mycoses

Diagnosis,Therapy and Prophylaxis of Fungal Diseases

Abstract

Oral Presentations Systemic Mycoses V02 MIAMI-Study: Micafungin in routine practice for the treatment of invasive candidiasis, oesophageal candidiasis or prophylaxis of candida infections in Germany O. Cornely,1 K. Hahnenkamp,2 A. Heininger,3 G. Langebartels,4 M. Kiehl,5 W. Knitsch,6 G. Silling7 and S. Wintersperger8 1 Klinik I fu€r Innere Medizin, Uniklinik Ko€ln, Ko€ln, Deutschland; 2 Klinik fu€r Ana€sthesiologie, operative Intensivmedizin und Schmerztherapie, Universita€tsklinikum Mu€nster, Mu€nster, Deutschland; 3Universita€tsklinik f. Ana€sthesiologie und Intensivmedizin, Universita€tsklinikum Tu€bingen, Tu€bingen, Deutschland; 4Klinik und Poliklinik fu€r Herz- und Thoraxchirurgie, Kliniken der Universita€t zu Ko€ln, Ko€ln, Deutschland; 5 Medizinische Klinik I, Klinikum Frankfurt/Oder, Frankfurt/Oder, Deutschland; 6Medizinische Hochschule Hannover, Klinik fu€r Allgemein-, Visceral- und Transplantationschirurgie, Hannover, Deutschland; 7Department of Medicine A, Haematology/ Oncology, University of Muenster, Muenster, Deutschland and 8 Medizinische Abteilung, Astellas Pharma GmbH, Mu€nchen, Deutschland Introduction Morbidity and mortality caused by invasive fungal infections remain a major concern in patient care. Micafungin is indicated for treatment of invasive and oesophageal candidiasis and as prophylaxis of Candida infection in patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) or patients expected to have neutropenia below 500 cells ll
1 for ≥10 days. MIAMI was the first nationwide, prospective, non-interventional study capturing data on safety and effectiveness of micafungin in patients treated in accordance with the approved label. Methods Safety and effectiveness data were captured at micafungin treatment initiation (Visit (V) 1); End of treatment (V2). Documentation included clinical/microbiological diagnosis at V1/V2, micafungin dose, treatment duration, adverse events. Liver function was monitored. Descriptive statistics were applied. Results Thirty five clinicians enrolled 219 eligible patients [138 (63.0%) male, 2 (0.9%) no data]. Mean age: 57.5 (18.5 SD, N = 215) years including 12 patients 1.0 ODI was added) and host factors (preexisting pulmonary disease was added). Diagnostic accuracies of the different methods for probable/proven IPA were evaluated. Results Thirty one patients (31 samples) had probable or proven IPA, 24 possible IPA (26 samples) and the remaining 167 patients (211 samples) did not fulfill IPA criteria. COPD (n = 11) was the most frequently observed primary underlying disease among those with probable or proven IPA, followed by bacterial pneumonia (n = 7), chronic autoimmune disease with lung involvement, bronchial carcinoma (n = 4 each), viral pneumonia (n = 2) and each one case of AIDS and TBC. Performances of LFD, GM, BDG and culutre are displayed in Table1.

doi:10.1111/myc.12216

Oral Presentation

Table 1

Test methods

Sensitivity

Specificity

PPV

BAL GM >0.5 ODI BAL GM>1.0 ODI BAL GM >3.0 ODI LFD BDG >80 pg ml
1 BDG >200 pg ml
1 Mycological Culture

97% 97% 61% 77% 90% 69% 19%

81% 93% 99% 93% 41% 61% 97%

42% 68% 86% 62% 19% 22% 60%

(30/31) (30/31) (19/31) (24/31) (26/29) (20/29) (9/31)

(170/211) (197/211) (208/211) (196/211) (76/186) (113/186) (205/211)

Diagnostic performance of BAL GM, culture, BDG, LFD and PCR for probable and proven IPA versus no IPA. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic odds ratio (DOR) displayed. Candida spp. in culture were interpreted as colonizations. Conclusion Thirty one cases of IPA were diagnosed among patients with underlying pulmonary diseases. GM followed by Aspergillus LFD testing were the most useful methods for diagnosing IPA in BAL. LFD represents a point of care test and may be a useful alternative in centers were GM testing is not available. Performance of conventional culture was limited by low sensitivity, while that of BDG was limited by low specificity among patients with underlying pulmonary conditions.

V04 Rare systemic mycoses – which should be kept in mind and how to be diagnosed? K. Tintelnot Robert Koch-Institut, FG 16, Berlin, Deutschland Since systemic mycoses have no pathognomonic clinical symptoms, the strategies to come to a clear diagnosis need a close collaboration of clinicians, pathologists and microbiologists. The group of rare infectious fungal organisms diagnosed in Europe include primary pathogens like Histoplasma capsulatum and Coccidioides species as well as opportunistic fungi. These belong to the Scedosporium apiospermum-complex, or for instance to the genera Fusarium, Mucor, Cryptococcus, Trichosporon and others. The diagnostic options depend on the clinical manifestation, adequate clinical samples, the immune status, stage of infection and the pathogen itself. Serological tests, e.g. for cryptococcosis, histoplasmosis or coccidioidomycosis, are rarely available or do not necessarily explain clinical symptoms. Extensive microscopic examination of clinical samples is of high relevance for direct detection of a potential pathogen, combined with culture and molecular methods. Since systemic mycoses are often diagnosed accidentally and the identification of the pathogen in histological sections is rarely possible, molecular examination of formalin fixed paraffin embedded (FFPE)-tissue is a promising way to come to a diagnosis.

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NPV (30/71) (30/44) (19/22) (24/39) (26/136) (20/93) (9/15)

99% 99% 95% 97% 96% 93% 90%

DOR (confidence interval) (170/171) (197/198) (208/220) (196/203) (76/79) (113/122) (205/227)

125 (16.5–938.5) 422.5 (53.5–3328) 109.5 (28.5–423.3) 44.8 (16.6–120.9) 6 (1.75–20.5) 3.4 (1.5–8) 14 (4.5–43)

Diagnostics I V06 (1?3)-b-D-Glucan kinetics in patients with candidemia J. Held,1,2 A. Busse Grawitz,3 T. Epting,3 R. Friedrich,2 S. Dr€ager2 and E. Rappold2 1 Mikrobiologisches Institut, Uniklinik Erlangen, Erlangen, Deutschland; 2Medizinische Mikrobiologie und Hygiene, Uniklinik Freiburg, Freiburg, Deutschland and 3Klinische Chemie, Uniklinik Freiburg, Freiburg, Deutschland Objectives Empirical therapy in febrile ICU-patients with candidemia contains an antifungal drug in only 5.7% of cases. As a consequence, antifungal therapy is usually started at the time of blood culture positivity, i.e. with a delay of 2–3 days after the onset of symptoms. However, a delay in the initiation of adequate treatment is associated with an increased mortality and therefore therapy should be guided by rapid diagnostic tests. A question of debate is how fungal antigen assays should be used. Specifically, it remains unclear whether a prophylactic screening approach (testing all patients 2–5 times per week) or a symptom-driven approach (testing only after the onset of symptoms) should be implemented. It is also unknown, whether and for how long the appearance of fungal antigens in serum precedes the onset of symptoms and for how long these antigens continue to circulate in the blood. Therefore, we initiated a study to determine (1?3)-b-D-Glucan (BDG) kinetics before and after diagnosis of candidemia. Methods We prospectively collected serum samples from patients with culture-proven candidemia at the University Medical Center Freiburg between March, 2013 and May, 2014. The sera were obtained from Clinical Chemistry which stores all samples for 5 days. This enabled us to get access to sera from the 3–4 days prior to the blood culture sampling (day 0). For follow-up purposes, serum samples were collected on day 2, 5, 9, 14 and then every 7 days until death or discharge. Furthermore, clinical information and data on antibiotic/antimycotic therapy of these patients was acquired. BDG levels were measured with the Fungitell assay according to the manufacturer’s instructions. Results In total, 512 serum samples (1–30 per patient) from 52 patients with candidemia were collected. The earliest serum was drawn 10 days before and the latest 120 days after day 0. Using the manufacturer0 s cut-off (80 pg ml
1) 45 patients tested positive for BDG at day 0 (sensitivity: 86.5%, 95% CI: 74.2–94.4). The median BDG level at this time point was 280 pg ml
1 (IQR 106–634). Thirty of the BDG-positive patients (88%) had elevated levels prior to day 0, with BDG levels being elevated up to 5 days before. Interestingly, in patients suffering from recurrent candidemia the relapse was accompanied by increasing BDG levels. In 7 out of 20 patients with a fatal outcome (35%) BDG levels increased drastically before death. None of the survivors showed such a kinetic. Conclusion Serum BDG proved to be a highly sensitive biomarker for diagnosis of candidemia. Because BDG positivity precedes the day of blood culture sampling, which is normally the day of the first symptoms, a screening approach would allow early diagnosis and initiation of therapy and therefore appears to be preferable over the

ª 2014 The Authors Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 2), 15–30

Oral Presentation

symptom-driven approach. Strongly increased BDG levels during follow-up are indicating relapse or imminent death.

V07 Clinical utility of a panfungal real time-PCR assay for detection of fungal infections B. Willinger,1 B. Selitsch,1 O. Hassan,1 G. Manhart,1 V. Dosch1,2 and C. Schabereiter-Gurtner1,3 1 Klinische Mikrobiologie, Medizinische Universita€t Wien, Wien, 2 € Universita€tsklinik fu€r Krankenhaushygiene und Osterreich; Infektionskontrolle, Medizinische Universita€t Wien, Wien, € € Osterreich and 3Ingenetix GmbH, Wien, Osterreich Objectives The rise in the incidence of fungal infections and the expanding spectrum of fungal pathogens make early and broad detection as well as accurate identification of fungal pathogens essential. While species- or genus-specific PCR assays assume a certain infection, panfungal real-time PCR enables the unspecific detection and quantification of any fungal DNA present in a clinical sample. In a panfungal PCR assay, universal primers should target sequences specific for all fungal species to guarantee the detection of all fungal taxa. For species identification, PCR amplicons have subsequently to be sequenced and phylogenetically analysed. Thus, in addition to improving turn-around time of microbiological identification, panfungal PCR can provide results which in some cases lead to potentially surprising diagnoses, as enabling diagnosis of also uncommon and rare fungal infections. Methods In this study a newly designed panfungal HybProbe realtime PCR assay was clinically evaluated. The panfungal real-time PCR assay targets the complete fungal ITS2 region using the LightCycler instrument (Roche
). Species are identified by subsequent phylogenetic sequence analysis (BLAST). After having developed the PCR assay 401 clinical samples derived from patients with clinically suspected invasive or superficial fungal infection were investigated. Results were compared to conventional methods (culture, KOH and histology) and clinical signs of infection and other molecular techniques. Results The samples consisted of 90 BALs and 6 bronchial secretions, 52 tissue samples, 66 samples of various sterile fluids, four paraffin tissue sections, 60 EDTA blood samples and 133 dermatological samples (90 nail samples and 43 skin scrapings). Out of these 401 samples 206 showed concordant positive or negative results. 20 samples showed positive results only by the panfungal PCR thus allowing for diagnosis which would have been missed if only culture would have been used. Especially, the use of PCR in blood and tissue samples showed better results than culture. However, there were cases when PCR detected airborne contamination or colonization In sum, fungal pathogens were properly identified by the panfungal assay. Conclusion The molecular approach helped to identify the species of culture negative but histologically positive samples. The assay was able to reduce time to detection and identification from 2 weeks down to 2 days. It was further able to successfully detect rare emerging pathogens, particularly in specimens from invasive infections. Its evident benefits make it a valuable tool especially where accurate and fast detection is necessary, such as the emergency setting, or where culture does not provide a clear or no result.

ª 2014 The Authors Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 2), 15–30

V08 A new DNA microarray allows the identification of all known pathogenic dermatophytes at species level C. Kupsch,1 M. Harder,2 M. Cavalar2 and Y. Gr€aser1 1 Institut fu€r Mikrobiologie und Hygiene, Charite Universita€tsmedizin Berlin, Berlin, Deutschland and 2 EUROIMMUN Medizinische Labordiagnostika AG, Lu€beck, Deutschland Dermatophytes are filamentous fungi, which cause superficial infections of keratinous tissues with a high prevalence worldwide. In immunocompromised patients they can occasionally trigger lifethreatening invasive infections. Nowadays, numerous specific and sensitive PCR-based methods are available for dermatophyte identification from clinical specimen, but, due to the high sequence identity among dermatophytes, none of these methods is able to distinguish all anthropophilic and zoophilic pathogens, so far. This, however, is of particular interest (i) for the efficient therapy of the infection, which differs for zoophilic and anthropophilic dermatophytes and (ii) to prevent and control endemic outbreaks, by tracing the source of infection. Therefore, in cooperation with EUROIMMUN Medizinische Labordiagnostika AG, we currently develop a DNA microarray, which allows the discrimination of all pathogenic dermatophytes by means of species specific polymorphisms within the ITS (internal transcribed spacer) regions and the TEF1 (translation elongation factor 1 alpha) gene, directly from clinical specimen.

V09 Identification of human pathogenic fungi via DNAMicroarray analysis for clinical applications L. S. L. Mayer,1 S. Hartmann,2 M. Cavalar,3 J. Weile,4 P. Rothacher,5 K.-H. Boven,6 S. Bailer1 and S. Rupp2 1 Universita€t Stuttgart, Stuttgart, Deutschland; 2FraunhoferInstitut fu€r Grenzfla€chen- und Bioverfahrenstechnik IGB, Stuttgart, Deutschland; 3EUROIMMUN Medizinische Labordiagnostik AG, Lu€beck, Deutschland; 4Herz- und Diabeteszentrum NRW, Bad Oeynhausen, Deutschland; 5Robert Bosch GmbH, Robert-Bosch-Platz, Gerlingen, Deutschland and 6 Multi Channel Systems MCS GmbH, Reutlingen, Deutschland Patients with a weak immune system like people receiving immunosuppressive treatment for cancer and organ transplantation or patients who suffer from AIDS or cystic fibrosis are representing a high-risk group for secondary infections with human pathogenic fungi. Those invasive fungal infections show a high morbidity and mortality rate between thirty to eighty percent. Deciding reasons may be inadequate medication due to inaccurate and time consuming classification of moulds and yeasts in clinical laboratories. For an increased life expectancy, an effective and early medication is necessary. Conventional molecular biological methods to identify human pathogenic species like PCR, qRT-PCR or sequencing preclude parallel detection resulting in material and sample intensity. To overcome these limitations we are developing a Fungal-Yeast-IdentificationChip as a fast and reliable device for the accurate identification of 55 human pathogenic moulds and yeasts. To this end we take advantage of DNA sequences of specific target genes which are representing evolutionarily conserved sequences variable enough to discriminate the relevant species. Sequence databases of ribosomal RNA genes as well as functional target genes are established to design probes with high discrimination power and primer pairs for the amplification of diagnostic target regions. To evaluate the DNA-Microarray in a first step individual PCRs with DNA of reference species were established. Up to now evaluation of the microarray has been completed with 55 reference species. After hybridization of labeled amplicons highly specific

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Oral Presentation

signals were obtained. Background fluorescence signals are very low and could be discounted. To increase specific signals of designed probes a probe-redesign took place. Furthermore, the microarray will be validated with patient samples like broncho-alveolar lavage or tracheal secretion provided by our clinical partner. For clinical application the Fungal-Yeast-Identification-Chip will be embedded in a Lab-on-a-chip-system. All required steps like cell lysis of human and pathogenic cells, amplification of target genes via PCR and the identification of 55 fungal pathogens via microarray are integrated in a disposable cartridge. Reliable and fast identification should be possible within a few hours and provides rapid therapeutic intervention in an early state of infection.

Pathogenecity & Resistance Factors I V10 Evolution of antifungal drug resistance in Candida albicans J. Morschha¨user Institut fu€r Molekulare Infektionsbiologie, Universita€t Wu€rzburg, Wu€rzburg, Deutschland The pathogenic yeast Candida albicans can develop drug resistance during antimycotic therapy. Resistance against the widely used antifungal drug fluconazole, which inhibits ergosterol biosynthesis, is caused by several mechanisms, including mutations in the target enzyme, upregulation of ergosterol biosynthesis genes, and overexpression of multidrug efflux pumps. In my lecture I will summarize the molecular basis of these resistance mechanisms, the genomic changes involved in the evolution of fluconazole-resistant strains, and their consequences for the fitness of the fungus.

V11 Identification of Candida albicans factors that modulate cytokine production in distinct epithelial cell types T. Pawlik,1,2,3 B. Hebecker,1,2,3 B. Hube2,4 and I. D. Jacobsen1 1 Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute (HKI), Research Group Microbial Immunology, Jena, Deutschland; 2Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute (HKI), Department of Microbial Pathogenicity Mechanisms, Jena, Deutschland; 3Jena University Hospital, Center for Sepsis Control and Care (CSCC), Jena, Deutschland and 4Friedrich Schiller University, Jena, Deutschland Disseminated candidiasis is a life-threatening nosocomial infection in intensive care patients. Systemic infection of mice is used as the ‘gold standard’ model to investigate this disease. In this model, internal organs differ in their ability to control Candida albicans: While the fungus proliferates in the kidneys, it is cleared from other internal organs such as liver and spleen. The kidney is characterized by a delayed cellular immune response, followed by massive production of proinflammatory cytokines and persistent accumulation of immune cells, which contributes to renal pathology. In contrast, the liver mounts only a moderate and transient proinflammatory response. The molecular mechanisms underlying these different organ responses are not yet understood. We hypothesize that C. albicans factors contribute to the organ-specific responses. To identify such factors, we used a large-scale screening approach. A collection of 1100 C. albicans single knock-out mutants was analyzed for their ability to damage human renal, intestinal and oral epithelial cells. In parallel, the host immune response was assed by quantification of the proinflammatory cytokines IL-6 and IL-8. As expected, the cytokine response to mutants with reduced damage potential was likewise reduced, demonstrating the

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feasibility of the screening approach. The identification of mutants with normal damage potential that caused a reduced cytokine response in all analyzed cell lines suggests that fungal factors indeed modulate the immune response independent of cell damage. We furthermore identified mutants with cell type-specific alterations in the host response. In total, 60 mutants in which damage potential and cytokine response were uncoupled were identified by the screening. Of these, eleven genes (mnn9, ric1, suv3, rfg1, pho23, mcm1, sch9, orf19.3035, orf19.422, orf19.764, orf19.3809) were selected for further analysis. The construction of homozygous deletion mutants for these genes is currently under way. These mutants will be phenotypically characterized in detail for morphology, growth properties and stress resistance under various conditions. Furthermore, we plan to analyze the interaction of these C. albicans mutants with different human epithelial cell lines and immune cells in more detail, including recognition by the host cells and activation of signaling pathways that lead to cytokine production. In summary, by using a large-scale screening approach we identified several candidate genes that might modulate epithelial cell responses to infection and might contribute to the organ-specific host responses observed in vivo.

V12 A large-scale collection of gene deletion mutants in C. glabrata €ller,1 W. Glaser,1 B. Willinger2 and F. Istel,1 T. Schwarzmu K. Kuchler1 1 Max F. Perutz Laboratories, Medical University Vienna, Vienna, € Osterreich, and 2Klinische Abteilung fu€r Klinische Mikrobiologie, € Medical University Vienna, Vienna, Osterreich Within the last decade, there has been a steady increase of invasive fungal infections in immunocompromised patients. The most common of these infections are caused by Candida spp., which has a high mortality in its disseminated form. C. glabrata (C.g.) now is the second most frequent cause and accounts for 15–20% of all cases of Candidiasis. Importantly, C.g. is inherently tolerant to azole antifungals when compared to most other Candida spp. This is of clinical importance because of the wide use of azole therapy in fungal infections. Moreover, C.g. is unable to form true hyphae or secrete proteases, which are considered important virulence factors of C.a. These differences, as well as the high tolerance to conventional fungal therapies, leave the nature of virulence factors largely unknown. To identify candidate virulence and drug resistance factors, we initiated the construction of a large-scale collection of C.g. deletion mutants. This collection enables studies on the molecular functions of genes implicated in virulence and drug resistance, as well as those modulating signaling pathways and stress response. Hence, we use a reverse genetic approach to generate a bar-coded C.g. gene deletion collection, which is subsequently analyzed in vitro for their sensitivity to different environmental stress conditions including heat, pH and osmotic stress, as well as a variety of other conditions including antifungal drug susceptibility. The collection now comprises more than 700 deletion strains. We shall present a comprehensive data set on the phenotypic profiling of the deletion collection in comparison to data from clinical patients isolates. Interestingly, a number of C.g. clinical isolates display high-level resistance to azoles (Fluconazole, Voriconazole and Posaonazole) as well as echinocandins (Caspofungin). Based on the phenotypic data from the deletion collection, we have also generated deletion mutants in clinical isolates to delineate the contributions of specific genes to clinical drug resistance phenotypes.

ª 2014 The Authors Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 2), 15–30

Oral Presentation

V13 Characterization and pathogenicity of Exophiala dermatitidis M. Olsowski, P.-M. Rath and J. Steinmann Institut fu€r Medizinische Mikrobiologie, Universita€tsklinikum Essen, Essen, Deutschland The black yeast-like fungus Exophiala dermatitidis is one of the causes of phaeohyphomycosis and is found in 5-10% of respiratory samples of patients with cystic fibrosis (CF). Furthermore, infections involving the central nervous system have been reported in Asia. The pathogenicity of this fungus from a variety of isolates and sources was analysed using the Caenorhabditis elegans survival model. A total of 22 isolates, compared of seven isolates from invasive infections in Asia, seven isolates from CF patients and eight isolates from environmental sources (Europe and Asia), were characterized by phenotypic and genotypic methods. Six isolates from each of the aforementioned groups were tested using the C. elegans survival model, as well as two melanin-deficient mutants of E. dermatitidis and their corresponding wild type strain. Species identification was done by sequencing of the ITS 1 and ITS 2 region. Molecular typing was performed by repetitive-sequence-PCR (Diversilab). Micro- and macromorphological characteristics were investigated by plating conidia cells onto different media (incubated at 35 °C). Synchronized C. elegans (N2 Strain) L4 and young adult stages were infected by feeding them E. dermatitidis. Cryptococcus neoformans was used as a positive control of infection and E. coli OP 50 (used as non-virulence food source)as a control of nematode lifespan. Infected C. elegans populations were observed daily to count dead and living nematodes over a period of seven days. E. dermatitidis-pathogenicity tests were performed in duplicate and all assays were repeated independently. The analysis of the survival data was performed using the KaplanMeier-Sch€ atzer statistical test. The invasive isolates from Asia showed a rapid growth of hyphal structures within the first 24 h, whereas the isolates from CF patients and the environment only formed a comparable amount of hyphe after 48 h. Genotyping revealed that isolates from CF patients clustered together whereas the isolates from other sources were highly diverse. E. dermatitidis showed pathogenicity in the C. elegans-assay, with varying mortality rates between fungal strains. One of the melanin deficient E. dermatitidis mutants was less efficient in killing C. elegans when compared to its wild type counterpart. Invasive isolates showed a similar pathogenicity when compared to isolates from CF patients and to environmental strains. The C. elegans survival model can be used to analyse the pathogenicity of different E. dermatitidis isolates and their contributory virulence factors. The isolates from Asian patients can be separated from the isolates from CF patients and those of the environment, by means of growth velocity of hyphal structures, therein indicating higher level of pathogenicity.

several medically relevant filamentous fungi. Thus, targeting the HOG pathway may be a promising option for the future treatment of invasive fungal infections.

Pathogenecity & Resistance Factors II V15 Echinocandin resistance in Candida albicans – how point mutations at different positions influence in vitro and in vivo susceptibility to echinocandin treatment M. Lackner,1 M. Tscherner,2 M. Schaller,3 K. Kuchler,2 C. Mair,1 €rl1 B. Sartori,1 F. Istel,2 M. C. Arendrup4 and C. Lass-Flo 1 Division of Hygiene and Medical Microbiology, Innsbruck 2 € Department for Medical University, Innsbruck, Osterreich; Medical Biochemistry, Medical University of Vienna, Vienna, 3 € Eberhard-Karls-Universita€t Tu€bingen, Universita€tsOsterreich; Hautklinik Tu€bingen, Tu€bingen, Deutschland and 4Unit of Mycology, Microbiology and Infection Control, Statens Serum Institute, Copenhagen, Da€nemark Candidemia is the fourth most common microbial bloodstream infection, with Candida albicans being the most common agent. The echinocandins are employed as first line treatment for invasive candidiasis until fungal speciation is confirmed by clinical diagnosis. Echinocandins block the FKS glucan synthases responsible for embedding b-(1,3) D-glucan into the cell wall. The increasing use of these drugs has led to the emergence of antifungal resistance, and elevated MICs have been associated with single residue substitutions in specific hot spot regions of FKS1 and FKS2. Here, we show for the first time, the caspofungin-mediated in vivo selection of a double mutation within one allele of the FKS1 hot spot 1 in a clinical isolate. We created a set of isogenic mutants and used a haematogenous murine model to evaluate in vivo outcome of echinocandin treatment. Heterozygous and homozygous double mutations significantly enhance the in vivo resistance of C. albicans when compared with heterozygous single mutations. The various FKS1 hot spot mutations differ in their MIC increase, substance-dependent in vivo response, and impact on virulence. Our results demonstrate that echinocandin EUCAST breakpoint definitions correlate with in vivo response at standard dosing regimen, but cannot predict in vivo response at dose escalation. Moreover, patients colonized by a C. albicans strain with multiple mutations in FKS1 have a higher risk for therapeutic failure.

V16

The HOG Pathway – a Potential New Drug Target for Anti-Fungal Therapy? €we and F. Ebel A. Wiedemann, A. Lo Max-von-Pettenkofer-Institut/LMU, Mu€nchen, Deutschland

Point mutation in FKS1 gene of Candida albicans leading to echinocandin resistance after echinocandin treatment €nigl,2 B. Ko €lli1 and W. Buzina1 K. Heidrich,1 R. Krause,2 M. Ho 1 Institute of Hygiene, Microbiology and Environmental Medicine, € Medical University of Graz, Graz, Osterreich and 2Department of Internal Medicine, Section of Infectious Diseases and Tropical € Medicine, Medical University of Graz, Graz, Osterreich

The high osmolarity glycerol (HOG) pathway enables fungi to adapt to hyperosmotic conditions. In Aspergillus fumigatus, we have recently identified the type III two-component signalling kinase TcsC as a key player in this signalling pathway. In an ongoing study we have characterized agents that induce a constitutive activation of the HOG pathway and thereby attack the fungus. The biochemical structures of the compounds are surprisingly diverse and in A. fumigatus we observed a complex pattern of cellular responses. A dramatic swelling of the fungal cells is the most obvious consequence of treatment and results in lysis and killing of A. fumigatus hyphae. Interestingly, this anti-fungal activity is not restricted to Aspergillus, but found in

Background The development of pan-echinocandin resistance of Candida albicans isolates with the same MLST (multilocus sequence typing) profile isolated from a candidemic patient prior and after echinocandin treatment is shown. Method Two isolates of C. albicans obtained from the same patient prior and after echinocandin treatment were investigated. Antifungal susceptibility tests were performed both with Etest
and MICRONAUT susceptibility testing system. The ß-1,3-glucan synthase catalytic subunit 1 (FKS1) gene, which is known as a hotspot for mutations leading to reduced susceptibility to echinocandins, was amplified and sequenced. MLST profiles were examined by comparison of the

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ª 2014 The Authors Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 2), 15–30

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Oral Presentation

sequence of 7 DNA sites encoding housekeeping genes: AAT1a, ACC1, ADP1, PMI1b, SYA1, VPS13, and ZWF1b. Results The colonizing isolate obtained after echinocandin treatment showed reduced echinocandin susceptibility whereas the pre-treatment invasive isolate was susceptible to all antimycotics tested. Both isolates shared the same MLST profile, suggesting a high clonal homology. The pre-treatment isolate had no mutations of the FKS1 gene compared to wild type strains, the post treatment isolate showed the S645F mutation in hot spot 1. Conclusions This report documents the development of panechinocandin resistance caused by a point mutation in FKS1 gene after echinocandin treatment.

rare AmB resistance. Heat shock proteins (HSP) are molecular chaperones found in virtually all organisms. They are constitutively expressed, however their expression increases considerably upon stress. A. terreus possesses nine HSP70 family members which are strongly up-regulated by AmB treatment. This study investigated the transcriptional response of HSP70 genes in AmB-resistant and susceptible A. terreus strains. Interestingly AmB-susceptible A. terreus isolates display a different HSP70 response, indicating a function in AmB resistance. Furthermore HSP70 inhibition results in a decreased AmB tolerance in resistant isolates, demonstrating synergistic effects between AmB treatment and HSP70 inhibition.

V19 V17 Targeting mitochondrial respiratory chain and redox status potentiates Amphotericin B-mediated antifungal activity E. Jukic,1 M. Blatzer,1 G. Blum,1 W. Posch,1 H. Hackl,2 €rl1 and D. Wilflingseder1 C. Lass-Flo 1 Hygiene und Medizinische Mikrobiologie, Medizinische € Universia€t Innsbruck, Innsbruck, Osterreich and 2Division of Bioinformatics, Medizinische Universia€t Innsbruck, Innsbruck, € Osterreich Invasive fungal infections have significantly increased over the past decades in immune-compromised individuals and high-risk patients. The first-line drug Amphotericin B (AmB) exerts a powerful and broad activity range against a vast array of fungi and has a remarkably low rate of microbial resistance. Nevertheless, some fungal species are resistant against AmB. Here, we focussed on the intrinsic resistance of Aspergillus terreus against AmB by comparing resistant and sensitive clinical A. terreus isolates. Since AmB-fungicidal action was described to involve production of reactive oxygen species (ROS), we studied the impact of the mitochondrial oxidative phosphorylation (OXPHOS) system upon AmB exposure and subsequent ROS generation. We here illustrated that particularly sensitive strains of A. terreus up-regulated mitochondrial OXPHOS gene expression levels upon AmB treatment, which resulted in an overproduction of ROS to toxic levels. In contrast, upon AmB treatment resistant A. terreus isolates showed decreased mitochondrial DNA contents and ROS levels associated with a better adaption to oxidative stress. Additionally, we examined the impact of N-acetylcysteine and L-ascorbic acid on AmB fungicidal activity, since redox-balancing agents influence detoxification mechanisms. We demonstrated that N-acetylcysteine rescued AmB-induced ROS increase and thus rendered susceptible A. terreus isolates more resistant to the fungicidal actions of AmB. In contrast, L-ascorbic acid exhibited pro-oxidative capacities and ROS levels in resistant A. terreus strains doubled. This was accompanied by a significant decrease of AmB minimal inhibitory concentrations, thereby rendering resistant strains highly susceptible to the drug. Importantly, these results provide novel therapeutical targets to effectively treat drug-resistant fungi.

V18 Members of the Heat Shock Protein 70 family are involved in Amphotericin B resistance in Aspergillus terreus € rtnagl, G. Blum and C. Lass-Flo €rl M. Blatzer, E. Jukic, C. Ho Sektion fu€r Hygiene und Medizinische Mikrobiologie, Department fu€r Hygiene und Medizinische Mikrobiologie/Medizinische € Universita€t Innsbruck, Innsbruck, Osterreich Amphotericin B (AmB) is a first line antifungal polyene drug widely used in clinics to treat life-threatening fungal infections. The mode of AmB action is multifactorial and still has to be elucidated in detail. We investigated the impact of heat shock protein 70 family members in A. terreus, an opportunictic fungal pathogen, which possesses a

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New insights into echinocandin activity against Aspergillus fumigatus K. Dichtl, S. Samantaray, Z. Zhu, F. Ebel and J. Wagener Max von Pettenkofer-Institut, Ludwig-Maximilians-Universita€t Mu€nchen, Mu€nchen, Deutschland Aspergillus fumigatus is an opportunistic pathogen that causes fatal systemic infections in the immunocompromised host. Systemic Aspergillus infections are treated with azoles, polyenes and, possibly, with echinocandins. These three antifungal drug classes directly or indirectly attack the fungal cell wall or associated cell membrane. We characterized a stress response pathway in A. fumigatus, the cell wall integrity (CWI) pathway, which we show to be important for growth, virulence and antifungal drug susceptibility. Interestingly, the echinocandin susceptibility of certain CWI signaling mutants is drastically increased. This suggests a link between the CWI pathway and the glucan synthase, the target of echinocandins. Recently, we characterized the role of the glucan synthase for cell wall biogenesis and viability. Based on our results, we propose a new target structure to increase the antifungal activity of echinocandins against Aspergillus.

Fungus-Host Interaction I V20 Platelets as an innate immune weapon in fungal infections €rl and C. Speth G. Rambach, C. Lass-Flo Sektion fu€r Hygiene und Medizinische Mikrobiologie, € Medizinische Universita€t Innsbruck, Innsbruck, Osterreich In the recent years, platelets have been shown to exert immunological functions and thus to participate in the critical interaction between fungal pathogen and host defence. Platelets can sense Aspergillus (A.) conidia and invading hyphae as well as Aspergillusderived factors and Aspergillus-induced inflammation. As a consequence, platelets undergo activation, they exert direct antifungal effector mechanisms and crosstalk with other arms of the innate and adaptive immunity networks. Nevertheless, there is a fragile balance between beneficial antimicrobial effects and detrimental reactions like thrombosis and exaggerated inflammation, which can modulate the outcome of invasive aspergillosis. Detailed studies were performed to identify the surface structures of A. fumigatus that are responsible for the interaction with platelets. Platelet activation by conidia is at least partly due to melanin, whereas the conidial hydrophobin layer is partly masking relevant components of platelet stimulation. A. fumigatus hyphae also contain surface compounds that interact with platelets. Here, the newly identified fungal polysaccharide galactosaminogalactan was identified to potently trigger platelet activation. Furthermore, A. fumigatus was proven to secrete factors that are capable to induce strong stimulation of the platelets. Two active components in the fungal culture supernatant could be identified: a fungal serine protease and the mycotoxin gliotoxin seem to be involved

ª 2014 The Authors Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 2), 15–30

Oral Presentation

in platelet activation; secreted cell wall polysaccharides might also play a role. Antimycotic drugs may further modulate and influence the interplay of platelets and fungi during invasive aspergillosis.

V21 Role of platelets in invasive Candida infections C. Speth,1 D. Gr€ assle,1,2 A.-L. Krieb,1 K. Pfaller,3 M. Hagleitner,1 1 € C. Lass-Florl and G. Rambach1 1 Sektion fu€r Hygiene und Medizinische Mikrobiologie, € Medizinische Universita€t Innsbruck, Innsbruck, Osterreich; 2 € Medizinische Universita€t Innsbruck, Innsbruck, Osterreich and 3 Sektion fu€r Histologie und Embryologie, Medizinische Universita€t € Innsbruck, Innsbruck, Osterreich Objectives Platelets are part of the innate immunity; their activation after contact with fungal pathogens might result in multifaceted antimicrobial functions, but also in thrombosis and inflammation. Candida species as common inducers of fungal septicaemia can come in close contact with platelets. The subsequent processes of mutual interaction, activation and antimicrobial defence reaction might profoundly influence the outcome of the infection. We therefore aim to elucidate the role of platelets in candidemia. Methods Human platelets were incubated with clinical isolates of different Candida species (albicans, glabrata, parapsilosis, tropicalis, dubliniensis, lusitaniae, rugosa). Candida-platelet-interaction was studied by electron microscopy (SEM), immunofluorescence, and FACS. Platelet activation was investigated by FACS analysis using different markers. Results Platelets are able to interact with Candida both in its yeast and pseudohyphal/hyphal form. The extent varied considerably between different Candida species, with C. glabrata showing the highest interaction rate, whereas platelets barely adhered to C. tropicalis and C. rugosa. Generally, platelets bound to Candida to a lesser extent than to Aspergillus species or mucormycetes. The interaction of platelets with Candida resulted in no or only marginal activation and granule release. Similarly, isolated wall components of C. glabrata stimulated the platelets only in high concentrations. Since many body sites have low oxygen concentrations, we cultivated Candida species under normoxic versus hypoxic conditions; however, their capacity to stimulate platelets did not change. Previous own results had revealed that Aspergillus fumigatus secretes various compounds that stimulate platelets; Candida culture supernatants, however, had no effect on the platelet activity. Conclusion Whereas platelets react on contact with Aspergillus by undergoing activation, only a moderate effect is visible with Candida species. This missing stimulation might result in a lack of platelet-driven antimicrobial reaction. Therefore, our results might at least partly explain the capacity of Candida to survive in the blood stream and to induce fungal sepsis.

V22 Immunological Crosstalk between C.albicans, PMNs and epithelial cells C. Braunsdorf,1 D. Mail€ander-Sanchez,1 J. Wagener2 and M. Schaller1 1 Molekulare Mykologie, Universita€ts Hautklinik Tu€bingen, Tu€bingen, Deutschland and 2Institute of Medical Science, University of Aberdeen, Aberdeen, Vereinigtes Ko€nigreich

depending largely on the host immune system. When epithelial cells are damaged during infection, they release cytokines to recruit and activate PMNs. Therefore immunocompromised people and especially patients with leukopenia have an increased risk for mucosal as well as systemic fungal infections. Using oral RHE model infected with Candida, we observed that PMNs integrated into the Model mediate a protective effect concerning cell death and inflammation. As the PMNs are separated form the infected epithelial cells through a membrane, the effect can only be mediated by soluble factors, released by PMNs. During Candida infection high amounts of LL-37 an important Antimicrobial peptide (AMP) are released by PMNs. AMPs, like LL-37 and Defensins are effector molecules of the host immune system. They often have multifunctional properties being directly antifungal and showing immunomodulatory actions on epithelial cells at the same time. Furthermore they influence cellular proliferation and differentiation and activate the adaptive Immune system, mediating wound healing and control of infection and inflammation.

V23 Cryptococcus neoformans capsule size is associated with the innate immune response during ex vivo whole blood infection € nniger and O. Kurzai J. Dietrich, K. Hu Septomics Research Centre, Friedrich Schiller University Jena, Jena, Deutschland Cryptococcus neoformans is the causative agent of cryptococcal meningitis and disseminated cryptococcosis, which mainly occurs in immunocompromised patients. A key virulence factor of C. neoformans is its polysaccharide capsule which protects the fungus against phagocytosis and complement opsonization. In this study we comparatively investigated the influence of two different capsule sizes on innate immune activation using a human whole blood model of infection. During in vitro culture conditions like YPD, capsule size of C. neoformans is small (Cpslow) compared to a condition reflecting the in vivo situation (RPMI, 5% CO2) that induces a larger capsule (Cpshigh). FACS-based association studies of infected blood revealed that a strong capsule expression prevented phagocytosis of C. neoformans by neutrophils (polymorphonuclear neutrophilic granulocytes, PMN). Whereas Cpslow cryptococci were directly engulfed by PMN after contact Cpshigh fungi were only associated with PMN during the early stages of infection and taken up later. In contrast, interactions of C. neoformans with monocytes were independent of the capsule size. In addition, we analyzed activation of the innate immunity and found a strong and rapid activation of the complement in response to Cpshigh C. neoformans, resulting in a significant increase in C5a already after 10 min of infection. In line with this, formation of reactive oxygen species within PMN and the release of neutrophilic granules were more pronounced in response to Cpshigh fungi. In contrast, levels of pro-inflammatory cytokines like IL-6, TNF-a and IL-1b were significantly elevated in the presence of Cpslow but not Cpshigh cryptococci. Additionally, the larger capsule protected C. neoformans against killing by effector mechanisms of innate immune responses whereas survival of Cpslow was substantially decreased during blood infection. In conclusion, the obtained data demonstrate that the innate immune response strongly depends on the capsule size of C. neoformans with regard to phagocytosis by PMN, innate immune cell and complement activation. Interestingly, capsule size had no influence on the phagocytosis by monocytes.

Cells of our innate immune system recognise invading fungal pathogens via Pattern recognition receptors (PRRs). In the case of fungal infection with the yeast C. albicans, recognition is dependent on TLR2, TLR4, Dectin1,Mannose Receptor, Nod as well as co-receptors like CD14. As Candida can be both Commensal and Pathogen in mucosal sites of the host, immune responsiveness or tolerance are

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Oral Presentation

V24 Iron acquisition of Candida glabrata in the host F. Gerwien,1 L. Kasper1 and B. Hube1,2,3 1 Hans Kno€ll Institute (HKI), Mikrobial Pathogenicity Mechanisms (MPM), Jena, Deutschland; 2Center for Sepsis Control and Care, University Hospital, Jena, Deutschland and 3Friedrich Schiller University, Jena, Deutschland The fungus Candida glabrata is an emerging pathogen with increasing importance in hospital-acquired systemic infections. For survival in the host the exploitation of sequestered trace elements, in particular iron, is essential to sustain cellular processes such as the respiratory chain, oxidative stress response and drug metabolism, where iron is needed as an important co-factor. Hence, restriction in iron availability leads to higher susceptibility to oxidative and osmotic stress. At the same time excess of iron can be toxic. Therefore a tightly regulated network controlling iron metabolism is necessary to prevail in the host and cause disease. Saccharomyces cerevisiae, the best studied relative of C. glabrata, and the less related species C. albicans greatly differ in their iron acquisition systems and regulatory networks. Regarding the use of host iron sources C. glabrata seems to mimic S. cerevisiae, since it is not able to obtain iron from hemoglobin, hemin and transferrin. Ferritin can only be used in an acidic environment. In contrast, the transcription factor Sef1 seems to be important for iron uptake as it is the case for C. albicans, but not for S. cerevisiae. Thus, C. glabrata seems to engage a distinct acquisition and regulation system for iron. We are therefore interested in identifying genes necessary for iron exploitation via screening a C. glabrata mutant library for growth defects under low and near toxic iron conditions. Furthermore the fungus has recently been shown to employ strategies to avoid killing after phagocytosis and to even replicate within macrophages. In this context we aim to unveil the role of host iron sources and C. glabrata -iron metabolism for intracellular survival within macrophages.

still able to cause lethal infection if intravenously applied to mice. This raises the question, to which extend and how C. albicans yeast cells contribute to pathogenesis. Finally, the feasibility of genetic manipulation in mice significantly contributed to our current understanding of how the immune system recognizes C. albicans and which factors are essential for host defence. For example, recent studies revealed that exposure of cell wall components of C. albicans dynamically changes during infection, an effect that cannot be recapitulated in in vitro experiments and underlining the importance of in vivo models for understanding host-pathogen interactions. However, it should also be noted that we do not yet understand to which extend physiological differences between mice and man affect the course and outcome of systemic candidiasis. During bloodstream infection, C. albicans likely interacts with different cellular and soluble components in blood, including neutrophilic granulocytes. Neutrophils are important for the host defence in both human and murine candidiasis. However, the relative proportion of neutrophils in murine blood is significantly lower than in human blood and in vitro studies suggested a lower candicidal efficacy of murine neutrophils. We therefore recently started to analyse the interaction of C. albicans with murine blood and identified several differences to human blood: While C. albicans was killed to a similar extend in blood of both hosts, biphasic killing kinetics with rapid initial killing followed by a late second inactivation phase was observed in murine blood. Furthermore, extracellular killing contributed to a larger extend to killing in murine blood. In addition, murine, but not human, lymphocytes associated with C. albicans, the effects of which are yet unknown. In summary, systemic infection of mice has been a valuable model to better understand pathogenesis of and host-pathogen interaction during candidiasis. The model is generally well characterised, although our understanding of the cellular and molecular mechanisms underlying candidiasis in mice is not yet complete.

V26 Fungus-Host Interaction II V25 Murine models for systemic candidiasis – recent insights into pathogenesis and open questions I. D. Jacobsen Leibniz-Institut fu€r Naturstoff-Forschung und Infektionsbiologie – Hans-Kno€ll-Institut, FG Mikrobielle Immunologie, Jena, Deutschland Murine models have been used for decades to study the different forms of Candida infections. Intravenous infection of mice has become the ‘gold standard’ model to investigate systemic candidiasis and to determine treatment efficacy. Following intravenous application, C. albicans initially infects all organs but is cleared from liver, spleen and lung over time. In contrast, infection progresses in the kidney. It has been suggested that faster recruitment of immune effector cells mediate the protective response whereas the relatively delayed recruitment to the kidney facilitates fungal proliferation. At later time points, infection of the kidney is characterised by massive immune cell infiltration and production of pro-inflammatory cytokines. This contributes to renal pathology but fails to control fungal growth. Distinct macrophage subsets are important for the response in the kidneys; however, which immune components mediate protection in other organs and why hyphae formation occurs in the kidney and brain but not in liver and spleen remains to be determined. Systemic infection of mice has been successfully applied to identify a number of virulence-associated C. albicans factors, with the ability to switch between yeast and hyphal growth as a prominent example. Although mutants locked in either the yeast or hyphal form are generally attenuated in the systemic mouse model and while filamentation is essential for invasion into cells and tissue in vitro and in intraperitoneal murine infection, some filament-deficient mutants are

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Galleria mellonella as a host model to study Aspergillus terreus virulence and amphotericin B resistance €rl3 E. Maurer,1 N. Browne,2 C. Surlis,2 K. Kavanagh,2 C. Lass-Flo and U. Binder1 1 Sektion fu€r Hygiene und Medizinische Mikrobiologie, € Medizinische Universita€t Innsbruck, Innsbruck, Osterreich; 2 Medical Mycology Unit, NUI Maynooth, Maynooth, Ireland and 3 Sektion fu€r Hygiene und Medizinische MIkrobiologie, € Medizinische Universita€t Innsbruck, Innsbruck, Osterreich Infections with Aspergillus terreus are of major concern, due to its high likelihood of dissemination and its intrinsic resistance to amphotericin B (amB). The reason for this resistance is not known yet. Recently, three clinical isolates, with distinct morphological variations, have been found to be amB susceptible in vitro. Efficacy of amB treatment and its influence on the larval immune system were investigated in the invertebrate model G. mellonella. Proteomic analysis of larval haemolymph, haemocyte counts and post-treatment infection studies were performed. Additionally, putative difference in virulence potential of the respective isolates was analyzed by correlating survival rates with physiological attributes and in vitro killing ability of larval haemocytes. Treatment with amB only showed success in the groups infected with amB-susceptible strains, which reflected our in vitro data. Furthermore, amB administration resulted in an increased number of circulating haemocytes. Proteomic studies showed different protein expression of proteins which have immune function. Pretreatment of larvae with amB also increased their resistance to Staphylococcus aureus infection, indicating a general ability of amB to prime the insect0 s immune system. Larval survival rates differed in the early time points of infection for the various isolates tested. The amB-resistant isolate T90, showed lowest mortality rates in the early time points of infection. This is in correlation with a slower germination rate of T90, and the highest fungal damage caused by haemocytes in vitro.

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Oral Presentation

This work demonstrated that G. mellonella is a useful model to determine the in vivo efficacy of amB against A. terreus isolates, and study the virulence potential of different isolates. Our results showed that antifungal treatment in vivo correlates with in vitro susceptibility data. Furthermore, one has to be aware of a non specific influence of antifungal drugs on the larval immune response.

V27 The two major fungal sepsis pathogens C. albicans and C. glabrata trigger different innate immune responses in a human whole blood model €nniger and O. Kurzai L. Bauer, K. Hu Septomics Research Centre, Friedrich Schiller University Jena, Jena, Deutschland Candida albicans and Candida glabrata are the most important pathogens in nosocomial fungal sepsis. Both species are commensals of the same niches and opportunistically pathogenic under similar conditions. Despite various similarities, these strains differ on key attributes as C. albicans is able to morphologically switch between the yeast and filamentous form which mediates tissue invasion and governs activation of immune cells. In contrast, C. glabrata does not form filaments in vivo. In this study, a human whole blood model was used to compare innate immune activation in response to C. albicans and C. glabrata in a situation similar to in vivo. During blood infection, both fungal pathogens predominantly associated with neutrophils (polymorphonuclear neutrophilic granulocytes, PMN). Microscopic analyses showed that most PMN which had phagocytosed contained a single C. albicans cell which was in contrast to C. glabrata infected samples where PMN often contained more fungal cells. Although low interactions with monocytes could be observed, the median association of C. glabrata with these phagocytes was higher than of C. albicans. Analyses of the early innate immune activation in response to both pathogens revealed significantly differences. C. albicans resulted in a more rapid and pronounced generation of C5a compared to C. glabrata. This was accompanied by a stronger up-regulation (CD66b, CD11b) and down-regulation (CD16) of activation markers on PMN surface after confrontation with C. albicans. Differences in PMN activation were also reflected by secreted antimicrobial effector proteins and cytokines. Levels of myeloperoxidase and elastase-2 from primary and lactoferrin from secondary neutrophilic granules as well as chemokines preferentially secreted by PMN (IL-8, MIF) were higher in response to C. albicans. In contrast, during C. glabrata infection we could detect higher release of pro-inflammatory (IL-1b, TNF-a) and regulatory cytokines (IFN-c, IL-12p70). Despite the strong PMN activation after contact to C. albicans, C. glabrata was more sensitive to innate immune activation and became more rapidly and efficiently killed. These data clearly reveal a differential activation of innate immunity in response to the two major pathogenic Candida species.

V28 The absence of NOD1 protects mice against invasive aspergillosis through enhanced fungal killing M. Gresnigt,1 A. Ammerdorffer,1 D. De Jong,2 T.-D. Kanneganti,3 O. Ibrahim-Granet4 and F.van de Veerdonk1 1 Department of Internal Medicine, Radboud University Nijmegen Medical Center, Nijmegen, Niederlande; 2Department of Gastroenterology and Hepatology, Radboud Universtity Nijmegen Medical Center, Nijmegen, Niederlande; 3Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Vereinigte Staaten von Amerika and 4Devision de Cytokines et Inflammation, Institut Pasteur, Paris, Frankreich Invasive aspergillosis is remains one of the most severe complications in immunocompromised patients. New insights in immunological

ª 2014 The Authors Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 2), 15–30

pathways are required to enable the design of targeted immunotherapy to improve the host antifungal immune responses. Several studies demonstrate that the intracellular pattern recognition receptor nucleotide-binding oligomerization domain (NOD)2 plays an important role in the host defense against Aspergillus. We investigate how human NOD2 deficiency influences the host response to A. fumigatus. Additionally, we investigated how deficiency of NOD2, and the other NOD receptor, NOD1 influences the susceptibility to invasive aspergillosis. We found that PBMCs of NOD2
/
patients responded with both decreased innate and T-cell cytokine responses to Aspergillus stimulation compared to healthy individuals. This decreased production of Tcell cytokines correlated with a decreased proliferation of IL-17+, IL22+, and IFN-gamma+ T-cells upon stimulation with Aspergillus. Suggesting that NOD2 plays an important role in the human host response to A. fumigatus. To investigate the importance of NOD2 and NOD1 in the antifungal host defence against Aspergillus, we infected deficient mice with bioluminescent A. fumigatus. Compared to wildtype, both NOD1 and NOD2 deficient mice were protected against invasive aspergillosis. However, NOD2 deficient mice demonstrated severe illness and inflammatory responses, while NOD1 deficient mice rapidly cleared the infection. We subsequently found that NOD1 deficient cells are hyperinflammatory and more efficient at killing A. fumigatus. Collectively, our data demonstrates that the NOD receptors are differentially regulating cytokine signaling in response to Aspergillus. On the one hand NOD2 is required for induction of responses while on the other hand NOD1 seems to be inhibitory for cytokine responses and fungal killing. These data suggest a potential for immunomodulatory therapies that target NOD1 in order to enhance the host response for more efficient fungal clearance.

V29 Candida glabrata virulence and stress resistance determinants C. Schu¨ller Universita€t fu€r Bodenkultur, Institut fu€r angewandte Genetik und € Zellbiologie (DAGZ), Tulln a.d. Donau, Osterreich Candida glabrata as a human opportunistic pathogen has acquired specific properties to support its proliferation in various host environments. Furthermore, the organism has gained the attribute of beeing stress resistant - compared to its relative baker0 s yeast. This is due to the contribution of canonical stress response pathways and mechanisms as well as a number of specific properties such as adhesion, which will be discussed. Recently, insights from genomic and reverse genetic analysis revealed novel virulence determinants. Exiting new forward genetic approaches will soon be possible once the organism has been transformed to undergo a sexual cycle. The contribution of stress response mechanisms to the Cg lifestyle will be presented from a yeast perspective.

Diagnostics II V30 Diagnostic potential of serologic markers and the Aspergillus lateral flow device test for invasive fungal infections M. Hoenigl Sektion fu€r Infektiologie und Tropenmedizin, Medizinische Universita€t Graz, Graz Galactomannan (GM) is a polysaccharide component of the cell wall of Aspergillus spp. that is released into the bloodstream by growing hyphae and germinating spores/ conidia. GM is clinically used in

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Oral Presentation

serum, bronchoalveolar lavage (BAL) fluid specimens, lately also urine, and CSF samples. Another target antigen for IFI diagnosis is ß-D-glucan (BDG), a cell wall component of most pathogenic fungi (e.g., Aspergillus spp., Candida spp.), except Mucorales and Cryptococci. Mannan antibody and Mannan antigene or combination of both is another serological marker, which turns positive exclusively in Candida infections (in particular invasive candidiasis and disseminated forms of Candida infections). One of the major limitations of both the GM and the BDG tests is that time to results varies between centers (ranging from less than a day to several days), depending on the number of specimens to be tested and the distance/duration of transport between the clinical setting and the laboratory where the test is performed. These limitations are overcome by the lateral-flowdevice (LFD) test, a new point-of-care test for IPA diagnosis developed at the University of Exeter, United Kingdom. This single-sample test, based on the detection of Aspergillus antigen by the monoclonal antibody (MAb) JF5, can be performed easily in every laboratory using BAL fluid or serum specimens, and it has a time to results of approximately 15 min. Recent studies have shown the greatpotential of this test with human BAL fluid and serum samples.

V31 Serum and Urine Galactomannan testing for screening in patients with hematological malignancies € ttmann,2 C. Koidl,3 S. Eigl,1 L. Fischbach,1 J. Prattes,1 W. Du 2 2 € lfler,2 B. Huber-Krassnitzer, P. Neumeister, J. Wagner,1 A. Wo R. Krause1 and M. Hoenigl1 1 Section of Infectious Diseases and Tropical Medicine, Medical 2 € Division of Hematology, University of Graz, Graz, Osterreich; € Medical University of Graz, Graz, Osterreich and 3Institute of Hygiene, Microbiology and Environmental Medicine, Medical € University of Graz, Graz, Osterreich Background Serum galactomannan (GM) has been established as an important method for diagnosing invasive aspergillosis (IA). In this study we evaluated performance of GM test in urine specimens and compared results with those obtained in serum. Methods The study was performed from July 2012–May 2013 in adult patients with underlying hematological malignancies hospitalized at the Department of Haematology at the Medical University of Graz in Austria and was approved by the local ethics committee. Serum and urine samples were collected and tested twice weekly (always on the same day). For serum samples an optical density index (ODI)⊇ 0.5 was considered positive. Results Each 253 serum and urine samples were collected in 81 cases. 23/253 (9.1%) serum samples from 13 patients resulted GM positive. Sensitivity, specificity, PPV and NPV for different cut-offs for urine samples (compared to serum results) were as follows: 0.20 ODI: 17.4%, 93.9%, 22.2%, 91.9%; 0.15 ODI: 30.4%, 90.7%, 25%, 92.8%; 0.1 ODI: 47.8%, 85.2%, 24.4%, 94.2%. A cutoff of 0.1 ODI was chosen for urine samples. 12/23 positive serum GM samples gave negative results in urine; 4 of those were derived from a patient with increasing positive serum GM levels in whom urine samples became positive with a 2 week delay. In 2 probable IA cases initially positive urine samples became negative under appropriate antifungal therapy while decreasing serum samples remained positive. Four serum samples were considered false positives, two urine sample false negatives. Urine resulted positive in 34/230 negative serum GM samples. 28 of those were considered potential false positives, 14/28 were obtained in patients with possible IA. In the other six samples urine GM resulted either positive before serum GM (2 patients) or remained positive while serum GM was already negative again. Conclusions A significant positive correlation was found between urine and serum GM results. Further studies are needed to evaluate the potential role of urine GM testing in IA diagnosis.

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V32 The actual role of PCR in the diagnostics of invasive fungal infections M. Lengerova,1,2 I. Kocmanova3 and Z. Racil1,2 1 Department of Internal Medicine - Hematology and Oncology, University Hospital Brno, Brno, Tschechische Republik; 2Faculty of Medicine, Masaryk University, Brno, Tschechische Republik and 3 Department of Clinical Microbiology, University Hospital Brno, Brno, Tschechische Republik PCR diagnosis of invasive fungal infections is still a hot topic. Despite the great efforts made in the past twenty years, there is still the challenge of finding a suitable clinical material, efficient DNA isolation methods and the correct interpretation of the results. Interesting topics include correlation of PCR results with traditional microbiology methods (culture, microscopy) and detection of other biomarkers (galactomannan, glucan) or early PCR detection and identification of rare etiological agents of IFD. A special chapter consists of the use of PCR for the detection of mutations leading to resistance to antifungal agents. The aim of this lecture is to summarize current knowledge about variants PCR used for the diagnosis of IFD, the critical points of this methodology, expert recommendations and our own experience. Supported by Ministry of Health, Czech Republic - conceptual development of research organization (FNBr, 65269705).

V33 Secretome analysis of human pathogenic dermatophyte species and identification of immunoreactive proteins as potential candidates for novel diagnostic tools C. Eymann,1 G. Wachlin,1 D. Albrecht,1 S. Tiede,2 U. Krummrei,3 €nger,4 M. Hecker1 and G. Daeschlein4 M. Ju 1 Institut fu€r Mikrobiologie, Ernst-Moritz-Arndt-Universita€t Greifswald, Greifswald, Deutschland; 2Institut fu€r Experimentelle Immunologie, EUROIMMUN AG, Lu€beck, Deutschland; 3 EUROIMMUN Medizinische Labordiagnostika AG, Lu€beck, Deutschland and 4Klinik und Poliklinik fu€r Hautkrankheiten, Ernst-Moritz-Arndt-Universita€t Greifswald, Greifswald, Deutschland Introduction Dermatophytes are highly specialized pathogenic fungi that cause the most common superficial mycoses in humans. These microorganisms only infect and multiply within keratinized host structures - for example, the skin’s epidermal stratum corneum, nails or hair - a characteristic that is putatively related to their keratinolytic activity. An increasing incidence of onychomycosis and tinea pedis caused primarily by Trichophyton rubrum and T. interdigitale strains and insufficient diagnostic and therapy require fast and specific diagnostic tools and effective therapy monitoring to prevent relapses, distribution, and to cure also chronically infected patients. Results Pure cultures of different Trichophyton species sampled from affected individuals were made and characterized genetically. Subsequently we analyzed the secretome of different T. rubrum- and T. interdigitale anthropophil-strains as well as one T. verrucosum-strain under defined in-vitro conditions to identify secreted putative virulence factors or antigens that react with the patients’ serum antibodies. Secreted proteins from these strains were separated via High-Performance-Electrophoresis-gels (HPE, SERVA) and identified by mass spectrometry. More than 100 different proteins including important virulence factors such as several known peptidases (keratinases) and hydrolases (e.g. lipases, glucosidases, one ceramidase) as well as uncharacterized proteins could be identified. In addition to typical secreted proteins (predicted by PSORT, SignalP) several putative intracellular proteins were also detected. Thereby significant similarities of protein patterns within the strains of T. rubrum and T. interdigitale anthropophil respectively and significant differences between the three species were monitored. The same protein extracts

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Oral Presentation

were analyzed by Western blotting with the respective patients’ serum antibodies to detect immunogenic proteins. In comparison to control sera up to 16 proteins showed significant antigen-antibodyreactions. Among them are peptidases as well as several oxidoreductases such as the putative extracellular thioredoxin reductase F2STQ1. One protein, the beta-glucosidase F2SZI9, seems to be a common immunoreactive antigen in all Trichophyton infections. Outlook The cross reactivity within the strains of one species, between the different species and between anthropophil and zoophil strains has to be tested as well as promising candidate proteins should be validated for new diagnostic methods based on antigenantibody-reactions in future serological tests.

Indoor & Environmental Mycology V35 Distribution of fungal damages in houses after severe water damage J. Rainer Institut fu€r Mikrobiologie, Leopold-Franzens Universita€t, € Innsbruck, Osterreich Damages after water pipe bursts or mismanagement of rainwater cause severe problems with fungal contamination in many occasions. Especially health affecting fungi like Stachybotrys chartarum, Aspergillus fumigatus and A. versicolor play a major role in this context. But also wood degrading fungi, i.e. Serpula lacrymans or Coniophora puteana may not be overlooked. In larger buildings with complex water entry the question concerning predictable distribution patterns of fungi may arise, as excessive opening of building elements is expensive and time consuming. The distribution of fungi throughout buildings after water damages will be discussed retrospectively on the basis of three cases. The first case was connected to water entrance in the basement from an unknown source in a new building and the contamination of drywall installations. This installations were contaminated with S. chartarum and other fungi belonging to the genera Aspergillus and Penicillium. The second case was caused by rainwater entering from a terrace into the floor construction. This led to massive increase of the fungal burden in the air of the apartments and the floor construction. Main contaminants were A. versicolor and Pencillium spp. The third case deals with a mixed infestation of a wooden house affecting three floors with a wood degrading fungus, Gloeophyllum sp., and strains of Chaetomium due to insufficient sealing of exit pipes. The distribution patterns of the fungi will be presented and probable factors affecting growth are dicussed.

V36 Interaction between Airborne Microorganisms and Particulate Matter D. Haas,1 H. Galler,1 J. Luxner,1 G. Zarfel,1 W. Buzina,1 H. Friedl,2 A. Grisold,1 J.-S.-M. Habib1 and F. F. Reinthaler1 1 Institute of Hygiene, Microbiology and Environmental Medicine, € Medical University of Graz, Graz, Osterreich and 2Institute of € Statistics, Graz University of Technology, Graz, Osterreich Background Airborne culturable microorganisms occur and adhere to particulate matter in the ambient air. This study was conducted to examine the correlations between particle counts, culturable fungi and bacteria concentrations, and their dependence on air temperature and relative humidity. Seasonal variations and the concentrations of the fungal genera Cladosporium, Aspergillus and Penicillium were also evaluated. Method Air samples were collected with the particle counter APC M3 and the one stage impactor MAS
100 with a flow rate of 100 l min
1.

ª 2014 The Authors Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 2), 15–30

For fungal examination the total of 354 samples were incubated on Dichloran Glycerol agar at 25 °C for 7 days. For bacteria the samples were incubated on Tryptic soy agar at 30 °C for 48 h. Fungal colonies were identified by macro-morphology and microscopically. Result The mean concentrations of particulate matter were the highest in January and February with 8.9 9 107 particles m
³ and decreased continuously to a minimum in July and August. The fungal spore concentrations were between 3.0 9 101 and 2.3 9 10³ cfu (colony forming units) m
³ air and reached the maximum in the summer months. It was found that the fungal spore concentrations were high at a temperature of 30 °C and a relative humidity of 70–80%. The attained concentrations of bacteria were between 0 and 2.5 9 10³ cfu m
³ and were positively correlated with the particulate matter. The spore concentrations of Cladosporium spp. increased with rising air temperature but were independent of relative humidity and did not correlate with the particulate matter. The spore concentrations of Penicillium spp. and Aspergillus spp. increased with increasing concentrations of particles and reached their maximum at a relative humidity of 70%, whereas the air temperature did not show a significant impact. Conclusion Airborne microorganisms interact continuously with particulate matter in the ambient air. The season, temperature and relative humidity play an important role in the release and distribution of particles and fungi in the air.

V37 Sequence analysis of the rDNA-ITS region of 50 wood decay fungi for development of probes for LCD-Array diagnostics N. Rangno,1 V. Heiser,2 G. Thiele,2 K. Jacobs,1 P. Langensiepen1 and W. Scheiding1 1 Mykolabor Dresden im IHD, Mykologie, Dresden, Deutschland and 2Chipron GmbH, Berlin, Deutschland Due to their dominant role during wood decay and degradation wood decaying fungi (Basidiomycota and Ascomycota) are of great ecological and economical importance. These fungi can attack and degrade standing trees and stored wood in the forest, like Heterobasidion annosum, can cause extensive damage on loadbearing constructions of buildings, like Serpula lacrymans, and some of them can even cause dangerous diseases in humans (allergic bronchopulmonary mycosis, pulmonary fungus ball, meningitis, brain abscess and onychomycosis), like Schizophyllum commune. Additionally, some wood decaying Ascomycota (Chaetomium and Paecilomyces species) are known to produce mycotoxins. A correct identification of wood decay fungi on species or genus level by conventional methods (e.g. macroscopic and microscopic analysis) requires outstanding expert knowledge and will not always lead to unambiguous results. Meanwhile, modern methods used in molecular diagnostics like DNA sequencing, PCR, RFLP, SSCP, DHPLC and DNA Microarrays have significantly contributed to improve and fasten species identification of wood decay fungi. However, analysing samples from building environments or natural microbiotopes in routine set-ups is still challenging because of the complexity of the starting material. The Mykolabor Dresden of IHD and Chipron GmbH Berlin are developing the preconditions for an inexpensive and robust, non fluorescent PCR-DNA macroarray (LCD-Array) for the identification of 50 wood decay fungi on species or genus level. This paper describes the analysis of sequence data from the nuclear internal transcribed spacer regions (ITS1 + ITS2) of the 50 most common wood decay fungi and its potential for the development of specific hybridization probes on LCD-Arrays. First results using reference material and a broad selection of samples from the built environment will be presented.

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Oral Presentation

V38 Evaluation of antimicrobial surface coatings S. Finger,1 M. Zieger,1 C. Wiegand,1 C. Rode,2 R. Wyrwa,2 €nler2 and U.-C. Hipler1 B. Gru 1 Klinik fu€r Hautkrankheiten, Universita€tsklinikum Jena, Jena, Deutschland and 2INNOVENT e. V., Jena, Deutschland

Behandelbarkeit der meist immunsupprimierten Patienten mit hoher assoziierter Sterblichkeit macht Ausbr€ uche nosokomialer Mykosen zu einem krankenhaushygienischen Ausnahmesituation, die ein rasches Eingreifen erfordert.

V40 Antimicrobial coatings are commonly used in household, food industry and hospital settings and can be applied to various materials using different antimicrobial substances. Subsequently, the functionality of objects with such special finishes has to be determined. Different methods are available for this and there is a wide range of national and international standard testing methods. The International Standard ISO 22196 e.g. describes the measurement of antibacterial activity on plastics surfaces. But it can be modified to test other materials employing a variety of test organisms including fungi. In this study, the ISO 22196 standard was modified to determine the antimicrobial activity of functionalized ceramic surfaces against the yeasts Candida albicans and Candida glabrata, the dermatophyte Trichophyton rubrum and the mould Aspergillus fumigatus. Samples with ZnO coating were tested according to the modified ISO 22196 for their antifungal activity. In brief, samples of 50 mm 9 50 mm were inoculated with Candida albicans (DSM 1386), Candida glabrata (DSM 11226), Trichophyton rubrum (70K10) and Aspergillus fumigatus (DSM 819), respectively. Basic materials without coating were used as control. Samples were incubated for 24 h at 37 °C under aerobic conditions. Thereafter, the samples were washed and serial dilutions were plated on agar plates. Colonies were counted after 24 h at 37 °C. It was shown, that antimicrobial coatings on ceramics exhibit antifungal activity against Candida albicans, Candida glabrata and Aspergillus fumigatus. However, the coatings tested here demonstrated no antifungal effect on Trichophyton rubrum.

Epidemiology & Prevention V39 Outbreaks of nosocomial mycoses C. Wendt MVZ Labor Limbach, Heidelberg, Deutschland Eine Suche bei Medline mit den Stichworten ‘nosomial outbreak’ ergibt nahezu 2800 Treffer, jedoch finden sich darunter nur etwas 100 Beschreibungen von Ausbr€ uchen nosokomialer Mykosen. Eine Abfrage der outbreak database (www.outbreak-database.com) ergibt 164 beschriebene Ausbr€ uche von Mykosen unter mehr als 3000 dort zusammengestellten Ereignissen. Am h€ aufigsten sind Ausbr€ uche durch Candida und Aspergillen beschrieben, gefolgt von Pneumocystis, Pichia und Trichophyton. Dabei waren die h€ aufigsten Infektionen die Sepsis, Pneumonien und Haut und Weichteilinfektionen. Die Anzahl der betroffenen Patienten war in der Regel gering und betraf zwischen 2 und 6 Patienten zumeist aus der H€amatologie/Onkologie, der Chirurgie und der Neonatologie. Risikofaktoren umfassten die Grunderkrankung der Patienten, Antibiotische Vorbehandlung und anderes Therapien, liegende € Zug€ ange und Prozeduren. Als h€aufigster Ubertragungsweg wurde der direkte oder indirekte Kontakt gefolgt von Inhalation und invasiven Prozeduren beschrieben. H€ aufig eigesetzte Maßnahmen der Bek€ amp€ fung der Ausbr€ uche waren Anderungen in der antibiotischen Thera€ pie, Anderungen in Behandlung oder Ausr€ ustung, Surveillance, H€ andedesinfektion und Fl€achendesinfektion. Damit unterscheiden sich die zusammengefassten Mykosen nicht wesentlich von anderen nosokomialen Infektionserregern. Es f€ allt jedoch auf, dass es in mehr als der H€ alfte der beschriebenen Ausbr€ uche zu Todesf€ allen unter den Patienten kam, die in einem Viertel der Ausbr€ uche mehr als 2 Patienten betrafen. Die schlechte

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Epidemiology of fungal species in respiratory samples of cystic fibrosis patients over a 5 year period – data from a national reference center L. Sedlacek, S. Suerbaum and S. Ziesing Medizinische Hochschule Hannover, Institut fu€r Med. Mikrobiologie und Krankenhaushygiene, Hannover, Deutschland Background Cystic fibrosis (CF) patients are often colonised with fungal species in the respiratory tract. Due to an enhanced awareness of rare moulds fungal diversity in cystic fibrosis microbiological samples seems to increase. The aim of this study was to give a retrospective overview about the detected fungi and their diversity in microbiological samples of CF patients in a CF reference laboratory over a 5-year period. Materials/Methods All fungal isolates in microbiological specimen from CF-patients during 2009–2013 were analysed. Beside standard media for bacterial culture, a non selective fungal medium (yeastpeptone-agar) and an additional selective Scedosporium-agar (SceSel+- agar) were inoculated and incubated for 10 days at 36 °C. Yeasts were identified by microscopy, biochemical profiling, MALDITOF analysis or DNA-sequencing methods. Moulds were identified by morphological criteria according to standard macroscopic and microscopic descriptions. If required, species identification procedures further included sequencing and analysis of the fungal ITS/beta-tubulin region. Each species has been counted only once per year and patient. Results In total 25 975 clinical samples from CF-patients during the observed time period were examined. In average there were 5195 samples from 637 CF patients annually. In mean about 1979 samples were collected from in-house patients at Hannover Medical School, 3400 from outpatients per year. Overall 75% of CF-patients were colonized by yeasts, mainly Candida albicans(38%), Candida dubliniensis (12%), Candida glabrata (9%), Candida parapsilosis (3%), Candida lusitania (2%) and Candida krusei (1%). Further data for rarely found yeasts (e.g. Trichosporon spp.) will be shown. Moulds: 35% of CF-patients were tested positive for growth of Aspergillus spp. [Aspergillus fumigatus (29%), followed by Exophiala dermatitidis and Scedosporium/Pseudallescheria boydii complex isolates (4% each)]. Data on the occurrence of rare moulds (e.g. Rasamsonia argillacea) will be presented. Annually frequencies of each yeast and mould species detected in 2009–2013 will be shown. Conclusion This retrospective analysis of yeasts and moulds in microbiological samples of CF patients gives an overview of fungal colonisation in CF-airways over a 5 year period. We couldn’t detect any new emerging fungal pathogens in CF-patients nor did we find any epidemiological changes. Nevertheless, microbiological laboratories should be aware of uncommon species. The clinical relevance of rare isolates has to be evaluated critically in concordance with clinical symptoms, immune status of the patient and available data regarding the pathogenicity of these fungal species.

ª 2014 The Authors Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 2), 15–30

Oral Presentation

V41 Molecular Epidemiology of Cryptococcosis in Germany: 2004–2010 A. Sanchini,1 I. McCormick Smith,1 L. Sedlacek,2 R. Schwarz,3 K. Tintelnot1 and V. Rickerts1 1 Robert Koch Institut, FG 16, Berlin, Deutschland; 2MHHannover, Mikrobiologie, Hannover, Deutschland and 3MVZ Labor Stein und Kollegen, Mo€nchengladbach, Deutschland Background Cryptococcosis is a fungal infection mostly caused by Cryptococcus neoformans. We identified agents of cryptococcosis diagnosed in Germany from 2004 to 2010. and used multi-locus sequence typing (MLST) to understand the molecular epidemiology of cryptococcosis. Methods Sero- and mating types of individual patient isolates were determined by PCR. MLST was performed using a seven-locus ISHAM scheme. Allele- and nucleotide diversity was calculated for each locus of C. neoformans var. grubii and C. neoformans var. neoformans. Phylogenetic relations were assessed by dendrograms. Clinical data were compared between infections caused by the two variants. Results We studied 101 isolates. Eight were identified as hybrids (8%). All non-hybrids were of the alfa-mating type. Among 78 C. neoformans var. grubii (77%), 16 sequence types (ST) were identified including three novel STs. They clustered in four groups, previously isolated in Asia, Europe or worldwide. Among 15 C. neoformans var. neoformans (15%), 10 STs were identified, without clustering. These isolates showed higher allele and nucleotide diversity compared with C. neoformans var. grubii. C. neoformans var. neoformans was more likely to cause soft-tissue infections (3/9, 33% vs. 1/63, 2%, P = 0.005) and to affect non-AIDS patients (7/14, 50% vs. 15/76, 20%, P = 0.036). Conclusions C. neoformans var. grubii is the predominant agent of cryptococcosis in Germany. MLST suggests that a part of these cases are acquired abroad by immigrants or tourists. C. neoformans var. neoformans isolates represent a greater genetic diversity and are associated with more variable clinical presentations.

V42 Vaccination against opportunistic fungi: chance or utopian dream? C. Speth Sektion fu€r Hygiene und Medizinische Mikrobiologie, € Medizinische Universita€t Innsbruck, Innsbruck, Osterreich Despite available antifungal compounds, morbidity and mortality of invasive fungal infections are still unacceptably high in immunocompromised patients. An efficient strategy to protect these at-risk patients from fungal infections could involve the induction of a specific immune response with an antifungal vaccine. Numerous animal trials performed in the last years with vaccination strategies against Aspergillus and Candida imply that immunoprotection against the fungal pathogens is possible and can be achieved with defined fungal antigens. The present understanding of antifungal immune responses as well as the state of the art for translation of this understanding into vaccine approaches are reviewed.

ª 2014 The Authors Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 2), 15–30

Dermatomycoses I V43 Dermatomycology – File Number XY solved – Have you already known? M. Schaller Universita€ts-Hautklinik, Tu€bingen, Deutschland Whereas typical mycotic infections are easy to diagnose, there are also patients with uncharacteristic manifestations of fungal diseases of skin, hair and nails which are difficult to identify. Such diagnostic difficulties are mainly caused by pre-treatment or immunosuppression. Identifying such diagnostic situations in order to guarantee adequate treatment is the intention of this lecture. Identifying fungal pathogens, especially the rare non dermatophytic pathogens, which cause nail fungus, is another problem. An example is the detection of mould as causative pathogen in onychomycosis. Sophisticated diagnostic measurements are indispensable prerequisites for a successful differentiation between mould and dermatophytes. Furthermore, to identify mould either as simply impureness or pathogenetic agent that causes disease, laboratory results and clinical findings must be evaluated professionally and precisely.

V44 The paradigm with Candida and the nail G. Ginter-Hanselmayer € Univ. Klinik fu€r Dermatologie und Venereologie, Graz, Osterreich Fungal nail infection may be caused by dermatophytes (Tinea unguium), yeasts and non-dermatophyte moulds (NDM). Because yeasts are opportunistic fungi their isolation needs to be interpreted contrary with concern to the state of colonization vs. true infection. In general yeast pathology remains defined to fingernails and is without therapeutic sequelae when isolated from toenails. Due to the multifactorial etiopathogenesis and different features there is no standardized classification for Candida-associated nail infections. Clinical presentations comprise nail infection (Candidal onychomycosis sive granuloma), Candidal paronychia and Candidal onycholysis, respectively. Candida onychomycosis is a scarce condition with only the immunosuppressed – f.e. patients suffering from chronic mucocutaneous candidosis (CMC), or female patients with Raynaud0 s disease or endocrine disturbances seem to be affected. Onychomycosis due to Candida may present with different clinical features like subungual onycholyses (DLSO) with accompanying paronychia or erosion of the distal and lateral nail plate. Both conditions will lead to a totally dystrophy oft he nail plate with invasion oft he entire nail plate by yeasts, in general by Candida albicans. Other Candida-spp. capable to infect the nail unit are Candida krusei, Candida parapsilosis, Candida tropicalis, Candida guilliermondii as well as Candida glabrata. Diagnosis of candidal onychomycosis depends on history and clinical examination, mycology and nail biopsy. Candidal paronychia presents in general a chronic course. One or several, in severe cases all fingers are affected. The isolation of yeasts in patients suffering from paronychia remains to be interpreted contradictory. Because the hands of these patients are frequently exposed to water traumatic alteration of the proximal nailfold allows penetration of bacteria or environmental irritants. Beside that other pathogenetic aspects like an immediate hypersensitivity reaction (contact dermatitis) to food allergens are discussed. In the background of this scenario true (primary) Candida infection seems to be rare with Candida hypersensitivity more reliable. Because isolation of Candida may be secondary (colonization), antifungal treatment does not usually produce any improvement. Topical steroids as well as avoidance of irritants and consecutively hand protection will result in a much better outcome and should

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Oral Presentation

therefore be taken into consideration. Successful treatment remains a challenge for the patients affected as well as the for the mycologist. References 1. Jayatilake JAMS, Tilakaratne WM, Panagoda GJ. Candidal onychomycosis: a Mini-Review. Mycopathologia 2009;168: 165–173. 2. Elewski BE. Onychomycosis: pathogenesis, diagnosis, and management. Clin Microbiol Rev. 1998;11:415–429. 3. Robert DT, Taylor WD, Boyle J. Guidelines for treatment of onychomycosis. Br J Dermatol. 2003;148:402–410.

V45 Cutaneous candidiasis – current state and perspectives M. Dolenc-Voljc Department of Dermatovenereology, University Medical Centre Ljubljana, Ljubljana, Slowenien Cutaneous candidiasis is one of the most common fungal skin infections. In the last decade, we have observed a rising incidence of cutaneous candidiasis in patients treated at our department. As an opportunistic skin infection, it can appear on the background of many clinical conditions. Aging of the population, HIV infection, systemic and local immunosuppression, diabetes, obesity, and treatment with systemic antibiotics are the most common predisposing factors. Local factors that create a warm and moist milieu predispose women more often to candidiasis of the hands and fingernail onychomycosis. Clinically, it can present with diverse clinical forms, from mild to severe candidial intertrigo, as a diaper dermatitis, angular heilitis, paronychia, onychomycosis, folliculitis, and as a genital candidiasis. It can be accompanied by infection of the mucous membranes or a manifestation of chronic mucocutaneous candidiasis. In immunosuppressed patients, unusual and atypical clinical manifestations have been observed. Occasionally, the skin can present the site of entry for systemic candidiasis. Most commonly, cutaneous candidiasis is caused by Candida albicans. Some other yeasts of the genus Candida, especially Candida parapsilosis, Candida guilliermondii, Candida tropicalis, and Candida krusei have become of significant importance as well. Non-albicans yeasts may be more resistant to azole antimycotic drugs and therefore a matter of concern. Mycological diagnosis should include identification of the causative yeast. Since positive culture does not always indicate the presence of Candida infection, especially in nail diseases, mycological results should be interpreted cautiously and in correlation with the clinical picture. Sensitivity testing against antimycotic agents is advised in cases, resistant to standard treatment. Current treatment recommendations include topical treatment with polyene and azole antifungal drugs. In chronic or widespread infections, systemic treatment with fluconazole and itraconazole is needed. Infections with non-albicans yeasts, resistant to standard treatment, may need a modified treatment approach. To prevent recurrences, treatment strategy should be individualised, considering also the patient’s predisposing factors.

V46 Vestibulodynia can be triggered by Candida albicans vulvovaginitis W. Mendling Deutsches Zentrum fu€r Infektionen in Gyna€kologie und Geburtshilfe, Wuppertal, Deutschland Vestibulodynia (VD), formerly named burning vulva syndrome or vulvar vestibulitis syndrome, is a subgroup of vulvodynia and occurs in more than 5% of women. It is nearly not mentioned in gynecologigical or dermatological textbooks. Primary VD begins with the first sexual experiences, secondary mostly in women of 20–30 years. They report of strong burning or pain ‘like a knife or fire’ by a touch or a penetration, and also tampon use or bycicling can be impossible.

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The diagnosis is based on clinical exclusion of infections (like vulvovaginal candidosis or herpes genitalis), neurologic disorders (like spinal or pudendal nerve compression), dermatoses (like lichen sclerosus), or vulvar intraepithelial neoplasia. Women with VD are commonly correctly diagnosed only after visiting several physicians within several years. Typical co-morbidities of VD are fibromyalgia, interstitial cystitis, irritative bowel syndrome and craniomandibular dysfunction. There are hints, that VD is influenced by psychosomatic problems and depression. Many women report, that an antibiotic therapy followed by vulvovaginal candidoses (VVC) preceded the beginning of VD. This is supported by following scientific results: 1. Women with VD and VVC have sinificantly more frequent a polymorphism of a component (NALP3) of the inflammasome and structures of the cytoplasma, which influence the interleukin - 1 beta- production. 2. Women with VD react significantly more frequent hyperergic to Candida (C.) albicans in skin tests than atopic women or those with non - atopic dermatitis. 3. If mice were challenged by recurrent VVC in an established mouse model, a significant number of them reacted highly sensible in their vulva region on touches, although the VVC was cured. Histochemical investigations of their vestibular skin showed 300% more and thicker nerve fibers in the lamina propria, 400% more ‘peptidergic fibers (calcitonin gene-related peptide immunoreactivity)’ and also sympathic nerve fibers with invasion in the superficial layers of the epithelium. This conditions could also be set off by Candida surface antigenes. More and thicker nerve fibers were also significantly more frequent found in women with VD in their vestibular epithelium. 4. Oral long time suppression with fluconazole can result in good cure rates of women with VD and VVC. This troubling and frequent condition of VD needs more intensive interdisciplinary research in future.

Dermatomycoses II V47 Photodynamic therapy (PDT) – treatment for fungal infections of the skin and nails B. Farkas Department of Dermatology, Medical Centre of HDF, and Hospital Keszthely, Budapest, Ungarn PDT is a non-invasive, selective, oxygen-dependent phototoxic reaction that can be used to treat different diseases. PDT combines the use of a photosensitizer and its excitation with visible light at an appropriate wavelength. This process results in photodamage and subsequent cell death. The photosensitizer may be administered intravenously (systemic PDT) or applied to the target lesion topically: topical PDT. Topical PDT is now increasingly used in dermatology for the treatment of a wide range of neoplastic, inflammatory and infectious cutaneous conditions, but the clinical indications established from evidence-based studies are much narrower and are open to continuing evaluation. The growing resistance to antifungal drugs has led to the investigation of PDT as a treatment for mycotic infections. This overview concentrates on the clinical applications of topical antifungal PDT for mycotic infections of the skin, the nails and the oral cavity. In this overview, conclusions of clinical trials and case series will be presented with respect to the following areas: the topical photosensitizing agent and the duration of its application, the employed light source and the light dose regimen, various strategies to minimize discomfort associated with treatment and the frequency of treatment. Literature, relating to the efficacy and safety of topical antifungal PDT used for the treatment of onychomycosis, superficial mycoses of the skin and mucosa, suggests that antifungal photodynamic therapy might be an alternative to traditional drug therapy; either as a therapy in itself or as a combination with antifungal compounds.

ª 2014 The Authors Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 2), 15–30

Oral Presentation

Further randomized clinical trials are needed before consensus protocols shall be established.

V48 Morphologic and physiologic features of Arthroderma benhamiae J. Brasch and S. Wodarg Klinik fu€r Dermatologie, Venerologie und Allergologie, Universita€tsklinikum Schleswig-Holstein, Campus Kiel, Kiel, Deutschland Arthroderma (A.) benhamiae is a teleomorph of a dermatophyte that belongs to the Trichophyton mentagrophytes-complex. Up to now its zoophilic anamorph has no name of its own and is usually simply called A.benhamiae as well. Whereas an identification of A. benhamiae by use of genetic methods is well established it is still difficult to unambiguously recognize this species by use of conventional techniques only. Since in most mycological labs run in dermatological offices genetic methods are not available many strains of A. benhamiae may therefore remain undetected. A better knowledge of the morphological and physiological criteria of A. benhamiae should help to improve this situation. For this purpose strains of A. benhamiae isolated in our lab were carefully assessed. Seven strains of A.benhamiae were used that had been unequivocally identified by different genetic methods (Prof. Gl€aser, Berlin; Dr. Ragno, Dresden) and that are deposed in the public German collection of microorganisms and cell cultures (DSMZ, Braunschweig). Growth of all strains was evaluated on various mycological agars and the micromorphology was assessed by use of microcultures. Furthermore, all strains were tested for vitamin dependence, enzyme release, growth on hair from different sources, hair perforation, growth on stratum corneum, temperature tolerance, salt tolerance and resistance to cycloheximide. As a result all A.benhamiae-isolates showed some largely consistent morphological and physiological characteristics. Noteworthy were the complete destruction of rabbit hairs by all strains. Uniformly no growth resulted on Trichophyton (T.) agars 6 and 7, limited growth was seen on T. agars 1, 2 and 5 and on potato dextrose agar and strong growth occurred on T. agars 3 and 4. In microcultures all strains developed peculiar loop-like mycelial junctions. The urease test and hair perforation test gave some inconsistent results with the various strains. Our findings should help to identify A.benhamiae by conventional methods. In particular the formation of loop-like mycelial junctions appears to be a distinctive criterion of A.benhamiae that can easily be checked in all labs without need of any special technical equipment as well as the growth-enhancing effect of thiamine. We want to encourage further verifying of these features.

V49 Microsporum canis and Trichophyton anamorph of Arthroderma benhamiae - a comparison of the prevalence of the zoophilic dermatophytes in Central Germany € ger S. Uhrlaß, P. Nenoff and C. Kru Labor fu€r medizinische Mikrobiologie, Partnerschaft Prof. Pietro Nenoff und Dr. Constanze Kru€ger, Mo€lbis, Deutschland Introduction Microsporum (M.) canis is considered as the most frequent occurring zoophilic dermatophyte in Germany and Europe. Trichophyton (T.) anamorph of Arthroderma (A.) benhamiae which is likewise a zoophilic dermatophyte seems to be a frequent and emerging pathogen of dermatophytoses at least in distinct regions of Germany. Methods Over a period of 3 years – 3/2010 until 3/2013, all samples sent into the lab for investigation of dermatophyte infections of the scalp, face, trunk, extremities, were examined for dermatophytes,

ª 2014 The Authors Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 2), 15–30

both by culture and by PCR. Materials from feet, toe nails and finger nails were not included in the study. The materials originated from the area of Leipzig and southern Saxony, Thuringia and Saxony-Anhalt, however, in particular also from other areas of Germany. Skin scrapings, hairs, hair roots, and swabs from the skin have been cultured on Sabouraud´s 4% dextrose agar with and without cycloheximide for 3 weeks. A uniplex PCR-Elisa assay was used for dermatophyte DNA detection. This method allows detection of T. anamorph of A. benhamiae, T. rubrum, T. interdigitale, Microsporum canis, and E. floccosum separately and directly from skin, nail and swabs. Results Over 3 years 8464 samples from 7680 patients were investigated. In 114 (1.5 %) of these 7680 patients M. canis was found using culture and/or PCR. In particular, M. canis was detectable in 100 samples by culture, in 107 samples by PCR, in 91 samples both by culture and by PCR. For 12 patients, only the cultural detection (without PCR) was done. A tinea corporis due to M. canis was present in 59 patients, tinea capitis in 8, tinea faciei in 5, and tinea manus in 2 patients. In 40 patients, the localization of the dermatomycosis was not declared. 45 % of patients (n = 5) were younger than 20 years old, 42 % 20 to 49 years, and 13 % 50 years and older. In contrast, T. anamorph of A. benhamiae could be detected by culture and/or PCR in 231 out of 7680 patients (2.9 %), whereby M. canis followed as second frequent zoophilic dermatophyte as the next. Conclusion M. canis represents still a frequent isolated zoophilic dermatophyte in Germany. It should be noticed, however, that an increase of the prevalence of infections due to T. anamorph of A. benhamiae occurred. This zoophilic dermatophyte, which is transferred from guines pigs to human beings, actually seems to be the most frequent causative agent of dermatophytosis in children and adolescents in some areas of Germany. Dermatologists, pediatricians, physicians in public health offices, veterinaries, and last but not least the stuff of pet shops should draw the consequences from these new epidemiological data.

V50 € rztekammer: Laws for Fungi – a Richtlinien der Bundesa useful norm? K.-D. Reinel Praxis fu€r Dermatologie, Hamburg, Deutschland In some medical faculties, for instance in Dermatology, the clinical diagnosis Mycosis can be proven by laboratory investigations (native preparation, culture, PCR) by the Dermatologist himself ‘in one hand’. It is an advantage for patients, that the same specialist he went to show his skin signs is the one who makes the definite diagnosis and will start the regarding antimycotic therapy. In Germany mycologic investigations are ruled by the ‘Richtlinien der Bundes€ arztekammer zur Qualit€ atssicherung laboratoriumsmedizinischer € Untersuchungen (RiLiBAK)’. These are Guidelines for securing the necessary quality in laboratory investigations. Part A, the general part, is law since 30.06.2013, meaning that these rules are already in existence and should be written down in the Book of Qualitymanagement in each private practice and in hospitals. Part B, containing standardization demands for internal and external Qualitysecuring is in existence since 01.04.2013 but there is an interimstime ending 31st of march 2014. At this time every specialist still wanting to do € mycologic laboratory investigations has to obey the rules of RiLiBAK Part B. Some of these rules are useful and necessary as for instance exact documentation of patient identity, skin localization where the material was collected, day of investigation and day of final laboratory result. Also of course the identification of the personal who did the identification. other rules, like special controls of the used standard mediums (normally produced by specialized pharmacological industry) with each new bought charge (of every medium used) and examination of these media regarding the growth of control strains of dermatophytes and /or yeasts and/or moulds seem to be just a

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Oral Presentation

clinging to the rules as means of standardizations (‘The norm is king’, even if its not useful in dermatology). € are comparable to laws, the dermatologists But as these RiLIBAK of course have to stick to these rules, even when some of them dont seem practical or at least useful. It is our hope, that all german dermatologists, who have learned and used to do mycologic investigations, will do mycology also in the future. This would be to the advantage of patients with dermatomycoses.

shaped by thick-walled chlamydospores, whereas rather strait fertile hyphae of Onychocola often show ellipsoidal two-celled arthroconidia. References 1. De Hoog et al. (2000) Atlas of Clinical Fungi. Centraalbureau voor Schimmelcultures, pp.790–791, 989–991. 2. Sigler L, Congly H (1990) Toenail infection caused by Onychocola canadensis gen. et sp. nov. J Med Vet Mycol 28, 405–417. 3. Nenoff et al. (2014)Infektionen an Finger- und Zehenn€ ageln durch Pilze und Bakterien. Hautarzt, 65(4), 337–348.

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Quality Control & Reference Center

Dermatomycosis of a female Rider: Who was the source? K.-D. Reinel Praxis fu€r Dermatologie, Hamburg, Deutschland A female Rider develloped a typical targetoid lesion on one upper arm. The father, a dermatologist, treated with a topical antimycotic substance, the lesion healed. Unfortunately no mycologic investigation was made at this time. Simultaneously the horse develloped a lesion in his hide. This time material (mostly hair and scales)was collected and cultured. The resulting fungal growth could not be differentiated by the father and was sent to the author some time later. The received culture was wooly and of greyish colour, in subcultures only moulds survived. But in the primary culture another fungus seemed to be at the bottom under the mould. This structure was investigated with a microscope. The picture resembled ‘mature microconidia with scattered macroconidia and inflated cells of Trichophyton equinum’ (Atlas of Clinical Fungi - GS DE HOOG et al). But the structures had a more uniform appearance regarding their size. So they also looked very similar to the ‘conidial apparatus with macroconidia’ of Microsporum nanum in the same book.The hobby-horse of the rider normally grazed on a meadow neighboring a farm and contacts with pigs were possible. Microsporum nanum causes lesion on pig ears, but lesions in humans are known and published. Trichophyton equinum is a subspecies(?) of Trichophyton tonsurans. It is zoophylic, transmission from horses to humans is known.

V52 Uncommon Cases of Dermatomycoses/Onychomycoses H. Mittag Fachbereich Medizin, Klinik fu€r Dermatologie und Allergologie Marburg UKGM, Philipps-Universita€t Marburg, Marburg, Deutschland Case 1: Focal tinea corporis was present on the skin of a student of veterinary medicine. Clinical and mycological aspects pointed towards Trichophyton verrucosum infection. Case 2: Tinea unguium due to the non-dermatophyte-mold Onychocola canadensis is not very well known and reports about this type of onychomycosis are rather limited in Germany. This presentation will show the clinical picture as well as macroscopic and microscopic features of the fungus. Discussion Both fungi – Trichophyton verrucosum and Onychocola canadensis - develop as white/off-white, small, slow growing colonies in vitro and both species are microscopically characterized by chains of spores. But in detail, these chains of Tr. verrucosum are irregularly

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V53 The National Reference Center NRZMyk as a Partner in Diagnosis and Management of Invasive Fungal Infections K. Voigt,1 M.von Lilienfeld-Toal,1 K. Kaerger,1 G. Walther,1 R. Martin1,2 and O. Kurzai1,2 1 Leibniz Institut fu€r Naturstoff-Forschung und Infektionsbiologie, Hans-Kno€ll-Institut, Nationales Referenzzentrum fu€r Invasive Pilzinfektionen NRZMyk, Jena, Deutschland and 2FriedrichSchiller-Universita€t, Septomics Forschungszentrum, Jena, Deutschland The German National Reference Center for Invasive Fungal Infections (NRZMyk, www.nrzmyk.de) appointed and funded by the RobertKoch Institute is situated at the Leibniz-Institute for Natural Product Research and Infection Biology (Hans Knoell Institute) in Jena since 2014. We provide specialized diagnostic tools for the detection of invasive mycoses and advise both clinical colleagues and public health services in all questions related to invasive fungal infection. Furthermore, experimental techniques are used to aid in the diagnosis of these infections. The NRZMyk hosts the Jena Microbial Ressource Collection as the major national strain collection for fungi, operating in close collaboration with the German Collection of Microorganisms and Cell Cultures (DSMZ). Scientists of the NRZMyk have actively participated in the Fungal Barcoding Consortium, a multi-laboratory, multi-national consortium that evaluated six genes as potential DNA barcodes for the kingdom Fungi. Within this project, a database of molecular barcodes was established which serves as a global reference for the molecular identification of Mucorales. Based on these findings the direct analysis and identification of Lichtheimia sp. was established and validated using MALDI-TOF. We also have experience in evaluating molecular diagnostic tests for invasive fungal infections. These activities are all closely linked to research on human fungal pathogens. Thereby the NRZMyk contributes to national research initiatives like the collaborative research center FungiNet (SFB/TR124) and the BMBF funded consortium InfectControl 2020 as well as to the Jena research focus Sepsis and Sepsis Sequelae. Recently we finished genome-sequencing of L. corymbifera, representing the most basal mucoralean pathogen sequenced so far, which opens new ways for comparative genomics. Together with international partners we have identified a novel genetic risk factor for invasive aspergillosis and characterized innate immune responses in invasive fungal infections. We coordinate the first genome-wide study on genetic risk factors for invasive aspergillosis. Based on these activities and in close cooperation with all mycologists and clinicians interested in mycology we hope to contribute to a continuous improvement of the clinical management of invasive fungal infections.

ª 2014 The Authors Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 2), 15–30