METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hateld, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Fungal Diagnostics
Methods and Protocols

Edited by

Louise OConnor and Barry Glynn

Molecular Diagnostics Research Group, NCBES, National University of Ireland, Galway, Ireland

Editors Louise OConnor Molecular Diagnostics Research Group NCBES National University of Ireland Galway, Ireland

Barry Glynn Molecular Diagnostics Research Group NCBES National University of Ireland Galway, Ireland

ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-62703-256-8 ISBN 978-1-62703-257-5 (eBook) DOI 10.1007/978-1-62703-257-5 Springer New York Heidelberg Dordrecht London
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Preface
Invasive fungal infections are a signicant cause of morbidity and mortality. Over the past decade there has been a concerted effort to develop reliable methods for the detection of such infections. Early diagnosis of IFI is critical, allowing timely administration of appropriate antifungal therapy. The increased use of antifungal drugs has led to a parallel emergence of resistant and less-susceptible strains. This emergence has increased the importance of prompt and accurate identication of the causative agents of fungal infections. Rapid diagnosis is perhaps the most critical factor in ensuring a positive patient outcome. In this respect it is our hope that this volume may provide ideas for those intending to introduce novel technologies for fungal detection into their laboratories. Key factors in introducing any new technology into a diagnostic setting include ease of use and rapid turnaround time without compromising sensitivity and specicity. To this end, we have focused on including in this volume methods which offer these characteristics with the potential to be adapted in the routine diagnostic setting. While focusing specically on fungal detection in clinical settings, the methods described are applicable to all areas utilizing fungal diagnostics including, environmental testing, agriculture and food production, and veterinary diagnostics. In addition, some of the methods described represent a signicant practical demonstration of state-of-the-art molecular methods which are suitable for detection of non-fungal microorganisms and infectious agents. Included in this volume also are comprehensive reviews of fungal infections, a review of commercially available systems, as well as chapters concerned with sample preparation and a broad range of interesting methods for detection. We would like to thank Professor John Walker and David Casey of Humana Press for this opportunity to become involved in this worthwhile project. Sincere thanks to the authors who gave of their own time to enlighten the broader scientic community of their expertise in this area of biology. Galway, Ireland Louise OConnor Barry Glynn

Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Overview of Invasive Fungal Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nina L. Tuite and Katrina Lacey 2 Fungal Diagnostics: Review of Commercially Available Methods . . . . . . . . . . . Javier Yugueros Marcos and David H. Pincus 3 Rapid Methods for the Extraction and Archiving of Molecular Grade Fungal Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andrew M. Borman, Michael Palmer, and Elizabeth M. Johnson 4 APEX DNA Microarray for the Identication of Pathogenic Fungi . . . . . . . . . Arianna Tavanti, Stefano Landi, and Sonia Senesi 5 Microscopic Detection of Yeasts Using Fluorescence In Situ Hybridization . . . Joo Incio and Maria da Luz Martins 6 Quantitative Detection of Aspergillus spp. by Real-Time Nucleic Acid Sequence-Based Amplication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yanan Zhao and David S. Perlin 7 Differentiation of Fungi Using Hybridization Probes on the LightCycler . . . . Stephan Fricke, Nadja Hilger, Christopher Oelkrug, Arne C. Rodloff, and Christian Fricke 8 Quantitative and Multiplex Detection of Pathogenic Fungi Using Padlock Probes, Generic qPCR, and Suspension Array Readout . . . . . . . . . . . Magnus Jobs, Ronnie Eriksson, and Jonas Blomberg 9 Species-Specic Identication of a Wide Range of Clinically Relevant Fungal Pathogens by the Luminex xMAP Technology. . . . . . . . . . . . . . . . . . . . . . . . S. Preuner and T. Lion 10 Applied Gene Histopathology: Identication of Fusarium Species in FFPE Tissue Sections by In Situ Hybridization . . . . . . . . . . . . . . . . . . . . . . Yoichiro Okubo, Minoru Shinozaki, Megumi Wakayama, Haruo Nakayama, Daisuke Sasai, Takao Ishiwatari, Tetsuo Nemoto, Tochigi Naobumi, and Kazutoshi Shibuya 11 Application of Chip-Based Flow Cytometry for Amphotericin B and Fluconazole Susceptibility Testing on Candida Strains . . . . . . . . . . . . . . . Orsolya Bouquet, Bla Kocsis, Ferenc Kilr, and Ildik Kustos 12 Surface Plasmon Resonance Genosensor for the Detection of Fusarium culmorum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michelangelo Pascale, Francesco Zezza, and Giancarlo Perrone v ix 1 25

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13 Hyperbranching Rolling Circle Amplication, An Improved Protocol for Discriminating Between Closely Related Fungal Species. . . . . . . . . . . . . . . Jiufeng Sun and Sybren de Hoog 14 Loop-Mediated Isothermal Amplication-Based Detection of Fusarium graminearum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ludwig Niessen 15 Multiplex-Tandem PCR for Fungal Diagnostics . . . . . . . . . . . . . . . . . . . . . . . Anna Lau, Keith Stanley, and Tania Sorrell 16 Specic Detection of Pneumocystis jirovecii in Clinical Samples by Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Joana Barbosa, Soa Costa-de-Oliveira, Ana Teresa Silva, Accio Gonalves Rodrigues, and Cidlia Pina-Vaz Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors
JOANA BARBOSA Department of Microbiology, Faculty of Medicine, Cardiovascular Research Unit, University of Porto, Porto, Portugal; Escola Superior de Sade Jean Piaget/ Vila Nova de Gaia, Gulpilhares, Vila Nova de Gaia JONAS BLOMBERG Department of Medical Sciences, Clinical Virology, Uppsala University, Uppsala, Sweden ANDREW M. BORMAN Mycology Reference Laboratory, Health Protection Agency, South-West Regional Laboratory, Bristol, UK ORSOLYA BOUQUET Department of Analytical and Environmental Chemistry, Faculty of Sciences, University of Pcs, Pcs, Hungary MARIA DA LUZ MARTINS Instituto de Higiene e Medicina Tropical/Centro de Recursos Microbiolgicos, Universidade Nova de Lisboa (IHMT/UNL), Lisboa, Portugal SOFIA COSTA-DE-OLIVEIRA Department of Microbiology, Faculty of Medicine, Cardiovascular Research Unit, University of Porto, Porto, Portugal SYBREN DE HOOG CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands RONNIE ERIKSSON Department of medical sciences, Clinical virology, Uppsala University, Uppsala, Sweden CHRISTIAN FRICKE Medizinische Dienste SRO, Langenthal, Switzerland STEPHAN FRICKE Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany; Institute of Clinical Immunology, University of Leipzig, Leipzig, Germany; Department of Hematology and Oncology, University of Leipzig, Leipzig, Germany NADJA HILGER Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany JOO INCIO Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisboa, Portugal TAKAO ISHIWATARI Department of Surgical Pathology, Toho University School of Medicine, Ota-Ku, Tokyo, Japan MAGNUS JOBS School of Health and Social Studies, Dalarna University, Falun, Sweden ELIZABETH M. JOHNSON Mycology Reference Laboratory, Health Protection Agency, South-West Regional Laboratory, Bristol, UK FERENC KILR Institute of Bioanalysis, Faculty of Medicine, University of Pcs, Pcs, Hungary; Department of Analytical and Environmental Chemistry, Faculty of Sciences, University of Pcs, Pcs, Hungary BLA KOCSIS Department of Medical Microbiology and Immunology, Faculty of Medicine, University of Pcs, Pcs, Hungary ILDIK KUSTOS Department of Microbiology, Alder Hey Childrens NHS Foundation Trust, Liverpool, UK; Department of Medical Microbiology and Immunology, University of Pcs, Pcs, Hungary KATRINA LACEY Molecular Diagnostics Research Group, National University of Ireland, Galway, Ireland

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Contributors

STEFANO LANDI Department of Biology, Genetic Unit, University of Pisa, Pisa, Italy ANNA LAU Centre for Infectious Diseases and Microbiology, The University of Sydney, Sydney, NSW, Australia Westmead Millennium Institute, Westmead Hospital, Westmead, Australia Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA T. LION Childrens Cancer Research Institute (CCRI), Labdia Labordiagnostik, Vienna, Austria JAVIER YUGUEROS MARCOS Research and Validation of Biomarkers, bioMrieux SA, Marcy lEtoile, France HARUO NAKAYAMA Department of Neurosurgery, Toho University Ohashi Medical Center, Meguro, Tokyo, Japan TOCHIGI NAOBUMI Department of Surgical Pathology, Toho University School of Medicine, Ota-Ku, Tokyo, Japan TETSUO NEMOTO Department of Surgical Pathology, Toho University School of Medicine, Ota-Ku, Tokyo, Japan LUDWIG NIESSEN Weihenstephan Center of Food and Life Sciences, TU-Mnchen, Lehrstuhl fr Technische Mikrobiologie, Freising, Germany CHRISTOPHER OELKRUG Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany YOICHIRO OKUBO Department of Surgical Pathology, Toho University School of Medicine, Ota-Ku, Tokyo, Japan MICHAEL PALMER Mycology Reference Laboratory, Health Protection Agency, South-West Regional Laboratory, Bristol, UK MICHELANGELO PASCALE Institute of Sciences of Food Production (ISPA), National Research Council of Italy (CNR), Bari, Italy GIANCARLO PERRONE Institute of Sciences of Food Production (ISPA), National Research Council of Italy (CNR), Bari, Italy DAVID S. PERLIN Public Health Research Institute Center, UMDNJ-NJMS, Newark, NJ, USA CIDLIA PINA-VAZ Department of Microbiology, Faculty of Medicine, Cardiovascular Research Unit, University of Porto, Porto, Portugal DAVID H. PINCUS R&D Microbiology, bioMrieux, Inc., Hazelwood, MO, USA S. PREUNER Childrens Cancer Research Institute (CCRI), Labdia Labordiagnostik, Vienna, Austria ARNE C. RODLOFF Institute for Microbiology and Infectious Epidemiology, University of Leipzig, Leipzig, Germany ACCIO GONALVES RODRIGUES Department of Microbiology, Faculty of Medicine, Cardiovascular Research Unit, University of Porto, Porto, Portugal DAISUKE SASAI Department of Surgical Pathology, Toho University School of Medicine, Ota-Ku, Tokyo, Japan SONIA SENESI Department of Biology, Genetic Unit, University of Pisa, Pisa, Italy KAZUTOSHI SHIBUYA Department of Surgical Pathology, Toho University School of Medicine, Ota-Ku, Tokyo, Japan Department of Dermatology, Peking University First Hospital, Beijing, China

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MINORU SHINOZAKI Department of Surgical Pathology, Toho University School of Medicine, Ota-Ku, Tokyo, Japan ANA TERESA SILVA Department of Microbiology, Faculty of Medicine, Cardiovascular Research Unit, University of Porto, Porto, Portugal TANIA SORRELL Centre for Infectious Diseases and Microbiology, The University of Sydney, Sydney, NSW, Australia The Westmead Millennium Institute, Westmead Hospital, Westmead, Australia KEITH STANLEY AusDiagnostics Pty. Ltd., Alexandria, NSW, Australia JIUFENG SUN Department of Parasitology, The Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China ARIANNA TAVANTI Department of Biology, Genetic Unit, University of Pisa, Pisa, Italy NINA L. TUITE Molecular Diagnostics Research Group, National University of Ireland, Galway, Ireland MEGUMI WAKAYAMA Department of Surgical Pathology, Toho University School of Medicine, Ota-Ku, Tokyo, Japan FRANCESCO ZEZZA Institute of Sciences of Food Production (ISPA), National Research Council of Italy (CNR), Bari, Italy YANAN ZHAO Public Health Research Institute Center, UMDNJ-NJMS, Newark, NJ, USA