The future of barcoding:

getting closer, or drifting further away from clinical practice? 12-13 April 2013

Abstracts Program book 1F = ?G 10–11 April 2013

CBS Symposium One Fungus = Which Genes 10-11 April 2013

The future of barcoding:

getting closer, or drifting further away from clinical practice?

A workshop by the ISHAM Working Group Medical Barcoding Utrecht, The Netherlands, April 12-13, 2013

Programe Introduction Welcome to the ISHAM Workshop on Barcoding of Medical Fungi. We are happy to announce that we have a fantastic panel of speakers. If you have any questions, please contact Sybren de Hoog ([email protected]) or to Wieland Meyer ([email protected]). Venue CBS-KNAW Fungal Biodiversity Centre, Utrecht, Uppsalalaan 8, The Netherlands. The institute is easily reached from Amsterdam Schiphol Airport within one hour by public transportation, please check www.cbs.knaw.nl. The weather in The Netherlands early April is still fresh, mostly around 10 – 15 °C, and rain may be expected. The workshop including lunches and dinners is provided at no cost. Accreditation The meeting has been accredited by the UEMS (European Union of Medical Specialists) with 12 CME accreditation points. These points are also recognized and valid in the United States (by the AMA). All participants will receive an obligatory Evaluation form at the end of the workshop, as part of the accreditation procedure. Certificates of attendance will be handed at the registration desk along with your congress information and abstract book. Sponsors We are grateful to our sponsors Thermo Fisher Inc., Elsevier Publishing Co. and the International Society for Human and Animal Mycology (ISHAM) for generous support of this meeting. Do become a member of ISHAM at www.ISHAM.org! For free membership of the ISHAM Working group on Barcoding, contact Wieland Meyer [email protected].

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The future of barcoding:

getting closer, or drifting further away from clinical practice?

A workshop by the ISHAM Working Group Medical Barcoding Utrecht, The Netherlands, April 12-13, 2013

Friday 12 April • 08.00 – 09.00 Registration, congress bags & badges, uploading oral and poster presentations; coffee • 09.00 – 09.10 Welcome and introduction • 09.10 – 09.30 Sybren de Hoog (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands): Some concepts – barcode, validated barcode, barcode identifier, species delimitations, and the genomic future. • 09.30 – 12.30:

Session 1: Next generation barcoding. Conveners: Benjamin Stielow and Vincent Robert. • 09.30 – 09.25 Benjamin Stielow (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands): Novel barcodes and roboted barcoding of medical fungi at CBS. • 09.25 – 10.20 Sylvie van Loon (Life Technologies, Ghent, Belgium): Rapid high resolution typing of organisms by multiplex PCR and semiconductor sequencing. • 10.20 – 10.45 Vincent Robert (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands): Handling massive amount of data. • 10.45 – 11.15 Break • 11.15 – 11.40 Markus Göker (DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany): Bioinformatic strategies for assessing the suitability of genes as universal fungal barcodes. • 11.40 – 12.05 Gianluigi Cardinali (Università degli Studi di Perugia, Perugia, Italy): New challenges and opportunities from the barcode of life and environmental metagenomics studies. • 12.05 – 12.30 Guy Cochrane (EMBL-EBI, Cambridge, UK): Sequence data compression in the world of identification and typing. • 13.00 – 14.00 Lunch at the Botanical Garden • 14.00 – 17.00:

Session 2: Virtual reality in the reference lab: Networking in eastern and western worlds. Conveners: Ruo-yu Li and Wangqing Liao. • 14.00 – 14.20 Sybren de Hoog (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands): ISHAM Working Groups. • 14.20 – 14.40 Fei-yan Bai (State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China): Candida MLST network. • 14.40 – 15.00 Weida Liu, Shuwen Deng (Dept. of Mycology, Institute of Dermatology, Chinese Academy of Medical Sciences, Nanjing, China; Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China): Chinese-CBS web accessible database. • 15.00 – 15.20 Daniel Wagner, Flavio Queiroz Telles (Hospital de Clinicas, Curitiba, Brazil; Daniel Wagner de Castro Lima Santos, Comissão de Controle de Infecção Hospitalar IIER, Grupo de Micologia Clínica e Infecção em Transplantes Unifesp, Sao Paulo, Brazil): Brazilian black yeast network. • 15.20 – 16.00 Break

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The future of barcoding:

getting closer, or drifting further away from clinical practice?

A workshop by the ISHAM Working Group Medical Barcoding Utrecht, The Netherlands, April 12-13, 2013

• 16.00 – 16.20 Anuradna Chowdhary (Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, India): Multidisciplinary approach with sterile fungi. • 16.20 – 16.40 Wieland Meyer (Molecular Mycology Research Laboratory, Sydney Medical School, Westmead Hospital, University of Sydney, Sydney, Australia): The MLST Scedosporium network. • 16.40 – 17.00 Stephane Bretagne (Parasitologie-Mycologie, Université de la Méditerranée CHU Timone, Marseille, France): Candida network at the NCRMA. • 17.00 – 18.00 Poster session and drinks • 18.00: Fungal BBQ at CBS

Saturday 13 April • 08.00 – 09.00 • Uploading oral presentations; coffee • 09.00 – 12.30:

Session 3: Evolution and the gold standard. Conveners: Wieland Meyer and Dea Garcia Hermoso. • 09.00 – 09.15 Kittipan Samerpitak (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands): Taxonomy of Ochroconis and Scolecobasidium, members of the novel order Ochroconiales. • 09.15 – 09.30 Anderson Rodrigues (Disciplina de Biologia Celular, Depto. de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Brazil): Barcoding of Sporothrix. • 09.30 – 09.45 Somayeh Dolatabadi, Grit Walther (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; Leibniz Institute for Natural Product Research and Infection Biology, Friedrich-SchillerUniversity Jena, Jena, Germany Germany): Heterogeneity of barcoding genes in Mucorales. • 09.45 – 10.00 David Geiser (Department of Plant Pathology, Buckhout Lab, Pennsylvania State University, USA): Fusarium. • 10.00 – 10.15 Wangqing Liao (Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China): New medical Penicillium. • 10.15 – 10.30 Neriman Yilmaz (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands Netherlands): Talaromyces covering pathogenic Penicillium species. • 10.30 – 11.00 Break • 11.00 – 11.15 Mark Stadler (Dept. Microbial Drugs, Helmholtz Centre for Infection Research, Braunschweig, Germany): Barcoding and drug discovery in Xylariaceae and basidiomycetes. • 11.15 – 11.30 Marcus Teixeira (Laboratório de Biologia Molecular, Universidade de Brasília, Brazil): Diversity in Paracoccidioides using novel genes. • 11.30 – 11.45 Peiying Feng (Department of Dermatology, 3rd Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China): Barcoding markers for cutaneous black yeasts. • 11.45 – 12.00 Sarah Abdallah Ahmed (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands): Pandora’s Box of agents of mycetoma. • 12.00 – 12.15 Lizel Mostert (Department of Plant Pathology, Stellenbosch University, Matieland, South Africa): Phaeoacremonium. • 12.15 – 13.30 Lunch at the Botanical Garden

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The future of barcoding:

getting closer, or drifting further away from clinical practice?

A workshop by the ISHAM Working Group Medical Barcoding Utrecht, The Netherlands, April 12-13, 2013

• 13.30 – 18.00:

Session 4: Novel non-DNA-based methods. Conveners: Stephane Ranque and Jens Frisvad. • 13.30 – 13.50 Markus Kostrzewa (Bruker, Bremerhafen, Germany): Maldi-Tof techniques. • 13.50 – 14.10 Victoria Girard, Valerie Monnin (BioMerieux, La Balme-les-Grottes, France): Maldi-Tof techniques. • 14.10 – 14.30 Teun Boekhout (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands): MaldiTof in yeasts. • 14.30 – 14.50 Wieland Meyer (Molecular Mycology Research Laboratory, Sydney Medical School, Westmead Hospital, University of Sydney, Sydney, Australia): Cryptococcus subspecies ID using Maldi-Tof. • 14.50 – 15.10 Ali Abdul Hussain (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands): Maldi-Tof in dermatophytes. • 15.10 – 15.30 Carine Marinach-Patrice (Pitié-Salpêtrière Hospital / Pierre & Marie Curie University, Paris): Maldi-Tof applied to medical mycology. • 15.30 – 15.50 Renaud Piarroux (Parasitologie-Mycologie, Université de la Méditerranée CHU Timone, Marseille, France): Current state of the art of Maldi-Tof for the identification of filamentous fungi. • 15.50 – 16.20 Break • 16.20 – 16.40 Stephane Ranque (Parasitologie-Mycologie, Universitéde la Méditerranée CHU Timone, Marseille, France): Monitoring fungal epidemiology with Maldi-Tof. • 16.40 – 17.00 Roger Grist, Joanna Freeke (Thermo Fisher Scientific, East Grinstead, UK; CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands): Next generation proteomic diagnostics using LC-MS-MS. • 17.00 – 17.20 Jens Frisvad (Center for Microbial Biotechnology, Technical University of Denmark, Lyngby, Denmark): Secondary metabolites: can they be used for barcoding?. Concluding remarks: • 17.20 – 17.40 Wieland Meyer: What does barcoding mean to clinical practice? • 17.40 – 19.00 Poster session, drinks and excursion to CBS collection • 19.30 Drinks and dinner at Chez Willy at Oude Gracht cellars (bus 11, busstop Neude)

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The future of barcoding:

getting closer, or drifting further away from clinical practice?

A workshop by the ISHAM Working Group Medical Barcoding Utrecht, The Netherlands, April 12-13, 2013

Pandora’s Box of agents of mycetoma in Pleosporales Sarah Abdallah Ahmed CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands

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umycetoma is a chronic fungal infection of subcutaneous tissue characterized by slow expansion and the presence of sinuses discharging coloured grains. Black grain mycetoma is only caused by fungi, but with quit diverse etiologic agents mainly belong to the fungal orders; Sordariales, and Pleosporales. The taxonomy of most of these pleosporalean mycetoma agents is still unclear. The aim of the present study was to clarify the taxonomy of black grain Pleosporales mycetoma including Madurella grisea, Medicopsis romeroi (syn.: Pyrenochaeta romeroi), Nigrograna mackinnonii (syn.: Pyrenochaeta mackinnonii), Leptosphaeria species, Pseudochaetosphaeonema larense. Five genes phylogeny based on ITS, SSU, LSU, RPB2, and TEF1 was performed, in addition to morphology and physiological characterization of the species. Taxonomic revision of the above mentioned species revealed new species names including Trematosphaeria grisea, Falciformispora senegalensis, Falciformispora tompkinsii, and new species of Arthopyrenia was described. All the species were coelomycetes producing asexual fruit body while Falciformispora produces ascomata with ascospores. Oral

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The future of barcoding:

getting closer, or drifting further away from clinical practice?

A workshop by the ISHAM Working Group Medical Barcoding Utrecht, The Netherlands, April 12-13, 2013

Correlation of MLST with microsatellite typing and infection forms of Candida albicans Feng-Yan Bai

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State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing China

andida albicans is the most common opportunistic fungal pathogen of humans. It causes from benign infections such as oral and genital candidiasis to fatal, systemic diseases in immunocompromised or critically ill patients. In addition to improved therapy, rapid and accurate identification of the disease-causing strains is of special value for diagnosis, clinical treatment and epidemiological studies. A variety of methods for strain typing of C. albicans have been described. Among the commonly used methods in recent years, microsatellite typing using a locus called CAI and multilocus sequence typing (MLST) have been shown to be highly discriminative and reproducible for unambiguous strain characterization of C. albicans. We performed CAI genotyping and MLST on a large set of C. albicans isolates with different clinical and geographic origins in China. We found overall fine correlation and complementary nature between the two strain typing methods. Generally, isolates with different CAI genotypes usually possessed different DSTs while isolates with the same CAI genotypes mostly possessed the same DSTs. In some cases, isolates that were indistinguishable by CAI typing could be differentiated by MLST and vice versa. Interestingly, when isolates with the same CAI genotypes exhibited different DSTs, these DSTs were usually closely related in phylogeny. We also showed significant association of specific genotypes with certain infections of C. albicans, especially genital candidiasis. We found that while the CAI genotypes and DSTs of C. albicans isolates from extragenital sites were highly diverse, isolates associated with vulvovaginal candidiasis and balanoposthitis from unrelated patients belonged to only a few genotypes, with CAI 30-45 and CAI 32-46 as the most dominant ones. In the Phylogenetic tree constructed from MLST data set, the two dominant CAI genotypes and their close derivatives were mostly concentrated in a specific cluster with DST 79 as the primary founder. Oral

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The future of barcoding:

getting closer, or drifting further away from clinical practice?

A workshop by the ISHAM Working Group Medical Barcoding Utrecht, The Netherlands, April 12-13, 2013

MALDI-TOF MS identification of pathogenic yeast species Teun Boekhout1, Anna Kolecka1, Kantarawee Khayhan1,2, Marizeth Groenewald1, Markus Kostrzewa3 1

CBS-KNAW, Fungal Biodiversity Centre, Utrecht, the Netherlands. 2 Department of Microbiology and Parasitology, Faculty of Medical Sciences, University of Phayao, Phayao, Thailand. 3 Bruker Daltonics, Bremen, Germany

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he MALDI-TOF MS Biotyper 3.0 system (Bruker Daltonics, Germany) was used to identify pathogenic yeasts using reference strains from the CBS-KNAW Yeast Collection as well as clinical test sets. The yeasts included species of the Candida parapsilosis complex (ortho-, meta-, para-), the Candia haemulonii complex, the Cryptococcus neoformans complex (including varieties and hybrids), Malassezia spp., and arthroconidial yeasts belonging to Trichosporon and Geotrichum, Galactomyces, Dipodascus and Magnusiomyces. MALDI-TOF MS proved to be an excellent diagnostic tool that allowed discrimination among almost all tested strains and identified the strains at the species level with high accuracy. The majority of strains was correctly identified at the species level with good scores (>2.0) but also with log-values ranging 2.0 >score >1.7. The obtained MALDI-TOF MS results were consistent with the current golden standard of ITS and/or LSU ribosomal DNA sequencing. Results of a ring test performed in 12 laboratories in The Netherlands and abroad will also be presented and discussed. Oral

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The future of barcoding:

getting closer, or drifting further away from clinical practice?

A workshop by the ISHAM Working Group Medical Barcoding Utrecht, The Netherlands, April 12-13, 2013

Multidisciplinary Approach with Sterile Fungi Anuradha Chowdhary Department of Medical Mycology, V. P. Chest Institute, University of Delhi, Delhi

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he clinical significance of rapidly growing non sporulating filamentous molds is poorly understood. Conventional identification based on their phenotypic characteristics is problematic as many isolates remain sterile in culture (Mycelia sterilia) due to lack of observable reproductive structures. Thus inconclusive culture report adversely affects treatment strategies. Classically, nonsporulating molds were of environmental origin without clinical significance and in the past, these have often been discarded as purported contaminants but this scenario has changed in recent years. Today, we know that many of these cultures are of basidiomycete affinity. The most common of these is Schizophyllum commune, Hormographiella aspergillata, Coprinopsis cinereus. Recently some less-known basidiomycete species, such as Cereporia lacerata, Perenniporia sp., Cyclomyces tabacinus, Irpex lacteus, Inonotus (Phellinus) tropicalis, Oxyporus corticola, and Volvariella volvacea have also been reported sporadically. Although the number of these species reported from clinical and environmental sources is growing rapidly, molecular data are available for only a fraction of these species. Sequencing of the ribosomal DNA internal transcribed spacer (ITS) region may offer a match in GenBank; therefore, an increasing number of taxa are being introduced in medical literature. We identified 50 white non-sporulating isolates originating from respiratory specimens of patients with bronchopulmonary mycoses during (2010-2013) by sequencing of ITS and D1/D2 regions. Also their antifungal susceptibility pattern, patients’ therapy and outcome were determined. Of 50 isolates, 26 (52%) were found to be Schizophyllum commune, 11 (22%) Cereporia lacerata, 4 (8%) Porostereum spadiceum, 3 (6%) Phanerochaete stereoides, one (2%) each Marasmiellus palmivorus and Perenniporia species and 3 (6%) remained unidentified. All of the isolates were sterile in culture on PDA after 8 weeks of incubation at 280C, except 15% (4/26) of S. commune and 9% (1/11) of C. lacerata isolates that developed basidiocarp formation typical of the species. All the isolates showed spicules and hyphal pegs, however, 39 (78%) of the isolates also revealed clamp connections characteristic of basidiomycetes. Furthermore, 63% of C. lacerata isolates showed basidium with basidiospore formation. Of 50 isolates, 35 (70%) were confirmed by both ITS and D1/D2 sequencing (>99% identity) and 12 (24%) by either ITS or D1/D2 sequencing. D1/D2 regions and ITS rDNA sequencing identified 92% and 70% of non-sporulating molds up to species level. Posaconazole (MIC50, 0.125µg/ml) exhibited excellent activity against all the species tested followed by isavuconazole (MIC50, 0.25µg/ml) and amphotericin B (MIC50, 0.5µg/ml). Twelve percent of the isolates showed higher MICs of itraconazole (>0.5 µg/ml) and voriconazole (>1 µg/ml). MICs of fluconazole and flucytosine ranged from 2-64 µg/ml. Caspofungin MEC ranged from 0.125-8 µg/ml. S. commune was isolated from 45% patients with a clinical diagnosis of allergic bronchopulmonary mycosis (ABPM), 11.5% each of pulmonary fungal ball and invasive pulmonary mycosis and 7.6% of allergic fungal sinusitis. On the other hand C. lacerata and P. spadiceum was implicated in a wide spectrum of clinical manifestations ranging from saprobic colonization to fungal pneumonia. The patients of ABPM treated with itraconazole had no relapse after 6 to 24 months of follow up. Also, invasive and fungal ball cases were treated successfully with voriconazole and itraconazole. It may be emphasized that, though the clinical significance of the isolation of basidiomycetes in healthy patients is probably negligible, a pathological role in debilitated patients cannot be excluded. The present study is one of the largest series of basidiomycetes characterized by ITS and D1/D2. Furthermore, Perenniporia and Cereporia species identified are reported for the first time as human pathogens. Oral

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The future of barcoding:

getting closer, or drifting further away from clinical practice?

A workshop by the ISHAM Working Group Medical Barcoding Utrecht, The Netherlands, April 12-13, 2013

In vitro antifungal susceptibility profiles of 77 molecularly characterized clinical mucormycete isolates from India Anuradha Chowdhary1, Shallu Kathuria1, Pradeep K Singh1, Ziauddin Khan2, Sohail Ahmad2, G Sybren de Hoog3, Jacques F. Meis 4,5 1 Department of Medical Mycology, V. P. Chest Institute, University of Delhi, Delhi, India, 2CBS- KNAW Fungal Biodiversity Center, Utrecht, the Netherlands, 2Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait, 3Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, the Netherlands, 4Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

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ucormycosis is a highly aggressive fungal disease, usually fatal in immunocompromised patients. The clinical manifestations include primary rhino-orbital-cerebral mucormycosis in patients with diabetes mellitus, and pulmonary infection in transplant recipients and haematological malignancy. Species identification is epidemiologically and clinically important because it impacts the choice of specific antifungal therapy. We report molecular characterization and in vitro antifungal susceptibility profiles of 77 clinical isolates of mucormycetes determined by microdilution with the CLSI method for 8 antifungals and with Etest for posaconazole and amphotericin B. The isolates originated from 70 patients investigated in tertiary care hospitals in Delhi during 2004-2012. Sixty (80%) of the isolates were from pulmonary, 8 (10%) cutaneous and 9 (11%) rhino-orbital-cerebral mucormycosis patients. Identification was done by standard mycological procedures and DNA sequencing of ITS and D1/D2 regions of rDNA. The mucormycetes investigated were: Rhizopus oryzae (n=34), R. microsporus (n=16), R. arrhizus (n=3), R. stolonifer (n=3), R. azygosporus (n=1), Syncephalastrum racemosum (n=10), Apophysomyces elegans (n=4), Lichtheimia corymbifera (n=2), L. ramosa (n=2), and Mucor circinelloides (n=2). Molecular characterization revealed phenotypic misidentification in 8 (10%) isolates. AFLP classified 52 isolates into 8 clades. Amphotericin B was found to be the most potent antifungal (GM MIC, 0.06µg/ml). Of the azoles, posaconazole exhibited the highest activity (GM MIC, 0.4µg/ml). Itraconazole and isavuconazole showed low MICs in 47% (36/77) and 36% (28/77) of the isolates (MIC,