PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/128930 Please be advised that this information was generated on 2021-10-10 and may be subject to change. Cryptococcus neoformans Cryptococcus complex: molecular, clinical and clinical molecular, complex: Cryptococcus neoformans complex: molecular, clinical and in vitro studies in vitro studies M.T. Illnait Zaragozí 2014 Zaragozí Illnait M.T. studies María Teresa Illnait Zaragozí Cryptococcus neoformans complex: molecular, clinical and in vitro studies ISBN: 978-94-6203-627-7 Printed by CPI Wöhrmann Print Service, Zutphen, The Netherlands. Layout by Ferry Hagen. Copyright © 2014 by Maria Teresa Illnait Zaragozí, All rights reserved. No part of this thesis may be reproduced or transmitted in any form or by any means without written permission of the author. The work described in this thesis was performed in several periods from 2007-2012 at the Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands Cover: Front: Almond tree, Havana, Cuba Back: Mango tree and pigeon, Havana, Cuba Pictures: María Teresa Illnait Zaragozí Cryptococcus neoformans complex: molecular, clinical and in vitro studies Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. mr. S.C.J.J. Kortmann, volgens besluit van het college van decanen in het openbaar te verdedigen op woensdag 27 augustus 2014 om 10.30 uur precies door María Teresa Illnait Zaragozí geboren op 10 maart 1967 te Havana, Cuba Promotoren Prof. dr. A. Voss Prof. dr. P.E. Verweij Copromotoren Dr. J.F.G.M. Meis (Canisius-Wilhelmina Ziekenhuis, Nijmegen) Dr. F. Hagen (Canisius-Wilhelmina Ziekenhuis, Nijmegen) Manuscriptcommissie Prof. dr. M.G. Netea Prof. dr. B.E. de Pauw Prof. dr. G.S. de Hoog (CBS-KNAW Fungal Biodiversity Centre, Utrecht) Paranimfen Dr. Ph.A. Van Damme N. Manraad Cryptococcus neoformans complex: molecular, clinical and in vitro studies Doctoral Thesis to obtain the degree of doctor from Radboud University Nijmegen on the authority of the Rector Magnificus prof. dr. S.C.J.J. Kortmann, according to the decision of the Council of Deans to be defended in public on Wednesday, August 27, 2014 at 10.30 AM by María Teresa Illnait Zaragozí Born on March 10, 1967 in Havana, Cuba Supervisors Prof. dr. A. Voss Prof. dr. P.E. Verweij Co-supervisors Dr. J.F.G.M. Meis (Canisius-Wilhelmina Ziekenhuis, Nijmegen) Dr. F. Hagen (Canisius-Wilhelmina Ziekenhuis, Nijmegen) Doctoral Thesis Committee Prof. dr. M.G. Netea Prof. dr. B.E. de Pauw Prof. dr. G.S. de Hoog (CBS-KNAW Fungal Biodiversity Centre, Utrecht) Paranymphs Dr. Ph.A. Van Damme N. Manraad Sometimes we feel that what we do is just a drop in the sea, but the ocean would be less without a drop. Mother Teresa of Kolkata Table of contents Chapter 1. General Introduction 11 Part I Molecular characterization of clinical and environmental C. neoformans species complex isolated from Cuba and The Netherlands Chapter 2. Microsatellite typing of clinical and environmental 35 Cryptococcus neoformans var. grubii isolates from Cuba shows multiple genetic lineages Chapter 3. Extensive genetic diversity within the Dutch clinical 43 Cryptococcus neoformans population Chapter 4. Microsatellite typing and susceptibilities of serial 53 Cryptococcus neoformans isolates from Cuban patients with recurrent cryptococcal meningitis Part II Antifungal susceptibility of Cuban and worldwide cryptococcal strains Chapter 5. In vitro activity of the new azole isavuconazole (BAL 61 4815) compared with six other antifungal agents against 162 Cryptococcus neoformans isolates from Cuba Chapter 6. In vitro antifungal susceptibilities and amplified 65 fragment length polymorphism genotyping of a worldwide collection of 350 clinical, veterinary, and environmental Cryptococcus gattii isolates Chapter 7. Cryptococcus neoformans - Cryptococcus gattii 73 species complex: an international study of wild- type susceptibility endpoint distributions and epidemiological cutoff values for fluconazole, itraconazole, posaconazole, and voriconazole Part III Clinical and environmental studies Chapter 8. Environmental isolation and characterization of 83 Cryptococcus species from living trees in Havana city, Cuba Chapter 9. Reactivation of a Cryptococcus gattii infection in a 91 cheetah (Acinonyx jubatus) held in the National Zoo, Havana, Cuba Chapter 10. Fatal Cryptococcus gattii genotype AFLP5 infection in 97 an immunocompetent Cuban patient Chapter 11. Cryptococcus gattii induces a cytokine pattern that is 103 distinct from other cryptococcal species Chapter 12. General discussion and future perspectives 115 Chapter 13. Summary - Samenvatting - Resumen 133 Chapter 14. Acknowledgments 145 Curriculum vitae List of publications Chapter General Introduction 1 Chapter 1 The incidence of cryptococcosis, initially considered unusual, began to increase with the arrival of acquired immunodeficiency syndrome (AIDS) in the 1980s to become one of the most important mycoses worldwide. After the advent of highly effective antiretroviral therapy in the mid-1990, its frequency started to decline in developed countries. However, cryptococcal meningitis remained the most common life- threatening opportunistic fungal infection with an estimated annual mortality of 625,000 persons, which ranks cryptococcosis as fourth among the most common causes of death due to infections in sub-Saharan Africa, higher than tuberculosis. (Armstrong et al. 2014; Park et al., 2009; La Hoz and Papas, 2013). Additionally, it has been reported that up to 6% of people with impaired cellular immunity due to other causes, are at risk of developing clinically apparent infections due to Cryptococcus (Bratton et al., 2012). From discovery of the causative agent of cryptococcosis to its current taxonomy Probably the first case of cryptococcosis was descrited in 1861 by Zenker. However because of the lack of microbiological evidence, the first case description is usually attributed to doctors Otto Busse and Abraham Buschke from Germany. In 1894 they reported the isolation of a microorganism from an injury in a 31-year-old woman and named it Saccharomyces hominis. Almost simultaneously, Sanfelice described the presence of encapsulated yeasts obtained from fermented peach juice; he demonstrated its pathogenicity in laboratory animals and called it Saccharomyces neoformans. Sanfelice shortly afterwards recovered a similar agent from the lymph node in an ox and called it Saccharomyces lithogenes because there were particular differences between this organism and the first one (Casadevall and Perfect, 1998, Bovers et al., 2008a). A year after the first cryptococcosis case report by Busse and Buschke, Curtis Ferdinand in France, described a “plant parasite” yeast as an etiologic agent of an ulcerating abscess in a young Frenchman with an unremarkable medical history. Based on different morphological features in comparison with the previous described isolates (elongated or oval yeasts) and its affinity for subcutaneous tissue shown in animal models, Curtis labelled this agent Saccharomyces subcutaneous tumefaciens. Despite the absence of epidemiological data, it is currently considered the first report of an autochthonous infection by C. gattii in France (Hagen et al., 2012; Kwon-Chung et al., 2011). These findings demonstrated three cardinal aspects: i) the ability to cause infection in humans and in animals, ii) this, added to the demonstration of Koch’s postulates by Sanfelice and Curtis, showed its pathogenic potential and iii) the fact that it can be recovered from natural sources revealed its saprophytic nature. However, the different denominations received yielded confusion about the true nature of this agent (Casadevall and Perfect, 1998; Bovers et al., 2008a). The genus Cryptococcus was first established in 1833 by Kützing to “accommodate” a new microorganism described as “globose hyaline microscopic organism with undetermined taxonomic position that produces whitish mucoid colonies”. Later, in 1901, Vuillemin argued that the microorganisms isolated by Busse and Sanfelice in 1894 actually belong to this genus. Thereafter, its definition underwent multiple 12 General Introduction proposed amendments until the gender name Cryptococcus Vuillemin and pointed C. neoformans as the type species. Nowadays, the genus includes about 100 pathogenic and non-pathogenic species (Kwon-Chung et al., 2011). 1 One of the main contributions to the current taxonomy of C. neoformans, was the recognition of its sexual stage. In 1975 the first teleomorph, Filobasidiella neoformans, was obtained by mating compatible isolates of serotypes A and D. The second species, Filobasidiella bacillispora was achieved by crosslinking compatible cells of serotypes B and C (Kwon-Chung, 1975). The correlation between the different serotypes and teleomorph states indicated that differences between isolates were not restricted to their antigenic properties. Subsequent studies showed that both species differ in their ecological niches, epidemiology, biochemistry, association with different host immune states, phylogeny and genomic arrangements (Kwon-Chung and Varma, 2006; Kwon- Chung et al., 2011). Studies about this agent and the disease it causes, led to its current classification. What once was considered a “complex species”, is now recognized as a “species complex” (Fonseca et