 66.0% [56.2, 75.0], and 61.9% [51.9, 71.2], at 5-12 days, 1 month, 3 months and 6 months post-treatment. Microbiological eradication rates (in the microbiologically assessable population) were 83.7% (82/98) at 1 month and 91.2% (52/57) at 6 months post-treatment. Levofloxacin was well tolerated. Three patients discontinued therapy for adverse event and 15 patients (12.8%) experienced at least one treatment-emergent adverse event (mainly gastrointestinal (n=7) and musculoskeletal and connective tissue disorders (n=6)). Conclusions: Levofloxacin 500mg once daily given orally for 28 days is clinically and microbiologically effective in the treatment of chronic bacterial prostatitis and is well tolerated.

Plasma protein binding DOES influence bacterial killing by fluoroquinolones -investigation by use of an improved method.

M. Zeitlinger, R. Sauermann, M. Fille, J. Hausdorfer (Vienna, AT) Introduction: Doubt on the influence of plasma protein binding (PPB) on bacterial killing of fluoroquinolones is based on the lack of any relevant inhibitory effect by addition of 20 or 70% serum to standard growth media. We set out to investigate whether i) this model is appropriate to investigate PPB of fluoroquinolones, ii) to develop an improved model and to iii) use this model to study the influence of PPB on activity of two fluoroquinolones. Methods: Protein binding of moxifloxacin and trovafloxacin was determined in Mueller-Hinton-broth (MHB) containing different concentrations of human serum (20-100%) or different concentrations of human albumin (4-16%). Bacterial growth curves (n=5) of S. aureus and P. aeruginosa were performed in 100% serum, MHB and MHB containing either 70% or 20% serum or 12% albumin. Killing of S. aureus and P. aeruginosa was investigated for moxifloxacin and trovafloxacin at MICs in MHB and MHB containing 12% albumin. Results: Protein binding decreased from 38% and 78% to 5% and 38% in pure serum and 20% serum for moxifloxacin and trovafloxacin, respectively. PPB in 12% albumin was identical to pure serum. Growth curves in MHB and MHB containing 12% albumin were identical while growth of S. aureus and P. aeruginosa was significantly hampered in pure serum and MHB containing 20 or 70% serum. Addition of 12% albumin significantly reduced bacterial killing of both S. aureus and P. aeruginosa by 1 to 3 log10 cfu/mL as compared to MHB without albumin after 8 hours of exposure to fluoroquinolones. Conclusion: Investigating influence of PPB on activity of fluoroquinolones by addition of 20 or 70% serum is insufficient as these media neither reach the PPB achieved in pure serum nor allow appropriate bacterial growth. MHB containing 12% albumin is a promising medium in this context as PPB is identical to pure serum and bacterial growth shows no difference compared to MHB. Highly significant impact of PPB on antimicrobial activity of fluoroquinolones was observed in the present study.

Influence of impaired Liver Function on the Pharmacokinetics of Amphotericin B Colloidal Dispersion S. Weiler, R. Bellmann-Weiler, M. Joannidis, R. Bellmann (Innsbruck, AT) Introduction: Investigations on the pharmacokinetics and elimination of amphotericin B (AMB) lipid formulations in liver impairment have been lacking so far. In this clinical study the pharmacokinetics of AMB colloidal dispersion (ABCD) was assessed in critically ill patients with cholestatic liver failure. Patients and Methods: Time-concentration profiles were determined in critically ill patients with cholestatic liver failure and in critically ill patients with normal hepatic function requiring ABCD for invasive fungal infections. The lipid-associated and liberated fraction of AMB was separated by solid phase extraction and subsequently quantified by high performance liquid chromatography. Results: Three patients with impaired and three patients with normal hepatic function on day one of ABCD therapy have been enrolled so far. After a single dose of ABCD (2.46 ± 0.54 mg vs. 2.94 ± 1.47 mg/kg in the impaired-liver group compared to the control group), the maximum concentration in patients with impaired liver function was four fold increased compared to the control group ( 

The elimination of ABCD appears to be delayed in cholestatic liver failure. Particularly the differences of the lipid-associated fraction are pronounced. More pharmacokinetic data are required to establish reliable dose recommendations for ABCD in patients with liver failure.

Effect of N-Chlorotaurine and Ammonium Chloride on Bacterial Proteins B. Moser, R. Arnitz, B. Sarg, H. Lindner, M. Nagl (Innsbruck, AT) Introduction: N-Chlorotaurine (NCT), a product of human leukocytes, is a promising antiseptic with very good tissue tolerability. It is a mild oxidative agent which triggers modification of the protein pattern in bacteria. A combination with ammonium chloride (NH4Cl) leads to the formation of monochloramine (NH2Cl), which penetrates bacteria more rapidly and thereby enhances the microbicidal activity. The aim of this study was to investigate the influence of the combined application of NCT and NH4Cl on the extend of the protein pattern change in different bacterial strains after a very short incubation time.

Smith diffuse-cultures were incubated in buffer, 1% NCT or 1% NCT plus 1% NH4Cl for 1 minute. To detect differences in protein pattern the extracted baterial proteins were seperated by 2D-PAGE and detected by silver staining. Results: After a 1 minute incubation of both bacterial strains in 1% NCT solution several proteins were separated into a series of spots with a different isoelectric point. The combined incubation of NCT and NH4Cl led to an enhanced destruction of proteins already after 1 minute, which was shown in additional separation of protein spots. Conclusions: These data fit very well to data showing a faster microbicidal effect of NCT plus NH4Cl, which can be explained by formation of the more lipophilic and microbicidal monochloramine.

Microbicidal activity of Monochloramine and Chloramine T Compared R. Arnitz, M. Nagl, W. Gottardi (Innsbruck, AT) Introduction: Chloramine T (CAT) and monochloramine (NH2Cl) are active chlorine compounds and well-known antiseptics with broadspectrum microbicidal activity. CAT has stronger oxidative activity than NH2Cl, while the latter one is a smaller molecule and more lipophilic. The question arose if lower oxidative activity can be compensated by higher lipophilicity regarding effectiveness. To address this problem, we investigated the bactericidal and fungicidal activity of pure NH2Cl compared to CAT. Methods: Bacterial strains (Escherichia coli ATCC 11229, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853) and clinical isolates of Aspergillus fumigatus, Aspergillus flavus and Candida albicans were taken for quantitative killing assays. All microorganisms were tested separately in equimolar solutions of CAT and NH2Cl, respectively. Pathogens treated in buffer without antiseptics served as controls. Results: At a concentration of 0.011 mM, CAT exerted no microbicidal activity, while NH2Cl killed all test bacteria within 30 min. At a concentration of 0.036 mM and 0.107 mM, a 3-4 log reduction occurred approx. 100 times earlier with NH2Cl than with CAT in E. coli, and 3-4 times earlier in S. aureus and P. aeruginosa. NH2Cl (0.355 mM) caused a 3 log reduction of C. albicans within 1 min compared to a 2 log reduction by 0.355 mM CAT. The difference between both antiseptics was even more pronounced in aspergilli.

The results show a significantly stronger bactericidal and fungicidal activity of monochloramine compared to chloramine T despite its lower oxidative activity. This phenomenon can be attributed to its lipophilicity and smaller molecular weight.

Microbicidal activity of N-chlorotaurine in the presence of heparin C. Martini, W. Gottardi, M. Nagl (Innsbruck, AT) Introduction: The sodium salt of N-chlorotaurine (NCT) is an endogenous, long-lived chloramine compound with broad-spectrum antimicrobial activity. It can be conceived for disinfection of catheters in human medicine. Heparin is applied to prevent blood coagulation in catheter systems (e.g. Port-A-Kath-systems). Heparin combined with NCT could be of advantage for irrigation of catheters to avoid coagulation as well as infection. It was the aim of this study to investigate the compatibility of both components regarding anti-coagulative and antimicrobial activity. Methods: In quantitative suspension tests 1% and 0.1% NCT plus 125 I.E./ml heparin was tested against bacteria (Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes, Staphylococcus aureus, Proteus) at pH 7.1 and 37°C. Results: All test bacteria were killed after 5 -10 min (1% NCT) and 30 -60 min (0.1% NCT). The combination of 1% and 0.1% NCT with 125 I.E./ml heparin was equally effective. Spectrophotometric investigations revealed no hints for a chemical reaction of NCT with Heparin. Conclusions: Addition of heparin does not impact the microbicidal activity of NCT and could be suitable for clinical application.

Antimicrobial efficacy of fosfomycin in cerebrospinal fluid R. Sauermann, M. Fille, M. Camuz Ligios, R. Schwameis, M. Zeitlinger (Vienna, Innsbruck, AT) Introduction: Treatment of shunt-associated ventriculitis (SAV) requires administration of an effective antibiotic regimen to avoid lifethreatening sequelae. Due to its activity against gram-positive pathogens, fosfomycin is considered for treatment of SAV. The present study aimed at a) evaluating if the fosfomycin concentrations measured in vivo at the target site, i.e. in cerebrospinal fluid (CSF) are sufficient to kill clinically relevant bacteria, b) evaluating if human CSF is an eligible medium for bacteriologic experiments and for simulation of SAV. Methods: CSF was collected from over 700 patients who did not receive antibiotics. Time-kill curves were performed using fosfomycin concentrations of 0.25, 0.5, 1, 2, 4, and 8-fold the MIC of the Staphylococcus aureus test strain for 24 hours. In order to evaluate different media the kill curves were preformed in Muller-Hinton-broth (MHB) plus glucose-6-phospate (G-6-P), in CSF, and in CSF incubated with 5% CO2. Ambient CO2 served to adjust the pH of CSF to physiological values. The fosfomycin concentration-time profile previously measured in vivo in CSF in the patient with the lowest drug levels was simulated in vitro using pooled CSF. Results: As expected fosfomycin concentrations above the MIC led to effective bacterial killing in MHB. Addition of fosfomycin to CSF did not induce any inhibition of bacterial growth if incubated at ambient air. Experiments performed in CSF with physiological pH achieved by 5% CO2 incubation showed sustained growth inhibition only at fosfomycin concentrations of 8-fold the MIC, while re-growth was observed for all lower fosfomycin concentrations. In-vitro exposure of two S. aureus strains with an MIC of 2 and 16 mg/ml to the lowest fosfomycin concentrations measured in vivo in CSF induced bacterial

killing for the first 10 hours, whereas bacterial re-growth was observed after 24 hours in CSF incubated with CO2. Conclusions: CSF incubated with 5% CO2 is eligible for performing bacteriologic experiments with fosfomycin. Fosfomycin exerted bacterial killing for at least 10 h in CSF, even in the patient with the lowest measured drug levels. CSF and MHB are substantially different bacteriologic media, which differ with regard to bacterial growth and killing. In CSF bacterial re-growth occurs at fosfomycin levels above the MIC. Thus, the use of MHB might overestimate the efficacy of antibiotics in infected CSF.

Activity of daptomycin and azithromycin against established biofilms formed by Staphylococcus epidermidis isolated in cardiac device infections E. Presterl, C. Kratzer, S. Reichmann, A. Lassnigg, W. Graninger (Vienna, AT) Introduction: Implanted devices are increasingly used to sustain cardiac function in acute and chronic heart failure. Bacterial biofilms are fundamental in the pathogenesis of implant infections where the predominant pathogen is S. epidermidis. The activity of the novel lipopeptid antibiotic daptomycin is tested against established biofilms formed by clinical isolates from patients with cardiac implant infections. Methods: Biofilms of 54 S. epidermidis isolated in 19 patients with cardiac implant infections (including pacemakers, implanted defibrillators, ventricular assist devices, prosthetic valves), in 20 patients with catheter-related bacteremia and 15 S. epidermidis skin isolates from healthy volunteers, were treated with daptomycin or vancomycin in increasing concentrations according to the respective MIC (1-128 x MIC) alone and only three S. epidermidis ísolated from cardiac implant infections were tested in combination with azithromycin (512 mg/l and 1024 mg/l) or N-acetylcystein (NAC 20 mg/l) in a static microtiter plate biofilm model. The optical density (OD) was determined at a wavelength of 550 nm. The OD of the untreated biofilm of the respective isolate was set as 1. To test for viable S. epidermidis in the biofilms, the unfixed biofilms were scraped off and seeded to Columbia agar. Results: Treatment with daptomycin alone reduced the OD of the biofilms significantly beginning with a concentration of 8 x MIC (OD_ DAP_MIC8: 0.86 ± 0.31; p <0.001). In contrast, treatment with vancomycin did not reduce the OD of the biofilm up to a concentration of 128 x MIC (OD_VAN_MIC128: 1.15 + 0.64). However, both daptomycin and vancomycin were not bactericidal even at the highest concentration. Azithromycin at both concentrations or NAC did not reduce the OD of the biofilms, neither in combination with DAP nor with VAN. However, the combination of DAP or VAN with AZI resulted in significant decrease of > 5 log count of viable bacteria beginning with a concentration of 1 x MIC of the respective antibiotic in one test strain. Conclusions: Daptomycin is a promising new option for the treatment of implant infection associated with biofilm formation. Azithromycin at high concentration might enhance bactericidal effects of daptomycin and vancomycin in selected strains isolated from cardiac implant infections.

Emerging respiratory infections in hematopoietic stem cell and solid organ transplant recipients in Germany T. Schenk, B. Huck, G. Peyerl-Hoffman, W. Kern, M. Hufnagel, B. Ehrenstein, A. Plentzt, D. Neumann-Haefelin, V. Falcone (Freiburg, Regensburg, DE) Introduction: Respiratory viruses represent a frequent cause of morbidity in immunocompromised patients. Recently discovered viruses, such as human metapneumovirus (HMPV), human bocavirus (HBoV) and human coronavirus NL-63 (HCoV-NL63), have been suggested as etiologicl agents of acute respiratory tract infections. The aim of our study was to investigate the relative contribution of these viruses to morbidity in pediatric as well as adult haematopoietic stem cell and solid organ transplant recipients. Methods and Results: Nasopharyngeal aspirates from 69 symptomatic children (53% with symptoms of upper respiratory tract infection, 20% with tracheobronchitis, 14 % with pneumonia, and 7% requiring artificial ventilation) were collected during 135 separate respiratory episodes. All samples were prospectively investigated by real-time PCR for HMPV, respiratory syncytial virus (RSV), influenza virus A and B (Flu A and B), rhinovirus (RV), parainfluenza virus 1-3 (PIV1-3), and adenovirus (ADV). Retrospectively, they were also analysed for HBoV DNA and HCoV-NL63 RNA. A total of 66 (49%) of the analysed samples were found positive for at least one of the viral agents. In particular, 16 (12%) ADV-, 15 (11%) RV-, 7 (5%) HBoV-, 4 (3%) RSV-, 4 (3%) Flu A and B -, 4 (3%) PIV1-3-, 4 (3%) HCoV-NL63-, and 3 (2%) HMPV-positive samples were detected. Prospective analysis of respiratory samples from 32 adult patients (41% with symptoms of upper respiratory tract infection, 6% with tracheobronchitis, 26% with pneumonia and 6% requiring artificial ventilation) could not identify HBoV or HCoV NL63, whereas one severe case of HMPV infection was found (Huck et al. J Clin Microb. 2006 , 44:2300 -2303 . Moreover, retrospective analysis of 186 different respiratory samples and 121 BALs from transplanted patients revealed low incidence of HMPV (less than 1%) and confirmed the absence of HBoV and HCoV-NL63 among adult patients. Partial sequencing and phylogenetic analysis of the HMPV, HBoV, and NL-63 amplicons was performed and showed that viral strains circulating among immunocompromised patients cluster within the known different genotypes (A1, A2a, A2b, B1, and B2 for HMPV and 1 and 2 for HCoV NL63 and HBoV). Conclusions: Our results confirm the occurrence of HMPV infection among transplant recipients, although at a low incidence rate. Moreover, they suggest a role for HBoV and HCoV-NL63 in the etiology of respiratory tract infections among immunocompromised children, but not adults.

Herpes Viral (VZV and HSV) reactivation in patients treated with Bortezomib G. Härter, P. Liebisch, H. Döhner, P. Kern (Ulm, DE) Introduction: Bortezomib (BTZ, Velcade ® ) is a proteasome inhibitor, which is increasingly used in patients (pts.) with hematological malignancies, especially in pts. with relapsed or refractory multiple myeloma (MM). Reactivation of varicella zoster virus (VZV) during or after BTZ therapy was reported to occur in ~10 -15% of cases. To elucidate the efficacy of antiviral prophylaxis, we determined the incidence of herpes virus reactivation (HVR) in subjects treated with BTZ before and after the introduction of antiviral prophylaxis. Methods: A retrospective analysis of HVR was conducted in 44 pts. with MM who received BTZ (n=25), BTZ/dexamethasone (n=10), or BTZ, dexamethasone, and cyclophosphamide (n=9) at our institution.

Results: A range of 1 to 11 (median: 4) cycles of BTZ were applied. Overall, 10 pts. developed HVR (22.7%), among them 4 pts. treated with BTZ, 2 pts. treated with BTZ/dexamethasone, and 4 pts. treated with BTZ, dexamethasone, and cyclophosphamide. 8 pts. developed VZV (18.2%) and 2 pts. HSV reactivation (4.5%). Median time to onset of HVR was 33 days (range: 23-365). Among pts. not receiving antiviral prophylaxis, the incidence of HVR was 35.0%. In pts. receiving prophylaxis, the incidence of viral complications was 13.6%. The difference in the incidences of viral complications was statistically not significant (Fisher´s exact test p=0.12). Conclusions: In our cohort of pts., treatment with BTZ was associated with a high risk of HVR, reaching up to 35% in pts. not receiving antiviral prophylaxis. However, primary prophylaxis with nucleosideanalogons can reduce the risk of this complication. Thus, we recommend antiviral prophylaxis for all pts. receiving BTZ. Notably, despite prophylaxis, HVR was still common (13.6%). To elucidate possible risk factors for infectious complications in pts. receiving small molecules, prospective and systematic surveillance for infectious complications is warranted.

Prospective studies from Asia showed that hepatitis B (HBV) viremia and HBV genotypes (GT) were independent predictive factors for the severity of liver disease and development of hepatocellular carcinoma (Kao et al., Gastroenterology 2002) . Similar results have been published in a recent longitudinal survey of a large series of Spanish HBV patients demonstrating a significantly more favorable outcome in patients with GT A compared to D and F (Sanchez-Tapias et al., Gastroenterology 2002) . The aim of our study was to analyze the correlation between phenotypic (inactive carriers, viremia) and genotypic (genotype) parameters in our cohort of patients. In total, 108 HBV patients (mean age: 43 [range:12-77] ys.), who have been referred to our center within the last two years, were included in this analysis. Interestingly the majority of patients (76%) were male. HBV genotype were performed by using the INNO-LiPA HBV genotyping test (Innogenetics, Austria) and the viral load was measured with the TaqMan test (Roche, Austria). Patients were divided into two groups: low (<105 copies/ml = inactive carrier) and high (>105 copies/ml) viremic. No difference was seen between both groups concerning age and gender. The prevalence of GT A, D, B, C and F was 21%, 69%, 5%, 4% and 1%, indicating that two thirds of the patients were from the Mediterranean area and one forth from local regions. All GT B patients were within the high viremic group. Regarding GT A and D, the majority of patients, in particular with GT D, presented with high viral loads (A: 56% vs. 44%; D: 65% vs. 35%). GT C patients were equally distributed between both groups. Moreover, the highest mean viral loads could be observed in GT C patients (640 x 106 copies/ml) followed by GT B (290 x 106), D (110 x 106) and A (57 x 106) patients (p < 0.01). As expected, no difference in mean viral loads was seen between the different GTs in the low viremic (inactive carrier) group. Our study shows a strong association between viremia and genotype in our cohort of HBV patients. This indicates that the lower viremia in GT A patients might be responsible for the better response to interferon therapy as well as the more favorable clinical outcome seen in GT A patients. In contrast, the significantly higher viremia in GT D patients seems to be the responsible parameter for the more aggressive courses of HBV infection in these patients.

Patients with Acute Exacerbation of COPD F.C. Ringshausen, A.M. Tan, A. Wagner, G. Schultze-Werninghaus, G. Rohde (Bochum, DE) Background and Aims. In 2005 a novel parvovirus was discovered. Initially it has been detected in children with acute lower respiratory tract infection and was named human bocavirus (HBoV). Its clinical relevance as a causative agent of acute respiratory tract disease is uncertain, its epidemiological pattern unknown. HBoV has a global distribution and shows a seasonal accumulation in winter and spring months. Clinical courses consist of wheezing episodes, asthma exacerbations, obstructive bronchitis, bronchiolitis, pneumonia and gastroenteritis. Patients with structural pulmonary diseases or immune disorders seem to be afflicted predominantly. The aim of this study was to determine the frequency and the potential clinical relevance of HBoV in adult patients with acute exacerbation of chronic obstructive pulmonary disease (COPD). Methods. We retrospectively investigated the presence of HBoV DNA by qualitative polymerase chain reaction (PCR) of 7 nasopharyngeal aspirate specimens and 145 induced sputum samples obtained from 146 different adult patients between October 1999 and April 2003. Clinical data were collected in a standardized manner and all samples were analyzed for other common respiratory viruses (respiratory syncytial virus [RSV], rhinovirus, influenza A virus, parainfluenza virus type 3, human metapneumovirus) by PCR. Results. 96 of 146 patients (66%) had an acute exacerbation of COPD, 50 patients (34%) had a stable COPD or were smokers without COPD, respecively. HBoV DNA was detected in induced sputum samples in 2 of 146 patients (1,4%). One Patient positive for HBoV had an acute exacerbation of COPD and an episode of acute gastroenteritis. Thus, the prevalence of HBoV in acute exacerbations of COPD in adults demonstrated to be low (1,0%). The other patient positive for HBoV had a severe but stable COPD. In both patients HBoV was only detected in induced sputum samples, corresponding nasopharyngeal aspirates were negative. Both patients had a chronic ambulatory steroid medication, viral coinfections (RSV, RSV and influenza A virus, respectively) and presented in the winter/spring season (March and November, respectively). Conclusions. It is unlikely that HBoV acts as a relevant pathogen in acute exacerbations of COPD in adult patients. The fact that we only detected HBoV in induced sputum samples, suggests infection of the lower respiratory tract. Specifity of positive HBoV results remains to be confirmed by sequencing of PCR products.

Excellent long-term outcome of hepatitis B patients after liver transplantation K. Kopf, I. Graziadei, K. Nachbaur, W. Mark, R. Margreiter, W. Vogel (Innsbruck, AT) Liver Transplantation (LT) is the only effective therapy for end-stage liver disease due to Hepatitis B (HBV). Before introduction of passive immunoprophylaxis with hepatitis B immunoglobulin (HBIg) and new antiviral nucleos(t)ide analogues HBV recurrence was seen in the majority of the patients and the course of hepatitis B infection of the allograft was highly progressive resulting in an inferior graft survival. In this study we analyzed the different clinical courses of HBV recurrence, the impact of HBV recurrence on patient and graft survival and potentially (non)viral contributing factors for long term outcome of HBV patients after LT. Between 1983 and 2006, 62 HBV patients (55m/7f; mean age 50ys) were transplanted at our center; 8 were coinfected with hepatitis C and 5 with hepatitis D. The mean follow-up was 44 (range: 1-246) months. Genotyping of HBV was performed in 23 patients: A (n=8), D (n=16). Twenty patients received no or short term HBIg (prior to 1995), 6 Infection Abstracts KIT2008 patients received long-term HBIg alone and 39 patients in combination with Lamivudine(LAM) or Adefovir (ADV). The actuarial overall 1-,5-,10-,15-year survival rates were 88%, 74%, 69% and 69%, respectively, which were comparable to those of other indications. Patients with HBIg alone or in combination with LAM/ ADV showed significantly better 1-, 5-, 10-year survival rates (100%, 100%, 80% and 95%, 79%, 79%, respectively) compared to patients treated without HBIg (70%, 55%, 50%). Six patients required one ReLTx and one patient two ReLTx. In total 18 patients (27.7%) developed recurrent HBV-infection after LT: 40% in the non HBIg group and 22% in the HBIg±LAM/ADV group. Six of the 10 patients in the HBIg/LAM group were HBV DNA positive prior to LT, four presented with LAM mutants. HBV recurrence, however, did not negatively impact patient outcome. All patients with recurrent disease were treated with antivirals (LAM, ADV and Tenofovir). Only one patient was retransplantated due to HBV recurrence. A significant better survival post LT was seen in genotype D patients; otherwise no further predictive parameters could be observed. Our study showed that HBV patients had excellent long term survival following LT, in particular patients with combined HBIg/LAM prophylaxis. Recurrent HBV infection in the allograft (28% of patients) could be effectively treated in the majority of patients and did not negatively impact long term outcome after LT.

Infection with polyomavirus WU in children with acute respiratory tract diseases F. Neske, K. Blessing, F. Ullrich, A. Pröttel, H.W. Kreth, B. Weissbrich, B. Weissbrich (Würzburg, DE) Background: The human polyomavirus WU (WUV) has been recently described as a novel virus in respiratory tract samples. The aim of this study was to investigate the frequency and the epidemiological pattern of WUV in pediatric patients. Methods: We tested nasopharyngeal aspirates (NPA) obtained between 2002 and 2007 from pediatric in-patients with acute respiratory tract diseases at the University of Würzburg for the presence of WUV DNA. The specificity of positive PCR reactions was confirmed by sequencing. Results: WUV DNA was found in 4.8 % of the NPA. The median age of the infants and children with WUV infection was 3.0 years. Infections with WUV were found year-round, though most occurred in the winter months. Coinfections were found in 54.8 % of the WUV positive samples. Influenza A, adenoviruses, and hBoV were most frequently detected as coinfecting agents. Sequence determination of the PCR products in the large T region revealed high identity (99 %) between the nucleotide sequences obtained in different years and in comparison to published sequences. Conclusions: WUV is frequently found in NPA of hospitalized children with acute respiratory tract diseases. Proving the clinical relevance of WUV is challenging, because application of some of Koch's revised postulates is not possible. Further studies are necessary in order to determine whether WUV is indeed a human pathogen.

Tulavirus in Germany -a neglected hantavirus? S. Essbauer, J. Schmidt-Chanasit, S. Friedewald, G. Dobler, W. Splettstößer, M. Pfeffer, A. Thomas, M. Mertens, M. Schlegel, G. Heckel, R. Ulrich (Munich, Hamburg, Greifswald-Insel Riems, DE; Bern, CH) Introduction: European hantaviruses are rodent-borne zoonotic pathogens that can cause haemorrhagic fever with renal syndrome (HFRS) of different severity. In Germany the predominant hantavirus species is Puumala virus. However, Tulavirus (TULV) has been described as the possible cause of fever, renal syndrome, and pneumonia in a German patient (Klempa et al., 2003 , Schultze et al. 2002 . Despite this report, the role of TULV in human infections as well as the spatial and temporal occurrence of TULV in Germany are quite unclear . Methods: The objective of our study was to investigate the prevalence of TULV in natural reservoir hosts in several regions of Germany including also military training areas in South-West Germany. RT-PCRs targeting the S-and M-segments of TULV were used to screen the lungs of trapped Microtus species. Assays based on recombinant TULV nucleocapsid protein were used to investigate hantavirus-specific antibodies in the rodent sera. Results: In some of the areas in central and south-eastern Germany (e.g. Göttingen, Sigmaringen) a high TULV prevalence with up to 7% was observed in common voles. At two different trapping sites in the federal state Brandenburg and in Göttingen TULV was not only found in its designated host, the common vole, but also in field voles. RT-PCR amplification and subsequent phylogenetic analysis of S-and M-segment genomic sequences demonstrated significant differences between TULV originating from different regions in Germany. A ntsequence divergence of about 20% was observed in S-and M-segments between the TULV strains. Conclusions: Our investigations demonstrated a broad geographical distribution of TULV in Germany. In addition, genetically different TULV were found to presently circulate in voles in several regions of Germany. Embedded in the German-wide network "Rodent-borne pathogens" future rodent trapping activities are dedicated to monitor the population density of rodents as well as the prevalence and molecular variation of TULV in both Microtus species of different populations.

Limited specifity of the Mikrogen Anti-HEV-IgG/IgM RecomBlot for the detection of acute Hepatitis E Virus infections S. Scheithauer, K. Ritter, M. Häusler, M. Kleines (Aachen, DE) Introduction: Acute hepatitis E (HEV)is a sporadic and endemic infection of the liver, ranging from asymptomatic to fatal illness. Clinically it cannot be distinguished from the acute course of another viral hepatitis. Due to the transmission routes the disease appears only sporadically in Europe. However, IgG antibodies can be detected in 2,1% of the German population, raising the question whether this reflects an asymptomatic past infection or is due to a lack of specifity of the commercially available tests. To address this question we studied 18 in-patients with underlying infectious diseases accompanied by elevated liver enzymes and healthy individuals for the presence of HEV antibodies using the Mikrogen ® Anti-HEV-IgG/IgM RecomBlot (Mikrogen, Germany). Methods: 18 hospitalized patients aged 15 to 70 years with an acute infection caused by the hepatitis A virus (HAV, n=2), the Epstein-Barr virus (EBV, n=5), the Puumalavirus (n=4), the hepatitis B virus (HBV, n=1), the Cytomegalovirus (CMV, n=1) or gram-negative sepsis (n=5)and 12 healthy individuals were tested for HEV antibodies using the Mikrogen immunoblot, which is based on antigenic material from HEV open reading frames 2 and 3. The primary diagnosis was established using HAV-, HBV-, and HCV-specific assays on the Architect ® analyzer (Abbott, USA), EBV-and CMV-specific assays on the BEP III processor (Dade, Germany) and the Recomline Bunyavirus (Mikrogen), respectively. To rule out an acute HEV coinfection in the cases of an HAV-infection, the specimens were tested at the German reference laboratory for HEV-RNA by RT-PCR and for HEV-antibodies using an ELISA format with overall negative results.

Conclusion: Laboratory diagnosis of an acute HEV infection according to the German case definition (RKI) is based on the detection of HEV-RNA or IgM anti-HEV antibodies or on a significant increase in total/ IgG anti-HEV antibody levels. Applying this definition to the results gained by the Mikrogen Anti-HEV-IgG/IgM RecomBlot will lead to a significant overestimation of acute HEV-infections. Furthermore, epidemiologic data based on this assay will overestimate the prevalence of anti HEV antibodies. Increasing the cutoff level leads to a better specificity for IgG only as shown recently in the literature. According to our data the Mikrogen RecomBlot is no reliable tool on its one for the diagnosis of acute hepatitis E at least in cases of another underlying infectious disease.

VZV serology: Evaluation of new anti-VZV IgG and IgM assays B. Schenk, M. Radtke, U. Bartelt, G. Enders, D. Franke (Hamburg, Stuttgart, DE) Introduction: Diagnosis of Varicella zoster virus (VZV) latent and acute infection is frequently done by serology. Routine diagnosis mainly includes confirmation of suspected acute infection or confirmation of VZV-specific immunity. In this study we evaluated two new commercial immunoassays regarding technical and diagnostic performance: VZV-IgG-ELISA PKS medac and VZV-IgM-ELA Test PKS medac. Material and methods: Both evaluated assays are using purified virus for antibody detection. The assay for IgG determination is a quantitative indirect ELISA applying single-point quantitation (SPQ). The assay format for IgM detection is m-capture using enzyme-labelled antigen (ELA). Enzygnost Anti-VZV/IgG and Enzygnost Anti-VZV/ IgM (Dade Behring Marburg GmbH, Marburg, Germany) were used as reference assays. 150 sera pre-defined as seronegative, from patients with latent, and acute infection (n = 50 each) were used for diagnostic evaluation. In addition, 150 sera from blood donors were measured in parallel to the reference assays to determine the antibody prevalence. The technical performance was investigated by using predefined sera of different reactivity. Concerning precision intra-and interassay variation were investigated manually as well as using an automatic ELISA processor. Furthermore person-to-person variation, lot-to-lot variation, dilution linearity, and correlation between SPQ and standard curve were investigated. Suitability for automation was investigated using two different automatic devices parallel to manually performed test runs. Results: The overall agreement with the interpretation of the pre-defined sera obtained with the medac assays was 99 % (IgG) and 96 % (IgM), respectively. Regarding the blood donor sera an IgG prevalence of 98 % (0.7 % borderline, 1.3 % negative) compared to 98 % (1.3 % and 0.7 %) with the reference assay was determined. IgM prevalence was found to be 0.7 % (0 % borderline, 99.3 % negative) with the medac test and 1.3 % (6.0 %, 92.7 %) with the reference assay. All precision experiments revealed coefficients of variation < 16 % (reactive samples). Dilution linearity showed a correlation between expected and obtained values of R^2 = 0.84. The correlation between SPQ and standard curve was 0.98 (y = 0.91, n = 306). Manually and automatically obtained test results showed a good concordance (R^2 > 0.97).

The results of the evaluation document the suitability of the medac assays for routine diagnosis of VZV infection.

Comparison of commercial indirect micro-immunofluorescence test; elisa assay and dip stick assay for the detection of igg antibodies against rickettsiae G. Dobler, P. Klein, S. Essbauer, M. Faulde, R. Wölfel (Munich, Koblenz, DE) Rickettsial diseases are emerging infections in many parts of the world. So far the golden standard for detection of anti-rickettsial antibodies is indirect micro-immunofluorescence (MIFT). Epidemiological studies are hampered because the MIFT is labour-intensive, time-consuming and subjective in reading results. Recently, ELISA assays and field applicable dip stick tests became commercially available. So far, few data on sensitivities and detection limits of these assays are available. Methods: 139 sera from an Afghan population were used in the study. Sera were tested for anti-typhus and anti-spotted fever rickettsial IgG antibodies using a commercial anti-spotted fever rickettsia IgG MIFT, IgG ELISA and IgG dip stick test and to an anti-typhus rickettsia MIFT and dip stick test. All tests were purchased by PanBio (Brisbane, Australia) and conducted according to the manufacturer's instructions. Results: 26/139 sera (18.7%) reacted positive in the spotted fever MIFT with titres > 1:32. By ELISA only 10/139 sera (7.2%) showed positive results. All ELISA positive sera also reacted in MIFT (100% concordance). However, 16 sera positive in MIFT were negative in ELISA. ELISA positive sera exclusively exhibited titres in MIFT of => 1:128. Only 5/26 MIFT positive sera also showed positive reaction by the dip stick test. The sensitivity of the dip stick test was determined at 19.2%. 69/139 sera (49%) showed positive results in antityphus MIFT. Comparison with the commercial anti-typhus dip stick test showed that 18/25 sera positive in anti-typhus MIFT also reacted in the dip stick test, resulting in a sensitivity of 72 % for this test. Conclusions: By MIFT anti-spotted fever rickettsial IgG antibody titers => 1:64 were found in 18.7% and anti-typhus rickettsial IgG antibodies were detected in 49% in the population tested. MIFT was the most sensitive test for detection of anti-spotted fever and anti-typhus rickettsial IgG antibodies. By ELISA, only sera with anti-spotted fever IgG titres of => 1:128 could be detected. Dip stick test for detection of anti-spotted fever IgG were found to have low sensitivity. In contrast the dot spot test for anti-typhus antibodies was found to exhibit adequate sensitivity. Consequently, the ELISA assay and the dip stick test in its present form cannot be recommended for epidemiological studies. The usefulness of the ELISA used in this study for the detecting of acute rickettsial infections remains to be determined.

Diagnostic determination of immunity to varicellazoster virus using RIDASCREEN® VZV IgG EIA A. Sauerbrei, P. Wutzler (Jena, DE) Introduction: Determination of specific immunoglobulin G (IgG) is of great significance for serological proof of immunity to varicella-zoster virus (VZV) in case of negative patients' histories of varicella and in Infection Abstracts KIT2008 special groups of persons after varicella vaccination. The objective of this study was to assess the performance of the RIDASCREEN® VZV IgG EIA (R-Biopharm, Darmstadt, Germany), a novel VZV glycoprotein-based enzyme immunoassay (EIA). Methods: Different panels of sera from seronegative persons, patients with latent VZV infection and vaccinees were included. Fluorescent antibody to membrane antigen (FAMA) test was used as reference method. In addition, the results were compared with that of the SERION ELISA classic VZV IgG (Virion\Serion, Würzburg, Germany) and the Enzygnost ® Anti VZV/IgG (Dade Behring, Marburg, Germany). Results: After testing of VZV antibody-negative sera, the specificity of the RIDASCREEN ® VZV IgG EIA was calculated as 98% with borderline results classified as correct and 88% when borderline results were classified as incorrect. Testing of sera from latently infected persons revealed an excellent sensitivity of 94.2 -100%. For measuring immunity in response to varicella vaccination, the sensitivity of the RIDASCREEN ® VZV IgG EIA in comparison with FAMA was between 88.6% with borderline results classified as correct and 57.1% when borderline results were classified as incorrect. For determination of natural immunity, the RIDASCREEN ® VZV IgG EIA revealed an equal sensitivity as the SERION ELISA classic VZV IgG whereas the sensitivity of both EIAs was higher than the sensitivity of the Enzygnost ® Anti VZV/IgG. When VZV immunity after varicella vaccination was determined, the sensitivity of the RIDASCREEN ® VZV IgG EIA was between the sensitivity of the SERION ELISA classic VZV IgG and the Enzygnost ® Anti VZV/IgG. Conclusions: The RIDASCREEN ® VZV IgG EIA can be recommended for the reliable determination of natural VZV immunity. The immunity in vaccinees should only be evaluated when a negative test result prior to the vaccination is available.

Protease inhibitor resistance mutations influence recognition of protease by HIV-1-specific CTL S. Mueller, B. Schätz, K. Eismann, S. Bergmann, H. Walter, K. Korn, B. Schmidt, B. Spriewald, E. Harrer, T 

Introduction: Considering the various pathways which have been observed in the development of drug resistance against protease inhibitors, it is likely that immunological host factors like the HLA system interact with the emergence of resistance mutations. We analyzed HIV-1 protease sequences and protease-specific cytotoxic T lymphocyte (CTL) responses in a HLA class-I-typed cohort of 94 HIV-1-infected patients in order to determine the influence of HIV-1-specific CTL on the development of drug resistance mutations in HIV-1 protease.Univariate statistical analyses were performed to identify correlations between amino acid substitutions and HLA alleles. T-cell activity was measured by ELISPOT and Cr51-release assays using peptide stimulated T-cell lines. Results: Several major and minor drug mutations showed associations to HLA class-I alleles. Based on these associations we defined several new CTL epitopes, which could be verified in biological assays. Major (L33F, M46I) and minor (L10I, E35D, I54V, I62V, A71V, and I93L) drug resistance mutations were located in CTL epitopes that were restricted by the associated HLA class-I alleles. In most patients, these drug resistance mutations reduced CTL recognition, indicating that these mutations could be selected as CTL escape mutations in patients with the corresponding HLA class-I alleles. Interestingly, some patients were able to generate specific CTL against certain drug resistance mutations, such as the A71V mutation. This is suggesting that CTL could influence the development of drug resistance mutations.

Prospective studies are needed to find out whether CTL targeting drug resistance mutations in the HIV-1 protease influence the emergence of resistance to protease inhibitors. The interaction between the CTL response and the development of drug resistance mutations could have important clinical implications for the sequencing of PIs in antiretroviral therapy, the understanding of drug resistance pathways, and the design of therapeutic vaccines.

Role of CTL Mediated Immunselection in a dominant Nef CTL Epitope

Introduction: To study the role of CTL escape for disease progression in HIV-1-infection we analyzed the CTL response to the dominant HLA-B8-restricted CTL epitope FLKEKGGL (FL8) in HIV-1 Nef. Methods: HIV-1 nef genes derived from 56 patients were analyzed by PCR based sequencing. T cell responses against FL8 and mutated FL8-variants were detected by g-IFN-ELISPOT. Results: The longitudinal analysis of an HIV-1-infected patient with good control of HIV-1 viremia for several years demonstrated an association of rising viremia with the emergence of CTL escape mutations within the HLA-B8-restricted Nef-specific CTL epitopes FLKEKGGL and WPAIRERM. Analysis of nef genes in 56 HIV-1infected patients demonstrated a significant correlation between the occurrence of mutations in the FL8 epitope and the presence of HLA-B8. The mutations within the FL8 epitope could decrease CTL recognition, however, there was a strong variation regarding the recognition of viral variants between individual donors. The presence of FL8 mutations was associated with lower CD4 counts and higher viral loads. Conclusion: Our data demonstrate a strong CTL selection pressure on the immunodominant HLA-B8-restricted CTL epitope FL8 in HIV-1 Nef. The association of FL8 mutations with lower CD4 counts indicate an important role of CTL escape mutations for disease progression.

Cost-savings of prospective HLA-B*5701 screening prior to ABC/3TC FDC use in Germany E. Wolf, M. Stoll, M. Becker-André, M.C. Müller, W. Becker, A. Gorriahn, H. Jäger, R. Welte, M. Baudewig, R. Walli (Munich, Hannover, DE) Introduction: The presence of HLA-B*5701 is strongly associated with an increased risk for an Abacavir (ABC) hypersensitivity reaction (HSR). Several guidelines on the management of HIV-patients (e.g. the German-Austrian) have included a recommendation for prospective HLA-B*5701 screening prior to initiation of ABC. To date, there is no data on the costs of an ABC HSR or the cost-savings of prospective HLA-B*5701 screening prior to ABC/3TC fixed-dose combination (FDC) use in Germany. Methods: A decision tree model was used to estimate cost-savings per patient for prospective HLA-B*5701 screening of all patients initiating ABC/3TC FDC. Direct medical costs and indirect costs (productivity loss) were included with 2007 as reference year. A standardized questionnaire was used to assess the clinical presentation and resource utilisation of clinically suspected ABC HSR cases without prior HLA-B*5701 screening in 5 HIV centres in Germany. Resource utilisation was valued with the outpatient fees of the social health insurance (EBM) and the G-DRG system for inpatient services. Productivity loss was valued with age and sex specific labour costs. Estimates for hospitalization rate and incidence of clinically suspected ABC HSR with and without prospective HLA-B*5701 screening were based on data from recent clinical trials (e.g. PREDICT-1) and cohorts. Results: 32 cases of clinically suspected HSR between 1998 and 2007 were included in the analysis. Mean direct costs for inpatient and out-Abstracts KIT2008 patient care were € 6.904 and € 74. Additional medication costs for discarded ABC/3TC FDC were € 426 per case. With a 12% hospitalization rate and a 15% private health insurance rate, overall mean direct costs per case were € 1.361. Including € 513 for indirect costs, mean total costs per case were € 1.874. Assuming that withholding ABC/3TC FDC from HLA-B*5701 positive patients results in a reduction of clinically suspected ABC HSR incidence from 10% to 0.5%, the estimated thresholds for cost-savings were estimated at € 130 (direct costs) and € 179 (total costs) per patient. Conclusions: According to our German model, prospective HLA-B*5701 screening prior to ABC/3TC FDC initiation results in costsavings to the health care payer and the society if the screening costs are below € 130 and € 179 per patient, respectively. Irrespective of economic considerations, there is a clear medical benefit of prospective HLA-B*5701 screening prior to ABC use.

High incidence of HIV-associated Hodgkin's Lymphoma in patients on HAART -evidence from the prospective German ARL Cohort Study D. Gillor, M. Hentrich, C. Wyen, M. Oette, A. Plettenberg, J. Rockstroh, J. van Lunzen, C. Mayr, S. Esser, H.A. Horst, H. Jäger, G. Haerter, F. Mosthaf, B. Schaaf, G. Fätkenheuer, C. Hoffmann (Cologne, Munich, Düsseldorf, Hamburg, Bonn, Berlin, Essen, Kiel, Ulm, Karlsruhe, Lübeck, DE) Background: Recent epidemiological data reported on relatively high numbers of HIV-HL cases in the era of HAART. This led to the speculation that the incidence of HIV-HL may increase in the setting of an improved immunity. Methods: This prospective, ongoing multicenter cohort study includes all patients with new or recurrent Non-Hodgkin's lymphoma (NHL) and HIV-HL, diagnosed since January 2005. All relevant data on HIV and lymphoma characteristics, treatment and outcome is collected. After enrolment, patients are followed every six months. Results: As of May 2007, 136 patients, among them 20 patients with HIV-HL, were included in the cohort. When compared to the most common NHL subtypes, namely diffuse large B-cell lymphoma (DL-BCL) and Burkitt-Lymphoma (BL), patients with HL were more likely to be treated with HAART at the time of lymphoma diagnosis (85 % versus 24 % BL and 37 % DLBCL, respectively, p=0.0001). Patients with HL were also more likely to have a plasma viremia below 50 HIV-RNA copies/ml at the time of diagnosis (53 % versus 13 % BL and 25 % DLBCL, respectively, p=0.01). There were a lower rate of patients with low CD4-cells (< 100 cells/ul) among HL cases (6 % versus 22 % BL and 40 % DLBCL, respectively, p=0.01). We found no differences with respect to demographical data, lymphoma stage or previous AIDS diagnosis. After a median follow-up of 6.0 months, there was a trend towards better overall survival for patients with HL when compared to NHL, although this difference did not reach statistical significance (p=0.26). Conclusions: Preliminary data of this ongoing, prospective study suggest that there is a high incidence of HIV-HL in patients on HAART, including those with sufficient viral suppression. HAART does not appear to have any protective effect on the incidence of HIV-HL.

Transient worsening of liver function upon introduction of ART in HIV/HCV coinfected patient T. Kümmerle, C. Lehmann, E. Rund, G. Fätkenheuer (Cologne, DE) Case: A 45 year old man with HIV/HCV-coinfection was biopsied and showed substantial liver damage (G4 inflammation,G1 fibrosis).Initiation of IFN/RBV treatment wasn't able to control HCV disease. Due to deterioration of immune function with CD4-cells below 100/ ul,PI/r-based ART was started. Over the following weeks,HIV-RNA sharply declined,while simultaneously massive hepatocellular injury with elevation of LFTs occurred. A quantitative assay failed to show any increase in HCV-viral load. ART was stopped immediately. Consequently,the elevated LFTs slowly returned to baseline levels. Due to the persistent massive immune deficiency,reappliaction of ART with a different regimen was decided.Again,virologic control with a decline of HIV-PCR greater 1 log was achieved. Simultaneously,we could see a rapid 10 to 15fold increase in TA and bilirubin levels compared to baseline. As before,interruption of HIV treatment was decided,which led to the normalization of liver function tests.A consecutive liver biopsy showed major disease progression (inflammation G3,fibrosis G3). A difficult therapeutic decision needed to be made as in our opinion due to the lack of other therapeutic options,only control of HIV disease could help reduce the activity of HCV-infection and slow down inflammatory liver disease.With reintroduction of the same regimen as before,a sharp rise in both TA,g-GT and Bilirubin was detected. Our patient was in good physical condition,and this time we decided to keep him on ART. Decreasing HIV-viral load and rising CD4-cells were seen. Over time,liver function tests steadily improved.Now for about 20 months,our patient has had good control of his HIV infection under steady ART.No further flares of hepatitis or increases of LFTs were seen. Sonographic imaging of the liver showed diffuse parenchymal damage and hepatomegaly but no signs of cirrhosis.The results of a current liver biopsy are pending. Conclusion/Summary: In HIV/HCV-coinfection,good control of HIVdisease to prevent rapidly progressive liver disease is imperative. In the setting of a weakened immune system,immune restoration disease upon introduction of ART can lead to a temporary worsening of liver disease. However,in our case,continuation of ART led to normalization of elevated liver function parameters to baseline levels and a slow-down of hepatic inflammatory activity probably due to increased immune competence.

Switching to Nevirapine -Risk of hepatic failure and effects on metabolic and immunologic parameters J.A. Rump, S. Usadel (Freiburg, DE) Introduction: There are reasonable concerns when switching patients with high CD4 cell counts to nevirapine (NVP). The aim of this retrospective analysis was to investigate the safety and efficacy of NVP based HAART after switching from Protease inhibitors (PI) or Efavirenz (EFV). Methods: In a retrospective study between 2000 and 2007 97 patients (32 female, 65 male) pretreated with different PI regimens (n=70, 21 female, 49 male) or Efavirenz (n=27, 11 female, 16 male) and switched to NVP were analysed. Biochemistry, lipid profile, CD4 count and viral load were investigated 96 weeks prior to the switch, at baseline, week 4, 12, 24, 48, 72 and 96 weeks. The reasons for switching were side effects and deescalation of therapy. Results: At baseline the avarage CD4 cell count was 636/µl ± 298/µl and the viral load was below detection in the study group. No viral rebound was seen after the switch. GGT rised from 34 U/l ± 31 U/l to 77 U/l ± 63 U/l, transaminases remained stable during the observation period (GOT 22 U/l ± 9.7 U/l, GPT 24 U/l ± 13.7 U/l at baseline and 26 U/l ± 11.5 U/l and 31 U/l ± 18.1 U/l at week 96 respectively). None of the patients developed a WHO grade II or more liver complication. Cholesterol levels did not change in the PI-group during the whole observation period. A slight change was observed in the EFV-group (217 mg/dl ± 35.7 mg/dl at baseline and 178 mg/dl ± 88.7 mg/dl at week 96). Patients switched from PI to NVP had an improvement of gastrointestinal side effects. Conclusions: Switching to NVP in patients with high CD4 cell-count, male and female, caused no significant case of liver damage. Effects on lipids are neglectable. PI induced diarrhea was significantly reduced. CD4 count remained stable, no viral rebound was seen.

Successful treatment of extensive condylomata acuminata of the urethra and urinary bladder with intravenous cidofovir in an HIV-positive patient B. Jensen, R. Winzer, M. Oette, S. Koch, D. Häussinger (Düsseldorf, DE) Introduction: Condylomata acuminata (CA) are anogenital lesions caused by human papillomavirus infection. Involvement of the urethra and/or urinary bladder is rare but often difficult to treat. Methods: Case report. Results: A 42-year old, male, HIV-positive patient presented with recurrent urinary tract infections in our outpatient department. He reported having urinary tract infections every 4-6 weeks for about 3 years. The patient was on successful antiretroviral therapy (HI-VL <50 Eq/ml; CD4-cells 357/mcl, 21%) with a quadruple NRTI-regimen (AZT/3TC/ABC/TDF). Clinically he had some small penile CA, but cystoscopy also revealed a extensive affection of the urethra and the urinary bladder with multiple CA affecting large areas of the urinary bladder. The penile and urethral CA were laser-ablated, additionally a transurethral resection of some CA in the urethra and urinary bladder was performed. Unfortunately the laser ablation caused a dorsal penile urethral fistula, therefore laser ablation or transurethral resection was not chosen for further treatment of the remaining CA. Because of case reports describing successful treatment of recurrent respiratory papillomatosis or anogenital CA with topical, but also intravenous Cidofovir we started a systemic therapy with Cidofovir, which is primarily excreted renal as unchanged drug. After five applications of 360 mg (5 mg/kg body weight) of cidofovir intravenously over the next four months a cystoscopy was performed again and showed no CA in the proximal urethra and urinary bladder. There were only few persisting CA at the distal urethra. The treatment with cidofovir intravenously was very well tolerated, no side effects were observed. Conclusions: To our knowledge this is the first reported case of a successful treatment of condylomata acuminata in the urinary bladder with cidofovir. Intravenous treatment with cidofovir could be a promising therapeutic option in patients with condylomata acuminata of the lower urinary tract.

Immunogenicity and Efficacy of an MVA-nef Vaccine in a Randomized Controlled Phase-II-Study in HIV-1 infected patients with CD4 Counts > 250/µl followed by Structured Treatment Interruption (STI)

Introduction: To assess immunogenicity and safety of an MVA nef vector in dependence of dose and in comparison to MVA-BN (Modified Vaccinia Ankara-Bavarian Nordic) alone we performed a randomized phase II study in HIV-1 infected patients on HAART followed by a structured treatment interruption (STI). Methods: 77 patients were included in this single blind, randomized controlled phase II study. 22% of subjects were vaccinia naive, 78% of subjects were pre-vaccinated against smallpox. Subjects received three s.c. vaccinations of either 1E8 TCID50 MVA-BN (IMVA-MUNE) 1E8 TCID50 or 5E8 TCID50 MVA-nef (n=26, 25 and 26, respectively) at week 0, 8 and 16. At week 20 patients were offered a STI with close monitoring of viral loads. HIV-1-specific T-cells were monitored by g-IFN-ELISPOT. Nef-specific T-cell reactivity was tested against four different peptide pools containing 9-17 overlapping 15-mers spanning the whole nef gene. Responders to the vaccine were defined as the de novo appearance of nef-specific T-cells or as an at least 1.7 fold increase of a pre-existing nef-specific T-cell response in the same pool at two time points. Results: After three vaccinations a de novo induction or a relevant increase (>1.7 fold of a pre-existing response) of nef-specific T-cells was observed in 3/26 (12%) of patients in the MVA-BN arm, in 11/25 (44%) in the 1E8 TCID50 MVA-nef arm and in 16/26 (62%) patients in the 5E8 TCID50 arm. A MVA-specific T-cell response could be detected in 16/26 (62%) of patients in the MVA-BN arm, in 19/25 (76%) in the 1E8 TCID50 MVA-nef arm and in 23/26 (89%) patients in the 5E8 TCID50 arm. In total 37/77 subjects decided to stop HAART after visit 9 (week 20). All patients experienced an increase in viral load within week 0 to 8 after HAART interruption followed by a decrease of the viral load for the subsequent period. At week 52 viral loads were significantly lower in the patients in the 5E8 TCID50 arm than in the patients in the MVA BN arm. Conclusions: In this ongoing study MVA-Nef proved to be safe and immunogenic in HIV infected patients. Viral load analyses in the subgroup of patients with STI show a better viral control in the patients vaccinated with the high dose MVA-nef vaccine. Additionally, the strong induction of specific antibodies and T-cells against MVA-BN® confirms the potential as a safe and potent smallpox vaccine in HIVinfected subjects. Acknowledgement: Funded by NIAID (contract No. N01-AI-40072) and Bavarian Nordic A/S.

K. Kurz, R. Zangerle, M. Sarcletti, C. Winkler, B. Mumelter, G. Weiss, D. Fuchs (Innsbruck, AT) Introduction: Depression and impaired quality of life (QoL) are frequently observed in patients suffering from HIV infection. As an enhanced degradation of the serotonin precursor tryptophan is well documented in HIV infected patients, disturbances in tryptophan metabolism may be involved in the pathogenesis of HIV related depression. In this study, the relationship between QoL, depression, different lab parameters and tryptophan metabolism was investigated. Methods: One hundred-fifty-two HIV infected patients (classified according to CDC-criteria) completed psychological questionnaires (BDI and MQoL-HIV) to estimate their QoL and mood. Disease progression was monitored by determination of viral load (VL), CD4+ cell counts, haemoglobin and urinary/plasma neopterin, tryptophan and kynurenine concentrations. Disease progression was reflected by increasing VL, decreasing CD4+ cell counts, and also by enhanced tryptophan degradation. Results: Fourty-one patients presented with mild, 22 with moderate depression and 14 patients were severely depressed. BDI-scores and MQoL-scores were associated strongly with each other (rs= -0.838; p <0.001). Patients without depression (and higher QoL, respectively) had significantly lower plasma neopterin concentrations, higher CD4+ cell counts and haemoglobin concentrations and better QoL scores (all p <0.01) than depressive patients. Furthermore they showed lower rates of tryptophan degradation (p <0.05). Significant associations existed between tryptophan degradation and immune activation. Haemoglobin and viral load were predictive for impaired QoL, while high urinary neopterin concentrations and low haemoglobin were the best predictors for depression. Conclusions: Depressive mood and impaired QoL appear to be related to clinical parameters like immune activation, haemoglobin values and viral load in HIV infected patients.

Phenotype Frequency of HLA-B*5701 in HIV-infected patients in Germany S. Esser, J. van Lunzen, M. Baudewig, B. Thiele, M. Mösmang, N. Banik, H. Pearce, R Walli (Essen, Hamburg, Munich, Kaiserslautern, DE; Brentford, UK) Introduction: The MHC class I allele HLA-B*5701 is strongly associated with an increased risk for an abacavir hypersensitivity reaction (HSR). It has been shown that prospective HLA-B*5701 screening can significantly reduce the risk to develop an HSR. The clinical benefit of prospective HLA-B*5701 screening, however, is strongly influenced by the phenotype frequency in a population of interest. To date, there is no data on the frequency of HLA-B*5701 phenotype in routinely treated HIV patients in Germany. Methods: Prospective, epidemiological study in 33 hospital-based (n=11) and office-based (n=22) HIV-centres in Germany. Based on an assumed phenotype frequency of 5%, a minimum of 1.825 patients were required for a 1% accuracy of the 95% CI. Consecutive HIVinfected patients regardless of ART status were enrolled (30-90 per centre). Ethnicity of all patients and detailed geographic descent of Europeans were assessed using standardized questionnaires. HLA-B*5701 was determined in 7 local laboratories from EDTA blood samples using amplification (SSP, SSO) or sequencing techniques (60 to 598 samples per lab). In addition, buccal swabs were collected for concordance analyses in a central reference laboratory. Results: 1.885 of 2.079 screened patients consented and were enrolled, 1.879 subjects were evaluable. The overall HLA-B*5701 phenotype prevalence was 7.3% [6. 16-8.56 ]. The majority of subjects were White/ Caucasian and of Central European descent (GER, FRA, BEL, NL, CH, AUT, CZ, POL). Details for observed HLA-B*5701 frequency according to selected ethnicity and geographic origin (n> 20) are shown in the table below.

The highest frequencies of HLA-B*5701 in this German cohort was observed in White/Caucasian subjects of Central European descent and was higher than previously reported. The data indicate that screening of these patient populations is of highest value and interest. Conclusions regarding observed HLA-B*5701 frequency in patients of other ethnic or geographic subcategories warrant caution and further investigation due to small sample sizes.

Glatiramer acetate reduces the rate of cerebral malaria in C57BL/6 mice infected with Plasmodium berghei P. Lackner, A. Reisinger, R. Beer, C. Burger, A. Dietmann, G. Broessner, R. Helbok, E. Schmutzhard (Innsbruck, AT) Cerebral malaria (CM) is still associated with high mortality and a high rate of transient or persistent neurological sequelae. Studies on immune modulation and/or neuroprotection as ancillary treatment in murine CM implicate promising potentials. Therefore we investigated the effect of Glatiramer acetate (Glat), a drug for treatment of relapsing-remitting multiple sclerosis, in a mouse model of cerebral malaria. A total of seventy-two C57BL/6 mice were used for this study. Mice were infected with Plasmodium berghei ANKA blood stages and either treated with Glat or vehicle beginning with the day of infection. Parasitemia was measured every day. The clinical course of the disease was assessed by a battery of 40 standardized tests for evaluating neurological functions in mice. Samples of animals on day 4 post infection and of moribund animals were analyzed. Cryostat sections of brains were assessed for hemorrhage and caspase-3 activation. Tumor necrosis factor-alpha, interferon-gamma, IL-5, IL-4 and IL-2 were measured in sera using a commercially available cytometric bead array kit. In the control group 15.4% of the animals survived the CM vulnerable phase between day 5 and day 9 and did not develop CM. In the treatment group 42.3% of the animals survived the CM vulnerable phase, yielding a statistically significant survival benefit (p < 0.05). The treatment did not affect parasitemia. Stereological analysis did not show statistically significant differences in the estimated area of hemorrhage or apoptosis as assessed by the estimated number of parenchymal cells immunopositive for activated caspase-3. Univariate analysis of cytokine levels yielded a significantly lower level of interferongamma in Glat treated animals. The current data indicate that Glatiramer acetate could be a potential new ancillary treatment in CM.

Complement factors C1q, C3 and C5 in the brain and serum of mice with cerebral malaria P. Lackner, C. Hametner, R. Beer, C. Burger, G. Broessner, R. Helbok, C. Speth, E. Schmutzhard (Innsbruck, AT) The pathomechanisms leading to brain damage due to cerebral malaria (CM) are yet not fully understood. Immune mediated and ischemic mechanisms have been accused. The role of complement factors C1q, C3 and C5 for the pathogenesis of CM were investigated in this study. C57BL/6 mice were infected with Plasmodium berghei ANKA blood stages. The clinical severity of the disease was assessed by a battery of 40 standardized tests for evaluating neurological functions in mice. Brain homogenates and sera of mice with CM, infected animals without CM and non infected control animals were analyzed for C1q, C3 and C5 up-regulation by Western blot. To control and correct for equal loading, densitometric values of complement factors were normalized by the respective values for alpha-tubulin. Non-parametric methods were used for statistical analysis. Densitometric analysis of Western blot experiments of brain homogenates yielded statistically significant differences in the levels of C1q and C5 in the analyzed groups. Correlation analysis showed a statistically significant association of C1q and C5 levels with the clinical severity of the disease in mice with CM. More severely affected animals showed higher levels of C1q and C5. No statistically significant differences were found between the complement levels of frontal and cau-

dal parts of the brain. Densitometric analysis of Western blot experiments of sera yielded statistically significant differences in the levels of C1q in the analyzed groups. Infected animals without CM showed lower levels of C1q compared to animals of the control group. The current study provides direct evidence for up-regulation of complement factors C1q and C5 in the brains of animals with CM. No differences in complement levels were observed between frontal and caudal parts of the brain. In conclusion local complement up-regulation is a putative mechanism for brain damage due to experimental cerebral malaria. Methods: A German traveler presented with a typical arthralgia and fever two days after she returned from a fortnight vacation to Rajasthan, northern India. A serum sample of her was investigated by means of real-time RT-PCR, virus isolation, and nucleotide sequencing. Results: Real-time RT-PCR results indicated an acute CHIKV infection and the virus was isolated in Vero cells. Sequencing of the entire viral genome was performed and revealed a high level of nucleotide sequence identity to CHIKV isolates from the recent epidemic in the Indian Ocean islands. Phylogenetic analyses placed our CHIKV isolate in one clade together with the 2005 and 2006 isolates from Mauritius, Reunion, and Mayotte but not into the neighborhood of any CHIKV from Asia. In detail, we found only 20 nucleotide exchanges to a recent isolate we made from a patient returning with a CHIKV infection from Mauritius, resulting in only five amino acid changes (nsP1 T128K, T376M, nsP3 S472N, and capsid P23S, V27I). Conclusions: This is the first proof of a non-Asian CHIKV subtype occurring and causing disease in Asia. Although the excessive dimension of the 2005/2006 outbreak in the Indian Ocean islands was at least in part accounted to the naïve population affected, our results indicate that the emergence of this CHIKV subtype may rather be a result of a better viral fitness. Whether this is reflected by higher viremia in humans or an enhanced adaptation of the vector population resulting in increased transmission rates warrants further investigations. However, the occurrence of an African CHIKV in Asia further demonstrates how fast viruses can emerge and establish in places where competent vectors are prevalent.

S. Zimmermann, A. ter Meulen, E. Fichet-Calvet, L. Koivogui, O. Sylla, M. Goris, R. Hartskeerl, J. ter Meulen (Heidelberg, Frankfurt, DE; Paris, FR; Conakry, GN; Amsterdam, Leiden, NL) Introduction: Acute leptospirosis is a prime example for a neglected disease in Africa. To confirm this hypothesis, a pilot study was undertaken in 2004 in Conakry (Guinea) with an investigation of both humans and small mammals. Sera from 1200 human subjects were screened for leptospi-rosis antibodies to estimate the incidence and identify risk factors for transmission. In parallel, rodents were trapped in households to identify the reservoir animals of the disease. Methods: A cross-sectional serologic survey was carried out in 5 resource poor urban neighbourhoods in Conakry, Guinea. A detailed questionnaire was completed to document demographic and environmental risk factors. Leptospirosis specific IgM and IgG levels were detected by ELISA and confirmed with MAT testing. The trapped rodents were taxonomically identified and one kidney was collected for detection of leptospires by culture and PCR testing. A nested PCR with a high sensitivity was performed. PCR positive samples were confirmed using different typing PCRs. Results: 6 percent of study subjects were positive for leptospira antibodies. Preliminary epidemiological analysis revealed as risk factors for leptospira IgM antibodies: (i) living in a neighbour-hood from which leptospirosis cases were reported in 2001 (ii) use of tap water for washing and bathing (iii) living close to a waste pipe (iv) history of hospitalisation during the past rainy season. 330 rodents were trapped within the 5 neighbourhoods. Rattus rattus and Mus musculus were the most frequent species, but in addition some Crocidura and Mastomys spp. were identified. In five of the kidney samples leptospiral DNA was detected by nested PCR. Conclusion: This survey shows that a significant percentage of the population of Conakry is exposed to leptospirosis. Transmission probably occurs through leptospira infested water on the domestic compounds, with rodents as one possible reservoir. Leptospirosis is a neglected disease in Africa and there is an urgent need for further identification of the main reservoirs of the disease and effective environmental control measures. Olsen, B. Sharp, J.D. Kvalsvig, C.C. Appleton, I. Kleinschmidt (Munich, DE; Charlottenlund, DK; Durban, ZA) Introduction: Geographic information systems (GIS) are increasingly being used for the prediction of parasitic diseases and for research into their environmental epidemiology. However, there have been few GIS based studies into the ecology of geohelminth infection. This study examines associations of selected ecological factors with hookworm infection in order to assess their potential for disease prediction and to further investigate hookworm ecology. Methods: Approximately 800 primary schoolchildren from a small area in rural KwaZulu-Natal were examined for geohelminth infection at baseline and re-examined 3 and 29 weeks after treatment with 400 mg albendazole. We recorded the geographical positions of their homes and interviewed the pupils in order to assess their relevant behaviour and socio-economic background. This information was combined with remotely sensed, cartographic and other data to form a GIS database. Results: Prevalence maps and spatial statistics revealed considerable spatial clustering of infection in the relatively small study area (~28 x 16 km). Multivariate logistic regression models showed a strong positive association of infection at baseline (baseline prevalence =83.2%) with settlement density (odds ratio [OR] = 1.24, 95% confidence interval [95%CI] = 1.10 -1.38) and vegetation density as characterized by the normalized difference vegetation index (NDVI) (OR = 1.66, 95%CI = 1.25 -2.22) and a strong negative association with the clay content of the soil (OR = 0.67, 95%CI = 0.62 -0.73). Socio-economic status and behaviour, although correlated with hookworm infection, did not seem to confound the above associations. Spatial analysis of the model residuals suggested that since the models accounted for most of the spatial pattern, the model standard errors were not affected by spatial clustering. Conclusion: Our study shows that the pattern of hookworm infection is strongly influenced by several environmental factors. The GISaided prediction of areas in need of treatment is therefore a promising tool to guide control efforts when epidemiologic information is insufficient.

E. Saathoff, A.

Genes from Chagas Susceptibility Loci that are Differentially Expressed in T. cruzi-Resistant Mice are Candidates Accounting for Impaired Immunity S. Graefe, T. Streichert, B. M. Budde, P. Nürnberg, C. Steeg, B. Müller-Myhsok, B. Fleischer (Hamburg, Cologne, Munich, DE) Variation between inbred mice of susceptibility to experimental Trypanosoma cruzi infection has frequently been described, but the immunogenetic background is poorly understood. Outcross mice (B6D2F1, (F1)) of the two susceptible parental mouse strains C57BL/6 (B6) and DBA/2 (D2) are highly resistant to this parasite. In an attempt to identify putative susceptibility genes, we framed the kinetics of early infection in B6 and in F1 mice and analysed gene transcription differences at the stage at which the course took divergent directions in these differentially susceptible mice. We monitored the infectious course by determining parasite tissue burdens by real-time PCR. The spleen was analysed histologically and immuno-histochemically and by flow cytometry to assess changes of its anatomy and cellular composition. On day 11 of experimental infection, differential gene expression was analysed in spleen cells from B6 and F1 mice with microarrays. Genes from previously mapped susceptibility loci on Chromosomes 5, 13 and 17 were specifically observed with respect to their expression patterns. We found that the increase of tissue parasitism during the early phase of infection was comparable up to day 11 between susceptible B6 and resistant F1 mice. A reduction of spleen parasite burdens was observed thereafter in both strains, but it was comparatively retarded in susceptible mice. In B6 mice (but not in F1 mice), splenic microarchitecture was progressively disrupted after this time point. By comparison, we observed loss of follicles and B lymphocytes and an increase of macrophages in the spleens of susceptible B6 mice. Analysis of gene expression on day 11 of spleen cells from infected B6 and F1 mice with microarrays identified about 0.3 % of transcripts that were differentially expressed.

Our results indicate that innate mechanisms are not of primary relevance to resistance of F1 mice to T. cruzi infection, and that differential susceptibility to experimental infection with this protozoan pathogen is not paralleled by extensive variation of the transcriptome. Assuming that differential susceptibility is mediated by altered gene expression, we propose that the following differentially expressed transcripts from the susceptibility loci are strong candidates for the observed phenotypic variation between B6 and F1 mice: H2-D1, Ng23, Msh5, H2-Ea and Tubb5 from Chromosome 17; and Cxcl11, Bmp2k and Spp1/Opn from Chromosome 5.

Identification of Three Early Proteins of Mycobacteriophage L5 Leading to Host Shut-off in Myco bacterium smegmatis and Escherichia coli J. Rybniker, G. Plum, N. Robinson, P. Hartmann (Cologne, DE) Background: Mycobacteriophage L5 is a temperate phage with a broad host range among the mycobacteria including Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium ulcerans and M. smegmatis. Though the complete DNA sequence of the L5 genome is available, the function of many genes and their products is unknown. It has recently been shown that L5 switches off host protein synthesis during early stages of lytic growth. This phenomenon called host shutoff has also been described for bacteriophages of other species such as Staphylococcus aureus and E. coli. The identification of host shutoff inducing gene products of mycobacteriophage L5 was the object of this work. Methods: A set of L5 early genes of unknown function was cloned into the acetamide inducible expression vector pSD24 and expressed in M. smegmatis. Colonies that failed to grow on agar-plates containing acetamide were further investigated using growth curves and serial dilutions of acetamide.

We were able to show that gp77, gp78 and gp79 of mycobacteriophage L5 have growth inhibitory effects on M. smegmatis upon expression even with very low concentrations of acetamide (0.02%). The ORFs of these genes are situated in the right arm of the phage genome. This region, which is transcribed leftwards towards the attP integration site, encodes for early regulatory proteins such as DNA primases and helicases and the L5 repressor (gp71). Interestingly, the growth inhibitory effect of all three proteins was also seen in E. coli when expression was induced from an IPTG inducible promoter. We also identified a putative promoter in the L5 genome which probably regulates the expression of these newly identified proteins during lytic growth. It is situated within the sequence of gp83 and seems to transcribe the sequences of gp82 to gp77. Conclusion: To our knowledge, this is the first identification of mycobacteriophage-derived early proteins that are involved in suppression of bacterial growth. The further analysis of these and other mycobacteriophage derived toxic polypeptides together with the identification

of their cellular target might represent a tool for the rapid identification of promising drug targets in the emerging and re-emerging mycobacterial pathogens.

Susceptibility/Resistance against innate defense antimicrobial peptides is independent of E. coli acrAB and P. aeruginosa mexAB RND efflux pumps S. Rieg, A. Huth, H. Kalbacher, W.V. Kern (Freiburg, Tübingen, DE) Introduction: Antimicrobial peptides (AMPs) are multifunctional molecules of innate immunity. In the course of host-pathogen co-evolution bacterial evasion strategies have emerged to alleviate AMP effects. In the present study we investigated the contribution of E. coli and P. aeruginosa RND type efflux pumps on resistance against AMPs. Methods: Testing of AMP susceptibility was performed with E. coli acrAB and P. aeruginosa mexAB-oprM RND efflux pump overexpressing strains and compared to respective RND knockout mutants. Standard CFU assays and measurement of MIC (microbroth dilution) were performed. A significant effect on AMP susceptibility/resistance was defined as an at least fourfold reduction in LD90 or MIC values of efflux pump deficient strains. Results: LD90 values of human cathelicidin LL-37 proved to be 8 µg/ ml in either E. coli strain. CFU testing using human defensins HNP1-3, hBD-2 and HD-5 likewise yielded no significant differences in LD90-values between the E. coli acrAB overexpressing strain and the respective knockout mutant. LD90 values of polymyxin B which was previously found to be a substrate of the mtrCDE RND pump of N. gonorrhoeae did not differ significantly between the two E. coli test strains (2 µg/ml vs. 1.5 µg/ml). Similarily, microbroth dilution assays with LL-37, HNP1-3 and hBD-2 yielded comparable MIC values between the two E. coli strains. CFU assays were also performed using P. aeruginosa PA1426 overexpressing mexAB-oprM and results were compared with those obtained with the respective oprM knockout mutant PA1425. LD90 values of LL-37, HNP1-3, hBD-2 and hBD-3 and HD-5 again were not affected. Consistent with these findings, the LD90 of polymyxin B was not altered by loss of oprM and proved to be 2 µg/ml in either P. aeruginosa test strain. Conclusions: Multidrug resistance (MDR) efflux pumps are capable to confer resistance to structurally unrelated toxic compounds, and for both N. gonorrhoeae and N. meningitidis, active transport of AMPs out of the bacterial cytoplasm and periplasmic space has been reported to confer resistance to AMPs. In the current study using CFU and MIC assays, E. coli acrAB and P. aeruginosa mexAB RND type efflux pumps were not found to have a significant impact on susceptibility/resistance against AMPs. Thus, human AMPs LL-37, HNP1-3, hBD-2/-3 and HD-5 are not included in the subsrate profile of the two clinically relevant RND efflux pumps E. coli acrAB and P. aeruginosa mexAB.

Nramp1-functionality increases iNOS expression via repression of IL-10 formation.

G. Fritsche, M. Nairz, E. Werner, H. Barton, G. Weiss (Innsbruck, AT; Southampton, UK) In mice, resistance to certain intracellular microbes depends on the expression of a late phagosomal protein termed natural-resistance associated macrophage protein 1 (Nramp1, Slc11a1). Nramp1 functionality is associated with a sustained pro-inflammatory immune response, including the formation of the antimicrobial effector molecule nitric oxide via induction of inducible nitric oxide synthase (iNOS).

To investigate the putative mechanism underlying this latter phenomenon we used RAW264.7 murine macrophage cells stably transfected with a functional (RAW-37) or non-functional (RAW-21) Nramp1. We then analysed the expression of IL-10 and other cytokines by quantitative realtime PCR and investigated the influence of IL-10 on growth of S. typhimurium whithin macrophages and the production of pro-inflammatory cytokines and effector molecules. We found that the production of and signaling by the anti-inflammatory cytokine IL-10 was significantly enhanced in macrophages lacking functional Nramp1. Upon infection of these macrophages with S. typhimurium pathogen survival was significantly better in RAW-21 than in RAW-37 cells and inversely correlated to nitric oxide (NO) and TNF-a formation. Addition of an anti-IL-10 antibody to RAW-21 cells led to a significant reduced survival of S. typhimurium within these cells, which was paralleled by enhanced the formation of NO and TNF-a reaching levels comparable to that observed in cells bearing functional Nramp1. Thus, Nramp1 mediates effective host defense in part via supression of excessive IL-10 production which finally leads to strengthening of antimicrobial effector mechanisms.

Role of indoleamine 2,3-dioxygenase in infection control and immunodeficiency K. Schroecksnadel, G. Brandacher, G. Weiss, D. Fuchs (Innsbruck, AT) Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme, which is inducible by interferons. Degradation of the essential amino acid represents an effective anti-proliferative strategy, which is established during immune response. It is directed to halt growth of pathogens in infected cells and also to stop malignant rowth. Enhanced tryptophan degradation has been described in a variety of disorders including infections with viruses, e.g., HIV, and intracellular bacteria, and close associations were observed with immune activation markers like neopterin. Accelerated tryptophan degradation reflects the extent, the activity and the course of disease: in HIV infection it predicts shorter survival, the loss of immunocompetence and the development of cachexia and anemia. Data suggest that tryptophan deprivation is involved in these typical symptoms of chronic inflammatory conditions. Enhanced degradation of tryptophan is also related to impaired quality of life, and this association is linked with the close relationship between tryptophan and the biosynthesis of neurotransmitter serotonin: tryptophan deficiency could represent an important aspect in the pathogenesis of cognitive impairment and depression in patients with infections. In 1996 we demonstrated accelerated tryptophan degradation together with immune activation also in normal human pregnancy, and in 1998 enhanced IDO activity was found to be critical in the induction of immunotolerance during pregnancy (Munn DH, et al. 1998) . Subsequent studies were able to demonstrate the central place of IDO activation in the development of immunodeficiency and immunotolerance. Thereby IDO expression in dendritic cells is crucial in the control of regulatory T-cells. In summary, IFN-g-induced IDO activity in patients with infections is part of the anti-microbial immune defense strategy, which in the long run is detrimental to the host, when tryptophan deprivation and production of tryptophan catabolites, some of which with pro-apoptotic potential, becomes strong enough to also counteract T-cell responsiveness. Data on IDO provide a basis for the better understanding of the complex interplay between immune activation cascades and the development of immunodeficiency, or in other words, of the pro-and anti-inflammatory consequences of Th1-type cytokine IFN-g, among which IDO is a central component.

P230 THP-1 cells infected with M. avium release a potent chemoattractant for human neutrophils presumably bound to a molecule of the HSP-70 family P. Hartmann, N. Robinson, J. Rybniker, K. Brandl, I. Fink, G. Plum (Cologne, Regensburg, DE) Background: Mycobacterium avium complex (MAC) causes lifethreatening bacteremia in immunocompromised patients. We among others have previously shown that not only macrophages but also human neutrophils ( 

Differential activity of innate defense antimicrobial peptides against Nocardia asteroides (sensu stricto)

B. Meier, S. Rieg, H. Kalbacher, E. Fähnrich, A. Huth, D. Wagner, W.V. Kern (Freiburg, Tübingen, DE) Introduction: Members of the genus Nocardia are aerobic, gram positive, filamentous rods. Human infection occurs after inhalation or direct inoculation and presents as pulmonary, CNS, cutaneous/lymphocutaneous or disseminated disease. To investigate the contribution of the innate immune defense, we investigated the susceptibility of Nocardia asteroides against human defensin and cathelicidin antimicrobial peptides (AMPs). Methods: A colony forming unit (CFU)-assay with Nocardia asteroides sensu stricto (ATCC 19247 ) was established. AMPs tested in the our study were human neutrophil peptides (HNP)1-3, human betadefensin (hBD)-3 and human cathelicidin LL-37/hCAP-18. Levofloxacin was used as control. Results: After 16 hours incubation with 8 µg/ml levofloxacin, a killing rate of 94% was observed in the CFU assays. Using HNP1-3, an 89% CFU reduction was detected at a concentration of 32 µg/ml. In contrast to HNP1-3, hBD-3 and LL-37 did not exhibit killing activity on Nocardia asteroides when tested in concentrations up to 64 µg/ml (hBD-3) or 128 µg/ml . Surprisingly, significantly higher CFUcounts could be observed after incubation with hBD-3 and LL-37. Conclusion: CFU-assays using Nocardia asteroides revealed differential activity of human AMPs. Whereas HNP1-3 exhibited potent activity against the tested Nocardia strain, hBD-3 and LL-37 failed to kill Nocardia in our CFU-assay. Further investigations with different Nocardia species are currently undertaken to elucidate the role of AMPs within innate immunity in nocardiosis.

Galanin-message-associated peptide provides innate defence against Candida sp.

I. Rauch, S. Holzmeister, M. Hell, W. Sperl, B. Kofler (Salzburg, AT) Introduction: Several epithelial surfaces are continuously exposed to potential pathogens but rarely become infected, because specific secretions protect them. Antimicrobial peptides (AMPs) participate in this innate immune response by providing a rapid first line defence against infection. Neuropeptides are expressed by neuronal and nonneuronal tissues in various organs including the skin, which constitutes the first barrier against external stress. These neuropeptides exert potent growth-and immunomodulatory effects and few have also been shown to possess antimicrobial activity. The galanin precursor peptide mRNA (ppGAL) is one of the few neuropeptide mRNAs expressed in keratinocytes of the human skin. The galanin gene encodes a precursor peptide that is processed into the 29 amino acid peptide galanin and the 59 amino acid galanin-message-associated peptide (GMAP). Our objectives where to test whether one of these two peptides possesses antimicrobial activity. Methods: A cell viability assay and microscopical evaluation were used to examine microbial growth. Influence of GMAP on C. albicans adhesion to keratinocytes was determined using a fluorescence-based adherence assay. The effect of co-culture of C. albicans and keratinocytes on ppGAL expression was assessed by qPCR.

We could demonstrate that GMAP possesses growth-inhibiting activity against different Candida species. GMAP inhibits the yeast-to-hyphal transition of C. albicans, which is an important virulence adaptation of this pathogen. Preliminary data suggest that GMAP also reduces the ability of C. albicans to adhere to epithelial cells. Exposure of keratinocytes to C. albicans increased the GMAPprecursor expression 2-fold. Conclusion: These studies establish GMAP as a new component of the innate immune system, which has implications for prophylactic and therapeutic strategies of Candida infections. Supported by grants of the Jubiläumsfonds (Nr. 11996) and the Paracelsus Private Medical University (Nr.05/01/003).

A macrophage -epithelial network regulates pulmonary host defense C. Hess, R. Bals (Marburg, DE) Introduction: The lung is continuously exposed to microorganisms. The innate host defense system provides a basic host defense that eliminates bacterial pathogens. Airway epithelial cells (AECs) have a role as structural barrier tissue and in addition can engage in inflammation and immunity. Here we report a mechanism whereby macrophages regulate the sensitivity of AECs to microbial patterns.

Whereas native AECs are largely insensitive to ligands of TLR2 or TLR5, exposure to secretory products of macrophages results in increased reactivity. This augmented sensitivity is associated with increased expression of TLR genes and surface expression of the protein. The effect of macrophages depends on secreted mediators including TNF-alpha. Animals lacking myeloid

RelA/p65 showed significantly decreased epithelial responses to pulmonary infection. Conclusions: Our data provide a case for lung-specific regulation of surface immunity where epithelial innate immunity is regulated by the influence of macrophages. This mucosal host defense network allows adjusting the local defense and inflammatory responses to the local needs.

The regulation of lipocalin 2 by iron status and the regulation of iron status by lipocalin 2 I. Theurl, M. Theurl, M. Seifert, M. Nairz, J. Pinto, G. Weiss (Innsbruck, AT; Porto, PT) Background: NGAL, Lipocalin 2 (24p3) is a monocyte and neutrophile derived peptide which exerts anti-microbial effects by binding bacterial siderophores, but it may also affect cellular iron uptake into mammalian cells.

We measured the expression of mRNA and protein levels of lipocalin-2 in different mouse models of iron overload and biopsy specimen from patients suffering from hemochromatosis and ACD. Moreover, we performed primary cell culture experiments using freshly isolated mouse hepatocytes and human liver cell lines overexpressing different HFE mutants. Result: Secondary iron overload of mice resulted in decreased lipocalin-2 expression in several tissues. In contrast, lipocalin expression was significantly increased in liver specimens of Hfe-/-mice and patients suffering from hemochromatosis (C282Y) as compared to HFe+/+ mice and controls, respectively. In addition we observed a significant increase of lipocalin -2 levels in Hfe -/-primary hepatocytes as compared to cells obtained from Hfe +/+ mice and importantly, in contrast to Hfe+/+ mice lipocalin levels were not regulated by iron. Importantly lipocalin expression in HepG2 cells is regulated by Hfe. Conclusion: Our data provide evidence that lipocalin-2 may play a role in the pathophysiology of hemochromatosis as lipocalin-2 is able to transport iron across cell membranes.

Autocrine formation of hepcidin induces iron retention in human monocytes I. Theurl, M. Theurl, M. Seifert, S. Mair, M. Nairz, H. Rumpold, H. Zoller, R. Bellmann-Weiler, H. Niederegger, H. Talasz, G. Weiss (Innsbruck, AT) Hepcidin, a master regulator of iron homeostasis, is produced in small amounts by inflammatory monocytes/macrophages. Chronic immune activation leads to iron retention within monocytes/macrophages and the development of anemia of chronic disease (ACD). We questioned whether monocyte derived hepcidin exerts autocrine regulation of cellular iron metabolism. Monocyte hepcidin mRNA expression was significantly induced within three hours after stimulation with LPS or IL-6, and hepcidin mRNA expression was significantly higher in monocytes of ACD patients than in controls. In ACD patients monocyte hepcidin mRNA levels were significantly correlated to serum IL-6 concentrations, and increased monocyte hepcidin mRNA levels were associated with decreased expression of the iron exporter ferroportin and iron retention in these cells. Transient transfection experiments employing a ferroportin/EmGFP fusion protein construct demonstrated that LPS inducible hepcidin expression in THP-1 monocytes resulted in internalisation and degradation of ferroportin. Transfection of monocytes with siRNA directed against hepcidin almost fully reversed this LPS mediated effect. Using ferroportin mutation constructs we found that ferroportin is mainly targeted by hepcidin when expressed on the cell surface.

Our results suggest that ferroportin expression in inflammatory monocytes is negatively affected by autocrine formation of hepcidin, thus contributing to iron sequestration within monocytes as found in ACD.

Polymorphonuclear leukocyte function cannot predict the occurrence of infection after renal transplantation S. Blaas, B. Salzberger, P. Hartmann, I. Fink, T. Glück (Donaustauf, Regenburg, Cologne, Regensburg, Trostberg, DE) Objective: Infections are a major cause of morbidity and mortality after renal transplantation. Early therapy has the potential to reduce the risk of complications. Previous reports have suggested that impaired chemotactic ability may be able to predict infections in the early postoperative period after solid organ transplantation. We examined the role of various tests of polymorphonuclear leukocyte function as predictors of nfections in renal transplant patients under immunosuppressive therapy in a prospective observational study. Methods: At fixed time intervals during the first year after kidney transplantation phagocytic activity and the release of reactive oxygen species was measured by commercially available assays (Phagotest, Phagoburst, Orpegen Pharma), and the neutrophil chemotactic ability was tested using a chemotaxis chamber assay (Neuroprobe). Bacterial and viral infections 14 days prior and 60 days after every measurement were monitored. Results: In 72, 51 and 54 patients transplanted between June 2003 and January 2005 polymorphonuclear leukocyte function assays were performed in periods I (day 14 -50), II (day 60 -150) and III (day 180 -380) after transplantation, respectively. There were 6, 3, and 0 bacterial and 13, 1, and 1 viral infections in time periods I, II and III in patients without an infection 14 days prior to the test performance. There was no statistically significant difference in phagocytic activity, the ability to release reactive oxygen species, and in neutrophil chemotactic ability between patients developing a bacterial or viral infection within the following 60 days and those without infection. Conclusion: While the impairment of neutrophil activity may have some potential to predict infections early after solid organ transplantation, we did not find a predictive value of polymorphonuclear leukocyte functional assays for bacterial or viral infections during the first year after renal transplantation.

Nramp1 limits the intracellular growth of Salmonella typhimurium by impairing bacterial iron uptake and strengthening macrophage effector functions M. Nairz, G. Fritsche, I. Theurl, M. Theurl, S. M. Mair, R. Bellmann-Weiler, H. C. Barton, G. Weiss (Innsbruck, AT; Southampton, UK) Introduction: Natural-resistance associated macrophage protein 1 (Nramp1) is a phagolysosomal transmembrane protein which acts as a transporter for protons and divalent ions including iron. Nramp1 functionality is associated with an increased resistance against various intracellular pathogens including leishmania, mycobacteria and salmonellae.

We investigated the regulation of iron homeostasis and immune function in RAW264.7 macrophages stably transfected with functional or non-functional Nramp1 in response to infection with Salmonella typhimurium. Results: Macrophages bearing functional Nramp1 displayed a reduced expression of transferrin receptor 1, resulting in the decreased acquisition of transferrin-bound iron. In contrast, the expression of the transmembrane iron-exporter, ferroportin 1, and in parallel cellular iron release was significantly higher in Salmonella-infected Nramp1functional macrophages as compared to phagocytes lacking Nramp1.

Conceivably, Nramp1 functionality caused a reduction in the cytoplasmatic iron pool within macrophages. Additionally, Nramp1-expressing macrophages were capable of limiting the intracellular persistence Infection Abstracts KIT2008 of S. typhimurium significantly better as compared to cells non-functional for this protein. This antimicrobial effect of Nramp1 was characterized by the reduced iron access by engulfed Salmonella and the increased formation of nitric oxide, tumour necrosis factor and interleukin-6 by infected phagocytes. Conclusions: Our data suggest that the protective effects of Nramp1 may be partly attributable to its capacity to reduce the iron availability within phagocytes, an effect which limits microbial iron access while concomitantly strengthening macrophage immune effector functions.

High prevalence of antibodies to human liver calreticulin in malaria. Epitope mapping and potential implications for diagnosis.

W. Kreisel, W. Mössner, M. Pautsch, P. Deibert, K. Rösler, J. Riedel, H. Hoschützki (Freiburg, Teningen, DE) Background: Antibodies to stress proteins can be found in various infectious or autoimmune diseases. Antibodies to the endoplasmic reticulum stress protein calreticulin (CRT) have been demonstrated in systemic lupus erythematosus and infection with Onchocerca. We found antibodies to CRT in autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), alcoholic liver cirrhosis (ALC) and yersiniosis. We aimed: 1. To determine antibodies to CRT in malaria. 2. To investigate their epitope specificities in malaria, AIH, PBC, and ALC. 3. To calculate whether results of a rapid immunochromatographic detection test using HRP2 and malaria specific aldolase could be improved by determination of anti-CRT antibodies.

We determined antibodies to CRT of the classes IgA, IgG, and IgM by western blotting and perfomed the binaxTM test in 77 patients with malaria and138 healthy controls (HC). 138 overlapping octapeptides were synthesized covering the entire sequence of human CRT including the signal peptide. Using a blot technique the prevalence of antibodies to the peptides was determined in healthy controls (HC), ALC, AIH, and PBC (n=10, each). A serum was regarded as containing epitope specific antibodies if at least one of the antibody classes IgA, IgG, or IgM yielded a positive result.

Results: There was a high prevalence of antibodies to CRT in malaria (anti-CRT IgG: 85.7%, anti-CRT IgA 32.5%, anti-CRT IgM 70.1%). The binaxTM test yielded a sensitivity of 85.7% and a sensitivity of 100%. Including anti-CRT IgM into serological testing led to a sensitivity of 94.8% and a specificity of 96.4%. 15 linear peptide epitopes were identified as targets of antibodies. Disease-unspecific epitopes were covered by the peptides 46-49 (ICGPGTKKVHVIFNYKG) in the N-domain (amino acids 1-180) and by peptides 68-70 (FLPPK-KIKDPDASK) in the P-domain (amino acids 181-290) of CRT. Peptide 125 (DKKRKEEE) localized near to the C-terminus strongly reacted with all malaria, ALC, and PBC, 8/10 ALC and 4/10 HC sera. The P-domain contained two epitopes, covered by peptides 83-84 (AKKPEDWDEEM) and 91-94 (YKGEWKPRQIDNPDYKG), respectively, which were nearly exclusively recognized by sera from malaria and PBC patients. Malaria: 9/10 and 10/10, PBC: 9/10 and 10/10, AIH: 2/10 and 1/10, ALC: 1/10 and 2/10, HC: 0/10 and 0/10. Conclusion: Antibodies to the endoplasmic reticulum stress protein calreticulin including epitope-specific antibodies may improve rapid serological testing for malaria.

Opsonic antibodies to Enterococcus faecalis Strain Type 2 and 5 are directed against a novel capsular polysaccharide C. Theilacker, I. Toma, Z. Kaczynski, I. Sava, M. Halima, O. Holst, J. Hübner (Freiburg, DE; Gdansk, PL; Borstel, DE) Introduction: Enterococci are a frequent cause auf nosocomial bacteremia. Opsonophagocytic killing of Enterococcus faecalis is mediated by antibodies directed against carbohydrate antigens of the cell wall and bacterial capsule. In strains of the CPS-A and CPS-B serotype, opsonic antibodies are specific for lipoteichoic acid (LTA). However, serum raised against purified LTA does not kill strains of the CPS-C and CPS-D serotype. Methods: Capsular polysaccharide was isolated from E. faecalis type 2 and type 5, a CPS-C and CPS-D strain, respectively. The isolated polysaccharide was studied by sugar analysis, one-dimensional and two-dimensional homonuclear and heteronuclear 1H and 13C NMR spectroscopy. The binding of rabbit antiserum to capsular polysaccharide and LTA was assessed by ELISA and the specificity of opsonic antibodies was determined in an inhibition opsonophagocytic killing assay. Results: Capsular material released by enzymatic digestion contained small amounts of a novel, high molecular weight capsular polysaccharide. This material was successfully separated from contaminating LTA by gel-permeation and anion-exchange chromatography. Purity was confirmed by SDS-PAGE analysis. The novel polysaccharide contained an unusual -> 6)-3-O-[1-carboxyethyl]-beta-D-Galf-(1 -> residue in the repeating unit. Polysaccharide isolated from two distinct serotypes differed by the O-acetylation in position 5 of the Galf residue in the type 2 strain. Rabbit antiserum raised against purified type 2 polysaccharide contained high titers of polysaccharide-specific antibodies and and mediated opsonophagocytosis of E. faecalis type 2. Conclusion: A novel capsular polysaccharide was found in E. faecalis CPS-C and CPS-D strains which is immunogenic in vivo and a target of opsonic antibodies. It may represent a structural basis of serodiversity of E. faecalis strains.

Biofilm production by Candida spp isolated from cardiac surgery patients of an intensive care unit S. Tobudic, C. Kratzer, W. Granninger, A. Lassnigg, E. Presterl (Vienna, AT) Introduction: Patients undergoing cardiac surgery have a very high risk of invasive Candida infection, particularly if assist devices supporting cardiac function are used to bridge longer periods of cardiac failure. The aim of this study was to evaluate the biofilm formation of superficial Candida isolates from patients with cardiac assist devices admitted to the cardiothoracic intensive care unit (ICU).

Material and methods: The tested Candida species were isolated from skin swabs and medical devices of cardiac surgery patients and included Candida albicans (10), Candida parapsilosis (10), Candida glabrata (6), Candida tropicalis (1), Candida guilliermondii (1), Candida famata (1), Candida rugosa (1), and Candida norvegensis (1). Ten Candida blood strains isolated from blood of non-intensive care patients were used as controls and included C. albicans (8), C. parapsilosis (1), C. glabrata (1). Biofilms were grown for 8, 24, 48, 72, 96 and 120 hours in 96-well microtiter plates, fixed and stained with crystal violet. For quantification of biofilms the mean optical density (OD) was determined at a wavelength of 620 nm. Results: All tested isolates from the ICU patients were able to form a biofilm (OD > 0.5): 3 strains after 8 hours (OD 0.79 ± 0.26), 20 strains after 24 hours (OD 1.13 ± 0.7) 1 strain after 48 hours (1.02 ± 0.13), 3 strains after 72 hours (OD 0.66 ± 0.14), and 4 strains after 96 hours (0.89 ± 0.26). No difference in OD measurement was detected between several Candida species. Strains able to form significant par-

ticularly thick biofilm (1.84 ± 1.22, p<0.02) were isolated from implanted devices. Candida bloodstream produced significantly less biofilm (OD 0.342 ± 0.29, p< 0.05), and OD > 0.5 showed only two strains. Conclusion: In contrast to blood, superficial Candida strains of ICU patients showed a high ability to form biofilms. Particularly Candida strains with early biofilm formation (after 8 hours) seem to have high potency to colonize devices and cause systematic infections Thus, to test the biofilm formation may help to identify the patients with high risk to develop infections.

The role of glycolipids in biofilm formation in Enterococcus faecalis C. Theilacker, P. Sanchez-Carballo, I. Toma, F. Fabretti, O. Holst, J. Hübner (Freiburg, Borstel, DE) Introduction: Biofilm production is a critical step in enterococcal infections. In other Gram-positive bacteria, composition of membrane glycolipids has been implicated in biofilm formation and anchoring of lipoteichoic acid in the cell wall. Methods: A non polar deletion mutant was created by homologous recombination. Glycolipids were isolated by preparative layer chromatography and their structure determined by nuclear magnetic resonance spectroscopy. The deletion mutant was characterized by ELISA of LTA content, biofilm assay, adherence to Caco-2 cells, opsonophagocytosis killing assay, hydrophobicity assay, and a mouse bacteremia model. Results: Homology search revealed a gene, designated biofilm associated glycolipid synthesis A (bgsA) within the sequence of E. faecalis V583 sharing high similarity with cpoA, and iagA, two glucosyltransferases in Streptococcus pneumoniae and Streptococcus agalactiae, respectively. We constructed a non polar deletion mutant of bgsA in E. faecalis 12030. Analysis of major glycolipids by nuclear magnetic resonance spectroscopy revealed that the cell membrane of 12030Del-tabgsA was devoid of diglycosyl-diacylglycerol (DGlcDAG) while monoglycosyl-diacylglycerol (MGlcDAG) was overrepresented. The content of LTA of cell walls of 12030DeltabgsA was 3,3-fold higher than in the wild type control. In addition, 12030DeltabgsA was significantly less hydrophobic than wild type bacteria. Deletion of bgsA led to a almost complete abolishment of biofilm formation on plastic surfaces. Also, E. faecalis DeltabgsA was impaired in adherence to Caco-2 cells. Cell growth, autolysis, and sensitivity to antimicrobial peptides were not affected in the mutant. The 12030DeltabgsA mutant was significantly more susceptible to opsonophagocytic killing mediated by rabbit antibodies against cell surface antigens of E. faecalis. In a mouse bacteremia model, virulence of E. faecalis 12030Del-tabgsA was attenuated compared to the wild type strain.

The composition of membrane glycolipids in E. faecalis leads to a retention of LTA in the cell wall and has major impact on biofilm formation on polystyrene surfaces, resistance to antibody-mediated opsonophagocytosis, and adherence to epithelial cells. This knowledge maybe helpful in understanding mechanisms of biofilm formation and the engineering of surface properties of implanted medical devices less prone to biofilm infections.

S. saprophyticus autolysin adhesin mediates internalization into urothelial cell line 5637 F. Szabados, B. Kleine, M. Kaase, B. Heck, S. Rodepeter, T. Sakinc, I. Schmitz, S. Gatermann (Bochum, DE) Introduction: Uropathogenic E. coli (UPEC) and S. saprophyticus may cause acute and recurrent urinary tract infections. Certain UPEC strains are known to be internalized into urothelial cell lines and is described as an important pathogenicity factor of acute and recurrent infections. Within a cell, the bacteria are usually pro-tected from anti-infective therapy and the bacteria can maintain the infection from there. S. saprophyticus shows strong adhesion to uroepithelial cells, we therefore investigated the ability of S. saprophyticus for internalization into the urinary bladder carcinoma cell line 5637 using a FACS-based method. Methods: S. saprophyticus ATCC 15305, wildtype strain 7108, S. aureus Cowan I, S. carnosus TM300 were tested in a modified FACS assay and in a classical gentamicin protection assay described previously. Isogenic knockout mutants of several candidate proteins (collagen binding SD-repeat protein SdrI, staphylococcal surface protein Ssp with a lipase activity and fibronetin binding autolysin adhesin Aas) were constructed and tested for internalization. These results were validated in histological stains and transmission electron micrographs.

We could show internalization of S. saprophyticus strain ATCC 15305 into urinary bladder carcinoma cell line 5637 in contrast to noninvasive S. carnosus strain TM300. Internalization was blocked in an isogenic knockout strain and in an Aas-antibody treated strain in contrast to S. saprophyticus wild type strain. Conclusion: We found strong evidence for internalisation of S. saprophyticus into urinary bladder carcinoma cell line. We found further evidence that fibronectin binding autolysin adhesin plays a major role in S. saprophyticus internalization. These results bear striking similarities to mechanims described for S. aureus fibronectin binding proteins. Recurrent urinary tract infections due to S. saprophyticus could be explained in analogy to uropathogenic E. coli by internalization into uroepithelial target cells.

PCR for detection of free Schistosoma-DNA in human plasma D. Wichmann, M. Panning, T. Quack, S. Kramme, G.-D. Burchard, C. Grevelding, C. Drosten (Hamburg, Giessen, DE) Introduction: Schistosomiasis presents with a large clinical spectrum. Inhabitants of endemic regions and immigrants are more likely to suffer from chronic urogenital or intestinal schistosomiasis whereas returning travellers present with an acute illness called Katayama fever. The diagnostic gold standard is microscopic demonstration of eggs in stool or urine specimens, but these direct methods are limited by the facts that egg shedding fluctuates widely, that sensitivity is poor in patients with low parasite burden and that eggs are not yet shed in patients with Katayama fever. Serological methods have a high sensitivity but cannot discriminate between active and past disease and can still be negative in Katayama fever. Polymerase chain reaction (PCR) methods for detection of DNA from urine or stool samples have also been established, but unfortunately, the low equivalent volume of the samples which can be analysed in PCR (and microscopy) is a major limitation. To overcome these obstacles we have chosen a different approach. Detection of non-physiological DNA in human plasma by PCR has been followed up in several fields of medicine, e.g. early sexdetermination in pregnancy by detection of Y-chromosomal DNA in maternal plasma. A single schistosome contains DNA copies in vast stochiometrical excess over parasite count, and free DNA will be randomly distribute through the plasma, by this the obstacle of sampling errors due to statistical distribution phenomena are abrogated. However, such a concept has not been followed up to now. We established a real-time PCR and optimised it for detection of Schistosoma-DNA from large volumes of plasma. Method: Real-time PCR with internal control for detection of free parasite DNA in human plasma.

Results: The applicability of the assay for detection of parasite DNA and for controlling the success of anti-parasite treatment was demonstrated in a Balb/C mouse model. In a second step the method was applied to patients with different stages of infection. PCR was clinically superior to classical methods in all groups, with 100% detection rates in Katayama fever and overt disease and a clear discriminatory power between different clinical stages on the basis of DNA quantification. Conclusion: Detection of free parasite DNA in human plasma by realtime PCR is superior to conventional diagnostic methods in patients with Katayama fever and chronic disease. In addition it is a valuable tool for the control of treatment success.

Evaluation of the GenoType MTBDRplus assay for rapid molecular detection of isoniazid and rifampicin resistance of Mycobacterium tuberculosis M. Fritz, H. Lay, V. Kempf (Tübingen, DE) Isoniazid (INH) and rifampicin (RMP) are both first line regime drugs recommended for treatment of tuberculosis. A world-wide increase of drug resistant Mycobacterium tuberculosis (MDR) strains requires rapid and reliable diagnostic methods for detection of such resistant strains for therapeutic and epidemiological reasons. Susceptibility testing of M. tuberculosis is usually done by phenotypical drug susceptibility testing from liquid cultures using, e.g., the BACTEC MIGIT 960 system (Becton Dickinson). However, due to the low growth rates of M. tuberculosis, phenotypic testing normally takes several weeks. In contrast, the novel molecular GenoType MTBDRplus assay (Hain Lifescience GmbH) verifies genotypic resistance to INH and RMP due to the detection of encoded mutations or deletions in the rpoB, katG and inhA genes causative for the respective resistance patterns.

As there is evidence that this assay can also be performed directly from Ziehl-Neelsen smear positive respiratory specimens, we compared the results of the phenotypic and genotypic detection of antibiotic resistance patterns. Our results show that rapid molecular detection of the genotypes revealed comparable results as when tested phenotypically. Remarkably, molecular testing revealed results in approximately 6 hours after the specimen arrived in the laboratory whereas phenotypic susceptibility testing took in average four weeks.

Our results indicate that the new molecular GenoType MTBDRplus assay is useful test for rapid detection of MDR tuberculosis, particularly for patients from regions with known risk of MDR-TB.

Specificity of GenoType® MRSA Direct is increased when PCR result interpretation is based on hybridization reaction and agarose gel electrophoresis S. Borgmann, A. Kick, M. Schuierer, A. Reber, K. Beyser, H. Gruber, R. Abel, A. Lindauer (Weiden, Bayreuth, DE) Introduction: Identification of MRSA bearing patients might be accelerated by performing PCR from primary materials (swabs, etc.) circumventing bacterial culture. In our lab GenoType® MRSA Direct test (Hain, Nehren, Germany) has been performed since January 2006 servicing more than 40 hospitals in South-eastern Germany (Northern Bavaria) with microbiological analyses. Here we present data covering all PCR examinations collected over a time period between April and October 2006. Methods: MRSA PCR and hybridisation of PCR products against specific probes linked to nitrocellulose strips were performed according to the manufacturers´ recommendations. In addition, all PCR products were examined by agarose gel electrophoresis. All results were documented and compared to those of bacterial cultures performed within the following 7 days.

A total of 861 samples was examined by PCR out of which 457 samples were grown and characterized through microbiological routine within the next 7 days. Among the cohort of PCR negative MRSA samples only 2 % showed bacterial growth. Hybridization positive MRSA without detectable PCR products on agarose gels could be grown in bacterial cultures in only 14% of the samples, suggesting a high percentage of false positive PCR results. Even with a detectable PCR product on an agarose gel combined with a positive hybridisation signal growth of MRSA was observed in only 71% of the samples. Since this result also indicates a low specificity these 25 cases were backtracked in more detail. Eventually, there were only 5 cases left for a fair comparison of positive PCR results versus negative bacterial growth results suggesting specificity higher than 90%. Conclusion: GenoType ® MRSA Direct test is a sensitive tool for identifying MRSA bearing patients. However, interpretation of PCR results needs to be based on hybridisation combined with agarose gel electrophoresis.

Sensitive detection of Helicobacter pylori-DNA from stool with a PCR-"Lateral Flow Dipstick" M. Weizenegger, U. Eigner (Heidelberg, DE) Introduction: Helicobacter pylori (H. pylori) is the major cause for peptic ulcer and gastric cancer. We compared a highly sensitive PCRdipstick assay for detection of H. pylori-DNA from stool with data obtained from the RIDASCREEN ® FemtoLab H. pylori Enzyme immunoassay assays (EIA) (r-biopharm, Darmstadt, Germany). The EIA shows a sensitivity of 95.3% and a specificity of 97.1% when compared to culture and histology. Methods: PCR-Dipstick: The dipstick (Milenia Biotec, Bad Nauheim, Germany) is a lateral flow device and contains three lines. One for the detection of the H. pylori specific DNA, the second for detection of the amplification control (AC) and the third for monitoring the successful migration of the gold particles. DNA from 0,2g stool was extracted with the QIAmp DNA stool minikit (Qiagen, Hilden, Germany) as recommended by the manufacturer. 5µl of the eluated DNA and 1U of HotStar Taq polymerase (Qiagen, Hilden, Germany) was pipetted to 45µl of the amplification mix containing biotinilated primers and a FAM labelled probe. The PCRamplification protocol (15min, 95°C, 10 x 30sec 95°C, 2 min 58 °C , 30 x 30 sec 95°C, 40 sec 53 °C, 40 sec 95 °C, 8 min, 70°C and for final denaturation 2 min 95°C, 5 min 20°C) run in a PE 9700 thermocycler (Applera, Darmstadt, Germany). 100µl running buffer and 10µl amplicon were pipetted into a microtiterplate well. The dipstick was incubated for 10min and than evaluated by eye. Results: 32 EIA positive and 23 negative stool samples, sent for routine EIA diagnostic to the laboratory, were selected and frozen before PCR analysis for several weeks at -20°C. The PCR-dipstick was concordant positive for 29 and concordant negative for 18 specimens. Three stool samples were false negative and five false positive. The

sensitivity, specificity, positive predictive value and negative predictive value were 90.6%, 85.7%, 85.3% and 85.7%.

The PCR-Dipstick seems to be a sensitive and specific tool for detection of H. pylori -DNA. It can be an alternative to real time PCR as no expensive instrumentation is required. For a conclusion, wether such a PCR-test is of superior sensitivity and specificity in comparision to established methods more data from EIA-negative stool specimens of patients highly suspicious for H. pylori infections have to be investigated.

Rapid and specific diagnostics of ricketssial infections by a pcr-based hybridization chip assay R. Wölfel, G. Dobler (Munich, DE) Introduction: Rickettsiae, a group of fastidious intracellular gramnegative bacteria, are divided into two groups. These are the typhus group, which consists of Rickettsia typhi and R. prowazekii and the spotted fever group, which includes about 20 different species. Rickettsiae have been characterized conventionally and identified by serotyping and protein analysis. Furthermore several DNA-based techniques have been developed for the species identification of rickettsiae mainly based on sequencing of various rickettsial genes. However these methods are time-consuming, expensive and require sophisticated lab equipment. Therefore, a new identification method that counteracts these disadvantages is needed especially for rapid, sensitive and specific laboratory diagnostics of human rickettsioses. Methods: A method for the detection of rickettsia based on a PCR that amplifies a biotin-labelled fragment of the 23S-5S intergenic spacer region is presented. Initial amplification can be performed either on a real-time PCR system using a broad-range rickettsial probe or on a conventional PCR cycler under basic laboratory conditions. Amplification products are hybridized to low density arrays carrying universal, group-and strain-specific oligonucleotide probes. The arrays are based on plastic photo-slide frames and are processed in a simple and purely manual procedure. Bound PCR fragments are visualized with the naked eye using a blue precipitating peroxidase substrate. A competitive internal control is included in each assay to identify sample-derived PCR inhibition. Results: Several rickettsial species, among them Rickettsia (R.) conorii, R. africae, R. rickettsii, R. monacensis, R. sibirica, R. canadensis, and R. massiliae were tested in the hybridization chip assay. A high discrimination between rickettsial species and a high sensitivity was found for all rickettsial species tested. Conclusions: The method is highly sensitive and specific in identifying rickettsia species from both patient and arthropod samples. Additionally the generic approach used allows identifying up to now unknown pathogenic rickettsial species.

Comparison of different media for preservation and transport of living rickettsiae H. Frickmann, G. Dobler, R. Wölfel (Munich, DE) Introduction: Rickettsiae are obligate intracellular organisms so they tend to destabilization outside living cells. Therefore transport of samples containing rickettsiae for keeping them alive in order to grow them in cell culture or to detect them by antigen or nucleic acid detection systems is difficult due to their sensibility to environmental effects. Methods: In order to determine the capability to preserve viability of rickettsiae for transport purposese, the commercially available transport medium COPAN "LITM-RT transport medium for viruses, chlamydia, mycoplasma & ureaplasma was compared with minimal essential medium (MEM, GIBCO, invitrogen, Karlsruhe, Germany) and MEM + 10% bovine fetal serum. Rickettsia (R.) honei was used for the study because of its ability to induce cytopathic effects in Vero-E6 cell cultures. Cell culture supernatants containing R. honei were mixed with transport medium in a ratio of 1:3 ensuring that at least 1.000 tissue culture infecting doses (TCID) were present. The original titer and the titers after storage for various times and temperatures (4°C, 22°C) were determined in Vero-E6 cell monolayers. Cytopathic effect was determined by microscopic examination.

We were able to demonstrate that temperature is a more important factor for the viability of rickettsiae than the ingredients and composition of the medium. After two weeks of storage at room temperature no viable rickettsiae were detectable any more. However by storage at 4°C rickettsial titres decreased slowly and viable rickettsiae were detectable up to 4 weeks. The commercial COPAN medium showed the best stabilizing effect on rickettsiae. However, also MEM + 10 % bovine fetal serum showed a good stabilizing effect on the rickettsiae. Pure MEM gave the poorest but still acceptable results in the preservation of viability of rickettsiae.

Conclusions: It was shown that the transport medium COPAN "LITM-RT transport medium for viruses, chlamydia, mycoplasma & ureaplasma" gave the best results regarding storage and transport of rickettsiae. If possible, the sample-containing medium should be transported at 4°C. MEM + 10% bovine fetal serum can be used in the case that no COPAN medium is available with similarly good results. However it is important to transport and store the media with suspected rickettsiae at 4°C for prevention of inactivation.

S. Poppert, M. Riecker, A. Lackner, N. Wellinghausen, A. Essig (Ulm, DE) Rapid identification of pathogens from blood cultures is of major importance, because the timely initiation of an adequate therapy greatly influences patient outcome and in addition may lead to cost savings. As previously published rapid identification (1-2 hours) may be achieved by Fluorescence in situ Hybridization using fluorescently labelled probes complementary to specific sequences of the ribosomal RNA. The method has not yet been broadly introduced into routine diagnostics, because the number of covered pathogens is still limited and available probes have not yet been evaluated on sufficiently large sample collections.

In a first step we therefore evaluated 12 probes from the literature and 17 newly designed probes, including probes for Staphylococci, Streptococci, Enterococci, Pseudomonas, Acinetobacter, Propionibacterium and yeasts using a collection of more than 900 clinical isolates and reference strains. In the next step the FISH method was assessed on 892 positive blood cultures according to an algorithm based on the gram stain. Nearly all selected probes demonstrated a sensitivity of 100% and specificities of better than 95%, when tested on the strain collection.

In 838 (94%) of the investigated blood cultures the pathogen was correctly identified by FISH. In 52 cases (5,8%) no result was achieved because the respective rare pathogens were not covered by the probe set. In just 3 cases (0,2%) FISH lead to a false result. In conclusion FISH proved to be a highly sensitive and specific tool for fast identification of pathogens from blood cultures. As an easy performable and cheap method, FISH is suitable to accelerate pathogen identification in a routine laboratory and may thus improve patient outcome and reduce therapy costs.

Abstracts KIT2008

Identification and classification of microorganisms using the MALDI BioTyper T. Maier, M. Kostrzewa (Leipzig, DE) Introduction: Fast and secure identification of microorganisms is an essential requirement in many areas, e.g. clinical diagnosis, epidemiology, environmental investigation and food control. Currently, mainly methods based on morphological investigation and tests regarding biochemical capabilities are used in routine. Unfortunately, these methods have limitations in resolution, applicability and reliability. Frequently, they end up in genus determination. Modern molecular methods which have a higher resolution power generally are too laborious and costly for high-throughput routine usage. MALDI-TOF MS fingerprinting has been shown to be well suitable for the identification of microorganisms. Methods: Microorganisms were cultured on solid media and applied to a MALDI sample target, directly or after a short inactivation/extraction protocol. After addition of matrix, linear profile spectra were acquired in a MALDI-TOF MS instrument. Based on the acquired profile spectra an according reference data base of more than 1500 different microorganism species was created using the MALDI Bio-Typer software. This database could be used for analysis of microorganism profile spectra with dedicated BioTyper algorithms, i.e. pattern matching, weighted pattern matching, principle component analysis and correlation analysis. Results: MALDI fingerprinting combined with the standard pattern matching algorithm was found to be a very robust method for identification of bacteria. Simple sample preparation by direct smear of bacteria onto the target plate resulted in mass spectra of good quality in many cases. For more difficult samples, e.g. microorganisms with robust cell walls, a short extraction protocol increased the quality of results, significantly. Identification was possible on the genus and mostly also on the species level without any fine-adjustment. A weighted pattern matching approach which applies the selective analysis of differentiating features in closely related groups is able to resolve closely related species as well as subtypes of bacteria. Principle component analysis (PCA) followed by cluster analysis as well as correlation analysis can differentiate microorganisms on the species level. Partially, also differentiation on the subspecies level was possible but this was more dependent on rigorously standardized sample preparation and measurement compared to pattern matching. The technology was applied in several areas of microbiology and compared to standard methods.

Pathogen detection in bloodstream infections and kolpitis within a few hours: From classical DNA microarrays to Lab-on-a-Chip systems

We present a novel approach for rapid identification of infectious pathogens by combining PCR amplification with microarray analysis. In comparison to conventional diagnostic procedures, all depending on bacterial growth, this assay substantially accelerates diagnosis. For a start a classical oligonucleotide microarray (fluorescence readout) was established for the identification of 26 blood stream infection relevant pathogens. As expected from sequence similarity in the target genes (16S / 18S rRNA), members of the Enterobacteriaceae family turned out to be too conserved for accurate species identification based on single species specific DNA probes. However, typical hybridization patterns of each species could be successfully employed for accurate discrimination of each member of Enterobacteriaceae. Data from all identification experiments were collected to a hybridization matrix which served as base for statistical analysis. The combination of rank normalization and k-nearest-neighbor method enabled a correct determination of 100 % of the bacteria at genus level and 96.7% at species level. After thorough assay optimization (DNA isola-tion, PCR amplification, PCR target labeling) we succeeded to identify 10 E. coli cells per mL whole blood. A diagnostic chip for the identification of kolpitis causing pathogens including bacteria, yeasts, moulds, parasites and viruses is currently under development. Up to now a microarray was established which can identify kolpitis relevant bacteria and yeasts down to species level. Probe validation showed good agreements with conventional identification methods and further probes are already introduced for the detection of intracellular pathogens. Relying on our experiences from pathogen microarrays we currently develop a Lab-on-a-Chip (LOC) device aiming to miniaturize and accelerate the diagnostic assay further.

Detection of phlebovirus rna by pan-phlebovirus rt-pcr Results: Using the RT-PCR detection of the L segment RNAs of all tested viruses were detected. However by application of the additional internal primer pair (nested RT-PCR), only some of the viruses gave positive signals. These data show that the nucleotide variation in the segment tested is more variable than known so far. Therefore the nested RT-PCR does not detect all phleboviruses tested. However the primer regions of the first RT-PCR seem to be constant so that they can be used for a pan-phlebovirus RT-PCR.

Our results show that a pan-Phlebovirus RT-PCR can be used to detect phleboviruses from different species and from different continents. However it seems that the nucleotide sequences of the different viruses are highly variable so that the more sensitive nested RT-PCR cannot detect all phleboviruses. Sensitvity of the phlebovirus RT-PCR is lower than of the nested RT-PCR. Therefore, the pan phlebovirus RT-PCR has to be used with caution in clinical materials as for most of the human pathogenic phleboviruses no data on viral load in blood or cerebrospinal fluid are known.

Rapid and simple detection of ebola virus and marburg virus by pcr-based lateral flow dipstick assay R. Wölfel, M. Panning, G. Dobler (Munich, DE) Introduction: Ebola virus (EBOV) and Marburg virus (MBGV) cause severe hemorrhagic fever in humans and nonhuman primates with high lethality rates. Rapid identification of these viruses is required to prevent spread of the infection in outbreak situations. Methods: We develop and evaluate a one-step reverse transcription PCR assay for rapid detection of all human pathogenic Filo viruses. Detection of amplification products is based on the established immunological «lateral-flow dipstick» (LFD) technique which has been adapted to detect PCR products.

Results: This approach combines the sensitivity of molecular detection with the easy and safe handling of the LFDs. The specificity of the detection is increased by probe hybridization during the analysis. This internal sequence verification of the amplification product guarantees a reliable molecular detection. In addition an internal amplification control is integrated on the same test strip as the target reaction to detect any sample derived PCR inhibition and to reduce the rate of false negative test results. Preliminary evaluation data indicate that the assay could detect less than 20 copies of in-vitro transcribed EBOV and MBGV RNA per reaction. In addition, the assay was highly specific for both Filo viruses.

The Filo virus detection assay developed in this study is rapid, simple, specific, and sensitive for the detection of both EBOV and MBGV, and so may be an effective diagnostic tool for these hemorrhagic fevers. In contrast to other PCR detection platforms for molecular diagnostics the Filo LFD does not require sophisticated instrumentation and needs only 10 minutes for the read-out step. It seems very suitable for diagnosis in the field or laboratories in Ebola and Marburg outbreak areas such as Central Africa.

S. Richter, H. Huemer, S. Revilla-Fernández, W. Zenz, V. Strenger, M. Müller, H. Ellerbrock, A. Nitsche, F. Allerberger (Moedling, Vienna, Graz, AT; Berlin, DE) Introduction: The genus Orthopoxvirus contains four species that infect humans: Variola virus (VARV), monkeypox-(MPXV), vaccinia-(VACV), and cowpox virus (CPXV). VARV and MPXV cause often life-threatening diseases, whereas VACV and CPXV in immunocompetent people generally are associated with local lesions. We report on the diagnostic procedures employed in a case of cowpox occurring in Austria in 2007. Methods: Electron microscopy (EM) of lesion crust, subepidermal edema and vesicle fluid was performed by tissue sectionning and negative staining, including enrichment by ultracentrifugation (TEM: Zeiss 906,80kv). Sections of biopsy material, embedded in Epon, were contrasted with UA and lead citrate. Virus culture was performed using the monkey MA104 cell line and rabbit kidney cell RK13. Molecular diagnostics was performed using orthopoxvirus-specific primers for the hemagglutinin and A36R genes and species identity determined by restriction enzyme typing as well as direct DNA sequencing.

A 17 year old girl from a farm in Carynthia was hospitalised with a 10 days history of antibiotic treatment (i.v. clindamycin 600mg tid) for a local infection on the anterior neck (black crust of 2 cm diameter) with erythema and edema. CRP (5.6 mg/l) and leucocyte count (8.20 G/l) were normal. Electron microscopy of the biopsy material (crust, subepidermal edema) and vesicle fluid yielded orthopoxvirus. PCR showed the cleavage patterns typical for local CPXV strains, thus excluding VACV and MPXV as differential diagnosis. Conclusions: CPXV is still endemic in Austria; small rodents seem to be the main reservoir hosts. Most patients have only one lesion, which passes through macular, papular, vesicular, and pustular stages before forming a hard black crust. EM of vesicle fluid and biopsy material is particularly valuable as frontline technique because it allows rapid differentiation of parapox-, herpes-and presumptive orthopoxviruses by pattern recognition. In parallel, molecular diagnostics (PCR, RFLP, Sequencing) are essential to confirm the result and determine the virus species, especially if samples are in a suboptimal condition or the patient is in a convalescent phase of infection. CPXV like MPXV is mainly transmitted by contact with infected rodents and in middle Europe often brought to humans by domestic cats. Infection via cows is a rather rare event. Exclusion of MPXV is important as it has been acquired in the US most recently by imported African pet rats.

T. Krech, S. Castriciano, M. Chernesky (Kreuzlingen, CH; Brescia, IT; Hamilton, CA) Background: Several studies have shown equal sensitivity and specificity for vaginal swabs compared to endocervical swabs and first voided urine for the detection of sexually transmitted viruses and bacteria. Novel flocked swabs promise higher detection rates. Aims: We compared the detection rates of HPV and Chlamydia trachomatis vaginal and endocervical swabs by using a special dual flocked/Rayon swab to avoid sampling bias. Method: A total of 1252 samples from 494 asymptomatic patients where tested from vaginal dual swabs taken by the gynecologist during routine visits. In 129 of them also an endocervical dual swab was taken. Detection of HPV high risk types was done by the Digene ® HPV Test*, using Hybrid Capture ® 2 (hc2) technology. Detection of Chlamydia trachomatis was done by an in-house real-time PCR decribed elsewhere. Vaginal sampling gave 30% more positive results for HPV than traditional endocervical sampling. For C.trachomatis vaginal sampling was also more sensitive than endocervical sampling. The genotypes of HPV detected in vaginal samples were the same as detected from endocervical swabs. Conclusions: Sampling with novel flocked swabs doubled the positive rate for Human Papillomavirus detection from vaginal swabs compared to conventional Rayon swabs. Furthermore vaginal sampling gave significantly more positive HPV results than traditional endocervical sampling. C.trachomatis was also more often detected with flocked swabs than with conventional Rayon swabs and vaginal swabs gave also more positive results than endocervical sampling.

A novel, universal, automated biosensor system for rapid, nucleic acid based detection of emerging diseases D. Ecker, V. Harpin, G. Schwarz, W. Pusch, R. Sampath, V. Harpin (Carlsbad, US; Bremen, DE) Introduction: More than 1400 known pathogenic organisms are potential threats to human health, and newly emerging threats are likely to increase this number exponentially. To address the needs of human healthcare surveillance as well as requirements in other fields (e.g. animal health and food supply), new robust and automated systems are required to deliver solid answers rapidly. Method: The Ibis T5000 biosensor system is a novel pathogen detection system, which uses broad range primers for amplification of nucleotide target sequences followed by time-of-flight (TOF) mass spectrometry (MS) analysis. Based on this MS information the system derives base composition signatures to identify most organisms present in a sample without priory knowledge about the causative infectious agent(s).

Results: An eight primer set up panel targeting different influenza virus core segments detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, the validation of the assay showed correct identification of 656 clinical respiratory samples with sensitivity and specificity better than 97%. Conclusion: Rapid reverse transcription PCR/MS analysis can be used to simultaneously identify influenza viruses and moreover monitor global spread and emerge of novel viral genotypes (Sampath et al. PLoS ONE, 2007 May 30; 2(5) . The broad applicability of this technology was also demonstrated recently in an alpha virus study where 35 of 36 mosquito extracts were determined unambiguously (Eshoo et al.Virology, 2007 Jul 24) . In summary the described method promises high throughput capabilities well suited for routine survey and detection of unanticipated, potentially unknown and newly emerging infectious diseases.

Rapid routine detection of enterovirus RNA in cerebrospinal fluids by a one step real time RT-PCR assay A. Heim, U. Dierßen, GH Harste (Hannover, DE) A one-step reverse transcription/real-time PCR assay with a TaqMan probe (TM-PCR) was developed for the detection of enterovirus RNA in cerebrospinal fluid (CSF) employing newly designed primer and probe sequences. These sequences were selected by creating a multiple alignment of 63 enterovirus sequences, and by calculating melting temperatures for various mismatches. The amplicon included a small part of the domain IV and the main part of domain V of the highly conserved internal ribosomal entry site (IRES). Generic detection of enteroviruses was confirmed by testing a panel of 41 prototypes representing all human enterovirus species. Sensitivity defined as 95% detection limit was found to be 100 copies per run using in vitro transcribed coxsackievirus B3 RNA as a standard. Specificity of the assay was tested with laboratory strains of HSV, VZV, EBV, CMV, Influenza A and B, morbillivirus adenoviruses and parechovirus-1 and -2, which all gave negative results. The TM-PCR was evaluated against an established, in house nested PCR assay for the detection of enterovirus RNA in 112 CSF samples of patients suffering from meningitis and encephalitis. Concordant results were obtained in all samples (11 positive, 101 negative). For testing diagnostic specifity, 76 CSF samples of patients with chronic inflammatory diseases (encephalomyelitis disseminata) were tested and found all negative. Thus, the new real time PCR assay should be an excellent alternative to conventional PCR protocols for the diagnosis of enterovirus meningitis. As a one step protocol, hands on time and cross contamination risk are reduced to a minimum, and time required for reverse transcription, amplification and detection is only 100 minutes.

Introduction: Invasive bacterial disease continues to be a major cause of severe disease, death and long-term disability. Both for the optimal management of the patient and for epidemiological purposes it is important for diagnosis confirmation and species identification. All probable and confirmed cases of invasive bacterial disease must be notified to the public health authorities. In case of suspected bacterial meningitis prompt administration of antibiotics is evident to decrease the risk of a poor outcome. On the other hand early administration of antibiotics significantly reduces the chances for traditional microbiological isolation, culture and identification of the causing organism.

Because of this cross-purpose molecular biological diagnosis has gained increasing significance in maximising case ascertainment of disease.

The introduction of molecular diagnosis at the National Reference Centre for Meningococci, Pneumococci and Haemophilus influenzae (NRC) has resulted in a steady increase in the number of confirmed cases of bacterial meningitis in Austria. Methods: Since 2000 the NRC offers free of charge real time polymerase chain reaction (RT-PCR) as a screening method for Meningococci, Pneumococci and H. influenzae in clinical samples of cerebrospinal fluid (CSF), whole blood, serum, and other materials in suspected cases of bacterial meningitis and septicaemia. The gene target used to detect Neisseria meningitidis is the ctrA gene and for serogroup identification the siaD gene is used. Serosubtype identification is facilitated by amplification and sequence-specific probe detection of the porA gene. For S.pneumoniae the ply primers are used to amplify the pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, 33 For Haemophilus influenzae the p6omp gene is amplified. By all specimens DNA is extracted using the Roche MagNA pure system. 

Do ICUs with an MRSA problem also have a VRE problem?

E. Meyer, F. Schwab, P. Gastmeier (Freiburg, Berlin, DE) 

Analysis of needs in the area of prevention and control of antimicrobial resistance in Germany A Barger, M. Kramer (Berlin, DE) Introduction: The increase of antimicrobial resistant pathogens complicates the treatment of infectious diseases and causes additional medical costs. The main reason commonly is inappropriate use of antibiotics. Therefore, the EU Commission published a strategy on prudent use of antimicrobial agents in human medicine in 2001. The focus of this strategy is the establishment of national strategies in EU member states. In the process of the development of a German strategy on prevention and control of antimicrobial resistance (AMR) the shortcomings and needs in the area of AMR in Germany (DE) were investigated by interviewing experts in this field. Methods: Therefore, a structured questionnaire with 43 open questions in the area of surveillance, prevention, and control of AMR was set up. Multiple answers were possible. In total 14 German experts were interviewed from April 2006 until February 2007. Answers were classified into categories and frequencies were determined.

The main deficit in the area of prevention and control of AMR in DE is the (continuous) medical education of medical students and physicians according to 10 of 14 experts (10/14). Missing representative data on AMR and antibiotic consumption (6/14), shortcomings in standardization (5/14), and the influence of representatives of pharmaceutical industry on physicians (5/14) were pointed out as further problems. Therefore, surveillance systems on AMR and antibiotic use should be strengthened (9/14), physicians should be trained on appropriate antibiotic therapy (4/14), control measures on restricted antibiotic use in hospitals should be implemented (4/14), and the use of recommendations should be controlled (3/14). Inappropriate use of antibiotics via the population is not a problem in DE (5/14) . The overall aim of a German strategy should be the reduction of AMR and multi-resistant pathogens respectively (11/14).

The expert interviews give a summary on deficits and shortcomings, and describe the needs in the area of prevention and control of AMR in Germany. Therefore, results from the interviews were combined with results from literature researches, and are used as basic components in the process of the development of a strategy on prevention and control of AMR in DE.

F. Haamann, D. Boulkhemair (Hamburg, DE) Introduction: Legionellae are ubiquitous environmental bacteria; they are also detectable in warm water installations and in the cooling water of treatment units in dental practices. In the course of treatment, dental instruments may emit nebulised spray, as an infectious aerosol which can be breathed in by dental personnel. It must then be asked whether employees in dental practices are exposed to an increased risk of disease. Method: A literature search was performed, which identified two reviews and seven studies investigating microbial contamination in dental units and their water systems in dental facilities. In addition, 5 relevant studies from 1985 to April 2003 were analysed which dealt with the seroprevalence of legionella antibodies and respiratory diseases in dental personnel. Results: No case of respiratory disease in dental employees was identified which could be retrospectively linked to exposure to legionellae. Nevertheless, antibodies to legionellae could be detected in all groups of dental employees; dentists exhibited the highest titres. Conclusions: It may be deduced that dental personnel has an immunological response to legionellae. Even though microbial contamination of the dental units has been demonstrated, professional exposure does not represent a risk, if suitable protective measures are taken to ensure that the concentration of legionellae in drinking water is kept low. On the other hand, it can be assumed that occupational Legionella infection may not be identified or accepted as such. If legionellosis occurs in dentists and their personnel, it should be considered as an occupational disease. In the course of occupational medical care, it is recommended that extensive advice should be given on Legionella infection and that Legionella antibodies may be determined. Water hygiene is of primary importance. If suitable technical and organisational protective measures are combined with training of the dental team, microbial colonisation of dental water systems can be drastically reduced, thus avoiding the risk of health damage.

S. Rieg, G. Peyerl-Hofmann, K. de With, C. Theilacker, J. Hübner, D. Wagner, C. Schneider, H. Wisplinghoff, W.V. Kern (Freiburg, Cologne, DE) Background: Staphylococcus aureus is one of the most important pathogen causing both community-and hospital-acquired bacteremia with substantial morbidity and mortality. We assessed the recent clinical epidemiology of this disease by studying adult patients with Staphylococcus aureus bacteremia (SAB) who were admitted to a university hospital in southwestern Germany. (24%) were community-acquired, 31% were healthcare-associated, and 43% were nosocomial cases. MRSA was not observed among the community-acquired cases, compared with a rate of 17% in healthcare-associated and in nosocomially acquired cases. The majority of the infections (53%) were considered primary SAB (unknown portal of entry, or iv catheter-related). Complicated SAB with occurrence of metastatic deep-seated foci was observed in 178 (36%) patients, with pneumonia, bone infection, and soft tissue or visceral abscesses representing the most common manifestations. Endocarditis as defined by Duke criteria was confirmed in 25 patients (10%) during the retrospective study period and in 27 patients (11%) during the prospective evaluation period. Infectious diseases (ID) specialists were involved in 332 cases (66%). In-house mortality was 22%, and mortality at the 1-year follow-up was 169/353 (48%). In-house mortality was improved in cases with ID consultations (25% vs 37%, p=0.02).

Conclusions: SAB remains a frequent cross-specialty severe infection and represents an immense challenge for hospitals and medical centers to provide expert interdisciplinary management and resources for improved infection prevention. Clinical epidemiological findings in this study were similar to those reported previously from other sites. Our cohort study is ongoing and will be used to address further important issues in SAB, including quality of care indicators, relevance of adherence to infectious disease consultant recommendations after

adjustment for potential confounders, and new risk-adapted therapeutic approaches.

An analysis of potential quality indicators in Staphylococcus aureus Bacteremia management using the prospective INSTINCT Cohort A. Kaasch, H. Seifert, H. Wisplinghoff, K. Achilles, A. Langhorst, G. Peyerl-Hoffmann, S. Rieg, G. Fätkenheuer, W.V. Kern (Cologne, Freiburg, DE) Introduction: Staphylococcus aureus bacteremia (SAB) is a severe disease, but optimal diagnostic and therapeutic decisions are able to reduce mortality and secondary complications. Thus SAB is highly suitable to develop and validate instruments to develop and measure process of care quality indicators for optimal infection management.

Methods: Simple quality criteria were developed based on a literature review and in cooperation with the ABS International project group. The indicators chosen are meant to reflect critical but modifiable diagnostic or therapeutic decisions. A set of three preliminary indicators was examined in this study. One measures the percentage of patients with non-catheter related SAB who have undergone echocardiography within 10 days after onset of SAB since timely performance of echocardiography is crucial in detecting endocarditis as a source for bacteremia. Central venous catheters (CVC) that are in place are a possible source of SAB. The second assesses the removal of CVCs in patients with potential catheter-related infection within a specified time period. Adequate antimicrobial therapy in respect to duration, dosage, and susceptibility testing results is reflected by the third indicator, expressed by the percentage of patients with adequate antimicrobial therapy for more than 10 days within 14 days after SAB onset. Analysis of these potential indicators was carried out on a cohort of 231 patients with SAB monitored prospectively at two university hospitals. Data was collected based on assessment through infectious diseases consultations, patient charts, microbiological reports and interviews.

Of the 231 patients with SAB, 50% were hospital-acquired infections, 29% were healthcare-associated infections, and the remaining 21% were community-acquired infections. Of 159 patients with non-catheter-related SAB, 38% (61) underwent echocardiography within 10 days. In 35 cases with central venous catheters at onset of SAB, 80% (28) of central lines were removed within 10 days. Of the 210 patients that survived for more than 10 days, 80% (168) received adequate antimicrobial therapy.

The selected potential indicators were assessable using the INSTINCT cohort database. They should and can be evaluated and perhaps refined as predictors of different outcomes such as mortality or relapsing disease. Whether there is a critical target to be achieved to be relevant for improved outcomes remains to be determined.

Surgical-site infection after Spinal Fusion Surgery -First report of prospective ongoing outcome study C. Remschmidt, Y. Achermann, D. Grob, J. Wüst, M. Vogt (Zug, Zürich, CH) 

Legionnaires' disease in patients admitted to a German University Hospital between 1998 and 2005 P. Klare, D. Jonas, A. Conrad, W.V. Kern, M. Dettenkofer (Freiburg, DE) Introduction: While the majority of infections caused by Legionella occur sporadically, water supply systems in hospitals may be a relevant origin of Legionnaires' disease (LD). Often, patients (pts) with severe underlying disease are affected, but little is known about recent epidemiologic developments in Germany. The aim of the present study was to assess the origin and course of LD in pts admitted to University Medical Center Freiburg (UMCF). Methods: Cases were identified through a search in the diagnostic laboratory databases for the period between 1998 and 2005. We looked for pts with positive culture, positive urinary antigen test, and/ or evidence for the infection by direct immunofluorescent antibody detection of the organism in respiratory specimens. We assumed a nosocomial origin if the time between admission and first radiological signs of pneumonia had been >10 days, community acquisition was assumed if the time was <2 days; the other cases were considered indeterminate. Cases were evaluated retrospectively with regard to epidemiological data, potential risk factors and outcomes. Community acquisition versus nosocomial origin, gender and underlying disease were analysed with respect to their influence on outcomes by univariate (Fischer´s exact test) and multivariate (logistic regression) analysis.

Results: Forty-four adult pts of LD were treated during the period of study, 31 were male. Most had community-acquired legionellosis (32/44, 73%) while 7 pts had nosocomial disease (16%). 14 pts died during hospitalisation (32%). Most pts were initially treated with macrolides, and very few received initial therapy with a fluoroquinolone. More than half of the pts required intensive care admission. Univariate analysis showed that pts with nosocomially acquired LD had significantly higher in-hospital mortality than pts with community-ac-

quired infection (5/7 vs 9/32, p<0.05). In multivariate analysis the origin of infection was only marginally significant (p=0.10) for outcome.

Due to the small numbers, gender, malignancy, COPD or organ transplantation showed no significant impact on survival. Conclusions: Few of the cases were clearly nosocomially acquired, but, overall, we observed very severe disease with a high mortality. As expected, nosocomial cases tended to have poorer outcomes than community-acquired cases. We conclude that earlier diagnosis and a more aggressive therapeutic approach are needed to improve outcomes of this disease.

Evaluation of infection control practices in 30 haematopoietic stem cell transplant (HSCT) facilities and experience of the patients involved S. Wenzler-Röttele, A. Neuwöhner, A. Blaich, M. Dettenkofer (Freiburg, DE; Basel, CH) Objective: Haematopoietic stem cell transplant (HSCT) recipients are cared for in specialised facilities and versatile precaution measures are commonly practised in order to prevent fatal opportunistic nosocomial infections in these highly immuncompromised patients. However, evidence is lacking on whether many of these interventions efficiently reduce the incidence of these infections. Furthermore, most of the measures are cost-intensive and restrict patient comfort and reconvalescence. A survey was performed to assess the spectrum of measures commonly practised and to evaluate the precaution measures taken in HSCT-facilities in German-speaking countries. In addition, the patients` acceptance of these measures was evaluated.

Methods: A detailed questionnaire on infection control measures differing according to allogeneic and autologous HSCT recipients was compiled. This questionnaire was sent to 64 HSCT facilities in April 2005. Two additional questionnaires addressed to the nursing teams were sent to the 30 centres that had responded to the first questionnaire. One was to be filled out by the nurses themselves, the other by the HSCT-patients. Results: 30 questionnaires (47%) were completed and returned by the end of October 2005. Of the 30 centres, 16 were university hospitals, 9 teaching hospitals and 5 other institutions. It became apparent that hygiene measures differ greatly among the different institutions. Even evidence-based recommendations were not routinely followed in all facilities. Another 46 questionnaires were returned by the nursing teams and 27 by the patients involved. As a result several deficiencies became apparent with regard to hand hygiene measures. As for the patients, they felt they had been sufficiently informed about the necessary hygiene measures and acceptance was high. Although they suffered from the restrictions they felt their illness itself to be more confining.

The broad variety of different precautionary measures taken by the different facilities reflects the lack of evidence for many infection control measures that are commonly practised in the care of HSCT recipients. Training in hand hygiene for the health-care personal seems to be an efficient measure to improve hygiene in HSCTfacilities. Patients accepted the hygiene measures demanded of them, but would also benefit from a rational reduction in their number. This study was supported by the Robert Bosch Stiftung Stuttgart, Germany 

Coloring and perfume free products for hand hygiene: A straightforward Approach to improving Compliance A. Conrad, I. Poser, S. Wenzler-Röttele, A. Pfister-Wartha, M. Dettenkofer (Freiburg, DE) Introduction: Hand hygiene represents the most essential and uniformly accepted measure in infection control, and decontamination of healthcare-workers' hands using alcoholic hand rub is generally well tolerated. However, there is a certain risk of sensitization since many hand hygiene products contain dyestuffs and perfumes that are associated with this risk but do not contribute to the efficacy of the formulations. Methods: A dye-and scent-free hand hygiene product line was evaluated at the University Medical Center Freiburg in association with a campaign to improve hand hygiene compliance. The intervention was accompanied by short (15 min) training sessions on adequate indications and proper hand hygiene measures. After a 6-week implementation phase in 11 intensive care units and hemato-oncology wards, the products were evaluated by use of a structured questionnaire. Results: Of 611 questionnaires distributed, 41% were completed, the majority by nurses and about 7% by physicians. 96 of 226 of the respondents (42%) preferred the dye-and scent-free hand hygiene products, which were generally well tolerated. As personnel tend to stick to products they know (especially if used on their own skin), this is an interestingly high figure. Only 3% of the respondents complained about a malodour (alcohol without perfume). Proper hand hygiene was judged to be of high relevance to assure a high standard of patient safety. Conclusions: Since dyestuffs and scents are dispensable, in future only coloring and perfume free hand hygiene products should be promoted and used. The range of commercially available products that meet high standards of microbicidal activity is currently expanding.

Abstracts KIT2008

Efficacy of silver nanoparticles impregnated external ventricular drain catheters in patients with acute occlusive hydrocephalus P. Lackner, R. Beer, G. Broessner, R. Helbok, K. Engelhardt, B. Pfausler, C. Brenneis, K. Galiano, A. Obwegeser, E. Schmutzhard (Innsbruck, AT) Introduction: Catheter associated infection of cerebrospinal fluid (CSF) is a potentially life threatening complication of external ventricular drainage (EVD). The purpose of this pilot study was to address the efficacy of silver-impregnated EVD catheters in neurological and neurosurgical patients requiring external CSF drainage due to acute occlusive hydrocephalus. Methods: 19 consecutive patients were enrolled in the treatment arm of the study and data was prospectively recorded for these patients.

The control group consisted of 20 patients for whom data was retrospectively assessed. CSF samples were drawn at least three times a week and routine bacterial cultures and CSF analyses were done according to standard protocols. The primary endpoint of the study was the occurrence of catheter associated ventriculitis (CAV) proven by positive CSF culture. Secondary endpoints were bacterial colonization of the catheter tip and CSF pleocytosis.

In 20 control patients, 5 CAVs were microbiologically diagnosed. In contrast, no positive CSF cultures were found in the treatment group. This difference was statistically significant (p < 0.05). All CAVs occurred later than day 10 after catheter placement. Colonization of the catheter tip was found in 6 patients in the control group and in 5 patients in the treatment group (not significant).

This pilot study indicates that EVD catheters impregnated with silver nano-particles might be a new option for preventing CAV in neurocritical care patients and therefore evaluation in a large prospective randomized study is warranted.

E. Meyer, F. Schwab, B. Schroeren-Boersch, P. Gastmeier (Freiburg, Berlin, DE) Objective: To determine and to analyse antibiotic prescribing parameters of intensive care units (ICUs) which are not only measures for quantity but also for quality in the sense of picturing diversity of antibiotic use. Methods: In 2005, 43 ICUs participating in SARI (Surveillance of Antibiotic Use and Bacterial Resistance in Intensive Care Units) provided data and were included in the analysis. For antibiotic prescribing indicators we choose the following parameters: -The number of different antibiotics used -The most frequently antibiotic used of each ICU (top1) and the three and ten most frequently antibiotics used (top3, top 10) -% of total antibiotic use of penicillins, of quinolones, of cephalosporins, of carbapenems and of glycopeptides. Antibiotic use was expressed as defined daily doses (DDD) per 1000 patient-days.

The median of total antibiotic use was 1156 DDD/1000 pd and ranged from 450 to 1799 DDD/1000 patient days.

-Total antibiotic use and the percentage of different antibiotic groups used were extremely heterogeneous in single ICUs.

-The median number of different antibiotics used was in 27 (range 19 -37).

-The most frequently single antibiotic used per ICU (top1) accounted for almost 20% (median) of the total antibiotic use. The top 3 antibiotics used made up 40% of total use and the top 10 of 80% (range 62.5 -93.8). Maximum the top 3 single antibiotics used accounted for over 60% of total antibiotic use in an ICU.

-At single ICU level quinolones could make up more than 35%, broad spectrum cephalosporins almost 40% and carbapenems 30% of their total antibiotic use. However, at median penicillins and cephalosporins accounted each for a forth of total use. Conclusion: Prescribing parameters of antibiotic use differ considerably from ICU to ICU. The three most frequently used antibiotics account for 40% of the total antibiotic use. Prescribing parameters provide additional and easy to understand information for the analysis of antibiotic consumption and of the selective pressure applied by those drugs.

E. Meyer, F. Schwab, P. Gastmeier (Freiburg, Berlin, DE) Introduction: The proportions of methicillin-resistant S. aureus (MRSA), vancomycin-resistant E. faecium and extended spectrum ßlactamase (ESBL) producing enterobacteriaceae have increased worldwide in the recent years. We analysed the resistance situation for these pathogens in German ICUs. 

A. Nienhaus, A. Schablon, R. Diel (Hamburg, Düsseldorf, DE) Although health service employees are regularly examined for TB, there are no recent data on the frequency of latent tuberculosis infections (LTBI). However, these data are important when estimating the risk of infection and when providing advice about performing chemoprophylaxis for LTBI. In this context, the results of prophylactic investigations were evaluated for hospital employees.

The results of 440 investigations from 4 hospitals were examined in accordance with the ordinance on biological substances, either regularly or after TB contact. The data were classified by age, gender, BCG vaccination, land of birth and type of work, using a standardised form. The LTBI was determined with the Quantiferon Gold in Tube. Seven persons (1.5%) were excluded, as their tests could not be evaluated. Odds ratios (ORs) were calculated for various risk factors. The prevalence of LTBI in hospital employees was 7.9%. This is age dependent, being 2.4% for subjects aged under 40 and 21% for subjects aged over 50. The LTBI prevalence is greater in immigrants (OR 2.6; 95% CI 1.2-5.7). The LTBI is slightly higher for doctors and nurses than for other hospital employees (9.1% versus 6.0%, p=0.24). The prevalence of LTBI is unaffected by gender, BCG vaccination and the reason for the investigation (regular contact with TB patient vs. environmental investigation). The prevalence of LTBI is particularly low in young employees. This indicates that the risk of infection is low for hospital employees. As no comparable data are available for the general population, no statement can be made about the occupational-specific risk of infection. As the data indicate that the risk of infection is low, chemoprophylaxis after TB infection is reasonable.

BCG instillation -start of a chain reaction of iatrogenic side effects: a case report S. Rieg, K. de With, A. Serr, W.V. Kern, D. Wagner (Freiburg, DE) Introduction: Bacillus Calmette-Guérin (BCG) is a live attenuated strain of Mycobacterium bovis that is used for intravesical therapy of early-stage transitional carcinoma of the bladder. Fever and inflammatory cystitis are frequent side effects, but granulomatous prostatitis, epididymo-orchitis, hepatitis or pneumonitis, and, rarely, disseminated BCG sepsis have also been reported. We here present a patient with BCG-induced M. psoas abscess and infected aneurysm of the right iliacal artery. Case report:

The 74 year old male patient received 14 courses of BCGinstillation (strain Connaught) from 03/05 until 05/06 for recurrent bladder cancer. 3 months later he noted a swollen right leg. MRI showed an abscess of the right psoas muscle and multiple small abscesses in the small pelvis. The psoas abscess was drained for few days, microscopy of the pus revealed acid fast bacilli, PCR identified an MTB-complex organism. A tuberculin skin test was positive and a tuberculous abscess was suspected. Treatment with rifampin, isoniazid, ethambutol and pyrazinamide was started. Culture confirmed BCG infection and pyrazinamide was stopped. One month after treatment initiation the patient developed icterus and severe neuralgia of the right leg. The psoas abscess showed enlargement, compressing the right iliacal vein and extending ventrally of the aortic bifurcation. A second drainage was performed, without growth of BCG or detection of mycobacterial DNA. One week later, control imaging revealed a rapid progressive new aneurysm (3-4 cm) of the right iliacal artery with surrounding inflammation. Shortly before surgery, the patient developed severe back pain without evidence of rupture. The aneurysm was resected and a femoro-femoral cross-over bypass was necessary. BCG-DNA was present in the resected aneurysm. During surgery the right femoral nerve was damaged and the peroneal nerve was compressed. The patient is currently treated for 12 months, actually with isoniazid and ethambutol with slow regression of the abscesses. His neural deficits did not improve in spite of intensive physiotherapy.

Conclusions: Hematogenous dissemination of BCG may lead to abscesses and arterial aneurysms and should be suspected in any patient with history of BCG-instillation. Interferon-gamma release assays rather than tuberculin skin test may be helpful in the differential diagnosis of abscesses with acid-fast bacilli.

Unusual presentation of multibacillary leprosy in an immigrant from the Philippines A. Stich, A. Müller, U. Ziegler, C. Osiander (Wuerzburg, DE) Introduction: Although leprosy is the target of extensive control programmes in many countries, new cases still continue to be registered in considerable numbers. Leprosy has to be considered as a potential differential diagnosis of chronic skin lesions in immigrants in Europe. We discuss the case of a 26 year old patient originating from the Philippines. She developed extensive ulcerative skin lesions on the face, trunk and extremities shortly after her arrival in Germany. However, it took more than five months after the first contact with the German medical system, until the diagnosis was suspected for the first time.

We diagnosed multibacillary leprosy based on clinical findings and pathohistological results. Treatment was started according to WHO guidelines with a combination of Rifampicin, Dapsone and Clofazimine over a total period of 18 months. The administration of the drugs was monthly supervised.

The treatment was very well tolerated. No major treatment reactions were observed. The lesions healed with central scarring, permanent neural damage could be avoided.

The clinical presentation of leprosy is virtually unknown to many clinicians in industrialised countries. Patients with leprosy

remain often undiagnosed for lengthy periods of time. We discuss the major clues in the differential diagnosis of leprosy and the management of cases including the methods how the anti-leprosy drugs can be obtained.

Negative tuberculosis-specific interferon-gammarelease assay in a patient with pulmonary Mycobacterium kansasii infection despite ESAT-6 and CFP10-geneexpression in vitro M. Greiten, M. Vavra, W.V. Kern, D. Wagner (Freiburg, DE) Introduction: Mycobacterium kansasii (MKAN) causes pulmonary disease in immunocompetent patients clinically indistinguishable from pulmonary tuberculosis (TB). The genomes of both pathogens encode genes of the RD-1-region, esxA (encoding ESAT-6) and esxB (encoding CFP-10). It is therefore assumed that patients with MKAN-infection show cross-reactivity in TB-specific interferon-gamma release assays (TIGRA 

The 55 year old man had a history of hodgkin-lymphoma treated with splenectomy and radiochemotherapy. Consecutively he developed brachial plexus neuropathy, osteonecrosis of the right femoral head (treated with hip replacement) and persistent CD4-lymphocytopenia. Stage II pulmonary sarcoidosis was diagnosed, corticoid therapy resulted in clinical improvement. Fever recurred after 6 months, and azathioprin (AZA) was added for suspected persisting sarcoidosis. 2 months later, microscopy of BAL revealed few acid fast bacilli (no growth on Löwenstein Jensen-slants, negative PCR for mycobacteria belonging to MTB-complex). Granulomatous myelitis was interpreted as sarcoid infiltration, and AZA was substituted by leflunomid. The fever persisted another 3 months, the patient developed weight loss and severe pain affecting his replaced hip. An infected hip was diagnosed and a Girdlestone arthroplasty performed. Pus showed countless acid-fast bacteria identified as MGEN by 16-S-rDNA-PCR. MGEN was also detected in urine (PCR), duodenum and pancreas (histologically). Therapy with clarithromycin , rifampin and ethambutol was started. The clinical course was complicated over the next 6 months, when MRI revealed disseminated osteomyelitis of both humeri, ribs, pelvis, and vertebrae. Empyema around the girdlestone hip persisted, and an intramedullary abscess in the left femur was found. Pus from both sides grew MGEN. The empyema was treated surgically, and interferon-gamma 1b (IFN) was added. IFN-therapy was stopped after 6 months, when the patients condition deteriorated and mycobacteria again were found in the duodenum. Conventional therapy was changed to clarithromycin and moxifloxacin resulting in marked improvement of his clinical condition and anemia 4 months thereafter.

We describe the first foreign body infection and disseminated osteomyelitis due to MGEN. Infection and dissemination was likely related to persistent CD4-lymphocytopenia and the additional immunosuppressive therapy. Conventional therapy as well as addition of IFN was poorly effective, addition of moxifloxacin appeared to alter the clinical course and was associated with major improvement.

Leprosy (Hansen's Disease) as an imported disease in a German traveller I. Liebold, T. Weinke, W. Güthoff, M. Kuppinger (Potsdam, DE) Question:Which disease could be found in a patient with nodular skin lesions in both forearmes and lower legs ever since 6 months? Methods: In this case report a 40-year-old Caucasian male patient realized since about 6 months subcutaneous nodules on both forearms and lower legs and a decreasing fitness. Since one year he complained of paresthesia and pain on points of contact. Besides that he noticed a sore throat, an unproductive cough, and a loss of appetite. 

were supplied by the Leprosy Elimination Group of WHO free of charge.

A lepromatous reaction could not be observed, and treatment was tolerated well. In the follow-up examinations the patient reported an improvement of his general physical condition. Control smears of his nasopharynx and throat were negative after one year of treatment.

Overall the patient will be treated for 2 years. Conclusion: Leprosy has a long incubation period of 2 up to 40 years. It must be considered in the differential diagnosis of travellers with polyneuropathy or cutaneous lesions to avoid a dissemination in the individual patient and to avoid further spreading to the community.

Extensively drug resistant tuberculosis (XDR TB) in a high income country: A Report of four unrelated cases S. Blaas, R. Mütterlein, J. Weig, A. Neher, B. Salzberger, N. Lehn, L. Naumann (Donaustauf, Parsberg, Neustadt/Waldnaab, Munich-Gauting, Regensburg, Oberschleißheim, DE) Introduction: Multi drug resistance (MDR) of M. tuberculosis is a major threat to public health. This has been reinforced by recent reports about patients infected by XDR strains in South Africa. There is very little information about the clinical course of XDR TB patients in industrialised countries.

We evaluated all isolates of M. tuberculosis, in which drug susceptibility testing was performed at our institution since 1997, for multi and extensive drug resistance. Clinical courses of patients infected by strains fulfilling the recently revised criteria for XDR tuberculosis were analysed. Results: Four XDR M. tuberculosis isolates were identified during the last ten years. All patients had been immigrated from eastern Europe and Russia and none was HIV infected. All patients where treated for TB for several years and three received 4.5 to 6 years of inhospital treatment in Germany. Non-compliance was an important factor in all four patients, in three patients so severe, that they had to be treated in Germanys only locked facility for TB treatment. One patient with XDR TB died, one patient had still open pulmonary tuberculosis at last contact and 2 patients are considered to be cured. Discussion: Cases of XDR TB have been treated in our region for several years and fortunately it has not gained such a tremendous impact as suggested by the data from the recent report from HIV positive patients from South Africa. But even in our high income setting XDR TB has a tremendous impact on quality of live, outcome and the use of medical resources. Therefore, all reasonable efforts to prevent the spread of XDR TB must be made and maintained.

Concomitant tuberculosis in an African patient with sarcoidosis U. Ehehalt, S. Schmiedel, G.D. Buchard (Hamburg, DE) A 26-year old man, originating from Dschibuti, was admitted to another hospital because of headache and paresis of the left leg, additionally he suffered from relapsing bilateral uveitis. Former history was uneventful, but in the family history the mother of the patient suffered from sarcoidosis. The condition of the patient quickly deteriorated, with paresis of both legs and both arms, and alteration of the mental status. On MRI white-matter lesions were seen and a brain biopsy was performed showing a granulomatous inflammation. ACE was elevated. A diagnosis of sarcoidosis was made and treatment with corticosteroids initiated. However, the condition of the patient deteriorated further, he got respiratory problems and was tranferred to our hospital. On admission the patient was comatose, had signs of ARDS and had to be intubated. A chest CT scan showed small nodules disseminated throughout the lungs, in adition subcarinal lymphadenopathy and fibrotic alterations in the lower lobes. A PCR for M. tuberculosis from BAL was positive. Steroids were stopped and anti-tuberculous treatment was begun. The neurologic symptoms did not improve and a new MRT showed hemorrhagic arachnoidal signal alterations compatible with sarcoidosis. The patient was again treated with steroides in addition to TB treatment. Then the symptoms slowly got better, the patient could get extubated, the paresis gradually improved so that he finally could leave our hospital to start a rehabilitation. This patient obviously had concomitant neurosarcoidosis and tuberculosis. Sarcoidosis -which is prevalent in Africans -was suspected by typical neuroradiological findings and elevated ACE and proven by histopathological diagnosis in the stereotactic biopsy. Tuberculosis was suspected by CT findings of the lungs and a positive Quantiferontest and proven by detection of M. tuberculosis by PCR. Sarcoidosis occasionally develops in patients previously treated for tuberculosis. Less commonly, tuberculosis develops as an opportunistic infection in patients following corticosteroid treatment for sarcoidosis -as in our patient. Diagnosis can be very difficult because of the overlapping clinical and laboratory findings.

VAN SWIETEN -First nationwide pandemic exercise in Austria

Background: To optimise Austria's pandemic preparedness a national exercise was designed. The aim of the exercise was the evaluation of -communication between the MoH, other ministries and regional health boards -preparedness plans on national and regional level (pharmaceutical/ non-pharmaceutical interventions, command-control structures) -interoperability of plans Methods: VAN SWIETEN was a 2-days lasting command-exercise involving 193 persons on different levels of the health authorities including veterinarians, reference centres, other ministries and local hospitals. Exercises centre was at MoH. The pandemic scenario comprised three topics: preparation of measures, implementation of measures and vaccination. Players received 24 events, the results were provided -were adequate -as GIS maps by the exercise control. A player handbook, contact details of players and national/regional pandemic preparedness plans were provided. Communication tools were E-mail, telephone, fax and audio conferences. Media were not involved. Results: Several topics for further discussion were identified: closure of airports, medical care for workers from EU-countries versus "health care shopping", vaccination concept, surveillance in a pandemic, planning presumptions and business continuity. Conclusions: Successful exercises need a careful planning process and critical evaluation. Aim and type of the exercise have to be defined, scenarios have to be realistic and relevant for players and clear evaluation criteria have to be set up. After the exercise, a concrete working program including a road map with allocation of tasks to working groups has to be developed, the results have to be implemented into the crisis plan and follow-up exercises have to be performed to evaluate the improvements. Follow-up exercises must be spaced out accordingly containing only few repetition elements, otherwise compliance of players will decrease with negative consequences on the quality of the performance.

Abstracts KIT2008

Chikungunya Fever in Travellers -clinical presentation and course W. Taubitz, C. Walentiny, T. Löscher, G. Burchard, A. Kapaun, J. Kramer (Munich, Hamburg, Heidelberg, DE) Background: Chikungunya virus (CHIKV) infections emerged in western Indian Ocean islands in 2005, spread out to India causing an ongoing outbreak that involves over 1.5 million patients, including travellers who visited these areas. The study aimed to analyse the main symptoms, their duration and the best diagnostic methods.

The study included 69 travellers who returned home from epidemic countries and showed signs and symptoms compatible with a CHIKV infection (fever and/or arthralgia). 20 cases with confirmed infection by serology, PCR or culture were analysed. Results: All patients showed flu-like symptoms with fever and joint pain. 68.8% of the patients had arthralgia for more than two months, 12.5% for more than six months. No serious complications were observed. Slight positive Chikungunya IgM-antibodies were detected after three days of symptoms. PCR-detection of the virus was successful in four patients, a virus detection in culture was possible in two patients.

Conclusions: Due to the ongoing epidemic, the role of Chikungunya fever as differential diagnosis for febrile diseases is rising. Before travelling to Reunion, Mauritius, Seychelles or India travellers should be informed of the danger of a CHIKV infection and about prophylactic actions like mosquito protection. Serology seems to be the best diagnostic method after at least three days of symptoms.

Prevalence of rickettsiae in ticks in southern Germany R. Wölfel, S. Essbauer, M. Pfeffer, R. Terzioglu, A. Thomas, G. Dobler (Munich, DE) Introduction: Rickettsioses are emerging and re-emerging diseases. They occur worldwide. While data on rickettsial organisms in tropical and subtropical countries are accumulating, the occurrence of rickettsiae and their epidemiology in Central Europe is still far from known. The circulation of rickettsia species of the spotted fever group transmitted by ticks in Germany has not been studied in detail, nor has the prevalence rates of rickettsiae in ticks.

In order to elucidate the distribution of rickettsial species in Germany more than 3.000 ticks of several regions in Southern Germany were screened for rickettsial organisms by amplification of the part of the gltA gene by polymerase chain reaction (PCR). Positive ticks were further tested using different PCRs and nested PCRs to determine the species by sequencing the ompA, ompB and 16S RNA genes.

The minimal infection rates of ticks ranged from 5.2% to up to 14% depending on the respective tick stages. Tick larvae exhibited low rates of infection while adult ticks were PCR positive at significant higher rates. Identification of rickettsial species by sequencing resulted in the identification of Rickettsia (R.) helvetica, R. felis, R. monacensis resp. IRS4/3, and R. massiliae similar strain. The detailed study of one location showed that rickettsiae were not evenly distributed within one natural focus but that hotspots with high infection rates in ticks seem to exist within the focus.

In conclusion, our investigations demonstrated that at least 4 different rickettsial species are circulating in different areas of Southern Germany. Two of them (R. felis, R. massiliae) are proven human pathogens. One rickettsial species (R. monacensis) is closely related to other spotted fever rickettsiae and is therefore assumed to be pathogenic while the human pathogenicity of R .helvetica warrants further clarification. In the areas tested unclear feverish infections with lymphadenitis or exanthema should be tested also for rickettsial diseases.

Cerebral Venous Thrombosis Complicating Meningitis due to the Tick Born Encephalitis-Virus G Rieder, C. Blättner, M. Viermetz (Traunstein, DE) Tick born encephalitis (TBE) typically results in a 2-peak illness with flu-like symptoms, followed by lymphocytic meningitis. In a minority encephalitis may develop with cognitive or focal deficits and poor prognosis. Typical Magnetic Resonance Imaging (MRI) abnormalities have been described in severe TBE, but the value of MRI has been challenged due to the lack of specific treatment. A 40-year old, otherwise healthy carpenter was admitted to a hospital on 16-07-07 with a 5 day history of fever, joint pain, headache, nausea and diarrhoea, but no neurological deficit. A blood film showed mild leucocytopenia (2200/mcl), the cerebrospinal fluid (CSF) an elevated protein ( MRI revealed focal brain oedema in the left fronto-parasagittal cortex, a thrombotic occlusion of the superior sagittal, the right and (subtotal) of the left transverse sinus, but no abnormalities suggestive of TBE or Herpes encephalitis. A presumptive diagnosis of partially treated bacterial meningitis with cerebral venous thrombosis (CVT) was made, but antibiotics were stopped when serology and culture results were available. Treatment of CVT was immediately with i.v. heparin followed by phenprocoumon. 10 days later, complete resolution of the neurological deficits, normalisation of the EEG and recanalisation of the CVT without residual parenchymal lesion was demonstrated. CSF follow up was not feasible due to anticoagulation. We conclude that our patient had viral meningitis only, complicated by CVT, a complication not yet described in TBE. While there is no causal treatment for TBE, this case stresses the value of early MRI in meningoencephalitis in order to screen for complications with therapeutic consequences.

Severe mycoplasma pneumonia with thromboembolic complications and hemolytic anemia I. Liebold, T. Weinke, W. Güthoff (Potsdam, DE) Methods: We describe the case of a young adult patient with a severe course of a mycoplasma pneumonia including complications of thromboembolism and haemolytic anemia.

A 24 year-old male patient was hospitalized with community acquired pneumonia. Chest x-ray showed a diffuse infiltrate of the right lower lobe, but nearly normal signs of auscultation. Initial out-patient treatment consisted of clarithromycin. He was hospitalized severely ill, so that calculated antibiotic treatment was extended to i.v. ceftriaxon + clarithromycin. Serology confirmed a mycoplasma infection (HAT 1:10000, IgM assay 79,0). On initial presentation the patient displayed an erythema multiforme which we interpreted as an extrapulmonary manifestation of his disease. After 2 weeks the clinical condition deteriorated with pleuritis like symptoms and dyspnea. CT scan showed the typical finding of an embolus of the pulmonary arteries. Other thrombophilic factors for diathesis could be excluded. Treatment was started with heparin, followed by oral anticoagulants. Follow-up blood results showed a decreased level of haemoglobin due to hemolysis. Hemolysis could be 

Involvement of methicillin-sensitive PVL-producing staphylococcus aureus in a severe necrotising pneumonia of a healthy woman after incision of a bartholin's abscess N. Jung, C. Lehmann, M. Hellmann, H. Seifert, G. Fätkenheuer, M. Kochanek (Cologne, DE) Background: Panton-Valentine leukocidin (PVL)-producing Staphylococcus aureus is emerging as a serious problem worldwide. There has been an increase in the incidence of necrotizing lung infections in otherwise healthy young people associated with a very high mortality. Case Report: We present a 23-year-old apparently healthy female patient without any typical predisposing findings who developed severe sepsis with necrotizing pneumonia and multiple abscesses secondary to the incision of a bartholin´s abscess. Methicillin-sensitive Staphylococcus aureus was isolated from the blood and the smear of the wound, harbouring Panton-Valentine leucocidin genes. Aggressive antibiotic therapy with flucloxacillin, rifampicin and clindamycin lead finally to recovery. Conclusions: PVL-positive Staphylococcus aureus should early been included in the diagnostics especially when young immunocompetent persons develop a fulminant necrotising pneumonia. Diverse minor infections-as bartholin`s abscess -can proceed this life threating syndrome. Aggressive antibiotic treatment is recommended for treatment but no evidence-based guidelines are available so far.

Typhoid fever after ERCP in choledocholithiasis G. Jechart, R. Scheubel, H. Messmann, P. Sommer, M. Anthuber, H. Arnholdt, H. Jähnig, S. Braun, W. Ehret (Augsburg, DE) Aims: We report a 47-year-old men, who suffered from typhoid fever after endoscopic removal of gallstones in the ductus hepatocholedochus; this is an unusual case of secondary invasive infection with Salmonella enterica serotype Typhi after chronic carriage.

The patient presented with acute onset of abdominal cramps in the right upper quadrant and vomiting. He is an imigrant from India and lives in Germany since 11 years. The last visit in India was 4 months ago. The endoscopic examination ( ERCP ) revealed a 12 mm gallstone in the ductus hepatocholedochus, which was removed in several steps of cutting and crumbling. There was a selflimiting bleeding of the Papilla Vateri while opening the ostium. 5 h later in the evening the patient developed fever ( up to 40°C ). He had no more pain, but felt very ill. Blood cultures were taken. Results: There were gram negative isolates growing in two of three blood cultures, which were identified as Salmonella enterica serotype Typhi ( agglutination tests). The CRP level raised from 0,56 mg/dl on admission to 20,19 mg/dl ( norm 0 -0,5 mg/dl ) after 12 h, leucocytes falling from 10,6 to 5,4/nl at the same time. The liver enzymes and bilirubin level were slightly elevated. Intravenous administration of ciprofloxacin ( 2 x 400 mg ) resolved the fever in 3 days and the patient was in good health after 10 days, before he was send to cholecystectomy in the surgical department. We present 6 images of the radiographic findings of choledocholithiasis and chronic cholecystitis, the endoscopic view of the Papilla Vateri and the gall bladder after surgical removal. Conclusion: endoscopic procedures in symptomatic choledocholithiasis are a possible source of systemic infection with Salmonella enterica serotype Typhi ( typhoid fever ) in asymptomatic carriers.

Diagnosis of tertiary syphilis in a patient presenting with atypical skin tumour U. Ziegler, A. Müller, D. Ludin, S. Jandl, A. Stich (Wuerzburg, DE) Introduction: A 44 year old man was referred to the out patient department of our hospital. He had developed a solid, painless skin tumour on his left forearm over a period of ten weeks. A skin biopsy has revealed granulomatous tissue and a dense lymphocytic infiltration of the dermis. The pathologist suspected a cutaneous leishmaniasis (CL) or borreliosis. By taking his travel history we learnt that he frequently visits Thailand, last time 11 months ago. He had never been travelling to areas where CL is endemic. On direct interrogation, he denied sexual risk behaviour, in stead he is living in a stable homosexual partnership since many years. Methods: On clinical examination, we found the patient in good clinical condition. The skin lesion was found as previously described without significant inflammatory reaction. He had no lymphadenopathy, rash or fever. Results: Initial laboratory findings (full blood count, basic blood chemistry) were normal. An HIV test was offered (which was negative) as well as routine screening for syphilis. There, serological testing revealed a positive result for TPHA (1:5120) and VDRL (1:128). Neurosyphilis was ruled out by normal results of the cerebrospinal fluid.

We diagnosed a gummatous lesion as a rare manifestation of syphilis. The patient responded well to a treatment of Penicillin G 10 million units i.v. three times daily for three days, followed by intramuscular injections of Benzathin Penicillin 2.4 M weekly for three weeks. Conclusion: Syphilis can be a chameleon and can cause atypical skin lesions. However, today it is rarely seen. Therefore, the indication for serological testing should be applied generously.

C. Maurer, K. de With, S. Rieg, A. Serr, C. Schneider, M. Braun-Falco, K. Tintelnot, W.V. Kern, D. Wagner (Freiburg, Berlin, DE) Introduction: Histoplasmosis is endemic in the Americas and parts of Asia and Africa. The infection can be imported into non-endemic countries and present as acute or reactivating disease since the organism may persist intracellularly. We here present three cases diagnosed in the last three years. 

U/l) and ferritin (50,000 ng/ml), moderatedly increased ALT levels and Coombs-positive hemolytic anemia. Liver biopsy led to complications, pulmonary infiltrations developed thought to represent nosocomial pneumonia. Finally the patient died. Disseminated histoplasmosis was diagnosed post mortem by culture from blood and bone-marrow-biopsy. 3. A 29-year old HIV-neg. female native from Brazil presented with right-sided cervical lymphadenopathy, a paranasal skin-ulcer, left upper lobe infiltrates and adjacent hilar lymphadenopathy. Histology of transbronchial biopsy and of a right paranasal skin-ulcer revealed presence of PAS and Grocott positive small yeast cells suggestive for histoplasmosis. Diagnosis was confirmed by PCR and serology. Therapy with itraconazol led to clinical and radiological improvement and significant reduction of the CFT-antibody titre. The patient is under treatment for 4 months and well on last follow-up.

The clinical manifestation of histoplasmosis can vary from localized to disseminated disease. Histoplasmosis must be considered as a differential diagnosis not only in patients with underlying immunosuppression, but also with only history of residence or travel to endemic countries.

Bat-associated Histoplasmosis can be transmitted at entrance of bat caves and not only inside the caves M. Mueller, B. Jülg, J. Elias, A. Zahn, J. Bogner (Munich, Würzburg, DE) Several reports showed occasional and occupational instances of histoplasmosis contracted while bat-caves were visited by tourists or researchers or bat researchers. Up to now humid air contaminated with H. capsulatum spores was regarded a major epidemiological risk factor for new cases of histoplasmosis in travellers. We report about three bat researchers who acquired histoplasmosis. All three members of a scientific project did not enter bat-caves yet contracted acute pulmonary histoplasmosis. The only contact to bats happened in the close proximity of a bat cave where bats were observed while they left the cave in the evening. Because bat-caves sometimes are advertised as tourist attraction and because knowledge about transmission of human histoplasmosis seems incomplete we wish to communicate and stress the fact that even outside of caves bats may disperse enough organisms to transmit considerable numbers of infecting units.

Pneumatosis intestinalis? -An unusual course of an acute abdomen in a female ethiopian patient G. Birkenfeld (Regensburg, DE) A 32year old women from Addis Abeba was admitted to the department of gastroenterology with abdominal cramps in the epigastric region and lower abdomen. She reported severe consti-pation, nausea and vomiting during the last week along with a permanent dry cough. On examination she presented in a poor condition, pale, short of breath and with an enormous swollen abdomen, tender and painful on palpation with random,tinkle bowel sounds, RR 100/60 mmHg, pulse 126/min, temp. 38,2°, exsiccated and exhausted. Emergency laboratory results: sodium 126, potassium 3,0 mmol/l,leucocytes normal, eosinophiles 5 %, mild anemia, CHE 336 (N > 3000), normal renal function, 02-sat. 90%. Pregnancy test negative. TPHA, HIV-, hepatitis C, B and schistosomiasisserology were sent for labtests. X-rays: abdominal: extremely dilated small intestine and colon loops, a paralytic ileus was diagnosed. Chest:"Interstitial diffuse pneumonia"

Abdominal ultrasound: chain-like air reflexes within the wall of the gall bladder and along the 2 cm thickened, dilated gut walls. CT scan: "pneumatosis intestinalis". Clinical course: 24 hours after admission the patient had to be transferred to the intensive care unit due to progressive hypotension, oligury, respiratory distress and increasing abdominal pain. After intubation bronchoscopy revealed mucous membranes in all airways with diffuse slight bleeding, a comparable endoscopic picture plus many ulcers was seen in the upper gastro-intestinal tract. Biopsies were taken for microbiology and parasitolgy. The patient's condition detoriated rapidly. After a few hours the results from the depart-ment of parasitology returned, almost simultan-eously with the virological tests: burden of strongyloides stercoralis had been detected in all mucous membrane samples, PcP was negative, no acid fast bacilli were seen. HIV I antibodies were positive, -viral load 740.000 copies/ml(PCR), CD4 cell count 4/ul(8%), hepatitis B antibodies positive, anti HCV, TPHA and schistosoma antibodies were negative. A simultaneuosly started therapy via gastral tube with albendazol 400 mg bid plus HAART led within three days to a surprisingly complete recovery of all symptoms. Strongyloides stercoralis, a nematode parasitosis, can lead in immunocompromised patients to a hyperinfection syndrome with a mortality rate up to 87 %. It should be considered in patients from hyperendemic areas who present with unexplained abdominal symptoms or signs of "pneumatosis intestinalis".

Successful treatment of cutaneous leishmaniasis in a 4 year old child with Miltefosin A. Stich, A. Mueller, D. Tappe, U. Ziegler, C. Kohlhauser-Vollmuth (Wuerzburg, DE) Introduction: Leishmaniasis is a constant health problem in Mediterranean countries. We report of a 4 year old German girl, which was infected with Leishmania infantum during a stay on the island of Mallorca at the age of 18 months. She developed a large cutaneous lesion between the eyes with extensive ulceration and swelling. Methods: After the diagnosis had been confirmed by microscopy and PCR specification, the extent of the lesion forced a rapid therapeutic decision. We administered Miltefosin at a dose of 2,5 mg/kg b.w. over a period of 28 days.

The treatment was very well tolerated. Within only a few days, the healing process commenced. After the end of the treatment period, the ulcer was closed. Six months afterwards, the lesion was totally healed leaving an excellent cosmetic result. Conclusion: To our knowledge this is the first documented case of a successful administration of Miltefosin in the paediatric treatment of cutaneous leishmaniasis (CL). Our experience supports a leading role for Miltefosin in the treatment of CL even in children.

Successful treatment of disseminated cutaneous Leishmaniasis caused by L. (Viannia) braziliensis complex with Miltefosine M. Fischer, A. Nühs, K. Kuhls, J. Clos, R. Hagen, G. Burchard (Hamburg, Berlin, DE) Miltefosine is the first oral drug with proven efficacy rates of over 90% for Old World visceral leishmaniasis and also shows promising results in the treatment of New World cutaneous Leishmaniasis in patients from L. panamensis-endemic areas.

We report here on a 40 years old male patient, who was suffering from multiple mosquito bites with initial bacterial superinfection during a three week holiday on the Carribean coast near Puerto Viejo in Costa Rica. After a further two months, 14 deeply ulcerated purulent cuta-

neous lesions up to 4 cm in diameter developed simultaneously on his trunk. These were initially diagnosed as ecthymata but did not respond to any antiseptic and antibiotic treatment. Three months later leishmaniasis was finally diagnosed. Leishmania were found in various skin smears and were also detected histopathologically in a skin biopsy (Giemsa staining). Culture was positive for Leishmania (Medium 199, Sigma) and species specific PCR from the skin (specimen taken by punch-biopy) identified Leishmania braziliensis complex, with a 90% probability for L. guyanensis sensu strictu.

Due to the extent of the 14 lesions the patient was systemically treated with oral miltefosine (Impavido ® ) 3 x 50 mg capsules/day for 28 days. Screening laboratory tests (blood count, liver enzymes and urea) were monitored in blood but remained at normal levels. After 21 days of treatment, tissue repair was observed followed by rapid reepithelisation and complete healing within 10 days. No relapse was detected during a follow up of 5 months. Miltefosine (hexadecylphoshocholine), initially developed as an anticancer agent, was found to eliminate Leishmania promastigote forms in culture in the late 1980s. Oral administration was overwhelmingly efficacious in various clinical studies for the treatment of visceral Leishmaniasis in India. It was also approved for the treatment of cutaneous leishmaniasis in Germany in 2005. However, the sensitivity to miltefosine was variable among different species causing cutaneous leishmaniasis. Presently, the efficacy of miltefosine is studied in various clinical trials in all endemic areas of CL. This clinical case shows that even in complicated leishmaniasis with multiple lesions caused by Leishmania (Viannia) braziliensis complex, Miltefosine was highly efficient, led to a rapid healing and showed no side effects during or after treatment.

Cutaneous leishmaniasis in a German tourist after travel to Greece N. Berens-Riha, E. Fleischmann, G. Bretzel, F. von Sonnenburg, T. Löscher (Munich, DE) A 66 year old German male noticed multiple nodular and papular skin lesions at the end of a 3 week hiking holiday in Southern Greece, peninsula of Mani, Peloponnese. The painless and reddened infiltrations were partly covered with squamous or scaly scabs and extended at both extensor sides of the forearms and the right lateral thigh. Because of further spread and partial ulceration of the lesions the patient presented three months later at our clinic. General physical examination and routine laboratory tests were without pathological findings. The diagnosis of cutaneous leishmaniasis (CL) was made by cytological and histological investigation of multiple biopsies of lesions showing numerous intra-and extracellular Leishmania amastigotes. Differentiation by PCR/RFLP revealed Leishmania tropica. The strain could be isolated in culture and zymodeme sub-typing is underway. Immunofluorescence using L. tropica promastigotes as antigen showed low titres of antibodies (1:32) in the patient's serum. The patient declined systemic therapy with antimony drugs or miltefosine due to possible side effects and a self-limiting course in most cases. Topical treatment with an ointment containing 15% paromomycin and 10% urea applied by a cellophane occlusive technique led to a regression of all lesions during a period of 10 weeks. Leishmania parasites are transmitted by female phlebotomine sandflies. Most skin lesions caused by L. tropica are singular and evolve from a nodular to an ulcerative stage. However, multiple primary lesions as in this case, satellite lesions, lymphatic and sporotrichoid subcutaneous spread may also occur. Lesions usually heal spontaneously within 6-24 months, but chronic or relapsing CL (recidivans leishmaniasis) may evolve in some patients. To our knowledge, no cases of CL in traveller to Greece have been published so far. CL in travellers to European Mediterranean countries has been reported mostly from Italy and Spain where dermatotropic strains of L. infantum are the predominant agent. However, both visceral and cutaneous leishmaniasis are endemic in Greece and most strains isolated from Greek CL patients were L. tropica. CL is not notifiable in Greece and there are no data on the current distribution and incidence of CL which may allow for an assessment of the risk for travellers.

24-nor-ursodeoxycholic acid as novel therapeutic strategy for inflammation-induced liver fibrosis in a murine model of Schistosoma mansoni infection M. Sombetzki (Rostock, DE) Introduction: Schistosoma mansoni infection is characterized by inflammation-induced granulomatous liver fibrosis and thus the leading cause of liver fibrosis and portal hypertension worldwide. Current therapy is ineffective once hepatic fibrosis is established. The aim of our study was to test 24-nor-ursodeoxycholic acid (norUDCA) as a novel therapeutic strategy for liver fibrosis since it is known to have great anti-inflammatory and/or anti-fibrotic effects in a mouse model of biliary fibrosis. Methods: Female NMRI mice were infected with fifty S. mansoni cercariae percutaneously. The time course of hepatic fibrosis was studied at different time points (4, 8, 12, 24 weeks post infectionem, wpi; n=5 per group). To test therapeutic effects of bile acids (BA), animals received a 0.5% BA-diet 12 wpi (norUDCA and ursodeoxycholic acid, UDCA; n=5 per group) and were harvested 16 wpi. Effects on serum liver tests, collagen deposition, expression of fibrosis-related genes and cytokines (biochemistry, western blotting, TaqMan, SyberGreen) were compared to uninfected and untreated controls. Liver histology was assessed by hematoxylin/eosin-, Sirius red-and immunohistochemical stainings for inflammatory markers (CD4, CD8, CD11b). Results: During infection hydroxyproline (HP) levels were elevated (max. 7-fold, 24 wpi), although liver function remained unaffected at examined time points. Matrix metalloproteinase-2 (MMP-2) mRNA expression was elevated and peaked around 24 wpi (11.8-fold). Tissue inhibitor of matrixmetalloproteinase-1 (TIMP-1) mRNA followed MMP-2 expression to a much higher extent (48-fold). Bile duct proliferation became evident 24 wpi. NorUDCA-administration significantly reduced HP-content and granuloma size (33%) versus untreated controls, whereas UDCA had no effects. NorUDCA did not affect cell-composition of granulomas, but reduced alpha-smooth muscle actin expression. No effects on mRNA expression levels of transforming growth factor-beta (TGF-B) or tumor necrosis factor-alpha (TNF-A) were detectable. Conclusions: This study demonstrates (i) a dysbalance between MMP-2 and TIMP-1 induction as key players in progressive liver fibrosis due to schistosomiasis and (ii) norUDCA -administration reduces inflammation-induced liver fibrosis on non-cholestatic conditions without affecting cellular composition of granulomas. The clarification of direct anti-inflammatory and anti-fibrotic effects of norUDCA will be the subject of further investigations.

Severe Plasmodium falciparum malaria with peripheral gangrene -a case report R. Helbok, P. Lackner, R. Beer, G. Broessner, C. Brenneis, B. Pfausler, E. Schmutzhard (Innsbruck, AT) Objective: To describe the intensive care management of a case with severe Plasmodium falciparum malaria. Design: Case report and review of the literature. Setting: Neurological intensive care unit, University of Innsbruck, Innsbruck, Austria. Patient: A 61-year-old man was found non-responsive in his home two days after his return from long-term stay in Uganda. Blood smear revealed Plasmodium falciparum infection and intravenous quinine was initiated. The patient developed multi-organ failure, presenting as cerebral malaria, acute renal failure, respiratory failure, dissemi-

nated intravascular coagulation with thrombocytopenia, metabolic acidosis and hyperlactatemia. On day 3 acral discolorations of his toes (Panel A and B) were observed. Failure of macrohemodynamic parameters required aggressive fluid-resuscitation, hydrocortisone and catecholamines. After a 7-day course of quinine the patient could be extubated and fully recovered from malarial disease. The tips of all toes remained gangrenous requiring necrectomy and further reconstruction therapy. Conclusion: Peripheral gangrene is a rare complication of severe Plasmodium falciparum malaria secondary to disturbance of the microcirculation and disseminated intravascular coagulation. Patients requiring high doses of catecholamines and possibly those with peripheral arterial disease might be at particular risk.

An Austrian wide Salmonella Enteritidis phage type 1 outbreak in 2007 P. Much, J. Pichler, C. Lehner, H. Wildt, C. Kornschober, F. Allerberger (Vienna, Mattersburg, AT) Introduction: In Austria in 2006, Salmonella caused at least 390 food borne outbreaks. In the majority of outbreaks proof of the infectious food vehicle cannot be obtained, because the outbreak strain cannot be detected in the incriminated food. Frequently the infectious vehicle can be elucidated employing epidemiological studies and the source of infection can be confirmed by microbiological testing of environmental samples, e.g. fecal samples from incriminated flocks of laying hens. We report on an Austrian wide Salmonella Enteritidis phage type 1 outbreak in 2007 Methods: Descriptive epidemiology was done by local health authorities in cooperation with AGES. Salmonella serotyping was done according to the Kauffmann-White method, phage typing according to the Colindale protocol. Results: A Salmonella Enteritidis (SE) phage type 1 PT1)outbreak affecting 10 persons after consuming Schomlauer Nockerl at a heuriger was confirmed by the local authorities in July 2007. The eggs used for the preparation of this food item were bought at the local egg producing plant M, consisting of 20,000 hens. The laying hen flocks, vaccinated against SE (live vaccine 3 times administered via water) were tested for salmonella and in 18 of 20 pooled faeces samples SE PT1 was detected. Because the eggs of producer M were also distributed to the Austrian wide acting supermarket chain B, a questionnaire was sent via local authorities to all bacteriological confirmed PT1 cases in Austria in 2007 (n=73) to ascertain who could be linked to this outbreak. By defining eggs as the source of infection and the supermarket B as involved egg supplier, 10 additionally cases could be classified as outbreak cases. As required by the National Poultry Hygiene Order (Geflügelhygieneverordnung 2007) more than 1,300 eggs of egg producer M were tested by culture; salmonella could not be detected. Because of this negative result, the eggs were furthermore distributed as class A eggs (i.e. consumable eggs).

Epidemiological results revealing links of salmonella outbreaks to egg production plants and the detection of outbreak strains in faecal or dust samples of incriminated laying hen flocks indicating the reservoir of salmonella are often underestimated and do not lead to proper action by health authorities. As a consequence of the described outbreak, which occurred before Novemebr 1, 2007, a concept for sanitation of the holding concerning structural and hygiene measures had to be implemented. As of November 1st, 2007, European legislation mandates that when, as a result of epidemiological investigation of food borne outbreaks, Salmonella ssp. is identified in a flock of laying hens as the source of infection for humans, the food business operator is no longer allowed to market his products as class A.

Outbreak of Salmonella Typhimurium PT 46 in Austria, 2006 D. Schmid, C. Kornschober, A.-M. Pichler, C. Alfery, R. Fretz, M. Mann, M. Wassermann-Neuhold, A. Luckner-Hornischer, E. Gschiel, F. Allerberger (Vienna, AT) Introduction: In 1993, Salmonella Typhimurium phage type (PT) 46 was confirmed in Austria for the first time with a total of 291 cases of enteritis confirmed by culture. During the following 10 years, from 1994 to 2003, S. Typhimurium PT 46 occurred sporadically with an average of 31 cases per year. In the second half of 2004, 218 cases occurred in Austria; in 2005, 39 cases were confirmed by culture. We report on an outbreak investigation covering the cases of 2006. Methods: Data on the microbiologically confirmed cases of 2006 were provided by the national reference center for salmonella. The evaluation of outbreaks was done in collaboration with local public health authorities.

Results: In 2006, 205 cases of S. Typhimurium PT 46 enteritis occurred from May to July. Of these, 177 were confirmed by culture and 28 were epidemiologically probable cases (no lab confirmation). In the first quarter of 2006 there were no culturally confirmed cases. The surge of cases from May to July 2006 was caused primarily by 10 small outbreaks: 8 cases in Burgenland; in Styria 11 cases after a birthday celebration, 13 cases in a nursing home, two family outbreaks of 3 cases each, 23 cases after a birthday party and 3 cases after consuming food from a delivery service; 8 cases at a workshop for the handicapped in Tyrol; a family outbreak of 3 cases in Carinthia; 4 cases related to a birthday in Lower Austria. Tyrol experienced 7 cases associated with a barbecue in August and 5 cases related to cakes in a bakery in September. Altogether, 86 of the 290 cases could be assigned to the above-mentioned outbreaks. No common denominator could be identified for the remaining 204 cases. Eggs for direct human consumption from two Styrian commercial flocks of laying hens (including flock M -already suspected epidemiologically as a reservoir for an outbreak in 2004) could be identified as the highly probable source of infection or as carrier of the infectious agent in at least 37 of the 86 cases. S. Typhimurium PT 46 was identified in fecal samples from the hens and in dust samples of both flocks.

In 1993, no targeted epidemiological investigations were performed despite more than 200 cases of disease being confirmed by culture. This was due to unclear legal requirements concerning targeted procedures when suspecting food-borne disease outbreaks. A decrease in the number of disease cases should never be used to justify foregoing the search for the reservoir of infectious agents. There is the hypothesis -based on the epidemiological and microbiological test results -that at least 24 of the 86 outbreaks cases in 2006 were causally related to the commercial flock M. In principle, these cases could therefore have been avoided. Since November 2007, pursuant to a European Union directive, it is prohibited to place eggs for direct human consumption on the market when there is epidemiological evidence of a causal relationship with salmonelloses.

Amplified fragment length polymorphism of diverse Salmonella enterica serovars for serotype differentiation and identification of serotype specific genetic markers A. Pietzka, A. Stöger, C. Kornschober, J. Zeinzinger, W. Ruppitsch, F. Allerberger (Vienna, AT) Introduction: Salmonellosis is a major cause of bacterial enteric illness in both humans and animals. In 2006, 5379 cases of salmonellosis were confirmed by culture in Austria. Effective epidemiological surveillance requires the accurate subtyping of strains. We analyzed a strain collection of 82 arbitrarily chosen isolates belonging to the 41 most frequent serotypes in Austria by amplified fragment length was chosen for comparison of the isolates. Analysis of the AFLP gels was performed with the aid of appropriate AFLP analysis software (SAGA-MX, LI-COR). A total of 128 bands were scored for their presence or absence for subsequent phylogenetic analysis. The scored bands were translated into a binary code for generation of a phylogenetic tree using TREECON. Results: The chosen primer combination (E14/M0) resulted in 77 distinct AFLP band patterns, 34 serotype specific AFLP groups, and eight major AFLP clusters for the 82 tested Salmonella isolates. Isolates of the remaining seven serotypes (Cerro, Albany, London, Manhatten, Chester, Anatum, Oranienburg) did not cluster in serotype specific AFLP groups. Conclusions: Although serotyping is a standard method for differentiation of Salmonella strains, the testing with a complete set of sera is time consuming and requires a well trained technician. Therefore, DNA based serotyping methods are under development. The requirements for a diagnostic assay are speed, sensitivity, reliability and robustness, best fulfilled by a PCR method. We consider AFLP band pattern analysis to be a promising candidate with the potential to substitute classical serotyping for subtyping of Salmonella.

Prognostic factors for invasive aspergillosis in patients with hematological malignancies S. Reuter, W.V. Kern, B. Hay, P. Kern (Freiburg, DE) Introduction: Reliable data defining risk factors for invasive aspergillosis (IA) in patients with leukaemia, lymphoma or after peripheral blood stem cell transplantation (PBSCT) are limited. We evaluated patient outcomes after IA over an extensive period of 9 years. Analysis of factors influencing IA-associated death are specially valuable in the light of major progress in diagnosis and treatment of IA. Methods: In this single-center study conducted between 1997 and 2005 we included all inpatients with IA, comprising possible, probable and proven cases. For survival analyses, patients were classed according to cause of death. For univariate analyses the Wilcoxon and log-rank test were performed and for multivariate analysis a cox model of logistic backward regression was used. Results: Attributable mortality was 41% after 1 year (overall mortality 53%) and 52% after 5 years (overall mortality 70%). Most attributable deaths occurred early after diagnosis with 50% of all deaths occurring during the first 6 weeks after IA diagnosis. Various parameters significantly correlated with death after IA: 1. Uncontrolled status of the hematological malignancy (p=0.007, as compared to hematological remission or IA at the time of first diagnosis of malignant disease), 2. extrapulmonary disease (p=0.0003, as compared to exclusive pulmonary affection), 3. IA classified as stable disease, mixed response or progressive disease at the time of first radiological evaluation of treatment response (p=0.004, as compared to either complete remission or partial response), 4. patients with proven IA (p=0.02, as compared to probable or possible IA), 5. more than 110 days elapsed since PBSCT at the time of IA (p=0,0112). In patients with neutropenia, a relationship was noted between increasing duration of neutropenia and increasing mortality (p=0.0001). We observed a trend towards improved survival in patients with a diagnosis of IA during the most recent years (2003) (2004) (2005) . For the small number of patients with a definite diagnosis of IA (n=29), survival was significantly improved during more recent years (p=0.002).

In a multivariate analysis factors independently associated with attributable mortality included year of first diagnosis (p=0.0492), pulmonary vs. extrapulmonary IA (p=0.0353) and duration of neutropenia (p=0.0088). Factors associated with overall mortality were pumonary vs. extrapulmonary IA (p=0.02), duration of neutropenia (p=0.02), age (p=0.03) and decreased mortality after autologous PB-SCT (p=0.04). Conclusions: Factors affecting mortality attributable to IA may prove valuable for the definition of high risk situations with influence on prognosis. In these settings, increased efforts of prevention, early diagnosis and aggressive treatment of IA are warranted in order to improve survival of this frequent complication in patients with hematological malignancies.

Black Water Fever -severe hemolytic anemia and jaundice after quinine therapy of severe falciparum malaria S. Schmiedel, G.D. Burchard (Hamburg, DE) Blackwater fever (BWF) is a severe clinical syndrome, characterized by intravascular hemolysis, hemoglobinuria, and acute renal failure that is classically seen in patients exposed to Plasmodium falciparum and who are taking quinine. BWF virtually disappeared after 1950. A resurgence of this affection which occurred frequently during the days of colonization has lately been noticed. We report a case of a patient of West-African origin who now lives in Europe for many years who was diagnosed with severe malaria after returning from a short term travel to his home country. Ten days after quinine treatment of severe falciparum malaria, the patient presented himself with macroscopic hemoglobinuria, jaundice, haemolytic anemia and acute renal failure.

In this case report the presentation, diagnosis, differential diagnosis and clinical management and therapy of the syndrome will be demonstrated and possible underlying pathomechanisms will be discussed.

Human Lyssa virus Infection: a life vaccine strategy is save and immunogenic S. Schmiedel, M. Panning, A.W. Lohse, C. Drosten, G.D. Burchard (Hamburg, Bonn, DE) Rabies in humans is a rare disease in European countries. We report a case of a human rabies infection imported from Marocco. Our patient already showed first clinical signs of neurological impairment. We administered a rabies life vaccine for the first time. Our intention was to induce an earlier immune response than achievable with the WHO recommended postexposure-vaccination-scheme. We were able to show that the vaccine was strongly immunogenic and save. Significant antibody concentrations were detectable in the serum in immunofluorescence-and neutralisation tests after 8 days postvaccination only. We injected the life vaccine subcutaneously to monitor viral expansion and clearence with PCR in the tissue closely, simultaneously we monitored viral load in blood, cerebrospinal fluid, saliva and other tissues. RNA of the attanuated virus was detectable in the tissue around the intracutaneous vaccination sites for three days. In blood samples RNA of the virus was detectable for two days -indicating a short and self-limiting viremia -but not in brain tissue, in cerebrospinal fluid (CSF) or biopsies from muscle, peripheral nerves, liver, spleen or lymph nodes. No measurable clinical events or side effects after the vaccination have been noticed. Wild type lyssa virus was detectable in conjunctival fluid and saliva during the whole course of illness -but only in the CSF and serum shortly before death.

Abstracts KIT2008

Uncommon clinical and parasitological features of severe falciparum malaria in a patient from Cameroun after splenectomy -a case report V. von Kalckreuth, A. Baumann, E. Tannich, S. Schmiedel (Hamburg, DE) A 51-year-old Camerounian woman presented to a small hospital in Germany with fever, asthenia and left upper abdominal pain. She had come to Germany from Cameroun three weeks before. The physical examination revealed paleness and an enlarged spleen, but was otherwise unremarkable. Laboratory data were: hemoglobin (Hb) level 10.0 g/dL, white blood cell count (WBC) 5.8 Mrd/L, C-reactive-protein-(CRP) level 225 mg/L and transient thrombocytopenia. Malaria thick and thin film, as well as malaria antigen test were repeatedly negative. The CT-scan of the abdomen revealed an extensive splenomegaly with multiple calcifications and subcapsular haemorrhagies. As a definitive diagnosis could not be established and in order to exclude a malignant lymphoproliferative disorder splenectomy was performed six weeks after the admission of the patient. The spleen had 1.180 kg in weight and 16x15x8.5 cm in size. Histopathologically massive granulomatous lesions suggestive for tuberculosis were found. PCR for M. tuberculosis was negative. In the further postoperative course the patient suffered from persistent fever and thrombocytopenia. She was transfered to our department three weeks after surgery. Laboratory data at admission revelaed renal insufficiency, advancing anaemia and thrombocytopenia. The patient was awake, fully orientated and urine output was normal. The blood smear now performed showed an extensive parasitaemia of P. falciparum with an unusual high proportion of schizonts and gametocytes. Interestingly, as found by PCR these parasites carried no var-genes in their chromosome set. Additionally, as shown on CHO cells, adhesion of these parasites to endothelial ligands like ICAM-1 and CD36 did not take place. Consecutively, the parasites might have failed to sequester in the peripheral capillaries of the patients tissues. In culture, after a delay of 30 days, parasites with var-genes showed up and adhesion on CHO cells then took place. These results could explain the relatively mild clinical symptoms of the patient compared to the extensive parasitaemia found after splenectomy and the exceptionally high proportion of mature parasites found in her peripheral blood. Parasites that have failed to sequester in non-splenic tissues could not be removed from the circulation by the spleen anymore. Treatment with quinine and doxycycline was initiated. Parasite clearance during treatment was markely prolonged. The general condition of the patient improved, she reprised appetite, gained weight and was discharged three weeks after admission. For the outpatient follow-up examinations the patient presented in a good medical condition and tuberculostatic therapy is up to now well tolerated.

In conclusion, in this patient splenic tuberculosis led to splenectomy as differentiating between a lymphoproliferative disorder, hyperreactive malarial splenomegaly and tuberculosis was not possible. The uncommon parasitological features found afterwards and the relatively benignant clinical course are based on the fact that the patients parasites carried no var-genes and that consequently adhesion to endothelial cells could not take place.

* This session is supplemented by posters with the program numbers P188 / P189 / P199 / P227 / P267. Detailed information about these contributions can be found under the above listed program numbers in this publication.

Molecular and Functional Analysis of Naturally Occurring Variations in the Putative Negative Toxin Regulator Gene tcdC of Clostridium difficile J. Fritzsche, B. Waidner, T. Giesemann, B. Scherer, M. Kist (Freiburg, DE) Introduction: Since 2001 ongoing spreading of a highly virulent Clostridium difficile (Cd) strain (Ribotyp 027, Toxinotyp III, 18 bp-deletion in the putative negative regulator gene tcdC) is observed. The epidemic strain was first detected in the United States, from there spreading over Canada and England, and reaching Western Europe in 2005. In the meantime it has been detected in the Netherlands, Belgium, France, Switzerland, and recently in Austria and Poland. During the same period a case-control-study on epidemiological risk factors and molecular virulence of nosocomial Cd-associated diarrhea was carried out at the Freiburg University Medical Centre (granted by the BMBF), resulting in Cd isolates from 600 study patients, and 400 patients investigated routinely during the same time period. Methods: From all isolates (N=1000), including three epidemic strains from Belgium and Switzerland as positive controls, PCR-based ribotyping, and in addition, molecular analysis of the putative negative regulator gene tcdC was carried out using PCR and molecular sequencing. Isolates with respective variations were further analysed phenotypically applying a Vero cytotoxicity assay, and a quantitative ELISA toxin assay, which was adapted for this purpose. Results: In a total of 35 isolates the same 18 bp deletion as found in the epidemic strains was detected. However in no case those isolates belonged to the epidemic ribotype 027. A total of 70 isolates presented with an already known 39 bp deletion, but in 19 isolates a 54 bp deletion was detected, which had not been described before. All "novel" deletions were further investigated by molecular sequencing. Growth curves performed from truncated and wild type strains were not significantly different. The already known 18 bp deletion was associated with an up to 2.2 fold increase in toxicity measured by the ELISA, however the toxic effect was more pronounced (about 10fold) if the verocell cytotoxicity test was applied. Surprisingly, cytotoxin production of isolates with deletions of 39 and 54 bp was markedly reduced, reaching zero in some cases. Conclusion: Various deletions in the putative negative regulator gene tcdC resulted in partly contradictory effects on toxiciy of C. difficile isolates. The functional impact of this putative regulator gene needs further investigation including the clinical outcome in respective patients.

Risk Factors of Nosocomial Diarrhoea with and without detection of Clostridium difficile M. Kist, C. Schneider, B. Scherer, M. Bußmann, G. Peyerl-Hofmann, M. Steib-Bauert, G. Pfeifer, W.V. Kern, K. Aktories, F. Daschner (Freiburg, DE) Introduction: In cases of nosocomial diarrhoea in adults C. difficile is by far the most frequent infectious agent isolated from stool specimens. It is typically associated with a proportion of antibiotic-associated diarrhoea, a condition that is frequent but in many cases could not be linked with invasive or toxin-producing pathogens. Various case-control studies have identified epidemiological risk factors for symptomatic Clostridium difficile associated diarrhoea (CDAD) such as older age, intensive care, gastrointestinal surgery, spectrum and duration of antimicrobial therapy. However, only few studies describ-

ing risk factors included patients with nosocomial diarrhoea in whom C. difficile had been excluded as causative pathogen. Methods: Cases of nosocomial diarrhoea (ND) were prospectively investigated for enteric pathogens including C. difficile by microbiological culture and toxin ELISA of stool specimens. Patients with ND and with detection of C. difficile were defined as cases. As controls, for each case with CDAD two other patients without diarrhoea and one patient with C. difficile-negative ND were actively recruited, if ever possible from the same ward, as negative controls, and as controls with ND, respectively. Clinical and epidemiological data were collected from cases and controls by using a standardized questionnaire by the same interviewer, or by checking patients' data sheets, if necessary. All C. difficile isolates were further characterized by ribotyping and PCR analysis of the pathogenicity locus and by detection of the binary toxin ADP-Ribosyltransferase (CDT). Epidemiological data were analyzed by applying univariate and multivariate analysis methods.

Results: A total of 420 pts with CDAD and complete data could be compared with 189 controls with ND but without detectable C. difficile infection, and with a total of 420 controls without diarrhoea. Applying multivariate analysis of cases and healthy controls including a broad spectrum of parameters revealed a panel of well known risk factors often associated with CDAD, namely abdominal surgery, OR 10.6 (1.3-86.4); total number of antimicrobial drugs, OR 7.7 (4.1-14.6); intensive care OR 1.6 (1.1-1.3); underlying gastrointestinal diseases, OR 1.44 (1.3-1.6); female gender, OR 1.42 (1.0-1.9); other underlying diseases, OR 1.19 (1.1-1.3), and nursing score, OR 1.15 (1.1-1.2). If cases and controls with C. difficile negative ND were included in the multivariate analysis surprisingly no risk factors remained. To our further surprise the potential risk factor «total number of antimicrobial drugs» appeared negatively associated with CDAD. Conclusion: Identification of epidemiological risk factors specifically related to CDAD needs further investigation.

Giessen -Project Z: Microbial diagnostics in clinical alveolar space infection -biomaterial bank T. Chakraborty, J. Lohmeyer (Giessen, DE) In the majority of pneumonia patients the etiology remains unclear because even in clinical studies only in 30% a causing agent can be isolated. Therefore, more sensitive detection assays for known and new micro-organisms are necessary to define the clinically relevant host microbe interactions in pneumonia. Innovative molecular detection assays for potentially pneumonia causing microbes must be prospectively evaluated in comparison with conventional culture methods. The associated Z project will use the established infrastructure and advanced diagnostic spectrum of the department of laboratory diagnostics and pathology (ZLP) to professionally process and store patient samples (blood, sputum, bronchoalveolar lavage, biopsies, urine) following established pre-analytical and preparative standard operation procedures. Central aim of this project is the storage of sufficiently large, clinically well-characterized and quality controlled pneumonia patient biomaterials that are electronically linked to detailed clinical datasets.

Giessen -Project B: Use of synthetic toll-like receptor 7-and 9-ligands as novel therapeutic approach in experimental Pneumococcal and Klebsiella pneumonia H. Hackstein, W. von Wulffen (Giessen, DE) Synthetic TLR7-und 9-ligands produce both pro-inflammatory and immunmodulatory effects, mainly by inducing interferons. In animal models prophylactic application of TLR9-ligands attenuates the course of inflammation, e.g. in pneumonia induced by Klebsiella pneumoniae. In mouse models of LPS-induced lung injury as well as in gram-positive and gram-negative pneumonia the impact of TLR7and 9-ligands on pulmonary organ (gas exchange) and immune function (inflammatory response, host defence) will be evaluated. Aim is to develop therapeutic intervention strategies that dampen systemic inflammation, attenuate lung tissue damage and preserve local microbe elimination from lung parenchyma.

Giessen -Project C: Host inflammatory pathways as target structures for the inhibition of viral replication and inflammatory tissue injury in influenza virus pneumonia S. Herold, S. Pleschka (Giessen, DE) Influenza viruses (IV) can cause severe pneumonia with high lethality, presumably resulting from an unbalanced over-induction of the pulmonary innate immune response added to the direct cytopathic effect caused by IV replication. The IV-induced activation of various signaling pathways in infected cells results from a complex crosstalk between different alveolar cell populations and is followed by a strong release of proinflammatory cytokines/chemokines ("cytokine storm") and a significant upregulation of adhesion molecules at the endo-/ epithelial barrier, both mediating the massive alveolar recruitment of peripheral blood leukocytes, in particular of monocytes/macrophages. This "hyper-inflammatory" innate host response is accompanied by alveolar leakage, lung edema and consecutive lung failure. However, to date, the factors released by mononuclear phagocytes in the early stage of IV infection which directly damage the epithelial are not known. In parallel, some of the known IV-induced signaling pathways besides being important mediators of proinflammatory host responses are also essential for effective virus replication in infected lung cells in vitro and in vivo. Accordingly, inhibition of these pathways not only reduces IV titers in the lung in infected mice without generation of resistant virus variants, but might additionally attenuate damaging innate host responses. Therefore, the project aims at the functional analysis of signaling cascades which initiate proinflammatory host responses and the characterization of connected molecular pathways mediating monocyte/macrophage recruitment to the lung, thereby damaging the highly sensitive pulmonary gas-exchange tissue. Inhibition of such pathways is supposed to reduce viral replication and at the same time to attenuate severe lung pathology under preservation of host defense functions.

Giessen -Project A: Transcriptome and proteome analysis in a human model of alveolar inflammation K. Mayer, F. Grimminger (Giessen, DE) Aim of this project is the identification and characterization of lung specific regulatory processes that are induced by bacterial components in the human alveolar space. A strictly defined alveolar inflammation

is induced in healthy volunteers by inhalative application of pathogenicity factors derived from gram-positive and -negative bacteria. At baseline and defined time points post inhalation peripheral blood leukocyte subsets and corresponding inflammatory cell populations from the bronchoalveolar fluid are separated and profiled for their respective transcriptome/proteome. By comparison of differential gene-and protein expression profiles cell specific regulation pathways in circulating, alveolar recruited and lung resident immune cells will be identified. Prototypic cellular signatures for distinct microbial agents and their pathogenicity factors in different host compartments are expected to provide new insights into the molecular pathogenesis of pneumonia and support the development of causal molecular therapies.

Giessen -Project E: Impact of bacterial virulence factors on alveolo-capillary barrier function K.T. Preissner, M. Steinmüller (Giessen, DE) The translocation of bacteria or their products from local pneumonia infection sites into the pulmonary or systemic circulation is a major cause of septic respiratory failure in intensive care units. The severe and often lethal damage of the alveolo-capillary barrier function is frequently caused by bacteria such as S. aureus or S. pneumoniae and/ or their products interacting with host tissues. The underlying pathophysiologic mechanism in this cross talk, however, are poorly understood. Therefore the effect of surface and secreted virulence factors of staphylococci and pneumococci such as S. aureus SERAM («secretable expanded repertoire adhesive molecules») or pneumolysine (PLY) on lung barrier integrity will be evaluated by leakage assessement in vivo and transcriptome/proteome analysis in vitro. In addition to the investigation of leukocyte traffic, bacteria adhesion/invasion in mouse pneumonia models in vivo established organ models of isolated/perfused lungs will be employed. Intervention strategies using antibodies/inhibitors of virulence factors/induced host pathways will be evaluated for their therapeutic potential to attenuated pneumonic barrier failure.

Giessen -Project D: Role of junctional adhesion molecules (JAMs) in alveolar leukocyte recruitment and microbial translocation S. Santoso, H. Hossain (Giessen, DE) Under homeostasis conditions lung epithelial and endothelial cells preserve their structural integrity by intercellular contacts (junctions) that form a major physiologic barrier for the spread of micro-organisms, but also for the transportation of ions, proteins and lipids as well as for the migration of leukocytes. Bacteria such as the worldwide most frequent pneumonia causing agent S. pneumoniae and its exotoxin pneumolysin can profoundly disturb the junction integrity and thereby affect its barrier function for microbial agents and their products on one hand and influence the leukocyte traffic on the other hand. The molecular cross talk between microbes/toxins and junctional structures such as junctional adhesion molecules (JAMs) will be investigated in established lung endo-/epithelial coculture systems in vitro and in mouse pneumonia models in vivo and evaluated for their functional impact on microbial and leukocyte transmigration aiming at developing molecularly targeted intervention strategies.

Pharmacological interaction of organic cation transporters 1 and 2 with antiretroviral and concomitant drugs N. Jung, C. Lehmann, M. Knispel, P. Hartmann, G. Fätkenheuer, A. Rubbert, E. Schömig, D. Taubert (Cologne, DE) Background: Understanding antiretroviral drug interactions is essential for efficacy and tolerability in the therapy of HIV infection. The organic cation transporters (OCT)1 and 2 show a broad substrate selectivity and play an important role in the detoxification of organic kations in the kidney and liver. More than 50% of the antiretroviral drugs and concomitant therapeutics for opportunistic infections carry positively charged moieties. Therefore we assessed inhibitors and substrates of OCT1 and 2 among common cationic antiretroviral and concomitant drugs. Methods: HEK-293 cells were stably transfected with human (h) OCT1 and OCT2 subcloned into the eukaryotic expression vector pcDNA3. The 50% inhibition concentration IC 50 was determined by measuring the specific uptake of 3H-labeled 1-Methyl-4-phenylpyridinium (MPP+). Uptake experiments were performed with the same cell systems determing quantitatively the specific substrate by liquid chromatography electrospray-ionization-tandem mass spectrometry (LC-ESI-MS/MS). The substrate concentration at half maximal transport velocity KM and the maximum reaction velocity Vmax were calculated using the Michaelis Menten equation. Results: Pentamidine (IC50(hOCT1) = 0.4(1) µmol/l (mean (s.e.m.); (OCT2) 3.8(1)), Nelfinavir (7(1); 33 (11)), Ritonavir (14(2); 51 (22)), Lamivudine (17(3); 13 (3)), Trimethoprim (20(3); 131(12)), Zalcitabine (24(5); 25 (7)), Saquinavir (37 (7); 105(29)) and Indinavir (37(6); 275 (57)) showed significant inhibition of hOCT1 and hOCT2. Lamivudine and Zalcitabine were identified as substrates of hOCT1 and hOCT2 (Lamivudine: OCT1(Vmax=2.0(0.2) nmol/mg/min; KM=249(51) µmol/l) OCT2 (Vmax=1.1(0.2); KM=248(86)) Zalcitabine: OCT1(Vmax=1.0(0.1); KM= 242(56); OCT2(Vmax=0.6(0.1); KM=232(13)). Conclusions: Our data suggest, that antiretroviral substances and concomitant drugs used for opportunistic infections in HIV infection are potently involved in the transport kinetics of cationic drugs that are substrates of hOCT1 and 2. Further studies are needed to elucidate the relevance of these drug-drug interactions in order to provide safer and more efficient therapy.

Differential regulation of iron dependant genes in the mycobacterial D. Wagner, L. Dreiser, M. Vavra, W.V. Kern (Freiburg, DE) Background: The iron concentration in the mycobacterial phagosome during infection of resting macrophages is initially low but increases significantly within 24 hr in both M.tuberculosis and M.avium phagosomes. Using quantitative RT-PCR we tested the hypothesis that the short-lived iron limitation leads to concordant iron/IdeR-dependant gene regulation during the first day of infection of human macrophages with both mycobacterial species and compared the intraphagosomal expression profile to extracellular expression in broth. Results: We found a differential intracellular gene expression profile for both M.tuberculosis and M.avium, possibly representing adaptive changes to different environmental conditions: Consistent with low phagosomal iron concentrations early in the infection, genes involved in the siderophore synthesis were higher expressed in both, M.tuberculosis and M.avium, after macrophage infection. Gene expression of these genes was decreased after 24 hr of infection in M.tuberculosis, but not in M.avium. Despite high iron concentration in the mycobacterial phagosome after 24 hr of infection, expression of the bacterioferritin (bfrA) gene was not induced. The gene expression of the human ferritin homologue BfrB, not present in the M.avium

genome, was upregulated during intracellular growth of M. tuberculosis indicating different functions as compared to BfrA. Expression of fecB and fecB2 were both induced early on in the infection in M.tuberculosis whereas fecB-expression was only increased after one day of infection with M.avium. Changes in the iron concentration in vitro did not mimic changes in phagosomal iron/IdeR dependant gene expression in both mycobacterial species. Conclusion: Our results show that iron/IdeR-depending genes are differentially regulated in the phagosome of resting human macrophages infected with either M. tuberculosis or M. avium. They imply overriding regulatory mechanisms in the mycobacterial phagosome on the induction of an iron-related transcriptional response.

Die LPS-Trap, eine Janus-Waffe gegen die bakterielle Sepsis?

employed against other MDR organisms in an era in which effective antibacterial therapies are fading.

The main aim of the study is to investigate the pathogenomic events leading to carbapenem-resistance and the association with epidemicity and virulence of A. baumannii. Carbapenem resistance is mediated mostly through three mechanisms: enzymatic carbapenem-hydrolysis (to be studied in work package 2 (WP-2)), reduced membrane permeability through the loss of porins or efflux pumps, expressed as outermembrane-protein changes (to be studied in WP-3). In WP-1 clinical A. baumannii isolates will be characterized using clinical and epidemiological data and their clonality and antibiotic resistance will be determined. These isolates will be subject for further analysis in all other WPs. WP-2 will examine enzymes that hydrolyze carbapenems, and the genetic events leading to the spread of resistance, WP-3, examine outer-membrane protein (OMP) changes associated with carbapenem resistance and identification of the genetic events leading to these OMP changes, WP-4 determines the associated effects of various mechanisms of carbapenem resistance on epidemicity and virulence (respectively) and the genetic events relevant to it.

This session is supplemented by posters with the program numbers P188 / P189 / P199 / P227 / P267. Detailed information about these contributions can be found under the above listed program numbers in this publication. 

S177, P254, P297

P187, P243, P282, P293

P238 Dettenkofer, M. O125, S147, P265

O87, P205, P208, P222, P247, P248, P252

O87, P205, P208

O165, P213, P214, P263

O72, S179

S57, O122, S148, P259

O143, P220, P221

S95, P239

S7, S109

O32, P228, P234

S21 Peyerl-Hofmann, G. . O125, O165, P199, P262

O87, P205

P227, P231, P262, P263, P274

O72, O165, P185, P236

O125, P262, P277

O122, O124, P259

O32, P218, P228, P229, P234

P208, P247, P248

P. Groß, B. Schnabl, W. Falk (Regensburg, DE) Das angeborene Immunsystem stellt ein über die Evolution konserviertes System zur Früherkennung und -abwehr von Mikroorganismen dar. Eine Schlüsselrolle als sogenannte Pattern-recognition-Rezeptoren (PRRs) nimmt die Familie der Toll-like-Rezeptoren (TLRs) ein. In einer Gram-negativen Sepsis führt die Stimulierung von TLR4 durch LPS zu einem "Zytokin-Sturm", der in einem Multiorganversagen enden kann. In bisherigen Arbeiten wurde ein lösliches Fusionsprotein aus MD-2 und dem extrazellulären Teil von TLR4 hergestellt, das in vitro und in vivo LPS-Effekte hemmen konnte (Brandl et al. 2005) Für in vivo Untersuchungen wurden IgG1-Fc-Chimäre des Fusionsproteins (LPS-Trap-Fc) hergestellt. Ebenso wurden verkürzte Varianten des Konstrukts -LPS-Trap-FcM und LPS-Trap-FcS -hergestellt, um das Protein auf die für die Bildung des TLR4/MD2-Komplexes notwendigen Domänen zu beschränken. Deren Produktion in Schneider S2 oder HEK 293T Zellen führte zu Proteinen mit den molekularen Massen 140kDa (LPS-Trap-Fc), 105kDa (LPS-Trap-FcM) und 100 kDa (LPS-Trap-FcS) unter reduzierenden Bedingungen. Die Bindung von LPS konnte durch Immunpräzipitationsexperimente an allen Konstrukten gezeigt werden. Experimente mit einem TLR4/MD2-Komplex-spezifischen Antikörper zeigten jedoch, dass nur die LPS-Trap-Fc in der richtige Konformation vorlag. Diese Ergebnisse wurden in Experimenten mit LPS stimulierten MonoMac6-Zellen bestätigt. Lediglich die LPS-Trap-Fc hemmte die Produktion proinflammatorischer Zytokine. Wegen Problemen bei der Isolierung von gereinigtem Produkt und zur Erleichterung von in vivo Experimenten wurden Adenoviren (Ad-LPS-Trap-Fc) eingesetzt, die in infizierten Zellen die Sezernierung der LPS-Trap-Fc hervorrufen. Die Expression der LPS-Trap-Fc konnte über Immunpräzipitation bestätigt werden. Die Serumspiegel des sezernierten Proteins konnten nach der Etablierung eines ELISA verfolgt werden. Hierbei stellte sich heraus, dass nach einem Maximum am Tag 3-4 p.I. die Proteinmengen im Serum abnahmen. Als ein Grund für die rasche Abnahme der Serumspiegel konnten LPS-Trap-Fc spezifische Antikörper ausgemacht werden. Nach Verabreichung von LPS am Tag 4 p.I. wurden Zytokinkonzentrationen im Serum untersucht. Die positiven Effekte auf die Zytokinausschüttung in vitro konnten in ersten Versuchen im Mausmodell nicht bestätigt werden. Die Ad-LPS-Trap-Fc infizierten Tieren zeigten leicht erhöhte Zytokinspiegel gegenüber den Kontrolltieren (Adbeta-Gal). Es besteht die Möglichkeit, dass die Bioverteilung von LPS durch nicht optimale Konzentrationen von LPS-Trap-Fc ungünstig beeinflusst wird. Um diese Effekte besser zu kontrollieren wird für die Zukunft der Einsatz von rekombinant hergestellten Proteinen angestrebt.

Cytomegalievirus Reaktivierungen bei Patienten mit refraktärer chronisch entzündlicher Darmerkrankung F. Hanses, H. Herfarth, C. Franzen, B. Salzberger (Regensburg, DE) Hintergrund: Erkrankungen mit CMV sind bei immunkompromittierten ein häufiges Problem. Bei Patienten mit chronisch entzündlicher Darmerkrankung (CED) sind nur wenige systematische Untersuchungen zur Häufigkeit, Ausmaß und klinischer Relevanz von CMV-Reaktivierungen vorhanden. Methoden: Zur Untersuchung dieser Fragestellung wurden hochsensitive PCR-Methoden zum Nachweis verschiedener Transkripte (RNA und DNA) von CMV entwickelt. Insgesamt 12 Patienten mit chronisch entzündlicher Darmerkrankung wurden bisher untersucht. Ergebnisse: CED Patienten mit aktiver Erkrankung unter moderater bis starker immunosuppressiven Therapie (n=7, mit Steroidtherapie, Methotrexate, Azathioprin oder TNF-alpha-Inhibitor -Therapie) wurden verglichen mit Patienten ohne Therapie oder nur mit Mesalazin (n=5). In der Patientengruppe mit immunsuppresiver Therapie fanden sich in Biopsien in 83% CMV-DNA im Vergleich zu 60% in der Gruppe ohne Immunsuppression und in 43% vs. 20% CMV mRNA. Der Nachweis von CMV-DNA und mRNA war auch in Bezug auf einzelne Biopsien deutlich hoher in der Gruppe der immunsupprimierten Patienten Zusammenfassung: CMV Reaktivierung bei Patienten mit CED ist assoziiert mit der Krankheitsaktivität und Schwere der Immunsuppression. Diese Assoziation und insbesonder die klinische Bedeutung muss in einer größeren Kohort überprüft werden.

Mechanismen der Carbapenem-Resistenz von Acinetobacter baumannii und ihre Bedeutung für die epidemische Ausbreitung P.G. Higgins, H. Seifert (Cologne, DE) Acinetobacter baumannii is an important opportunistic pathogen of increasing relevance in hospital infections. In the past, this microorganism has rarely been associated with clinical significance and was susceptible to most antimicrobial drugs. Now its incidence has increased dramatically in many parts of Europe and elsewhere, especially in intensive care units, accompanied by the emergence of multidrug resistance (MDR), and is associated with severe adverse patient outcomes. The spread of A. baumannii in affected hospitals appears to be very difficult to control, and recently this MDR organism has become endemic in many hospitals. Resistance to carbapenems, an antibiotic class of last resort, is increasing in A. baumannii nosocomial isolates and reaches 70% in some European hospitals. These data emphasize the ability of A. baumannii to adapt to new situations either by switching the expression of constitutive genes or by easily acquiring foreign pieces of DNA, allowing it to adapt and survive in hostile environments. This feature probably also reflects a great flexibility of the A. baumannii genome to adapt its regulation to new environmental signals. The main objectives of the present research project are to understand how these changes occur in terms of resistance to carbapenems, as this class is often the choice to treat severe infections caused by MDR A. baumannii, and in terms of virulence, trying to understand the molecular basis behind the transformation of a colonizing non-pathogenic A. baumannii strain into an invasive and virulent pathogen. In addition, we will investigate the observation that some A. baumannii isolates spread more efficiently than others in the hospital environment or even across frontiers, being disseminated among several countries, as has been reported with epidemic "European Author Index S: Symposium; O: Oral Presentation / Freier Vortrag; F: Fellow Program; P: Poster