Genetic Variability, Antigenicity and Antifungal Susceptibility of Madurella mycetomatis ISBN 978-90-8559-311-9 © Wendy van de Sande, 2007 All rights reserved. No part of this thesis may be reproduced or transmitted in any form, by any means, electronic or mechanical, without the written permission of the author, or where appropriate the publisher of the articles Printed by: Optima Grafische Communicatie, Rotterdam, The Netherlands Graphic design: Wendy van de Sande Genetic Variability, Antigenicity and Antifungal Susceptibility of Madurella mycetomatis Genetische variabiliteit, antigeniciteit en antifungale gevoeligheid van Madurella mycetomatis Proefschrift ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof.dr. S.W.J. Lamberts en volgens besluit van het College voor Promoties De openbare verdediging zal plaatsvinden op: Woensdag 24 oktober 2007 om 13:45 uur Door Wendy Wilhelmina Johanna van de Sande geboren te Breda Promotiecommissie Promotor: Prof.dr.dr. A.F. van Belkum Overige leden: Prof.dr. G.S. de Hoog Prof.dr. A.P. Oranje Prof.dr. H.A. Verbrugh Co-promotor: Dr. I.A.J.M. Bakker-Woudenberg The printing of this thesis was financially supported by: BD Diagnostic Systems, bioMérieux Benelux B.V., Eurogentec B.V., Janssen-Cilag, Merck Sharp & Dohme, Novartis Pharma and Pfizer B.V. Contents General introduction Chapter 1 Scope of the thesis 11 Chapter 2 Black grain myetoma, a neglected infection 15 Genetic variation in Madurella mycetomatis and its human host Chapter 3 Genotyping of Madurella mycetomatis by selective 39 amplification of restriction fragments (AFLP®) and subtype-correlation with geographical origin and lesion size Chapter 4 Polymorphisms in genes involved in innate immunity 53 predispose towards mycetoma susceptibility Antigenicity of Madurella mycetomatis Chapter 5 Translationally controlled tumour protein from 73 Madurella mycetomatis, a marker for tumorous mycetoma progression Chapter 6 Madurella mycetomatis compounds cross-reactive 93 with galactomannan are detectable in culture supernatant but not in serum Antifungal susceptibility of Madurella mycetomatis Chapter 7 In vitro susceptibilities of Madurella mycetomatis 101 to itraconazole and amphotericin B assessed by a modified NCCLS method and a viability-based 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay Chapter 8 In vitro antifungal susceptibility testing of 109 Madurella mycetomatis for six antifungal agents by the Sensititre system in comparison with a viability-based XTT assay and a modified NCCLS method Chapter 9 In vitro susceptibility of Madurella mycetomatis, 119 prime agent of madura foot, to tea tree oil and artemisinin Chapter 10 Melanin biosynthesis in Madurella mycetomatis 127 and its effect on susceptibility to itraconazole and ketoconazole General discussion and concluding remarks Chapter 11 Summarizing discussion 145 Chapter 12 Nederlandse samenvatting 159 Chapter 13 Dankwoord 175 Chapter 14 List of publications and curriculum vitae 179 General introduction Chapter 1 Scope of the thesis Wendy W.J. van de Sande Chapter 1 12 Scope of the thesis Introduction Mycetoma is a chronic, granulomatous, progressive inflammatory disease, characterized by a subcutaneous mass, tumefaction and the formation of sinus tracts. The sinuses discharge seropurulent material and fungal or bacterial grains. This disease, which can be caused by both bacteria and fungi, is encountered all over the world but is endemic in the so-called “mycetoma-belt”, between 30 oN and 15 oS of the equator. Sudan is one of the countries in which mycetoma has a high incidence. In this country the most common causative agent is the fungus Madurella mycetomatis. Although mycetoma was already described in 1842, still relatively little is known about this disease. Diagnostic assays and treatment are still suboptimal. Current diagnostic assays are time-consuming and not very reliable. Treatment, especially in eumycetoma, is difficult and usually involves both surgery and medical treatment. Adequate antifungal treatment may take years and is usually started without even knowing if the causative agent is actually susceptible to the chemotherapeutic agent applied. Scope of the thesis To understand the difficulties in diagnosing and treating mycetoma we first review this disease in chapter 2. The current knowledge about this disease, its prevelance, the diagnostic assays used, and the treatment given to the patients are highlighted. As will become clear in this chapter little is known about the disease. In the remaining of this thesis we will focus on three main themes: 1. Genetic variation of M. mycetomatis and its human host In the chapters belonging to this theme it was assessed whether genetic variability of both pathogen and host could be related to the outcome of the disease. In chapter 3 the selective amplification of restriction fragments (AFLP®) is used to devide 37 Sudanese clinical isolates of M. mycetomatis into two main clusters (clusters I and II) and one minor cluster (cluster III). The strains in cluster I mostly derived from patients in Central Sudan and were generally obtained from larger lesions than the strains found in cluster II. In chapter 4 several human genes involved in neutrophil function were studied with PCR-restriction analysis. It appeared that patients usually had a genotype correlated with a higher CXCL8 expression and a lower nitric oxide expression. This was confirmed phenotypically. 2. Antigenicity of M. mycetomatis In order to improve the diagnostic serology for mycetoma caused by M. mycetomatis the antigenicity of M. mycetomatis was assessed. In chapter 5 the first immunogenic antigen, a protein homologous to the translationally controlled tumour protein (TCTP) was discovered using a M. mycetomatis cDNA expression library. Both IgM and IgG responses could be measured for this protein, with the highest antibody levels found associated with the largest 13 Chapter 1 lesions. Unfortunately, not all patients formed antibodies against this protein. In chapter 6 it was shown that although M. mycetomatis secreted significant amounts of galactomannan-like compounds into the culture medium, these compounds were not detected in the patient serum. 3. Antifungal susceptibility of M. mycetomatis In order to improve the antifungal therapy in patients we tried to gain more insight in the in vitro susceptibility of M. mycetomatis against various antifungal agents. In chapter 7 a CLSI (formerly NCCLS) based assay was developed for M. mycetomatis. To facilitate endpoint reading a 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H- tetrazolium hydroxide (XTT) assay for fungal viability was used. In chapter 8 the assay described in chapter 7 was compared to a commercial Sensititre assay for the antifungal agents amphotericin B, ketoconazole, itraconazole, fluconazole and voriconazole. In chapter 9 the in vitro susceptibilities for the less commonly used antifungal agents artemisinin and tea tree oil were determined. In chapter 10 it was assessed what the effect of melanin produced by M. mycetomatis was on the in vitro susceptibilities to amphotericin B, ketoconazole, itraconazole, fluconazole and voriconazole. Finally in chapters 11 and 12 the findings reported in this thesis are summarized and discussed. 14 Chapter 2 Black grain mycetoma, a neglected infection Wendy W.J. van de Sande Adapted from: Abdalla O.A. Ahmed, Willem van Leeuwen, Ahmed Fahal, Wendy W.J. van de Sande, Henri Verbrugh and Alex van Belkum, Mycetoma caused by Madurella mycetomatis: a neglected infectious burden, The Lancet Infectious Diseases 2004, 4: 566-574. Abdalla O.A. Ahmed, Wendy W.J. van de Sande, Ahmed Fahal, Irma Bakker-Woudenberg, Henri Verbrugh and Alex van Belkum, Management of Mycetoma: Major Challenge in Tropical Mycoses with Limited International Recognition, Current Opinion in Infectious Diseases 2007, 20(7): 146-151. Chapter 2 16 Black grain mycetoma, a neglected infection Introduction Mycetoma is a chronic infectious disease, which remains localized, involves cutaneous and subcutaneous tissues, the fascia and bones (95). The disease is characterized by tumefaction, draining sinuses and the presence of grains (95). The oldest recorded case of possible mycetoma involves the skeleton found in Israel of an adult woman buried in the Byzantine period (300-600 AD) (95). This skeleton had morphologic changes in the bones of the distal portion of the lower extremities which are suggestive for mycetoma (95). The first written description of mycetoma was found in the Indian religious book Atharva Veda, where the disease was called “padavalmika”, meaning foot-anthill (95). In 1842 the first case was described in western literature by Dr. John Gill, a physician of the Madras Medical Service of the British Army in India (112). His colleague Dr. Colebrook proposed the name “Madura foot” in 1848 (95, 112). The term mycetoma was first used by Vandyke Carter in 1860 (112). He was the first to believe that mycetoma could be separated into two varieties depending on the colour of the grains associated with the disease. M. mycetomatis probably caused the black variety of mycetoma Carter described, whereas one of the aerobic actinomycetes caused the light-coloured variety (95). Epidemiology and causation As was already apparent from this short history lesson, mycetoma has a worldwide but uneven distribution. The disease is endemic in tropical and subtropical