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Biodiversity, pathogenicity and antifungal susceptibility of Cladophialophora and relatives

Badali, H.

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Download date:10 Oct 2021 Biodiversity, pathogenicity and antifungal susceptibility of

Biodiversity, pathogenicity and antifungal susceptibility of Cladophialophora and relatives Cladophialophora and relatives

Hamid Badali Hamid Badali

2010

Biodiversity, pathogenicity and antifungal susceptibility of Cladophialophora and relatives

Hamid Badali ISBN 978-90-70351-80-9 © Hamid Badali, 2010 [email protected] 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.

Cover: Cladophialophora bantiana Layout: K.F. Luijsterburg

Printed at Ponsen & Looijen bv, Ede, The Netherlands Biodiversity, pathogenicity and antifungal susceptibility of Cladophialophora and relatives

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof. Dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde commissie, in het openhaar te verdedigen in de Agnietenkapel op vrijdag 25 juni 2010, te 14.00 uur

door

Hamid Badali geboren te Zanjan, Iran Promotiecommissie

Promotores: Prof. dr. G.S. de Hoog Prof. dr. S.B.J. Menken

Overige leden: Prof. dr. P.H. van Tienderen Prof. dr. W. Admiraal Prof. dr. G. Haase dr. J.F.G.M. Meis dr. W. van de Sande

Faculteit der Natuurwetenschappen, Wiskunde en Informatica. Contents

Chapter 1 General introduction Black yeasts: their systematics and phylogeny 7

Chapter 2 Biodiversity of the genus Cladophialophora 21

Chapter 3 Cladophialophora saturnica sp. nov., a new opportunistic species of Chaetothyriales revealed using molecular data 47

Chapter 4 Cladophialophora psammophila, a novel species of Chaetothyriales with a potential use in the bioremediation of volatile aromatic hydrocarbons 61

Chapter 5 Use of amplified fragment length polymorphism to identify 42Cladophialophora strains, related to cerebral phaeohyphomycosis with in vitro antifungal susceptibility 77

Chapter 6 The clinical spectrum ofExophiala jeanselmei, with a case report and in vitro antifungal susceptibility of the species 91

Chapter 7 Rhinocladiella aquaspersa, proven agent of verrucous skin infection and a novel type of chromoblastomycosis 105

Chapter 8 First autochthonous case of Rhinocladiella mackenziei cerebral abscess outside the Middle East 115

Chapter 9 In vitro activities of antifungal drugs against Rhinocladiella mackenziei, an agent of fatal brain infection 121

Chapter 10 Discussion 125 Conclusion & Summary 136 Conclusies en samenvatting (Conclusion & Summary in Dutch) 139 Acknowledgements 143 About the author 144 List of publications 144 List of abstracts 145 Dedicated to Sahar and Ilya To my Father and Mother 1 Chapter 1

General introduction Black yeasts: their systematics and phylogeny 19 Chapter 1

Black Yeasts: their systematics and phylogeny Historically, major handbooks in ascomycete classification treated morphologically similar parts of the Fungal Kingdom, of which we now know that they belong together. A well-known example is the yeasts, which long time excluded Hemiascomycetes with hyphae, such as Geotrichum and Hyphopichia. Another example is the separation of lichenised and non-lichenised taxa (Acharius 1810, Lindau 1897, Zahlbruckner 1926). It was only in the 20th century that the latter classifications were merged (Luttrell 1951, 1955, Nannfeldt 1932). Today, it has been established that both lichenised and non-lichenised taxa belong to the subclass Chaetothyriomycetidae of Eurotiomycetes, specifically to three orders, viz., the Chaetothyriales, the Pyrenulales, and the Verrucariales. The subclass is characterised by a large ecological diversity (Geiser et al. 2006). The lichenised taxa are members of the Pyrenulales which are mainly found on trees, particularly in the tropics, and of the Verrucariales which predominantly colonize rocks. Previously (Luttrell 1951, 1955) the non-lichenised Chaetothyriales (family Herpotrichiellaceae) were attributed to the order Pleosporales based on their centrum development of teleomorph carpophores. Members of Pyrenulales and Verrucariales are generally found as teleomorphs, whereas members of Chaetothyriales are mostly known from their anamorphs, with the exception of species of Capronia. In the Chaetothyriomycetidae, teleomorphs are characterised by perithecial fruiting bodies and bitunicate asci with a dehiscence ranging from fissitunicate to evanescent. Lichenised taxa were first thought to belong to the orderXylariales , also based on their ascus type and centrum which was identified as Xylaria‘ -type’ (Luttrell 1951, 1955). Traditionally the most important morphological characteristicts used to define major groups in the Ascomycota were the type of ascus, septation of ascospores, the morphology and development of the ascoma, as well as the structure and organisation of the centrum. In Eriksson’s system (Eriksson 1982), in which the Euascomycetes are divided into four groups according to ascus type, the bitunicate ascus is enclosed in a double wall, which consists of a thin, brittle outer shell and a thick elastic inner wall. When the spores are ripe, the shell splits open so that the inner wall can take up water. As a consequence, this inner part of the ascus begins to extend with its spores until it protrudes above the rest of the ascocarp so that the spores can escape into free air without being obstructed by the bulk of the fruiting body. Bitunicate asci occur only in the Dothideomycetes and Chaetothyriomycetes.The orders Pyrenulales and Verrucariales were placed in the bitunicate group, whereas two non-lichenised families, Chaetothyriaceae and Herpotrichiellaceae, were included in the bitunicate order Dothideales (Eriksson 1982). Barr (1983) was one of the first to consider the three orders Verrucariales, Chaetothyriales and Pyrenulales (at that time included in the Melanommatales) as being closely related, based on ascus type (bitunicate or secondarily prototunicate) and hamathecium structure (insertion of sterile filaments in the centrum, either pseudoparaphyses or periphysoids). Molecular studies have now confirmed their close phylogenetic relationship. Dothideomycetes contains the majority of the fungal species with ascostromatic development and bitunicate asci and share a number of morphological characteristics with other fungal classes. It was recently formally described (Eriksson & Winka 1997) replacing in part the long- recognised Loculoascomycetes (Luttrell 1955). These early phylogenetic studies demonstrated that Loculoascomycetes, as it was defined, are not monophyletic, although contrasting views exist (Liu & Hall 2004). Nevertheless the majority of analyses have shown that some Loculoascomycete taxa, such as the “black yeasts” in the Chaetothyriales as well as the lichenised Verrucariales, reside within the Eurotiomycetes as subclass Chaetothyriomycetidae (Spatafora et al. 1995, Geiser et al. 2006, Gueidan et al. 2008) (Fig. 1). The majority of the remaining Loculoascomycete species are now placed in Dothideomycetes. Although finer morphological distinctions between the distantly related members of Loculoascomycetes can be made, their synapomorphies remain elusive (Lumbsch & Huhndorf 2007). These findings all point to the fact that a number of Loculoascomycete

10 Introduction morphological characters are either retained ancestral traits or that they exhibit convergence due to similar selection pressures. The name Chaetothyriomycetidae was first proposed to accommodate the order Chaetothyriales 1 within a subclass (Kirk et al. 2001). Subsequently, the order Verrucariales was shown to be sister to Chaetothyriales (Lutzoni et al. 2001) and was therefore included in the Chaetothyriomycetidae (Eriksson 2001), followed by the Pyrenulales (Eriksson 2006). At present, the order Chaetothyriales includes non-lichenised ascomycetes divided in two families, the Chaetothyriaceae and the Herpotrichiellaceae (Geiser et al. 2006). TheChaetothyriaceae are known as epiphytes, colonizing the leaves and the bark of trees in the tropics (Batista & Ciferri 1962). For many of the species it is still unclear whether they are saprophytic or biotrophic (Barr 1987). Species of the Herpotrichiellaceae are known either from their sexually reproducing stage (teleomorph), their clonally reproducing stage (anamorph), or both. Teleomorphs include small inconspicuous saprophytes, mainly growing on decaying wood and mushrooms (Barr 1987, Untereiner & Naveau 1999). Anamorphs are found as opportunistic pathogens on animals and humans, or are sometimes isolated from the environment using enrichment methods (Prenafeta-Boldú et al. 2006). The opportunistic black yeasts and filamentous relatives are frequently involved inskin, pulmonary and nervous system infections in healthy humans and only occasionally in immunocompromised patients (de Hoog et al. 2000, Horré & de Hoog 1999, Kantarcioglu & de Hoog 2004). Most of these species are known from their anamorphs only, and it is only recently, with the development of molecular techniques, that connections between anamorphs and teleomorphs were assessed (Untereiner 1997, 2000, Untereiner & Naveau 1999). Because little is known on the ecology of these organisms, the study of anamorph/teleomorph relationships and the isolation of strains from their putative primary habitats are a key issue to the understanding of the evolution of this group (de Hoog et al. 2000). Human-associated black yeasts are known since the end of the 19th century as a taxonomically quite heterogeneous group, sharing melanized cell walls and the formation of daughter cells by polar budding. Most black yeasts additionally exhibit hyphae and generate conidia from phialides with collarettes, from annelated cells, from sympodial rhachides, or from undifferentiated conidiogenous cells (de Hoog & Hermanides-Nijhof 1977). Conidia may be non septate or sometimes have up to three transversal septa. Furthermore, the formation of arthroconidia from fragmenting hyphae can occur in some genera. Only very few species, e.g. Phaeococcomyces exophialae, do not possess any hyphal states. Black yeast-like fungal pathogens are gaining importance because they are increasingly diagnosed in often life-threatening infections (Revankar 2007). Now that suitable isolation and identification techniques have been developed for these fungi (Wollenzien et al. 1995, Ruibal et al. 2005), they appear to be very common in arid climates, from Polar regions to the sub-tropics. Outside mammals black yeasts and relatives grow in extreme environments characterised by extreme environmental conditions include UV radiation, high temperatures, repeated freezing and thawing cycles, low water activity (Sterflinger 1998a, Zalar et al. 1999), acidity, nutrient deficiency (Sterflingeret al. 1999), matrix and osmotic stress and combinations of these factors (Sterflinger et al. 1999, de Hoog et al. 2005). The combined influence of these stress factors exerts a high selective pressure on the microbial community and as a consequence black yeasts are rarely found in complex microbial populations. Rather, they are solitary or thrive in spatial association with comparably stress-resistant organisms, such as lichens and cyanobacteria in very special habitats. Originally, melanized fungi with meristematic and/or yeast-like growth were described as inhabitants of living and dead plant material, including needles, leaves, bark, fruits and wood (de Hoog et al. 2000). The taxonomy and identification of black yeast and relatives is difficult due to a lack of phenetic characters and a high degree of morphological plasticity. Most of

11 Chapter 1

Fig. 1. Three-locus phylogeny of thePezizomycotina obtained from a Bayesian MCMC analysis depicting ancestral state reconstruction of lichenisation. Thick branches represent nodes supported by PP ≥ 95 % and ML bootstrap ≥ 70 %. Gueidan C, Ruibal C, de Hoog GS, et al (2008). Stud Mycol 61:111-9.

12 Introduction the human infections are restricted to the family of Herpotrichiellaceae (Haase et al. 1999, Badali et al. 2008). By means of molecular characteristics, a great number of previously unidentified taxa are described, rapidly increasing the number of species recognized in the Chaetothyriales. Black yeasts 1 and relatives are frequently encountered in abiotic human-made environments (de Hoog et al. 2000). For example, strains of Exophiala are frequently found in humidifiers as well as in bathing and steam-bath facilities (Matos et al. 2002). Species of Cladophialophora are causative agents of chromoblastomycosis (Badali et al. 2009), cerebral phaeohyphomycosis (Horré & de Hoog 1999; Kantarcioglu & de Hoog 2004, Badali et al. 2010f) and phaeohyphomycotic cysts and keratitis (de Hoog et al. 2000). They occasionally occur associated with sinusitis and have been isolated from subcutaneous infections (Fader et al. 1988, de Hoog et al. 2000). Fish can be infected with Exophiala pisciphila and E. salmonis, the latter causing the phenomenon of tumbling in salmon and trout owing to cerebral infections (de Hoog et al. 2000). The natural habitats described seem to be quite different at first sight, but in fact dead plant material, wood, biofilters, soil polluted with toxic hydrocarbons, rock and inert surfaces, and living mammalian tissue share some main ecological similarities, such as high temperature, osmotic stress, and oxygenic action (de Hoog et al. 2000, Badali et al. 2008). Stress tolerance is particularly enhanced by melanin (Butler et al. 1998, Jacobson 2000) and morphological adaptations (Horré & de Hoog 1999), capsules (Yurlova & de Hoog 2002), temperature tolerance (Sterflinger 1998b, Onofri et al. 2004), desiccation tolerance (Sterflinger 1998b), and physiology, i.e. they are potent degraders of monoaromatic compounds, due to which property they tend to accumulate in industrial biofilters (Cox et al. 1997, Prenafeta-Boldú et al. 2001, 2006, de Hoog et al. 2006). For recovery of black yeast like fungi selective isolation methods are required, e.g., the use of high temperature (Sudhadham et al. 2008), a mouse vector (Gezuele et al. 1972), extraction via mineral oil (Vicente et al. 2008) or enrichment on volatile aromatic hydrocarbons (Zhao et al. 2010). The success of the latter method, enabling isolation of black yeasts where direct plating previously had failed, has supported the hypothesis that herpotrichiellaceous black yeasts are potent degraders of aromatic hydrocarbons. Previously, the alkylbenzene enrichment technique has been applied in the biofiltration of air polluted with volatile aromatic compounds (Kennes & Veiga 2004, Cox et al. 1993, Weber et al. 1995).

Cladophialophora Although the genus Cladophialophora is polyphyletic, the majority of Cladophialophora species – characterised by one-celled, hydrophobic conidia arranged in long, branched, usually coherent chains and with nearly unpigmented conidial scars – are prevalently known from human infections. The genus was initially erected to accommodate a pathogenic species exhibiting Phialophora- like conidiogenous cells in addition to conidial chains (Borelli 1980), but thus far this feature is expressed in only a few strains of C. carrionii and thus has limited diagnostic value. Judging from multigene (nucLSU, nucSSU, RPB1) phylogenetic analyses, Cladophialophora is predicted to have teleomorphs in the ascomycete genus Capronia, but very few connections have thus far been established (Badali et al. 2008). Based on molecular data, anamorphs that are morphologically similar to Cladophialophora have been found in other groups of ascomycetes, particularly in the Dothideales / Capnodiales (e.g., Pseudocladosporium, Fusicladium; Crous et al. 2007). Braun & Feiler (1995) reclassified the dothidealean speciesVenturia hanliniana (formerly Capronia hanliniana) as the teleomorph of Fusicladium brevicatenatum (formerly Cladophialophora brevicatenata). Further distinction between Chaetothyriales and Dothideales / Capnodiales lies in their ecology, with recurrent human opportunists being restricted to the Chaetothyriales. Some species attributed to Cladophialophora may be found in association with living plants (Crous et al. 2007), but most of

13 Chapter 1 these are located at the base of the Chaetothyriales phylogeny, remote from the human opportunists (Badali et al. 2008). De Hoog et al. (2007) reported a cactus endophyte, Cladophialophora yegresii, as a sister species of C. carrionii, which is a major agent of human chromoblastomycosis. The latter fungus was believed to grow on debris of tannin-rich cactus spines, which were also supposed to be the vehicle of introduction into the human body. Crous et al. (2007) described several host- specific plant pathogens associated with the Chaetothyriales. Cladophialophora hostae caused spots on living leaves of Hosta plantaginea; C. proteae was a pathogen of leaves of Protea cynaroides (Proteaceae). In contrast, Cladophialophora species are frequently encountered in human disease, infections ranging from mild cutaneous lesions to cerebral abscesses (Li et al. 2009, de Hoog et al. 2007, Badali et al. 2008). Among the most virulent species are the neurotropes Cladophialophora bantiana and C. modesta, which are able to cause fatal infections in otherwise healthy individuals (Horré & de Hoog 1999, Kantarcioglu & de Hoog 2004). Cladophialophora carrionii and C. samoënsis are etiologic agents of the mutilating skin disorder chromoblastomycosis. Also this type of chronic infection occurs primarily in immunocompetent individuals (Yeguëz-Rodriguez et al. 1992, de Hoog et al. 2007). Cladophialophora devriesii and C. arxii rarely caused disseminated disease (de Hoog et al. 2000), whereas C. boppii, C. emmonsii and C. saturnica were involved in mild cutaneous infections (de Hoog et al. 2007, Badali et al. 2009). Within the Chaetothyriales, the natural niche outside humans is unknown for most species. Badali et al. (2009) described C. saturnica from plant debris in the environment, but the species was also found causing an interdigital infection in a Brazilian child with HIV infection. The species C. australiensis and C. potulentorum were found in soft drinks (Crous et al. 2007). If environmental species are able to provoke opportunistic infections, the question arises whether members of Chaetothyriales isolated from food products might imply a health risk. Cladophialophora psammophila was recently described as novel species from soil in a former gasoline station polluted with BTEX hydrocarbons (Badali et al. 2010e). This is of interest for the complete biodegradation of toluene and other related xenobiotics under growth limiting conditions, particularly in air biofilters, and dry and/or acidic soil. Phylogenetic reconstruction has shown that this fungus is closely related to the highly pathogenic species C. bantiana (Badali et al. 2010f). Below, we provide a brief overview of main clinical aspects due to Cladophialophora species and relatives.

Chromoblastomycosis Chromoblastomycosis is a chronic, progressive cutaneous and subcutaneous disorder histologically characterised by nodular and verrucous skin lesions. Tissue proliferation usually occurs around the area of traumatic inoculation of the etiologic agent, producing crusted, verrucose, and wart- like lesions eventually leading to emerging, cauliflower-like forms Queiroz-Telles( et al. 2009). Infections occur primarily in immunocompetent individuals. The most common etiological agents are Fonsecaea pedrosoi, F. monophora, Cladophialophora carrionii, and Phialophora verrucosa (Queiroz-Telles et al. 2009). However, Exophiala jeanselmei, E. spinifera, E. dermatitidis (Badali et al. 2010a, Padhye et al. 1996), Fonsecaea nubica (Najafzadeh et al. 2010), and Cladophialophora samoënsis (Badali et al. 2008) have also been reported. Chromoblastomycosis is currently classified into six clinical types (Queiroz-Telles et al. 2009): nodular, plaque, tumoral, cicatricial, verrucous and lymphatic, based on the descriptions of Carrión (1950). Chromoblastomycosis lesions are polymorphic and must be differentiated from those many other, non-fungal clinical conditions. The disease is histologically characterised by muriform cells (invasive form) in skin tissue (Mendoza et al. 1993), and easily recognized by direct examination (KOH 20-40 %). Chromoblastomycosis still is a therapeutic challenge for clinicians due to the recalcitrant nature of the disease, especially in the severe clinical forms. There are three treatment modalities, i.e., physical treatment,

14 Introduction chemotherapy and combination therapy, but their success is related to the causative agent, the clinical form and severity of the lesions. There is no treatment of choice for this infection. Most patients are treated with itraconazole, terbinafine, or a combination of both. 1

Cerebral phaeohyphomycosis Primary central nervous system (CNS) infection is characterised by black necrotic tissue due to several dematiaceous fungi, mostly by black yeasts and relatives. They are rare, but when caused by Cladophialophora bantiana, Exophiala dermatitidis, Fonsecaea monophora or Rhinocladiella mackenziei they have a very high mortality rate of up to 70 %, even despite the application of combined surgical and antifungal therapy (Li & de Hoog. 2009, Badali et al. 2010b, Badali et al. 2010d). The majority of reported CNS infections caused by melanized fungi have been found to be brain abscesses with no predisposing host factor or immunodeficiency. Symptoms include headache, seizures, cerebral irritation, fever and neurological deficits. The disease finally may result in black, necrotic brain tissue, black pus, and black cerebrospinal fluid. The pathogenesis may be haematogenous spread from an initial presumably subclinical pulmonary focus and occasionally through direct extension or accidental inoculation. It remains unclear why these fungi preferentially cause CNS disease in immunocompetent individuals, although some case reports and experimental data have suggested that immunodeficiency is a risk factor (Li & de Hoog. 2009, Horré & de Hoog 1999). Cladophialophora bantiana and Rhinocladiella mackenziei are extremely neurotropic black yeasts and affect the central nervous system and integuments despite surgical and antifungal therapy. Thus far they have not been isolated from sources other than living mammalian tissue (de Hoog et al. 2000) except for a single C. bantiana strain recovered via a mouse vector from sawdust (Dixon et al. 1980). Exophiala dermatitidis causes disseminated neurotropic infections; Cladophialophora bantiana and E. dermatitidis are distributed world-wide, but infections are most common in subtropical and humid climates of India and the Far East, respectively, while R. mackenziei is restricted to the arid climate of the Middle East and the Persian Golf region. Cerebral infections have thus far been diagnosed after CT-guided needle aspiration and were proven by positive histopathology and culture results. There is no standard therapy for this disease. Treatment as suggested in the literature has involved mostly (high-dose lipid) amphotericin B, itraconazole, and flucytosine or a combination of these drugs.

Cutaneous and subcutaneous phaeohyphomycosis In 1974 Ajello introduced the term ‘phaeohyphomycosis’, an umbrella term covering a heterogeneous group of cutaneous, subcutaneous and systemic fungal infections. Phaeohyphomycotic infections are caused by melanized fungi and relatives. The only common factor in phaeohyphomycosis is the presence of brownish fungal elements in tissue which can clearly identified with the Fontana- Masson staining, excluding Aspergillus, dermatophytes and yeasts. In subcutaneous cases, minor trauma, even when unrecognized by the patient, is supposed to be the inciting factor (Ajello 1974). Lesions eventually occur at the site of infection and often appear cystic or verrucous. Recently Harris et al. (2009) reviewed 17 cases of cutaneous and subcutaneous phaeohyphomycosis due to E. spinifera in different geographical locations and exhibiting a variety of clinical manifestations. Phaeohyphomycosis is an opportunistic infection that may have an adverse outcome with fatal disseminated disease in immunocompromised hosts such as kidney transplant recipients. Unfortunately, as with many other black yeast infections, there is no standard therapy for this disorder either. Surgical excision has successfully been applied in a number of cases (Badali et al. 2010c), and cryosurgery can be useful in some cases. Amphotericin B, ketoconazole, fluconazole,

15 Chapter 1 voriconazole, 5-fluorocytosine, terbinafine, griseofulvin, posaconazole, and particularly itraconazole have been used for phaeohyphomycotic infections (Harris et al. 2009).

Mycetoma Mycetoma is defined as a localized, chronic, granulomatous, suppurative, and progressive inflammatory disease of subcutaneous tissue and contiguous bone after a traumatic inoculation of the causative organism (McGinnis 1996). Etiologic agents are recovered from humans as well as from domestic animals (Hay et al. 2000). In advanced stages of the infection tumefaction, abscess formation and draining sinuses arise. The hallmark of a mycetoma is the presence of the fungus in the form of grains. In Africa the infection is particularly common from Sudan to Senegal, an area known as the ‘mycetoma belt’. The main etiologic agents areMadurella mycetomatis, Leptosphaeria senegalensis, Madurella grisea and several species of coelomycetes. Occasionally the black yeast-like fungi Cladophialophora bantiana, C. mycetomatis and Exophiala jeanselmei were reported to be involved in cases of eumycetoma (de Hoog et al. 2000, Werlinger et al. 2005, Bonifaz et al. 2009, Badali et al. 2008). Different antifungal agents have been used in the treatment of mycetoma, such as amphotericin B, fluorocytosine, itraconazole, fluconazole, and voriconazole (Ameen et al. 2008). In the past, amphotericin B was the most potent antifungal drug for severe fungal infections, but its use is associated with severe side effects, particularly nephrotoxicity and low efficacy and it therefore has been replaced by newer azoles and echinocandins. Unfortunately, again little data are available on the in vitro antifungal susceptibility of conventional and new antifungal agents against the black yeast-like agents of mycetoma.

Diagnostic methods Conventional methods to identify etiologic agents often has been based on clinical aspects, histological appearance, macroscopic and microscopic features of the fungus in culture, temperature tests, and a limited number of physiological and biochemical assays. Not only do these methods depend on experienced, skilled laboratory staff, but also they are time-consuming, non-quantitative, and may lead to contamination. Despite long experience with classical methods for fungal diagnostics, they may lead to problems in identification, and to incorrect interpretation and diagnosis, and ultimately inappropriate treatment. These methods have also proved unreliable in differentiating very similar species, particularly in the group of black yeast-likeCladophialophora and Exophiala species, which now can be identified by molecular methods only (Zenget al. 2007). Rapid screening techniques have recently been developed and are increasingly used in all aspects of fungal diagnostics, such as DNA/RNA probe and polymerase chain reaction (PCR) technology. This will become increasingly important, as treatment modalities have variable effectiveness across species (Vital & de Hoog. 2002). Several molecular methods have been described for typing black yeasts and relatives. Molecular data enhance insight into genetic and epidemiological relationships between environmental and clinical isolates. Understanding of pathogen distributions is essential for determining epidemiology and ecology. Subcutaneous Cladophialophora species had previously been united as C. emmonsii, primarily based on morphology and temperature tolerance. Phenotypic and genotypic studies proved that distinct species are concerned. With the development of new assays such as MLST and AFLP (Vos et al. 1995) the information content of strain data has drastically increased. AFLP analysis can be used both for identification of isolates to the species level in as well as for discrimination at the intra-species level, more commonly referred to as “typing”. With AFLP, most of the isolates can be differentiated by small differences within profiles with high overall similarity. This confirmation of species identifications

16 Introduction

Fig. 2. Schematic representation of the AFLP method. (Vos P, Hogers R, Bleeker M, et al (1995). Nucleic Acids Res 23: 4407-4414. is possible, still leaving room for precise strain typing, indicating the high discriminatory power of this technique. Sequence-based typing of multiple genes (MLST) can be used to distinguish existing species and to describe novel taxa in the black yeasts and relatives.

Amplified fragment length polymorphism (AFLP) Amplified fragment length polymorphism (AFLP) analysis is a highly effective method for fingerprinting of genomic DNA, visualizing DNA polymorphisms between samples. These fingerprints may be used as a tool for determining the identity of a specific DNA sample, or to assess the relatedness between samples. AFLP-analysis, first described by Vos et al. (1995), relies on selective amplification of restriction fragments from a digest of genomic DNA with three steps involving restriction of the DNA and ligation of oligonucleotide adapters, selective amplification of sets of restriction fragments, and gel analysis of the amplified fragments. The AFLP technique can be used for DNAs of any origin or complexity (Vos et al. 1995; Fig. 2). Two restriction enzymes, one with an average cutting frequency and a second one with a higher cutting frequency, are used to digest DNA. Double stranded adaptors are ligated to the obtained sticky ends to serve as primer binding sites in successive PCR amplicons. The ligated fragments are subsequently amplified in a PCR reaction using stringent PCR annealing temperatures. The number of fragments that will be generated can be modulated by extending the amplification primer(s) at the 3’ site with one or more selective nucleotides. The addition of each nucleotide reduces the number of amplified fragments by a factor four. The PCR primer that spansthe average-frequency restriction site is labeled. At first the primers were radioactively labeled; a major

17 Chapter 1 improvement was the switch to fluorescently labeled primers for detection of fragments on a high- resolution electrophoresis platforms. A highly informative complex DNA banding pattern of 50 to 500 bp is obtained. Variations between different isolates originate by differences in the number and location of restriction enzyme recognition sites in the genome. Reproducibility of major bands is excellent.

Antifungal susceptibility The need for new antifungal agents stems from the increased frequency of fungal infections in the highly immunocompromised patient and the inadequacy of current antifungal regimens. This is particularly acute in opportunistic black yeast and other filamentous fungal infections. For example, despite the long-standing availability of amphotericin B, a potent fungicidal agent acting principally at the level of the fungal cell membrane, mortality of patients is almost 100% for all reported cases of cerebral phaeohyphomycosis. In order to improve antifungal therapy, we tried to obtain more insight into the in vitro antifungal susceptibility of Cladophialophora and related species against various antifungal agents, tested according to Clinical and Laboratory Standards Institute (CLSI) Reference method for broth dilution antifungal susceptibility testing of filamentous fungi (Approved Standard M38-A2, 2nd ed., CLSI, Wayne, PA, U.S.A., 2008).

Aim and outline of the thesis The aim of this thesis was to evaluate the various taxonomic, morphological and ecological aspects of Cladophialophora and related species in general, and evaluation of specific pathogenicity. Additionally, we investigate the in vitro antifungal susceptibility of several antifungal drugs against different black yeasts and relatives, including Cladophialophora bantiana, Rhinocladiella mackenziei, and Exophiala jeanselmei, which are agents of mainly cerebral phaeohyphomycosis, chromoblastomycosis, and mycetoma. With an understanding of phylogenetic relationships, it is likely that the agents derived from common ancestors will prove to share a number of essential virulence factors which must have come about by similar processes of evolution. Nevertheless closely related species may differ in environmental ecology and, as a consequence, in pathogenic potential and geographic distribution. Phylogenetic kinship then suggests that very different types of ecology may be based on shared factors. Through these data we will obtain precise insight into the origin of clinical strains, and whether survival on and eventual transmission from a human patient to another human being – with the possibility of subsequent evolution – occurs in nature. Chapter one is an overview of melanized fungi (black yeasts and relatives) which are mainly restricted to the order Chaetothyriales in the family Herpotrichiellaceae, with emphasis on the genus Cladophialophora. In addition, discussions are provided on the current taxonomy and ecology of black yeasts and relatives. Chapter two is the main chapter of this thesis and presents ecological information combined with phylogenetic and taxonomic data of Cladophialophora species causing different disorders, and also interprets results in the light of potential health hazards of seemingly saprobic species that may occur in food products. A multigene phylogeny was applied from these isolates using the internal transcribed spacer region (ITS rDNA), the translation elongation factor 1-α (EF1-α), the partial beta tubulin gene (TUB), and the small subunit of the nuclear ribosomal RNA gene (nucSSU). Four novel species were discovered, originating from soft drinks, alkylbenzene-polluted soil, and infected patients. Membership of the carrionii and bantiana clades might be indicative of potential virulence to humans. In addition, evolution of pathogenicity in the genus of Cladophialophora is discussed considering adaptations to extreme environments, including melanin production,

18 Introduction

meristematic growth at low pH, as well as thermotolerance. Chapter three describes a novel species: Cladophialophora saturnica, an opportunistic species 1 of Chaetothyriales that caused an interdigital tinea nigra-like lesion in a HIV positive Brazilian child. It is separated from known Cladophialophora species on the basis of the divergence of ITS rDNA and partial EF1-α gene, as well as on a distinct ecology. The same species was recovered from the natural environment by using selective isolation techniques. Chapter four describes a novel species of Cladophialophora which was isolated from soil polluted with benzene, toluene, ethylbenzene, and xylene (BTEX). It proved to be able to grow on toluene and related alkylbenzenes as its sole carbon and energy source. This strain is of interest for the complete biodegradation of toluene and other xenobiotics under growth limiting conditions, particularly in air biofilters with dry and/or acidic soil. A preliminary MLST and AFLP analysis showed this fungus to be very closely related to the pathogenic species C. bantiana, sharing a C. bantiana-specific intron in SSU rDNA. However, it was unable to grow at 40 °C and proved to be non-virulent in mice. A clear phylogenetic and ecophysiological delimitation of the species is fundamental to prevent biohazard in engineered bioremediation applications. Chapter five evaluate the inter and intraspecific genomic variation of 42 Cladophialophora isolates from cases with cerebral phaeohyphomycosis and other infections that were identified by the high-resolution fingerprinting technique AFLP. Specific clinical preference is established, and thus a second objective of this chapter is the evaluation of in vitro susceptibility to eight conventional and new generation antifungal drugs against C. bantiana, to improve the antifungal therapy in patients. Chapter six addresses the re-identification of all clinical and environmental strains maintained under the name Exophiala jeanselmei by sequencing the ITS region of rDNA and to establish the species clinical manifestations. A new case of mycetoma due to this fungus is described. The species was involved in different pathologies ranging from cutaneous infection to subcutaneous. Given the high incidence of eumycetoma in endemic areas, the second goal of this chapter is evaluation of the in vitro susceptibility of E. jeanselmei to various antifungal drugs to improve antifungal therapy in patients. New antifungal agents with a better activity may help to improve the management of eumycetoma. Chapter seven of this thesis aims at re-classifying the clinical manifestation of chromoblastomycosis which was already characterised by Carrión in 1950 into five different types, viz. nodular, plaque type, tumoral, cicatricial, and verrucous lesions. We describe a novel type of chromoblastomycosis, which distributed in the lymphatic form. We prove that Rhinocladiella aquaspersa is an agent of chromoblastomycosis and phaeohyphomycosis. Given the difficulties treating chromoblastomycosis and other infections that are caused by R. aquaspersa, we evaluate the in vitro activities of extended-spectrum triazoles, amphotericin B, and echinocandins against clinical isolates of the fungus. Chapter eight reports the first case of cerebral infection in a middle-aged male in India, where Rhinocladiella mackenziei is not endemic. Cerebral phaeohyphomycosis due to R. mackenziei is a severe infection in the Middle East, resulting in nearly 100 % mortality despite the application of combined surgical and antifungal therapy. The species occasionally occurs in otherwise healthy patients. Chapter nine evaluates the in vitro activity of a new triazole antimycotic, isavuconazole, and seven comparators (amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, caspofungin and anidulafungin) against highly fatal isolates of Rhinocladiella mackenziei. Some R. mackenziei infections cause brain abscesses in patients without predisposing factors or

19 Chapter 1

immunodeficiency. Treatment regimens, which involve mostly amphotericin B, itraconazole and 5-flucytosine, and are associated with poor outcome in both animal and clinical studies. New drugs seem to be promising for effective treatment of the infection, although activity of isavuconazole against R. mackenziei needs to be confirmed in animal models. Chapter ten contains a summary of the results and conclusions of this thesis. The impact of the present study in understanding the morphology, ecology, taxonomy and pathogenicity of species in the genus Cladophialophora and related species is discussed. Furthermore, perspectives on future research are given.

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20 Introduction

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21 Chapter 1

Sterflinger K (1998a). Ecophysiology of rock inhabiting black yeasts with special reference to temperature and osmotic stress. Antonie van Leeuwenhoek 74: 271-281. Sterflinger K (1998b). Temperature and NaCl-tolerance of rock-inhabiting meristematic fungi. Antonie van Leeuwenhoek 74: 271-281. Spatafora JW, Mitchell TG, Vilgalys R (1995). Analysis of genes coding for small-subunit rRNA sequences in studying phylogenetics of dematiaceous fungal pathogens. J Clin Microbiol 33: 1322-1326. Sudhadham M, Dorrestein GM, Prakitsin S, et al (2008). The neurotropic black yeastExophiala dermatitidis has a possible origin in the tropical rain forest. Stud Mycol 61: 145-155. Untereiner WA, Naveau FA (1999). Molecular systematics of the Herpotrichiellaceae with an assessment of the phylogenetic positions of Exophiala dermatitidis and Phialophora americana. Mycologia 91: 67–83. Untereiner WA (1997). Taxonomy of selected members of the ascomycetes genus Capronia with notes on anamorph- teleomorph connections. Mycologia 89: 210-131. Untereiner WA (2000). Capronia and its anamorphs: exploring the value of morphological and molecular characters in the systematics of the Herpotrichiellaceae. Stud Mycol 45: 141-149. Untereiner WA, Straus NA, Malloch D (1995). A molecular-morphotaxonomic approach to the systematics of the Herpotrichiellaceae and allied black yeasts. Mycological Research 99: 897-913. Vicente VA, Attili-Angelis D, Pie MR, et al (2008). Environmental isolation of black yeast-like fungi involved in human infection. Stud Mycol 61: 136–144. Vitale RG, de Hoog GS (2002). Molecular diversity, new species and antifungal susceptibilities in the Exophiala spinifera clade. Med Mycol 40: 545-56. Vos P, Hogers R, Bleeker M, et al (1995). AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23: 4407- 4414. Weber FJ, Hage KC & de Bont JA (1995). Growth of the fungus Cladosporium sphaerospermum with toluene as the sole carbon and energy source. Appl Environ Microbiol 61: 3562-3566. Werlinger KD, Yen Moore A (2005). Eumycotic mycetoma caused by Cladophialophora bantiana in a patient with systemic lupus erythematous. J Am Acad Dermatol 52: 114-117. Yegüez-Rodriguez J, Richard-Yegres N, Yegres F, Rodríguez Larralde A (1992). Cromomicosis: susceptibilidad genética en grupos familiares de la zona endémica en Venezuela. Acta Cientifica Venezuelana43 : 98-102. Yurlova NA, de Hoog GS (2002). Exopolysaccharides and capsules in human pathogenic Exophiala species. Mycoses 45: 443-448. Zahlbruckner, A. 1926. Catalogus Lichenum Universalis. Leipzig: Borntraeger. 480 p. Zalar P, de Hoog GS, Gunde-Cimerman N (1999). Trimmatostroma salinum, a new species from hypersaline water. Stud Mycol 43: 57-62. Zeng JS, Sutton DA, Fothergill AW, et al (2007). Spectrum of clinically relevant Exophiala species in the U.S.A. J Clin Microbiol 45: 3713-3720. Zhao J, Zeng J, de Hoog GS, et al (2010). Isolation of black yeasts by enrichment on atmospheres of monoaromatic hydrocarbons. Microbial Ecol. DOI: 10.1007/s00248-010-9651-4. (in press). 22 2 Chapter 2

Biodiversity of the genus Cladophialophora

H. Badali 1, 2, 3, C. Gueidan 1, M.J. Najafzadeh 1, 2, A. Bonifaz 4, A.H.G. Gerrits van den Ende 1, G.S. de Hoog 1, 2

Published: Studies in Mycology 2008, 61: 175-191. 223 Chapter 2

Abstract Cladophialophora is a genus of black yeast-like fungi comprising a number of clinically highly significant species in addition to environmental taxa. The genus has previously been characterised by branched chains of ellipsoidal to fusiform conidia. However, this character was shown to have evolved several times independently in the order Chaetothyriales. On the basis of a multigene phylogeny (nucLSU, nucSSU, RPB1), most of the species of Cladophialophora (including its generic type C. carrionii) belong to a monophyletic group comprising two main clades (carrionii- and bantiana-clades). The genus includes species causing chromoblastomycosis and other skin infections, as well as disseminated and cerebral infections, often in immunocompetent individuals. In the present study, multilocus phylogenetic analyses were combined to a morphological study to characterize phenetically similar Cladophialophora strains. Sequences of the ITS region, partial Translation Elongation Factor 1-α and β-Tubulin genes were analysed for a set of 48 strains. Four novel species were discovered, originating from soft drinks, alkylbenzene-polluted soil, and infected patients. Membership of the both carrionii and bantiana clades might be indicative of potential virulence to humans.

Key words: Biodiversity, bioremediation, Cladophialophora, chromoblastomycosis, disseminated infection, MLST, mycetoma. Taxonomic novelties: Cladophialophora samoënsis Badali, de Hoog & Padhye, sp. nov., Cladophialophora subtilis Badali & de Hoog, sp. nov., Cladophialophora mycetomatis Badali, de Hoog & Bonifaz, sp. nov., Cladophialophora immunda Badali, Satow, Prenafeta-Boldú, Padhye & de Hoog, sp. nov.

Introduction Cladophialophora is a genus of black yeast-like fungi which are remarkably frequently encountered in human infections, ranging from mild cutaneous lesions to fatal encephalitis. The genus is morphologically characterised by one-celled, ellipsoidal to fusiform, dry conidia arising through blastic, acropetal conidiogenesis, and arranged in branched chains. The chains are usually coherent and conidial scars are nearly unpigmented (Borelli 1980, Ho et al. 1999, de Hoog et al. 2000). The genus was initially erected to accommodate species exhibiting Phialophora-like conidiogenous cells in addition to conidial chains (Borelli 1980) but thus far this feature is limited to the species C. carrionii. Teleomorphs have not been found, but judging from SSU rDNA phylogeny data, these are predicted to belong to the ascomycete genus Capronia, a member of the order Chaetothyriales (Haase et al. 1999). The type species of Cladophialophora, C. carrionii, is an agent of chromoblastomycosis, a cutaneous and subcutaneous disease histologically characterised by muriform cells in skin tissue. Muriform cells represent the invasive form of fungi causing chromoblastomycosis (Mendoza et al. 1993). Infections are supposed to originate by traumatic implantation of fungal elements into the skin and are chronic, slowly progressive and localised. Tissue proliferation usually occurs around the area of inoculation, producing crusted, verrucose, wart-like lesions. The genus has been expanded to encompass several other clinically significant species, including the neurotropic fungi C. bantiana and C. modesta causing brain infections (Horré & de Hoog 1999), C. devriesii and C. arxii causing disseminated disease (de Hoog et al. 2000) and C. boppii, C. emmonsii and C. saturnica causing cutaneous infections (de Hoog et al. 2007, Badali et al. 2008). Based on molecular data, anamorphs morphologically similar to Cladophialophora have been found in other groups of ascomycetes, particularly in the Dothideales / Capnodiales (e.g., Pseudocladosporium, Fusicladium; Crous et al. 2007). Distinction between chaetothyrialean and dothidealean / capnodialean anamorphs is now also supported by their teleomorphs. Braun

24 Biodiversity of the genus Cladophialophora

& Feiler (1995) reclassified the dothidealean species Venturia hanliniana (formerly Capronia hanliniana) as the teleomorph of Fusicladium brevicatenatum (formerly Cladophialophora brevicatenata). Further distinction between Chaetothyriales and Dothideales / Capnodiales lies in their ecology, with recurrent human opportunists being restricted to the Chaetothyriales. Braun (1998), summarizing numerous statements in earlier literature, separated Cladophialophora with Capronia teleomorphs (Herpotrichiellaceae, Chaetothyriales mostly as opportunistic or pathogens), from the predominantly saprobic or plant associated isolates in the Dothideomycetes. 2 Some species attributed to Cladophialophora may be found in association with living plants. De Hoog et al. (2007) reported a cactus endophyte, Cladophialophora yegresii, as the nearest neighbour of C. carrionii, which is a major agent of human chromoblastomycosis. The latter fungus was believed to grow on debris of tannin-rich cactus spines, which were also supposed to be the vehicle of introduction into the human body. Crous et al. (2007) described several host- specific plant pathogens associated with the Chaetothyriales. Cladophialophora hostae caused spots on living leaves of Hosta plantaginea, C. proteae was a pathogen of Protea cynaroides, and C. scillae caused leaf spots on Scilla peruviana. Finally, Davey & Currah (2007) described Cladophialophora minutissima from mosses collected at boreal and montane sites in central Alberta, Canada. Within the Chaetothyriales, most of these plant-associated species are found at relatively large phylogenetic distance from the main clade of Cladophialophora comprising most of the opportunistic species. The core of the genus Cladophialophora does comprise a number of environmental saprobes. Cladophialophora minourae and C. chaetospira occur in plant litter. Badali et al. (2008) described C. saturnica from plant debris in the environment, but the species was also found causing an interdigital infection in a Brazilian child with HIV infection. The species C. australiensis and C. potulentorum were found in soft drinks (Crous et al. 2007). If environmental species are able to provoke opportunistic infections, the question arises whether members of Chaetothyriales isolated from food products might imply a health risk. Understanding of the phylogeny and ecology of Cladophialophora is therefore essential. Many review articles incorrectly mention that black yeast-like fungi are commonly found on decomposing plant debris and in soil. In fact, Chaetothyrialean members are difficult to isolate from the environment as they seem to have quite specific, hitherto undiscovered ecological niches (Satow et al. 2008, Vicente et al. 2008). For their isolation, selective methods are required, e.g. by the use of high temperatures (Sudhadham et al. 2008), a mouse vector (Gezuele et al. 1972, Dixon et al. 1980), alkyl benzenes (Prenafeta-Boldú et al. 2006) or isolation via mineral oil (Satow et al. 2008, Vicente et al. 2001, 2008). An association with assimilation of toxic monoaromatic compounds has been hypothesised. Black yeasts and their filamentous relatives in theChaetothyriales are potent degraders of monoaromatic compounds and tend to accumulate in industrial biofilters (Coxet al. 1997, Prenafeta-Boldú et al. 2001, de Hoog et al. 2006). This might be a clue to dissecting their dual behavior as rare environmental oligotrophs as well as invaders of human tissue containing aromatic neurotransmitters. The present paper combines ecological information with phylogenetic and taxonomic data, and interprets them in the light of potential health hazards of seemingly saprobic species that may occur in food products. We applied multilocus sequence analysis and phenetic characterization to distinguish novel Cladophialophora species from various sources.

25 Chapter 2 Origin State Falcon Venezuela, State Falcon Venezuela, State Falcon Venezuela, State Falcon Venezuela, State Falcon Venezuela, State Falcon Venezuela, State Falcon Venezuela, State Falcon Venezuela, State Falcon Venezuela, – Virginia U.S.A., Brazil Dordrecht Netherlands, U.S.A. Africa, Pretoria, South Onderstepoort U.S.A., Bethesda Barbados del Grande Isla Uruguay, Queguay del Grande Isla Uruguay, Queguay Curitiba Paraná, Brazil, Source leg; female, Chromoblastomycosis; hand; male Chromoblastomycosis; arm; male Chromoblastomycosis; arm; female Chromoblastomycosis; lesion in human Skin human Chromoblastomycosis, griseus asymptomatic plant Stenocereus griseus asymptomatic plant Stenocereus griseus asymptomatic plant Stenocereus lesion, cat Sub-cutaneous human lesion right forearm, Sub-cutaneous male lesion, on limb, Skin Scales of face, male abscess, male Brain infection, dog Disseminated lesion, cat Skin dog Liver, tree Dead cut tree Trunk, toe lesion, child Interdigital α EF1 , TUB GenBank ITS, EU137267, EU137150, EU137211 EU137268, EU137151, EU137212 EU137269, EU137152, EU137213 EU137271, EU137154, EU137215 EU137292, EU137175, EU137234 EU137266, EU137201, EU137210 EU137323, EU137208, EU137263 EU137322, EU137209, EU137262 EU137324, –, U137264 EU103995, –, U140584 EU103996, –, U140583 EU103997, –, U140596 EU103998, –, U140597 EU103989, –, U140585 EU103994, –, U140591 EU103992, –, U140592 EU103993, –, U140587 EU103983, –, U140601 FJ385270, –, – EU103984, – , EU140602 ATCC 16264 ATCC C. carrionii) = Other reference Other UNEFM 82267 = dH 13261 UNEFM 9801 = dH 13262 UNEFM 2001/1 = dH 13265 UNEFM 2003/1 = dH 13267 44535 CDC B-1352 = FMC 282 ATCC of CDC A-835 =(ex-LT UNEFM SgSR1; dH 13275 dH 13276 UNEFM SgSr3; UNEFM SgSR1; dH 13274 CDC B-3634; NCMH 2248; 4991 CDC B-3875; NCMH 2247 FMC 292; dH 15357 det M-41/2001 56893; dH 12362 CBS 100433 – CDC B-3658; NCMH 2249; NIH B-3839 3830 UAMH dH 12333; IHM 1727 dH 12335; IHM 1733 157D; dH 12939 Status LT T T CBS CBS 114392 CBS 114393 CBS 114396 CBS 114398 CBS 260.83 CBS 160.54 CBS 114406 CBS 114405 CBS 114407 CBS 640.96 CBS 979.96 CBS 126.86 CBS 110029 CBS 173.52 CBS 444.96 CBS 678.79 CBS 648.96 CBS 109628 CBS 109630 CBS 118724 carrionii emmonsii boppii bantiana

Name Cladophialophora yegresii Cladophialophora Cladophialophora Cladophialophora Cladophialophora saturnica Cladophialophora Table 1. Isolation data of examined strains. 1. Isolation Table

26 Biodiversity of the genus Cladophialophora Origin Curitiba Paraná, Brazil, Wageningen Netherlands, Cayman U.S.A., Grand Island Curitiba Paraná, Brazil, Germany Chiba Yachimata, Japan, Shiroi Japan, Apeldoorn Netherlands, Kaiserslautern Germany, Atlanta U.S.A., Georgia, Colombo Paraná, Brazil, Sarandi Paraná, Brazil, U.S.A., Samoa Utrecht Netherlands, Netherlands Jicaltepec Mexico, (zoo) Netherlands Brazil Brazil America South –

2 Source cover/soil vegetable Litter, biofilter Toluene infection, male Disseminated cover/soil vegetable Litter, abscess, male Tracheal wood Rotting wood Decaying soil Gasolin-station inoculated with soil Biofilter male phaeohyphomycosis, Sub-cutaneous stem romanzoffianum, Syagrum vegetable cover Decaying skin lesion, male Chromoblastomycosis tea Ice contaminant Culture male Eumycetoma, aspiration-biopsy Lymphnode, Soil male Chromoblastomycosis, male Chromoblastomycosis, male Chromoblastomycosis, α EF1 , TUB GenBank ITS, – , EU140600 AY857508, –, EU140603 AY857507, EU103985, –, EU140595 FJ385275, –, – EU103986, – , EU140593 EU103988, – , EU140599 – , EU140598 AY251087, FJ385274, EU137207, EU137261 FJ385271, EU137206, EU137260 EU137318, EU137203, EU137257 FJ385269, –, EU137259 FJ385272, EU137205, EU137285 EU137291, EU137174, EU137233 FJ385273, –, – EU137293, EU137176, EU137235 FJ385276, –, – EU938554, – AY366925, EU938546, – AY366927, EU938550, – AY366926, EU938559, – AY366914, –, – AY366917, Other reference Other dH 11591; 4IIBPIRA 200384 ATCC 56280; CDC 82-030890 ATCC ISO 13F 5022 IFM 4701; UAMH 52853; IMI 298056 ATCC dH15250 dH 11474 CDCB-5680; de H.10680 dH 11588 dH 11601 CDC B-3253; dH 15637 dH 14614 dH 15898 dH 18909 dH 15691 dH 11602, 1PLE dH 11613 18658; IMI 134458; dH 15659 ATCC dH 15661 Status T T T T T T T T CBS CBS 102230 CBS 114326 CBS 147.84 CBS 118720 CBS 306.94 CBS 987.96 CBS 556.83 CBS 110551 CBS 109797 CBS 834.96 CBS 102227 CBS 102237 CBS 259.83 CBS 122642 CBS 454.82 CBS 122637 CBS 289.93 CBS 102238 CBS 102248 CBS 271.37 CBS 272.37 devriesii arxii minourae immunda samoënsis subtilis

monophora pedrosoi

Name Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora mycetomatis Fonsecaea Fonsecaea Table 1. (Continued). Table

27 Chapter 2 Origin Australia Denmark Australia Australia T = ex-type culture; LT = LT T = ex-type culture; Picea abies Picea Source drink Sports of Roots drink Sports drink Sports α EF1 , TUB GenBank ITS, EU035402, –, – EU035405, –, – EU035409, –, – EU035410, –, – Other reference Other CPC 1377 – CPC 1376; FRR 4946 CPC 1375; FRR 4947 Status CBS CBS 112793 CBS 491.70 CBS 112222 CBS 114772 Name Cladophialophora australiensis Cladophialophora chaetospira Cladophialophora potulenturum Pathogenic Institute for Research collection; IFM = Hoog private Netherlands; DH = G.S. de The Utrecht, Centre, Biodiversity Fungal U.S.A.; CBS = CBS-KNAW Collection, Manassas, Culture Type = American used: ATCC Abbrevations London, U.K.; IWW = Rheinisch Institute, Mycological IMI = International Uruguay; Montevideo, and Hygiene, of Epidemiology Institute Montevideo of Medicine, Faculty IHM = Laboratory of Mycology, Chiba, Japan; Fungi, Memorial Carolina NCMH = North Belgium; Louvain-la-Neuve, de Louvain, de l’Université collection; MUCL = Mycotheque private GHP = G. Haase Germany; an der Ruhr, Mülheim Wasserforschung, für Institut Westfählisches Texas of University Department of Pathology, Laboratory, Testing Canada; UTHSC = Fungus and Collection, Edmonton, Herbarium = Microfungus UAMH Germany; Berlin, Institute, Koch U.S.A.; RKI = Robert Chapel Hill, Hospital, Venezuela. Falcon, Coro, de Miranda, Francisco Experimental Nacional U.S.A.; UNEFM = Universidade Galveston, Center, Research Mycology Antonio, U.S.A.; UTMB = Medical Science Center at San Health ex-lectotype culture; Table 1. (Continued). Table

28 Biodiversity of the genus Cladophialophora

Table. 2. Primer sequences for PCR amplification and sequencing. Gene PCR primers Sequencing primers References

ITS rDNA V9Ga, LS266b ITS1c, ITS4c a de Hoog et al. (1998) b Masclaux et al. (1995) c White et al. (1990)

TUB Βt2a, Βt2b Βt2a, Βt2b Glass & Donaldson (1995)

EF1-α EF1-728F, EF1-986R EF1-728F, EF1-986R Carbone & Kohn (1999) 2 SSU rDNA NS1a, NS24b (BF83, Oli1, Oli9, BF951, BF963, BF1438, Oli3, a White et al. (1990) BF1419) c b Gargas & Taylor (1992) c de Hoog et al. (2005)

Materials and Methods

Fungal strains Strains used in this study were obtained from the CBS-KNAW Fungal Biodiversity Centre (Table 1). Stock cultures were maintained on slants of 2 % malt-extract agar (MEA, Difco) and oatmeal agar (OA, Difco) and incubated at 24 °C for two weeks (Gams et al. 1998). All cultures in this study are maintained in the culture collection of CBS (Utrecht, The Netherlands) and taxonomic information for new species was deposited in MycoBank (www.MycoBank.org).

DNA extraction The fungal mycelia were grown on 2 % (MEA) plates for 2 weeks at 24 °C (Gams et al. 1998). A sterile blade was used to scrape off the mycelium from the surface of the plate. DNA was extracted using an Ultra Clean Microbial DNA Isolation Kit (Mobio, Carlsbad, CA 92010, U.S.A.) according to the manufacturer’s instructions. DNA extracts were stored at −20 °C prior to use.

Amplification and sequencing Four genes were amplified: the internal transcribed spacer region (ITS), the translation elongation factor 1 alpha (EF1-α), the partial beta tubulin gene (TUB), and the small subunit of the nuclear ribosomal RNA gene (nucSSU). The primers used for amplification and sequencing are shown in Table 2. PCR reactions were performed on a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA) in 50 µL volumes containing 25 ng of template DNA, 5 µL reaction buffer (0.1

M Tris-HCl, pH 8.0, 0.5 M KCl, 15 mM MgCl2, 0.1 % gelatine, 1 % Triton X-100), 0.2 mM of each dNTP and 2.0 U Taq DNA polymerase (ITK Diagnostics, Leiden, The Netherlands). Amplification of ITS and nucSSU was performed with cycles of 2 min at 94 °C for primary denaturation, followed by 35 cycles at 94 °C (45 s), 52 °C (30 s) and 72 °C (120 s), with a final 7 min extension step at 72 °C. Annealing temperatures used to amplify EF1α and TUB genes were 55 and 58 °C, respectively. Amplicons were purified using GFX PCR DNA and gel band purification kit (GE Healthcare, Ltd., Buckinghamshire U.K.). Sequencing was performed as follows: 95 °C for 1 min, followed by 30 cycles consisting of 95 °C for 10 s, 50 °C for 5 s and 60 °C for 2 min. Reactions were purified with Sephadex G-50 fine (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and sequencing was done on an ABI 3730XL automatic sequencer (Applied Biosystems, Foster City, CA, U.S.A.). Sequence data obtained in this study were adjusted using the SeqMan of Lasergene software (DNAStar Inc., Madison, Wisconsin, U.S.A.).

29 Chapter 2

Alignment and phylogenetic reconstruction Phylogenetic analyses were carried out on three different datasets. The first dataset included a taxon sampling representative of the order Chaetothyriales (94 taxa) and the three genes nucLSU, nucSSU and RPB1. This dataset was used in order to assess the phylogenetic placement of diverse species of Cladophialophora within the Chaetothyriales. For this first analysis, sequences of nucSSU obtained from investigated strains of Cladophialophora were added to an existing dataset representing the Chaetothyriales (Gueidan et al. 2008). Alignments were done manually for each gene using MacClade 4.08 (Maddison & Maddison 2003) with the help of amino acid sequences for protein coding loci. Ambiguous regions and introns were excluded from the alignments. The program RAxML-VI-HPC v.7.0.0 (Stamatakis et al. 2008), as implemented on the Cipres portal v.1.10, was used for the tree search and the bootstrap analysis (GTRMIX model of molecular evolution and 500 bootstrap replicates). Bootstrap values equal or greater than 70 % were considered significant (Hillis & Bull 1993). The second and third datasets focused on two main monophyletic clades nested within the core group of Cladophialophora (Clade I and Clade II, Fig. 1). They included three genes, ITS, EF1α and TUB. The goal of these two analyses was to assess the delimitation of species ofCladophialophora . The second dataset (Clade I or carrionii-clade) comprised 15 taxa, and the third dataset (Clade II or bantiana-clade) 33 taxa. Phylogenetic reconstructions and bootstrap values were first obtained for each locus separately using RAxML (as described above). The congruence between loci was assessed using a 70 % reciprocal bootstrap criterion (Mason-Gamer & Kellogg 1996). The loci were then combined and analysed using RAxML (as described above). The phylogenetic trees were edited using Tree View v.1.6.6.

Morphological identification and cultural characterisation Strains were cultured on 2 % MEA and OA and incubated at 24 °C in the dark for two weeks (Gams et al. 1998). Identification was based primarily on macroscopic and microscopic morphology. Microscopical observations were based on slide culture techniques using potato dextrose agar (PDA) or OA because these media readily induce sporulation and suppress growth of aerial hyphae (de Hoog et al. 2000). Mounts of two-wk-old slide cultures were made in lactic acid or lactophenol cotton blue and light micrographs were taken using Nikon Eclipse 80i microscope with a Nikon digital sight DS-Fi1 camera.

Physiology Cardinal growth temperatures of strains were determined on 2 % MEA (Difco) (Crous et al. 1996). Plates were incubated in the dark for two weeks at temperatures of 6–36 °C at intervals of 3 °C; in addition, growth at 40 °C was recorded. Experiments consisted of three simultaneous replicates for each isolate; the entire procedure was repeated once.

Muriform cells All strains were tested for the production of muriform cells, i.e., the meristematic tissue form of agents of human chromoblastomycosis. Strains were incubated at 25 and 37 °C for one wk in a defined medium with low pH containing 0.1 mM/L calcium chloride. The basal medium was prepared by adding the following components to 1 L deionized distilled water: 30 g glucose, 3 g

NaNO3, 0.01 g FeSO4∙7H2O, 0.265 g NH4Cl, 0.003 g thiamin and 1 mM CaCl2; the pH was adjusted to 2.5 with HCl for all experiments (Mendoza et al. 1993).

30 Biodiversity of the genus Cladophialophora

Results

Phylogeny

Chaetothyriales dataset

Fig. 1 shows that, in the order Chaetothyriales, species of Cladophialophora belong to at least four 2 different lineages: one lineage corresponding to the family Herpotrichiellaceae (lineage 1, Fig. 1), and three basal lineages including mostly rock-inhabiting strains (lineages 2, 3 and 4, Fig. 1). Most of the human-opportunistic species (including the most virulent ones) belong to two well- supported clades within the Herpotrichiellaceae. Clade I (carrionii-clade, Fig. 1, 2) includes the pathogenic Cladophialophora species C. carrionii and C. boppii, as well as two pathogenic species of Phialophora (P. verrucosa and P. americana). Clade II (bantiana-clade, Fig. 1, 3) includes the pathogenic species C. bantiana, C. arxii, C. immunda, C. devriesii, C. saturnica, C. emmonsii, and C. mycetomatis, as well as the pathogenic genus Fonsecaea (F. pedrosoi and F. monophora). All the plant associated species of Cladophialophora belong to lineages 2, 3 and 4. Lineage 2 includes rock- inhabiting strains and the opportunistic pathogen C. modesta. Lineage 3 includes rock-inhabiting strains, the two opportunistic pathogens P. europaea, and C. laciniata, and three plant associated species of Cladophialophora (C. proteae, C. hostae, and C. scillae). Lineage 4 includes exclusively rock-inhabiting strains and plant leaving strains (C. minutissima, C. humicola, and C. sylvestris).

Carrionii-clade dataset Phylogenetic reconstructions of Clade I (carrionii-clade; Fig. 2) were first carried out for each gene separately (ITS: 542 characters, EF1-α: 117 characters, TUB: 402 characters). Topological conflicts were detected for all genes within both C. carrionii and C. yegresii. This incongruence involving only below species-level relationships, and therefore – in agreement with the genealogical recognition species concept (Taylor et al. 2000) –, the conflicts were ignored and the three loci were combined. In this combined analysis, C. boppii, an agent of cutaneous infection, was taken as an out-group for clade I (Fig. 2). The combined analysis shows that both the pathogenic species C. carrionii (represented by 6 strains) and the environmental species C. yegresii (represented by 3 strains) are monophyletic and well supported (100 % bootstrap, Fig. 2). The two species of Phialophora (P. verrucosa and P. americana) are sister taxa (100 % bootstrap, Fig. 2), and are nested among species of Cladophialophora. Two newly investigated strains (CBS 122642, isolated from soft drink; CBS 259.83, isolated from a patient with chromoblastomycosis) are shown to be phylogenetically distinct from other members of Clade I, and are described below as new species (C. samoënsis and C. subtilis).

Bantiana-clade dataset Phylogenetic reconstructions of Clade II (bantiana-clade, Fig. 3) were first carried out for each gene separately (ITS: 495 characters, EF1-α: 133 characters, and TUB: 355 characters). As topological incongruence was detected only within the species C. saturnica (relationships obtained with ITS differed from relationships obtained with EF1-α or TUB), the loci were combined. The resulting tree was rooted using C. mycetomatis, a species newly described here. A phylogenetic analysis at larger scale (Fig. 1) shows that this taxon is phylogenetically distinct from other species of Cladophialophora, and sister to all the other members of the bantiana-clade. The three species C. immunda, C. devriesii and C. saturnica all form monophyletic clades including both clinical and

31 Chapter 2 environmental strains. For C. immunda, strains from different geographical areas and ecological preferences all cluster together. The environmental species C. minourae is sister to the pathogenic species C. arxii. The truly pathogenic speciesC. bantiana is sister to C. emmonsii, although with no support. Finally, the genus Fonsecaea forms a well-supported monophyletic group nested within this clade of Cladophialophora.

Physiology The cardinal growth temperature test showed that all cultures obtained in this study had their optimal development at 27-30 °C, with growth abilities ranging between 9-37 °C. No growth was observed at 40 °C. For C. samoënsis, C. immunda and C. mycetomatis, the optimum growth temperature on MEA and PDA was 27 °C, with minimum and maximum of 15 and 37 °C, respectively. For all the other species, growth temperatures were identical except for the minimum temperature, which was 12 °C in C. subtilis. However, neither plant associated species nor strains isolated from sport drink nor apple juice (C. australiensis and C. potulentorum) had the ability to grow at 37 and 40 ° C (Fig. 4). Growth characteristics were studied at low pH after addition of

0.1 mM CaCl2 to the basal medium, inducing conversion of hyphae of Cladophialophora species into muriform cells when incubated at 25−37 °C (Table 3). Cladophialophora subtilis developed extensive mycelia and produced muriform cells at 25 °C after one wk incubation (Fig. 5). Hyphae were generally attached to these muriform cells in either terminal or intercalary positions (Fig. 5). However, no muriform cells were observed under the same conditions at 25 °C in other species (C. immunda, C. mycetomatis and C. samoënsis). Hyphae of C. subtilis, C. immunda and C. samoënsis converted to large numbers of muriform cells when incubated in the same conditions at 37 °C. Moreover, muriform cells were not observed for plant-associated species of Cladophialophora and for C. mycetomatis and C. yegresii neither at 25 nor at 37 °C (Table 3).

Taxonomy of Cladophialophora Cladophialophora carrionii (Trejos) de Hoog, Kwon-Chung & McGinnis, J. Med. Vet. Mycol. 33: 345 (1995). = Cladosporium carrionii Trejos, Revista de Biologia Tropicale, Valparaiso 2: 106 (1954). = Cladophialophora ajelloi Borelli, Proceedings of the 5th International Conference on Mycoses: 335 (1980). Type: Trejos 27 CBS H-18465, lectotype designated here; CBS 160.54 = ATCC 16264 = CDC A-835 = MUCL 40053, ex-type. Trejos (1954) introduced Cladophialophora carrionii but he did not indicate a holotype. For this reason, the isolate Trejos 27 = Emmons 8619 = CBS 160.54, the first strain mentioned by Trejos (1954), is selected here as lectotype for C. carrionii. The ex-type strain of Cladophialophora ajelloi, CBS 260.83, proved to be indistinguishable from C. carrionii based on both morphology and molecular data. This former species was also known to be able to produce phialides in addition to catenate conidia (Honbo et al. 1984). Cladophialophora ajelloi is here proposed as a taxonomic synonym of C. carrionii.

Cladophialophora samoënsis Badali, de Hoog & Padhye, sp. nov. MycoBank MB511809. Figs 6–7.

Etymology: Named after Samoa, the Pacific Island where the species was encountered in a human patient.

Coloniae fere lente crescentes, ad 30 mm diam post 14 dies, olivaceo-virides vel griseae, reversum olivaceo-nigrum.

32 Biodiversity of the genus Cladophialophora

Fig.1. Phylogeny obtained from a ML analysis of three combined loci (SSU, LSU and RPB1) using RAxML. Bootstrap support values were estimated based on 500 replicates, and are shown above the branches (thick branch for values ≥ 70 %). The tree was rooted using Verrucula inconnexaria, Placocarpus schaereri and the rock isolate TRN242. New species are highlighted with grey boxes.

33 Chapter 2

Fig. 2. Phylogeny of Clade I obtained from a ML analysis of three combined loci (ITS rDNA, EF1-α and TUB) using RAxML. Bootstrap support values were estimated based on 500 replicates, and are shown above the branches (thick branch for values ≥ 70 %). New species are highlighted using with grey boxes. Cladophialophora boppii (CBS 126.86 and CBS 110029) were taken as outgroup.

Cellulae gemmantes absentes. Hyphae leves, hyalinae vel pallide brunneae, 2–3 μm latae. Conidiophora semi- macronemata, septata, lateralia vel terminalia; stipites et ramoconidia denticulata. Conidia holoblastica, dilute brunnea, late fusiformia, unicellularia, levia, catenas longas ramosas cohaerentes formantia, cicatricibus dilute brunneis, 3–4 × 2–3 μm. Synanamorphe not visa. Chlamydosporae absentes. Teleomorphe ignota.

Description based on CBS 259.83 at 27 °C on MEA after 2 weeks in darkness.

Cultural characteristics: Colonies growing moderately slowly, reaching up to 30 mm diam, olivaceous-green to grey, with a thin, dark, well-defined margin, dry, velvety, darker after 4 weeks; reverse olivaceous-black. Cardinal temperatures: minimum 15 °C, optimum 27−30 °C, maximum 37 °C. No growth at 40 °C.

Microscopy: Budding cells absent. Hyphae smooth, hyaline to pale brown, branched, 2−3 µm wide, locally forming hyphal strands and coils. Conidiophores semi-macronematous, septate, lateral or terminal, with denticles on the stipe and on 0–1-septate ramoconidia. Conidia holoblastic pigmented, broadly fusiform, one-celled, pale brown, smooth-walled, forming long, cohering, branched acropetal chains of conidia; conidial scars pale. Conidia 3–4 × 2–3 µm. Synanamorph

34 Biodiversity of the genus Cladophialophora

Fig. 3. Phylogeny of Clade II obtained from a ML analysis of three combined loci (ITS rDNA, EF1-α and TUB) using RAxML. Bootstrap support values were estimated based on 500 replicates, and are shown above the branches (thick branch for values ≥ 70 %). New species are highlighted using with grey boxes. The tree was rooted with two strains of Cladophialophora mycetomatis (CBS 454.82 and CBS 122637). not seen. Chlamydospores absent. Teleomorph unknown.

Specimen examined: U.S.A., Samoa (Pacific), isolated from human patient with chromoblastomycosis, November 1979, CBS H-20113, holotypus; CDC B-3253 = CBS 259.83, ex-type.

Notes: This strain (CBS 259.83, from Samoa) was previously identified as C. ajelloi (Goh et al. 1982). In our multigene analysis, it clustered within the carrionii complex (Clade I; Fig. 2), but proved to be consistently different from all described species.

Case Report: According to the description by Goh et al. (1982), a healthy, 43-year-old male patient had a 5 × 3 cm erythematous, scaling lesion on his arm which was first observed about 3 yr earlier. The patient did not remember the circumstances under which the infection had been acquired. Muriform cells were revealed in superficial dermis and stratum corneum after skin scrapings. Histological examination showed segments of well-differentiated stratified squamous epithelium

35 Chapter 2

Fig. 4. Colony diameters of novel Cladophialophora species at different temperatures ranging from 6 to 40 °C, measured after two weeks on 2 % MEA. with moderate keratin production and underlying coarsely fibrillar dermal connective tissue. In this tissue, dense aggregates of chronic inflammatory cells, including lymphocytes, plasma cells and multinucleated foreign bodies (giant cells), were observed. Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) stained sections characterised dark brown, thick-walled, multiseptate muriform cells, measuring 6−12 μm in diameter, and dividing by fission. The histopathological observations led to the diagnosis of chromoblastomycosis, and the strain was identified asC. ajelloi (Goh et al. 1982). The taxon name C. ajelloi is not available for this taxon, as it was shown here to be a synonym of C. carrionii. Hence Cladophialophora samoënsis is described as a novel agent of chromoblastomycosis. Morphological observation showed branched chains of holoblastic conidia identical to those of Cladophialophora carrionii.

Cladophialophora subtilis Badali & de Hoog, sp. nov. MycoBank MB511842. Figs 8, 11.

Etymology: Named after thin-walled, conidial structures.

Coloniae fere lente crescentes, ad 30 mm diam post 14 dies, velutinae, olivaceo-virides vel griseae, reversum olivaceo-nigrum. Cellulae gemmantes absentes. Hyphae leves, hyalinae vel pallide brunneae, 2−3 μm latae. Conidiophora micronemata, septata, lateralia vel terminalia; stipites et ramoconidia denticulata. Conidia holoblastica, dilute brunnea, late fusiformia, unicellularia, levia, catenas longas ramosas cohaerentes formantia, cicatricibus dilute brunneis, 5−6 × 2−3 μm. Synanamorphe not visa. Chlamydosporae absentes. Teleomorphe ignota.

Description based on CBS 122642 at 27 °C on MEA after 2 weeks in darkness.

Cultural characteristics: Colonies growing slowly, reaching 30 mm diam (10 mm at 37 °C on PDA).

36 Biodiversity of the genus Cladophialophora

Fig. 5. Morphology of muriform cells in three species of Cladophialophora. A–B. Cladophialophora subtilis. C. Cladophialophora immunda. D. Cladophialophora carrionii. Scale bar = 10 µm.

Table. 3. Effect of calcium and temperature on production of muriform cells under the acidic conditions in basal medium for 13 species of Cladophialophora.

Cladophialophora species Muriform cells at 25 °C Muriform cells at 37 °C

C. carrionii + ++

C. samoënsis − +

C. subtilis + +++

C. mycetomatis – −

C. immunda − +

C. australiensis − −

C. potulentorum − −

C. yegresii − −

C. sylvestris − −

C. humicola − −

C. hostae − −

C. proteae − −

C. scillae − − No production of muriform cells is indicated by –, low production of small muriform cells by +, moderate production of large muriform cells by ++, and large production of large muriform cells by +++.

Colonies velvety, olivaceous-black, with a wide well defined margin darker than the colony centre, and a compact suede-like to downy surface; reverse olivaceous-black. Cardinal temperatures: minimum 12 °C, optimum 27−30 °C, maximum 37 °C. No growth at 40 °C.

Microscopy: Fertile hyphae septate, ascending to erect, smooth, thin-walled, hyaline to pale olivaceous, guttulate, branched, 2−3 µm wide, forming hyphal strands and hyphal coils. Conidiophores either micronematous, erect, sub-cylindrical, often reduced to a conidiogenous cell, or semi-macronematous, with stalks 60−80 µm long, guttulate, hyaline to pale olivaceous, cylindrical to sub-cylindrical, apically branched, 2.5−3 µm wide. Conidiogenous cells pale brown, slightly darker than conidia, sub-cylindrical to fusiform, with pigmented scars, smooth- walled, proliferating sympodially with 1−3, denticle-like extensions and guttulate. Ramoconidia present. Conidia one-celled, produced in long coherent chains, subhyaline to hyaline, smooth,

37 Chapter 2

Fig. 6. Cladophialophora samoënsis (CBS 259.83). A. Conidiophore. B–E. Conidial chains with ramoconidia and conidia. F. Conidiogenous loci (arrows). Scale bar = 10 µm. thin-walled, guttulate; conidia and ramoconidia ellipsoidal to ovoid, 5−6 × 2−3 µm, non-septate. Chlamydospores absent. Teleomorph unknown.

Specimen examined: The Netherlands, Utrecht, isolated from ice tea, December 2004, CBS H-20114, holotypus; CBS 122642 = dH 14614, ex-type.

Notes: The species is morphologically similar toC. carrionii, an agent of chromoblastomycosis. However, C. subtilis has distinct erect conidiophores which arise at right angles from fertile hyphae; conidiogenous cells are pale brown, slightly darker than conidia, sub-cylindrical to fusiform, with pigmented scars, proliferating sympodially with 1−3, denticle-like extensions. The species is known from a single strain originating from commercial ice tea. Several Cladophialophora species have been isolated from sugared drinks (C. potulentorum, C. australiensis), while pathogenic Exophiala species also have a preference for sugar-rich surfaces of fruits (Sudhadham et al. 2008). The association ofChaetothyrialean anamorphs with drinks is thus not surprising. The group is also associated with human disorders (de Hoog et al. 2000, Levin et al. 2004). Our species has the ability to grow at 37 °C and produces muriform cells when incubated at 25 and 37 °C at low pH (pH = 2.5). Further studies are required to evaluate its pathogenic ability

Cladophialophora mycetomatis Badali, de Hoog & Bonifaz, sp. nov. MycoBank MB511843. Figs 9, 12.

38 Biodiversity of the genus Cladophialophora

Etymology: Named after the clinical picture mycetoma caused by one of the strains.

Coloniae fere lente crescentes, ad 30 mm diam post 14 dies, velutinae, olivaceo-griseae, reversum olivaceo-nigrum. Cellulae gemmantes absentes. Hyphae leves, hyalinae vel pallide brunneae, 2.5–3 μm latae. Conidiophora solitaria micronemata, dilute brunnea, cylindrica, sursum ramosa, 3.0–3.5 µm lata. Cellulae conidiogenae dilute brunneae, leves, sympodialiter proliferentes. Ramoconidia 0–(1)-septata, cylindrica vel fusiformia, 2.5–4 × 2.5–3 µm. Conidia holoblastica, dilute brunnea, late fusiformia , unicellularia, levia in catenis longis ramosis cohaerentia, cicatricibus dilute 2 brunneis, 2.5−3 × 2−3. Synanamorphe not visa. Chlamydosporae absentes. Teleomorphe ignota.

Description based on CBS 122637 at 27 °C on MEA after 2 weeks in darkness.

Cultural characteristics: Colonies moderately expanding, reaching up to 30 mm diam, olivaceous- grey, velvety; reverse olivaceous-black. Cardinal temperatures: minimum 15 °C, optimum 27−30 °C, maximum 37 °C (maximum growth temperature 33 °C; CBS 454.82).

Microscopy: Budding cells absent. Septate hyphae creeping, ascending to sub-erect, smooth- walled, hyaline to pale olivaceous, guttulate, branched, 2.5−3.0 µm wide. Conidiophores solitary, micronematous, pale brown, cylindrical, apically branched, 3.0−3.5 µm wide. Conidiogenous cells pale brown, smooth-walled, sympodially proliferating. Ramoconidia cylindrical to fusiform, 2.5−4.0 × 2.5−3.0 µm. Conidia holoblastic, fusiform produced in long chains; subhyaline to pale olivaceous, guttulate. Chlamydospores absent. Teleomorph unknown.

Specimen examined: Mexico, Jicaltepec, isolated from human patient with mycetoma, 2006 CBS H-20116, holotypus; CBS 122637, ex-type.

Notes: It is remarkable that an environmental strain of C. mycetomatis (CBS 454.82) was isolated by W. Gams as a culture contaminant in the strain of Scytalidium lignicola Pesante (CBS 204.71) from the Netherlands. It was morphologically very similar to C. carrionii but was methyl-α-D- glucoside and melibiose negative and assimilated D-glucosamine and galactitol (de Hoog et al. 1995).

Case Report: A 49-year-old male farmer mainly growing corn and resident of Jicaltepec, in the semiarid zone Pinotepa Nacional Oaxaca, approximately 450 km south of Mexico City presented with a dermatosis localised to the left leg at the dorsum of the foot, affecting the third toe (Fig. 13A). The lesion consisted of a tumorous area, with deformation, and nodules with draining sinuses releasing thread-like material including black granules. The dermatosis had begun one and a half yr before, after a trauma with a thorn of cactaceous plant called nopal (Opuntia sp.). These led to progressive swelling of the region and occasional pain (Fig. 13A). Initial treatment included penicillin and sulfametoxazole-trimetroprim. The presumptive clinical diagnosis was that of mycetoma. Direct examination with KOH (10 %) showed some black granules approximately 500 µm in size. The granules were composed of branched, septate, dematiaceous hyphae (Fig. 13B). Clinical specimens cultured on Sabouraud Glucose Agar (SGA) with or without antibiotics (Mycosel) resulted in the growth of a dematiaceous fungus morphologically identified as Cladophialophora sp. Once the diagnosis of eumycetoma had been made, laboratory tests consisted of a complete blood count, blood chemistry and liver function tests; all of these were within normal limits. Foot radiographs (lateral and PA) showed no involvement of bones. Treatment with itraconazole, 200 mg/d was instituted, with significant clinical improvement at 8 mos. Liver and hematological function tests were monitored throughout the treatment period at 4-mo intervals, with no alterations.

39 Chapter 2

Fig. 7. Microscopic morphology of C. samoënsis (CBS Fig. 8. Microscopic morphology of C. subtilis (CBS 259.83). Branched conidial chains with ramoconidia and 122642). Fertile hyphae septate, ascending to erect. conidia. Conidiophores septate, lateral or terminal, with Conidiophores apically branched, cylindrical to sub- denticles on the stipe and on 0–1-septate ramoconidia. cylindrical Branched conidial chains with ramoconidia and Scale bar = 10 µm. conidia. Scale bar = 10 µm.

The province where the patient lived in the South of Mexico is a very poor regionina semi-arid area climate zone. The majority of inhabitants are farmers growing corn and other vegetables. Cactaceous plants are very common. The trauma with a thorn of a nopal cactus and the micromorphology of the fungus reminded of Cladophialophora carrionii, which is abundant under similar climatic conditions in Venezuela (de Hoog et al. 2007), but according to molecular data the fungus proved to be clearly separate. In addition to Cladophialophora, Exophiala species (particularly E. jeanselmei) are also known to cause mycetoma (de Hoog et al. 2003). Otherwise, subcutaneous infections by members of Chaetothyriales mostly lead to chromoblastomycosis-like infections which are characterised by muriform cells rather than granules, and do not lead to necrosis and draining.

Cladophialophora immunda Badali, Satow, Prenafeta-Boldú & de Hoog, sp. nov. MycoBank MB511844. Figs 10, 14.

Etymology: Named after its preference for polluted environments.

Coloniae fere lente crescentes, ad 30 mm diam post 14 dies 27–30 °C, velutinae, olivaceo-griseae vel olivaceo-virides, reversum olivaceo-nigrum. Cellulae gemmantes absentes. Hyphae leves, hyalinae vel pallide brunneae, adscendentes vel erectae, 2.5–4 μm latae, saepe circiter aggregatae. Conidiophora micronemata, septata. Conidia holoblastica, dilute brunnea, late fusiformia, unicellularia, levia, catenas cohaerentes formantia vel dilabentia, cicatricibus dilute brunneis, 3.0−4.5 × 2.5−4.0 μm. Synanamorphe no vista. Chlamydosporae vulgo absentes. Teleomorphe ignota.

40 Biodiversity of the genus Cladophialophora

Fig. 9. Microscopic morphology of C. mycetomatis Fig. 10. Microscopic morphology of C. immunda (CBS (CBS 122637 and CBS 454.82). Septate hyphae 834.96). Hyphae branched, septate, straight, ascending to creeping, ascending to sub-erect. Conidiophores solitary, erect. Hyphae giving rise to conidiophores. Lemon-shaped micronematous, cylindrical, apically branched. Conidia to pyriform to guttuliform, narrowed towards one or both holoblastic, fusiform produced in long chains. Scale bar = ends, coherent or deciduous. Scale bar = 10 µm. 10 µm.

Description based on CBS 834.96 at 27 °C on MEA after 2 weeks in darkness.

Cultural characteristics: Colonies growing moderately slowly, attaining up to 30 mm diam at 27−30 °C and 10 mm [20 mm in CBS 110551] at 37 °C. Colonies dark olivaceous-grey with a thin, dark, well-defined margin [wide grey margin in CBS 109797], spreading, downy, velvety; reverse olivaceous-black. Cardinal temperatures: minimum 15 °C, optimum 27−30 °C, maximum 37 °C. No growth at 40 °C.

Microscopy: Budding cells absent. septate hyphae , straight, ascending to erect, smooth, thin- walled, hyaline to pale brown, branched, 2−3 µm wide, frequently forming hyphal strands and coils [no hyphal coils in CBS 110551]. Hyphae giving rise to conidiophores which are pale brown, erect, mostly straight, branched or unbranched, long, sub-cylindrical and cylindrical to fusiform [fusiform to ellipsoidal in CBS 110551], 2.5−4 µm wide [with T-shaped foot cell in CBS 102227], with up to 6−8 septa. Conidiogenous cells branched, conspicuously denticulate, smooth-walled, guttulate, with pigmented scars. Conidia one-celled, acropetal, catenulate, sub-hyaline to pale brown, smooth [slightly verrucose in CBS 109797], thin-walled, lemon-shaped to pyriform to guttuliform, narrowed towards one or both ends, with pale pigmented scars. Conidia 3.0−4.5 × 2.5−4.0 µm, coherent or deciduous. Phialidic synanamorph not seen. Chlamydospores absent [present in CBS 109797]. Teleomorph unknown.

41 Chapter 2

Fig. 11. Cladophialophora subtilis (CBS 122642). A–D. Conidiophores with branched conidial chains and ramoconidia. E. Sympodial conidiogenesis. F–G. Conidiogenous loci (arrows). H. Conidia. Scale bars = 10 µm.

Specimens examined: U.S.A., Georgia, Atlanta, isolated from a sub-cutaneous ulcer on a 68-year- old female treated with long-term immunosuppressive therapy, (CBS H-20115, holotypus; CDC B-6580 = CBS 834.96, ex-type). CBS 109797. The Netherlands, isolated from hydrocarbon- polluted soil, CBS 110551 (Prenafeta-Boldú et al. 2006). Brazil isolated from hydrocarbon- polluted soil, CBS 122636, CBS 122255, CBS 122257, dH 18477, dH 18476, dH 18476, dH 18478 (Satow et al. 2008).

Case Report: According to the description by Padhye et al. (1999), a 68 yr-old-female who underwent long-term immunosuppressive therapy in view of a history of recurrent sinusitis, pneumonia, genital herpes, hysterectomy, chronic hypergammaglobulinemia, low grade lymphoma, Sjogren’s disease, rheumatois arthritis. She had not any history of predisposing factor such as truma. The pretibial lesion was non-responsive to cephalexin or ofloxacin. Due to the clinical manifestation of the lesion, a biopsy was performed which consisted of dermis and subcutaneous tissue. Biopsy tissue section was stained by PAS (Fig. 15A) and Gomori’s methanamine-silver (GMS, Fig. 15B) stains, showing septate hyphae, moniliform hyphae of different lengths, and thick-walled cells. The melanized fungal elements were within intense infiltrates of neutrophils and necrotizing granulomas with many giant cells. The histopathological observations led to the diagnosis of a subcutaneous phaeohyphomycosis infection. The strain was identified asCladophialophora species (Padhye et al. 1999) resembling C. devriesii and C. arxii. It formed dry conidia in branched acropetal chains inserted on pronounced denticles, fusiform to lemon-shaped conidia, and

42 Biodiversity of the genus Cladophialophora

Fig. 12. Cladophialophora mycetomatis (CBS 122637 and CBS 454.82). A–B. Conidiophores and conidial chains with ramoconidia and conidia. C–D. Cylindrical, septate conidiophores. G. Ellipsoidal to fusiform conidia. Scale bars = 10 µm.

Fig.13. Clinical manifestations of Eumycetoma caused by Cladophialophora mycetomatis. A. Deformed tumorous area of the foot, with nodules, draining sinuses and discharging purulent fluid. B. Branched black granule, approximately 500 µm in size (in 10 % KOH), with septate hyphae. being involved in a human infection. Unlike C. arxii, the species did not grow above 37 °C. We identified the strain with several environmental isolates by multilocus sequencing (Figs 1, 3 and Table 1). For the environmental strains, a striking association with aromatic hydrocarbons was observed: eight out of twelve strains of C. immunda originated from hydrocarbon-polluted soils. An association with assimilation of toxic monoaromatic compounds and infective potentials have been hypothesised (Prenafeta-Boldú et al. 2006). Black yeasts and their filamentous relatives in the ascomycete order Chaetothyriales are potent degraders of monoaromatic compounds and

43 Chapter 2

Fig. 14. Cladophialophora immunda (CBS 834.96). A–D. Conidiophores and conidial apparatus with T-shaped foot cell and cylindrical, septate, denticulate conidiogenesis. E. Chlamydospores. F–G. Thin-walled, lemon-shaped to pyriform to guttuliform conidia. H. Hyphal coil. Scale bar = 10 µm.

Fig. 15. Cladophialophora immunda (CBS 834.96). A. Gomori Methenamine-silver and B. Periodic acid-Schiff stained sections revealed septate hyphae, moniliform hyphae of different lengths, and thick-walled cells (GMS & PAS × 360).

44 Biodiversity of the genus Cladophialophora eventually tend to accumulate in industrial biofilters (Coxet al. 1997, Prenafeta-Boldú et al. 2001, de Hoog et al. 2006).

Discussion

Cladophialophora-like anamorphs are polyphyletic The genus Cladophialophora is morphologically characterised by poorly or profusely branched 2 chains of dry, rather strongly coherent, moderately melanized conidia. Our results show that this Cladophialophora anamorphic type is convergent, as the genus Cladophialophora is polyphyletic. In our combined phylogeny of the Chaetothyriales (Fig. 1), the plant-associated species (C. hostae, C. scillae, C. proteae, C. sylvestris, C. minutissima, and C. humicola) appeared to be closer to Ceramothyrium carniolicum (a putative member of the family of Chaetothyriaceae), and only distantly related to most remaining Cladophialophora species. It is interesting to note that Cladophialophora species that are consistently associated with pathology to humans mostly belong to the Herpotrichiellaceae, a family particularly diverse in human opportunists (e.g., Exophiala, Fonsecaea, Rhinocladiella, and Veronaea). The lineages to which the plant-associated species of Cladophialophora (lineages 2, 3 and 4) belong contain comparatively few human opportunists, all of them causing only mild cutaneous infections (e.g., Phialophora europaea, and Cyphellophora laciniata), or traumatic infection (Cladophialophora modesta). Similar Cladophialophora-like morphologies have also been observed in several unrelated environmental fungi, particularly in Cladosporium, Pseudocladosporium and Devriesia (Braun et al. 2003, Crous et al. 2007, Seifert et al. 2004). These genera are assigned to different families within the Dothideales and the Capnodiales, two ascomycete orders for which species are only exceptionally encountered in a clinical setting. Moreover, the conidial scars have a pale pigmentation in Cladophialophora, which is in contrast to members of the saprobic genera Cladosporium, Devriesia and Pseudocladosporium, where pronounced darker conidial scars are present. Furthermore, conidiophores in most Cladophialophora species are poorly differentiated, while those of Cladosporium species are usually erect and significantly darker than the rest of the mycelium. Finally, conidial chains of Cladophialophora species are coherent, while those of Cladosporium detach very easily.

Evolution of pathogenicity in Cladophialophora Melanin pigments are common to all Chaetothyriales, although little is known concerning the pathogenic mechanisms by which these fungi cause disease, particularly in immunocompetent individuals. However, the production of melanin was shown to be involved in pathogenicity. Melanins are pigments of high molecular weight formed by oxidative polymerization of phenolic compounds and usually are dark brown or black; in case of fungal pathogens melanin appears to function in virulence by protecting fungal cells against fungicidal oxidants, by impairing the development of cell-mediated responses, interfere with complement activation and reduce the susceptibility of pigmented cells to antifungal agents. In the environment, they protect organisms against environmental factors (Butler et al. 1998, Jacobson 2000). Another putative virulence factor is thermotolerance. According to de Hoog et al. (2000), species of Cladophialophora show a differential maximum growth at temperatures more or less coinciding with clinical predilections, species causing systemic infections being able to grow at 40 °C. The agents of chromoblastomycosis have a growth maximum at 37 °C, while a mildly cutaneous species, such as C. boppii, is not able to grow at this temperature (de Hoog et al. 2000).

45 Chapter 2

In the present study, all studied strains of Cladophialophora had an optimum growth around 27 °C, and were still able to grow at 37 °C, but not at 40 °C (Fig. 4). This observation agrees with the prevalent nature of these species as environmental saprobes, and their potential to cause superficial infections in humans, similarly to other opportunistic species. Tolerance of human body temperature is an essential requirement for pathogenicity, but this trait may have incidentally been acquired via adaptation to warm environmental habitants, such as hot surfaces in semiarid climates. Early experiments involving the inoculation of Cladophialophora in several species of cold- blooded animals have shown the abundant production of characteristic muriform cells in vivo (Trejos 1953). For C. yegresii and C. carrionii, the muriform propagules are also present in cactus spines (de Hoog et al. 2007); in this plant host, they can be regarded as an extremotolerant survival phase, and are likely to play an essential role in the natural life cycle of these organisms. The capacity of some Herpotrichiellaceae to grow in a meristematic form both in human hosts and in extreme environmental conditions supports the suggestion that the muriform cells may indeed be a main virulence factor in the development of the disease, representing an adaptation to the conditions prevailing in host tissues. The conversion of hyphae to muriform cells can be induced in vitro under acidic conditions (pH = 2.5) and low concentration of calcium. In the present study, we observed different morphogenetic responses between environmental and clinical species of Cladophialophora. C. subtilis isolated from ice tea, under the above circumstances formed structures resembling muriform cells at 27 °C which became larger when incubated at 37 °C. The pathogenic C. samoënsis and C. immunda also produced muriform cells at 37 °C. Muriform cells were not observed, neither on the plant-associated species (plant leaving) of Cladophialophora, nor in the C. mycetomatis isolated from both environment and a patient.

Conclusions Some members of Herpotrichiellaceae grow as ordinary filamentous moulds in pure culture, but can also be induced to form strongly melanized, isodiametrically expanding meristematic cells in vitro. This type of cells, dividing by sclerotic fission, is characteristically found in chromoblastomycosis. However, they can also be observed when these fungi grow in natural niches, in particular the ones characterised as extreme (e.g., F. pedrosoi within dried cactus thorns; Sterflinger et al. 1999). Vicente et al. (2008) have shown that the hydrocarbon-polluted environments yielded yet another spectrum of chaetothyrialean fungi. A generalized suite of adaptations to extreme environments, including melanin production and meristematic growth, as well as thermotolerance, is suggested to contribute to pathogenic potential (Gueidan et al. 2008). Many of the natural and artificial habitats that are associated with growth of Herpotrichiellaceae, such as decaying tree bark and creosoted poles and ties, are likely to favor fungi that, in addition to being able to break down the aromatic compounds occurring in these substrata, have a generalized set of adaptations to conditions that at least occasionally become highly stressful (extreme temperatures, pH, low availability of nutrients and growth factors, etc.). Many of these adaptations may incidentally predispose fungi towards human opportunistic pathogenesis.

Acknowledgements This research was financially supported by a grant of the Ministry of Health and medical education of Islamic Republic of Iran (No. 13081), which we gratefully acknowledge. The authors acknowledge Walter Gams for his comments on the manuscript and for providing Latin descriptions. Grit Walther is gratefully acknowledged for helping in part of description and

46 Biodiversity of the genus Cladophialophora drawing. We thank Marjan Vermaas for providing the photographic plates. Kasper Luijsterburg is thanked for technical assistance

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47 Chapter 2

Species diversity and polymorphism in the Exophiala spinifera clade containing opportunistic black yeast-like fungi. Journal of Clinical Microbiology 41: 4767–4778. Hoog GS de, Zeng JS, Harrak MJ, Sutton DA (2006). Exophiala xenobiotica sp. nov., an opportunistic black yeast inhabiting environments rich in hydrocarbons. Antonie van Leeuwenhoek 90: 257–268. Horré R, Hoog GS de (1999). Primary cerebral infections by melanized fungi: a review. Studies in Mycology 43: 176–193. Jacobson ES. Pathogenic roles for fungal melanins (2000). Clinical Microbiology Reviews 13: 708–717. Maddison WP, Maddison DR (2003). MacClade: Analysis of Phylogeny and Character Evolution, v. 4.6, Sinauer, Sunderland, MA. Masclaux F, Guého E, de Hoog GS, Christen R (1995). Phylogenetic relationships of human-pathogenic Cladosporium (Xylohypha) species inferred from partial LS rRNA sequences. Medical Mycology 33: 327–338. Mason-Gamer RJ and Kellogg EA (1996). Testing for phylogenetic conflict among molecular datasets in the tribeTriticeae (Graminae). Systematic Biology 45: 524–545. Mendoza L, Karuppayil SM, Szaniszlo PJ (1993). Calcium regulates in vitro dimorphism in chromoblastomycotic fungi. Mycoses 36:15–-164. Padhye AA, Dunkel JD, Winn RM, et al (1999). Subcutaneous phaeohyphomycosis caused by an undescribed Cladophialophora species. Studies in Mycology 43: 172–175. Prenafeta-Boldú FX, Kuhn A, Luykx D, et al (2001). Isolation and characterization of fungi growing on volatile aromatic hydrocarbons as their sole carbon and energy source. Mycological Research 105: 477–484. Prenafeta-Boldú FX, Summerbell R, Hoog GS de (2006). Fungi growing on aromatic hydrocarbons: biotechnology’s unexpected encounter with biohazard? FEMS Microbiology Reviews 30: 109–130. Satow MM, Attili-Angelis D, Hoog GS de, Angelis DF, Vicente VA (2008). Selective factors involved in oil flotation isolation of black yeasts from the environment. Studies in Mycology 61: 157-163 Seifert KA, Nickerson NL, Corlett M, et al (2004). Devriesia, a new hyphomycete genus to accommodate heat-resistant, Cladosporium-like fungi. Canadian Journal of Botany 82: 914 –926. Stamatakis A, Hoover P, Rougemont J (2008). A rapid bootstrap algorithm for the RAxML Web-Servers. Systematic Biology, in press. Sterflinger K, Hoog GS de, Haase G (1999). Phylogeny and ecology of meristematic ascomycetes. Studies in Mycology 43: 5–22. Sudhadham M, Dorrestein GM, Prakitsin S, et al (2008). The neurotropic black yeastExophiala dermatitidis has a possible origin in the tropical rain forest. Studies in Mycology 61: 145–155. Taylor JW, Jacobson DJ, Kroken S, et al (2000). Phylogenetic species recognition and species concepts in fungi. Fungal Genetics and Biology 31: 21–32. Trejos A (1954). Cladosporium carrionii n. sp. and the problem of cladosporia isolated from chromoblastomycosis. Revista de Biologia Tropical 2: 75–112. Vicente VA, Attili-Angelis D, Filho FQT, Pizzirani-Kleiner AA (2001). Isolation of herpotrichiellaceous fungi from the environment. Brazilian Journal of Microbiology 32: 47–51. Vicente VA, Attili-Angelis D, Pie MR, et al (2008). Environmental isolation of black yeast-like fungi involved in human infection. Studies in Mycology 61:136–144 White TJ, Bruns T, Lee S, Taylor J (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: a guide to methods and applications (Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds). Academic Press, San Diego, California: 315–322.

48 Chapter 3

Cladophialophora saturnica sp. nov., a new opportunistic species of Chaetothyriales revealed using molecular data

H. Badali 1, 2 , 3, V.O. Carvalho 4, V. Vicente 5, D. Attili-Angelis 6, I.B. Kwiatkowski 5, A.H.G. Gerrits van den Ende 1, G.S. de Hoog 1, 2

Published: Medical Mycology 2009; 47: 51- 62. 349 Chapter 3

Abstract While many members of the black yeasts genus Cladophialophora have been reported to cause diseases in humans, understanding of their natural niche is frequently lacking. Some species can be recovered from the natural environment by means of selective isolation techniques. The present study focuses on a Cladophialophora strain that caused an interdigital tinea nigra-like lesion in a HIV positive Brazilian child. The fungal infection was successfully treated with oxiconazole. Similar strains had been recovered from the environment in Brazil, Uruguay and the Netherlands. The strains were characterised by sequencing the Internal Transcribed Spacer (ITS rDNA) regions and the small subunit (nucSSU) of the nuclear ribosomal RNA gene, as well as the elongation factor 1-alpha (EF1α) gene. Since no match with any known species was found, it is described as the new species, Cladophialophora saturnica.

Keywords: black yeasts, Cladophialophora, cutaneous infection, taxonomy

Introduction The fungal genusCladophialophora currently contains seven species proven to be involved in human disease (de Hoog el al. 2000). Among the most virulent species is Cladophialophora bantiana, the main agent of a potentially fatal cerebral infection in transplant recipients and patients with leukemia, but also in immunocompetent individuals (Horre et al. 1999, Revankar et al. 2004, Kantarcioglu et al. 1999). Another member of the genus, Cladophialophora carrionii, is the etiologic agent of chromoblastomycosis, a common skin disease which is endemic in the arid climate zones of South America and Australia. This infection occurs primarily in immunocompetent individuals (Richard-Yegres et al. 1987, Yeguez-Rodriguez et al. 1992), and is supposed to result from the inoculation of plant debris contaminated with the fungus (de Hoog et al. 2007). The remaining clinically relevant species of Cladophialophora, viz. C. arxii, C. devriesii, C. emmonsii, C. boppii and C. modesta, are very rarely reported as etiologic agents of disease. Cladophialophora is an anamorph member of the Chaetothyriales, an ascomycete order that also comprises the black yeast genus Exophiala (de Hoog et al. 2000) and its filamentous relatives, Fonsecaea and Phialophora. The natural niche outside humans is unknown for most of these opportunists. In contrast to frequently expressed opinions, the fungi concerned are rarely isolated from dead plant material or rotten wood, and hardly ever from soil (Revankar 2007). A selective isolation method is required to recover these fungi, e.g., the use of high temperature (de Hoog et al. 2005), a mouse vector (Gezuele at al. 1972, Dixon et al. 1980), alkylbenzenes (Prenafeta-Boldu et al. 2006) or extraction via mineral oil (Iwatsu et al. 1981, Vicente et al. 2001). At the phylogenetic base of the Chaetothyriales is located a group of plant associated Cladophialophora species (Crous et al. 2007). The present paper describes a species of Cladophialophora, based on isolate CBS 118724 that caused an interdigital infection in an immunocompromised child in Curitiba, Brazil.

Case Report The patient, a 4-year-old girl living in an institution since she was one-year-old was perinatally infected with HIV. The HIV diagnosis was made at the age of 8 months. Her immunodeficiency status was classified as A2 according to the 1994 revised categories by the Center for Disease Control and Prevention (MMRW 1994). Her clinical category was characterised by mildly symptomatic features and moderate immunosuppression. Antiretroviral treatment was initiated at the age of 1 year and included zidovudine, lamivudine and ritonavir. She was treated at the hospital outpatient HIV clinic which she visited periodically for evaluation. At the moment of this report her weigh

50 Cladophialophora saturnica sp. nov., a new opportunistic species

was normal for her age, with T- CD4 values of 1,142/mm3 and T- CD8 of 1,116/mm3. The number of HIV RNA viral copies was 11,000 copies/ml. During physical evaluation, a non-symptomatic skin lesion was detected in the interdigital webs. The lesion was velvety, scaling, brownish black and sharply demarcated, non-macerated (Fig. 1) and clinically diagnosed as interdigital tinea nigra. To recover the etiologic agent, the area was soaked in 70% isopropanolol and scrapings from the skin were collected aseptically into 3 Fig. 1. A velvety, scaling, brownish black macular area in the a Petri dish using a sterile scalpel blade. interdigital webs. Treatment with topical oxiconazole was initiated as pigmented hyphae were found during microscopical observation of the interdigital scrapings. Culture plates of Sabouraud’s Dextrose Agar (SDA, Difco) containing chloramphenicol (50 mg/ml) and cycloheximide (500 mg/ml) were inoculated with portions of the scrapings and incubated at 25°C and 37°C. A search for similar strains in the patient’s environment was performed using the mineral oil technique. The melanized isolates obtained from this investigation, as well as four strains with molecular similarity found colonizing dead wood in Uruguay and developing in a biofilter in the Netherlands, were included in this study. Since no match of these strains was found with any described taxonomic entity, the fungus is reported here as a new species.

Materials and Methods

Fungal strains and morphology Strains studied are listed in Table 1. Stock cultures were maintained on slants of 2% malt extract agar (MEA, Difco) and oatmeal agar (OA, Difco) at 24ºC. For morphological observation, potato dextrose agar (PDA, Difco) slide cultures were prepared and mounted in lactophenol. To evaluate the natural niche, 10 g samples of vegetable cover found in the patient’s garden and from the surrounding area were added to 20 ml of sterile mineral oil in 100 ml of sterile saline, followed by the addition of antibiotics with subsequent vigorous shaking and incubation for 20 min at room temperature. Afterwards, the oil/saline interface was seeded on Mycosel agar plates and incubated at 30°C (Iwatso et al, 1981, Vicente et al, 2001). Physiology Growth temperatures of strains CBS 114326, 102230, 109628, 109630 and 118724 were determined by incubating MEA culture plates in the dark for 2 weeks at temperatures ranging from 6–36ºC at intervals of 3°C as well as at 40°C (Crous et al. 1996).

DNA extraction About 1 cm2 mycelium segments from 20 to 30-day-old cultures were transferred to a 2 ml Eppendorf tube containing 300 ml CTAB (cetyltrimethylammonium bromide) buffer and about 80 mg of a silica mixture (silica gel H, Merck 7736, Darmstadt, Germany/ Kieselguhr Celite 545, Machery, Duren, Germany, 2:1, w/w). Cells were mechanically disrupted for approximately 1 min with a tight-fit sterile pestle. Subsequently, 200 ml CTAB buffer was added, the mixture was vortexed and incubated for 10 min at 65°C. After addition of 500 ml of chloroform, the solution

51 Chapter 3 Origin del Queguay Grande Isla Uruguay, del Queguay Grande Isla Uruguay, Curitiba Paraná, Brazil, Curitiba Paraná, Brazil, Wageningen Netherlands, Cayman Island USA, Grand Curitiba Paraná, Brazil, Germany ---- Australia China China Chiba Yachimata, Japan, Shiroi Japan, Australia Australia ------Zoo Blijdorp Rotterdam, Netherlands, Brazil Brazil America South ---- USA Horizonte Belo Brazil, Thailand (seabear) Neotandia membranacea Neotandia Source tree Dead cut trunk, Recently toe lesion, child Interdigital cover/soil vegetable Litter, biofilter Toluene infection, male Disseminated cover/soil vegetable Litter, abscess, male Tracheal Male drink Sports bambusoides Phyllostachys Bamboo wood Rotting wood Decaying drink Sports drink Sports juice Apple australis Arctocephalus ------Soil male Chromoblastomycosis, Male male Chromoblastomycosis, abscess, male Brain abscess, male Brain victim Tsunami graft, Skin

α GenBank EF1 ITS, EU103983, EU140601 ------EU103984, EU140602 EU140600 AY857508, EU140603 AY857507, EU103985, EU140595 ------EU103986, EU140593 EU103987, EU140594 EU035402, ------EU035403, ------EU035404, ------EU103988, EU140599 EU140598 AY251087, EU035409, ------EU035410, ------DQ008141, ------AY366925, ------AY857511, ------AY366927, ------AY366926, ------AY366914, ------AY366917, EU103989, EU140585 EU103990, EU140586 EU103991, EU140589 Other reference Other dH 12333; IHM 1727 dH 12335; IHM 1733 157D; dH 12939 dH 11591, 4IIBPIRA 200384 ATCC 56280; CDC 82-030890 ATCC ISO 13F ----- 5881; dH 15849 UAMH HKUCC 10147 5022 IFM 4701; UAMH 52853; IMI 298056 ATCC CPC 1376, FRR 4946 CPC 1375, FRR 4947 CPC 11048, FRR 3318 dH 15691 dH 12659 dH 11602, 1PLE dH 11613 18658; IMI 134458; dH 15659 ATCC dH 15661 CBS 100433 dH 11331 1739/05; dH 14515 No. Lab. Solna strains examined. Status T T LT T LT T NT T T CBS 109628 109630 118724 102230 114326 147.84 ------306.94 409.96 112793 114747 115468 987.96 556.83 112222 114772 115144 289.93 269.37 102238 102248 271.37 272.37 173.52 102586 119719 Cladophialophora Cladophialophora Name saturnica Cladophialophora saturnica Cladophialophora saturnica Cladophialophora saturnica Cladophialophora saturnica Cladophialophora devriesii Cladophialophora devriesii Cladophialophora arxii Cladophialophora arxii Cladophialophora australiensis Cladophialophora chaetospira Cladophialophora chaetospira Cladophialophora minourae Cladophialophora minourae Cladophialophora potulenturum Cladophialophora potulenturum Cladophialophora potulenturum Cladophialophora monophora Fonsecaea monophora Fonsecaea monophora Fonsecaea monophora Fonsecaea pedrosoi Fonsecaea pedrosoi Fonsecaea bantiana Cladophialophora bantiana Cladophialophora bantiana Cladophialophora Table 1. Isolation data of 1. Isolation Table

52 Cladophialophora saturnica sp. nov., a new opportunistic species Origin USA Barbados Onderstepoort Africa, Pretoria, South Japan Washington USA, ------Virginia USA, State Falcon Venezuela, State Falcon Venezuela, State Falcon Venezuela, State Falcon Venezuela, State Falcon Venezuela, State Falcon Venezuela, State Falcon Venezuela, Uganda Australia Brazil Dordrecht Netherlands,

3 plant (Cactaceae) plant (Cactaceae) plant (Cactaceae) Source lesion, cat Skin dog Liver, infection, dog Disseminated infection, human Brain abscess, human Brain Brain lesion, cat Sub-cutaneous human lesion right forearm, Sub-cutaneous griseus Stenocereus griseus Stenocereus griseus Stenocereus leg lesion, female Chromoblastomycosis hand lesion, male Chromoblastomycosis arm lesion, male Chromoblastomycosis arm lesion, female Chromoblastomycosis lesion, male Skin male Chromoblastomycosis, lesion, on limb,male Skin Scales of face, male

α GenBank EF1 ITS, EU103992, EU140592 EU103993, EU140587 EU103994, EU140591 EU140588 AY857516, EU140590 AY857519, EU140582 AY857518, EU103995, EU140584 EU103996, EU140583 EU137323, EU137263 EU137322, EU137262 EU137324, EU137264 EU137267, EU137211 EU137268, EU137212 EU137269, EU137213 EU137271, EU137215 EU137292, EU 137234 AB109177/EU137266,EU13210 EU103997, EU140596 EU103998, EU140597 Other reference Other CDC B-3658; NCMH 2249; NIH 4992 B-3839; UAMH 3830 UAMH ------44223; CDC B-3426, dH 11313 ATCC 58040; CDC B-3466; dH10749 ATCC dH 13029 ; UTHSC 03-70 CDC B-3634; NCMH 2248; UAMH 4991 CDC B-3875; NCMH 2247; UAMH 4994a; dH16329 UNEFM SgSR1; dH 13275 dH 13276 UNEFM SgSr3; UNEFM SgSR1; dH 13274 UNEFM 82267; dH 13261 UNEFM 9801; dH 13262 UNEFM 2001/1; dH 13265 UNEFM 2003/1; dH 13267 CDC B-1352; FMC 282; ATCC44535 16264; CDC A-835; MUCL ATCC 4808; dH 15445 40053; IFA FMC 292, dH 15357 det M-41/2001 56893; dH 12362 C. Status T T T of ajelloi LT T CBS 678.79 648.96 444.96 101158 101252 ------640.96 979.96 114406 114405 114407 114392 114393 114396 114398 260.83 160.54 126.86 110029 Name bantiana Cladophialophora bantiana Cladophialophora bantiana Cladophialophora bantiana Cladophialophora bantiana Cladophialophora emmonsii Cladophialophora emmonsii Cladophialophora emmonsii Cladophialophora yegresii Cladophialophora yegresii Cladophialophora yegresii Cladophialophora carrionii Cladophialophora carrionii Cladophialophora carrionii Cladophialophora carrionii Cladophialophora carrionii Cladophialophora carrionii Cladophialophora boppii Cladophialophora boppii Cladophialophora Fungi, Pathogenic Institute for collection; IFMResearch Hoog private Netherlands; DHG.S. de The Utrecht, Centre, Biodiversity Fungal U.S.A.; CBSCBS-KNAW Collection, Manassas, Culture Type used: ATCCAmerican Abbrevations fu¨ Institut Westfa¨hlisches London, U.K.; IWWRheinisch Institute, Mycological IMIInternational Uruguay; Montevideo, and Hygiene, of Epidemiology Institute Montevideo of Medicine, Faculty IHMLaboratory of Mycology, hiba, Japan; U.S.A.; RKI Chapel Hill, Hospital, Memorial Carolina NCMHNorth Belgium; Louvain-la-Neuve, de Louvain, de l’Universite´ collection; MUCLMycotheque private GHP G. Haase Germany; an der Ruhr, Mu¨lheim Wasserforschung, r Antonio, Science Center at San Health Texas of University Department of Pathology, Laboratory, Testing Canada; UTHSCFungus and Collection, Edmonton, Herbarium UAMHMicrofungus Germany; Berlin, Institute, Koch Robert culture. NTex-neotype culture; LTex-lectotype culture; Tex-type Venezuela. Falcon, Coro, de Miranda, Francisco Experimental Nacional U.S.A.; UNEFMUniversidade Galveston, Center, Research Mycology U.S.A.; UTMBMedical Table 1. (Continued) Table

53 Chapter 3

Fig. 2. Colony diameters at different temperatures ranging from 6 ºC to 40 ºC, measured after two weeks on 2 % MEA were calculated for C. saturnica (CBS 102230, 109628, 118724, 114326, 109630).

was mixed and centrifuged for 5 min at 14,000 r.p.m. and the supernatant transferred to a new tube with 2 volumes of ice-cold 96 % ethanol. DNA was allowed to precipitate for 30 min at -20 ºC and then centrifuged for 5 min at 14,000 r.p.m. The pellets were washed with cold 70 % ethanol, dried at room temperature, re-suspended in 97.5 ml of TE-buffer with 2.5 ml of RNAse 20 U/ml-1, and incubated for 5 min at 37 °C, before storage at -20 ºC (Gerrits van den Ende et al. 1999).

Sequencing and phylogenetic reconstruction The ribosomal DNA Internal Transcribed Spacers (ITS rDNA) were amplified using primers V9G and LS266 and sequenced with the internal primers ITS1 and ITS4 (White et al. 1990). The translation elongation factor 1 alpha (EF1α) was amplified and sequenced using EF1-728F and EF1-986R (Carbone et al. 1999). Amplicons were cleaned with GFX PCR DNA and gel band purification kit (GE Healthcare, UK). Sequencing was performed on an ABI 3730XL automatic sequencer. Sequences were edited using the SEQMAN package (DNAStar Inc., Madison, USA) and aligned using BIONUMERICS version 4.61 (Applied Maths, Kortrijk, Belgium). The phylogenies were reconstructed with MrAIC (de Hoog et al. 2007) using a Neighbor-Joining criterion. Bootstrap values of ITS and EF1α trees were calculated with the program Treefinder using a parsimony criterion and 1000 replicates (Jobb et al. 2004). Small Subunit (nucSSU) rDNA amplicons were generated with primers NS1 and NS24 and were sequenced with primers BF83, Oli1, Oli9, BF951, BF963, BF1438, Oli3 and BF1419 (de Hoog et al. 2005). Nucleotide positions 96-1765 (with reference to Saccharomyces cerevisiae) of the SSU rDNA were aligned and a tree of Chaetothyriales was constructed with the package ARB developed by Ludwig et al. 2004, using a Neighbor-Joining criterion. For all analyses, the corrected Akaike Information Criterion (AICc) was used to select a substitution model with the program MrAIC (de Hoog et al. 2007). The results of this selection are presented in Table 2.

Results Cardinal growth temperatures showed that all cultures had their best development at 27 °C (Fig. 2), although the range spanned from 9–36 °C. No growth was observed at 40 °C. Using the phylogenetic marker nucSSU and a dataset representative of the Chaetothyriales, strain CBS

Table 2 Results from MrAIC using corrected Akaike information criterion (AICc)

Fragment/Gene Model df* lnl* AICc* wAICc*

rDNA ITS HKYG 78 -2617.4881 5415.8731 0.4995

EF1α K2PG 55 -1460.1768 3065.5536 0.7023 *df=degrees of freedom; lnl=Log likelihood; AICc=corrected AIC; wAICc=weighted corrected AIC.

54 Cladophialophora saturnica sp. nov., a new opportunistic species

102230 was found to be a member of a Clade (1) primarily containing Cladophialophora and Fonsecaea species. Members of this clade are primarily known to cause diseases in humans, but some may be recovered from the environment, e.g., C. minourae (Fig. 3). Clade (2), the sister group of Clade (1), contained Cladophialophora and Phialophora species, most of which are known to be involved in or associated with human skin disorders. Phialophora americana is known as the anamorph of Capronia semiimmersa. Basal to these clades was a group (3) of environmental, mainly waterborne Exophiala species, including the teleomorph species Capronia coronata. The remaining members of Chaetothyriales were found at considerable phylogenetic distance, including Cladophialophora modesta. Of several recently described species of Cladophialophora (Crous et al. 2007), three were included in this study (C. australiensis, C. chaetospira and C. potulentorum). The others were not considered because they were only remotely related from our species of interest (CBS 118724). For ITS sequences, MrAIC selected the HKY-G model. The base frequency of 3 ITS was T-0.2465, C-0.2874, A-0.2239, G-0.2420, TC-0.5339, AG-0.4660. An ITS rDNA tree composed of alignable members of SSU clades (1) and (2) showed considerable differences between species (Fig. 4); regions with ambiguous alignments were removed from the analysis. The ex-type strains of C. arxii, C. devriesii, C. emmonsii and C. minourae were found separated by 8.9, 10.4, 11.1 and 13.1% distance, respectively. Comparison in a local database on melanized fungi maintained at CBS showed four sequences identical to CBS 118724. These sequences were isolates from plant litter collected in the surroundings of the region where the patient lives in Brazil (CBS 102230), from a biofilter in the Netherlands (CBS 114326), and two from dead plant material in Uruguay (CBS 109628 and CBS 109630). The ex-type strains of Cladophialophora devriesii was the nearest neighbor at 6.5% ITS difference. Sequences of C. modesta, C. hostae, C. humicola, C. scillae and C. sylvestris were too distant for confident alignment and were therefore not included in the tree. TheEF1 α tree was built with substitution model K2P-G, the base frequency of which was; T-0.2688, C-0.2705, A- 0.2254, G-0.2351, TC-0.5394, AG-0.4605. With EF1α (Fig. 5) the ex-type strains of C. arxii, C. devriesii, C. emmonsii and C. minourae were found separated by 9.8, 10.4, 20.0 and 13.8% distance, respectively. The sequences could be aligned with confidence over almost their total lengths. Four strains had EF1α sequences nearly identical to CBS 118724 and were the same as the ones previously shown to be identical using ITS. The EF1α tree (Fig. 5) showed the same topology as the ITS tree. Cladophialophora bantiana, C. carrionii and C. yegresii formed a clade which was significantly different from CBS 118724. Fonsecaea species were found at a large phylogenetic distance. As the well-supported cluster including our strain of interest was located at a significant phylogenetic distance from the remaining taxa, we propose to recognize it as a new species with the following description:

Cladophialophora saturnica Badali, Carvalho, Vicente, Attili-Angelis, Kwiatkowski, Gerrits van den Ende & de Hoog, sp. nov. Figs 6, 7. MycoBank MB491920.

Etymology: named after Saturn-shaped conidia in face view.

Coloniae modice expandentes, siccae, velutinae, olivaceo- virides ad griseae, reversum olivaceo- nigrum. Cellulae gemmantes absentes. Hyphae septatae, dilute olivaceae. Conidiophora erecta, e denticulis prominentibus cellulas conidiogenas proferentia. Cellulae conidiogenae hyphis conidiophorisque paulo pallidiores, cylindricae, e denticulis hebetibus conidia limoniformia proferentes. Conidia unicellularia, olivacea, hilo Paulo obscuriore, levia, catenas breves (2-3), acropetales, cohaerentes formantia. Ramoconidia nonnumquam massa conidiorum disrupta oriuntur. Chlamydosporae et phialides absentes. Teleomorphe ignota. Optime crescit 27 °C,

55 Chapter 3

Fig. 3. Neighbor-joining tree based on positions 145-1640 of 115 SSU rRNA gene sequences generated with the ARB package. Cladophialophora modesta CBS 985.96 was used as out group.

Fig. 4. Neighbor-joining tree of the Cladophialophora species based on rDNA ITS, generated using the HKY-G model. The model was calculated using ML in MrAIC software. Bootstrap set to 1000 replicates and cut-off = 80 %. Cladophialophora boppii (CBS 126.86 and CBS 110029) were taken as out group.

56 Cladophialophora saturnica sp. nov., a new opportunistic species

Fig. 5 Neighbor-joining tree of the Cladophialophora species based on EF1α, generated using the K2P-G model. The model was calculated using ML in MrAIC software. Bootstrap set to 1000 replicates and cut-off = 80%. Cladophialophora boppii (CBS 126.86 and CBS 110029) were taken as out group. temperatura minima 9 ºC, maxima 36 ºC, 40 ºC haud crescit. Typus vivus et exsiccatus CBS 118724 in CBS praeservatur.

Colonies (OA, 30 ºC), moderately expanding, dry, velvety, olivaceous green to gray; reverse olivaceous black. Budding cells absent. Hyphae septate, pale olivaceous. Conidiophores erect, with prominent denticles bearing the conidiogenous cells. Conidiogenous cells slightly lighter than the mycelia and conidiophore, cylindrical, branched with small, blunt denticles bearing lemon- shaped conidia. Conidia one-celled, olivaceous with pigmented scars, smooth-walled, arising in short, cohering, acropetal chains of 2-3; conidial scars pale pigmented. Ramoconidia occasionally produced by disarticulation of the conidial apparatus. Chlamydospores absent. Phialides absent. Teleomorph is unknown. Optimal growth at 27 ºC, minimum and maximum 9 and 36 °C, respectively. No growth at 40 ºC According to phylogenetic reconstruction, C. saturnica was the nearest neighbor with C. devriesii with a genetic distance of 6.5 % and 10.4 % for ITS and EF1α genes, respectively. The five isolates of C. saturnica formed a strongly supported monophyletic group (bootstrap-93 % in ITS and 98 % in EF1α). Although one of these five isolates caused disease in a Brazilian child with HIV infection, pathogenicity of these strains has to be evaluated with immunocompromised animal models. Holotype: dried culture at CBS preserved as CBS H-19940; ex-type strain CBS 118724, isolated from interdigital, tinea nigra-like skin lesion of 4-year-old HIV-positive child, Curitiba, Brazil.

Discussion This report describes a skin lesion that although appearing clinically insignificant wasfound

57 Chapter 3

Fig. 6. Microscopic morphology of Cladophialophora saturnica. (A and B) CBS 102230, (C) CBS 109628, (D and E) CBS 114326, (F and G) CBS 118724, (H) CBS 109630. Scale bar10 µm. 58 Cladophialophora saturnica sp. nov., a new opportunistic species

Fig. 7. Line drawing of microscopic morphology of Cladophialophora saturnica, strain CBS 118724. Conidiophores erect, with prominent denticles bearing the conidiogenous cells. Scale bar10 µm.

59 Chapter 3 to be caused by a member of the genus Cladophialophora which contains numerous species causing disease in immunocompetent and immunocompromised individuals. This emphasizes the need for careful monitoring of patients with AIDS. The future patterns of emerging fungal opportunists in the twenty-first century will be influenced by immunocompromised hosts, permissive environmental conditions, and selective antifungal pressure (Walsh et al. 1999). Enhanced detection and identification of agents of infection in patient populations at risk are overdue. The genus Cladophialophora is morphologically characterised by poorly or profusely branched chains of dry, rather strongly coherent, moderately melanized conidia. The conidial scars have pale pigmentation, in contrast to those of members of the saprobic genus Cladosporium, where pronounced, black conidial scars are present. Conidiophores are poorly differentiated, while those of Cladosporium species are mostly erect, significantly darker than the rest of the mycelium. Conidial chains of Cladophialophora species are coherent, while those of Cladosporium detach very easily. Although species of Cladophialophora are predominantly involved in human disease, they might have their evolutionary origin in plant pathogens (de Hoog et al. 2000, de Hoog et al. 2007). A recent study has investigated the phylogenetic placement of some plant-pathogenic species of Cladophialophora (Crous et al. 2007). The results, although ambiguous, suggest that C. hostae, C. scillae, C. sylvestris and C. humicola are only distantly related from the clade including C. saturnica. Cladophialophora-like morphology is seen in several unrelated, environmental fungi, particularly in Pseudocladosporium/Fusicladium (Crous et al. 2007, Braun et al. 2003) and Devriesia (Seifert et al. 2004). These genera are assigned to different families within theDothideales and the Capnodiales, two ascomycete orders for which species are only exceptionally encountered in a clinical setting. Cladophialophora species in the core of the Chaetothyriales share a marked clinical potential with numerous members of the family. All anamorph genera described to date, viz. Cyphellophora, Exophiala, Fonsecaea, Rhinocladiella and Veronaea, have been reported from infections in humans and warm or cold-blooded vertebrates (de Hoog et al. 2000). Within the order, the family Herpotrichiellaceae is based on characters of the teleomorph genus Capronia. Most anamorph genera are poorly supported in phylogenies using ribosomal markers (Haase et al. 1999). Moreover, a similar morphology is observed in different subclades, and several clades are morphologically heterogeneous. In addition, Cladophialophora is polyphyletic, as for example, the causative agents of brain infection, C. bantiana and C. modesta, are clearly apart in SSU sequences (Fig. 3), and their ITS spacer regions are not even alignable. Cladophialophora saturnica is morphologically very similar to C. devriesii and C. arxii, which are comprise strains from the environment, as well as very rare agents of fatal dissemination in humans (Gonzalez et al. 1984, Tintelnot et al. 1995, and Padhye et al. 1999). The numbers of strains available for study is as yet too small to be certain about genetic delineation of these species. With the development of selective isolation methods, the number of known species tends to increase in the group of the black yeasts. The use of these new isolation methods and other investigations are currently carried out with the aim to improve the knowledge of the ecology of these fungi. Despite a limited taxon sampling, the differences observed for all phylogenetic markers are considered sufficient to propose that the investigated environmental strain represents a new species of Cladophialophora, C. saturnica, with a clinical potential. Species of Cladophialophora show a differential maximum growth at temperatures more or less coinciding with clinical predilection (de Hoog et al. 2000). While species causing systemic infections are able to grow at 40°C, those that are involved in chromoblastomycosis have a maximum growth at 37°C or in mildly cutaneous infections, such as C. boppii, are not able to grow at this these elevated temperatures. All strains of C. saturnica had an optimum growth around 27 °C, and were still able to grow at 37 °C, but not at 40 °C (Fig. 2). This observation matches with the prevalent nature of this species as an environmental saprobe, with the potential to cause superficial infection in humans, similarly to other opportunistic species.

60 Cladophialophora saturnica sp. nov., a new opportunistic species

Therefore, suggesting that potential pathogenesis in animal model should be evaluated for C. saturnica.

Acknowledgments The work of Hamid Badali was financially supported by a grant of the Ministry ofHealth and medical education of Islamic Republic of Iran (No. 13081), and the School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran which we gratefully acknowledge. The authors thank Cecile Gueidan for critical reading of the manuscript. Katia Cruz and Kasper Luijsterburg are acknowledged for help in building up part of the sequence database.

References 3 Braun U, Crous PW, Dugan F, et al (2003). Phylogeny and taxonomy of Cladosporium-like hyphomycetes, including Davidiella gen. nov., the teleomorph of Cladosporium s. str. Mycol Prog 2: 3-18. Carbone I, Kohn LM (1999). A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91: 553-556. Crous PW, Schubert K, Braun U, et al (2007). Opportunistic, humanpathogenic species in the Herpotrichiellaceae are phenotypically similar to saprobic or phytopathogenic species in the Venturiaceae. Stud Mycol 58: 185-217. Crous PW, Gams W, Wingfied MJ, Van Wyk PS (1996). Phaeoacremonium gen. nov. associated with wilt and decline diseases of woody hosts and human infections. Mycologia 88: 786-796. Centers for Disease Control (1994). Revised classification system for human immunodeficiency virus infection in children less than 13 years of age. MMRW 43: 1-19. De Hoog GS, Guarro J, Gené J, Figueras MJ (2000). Atlas of Clinical Fungi, 2nd ed. CBS-KNAW Fungal Biodiversity Centre, Universitat Rovira i Virgili, Utrecht, Reus, 1126 pp. De Hoog GS, Nishikaku AS, Fernández Zeppenfeldt G, et al (2007). Molecular analysis and pathogenicity of the Cladophialophora carrionii complex, with the description of a novel species. Stud Mycol 58: 219-234. De Hoog GS, Matos T, Sudhadham M, et al (2005). Intestinal prevalence of the neurotropic black yeast Exophiala (Wangiella) dermatitidis in healthy and impaired individuals. Mycoses 48: 142-145. De Hoog GS, GÖttlich E, Platas G, et al (2005). Evolution, taxonomy and ecology of the genus Thelebolus in Antarctica. Stud Mycol 51: 33-76. Dixon DM, Shadomy HJ, Shadomy S (1980). Dematiaceous fungal pathogens isolated from nature. Mycopathologia 70: 153-161. Gezuele E, Mackinnon JE, Conti-Diaz IA (1972). The frequent isolation of Phialophora verrucosa and Fonsecaea pedrosoi from natural sources. Sabouraudia 10: 266-273. Gonzalez MS, Alfonso B, Seckinger D (1984). Subcutaneous phaeohyphomycosis caused by Cladosporium devriesii, sp. nov. Sabouraudia 22: 427- 432. Gerrits van den Ende AHG, De Hoog GS (1999). Variability and molecular diagnostics of the neurotropic species Cladophialophora bantiana. Stud Mycol 43: 151-162. Horre´ R, De Hoog GS (1999). Primary cerebral infections by melanized fungi: a review. Stud Mycol 43: 176-193. Haase G, Sonntag L, Melzer-Krick B, De Hoog GS (1999). Phylogenetic inference by SSU-gene analysis of members of the Herpotrichiellaceae with special reference to human pathogenic species. Stud Mycol 43: 80-97. Iwatsu T, Miyaji M, Okamoto S (1981). Isolation of Phialophora verrucosa and Fonsecaea pedrosoi from nature in Japan. Mycopathologia 75: 149-158. Jobb G, Arndt von H, Korbinian S (2004). TREEFINDER: a powerful graphical analysis environment for molecular phylogenetics. BMC Evolutionary Biology 4: 18. Kantarcioglu S, De Hoog GS (1999). Infections of the central nervous system by melanized fungi: a review of cases presented between 1999 and 2004. Mycoses 47: 4-13. Prenafeta-Boldu´ FX, Summerbell RC, De Hoog GS (2006). Fungi growing on aromatic hydrocarbons: biotechnology’s unexpected encounter with biohazard. FEMS Microbiol Rev 30: 109-130. Padhye AA, Dunkel JD, Winn RM, et al (1999). Subcutaneous phaeohyphomycosis caused by an undescribed Cladophialophora species. Stud Mycol 43: 172-175. Ludwig W, Strunk O, Westram R, et al (2004). ARB: a software environment for sequence data. Nucleic Acids Res 32: 1363-1371. Richard-Yegres N, Yegres F (1987). Cladosporium carrionii en vegetacion xerofila: aislamiento en una zona endemica para

61 Chapter 3

la cromomicosis en Venezuela. Derm Venez 25: 15-18. Revankar SG (2007). Dematiaceous fungi. Mycoses 50: 91-101. Revankar SG, Sutton DA, Rinaldi MG (2004). Primary central nervous system phaeohyphomycosis: a review of 101 cases. Clin Infect Dis 38: 206-216. Seifert KA, Nickerson NL, Corlett M, et al (2004). Devriesia, a new hyphomycete genus to accommodate heat-resistant, Cladosporium- like fungi. Can J Bot 82: 914-926. Tintelnot K, von Hunnius P, De Hoog GS, et al (1995). Systemic mycosis caused by a new Cladophialophora species. J Med Vet Mycol 33: 349-354. Vicente VA, Attili de Angelis DA, Filho FQT, Pizzirani-Kleiner AA (2001). Isolation of Herpotrichiellaceous fungi from the environment. Braz J Microbiol 32: 47-51. Walsh TJ, Groll AH (1999). Emerging fungal pathogens: evolving challenges to immunocompromised patients for the twenty-first century.Transpl Infect Dis 1: 247-261. White TJ, Bruns T, Lee S, Taylor J (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DA, Sninsky JJ, White TJ (eds). PCR Protocols: A Guide to Methods and Applications. San Diego: Academic Press 315-322. YegÜez-Rodriguez J, Richard-Yegres N, Yegres F, Rodríguez Larralde A (1992). Cromomicosis: susceptibilidad genética en grupos familiares de la zona endémica en Venezuela. Acta Cient Venez 43: 98-102.

62 4 Chapter 4

Cladophialophora psammophila, a novel species of Chaetothyriales with a potential use in the bioremediation of volatile aromatic hydrocarbons

H. Badali 1, 2, 3, F.X. Prenafeta-Boldú 4, 5, J. Guarro6, C.H. Klaassen 7, J.F. Meis 7, G.S. de Hoog 1, 2, 8

1CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands;2 Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands; 3Department of Medical Mycology and Parasitology, School of Medicine/Molecular and Cell Biology Research Centre, Mazandaran University of Medical Sciences, Sari, Iran, 4GIRO Technological Centre, Mollet del Vallès, Spain; 5IRTA, Barcelona, Spain; 6Mycology Unit, Medical School, IISPV, Rovira i Virgili University, Reus, Spain; 7 Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands; and 8Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China

Submitted: (2010). 463 Chapter 4

Abstract Cladophialophora is a genus of asexual black yeast-like fungi with one-celled, hydrophobic conidia which is predicted to have teleomorphs in the ascomycete genus Capronia, a member of the order Chaetothyriales. Cladophialophora species are relatively frequently involved in human disease ranging from mild cutaneous lesions to cerebral abscesses. Although the natural niche outside humans is unknown for most opportunistic Cladophialophora species, the fungi concerned are rarely isolated from environmental samples such as dead plant material, rotten wood, or soil. The objective of the present paper is to describe a novel species of Cladophialophora which was isolated from soil polluted with benzene, toluene, ethylbenzene, and xylene (BTEX). It proved to be able to grow on toluene and other related alkylbenzenes as its sole carbon and energy source. This strain is of interest for the complete biodegradation of toluene and other related xenobiotics under growth limiting conditions, particularly in air biofilters, dry and/or acidic soil. A preliminary genetic analysis using MLST and AFLP showed that this fungus was closely related to the pathogenic species C. bantiana, sharing a C. bantiana-specific intron in SSU rDNA. However, it was unable to grow at 40 °C and proved to be non-virulent in mice. The clear phylogenetic and ecophysiological delimitation of the species is fundamental to prevent biohazard in engineered bioremediation applications.

Key words: Cladophialophora, black yeasts, air biofilters, ITS rDNA, AFLP, in vitro antifungal activity, pathogenicity, neurotropism.

Introduction Cladophialophora is a genus of asexual black yeast-like fungi with one-celled, hydrophobic conidia arranged in long, branched, usually coherent chains and with nearly unpigmented conidial scars. The genus was initially e!rected to accommodate a species exhibitingPhialophora -like conidiogenous cells in addition to conidial chains (Borelli 1980), but thus far this feature is expressed in only a few strains of C. carrionii and thus has limited diagnostic value. Judging from multigene phylogeny (nucLSU, nucSSU, RPB1) Cladophialophora is predicted to have teleomorphs in the ascomycete genus Capronia, a member of the order Chaetothyriales in the family Herpotrichiellaceae (Badali et al. 2008). Cladophialophora species are relatively frequently encountered in human disease, infections ranging from mild cutaneous lesions to cerebral abscesses (Li et al. 2009, de Hoog et al. 2007, Badali et al. 2009). At present the genus contains seven species proven to be involved in human disease (de Hoog et al. 2007, Badali et al. 2008). Among the most virulent species is the neurotrope Cladophialophora bantiana, which is potentially able to cause fatal infections in otherwise healthy individuals (Horré & de Hoog 1999, Kantarcioglu & de Hoog 1999). Cladophialophora carrionii and C. samoënsis are the main etiologic agents of the mutilating skin disorder chromoblastomycosis. Also this type of chronic infection occurs primarily in immunocompetent individuals (Yeguëz- Rodriguez et al. 1992, de Hoog et al. 2007). The natural niche outside humans is unknown for most opportunistic Cladophialophora species. In contrast to frequently expressed opinions (Revankar 2007), listing black yeasts as common saprobes, the fungi concerned are rarely isolated from environmental samples such as dead plant material, rotten wood, or soil. A number of plant- associated Cladophialophora species has been described (Crous et al. 2007), but most of these are located at the phylogenetic base of the Chaetothyriales, remote from the human opportunists (Badali et al. 2008). For recovery of herpotrichiellaceous fungi, selective isolation methods are required, e.g., the use of high temperature (Sudhadham et al. 2008), a mouse vector (Gezuele et al. 1972), extraction

64 Cladophialophora psammophila, with a potential use in the bioremediation of volatile aromatic hydrocarbons via mineral oil (Vicente et al. 2008) or enrichment on volatile aromatic hydrocarbons (Zhao et al. 2010). The success of the latter method, enabling isolation of black yeasts where previously direct plating had failed, has supported the hypothesis that herpotrichiellaceous black yeasts are potent degraders of aromatic hydrocarbons. Previously, the alkylbenzene enrichment has been applied in the biofiltration of air polluted with volatile aromatic compounds (Kennes & Veiga 2004, Cox et al. 1993, Weber et al. 1995). The objective of the present paper is to describe a novel species of Cladophialophora, based on the strain ATCC MYA-2335 (CBS 110553) which was isolated from soil polluted with benzene, toluene, ethylbenzene, and xylene (collectively known as BTEX). It proved to be able to grow on toluene and other related alkylbenzenes as its sole carbon and energy source (Prenafeta-Boldú et al. 2001). This strain is of interest for the complete biodegradation of toluene and other related xenobiotics under growth limiting conditions, particularly in air biofilters, dry and/or acidic soil (Prenafeta-Boldú et al. 2004, Prenafeta-Boldú et al. 2008). A preliminary genetic analysis showed that this fungus was closely related, perhaps even conspecific, to the pathogenic speciesC. bantiana (Prenafeta-Boldú et al. 2006). A clear phylogenetic and ecophysiological delimitation of the species is therefore fundamental to prevent biohazard in engineered bioremediation applications. 4 Materials and Methods

Fungal strain The isolate used in this study was obtained fromBS-KNAW theC Fungal Biodiversity Centre, Utrecht, The Netherlands (Table 1). Stock cultures were maintained on slants of 2 % malt-extract agar (MEA, Difco) and oatmeal agar (OA, Difco) and incubated at 24 °C for two weeks (Gams et al. 2007). The studied strain (CBS 110553) had been isolated from soil in a former gasoline station polluted with BTEX hydrocarbons, which was being treated by means of bioventing (Bennekom, The Netherlands). Soil texture was homogeneously sandy, and a sample was taken from about 2 m deep into the unsaturated zone of the contamination plume. About 10 g of soil were enriched in a solid state-like cultivation batch system that used perlite humidified with a mineral nutrient medium as carrier material (Prenafeta-Boldú et al. 2001). Toluene was supplied via the gas phase as the only carbon and energy source and the incubation was performed at 30 ºC under a relatively low water activity (0.9). Intense dematiaceous growth was observed on the top perlite layer after 5 weeks of incubation. The predominant fungal strain was isolated and purified by re-suspending colonized perlite granules and plating serial dilutions on mineral medium agar plates incubated under a toluene atmosphere.

Morphological and physiological characterization The isolate was cultured on 2 % MEA and OA and incubated at 24 °C in the dark fortwo weeks (Gams et al. 2007). Provisional identification was based on macroscopic and microscopic morphology. Microscopic observations were made with slide culture techniques using potato dextrose agar (PDA, Difco) or OA because these media readily induce sporulation and suppress growth of aerial hyphae (de Hoog et al. 2000). Mounts of two-week-old slide cultures were made in lactic acid or lactophenol cotton blue and light micrographs were taken using a Nikon Eclipse 80i microscope with a Nikon digital sight DS-Fi1 camera. Cardinal growth temperatures were determined by incubating MEA culture plates in the dark for 2 weeks at temperatures ranging from 21 to 40 °C at intervals of 3 °C (Crous et al. 1996). Experiments consisted of three simultaneous replicates for each isolate; the entire procedure was repeated once.

65 Chapter 4 Geography USA USA, Kentucky Carolina USA, North Chiba Japan, Netherlands The - Virginia USA, America South Netherlands Brazil Brazil del Queguay Grande Isla Uruguay, Wageningen Netherlands, Curitiba Paraná, Brazil, Curitiba Paraná, Brazil, del Queguay Grande Isla Uruguay, Apeldoorn Netherlands, Kaiserslautern Germany, Atlanta USA, Georgia, Colombo Parana, Brazil, Sarandi Parana, Brazil, Brazil UK Cayman Islands USA, Grand Curitiba Paraná, Brazil, Germany Source brain abscess Human, brain abscess Human, brain abscess Human, source unknown Human, soil Gasoline-polluted Cat, subcutaneous lesion phaeohyphomycosis Human, chromoblastomycosis Male, aspiration-biopsy Lymphnode, Soil chromoblastomycosis, Male, tree Dead biofilter Toluene toe lesion, child Interdigital cover/soil vegetable Litter, cut tree Trunk, soil Gasolin-station inoculated with soil Biofilter subcutaneous phaeohyphomycosis Male, stem romanzoffianum, Syagrum vegetable cover Decaying eumycetoma Male, phaeohyphomycosis Cutaneous disseminated infection Human, cover/soil vegetable Litter, abscess, male Tracheal GenBank number GenBank EF1, nucSSU ITS, EU103989, EU140585, AY554284 -,-, - -,-, - -,-, - -, AY150798 AY857517, EU103995, - , EU140584 EU103996, -, EU140583 -, - AY366914, - AY366925,-, - AY366927,-, - AY366926,-, EU103983, EU140601, - EU140603, - AY857507, EU103984, EU140602, - EU140600, - AY857508, FJ385270,-, - FJ385274, EU137261, - FJ385271, EU137260, - EU137318, EU137257, - FJ385269 , EU137259, - FJ385272, EU137258, - -, - -, - EU103985, EU140595, - FJ385275,-, - EU103986, EU140593, - Other reference Other 10958, CDC B-1940 (T) ATCC dH 12086 dH12082 IFM 41438 MYA-2335 (T) ATCC 4991 CDC B-3634; UAMH CDC B-3875; NCMH 2247 (T) 18658; dH 15659 ATCC dH 15691 dH 11602 dH 11613 dH 12333; IHM 1727 200384 ATCC dH 12939 dH 11591 dH 12335 dH15250 dH 11474 CDCB-5680; dH 10680 (T) dH 11588 dH 11601 dH 18473 - 56280; dH15405 (T) ATCC ISO 13F (T) CBS no. 173.52 110013 110009 644.96 110553 640.96 979.96 271.37 289.93 102238 102248 109628 114326 118724 102230 109630 110551 109797 834.96 102227 102237 122636 123977 147.84 118720 306.94 Taxon name Taxon bantiana Cladophialophora bantiana Cladophialophora bantiana Cladophialophora bantiana Cladophialophora psammophila Cladophialophora emmonsii Cladophialophora emmonsii Cladophialophora pedrosoi Fonsecaea monophora Fonsecaea monophora Fonsecaea monophora Fonsecaea saturnica Cladophialophora saturnica Cladophialophora saturnica Cladophialophora saturnica Cladophialophora saturnica Cladophialophora immunda Cladophialophora immunda Cladophialophora immunda Cladophialophora immunda Cladophialophora immunda Cladophialophora immunda Cladophialophora immunda Cladophialophora devriesii Cladophialophora devriesii Cladophialophora arxii Cladophialophora Table. 1. Isolation data of examined strains. 1. Isolation Table.

66 Cladophialophora psammophila, with a potential use in the bioremediation of volatile aromatic hydrocarbons

Molecular characterization Mycelia were grown on 2 % (MEA) plates for 2 weeks at 24 °C. A sterile blade was used to scrape off the mycelium from the surface of the plate and transferred to a 2 ml Eppendorf tube containing 400 µL TEX-buffer (Tris 1.2 % w/v, Na-EDTA 0.38 % w/v, pH 9.0) and glass beads (Sigma G9143). The fungal materials were homogenized by Mobio vortex for 5−10 min. Subsequently Geography Chiba Yachimata, Japan, Shiroi Japan, Jicaltepec Mexico, Netherlands The 120 µL SDS 10 % and 10 μl proteinase K were added and incubated for 30 min at 65 °C, the mixture was vortexed for 3 min. After addition of 120 μl of 5 M NaCl and 1/10 vol CTAB 10 % (cetyltrimethylammonium bromide) buffer, the material was incubated for 60 min at 55 °C. Subsequently 700 μl SEVAG (24:1, chloroform: isoamylalcohol) was slowly mixed, incubated for 30 min on ice water and centrifuged for 10 min at 14 000 r.p.m. The supernatant was transferred to a new tube with 225

μl of 5 M NH4-acetate, mixed carefully by inverting, incubated 4 Source wood Rotting wood Decaying eumycetoma Male, contaminant Culture for 30 min on ice water, and centrifuged again for 10 min at 14 000 r.p.m. The supernatant was transferred to another tube with 0.55 vol isopropanol and centrifuged for 5 min at 14 000 r.p.m. Finally, the pellet was washed with 1000 μl ice cold 70 % ethanol. After drying at room temperature, it was re-suspended in 100 μl TE buffer (Tris 0.12 % w/v, Na-EDTA 0.04 % w/v) plus 1.5 μl RNAse 20 U/mL and incubated for 15–30 min at 37 °C. DNA extracts were stored at −20°C until use (Gerrits van den Ende et al. 1999). GenBank number GenBank EF1, nucSSU ITS, EU103988, EU140599 EU140598, - AY251087, FJ385276,-, - EU137293, EU137235, - Three genes were amplified for phylogenetic characterization of the new species: the small subunit of the nuclear ribosomal RNA gene (nucSSU), the large subunit of the nuclear ribosomal RNA gene (nucLSU), and the largest subunit genes of RNA polymerase I (RPB1). For multilocus sequencing typing (MLST), the internal transcribed spacer region (ITS rDNA), the translation elongation factor 1 alpha (EF1-α), and the partial beta tubulin (TUB) genes were amplified. The primers used for

Other reference Other 5022 IFM 4701; UAMH 52853; IMI 298056 ATCC dH 18909 (T) dH 15898 amplification and sequencing were described previously (Badali et al. 2008, Gueidan et al. 2008). PCR reactions were performed on a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA) in 50 μL volumes containing 25 ng of template

CBS no. 987.96 556.83 122637 454.82 DNA, 5 μl reaction buffer (0.1 M Tris-HCl, pH 8.0, 0.5 M

KCl, 15 mM MgCl2, 0.1 % gelatine, 1 % Triton X-100), 0.2 mM of each dNTP and 2.0 U Taq DNA polymerase (ITK Diagnostics, Leiden, The Netherlands). Amplification of those genes were performed with cycles of 2 min at 94 °C for primary denaturation, followed by 35 cycles at 94 °C (45 s), 52 °C (30 s) and 72 °C (120 s), with a final 7 min extension step at 72 °C. Annealing temperatures used to amplify SSU, LSU, RPB1, ITS, EF1-α and TUB genes were 55 °C, 52 °C, 52 °C, 52 °C, Taxon name Taxon minourae Cladophialophora minourae Cladophialophora mycetomatis Cladophialophora mycetomatis Cladophialophora Atlanta, U.S.A.; DCU = Prevention, and Disease Control Netherlands; CDC = Centers for The Utrecht, Centre, Biodiversity Fungal U.S.A.; CBS = CBS-KNAW Collection, Manassas, Culture Type = American ATCC Abbreviations: of University = The UAMH Chiba, Japan; Chiba University, Toxicoses, and Microbial Fungi Center for Pathogenic collection; IFM = Research working dH = G.S. de Hoog Chiba, Japan; School of Medicine, Department of Dermatology, strain. Type T = Canada; Edmonton, Collection and Herbarium, Alberta Microfungus Table 1 (continued) Table 50 °C and 58 °C, respectively. Amplicons were purified using

67 Chapter 4

Cladophialophora bantiana CBS 173.52 100 Cladophialophora bantiana CBS 110013

85 Cladophialophora bantiana CBS 110009 Cladophialophora bantiana CBS 644.96 98 Cladophialophora psammophila CBS 110553 100 Cladophialophora emmonsii CBS 640.96 Cladophialophora emmonsii CBS 979.96 Fonsecaea pedrosoi CBS 271.37 100 100 Fonsecaea monophora CBS 289.93 Fonsecaea monophora CBS 102238 Fonsecaea monophora CBS102248 Cladophialophora saturnica CBS 109628 100 Cladophialophora saturnica CBS 114326 Cladophialophora saturnica CBS 118724 99 Cladophialophora saturnica CBS 102230 Cladophialophora saturnica CBS 109630 x10 100 86 Cladophialophora immunda CBS102237 Cladophialophora immunda CBS 122636 72 Cladophialophora immunda CBS 834.96 82 Cladophialophora immunda CBS 109797 Cladophialophora immunda CBS 110551 Cladophialophora immunda CBS 123977 Cladophialophora immunda CBS 102227 96 88 Cladophialophora devriesii CBS 147.84 Cladophialophora devriesii CBS 118720 76 Cladophialophora immunda dH18474 Fig. 1. Phylogenetic reconstruction

100 Cladophialophora immunda dH 18476 obtained from a ML analysis of three Cladophialophora immunda dH 18478 combined loci (ITS rDNA, EF1-α and Cladophialophora immunda CBS 122255 TUB) using RAxML. Bootstrap support Cladophialophora immunda dH 18477 values were estimated based on 500 Cladophialophora immunda CBS 122257 replicates, and are shown above the CBS 306.94 100 Cladophialophora arxii 100 Cladophialophora minourae CBS 987.96 branches (values ≥ 70 %). New species Cladophialophora minourae CBS 556.83 are highlighted using with grey boxes. Cladophialophora mycetomatis CBS 122637 The tree was rooted with two strains Cladophialophora mycetomatis CBS 454.82 of Cladophialophora mycetomatis (CBS 0.01 substitutions/site 454.82 and CBS 122637). QIAquick PCR purification Kit (Cat. No 28104, U.K.). Sequencing was performed as follows: 95 °C for 1 min, followed by 30 cycles consisting of 95 °C for 10 s, 50 °C for 5 s and 60 °C for 2 min. Reactions were purified with Sephadex G-50 fine (GE Healthcare Bio-Sciences, Uppsala, Sweden) and sequencing was done on an ABI 3730XL automatic sequencer (Applied Biosystems, Foster City, CA, U.S.A.). Sequence data obtained in this study were adjusted using the SeqMan of Lasergene software (DNAStar Inc., Madison, Wisconsin, U.S.A.). Alignments of DNA sequences were done manually for each gene using MacClade 4.08 (Maddison et al. 2003) with the help of amino acid sequences for protein coding loci. Ambiguous regions and introns were excluded from the alignments. The program RAxML-VI-HPC v.7.0.0 (Stamatakis et al. 2008), as implemented on the Cipres portal v. 1.10, was used for the tree search and the bootstrap analysis (GTRMIX model of molecular evolution and 500 bootstrap replicates). Bootstrap values equal or greater than 70 % were considered significant (Hillis et al. 1993). Data were analysed as Clade II (Badali et al. 2008). TheBantiana -clade comprised 36 taxa. Phylogenetic reconstructions and bootstrap values were first obtained for each locus separately using RAxML (as described above). The congruence between loci was assessed using a 70 % reciprocal bootstrap criterion (Mason-Gamer et al. 1996). The loci were then combined and analysed using RAxML (as described above). The phylogenetic trees were edited using Tree View v. 1.6.6. AFLP analysis was performed as described previously (Badali et al. 2010).

68 Cladophialophora psammophila, with a potential use in the bioremediation of volatile aromatic hydrocarbons

Fig. 2. Amplified fragments length polymorphism (AFLP) profile of Cladophialophora psammophila (CBS 110553) in relation to that of reference strains of Cladophialophora bantiana (CBS 173.52) and Cladophialophora minourae (CBS 556.83).

Fig. 3. Survival rates of OF-1 mice infected intravenously with 1x107 CFU/animal. T = type strain.

Table. 2. Minimum inhibitory (azoles) and effective (echinocandins) concentrations in mg/L of different antifungal drugs against Cladophialophora psammophila (CBS 110553) and other Cladophialophora reference strains

Name CBS no AmB FLU ITC VOR POS ISA CAS ANI

C. bantiana 173.52 (T) 0.25 16 0.016 0.125 0.016 0.031 1 0.125

C. bantiana 110009 1 16 0.031 0.25 0.031 0.031 8 0.125

C. bantiana 110013 0.5 32 0.063 1 0.063 0.5 2 0.063

C. psammophila 110553 (T) 0.25 32 0.016 0.25 0.016 0.125 2 0.031

C. minourae 555.83 (T) 1 32 0.063 0.25 0.031 0.25 1 0.063

Virulence testing The virulence of the new species was compared with that of three reference strains of Cladophialophora bantiana and a strictly non-pathogenic strain of C. minourae using a murine model, which was previously described by Mariné et al. (2009) determining the mortality rates of the infected animals. Briefly, six-week-old OF-1 male mice (ten per group) (Charles River, Criffa SA, Barcelona, Spain) weighing 28-30 g were used. Animals were housed five per cage in standard boxes with corncob redding and free access to food and water. Conditions were approved by the Animal Welfare committee of the Medical School of the University Rovira i Virgili. The isolates tested were Cladophialophora sp. CBS 110553, C. bantiana (CBS 110013, CBS 110009 and CBS 173.52 as ex-type strain), and C. minourae (CBS 556.83). They were stored on corn meal agar (CMA, 30 g corn and 15 g agar per litre) slants covered with paraffin oil, and prior to the study they were cultured on PDA for 5–8 days at 30 ºC. Inocula were prepared by flooding the surface of the agar plate with sterile saline, scraping the sporulating mycelium with a culture loop, and

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Fig. 4. Cladophialophora psammophila (CBS 110553). A–B. Culture on MEA and PDA) produced velvety to hairy, powdery and olivaceous-black with a wide well-defined margin. C. Hyphal coil. D–G. Conidiophores laterally or terminally on undifferentiated hyphae, ellipsoidal to fusiform or sub-cylindrical. F. One-celled and long conidia, produced inlong, strongly coherent chains. Scale bars = 10 μm.

70 Cladophialophora psammophila, with a potential use in the bioremediation of volatile aromatic hydrocarbons

Fig. 5. Line drawing of microscopic morphology of Cladophialophora psammophila (CBS 110553). Scale bar = 10 µm.

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Fig. 6. Colony diameters at different temperatures ranging from 27 °C to above 40 °C, measured after two weeks on 2 % MEA were calculated for Cladophialophora bantiana (CBS 173.52) and Cladophialophora psammophila (CBS 110553). drowning up the resultant suspension with a sterile Pasteur pipette. Suspensions were filtered once through sterile gauze to remove hyphae. Conidial suspensions were adjusted after counting with a haemocytometer. The viability of these inocula was verified by plating dilutions of the suspension on PDA plates. Infection was established intravenously with 0.2 ml of inoculum via the lateral tail vein. Inocula administered were 2 × 107 conidia/animal. Preliminary experiments with several reference strains of C. bantiana demonstrated that this concentration produced infections with subsequent 100 % mortality within 20 days of inoculation (data not shown).

Antifungal susceptibility In vitro antifungal susceptibilities of different drugs against CBS 110553 and reference strains (C. bantiana and C. minourae which are a neurotropic pathogen and a saprobe, respectively) were performed by microbroth dilution as described in the Clinical and Laboratory Standards Institute (CLSI) document M38-A2. Minimum inhibitory concentrations (MIC) for amphotericin B (AmB, Bristol-Myers Squib, Woerden, The Netherlands); fluconazole (FLU, Pfizer Central Research Sandwich, UK); itraconazole (ITC, Janssen Research Foundation, Beerse, Belgium); voriconazole (VOR, Pfizer); posaconazole (POS, Schering-Plough, Kenilworth, USA); isavuconazole (ISA, Basilea, Basel, Switzerland), and minimum effective concentrations (MEC) for caspofungin (CAS, Merck Sharp & Dohme, Haarlem, The Netherlands) and anidulafungin (ANI, Pfizer) were determined. As per the CLSI guidelines, stock solutions of the drugs were prepared in the appropriate solvent. The drugs were diluted in the standard RPMI-1640 medium (Sigma Chemical Co.) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma) with L-glutamine without bicarbonate to yield two times their concentrations were as follows: amphotericin B, itraconazole, voriconazole, posaconazole and caspofungin 0.016–16 mg/ml; fluconazole 0.063–64 mg/ ml; isavuconazole 0.002–2 mg/ml and anidulafungin 0.008–8 mg/ ml. Plates were stored at –70 °C until they were used. Methods for sporulation and preparation of conidial suspensions were those of Badali et al. (2010). Paecilomyces variotii (ATCC 22319), Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258) were used as quality controls.

72 Cladophialophora psammophila, with a potential use in the bioremediation of volatile aromatic hydrocarbons

Results Using the phylogenetic markers including nucSSU, nucLSU and RPB1, Cladophialophora species are predicted to belong to at least four different lineages. Most of the human-opportunistic species belong to two well supported clades within the Herpotrichiellaceae (Badali et al. 2008). Strain CBS 110553 was nested in lineage one corresponding to the family Herpotrichiellaceae (Tree not shown) that contained the pathogenic Cladophialophora species (C. carrionii, C. boppii, C. bantiana, C. arxii, C. immunda, C. devriesii, C. saturnica, C. emmonsii C. mycetomatis) as well as the pathogenic genus Fonsecaea (F. pedrosoi and F. monophora) and Phialophora (P. verrucosa and P. mericana). Phylogenetic reconstructions were first performed for each gene separately (ITS: 495 characters, EF1-α: 133 characters, TUB: 355 characters). Topological conflicts were detected only within the species C. saturnica, the incongruence involving only below species-level relationships, and therefore – in agreement with the genealogical recognition species concept (Taylor et al. 2000) – the conflicts were ignored and the three loci were combined. In this multilocus analysis, Cladophialophora mycetomatis, an agent of true mycetoma was taken as an outgroup (Fig. 1). This analysis showed that CBS 110553, originating from hydrocarbon polluted soil was phylogenetically Cladophialophora distinct from other species of , and a sister taxon (85 % bootstrap support) of the 4 pathogenic species C. bantiana. Hence, it is described below as a novel species of Cladophialophora. The AFLP profile of C. psammophila (CBS 110553) was compared with strains of C. bantiana, yielding similar data as obtained by sequencing analysis in that banding patterns were invariable within C. bantiana and deviated considerably from the CBS 110553 profile: no major bands were shared by the two entities (Fig. 2). Survival rates of mice experimentally infected with strains of Cladophialophora are shown in Fig. 3. All animals infected with three different strains of C. bantiana died between days 12 and 17; differences between strains were insignificant. Animals infected with C. psammophila and C. minourae survived and remained alive until the end of the experiment. Table 2 summarizes the MIC values of eight antifungal drugs against three highly neurotropic isolates (C. bantiana) and two non-pathogenic species (CBS 110553 and C. minourae). There was a uniform pattern of low MICs for itraconazole, posaconazole and isavuconazole. The highest MICs were for fluconazole followed by amphotericin B, the echinocandins and voriconazole.

Cladophialophora psammophila Badali, Prenafeta-Boldú, Guarro & de Hoog, sp. nov. MycoBank MB 515525. Figs 4, 5

Coloniae fere lente crescentes, ad 30 mm diam post 14 dies 33 °C, velutinae vel lanosae, conidiis maturis pulverulentae, olivaceo-nigrae, margine obscuriore circumdatae. Hyphae fertiles erectae, cylindricae, sursum ramosae, 2.0−2.5 µm latae. Conidia unicellularia, in catenis longis persistentibus, raro ramosis connexa, ex hyphis indistinctis orientia, pallide olivacea, levia, tenuitunicata, ellipsoidea vel fusiformia vel subcylindrica, 6.5−10.0 × 2.0−3.5 μm. Teleomorphosis ignota.

Description based on CBS 110553 at 27 °C after 2 wk in darkness.

Macroscopy on PDA: Colonies moderately expanding, with a daily growth rate of 2.1 mm (1.5 mm at 37 °C). Colonies velvety to hairy, powdery when sporulating, olivaceous-black with a wide well-defined margin which is darker than the colony centre; reverse olivaceous-black (Figs 4A−B). Microscopy on MEA: Fertile hyphae septate, ascending to erect, smooth- and thin-walled, hyaline to pale olivaceous, guttulate, branched, 2−3 μm wide, forming hyphal strands and hyphal coils.

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Conidiophores laterally or terminally on undifferentiated hyphae, pale olivaceous, smooth- and thin-walled, ellipsoidal, fusiform or sub-cylindrical, 6.5−10.0 × 2.0−3.5 μm, erect. Conidia one- celled, produced in long, strongly coherent, poorly branched chains (Figs 4C-H, 5). Teleomorph unknown.

Physiology: Cardinal temperatures: optimal development at 27−30 °C, with growth abilities between 9−37 °C. No growth at 40 °C (Fig. 6).

Specimen examined: The Netherlands, Bennekom, isolated from sandy soil in a former gasoline station polluted with BTEX hydrocarbons (benzene, toluene, ethylbenzene, and xylene), CBS H-20384 (Holotype) = CBS 110553 (ex-type culture).

Discussion The new species C. psammophila morphologically resembles C. bantiana, which is a virulent neurotropic agent of fatal cerebral phaeohyphomycosis in humans, causing granolumatous abscesses in the brain and having a maximum growth temperature of 42 °C. The multiple sequences analyzed in this study deviate sufficiently from those of C. psammophila to be sure that the two species can be considered to represent separate though highly related species. Members of C. bantiana are unique by consistently containing a distinctive 558-bp intron beginning at position 1768 in the small subunit of the ribosomal DNA gene (Gerrits van den Ende et al. 1999). Interestingly, this intron is also present in C. psammophila, underlining the close kinship of the two species. Cladophialophora bantiana has a worldwide distribution, but until now it could be isolated from the environment only by using a mammal vector (Conti-Diaz et al. 1977, Dixon et al. 1980). The natural niche of this organism remains unknown and it is presumably introduced via inhalation as the main infection route. In contrast, C. psammophila was isolated by hydrocarbon enrichment from a location where hydrocarbon pollution was prevalent. It does not grow at temperatures above 37 °C. Pathogenicity tests on a murine model indicated that C. psammophila is non-virulent, whereas C. bantiana kills mice within two weeks. Both Cladophialophora species, although at short phylogenetic distance, thus exhibit striking ecological differences. The ecophysiological divergence between C. bantiana and the newly described C. psammophila is puzzling. Future genome comparisons will serve to reveal the essential virulence factors involved in the pathogenicity of the Bio-Safety Level-3 species C. bantiana. Phylogenetically the genus Cladophialophora – morphologically consistently characterised by poorly or profusely branched chains of dry, rather strongly coherent, moderately melanized conidia – is polyphyletic within the order Chaetothyriales (Badali et al. 2008). In part the genus is closely related to members of genera that are consistently associated with human infection, such as Exophiala, Fonsecaea and Rhinocladiella (lineage 1 in combined phylogenetic reconstructions (nucSSU, nucLSU and RPB1) of Chaetothyriales published by of Badali et al. 2008). This part of the tree comprises a number of Capronia teleomorphs and may therefore be regarded to represent the family Herpotrichiellaceae. This family also encompasses several species that have been related to the metabolism of monoaromatic hydrocarbons and other related aromatic substrates, both in polluted and natural environments. Consequently, these organisms are of interest in biotechnological applications for the bioremediation of hydrocarbon pollution. The distantly related Cladophialophora species have been described as host-specific plant-associates (Crous et al. 2007) and are unlikely to belong to the Chaetothyriales. No association to aromatic substrates has ever been reported in this latter group. Enrichment on toluene as the sole sources of carbon and energy was a key factor for the isolation

74 Cladophialophora psammophila, with a potential use in the bioremediation of volatile aromatic hydrocarbons of the strain CBS 110553. The physiological and metabolic characterization of axenic cultures demonstrated that assimilation of aromatic hydrocarbons was limited to certain alkylbenzenes, such us toluene and ethylbenzene (Prenafeta-Boldú et al. 2001). This biodegradation pattern was confirmed in biodegradation assays of complex BTEX mixtures simulating gasoline pollution, in which only toluene and ethylbenzene were mineralized, while the xylene isomers were co- metabolized and benzene remained undegraded throughout the experiment (Prenafeta Boldú et al. 2002). Alkylbenzene degradation proceeded through the initial oxidation of the alkyl side chain, rather than the aromatic ring (Prenafeta Boldú et al. 2001). Growth of C. psammophila in liquid cultures under agitation was predominatly yeast-like. Furthermore, enzymological studies on the related Cladophialophora saturnica CBS 114326, initially misidentified as a Cladosporium sphaerospermum, showed the involvement of a cytochrome P450 monooxygenase in the initial oxidation of the alkyl group (Luykx et al. 2003). The exceptional metabolic and physiological capacities of CBS 110553 have been investigated in relation to the biofiltration of waste gases polluted with volatile aromatic hydrocarbons and for the bioremediation of BTEX pollution in soil microcosms (Prenafeta-Boldu et al. 2004 and 2008). The striking close phylogenetic proximity of strain CBS 110553 to the notorious human pathogen C. bantiana has put its applicability in bioremediation into question due to potential 4 biohazard (Prenafeta-Boldu et al. 2006). Cladophialophora bantiana has regularly been recognized in human disease by its high mortality rate despite the application of combined surgery and antifungal therapy, and has therefore been considered as one of the most dangerous pathogenic fungi known to date (de Hoog et al. 2003). The majority of reported CNS infections caused byC. bantiana were found to be brain abscesses with no predisposing factors or immunodeficiency (Horré & de Hoog 1999). The pathogenesis may be haematogenous spread from an initial, presumably subclinical, focus which may be pulmonary, subcutaneous or intestinal (Li et al. 2009). It remains unclear why this fungus preferentially causes CNS disease in immunocompetent individuals. It has been hypothesized that, in addition to other virulence factors that are common in herpotrichiellaceous fungi, such as thermotolerance, dimorphic yeast-like growth, wall melanization, etc., the tendency to infect central nervous tissue in mammals is also related to the capacity of metabolizing aromatic compounds, which is also conspicuous in the Herpotrichiellaceae (Prenafeta-Boldu et al. 2006). Being primary neurotropic, a characteristic from just a few species in the Herpotrichiellaceae, these fungi must also possess other, as yet undiscovered attributes conferring infective capacity towards the brain. An association between metabolism of aromatic hydrocarbons and opportunistic pathogenicity has also been observed in Cladophialophora immunda and C. saturnica, as well as in the related genus Exophiala, particularly E. oligosperma and E. xenobiotica. Representatives have been reported from opportunistic human infections and from hydrocarbon polluted sites (Badali et al. 2008, de Hoog et al. 2003, Vicente et al. 2008). However, infection patterns of these species tend to be coincidental, and are mostly limited to skin and nails. Cladophialophora psammophila thus far has never been reported from clinical cases, and virulence test on the strain CBS 110553 indicated that the new species is, in principle, non-pathogenic.

Acknowledgments This study and the work of H. Badali have been funded by a research grant (No. 13081) from the Ministry of Health and Medical Education of the Islamic Republic of Iran and the School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. The authors thank Walter Gams for his comments on the manuscript and for providing the Latin descriptions.

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References: Badali H, Gueidan C, Najafzadeh MJ, et al (2008). Biodiversity of the genus Cladophialophora. Stud Mycol 61: 175-191. Badali H, Carvalho VO, Vicente V, et al (2009). Cladophialophora saturnica sp. nov., a new opportunistic species of Chaetothyriales revealed using molecular data. Med Mycol 47: 51-62. Badali H, de Hoog GS, Breuker-Curfs I, et al (2010). Use of amplified fragment length polymorphism to identify 42 clinical Cladophialophora spp., related to cerebral phaeohyphomycosis with in vitro antifungal susceptibility. J Clin Microbiol . DOI: 10.1128/JCM.00653-10. (in press). Borelli D (1980). Causal agents of chromoblastomycosis (Chromomycetes) Proceedings of the 5th International Conference on Mycoses pp. 335-340. Clinical and Laboratory Standards Institute (2008). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved Standard M38-A2; 2nd edn: CLSI, Wayne, PA, USA. Conti-Díaz IA, Mackinnon JE & Civila E (1977). Isolation and identification of black yeasts from the external environment in Uruguay. Pan Amer Health Org Sci Publ 356: 109-114. Crous PW, Gams W, Wingfied MJ, Van Wyk PS (1996). Phaeoacremonium gen. nov associated with wilt and decline diseases of woody hosts and human infections. Mycologia 88: 786-796. Crous PW, Schubert K, Braun U, et al (2007). Opportunistic, human pathogenic species in the Herpotrichiellaceae are phenotypically similar to saprobic or phytopathogenic species in the Venturiaceae. Stud Mycol 58: 185-217. Cox HHJ, Houtman JHM, Doddema HJ, Harder W (1993). Enrichment of fungi and degradation of styrene in biofilters. Biotechnol Lett 15: 737-742. de Hoog GS, Guarro J, Gené J, Figueras MJ (2000). Atlas of clinical fungi, 2nd ed. CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands and Universitat Rovira i Virgili, Reus, Spain. de Hoog GS, Vicente V, Caligiorne RB, et al (2003). Species diversity and polymorphism in the Exophiala spinifera clade containing opportunistic black yeast-like fungi. J Clin Microbiol 41: 4767-4778. de Hoog GS, Nishikaku AS, Fernandez-Zeppenfeldt G, et al (2007). Molecular analysis and pathogenicity of the Cladophialophora carrionii complex, with the description of a novel species. Stud Mycol 58: 219-234. Dixon DM, Shadomy HJ, Shadomy S (1980). Dematiaceous fungal pathogens isolated from nature. Mycopathologia 70: 153-161. Gams W, Verkley GJM, Crous PW (2007). CBS Course of Mycology, 4th ed. CBS-KNAW Fungal Biodiversity Centre, Utrecht. Gerrits van den Ende AHG, de Hoog GS (1999). Variability and molecular diagnostics of the neurotropic species Cladophialophora bantiana. Stud Mycol 43: 151-162. Gezuele E, Mackinnon JE & Conti-Diaz IA (1972). The frequent isolation ofPhialophora verrucosa and Fonsecaea pedrosoi from natural sources. Sabouraudia 10: 266-273. Gueidan C, Ruibal Villaseñor C, de Hoog GS, et al (2008). A rock-inhabiting ancestor for mutualistic and pathogen-rich fungal lineages. Stud Mycol 61: 111-119. Hillis DM & Bull JJ (1993). An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. System Biol 42: 182-192. Horré R & de Hoog GS (1999). Primary cerebral infections by melanized fungi: a review. Stud Mycol 43: 176-193. Kantarcioglu S, de Hoog GS (1999). Infections of the central nervous system by melanized fungi: a review of cases presented between 1999 and 2004. Mycoses 47: 4-13. Kennes C & Veiga MC (2004). Fungal biocatalysts in the biofiltration of VOC-polluted air.J Biotechnol 113(1-3): 305-19. Li DM & de Hoog GS (2009). Cerebral phaeohyphomycosis-a cure at what lengths? Lancet Infect Dis 9: 376-383. Luykx DMA, Prenafeta-Boldú FX, de Bont JAM (2003). Toluene monooxygenase from the fungus Cladosporium sphaerospermum. Biochem Biophys Res Comm 312: 373-379. Maddison WP, Maddison DR (2003). MacClade Analysis of Phylogeny and Character Evolution, v. 4.6, Sinauer, Sunderland, MA. Mariné M, Pastor FJ, Guarro J (2009). Combined antifungal therapy in a murine model disseminated infection by Cladophialophora bantiana. Med Mycol 47: 45-49. Mason-Gamer RJ, Kellogg EA (1996). Testing for phylogenetic conflict among molecular datasets in the tribe Triticeae (Graminae). Syst Biol 45: 524-545. Prenafeta-Boldú FX, Luykx DMA, Vervoort J, de Bont JAM (2001). Fungal metabolism of toluene: Monitoring of fluorinated analogs by 19F nuclear magnetic resonance spectroscopy.Appl Environ Microbiol 67: 1030-1034. Prenafeta-Boldú FX, Kuhn A, Luykx D, et al (2001a). Isolation and characterisation of fungi growing on volatile aromatic hydrocarbons as their sole carbon and energy source. Mycol Res 105: 477-484 Prenafeta-Boldú FX, Vervoort J, Grotenhuis JTC, van Groenestijn JW (2002). Substrate interactions during the biodegradation of benzene, toluene, ethylbenzene, and xylene (BTEX) hydrocarbons by the fungus Cladophialophora sp strain T1. Appl Environ Microbiol 68: 2660-2665. Prenafeta-Boldú FX, Illa J, van Groenestijn JW, Flotats X (2008). Influence of synthetic packing materials on the gas

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dispersion and biodegradation kinetics in fungal air biofilters.Appl Microbiol Biotechnol 79: 319-327. Prenafeta Boldú FX, Ballerstedt H, Gerritse J, Grotenhuis JTC (2004). Bioremediation of BTEX hydrocarbons: Effect of soil inoculation with the toluene-growing fungus Cladophialophora sp. strain T1. Biodegradation 15: 59–65. Prenafeta-Boldú FX, Summerbell RC, de Hoog GS (2006). Fungi growing on aromatic hydrocarbons: biotechnology’s unexpected encounter with biohazard. FEMS Microbiol Rev 30: 109-130. Revankar SG (2007). Dematiaceous fungi. Mycoses 50: 91-101. Stamatakis A, Hoover P, Rougemont J (2008). A rapid bootstrap algorithm for the RAxML Web-Servers. Syst Biol 57: 758-771. Sudhadham M, Dorrestein GM, Prakitsin S, et al (2008). The neurotropic black yeastExophiala dermatitidis has a possible origin in the tropical rain forest. Stud Mycol 61: 145-55. Taylor JW, Jacobson DJ, Kroken S, et al (2000). Phylogenetic species recognition and species concepts in fungi. Fungal Genet Biol 31: 21-32. Vicente VA, Attili-Angelis D, Pie MR, et al (2008). Environmental isolation of black yeast-like fungi involved in human infection. Stud Mycol 61: 136-144. Weber FJ, Hage KC, de Bont JA (1995). Growth of the fungus Cladosporium sphaerospermum with toluene as the sole carbon and energy source. Appl Environ Microbiol 61: 3562-3566. Yegüez-Rodríguez J, Richard-Yegres N, Yegres F, Rodríguez Larralde A (1992). Cromomicosis: susceptibilidad genética en grupos familiares de la zona endémica en Venezuela. Acta Cient Venez 43: 98-102. Zhao J, Zeng J, de Hoog GS, et al (2010). Isolation of black yeasts by enrichment on atmospheres of monoaromatic hydrocarbons. DOI: 10.1007/s00248-010-9651-4. (in press). 4

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Use of amplified fragment length polymorphism to identify 42 Cladophialophora strains, related to cerebral phaeohyphomycosis with in vitro antifungal susceptibility

H. Badali 1, 2, 3, G.S. de Hoog 1, 2, I. Breuker-Curfs 4, C.H.W. Klaassen 4, J.F. Meis 4

Accepted: Journal of Clinical Microbiology (2010), DOI: 10.1128/JCM.00653-10. (in press). 579 Chapter 5

Abstract The AFLP technique has been applied to identify neurotropic chaetothyrialean black yeasts and relatives from clinical sources. C. bantiana, C. emmonsii, C. arxii, C. devriesii and C. modesta identified previously based on sequencing, phenotypic and physiological criteria were confirmed with the cluster analysis demonstrating a clear separation of C. bantiana as a rather homogeneous group from the other species. C. bantiana is a neurotropic fungus causing cerebral abscesses with a mortality of up to 70 %. Successful therapy consists of neurosurgical intervention and optimal antifungal therapy. Since the latter is not clearly defined in a large series we testedin vitro activities of eight antifungal drugs against clinical isolates of C. bantiana (n =37), C. modesta (n=2), C. arxii (n=1), C. emmonsii (n=1), and C. devriesii (n=1), all having caused invasive infections.

The resulting MIC90s for all neurotropic C. bantiana strains were as follows in increasing order: posaconazole, 0.125 µg/ml; itraconazole, 0.125 µg/ml; isavuconazole, 0.5 µg/ml; amphotericin B, 1 µg/ml; voriconazole, 2 µg/ml; anidulafungin, 2 µg/ml; caspofungin, 4 µg/ml; and fluconazole, 64 µg/ml. Based on these in vitro results and previous clinical and animal studies, posaconazole seems to be a good alternative for the standard use of amphotericin B in C. bantiana cerebral infections. The new agent isavuconazole, which is also available as an intravenous preparation, has an adequate activity against C. bantiana.

Keywords: Cladophialophora bantiana, C. modesta, C. emmonsii, C. devriesii, fungal brain abscess

Introduction The genus Cladophialophora represents anamorph members of the ascomycetes in the order Chaetothyriales in the family Herpotrichiellaceae comprising the black yeasts and relatives (de Hoog et al. 2000). These dematiaceous fungi are normally associated with soil or vegetative matter; however, they are being increasingly seen as causative agents of mycoses in humans (Horré et al. 1999, Li et al. 2009, Revankar et al. 2004) and domestic (Elies et al. 2003, Guillot et al. 2004) and wild animals (Janovsky et al. 2006). Cladophialophora carrionii is the type species and an agent of chromoblastomycosis, a cutaneous and subcutaneous disease. The genusCladophialophora encompasses several other clinically significant species, which are potentially able to cause severe fungal infections in otherwise immunocompetent patients. In human infections, the brain is involved frequently (Horré et al. 1999, Li et al. 2009, Revankar et al. 2004). Within the genus the majority of brain abscesses with fatal outcome is associated with Cladophialophora bantiana, a neurotropic fungus (formerly Cladosporium bantianum, Cladosporium trichoides, Cladosporium trichoides var. chlamydosporum, Torula bantiana, and Xylohypha bantiana), although severe phaeohyphomycotic infections are also caused by novel species like C. modesta (McGinnis et al. 1999), C. arxii (Shigemura et al. 2009, Tintelnot et al. 1995), C. emmonsii (Xylohypha emmonsii) (Padhye et al. 1988), C. devriesii (Gonzalez et al. 1984, Howard et al. 2006, Mitchell et al. 1990) C. saturnica (Badali et al. 2009), C. boppii (Lastoria et al. 2009) and C. immunda (Badali et al. 2008). Moreover, Exophiala dermatitidis and Rhinocladiella mackenziei, other members of the black yeast group, are also frequently isolated from cerebral infections (Chakrabarti 2007, Horré et al. 1999, Li et al. 2009). Central nervous system infection due to C. bantiana is reported worldwide, though a general preference for warmer climates with high humidity is apparent (Horré et al. 1999). Indeed many cases are reported from India (George et al. 2008, Jayakeerthi et al. 2004, Lakshmi et al. 2008, Sood et al. 2000) as opposed to cases from arid climatic zones (Chakrabarti 2007). The first case of C. bantiana (trichoides) was reported in 1952 when the fungus was isolated from a human brain abscess and demonstrated to be neurotropic in laboratory animals (Binford et al. 1952). A

80 Use of AFLP to identify Cladophialophora strains, related to cerebral phaeohyphomycosis with in vitro antifungal susceptibility review of 17 cases of brain abscess, published in the English language literature by the mid-1970s, reported that the majority of patients had no underlying disease (Middleton et al. 1976). The most recent series of 48 patients with brain abscess due to C. bantiana showed that 35 patients (72 %) had no risk factors and that only 13 patients (28 %) survived the infection, despite combined surgical and antifungal treatment (Revankar et al. 2004). The minority of immunocompromised patients are transplant recipients, i.v. drug abusers or those on steroids (Alhabib et al. 2003, Dixon et al. 1989, Emmens et al. 1996, Harrison et al. 2008, Keyser et al. 2002, Lee et al. 2003, Levin et al. 2004, Salama et al. 1997, Silveira et al. 2003, Trinh et al. 2003, Walz et al. 1997). The mode of infection is either by haematogenous spread from an unrecognized pulmonary focus, through direct extension from adjacent paranasal sinuses or penetrating trauma to the head, although the majority had had no recent evidence of pulmonary or sinus infections (Horré et al. 1999). Cladophialophora species are prone to identification problems (Badali et al. 2008, Gerrits van den Ende et al. 1999). Due to the high degree of phenotypic similarity between recently described new Cladophialophora species and C. bantiana, identification problems are imminent. For most cases published in the older literature, identification down to the species level cannot be repeated or confirmed by molecular methods due to the absence of original isolates; hence the etiological agent described in older publications may often have been misidentified. It is now well established that molecular identification methods, which have driven new developments in fungal taxonomy are more reliable than classical morphological methods (Badali et al. 2008, Gerrits van den Ende et al. 1999). Amplified fragment length polymorphism (AFLP) is a technique based on the detection of genomic restriction fragments by PCR amplification, which can be used on any organisms’ DNA 5 (Vos et al. 1995). The purpose of this study was to study the inter and intraspecific genomic variation of 42 Cladophialophora isolates stored in the CBS collection and recovered from cases with cerebral phaeohyphomycosis and other infections. Antifungal therapy is mainly based on the experience gathered from and published in isolated case reports which mostly involved amphotericin B, itraconazole and 5 flucytosine single or in different combinations (Revankaret al. 2004). Animal studies (Al-Abdely et al. 2005) and human experience suggested that amphotericin B had no value in treating cerebral infections probably due to poor penetration of the central nervous system but that triazoles might be of value (Bonifaz et al. 2009, Heney et al. 1989, Lyons et al. 2005). A recent study of antifungal therapy in a murine model of disseminated infection by C. bantiana confirmed the poor activity of amphotericin but found that posaconazole and 5 flucytosine extended survival (Mariné et al. 2009). This suggests that new antifungal drugs with broad-spectrum activity and suitable pharmacokinetic profiles compared with those of conventional antifungal agents might be more effective against C. bantiana (Al-Abdely et al. 2005, Mariné et al. 2009). Only limited data are available of in vitro antifungal activities against the neurotropic fungus C. bantiana. Therefore, with no standard therapy available, unfavourable results in animal experiments, and only a small published series of susceptibility testing of itraconazole and voriconazole (Radford et al. 1995), the second objective of this study was in vitro susceptibility testing for eight antifungal drugs, including the new tri-azole isavuconazole, of this large collection of Cladophialophora strains.

Materials and Methods

Fungal strains Table 1 summarizes and characterizes a total of 42 isolates of Cladophialophora spp. that originated

81 Chapter 5 from different human and veterinary clinical sources with cerebral phaeohyphomycosis or other infections. Strains were obtained from the reference collection of the CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands and were handled in Bio-Safety Level-3 conditions. Stock cultures for transient working collections initially were grown on oatmeal agar (OA) (Difco Oatmeal, Brunschwig Chemie, Amsterdam, the Netherlands) at 24 °C for one week, and identified to the species level by sequencing of the internal transcribed spacer regions of rDNA region, partial Translation Elongation factor 1-alfa and beta-tubulin genes (Badali et al. 2008).

DNA extraction The fungal mycelia were grown on 2 % (MEA) plates for 2 weeks at 24 °C. A sterile blade was used to scrape off the mycelium from the surface of the plate. DNA was extracted using an Ultra Clean Microbial DNA Isolation Kit (Mobio, Carlsbad, CA) according to the manufacturer’s instructions. DNA extracts were stored at −20 °C prior to use.

AFLP analysis Approximately 50 ng of genomic DNA was subjected to a combined restriction ligation procedure containing 50 pmol of HpyCH4 IV adapter, 50 pmol Mse I adapter, 2 U of HpyCH4 IV (New England Biolabs, Beverly, MA), 2 U of Mse I (New England Biolabs), and 1 U of T4 DNAligase (Promega, Leiden, The Netherlands) in a total volume of 20 μl of 1 × reaction buffer for 1 h at 20°C. Next, the mixture was diluted five times with 10mMTris- HCl (pH 8.3) buffer. Adapters were made by mixing equimolar amounts of complementary oligonucleotides (5’-CTCGTAGACTGCGTACC-3’ and 5’-CGGGTACGCAGTC-3’ for HpyCH4 IV; 5’-GACGATGAGTCCTGAC-3’ and 5’-TAGTCAGGACTCAT-3’ for Mse I) and heating to 95 °C, subsequently cooling slowly to ambient temperature. One microliter of the diluted restriction-ligation mixture was used for amplification in a volume of 25 μl under the following conditions: 1 μM HpyCH4 IV primer with one selective residue (underlined) (5’-Flu- GTAGACT GCGTACCCGTC-3’), 1 μM MseI primer with four selective residues (underlined) (5’- GATGAGTCCTGACTAATGAG-3’), 0.2 mM of each deoxynucleoside triphosphate, and 1 U of Taq DNA polymerase (Roche Diagnostics, Almere, The Netherlands) in 1× reaction buffer containing 1.5 mM MgCl2. Amplification was done as follows. After an initial denaturation step for 4 min at 94 °C in the first 20 cycles, a touchdown procedure was applied: 15 s of denaturation at 94 °C, 15 s of annealing at 66 °C, with the temperature for each successive cycle lowered by 0.5°C, and 1 min of extension at 72 °C. Cycling was then continued for a further 30 cycles with an annealing temperature of 56 °C. After completion of the cycles, incubation at 72 °C for 10 min was performed before the reaction mixtures were cooled to room temperature. The amplicons were then combined with the ET400-R size standard (GE Healthcare, Diegem, Belgium) and analyzed on a Mega BACE 500 automated DNA platform (GE Healthcare) according to the manufacturer’s instructions.

Data analysis Data was inspected visually and was also imported in BioNumerics v. 5.1 software (Applied Maths, Sint-Martens-Latem, Belgium) and analyzed by UPGMA clustering using the Pearson correlation coefficient. The analysis was restricted to DNA fragments in the range from 80-250 bp.

In vitro antifungal susceptibility testing

82 Use of AFLP to identify Cladophialophora strains, related to cerebral phaeohyphomycosis with in vitro antifungal susceptibility

Amphotericin B (AmB, Bristol-Myers-Squib, Woerden, The Netherlands), fluconazole (FLU, Pfizer Central Research Sandwich, UK), itraconazole (ITR, Janssen Research Foundation, Beerse, Belgium), voriconazole (VOR, Pfizer), posaconazole (POS, Schering-Plough, Kenilworth, USA), isavuconazole (ISA, Basilea Pharmaceutical Ltd. Swiss), caspofungin (CAS, Merck Sharp & Dohme BV, Haarlem, The Netherlands) and anidulafungin (ANI, Pfizer) were obtained from the manufacturers as pure powders. As described in the CLSI M38–A2 guidelines for in vitro susceptibility studies, additive drugs dilutions were prepared at 100 times the final concentrations in different solutions (CLSL, M38-A2). The drugs were diluted in the standard RPMI 1640 medium (Sigma Chemical Co.) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma) with L-glutamine without bicarbonate to yield two times at their concentrations were as follows: amphotericin B, itraconazole, voriconazole, posaconazole and caspofungin 0.016– 16 μg/ml; fluconazole 0.063–64 μg/ml; isavuconazole and anidulafungin 0.008–8 μg/ml. Plates were stored at -70 °C until they were used. Broth microdilution was performed as described by the Clinical and laboratory Standards Institute in accordance with documents M38-A2. Briefly, all clinical isolates were grown on Potato Dextrose Agar plates (PDA, Difco) at 35 °C for up to 7 days for sporulation. Inoculum suspensions were prepared under biosafety laboratory (level 3) regulations by slightly scraping the surface of mature colonies with a loop in sterile saline solution with Tween 40 (0.05%). After allowing heavy particles to settle, the homogenous conidial suspensions were transferred to sterile tubes and adjusted spectrophotometrically at 530 nm wavelength to optical densities (OD) that ranged from 0.17 to 0.15 (68 to 71 T %). The inoculum suspensions including mostly non-germinated conidia were diluted 1:50 in RPMI 1640 medium. The final concentration of the stock inoculum suspensions of the tested isolates ranged 5 from 0.5 ×104 – 3.1 × 104 CFU/ml as performed by quantitative colony counts to determine the viable number of colony forming units per milliliter. After inoculation, the microdilution plates were incubated at 35 °C and examined visually and spectrophotometrically at 420 nm after 72 h of incubation. The MIC (minimum inhibitory concentrations) endpoints were defined with the aid of a reading mirror as the lowest concentration of drug that prevents any recognizable growth (100 % inhibition) for amphotericin B, itraconazole, voriconazole, posaconazole and isavuconazole. For fluconazole a prominent reduction of growth inhibition (≥50 %) compared to drug-free growth control was used. The MEC (minimum effective concentrations) was defined microscopically as the lowest concentration of drug that leads to the growth of small, rounded, compact hyphal forms as compared with the long, unbranched hyphal clusters that were seen in the growth control. Paecilomyces variotii (ATCC 22319), Candida parapsilosis (ATCC 22019) and

Candida krusei (ATCC 6258) strains were used as quality control organisms. Values for MIC50 and

MIC90 were obtained by ordering MIC data for each antifungal in ascending arrays and selecting the median and 90th quartile, respectively, of the MIC distribution. Geometric mean MICs were computed using Microsoft® Office Excel 2003 SP3, for which purpose values “

Results Fig. 1 depicts a dendrogram of the AFLP analysis including the type strains of C. bantiana; C. emmonsii, C. devriesii, C. modesta and C. arxii demonstrating that C. bantiana is phylogenetically distinct from the other Cladophialophora spp. The AFLP pattern of C. bantiana strains, from different geographically regions (Table 1) such as U.S.A., Japan, India, Belgium, France, South- Africa, Mexico or Brazil, clustered together. The structuring of this dataset suggests that C. bantiana populations dispersed quickly across the world. There was one main cluster ofC. bantiana

83 Chapter 5 Geography USA USA Carolina USA, North USA, Maryland USA, Kentucky USA, Pennsylvania USA, Georgia USA DC Washington USA, Carolina USA, North Virginia West USA, USA, Missouri Virginia USA, USA Japan Japan Japan Japan Japan Japan Japan India Belgium Brazil Thailand unknown Mexico Source brain abscess Human, brain abscess Human, brain abscess Human, brain abscess Human, brain abscess Human, source origin, unknown Human brain abscess Human, brain abscess Human, brain abscess Human, origin, brain abscess Human source origin, unknown Human source origin, unknown Human source origin, unknown Human brain abscess Human, source origin, unknown Human source origin, unknown Human source origin, unknown Human source origin, unknown Human source origin, unknown Human source origin, unknown Human brain abscess Human, subcutaneous phaeohyphomycosis Human, brain abscess Human, brain abscess Human, subcutaneous phaeohyphomycosis Human, brain abscess Human, eumycetoma Human, Other reference Other 58037; CDC B-3112 ATCC 22649 ATCC dH 16810 46715; dH16022 ATCC dH 12086 dH 15506 58035; CDC B-1551 ATCC 10958 CDC B-1940 (T) ATCC 58040; CDC B-3466 ATCC dH12082 dH 12084 dH12081 dH 12083 dH 12080 IFM 41438 IFM 41439; dH 15162 IFM 41436; dH16119 IFM 41434; dH 16116 IFM 41437; dH16121 IFM 4820; DCU 651 44223; CDC B-3426 ATCC 44217 ATCC - dH 11331 dH 14515 24928; dH 10739 ATCC dH 16363 CBS no. CBS 101251 CBS 100428 CBS 119547 CBS 564.82 CBS 110013 CBS 194.54 CBS 100432 CBS 173.52 CBS 101252 CBS 110009 CBS 110011 CBS 110008 CBS 110010 CBS 110007 CBS 644.96 CBS 101253 CBS 642.96 CBS 641.96 CBS 643.96 CBS 646.96 CBS 101158 CBS 100431 CBS 155.53 CBS 102586 CBS 119719 CBS 100429 CBS 123392 bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana bantiana

Taxon name Taxon Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Table. 1. Isolation data of tested strains. 1. Isolation Table.

84 Use of AFLP to identify Cladophialophora strains, related to cerebral phaeohyphomycosis with in vitro antifungal susceptibility Geography Africa South Africa South Canada, Edmonton Sweden USA, California Canada, Edmonton Montpellier France, Africa South Antilles Netherlands USA Carolina USA, North Virginia USA, Virginia USA, USA, Florida Cayman Islands Grand

5 Source brain abscess Human, subcutaneous phaeohyphomycosis Human, brain abscess Human, leg subcutaneous phaeohyphomycosis Human, Cat, brain abscess abscess liver Dog, thorax tumefaction Dog, disseminated infection Dog, abscess liver Dog, sawdust experimentally infected by Mouse, brain abscess Human, subcutaneous phaeohyphomycosis Human, hand subcutaneous phaeohyphomycosis, Human, brain abscess Human, disseminated infection Human, Other reference Other SAIMR J-1872 W262 SAIMR 6501; dH16330 UAMH dH 16362 58039; CDC B-1897 ATCC 3830 UAMH CNRMA 2004/222 - CDC B-3394; NCMH 1168 CDC 3432; IFM 4821 4004; dH16331 (T)UAMH CDC B-3875; NCMH 2247 (T) CDC B-5420; dH 11918 CDC B-5887; dH11524 56280; dH15405 (T) ATCC CBS no. CBS 984.96 CBS 649.96 CBS 981.96 CBS 120376 CBS 100436 CBS 648.96 CBS 118738 CBS 444.96 CBS 328.65 CBS 647.96 CBS 985.96 CBS 979.96 CBS 102594 CBS 102461 CBS 147.84 bantiana bantiana bantiana bantiana bantiana bantiana

Taxon name Taxon Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora Cladophialophora bantiana Cladophialophora bantiana Cladophialophora bantiana Cladophialophora bantiana Cladophialophora modesta Cladophialophora emmonsii Cladophialophora emmonsii Cladophialophora arxii Cladophialophora devriesii Cladophialophora Netherlands; CDC = Centers for The Utrecht, collection, Hoog private Netherlands; dH = G.S. de The Utrecht, Centre, Biodiversity Fungal U.S.A.; CBS = CBS-KNAW Collection, Manassas, Culture Type = American ATCC Abbreviations: University, Chiba Toxicoses, Microbial Fungi and Pathogenic Center for Research Edmonton, Canada; IFM = Herbarium, Collection and Microfungus of AlbertaUniversity = The U.S.A.; UAMH Atlanta, and Prevention, Control Disease Chiba, Japan; School of Medicine, U.S.A; DCU = Department of Dermatology, Chapel Hill, Hospital, Memorial Carolina Africa; NCMH = North South Johannesburg, Research, for Medical African Institute SAIMR = South Chiba, Japan; T = ex-type culture. of Paris. Institute and Antifungals at the Pasteur Center for Mycoses Reference CNRMA = Collection of the National Table. 1. (continued) Table.

85 Chapter 5

Table. 1. Geometric mean MICs, MICs ranges, MIC50, and MIC90 performed by in vitro susceptibility testing of 8 antifungal drugs against 42 strains of Cladophialophora expressed in μg/ml.

Name/Number Cladophialophora bantiana C. modesta C. arxii C. devriesii C. emmonsii (n=37) (n=1) (n=1) (n=1) (n=1)

Drugs Range G mean MIC50 MIC90 MICs MICs MICs MICs MICs

Amphotericin B 0.125 - 2 0.700 1 1 1 1 2 0.5 1

Fluconazole 16 - 64 35.14 32 64 32 8 16 32 32

Itraconazole < 0.016 - 0.25 0.064 0.063 0.125 0.5 0.016 0.031 0.125 0.125

Voriconazole 0.125 - 4 0.769 1 2 2 0.125 0.25 2 0.5

Posaconazole < 0.016 - 0.25 0.044 0.031 0.125 0.25 0.016 0.031 0.063 0.063

Isavuconazole 0.008 - 1 0.259 0.25 0.5 2 0.063 0.031 1 1

Caspofungin 1-8 2.551 2 4 4 2 2 2 4

Anidulafungin 0.016 - 4 0.073 0.063 2 0.5 1 1 1 1

including the type strain (CBS 173.52) with similarities of more than 75 %. The C. emmonsii strains segregate in one cluster including the reference strain (CBS 979.96) and one clinical taxon originating from a subcutaneous lesion which had >50 % similarity to each other. The remaining taxa from clinical sources were represented by single strains and the AFLP pattern shows that they are completely distinct from the C. bantiana and C. emmonsii clade (<20 % similarity).

Table 2 summarizes the results of geometric mean MICs, MIC ranges, MIC50, and

MIC90 distribution of 37 C. bantiana isolates for eight antifungal agents. Five other clinical Cladophialophora species have similar individual MIC results compared to the C. bantiana

MIC90 data. For each antifungal the MIC50 and geometric mean MIC values differed by <1 log2 dilution step, indicating that in all cases the MIC50s obtained by inspection reasonably reflect the central tendency of antifungal susceptibility of the population. Overall, all of the isolates showed a uniform pattern of low MICs for itraconazole, posaconazole and isavuconazole. The widest ranges and the highest MICs were seen for fluconazole (range 16–64 μg/ml). Amphotericin B MICs ranged from 0.125–2 μg/ml and itraconazole, posaconazole and isavuconazole had MIC ranges of < 0.016–0.25 μg/ml, < 0.016–0.25 μg/ml and 0.008–1 μg/ml, respectively. There were no differences in the activity of itraconazole and posaconazole and they were generally more active than amphotericin B, fluconazole, voriconazole and isavuconazole. C. modesta, C. arxii, C. emmonsii (Xylohypha emmonsii), and C. devriesii were also inhibited by low concentrations of azoles similar as C. bantiana. Isavuconazole exhibited potent activity against C. bantiana and the other neurotropic strains with a MIC90 0.5 μg/ml. The voriconazole MIC90 (2 μg/ml) was 2 log2 dilution steps less active than isavuconazole (0.5 μg/ml ) which in turn was 2 log2 dilution steps less active than itraconazole and posaconazole (MIC90 0.125 μg/ml). The only available isolate of

C. modesta had a high MIC (2 μg/ml) for voriconazole and isavuconazole, similar as the MIC90 of voriconazole for C. bantiana. Of the two echinocandins, anidulafungin had the best activity with a geometric mean MEC which was 5 log2 dilution steps more active than caspofungin although the

MEC90 of 2 μg/ml would not qualify anidulafungin as an agent which can be used as monotherapy.

Discussion Primary cerebral phaeohyphomycosis in humans without obvious predisposing factors is rare, but increasingly recognized as an infectious disease associated with high mortality and a poor prognosis (Dixon et al. 1989, Horré et al. 1999, Li et al. 2009, Revankar et al. 2005). The most

86 Use of AFLP to identify Cladophialophora strains, related to cerebral phaeohyphomycosis with in vitro antifungal susceptibility common agent of cerebral phaeohyphomycosis that belongs to the order of Chaetothyriales is C. bantiana which has been reported in both healthy and immunocompromised hosts. In addition other species with similar clinical presentation have been described. Patients with bone marrow and solid organ transplantation or patients who are on steroid therapy are particularly affected (Alhabib et al. 2003, Dixon et al. 1989, Emmens et al. 1996, Harrison et al, 2008, Keyser et al. 2002, Lee et al. 2003, Levin et al. 2004, Salama et al. 1997, Silveira et al. 2003, Trinh et al. 2003, Walz et al. 1997). Cladophialophora bantiana, a neurotropic fungus, has rarely been isolated from sources other than clinical samples. C. bantiana is distributed world-wide but infections are especially encountered in subtropical and humid climates areas (Chakrabarti 2007). Although C. bantiana has been recovered from the environment (Dixon et al. 1997) and clinical infection linked to traumatic inoculation (Petrini et al. 2006), the environmental niche of C. bantiana is largely unknown and only one such strain of C. bantiana (CBS 647.96), recovered from sawdust, was available in this study. An unambiguous connection between a clinical and an environmental strain still has to be proven. Correct identification with mycological procedures remains difficult, due to the high degree of phenotypic similarity between these groups of fungi. For most cases published in the older literature, the etiological agent may often have been misidentified; however re-identification down to the species level cannot be performed by molecular methods due to absence of original isolates. Recently, molecular data have shown that Cladophialophora contains a number of hitherto unknown species closely related but significantly different from C. bantiana (Badali et al. 2010). A study of the variability and molecular determination of the neurotropic species C. bantiana and ITS sequencing data indicated a low degree of variability (Gerrits van den Ende et al. 5 1999). Interestingly, C. bantiana consistently contains an invariable intron of 558 bp at position 1768 in the SSU rDNA gene, while it was absent and present in all C. emmonsii isolates and C. psammophila, respectively, which are closely related to C. bantiana (Badali et al. 2010). Remarkably, this intron might be involved in evolutionary events, because it is not found outside the genetically homogeneous C. bantiana. Other neurotropic species of Cladophialophora closely related to C. bantiana are involved in brain infections. A fatal cerebral infection with C. modesta was reported in a 25-year-old black male after possible traumatic inoculation (McGinnis et al. 1999) and C. arxii was found as the cause of cerebral phaeohyphomycosis in a 30-year-old African-American woman who underwent cardiac transplantation for postpartum cardiomyopathy (Osiyemi et al. 2001). In contrast, C. devriesii was involved in a case of disseminated disease without involvement of the central nervous system (Mitchell et al. 1990), which years before presented as subcutaneous mycosis (Gonzalez et al. 1984). C. (Xylohypha) emmonsii was the cause of a subcutaneous mycosis (Padhye et al. 1988) and C. boppii was responsible for a pulmonary infection in a lung transplant recipient (Lastoria et al. 2009). AFLP is one of a series of techniques for phylogenetic studies, plant and animal genetic mapping and genotyping and is well suited for distinguishing closely related organisms at the species to strain level (Klaassen et al. 2007). The AFLP method relies on selective amplification of restriction fragments from a digest of genomic DNA with many advantages when compared to other marker technologies including randomly amplified polymorphic DNA, restriction fragment length polymorphism, and microsatellites. AFLP not only has higher reproducibility, resolution, and sensitivity at the whole genome level compared to other random amplification techniques, but it also has the capability to amplify between 50 and 100 fragments at one time. In addition, no prior sequence information is needed for amplification (Walz et al. 1997). Bakkeren et al. (2000) have shown that the phylogenetic trees obtained from AFLP analysis are quite similar to those obtained by ITS sequences in Ustilago species, and in addition permit distinction of closely

87 Chapter 5

Fig. 1. Dendrogram of AFLP analysis of 42 Cladophialophora isolates (CBS numbers to the right of the figure; the scale bar on the left indicates the percentage similarity).

88 Use of AFLP to identify Cladophialophora strains, related to cerebral phaeohyphomycosis with in vitro antifungal susceptibility related isolates that cannot be resolved by ITS sequence comparison. Our AFLP results are in line with previous sequencing data and also show obvious differences among clinically important Cladophialophora species as agents of cerebral infection. Based on the AFLP patterns we did not see any misidentification for those taxa. All strains in this study were originally identified as C. bantiana, C. emmonsii, C. arxii, C. devriesii and C. modesta based on sequencing, phenotypic and physiological criteria which is in the line with the cluster analysis demonstrating a clear separation of C. bantiana as a rather homogeneous group from C. modesta, C. emmonsii, C. arxii and C. devriesii. Although dematiaceous fungi as agents of central nervous system infections are generally susceptible to most antifungal agents in vitro, treatment is difficult because frequent relapses are observed (Revankar et al. 2004) Antifungal therapy is based on the experience of treatment in single patient case reports or small series which mostly involved amphotericin B, itraconazole and 5 flucytosine. In animal models, amphotericin B prolonged survival of mice infected with C. bantiana, but the infection did not completely disappear (Al-Abdely et al. 2005). Although in vitro activity of amphotericin B has been demonstrated for C. bantiana in this and previous studies, the drug was ineffective in many cases of cerebral phaeohyphomycosis with or without flucytosine (Emmens et al. 1996, George et al. 2008, Harrison et al. 2008, Revankar et al. 2004, Sekhon et al. 1992). In vitro results indicate that amphotericin B MICs for most non-dermatophyte opportunistic filamentous fungi isolates clustered between–2 0.5 μg/ml. Very little data are available concerning correlation between MIC and outcome of treatment with amphotericin B for dematiaceous fungi. Generally, filamentous fungi are not susceptible to fluconazole and most MICs were >16 μg/ml. Two case reports described improvement of C. bantiana brain abscess after 5 treatment up to 6 weeks fluconazole and surgical excision (Delfinoet al, 2006; Gomes et al. 2003). Probably surgical intervention was the cause of improvement. Fortunately, the antifungal armamentarium has been extended with new triazoles, potent agents active against drug-resistant strains and with less toxicity. There are only limited data in the literature regarding the susceptibility of C. bantiana isolates to antifungals (Radford et al. 1997). Less successful outcome of treatment with itraconazole (Osiyemi et al, 2001) and voriconazole (Hanieh et al, 2006 Roche et al, 2005, Fica et al. 2003) for cerebral abscess due to C. bantiana were observed, although itraconazole had low MICs (MIC90 0.125 μg/ml) in this study. The explanation might be a less optimal penetration in CNS. Whereas voriconazole has a good penetration into the

CNS the MICs of C. bantiana were in the higher ranges (MIC90 2 μg/ml). Some authors report successful treatment of C. bantiana brain abscess with voriconazole (Lyons et al. 2005) while others report clinical failure (Hanieh et al, 2006 Roche et al, 2005, Fica et al. 2003). Although an excellent drug for treatment of cerebral aspergillosis, voriconazole might not be a first choice forC. bantiana. In contrast to another study with only seven strains of C. bantiana (Radford et al. 1997), giving a MIC range of 0.12–1.0 μg/ml voriconazole we found a large range of activity. Therefore in vitro susceptibility testing may be warranted before using voriconazole for a CNS infection due to C. bantiana. Posaconazole and itraconazole (MIC90 0.125 μg/ml) demonstrated the best in vitro activities, followed by isavuconazole (MIC90 0.5 μg/ml). Some clinical experience suggests that therapy with itraconazole was used successfully in treating a C. bantiana cerebral infection (Harrison et al. 2008, Lee et al. 2003), eumycetoma (Bonifaz et al. 2009) and C. arxii osteomyelitis (Shigemura et al. 2009). Treatment with posaconazole is supported by our in vitro results, other in vitro data from small series (n=5, range 0.06-0.5 μg/ml) (Garzoni et al. 2008, Espinel-Ingroff 1998), a clinical case (Garzoni, et al. 2008) and data from murine infection models in which posaconazole prolonged the survival of mice and reduced the brain fungal burden compared to itraconazole and amphotericin B (Al-Abdely et al. 2005, Mariné et al. 2009). Moreover, the

89 Chapter 5

apparently good penetration into the CNS (Rüping et al. 2008) supports use of posaconazole for this difficult to treat infection. Most melanized fungi appear to be resistant to echinocandins, probably due to a reduced presence of β-glucan in the cell walls (Espinel-Ingroff 1998; Odabasiet al. 2004). We found poor activity of caspofungin but anidulafungin had activity for C. bantiana showing a geometric mean MIC of only 0.073 μg/ml although several isolates had a high MEC

(MEC90 2 μg/ml). Another echinocandin, micafungin was not active in animal studies when used as monotherapy but seemed to be promising in combination with posaconazole and 5 flucytosine (Mariné et al. 2009). The investigational agent isavuconazole shows broad-spectrum activity against many major opportunistic fungi and true pathogenic fungi (Gonzalez et al. 2005). Here we show that isavuconazole is also very active against C. bantiana but its true value needs to be confirmed in animal models.In conclusion, itraconazole, posaconazole, and isavuconazole demonstrated in vitro antifungal activity against neurotropic isolates of C. bantiana. Some positive and negative correlating clinical experiences are available for itraconazole, voriconazole and posaconazole but the most important intervention for cerebral phaeohyphomycosis caused by C. bantiana is probably remains complete neurosurgical excision of the abscesses.

Acknowledgments This study and the work of Hamid Badali were funded by the Ministry of Health and Medical Education of the Islamic Republic of Iran (grant number 13081) and the School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. This study was partially supported by an unrestricted grant from Basilea Pharmaceuticals, Basel, Switzerland.

References

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The clinical spectrum ofExophiala jeanselmei, with a case report and in vitro antifungal susceptibility of the species

H. Badali 1, 2, 3, M.J. Najafzadeh 1, 2, M. van Esbroeck 4, E. van den Enden 4, B. Tarazooie 1, J.F.G.M. Meis 5, G.S. de Hoog 1, 2

Published: Medical Mycology 2010; 48: 318–327 693 Chapter 6

Abstract Exophiala jeanselmei is clinically redefi ned as a rare agent of subcutaneous lesions of traumatic origin, eventually causing eumycetoma. Mycetoma is a localized, chronic, suppurative subcutaneous infection of tissue and contiguous bone after a traumatic inoculation of the causative organism. In advanced stages of the infection, one fi nds tumefaction, abscess formation and draining sinuses. The species has been described as being common in the environment, but molecular methods have only confi rmed its occurrence in clinical samples. Current diagnostics of E. jeanselmei is based on sequence data of the Internal Transcribed Spacer (ITS) region of ribosomal DNA (rDNA), which sufficiently refl ects the taxonomy of this group. The first purpose of this study was the re- identifi cation of all clinical (n=11) and environmental strains (n=6) maintained under the name E. jeanselmei , and to establish clinical preference of the species in its restricted sense. Given the high incidence of eumycetoma in endemic areas, the second goal of this investigation was the evaluation of in vitro susceptibility of E. jeanselmei to eight conventional and new generations of antifungal drugs to improve antifungal therapy in patients. As an example, we describe a case of black grain mycetoma in a 43-year-old Thai male with several draining sinuses involving the left foot. The disease required extensive surgical excision coupled with intense antifungal chemotherapy to achieve cure. In vitro studies demonstrated that posaconazole and itraconazole had the highest antifungal activity against E. jeanselmei and E. oligosperma for which high MICs were found for caspofungin. However, their clinical effectiveness in the treatment ofExophiala infections remains to be determined.

Key words: Exophiala jeanselmei, black yeasts, ITS rDNA, mycetoma, antifungal susceptibility testing.

Introduction Mycetoma is defined as a localized, chronic, granolumatous, suppurative and progressive inflammatory disease of subcutaneous tissue and contiguous bone after a traumatic inoculation of the causative organism (McGinnis 1996). Causative agents are recovered from human and also from domestic animals (Hay 2000). In advanced stages of the infection tumefaction, abscess formation and draining sinuses arise. Hallmark of a mycetoma is the presence of the fungus in the form of grains. In Africa the infection is particularly common from Sudan to Senegal, an area known as the “mycetoma belt”; main etiologic agents in that area are Madurella mycetomatis and Leptosphaeria senegalensis. Other recurrent fungal agents are Madurella grisea and several coelomycete species. Classically, the black yeast Exophiala jeanselmei has been reported to be involved in subcutaneous lesions of traumatic origin, eventually causing eumycetoma (de Hoog et al. 2000, Bittencourt et al. 1995). Members of the ascomycete order Chaetothyriales, are remarkably frequently encountered and reported as agents of disease. Many of these species are oligotrophic (Satow et al. 2008) and are therefore found in uncommon habitats. This property also seems to be a predisposing factor for human infection, since a large number of species are found as etiologic agents of disease. Several members of Chaetothyriales have been reported involved in eumycotic mycetoma in humans and animals. Werlinger et al. (2005) and Bonifaz et al. (2009) described cases of mycetoma caused by Cladophialophora bantiana. Guillot et al. (2004) reported on a case in a dog coused by the same species. Cladophialophora mycetomatis is a novel species recently described by Badali et al. (2008) as an agent of eumycetoma from Mexico. Black yeasts identivied as E. jeanselmei have also been reported from many other types of infection, such as chromoblastomycosis or mild cutaneous disease (Naka et al. 1986), disseminated infections, endocarditis and arthritis (Nucci et al. 2001). However, recent molecular studies have shown that the species is very heterogeneous. The original

94 The clinical spectrum ofExophiala jeanselmei morphological varieties have now been raised to species level (de Hoog et al. 1994), and additional, morphologically nearly indistinguishable species have been described (de Hoog et al. 2003). Hence, the clinical predilection of E. jeanselmei has to be re-evaluated on the basis of correctly identified material. With obsolete mycological procedures, identification of black yeasts remains difficult (de Hoog et al. 2000). Since the original isolates no longer ara aviable for most cases published in older literature, their identification down to the species level cannot be performed by molecular methods. It is posible that the etiological agent may often have been misdiagnosed. In this article a case of eumycetoma coused by E. jeanselmei matching the original clinical concept of the species is presented. Current diagnostics of E. jeanselmei is achievied through the use of sequence data of the Internal Transcribed Spacer (ITS) region of ribosomal DNA (rDNA), which reflects the taxonomy of this group (Desnos-Ollivier et al. 2006). Therefore, the first purpose of this study was the re-identification of all clinical (n=11) and environmental strains (n=6) preserved under the name E. jeanselmei in the collection of CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands, and to establish the species clinical manifestation. Given the high incidence of eumycetoma in endemic areas, the second goal of this study was the evaluation of in vitro susceptibility of E. jeanselmei to eight conventional and new generations of antifungal drugs to improve the antifungal therapy in patients. New antifungal agents with a better activity may help to improve the management of eumycetoma as sufficient knowledge is gained as to the in vitro activity of new antifungal agents.

Materiales and Methods

Fungal strains Strains used in this study were obtained from the CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands (Table 1), deposited and phenetically identified E. jeanselmei. Stock 6 cultures were maintained on slants of 2 % Malt Extract Agar (MEA, Difco) and Oatmeal Agar (OA, Difco) and incubated at 24 °C for two weeks (Gams et al. 2007). The identification of 17 strains was verified with sequences of the internal transcriber spacer regions (ITS) of the rDNA. An isolate from a recent case of mycetoma was added (CBS 122339).

DNA extraction and sequencing Mycelia were grown on 2 % MEA plates for 2 weeks at 24 °C (Gams et al. 2007). A sterile blade was used to scrape off the mycelium from the surface of the plate. DNA was extracted using an Ultra Clean Microbial DNA Isolation Kit (Mobio, Carlsbad, CA 92010, USA) according to the manufacturer’s instructions. DNA extracts were stored at −20 °C until ready for use (Badali et al. 2008). Internal Transcribed Spacers rDNA (ITS) were amplified using primers V9G and LS266 and sequenced with the internal primers ITS1 and ITS4 (white et al. 1990). PCR reactions were performed on a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, U.S.A.) in 50 µL volumes containing 25 ng of template DNA, 5 µL reaction buffer (0.1 M Tris-HCl, pH 8.0, 0.5

M KCl, 15 mM MgCl2, 0.1 % gelatine, 1 % Triton X-100), 0.2 mM of each dNTP and 2.0 U Taq DNA polymerase (ITK Diagnostics, Leiden, The Netherlands). Amplification was performed with cycles of 5 min at 94 °C for primary denaturation, followed by 35 cycles at 94 °C (45 s), 52 °C (30 s) and 72 °C (90 s), with a final 7 min extension step at 72 °C. Amplicons were purified using GFX PCR DNA and gel band purification kit (GE Healthcare, Ltd., Buckinghamshire U.K.). Sequencing was performed as follows: 95 °C for 1 min, followed by 30 cycles consisting of 95 °C for 10 s, 50 °C for 5 s and 60 °C for 2 min. Reactions were purified with Sephadex G-50 fine

95 Chapter 6

Table 1. Summary of source and identification of strains tested.

CBS number Other collection Source Country Reference Final name

CBS 122339 dH 18920 Man, mycetoma Thailand Current paper E. jeanselmei

CBS 119095 dH 13453 Man, foot skin USA, Dallas None E. jeanselmei

CBS 109635 dH 12305 Man, arm lesion USA, San Antonio [13] E. jeanselmei

CBS 148.97 dH 10808 Man, subcutaneous cyst Japan, Ibaraki [22] E. jeanselmei

CBS 507.90 dH 15933 (T) Man, mycetoma France [20] E. jeanselmei

CBS 116.86 dH 15309 Man, chromomycosis Japan [10] E. jeanselmei

CBS 677.76 dH 16163 Man, mycetoma Pakistan [21] E. jeanselmei

CBS 664.76 ATCC 34123 Man, skin infection None [23] E. jeanselmei

CBS 528.76 ATCC10224 Man, hand skin lesion None None E. jeanselmei

CBS 814.95 dH 16266 Soil biofilter Netherlands, Delft [13] E. oligosperma

CBS 634.69 dH 16108 Wood, ship resting Baltic Sea None E. oligosperma

CBS 538.76 dH 15984 Man, bronchus None None E. oligosperma

CBS 537.76 dH 15981 Man, eye infection Italy [24] E. oligosperma

CBS 642.82 dH 16117 Soft rot of preservative-treated wood Australia, Lucia None E. oligosperma

CBS 527.76 dH 15966 Culture contaminant of Hyphodontia breviseta Sweden, Bohuslan None E. xenobiotica

CBS 102241 dH 11605 Soil under coffee plant Brazil, Paraná None E. bergeri

CBS 663.76 dH 16149 Wood None [23] Capronia pilosella

Abbrevations used: ATCC = American Type Culture Collection, Manassas, U.S.A.; CBS = CBS-KNAW Fungal Biodiversity Centre, Utrecht, The netherlands; DH = G.S. de Hoog working collection; IFM = Research Institute for Pathogenic Fungi, Chiba, Japan; IHM = Laboratory of Mycology, Faculty of Medicine, Montevideo Institute of Epidemiology and Hygiene, Montevideo, Uruguay; BMU = Beijing Medical University. T = ex-type culture

(GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and sequencing was done on an ABI 3730XL automatic sequencer (Applied Biosystems, Foster City, CA, U.S.A).

Alignment and phylogenetic reconstruction Sequence data obtained in this study were adjusted using the SeqMan of Lasergene software (DNAStar Inc., Madison, Wisconsin, U.S.A.). ITS sequences were aligned manually using BioNumerics version 4.61 (Applied Maths, Kortrijk, Belgium). The program RAxML-VI-HPC v.7.0.0 (Stamatakis et al. 2008), as implemented on the Cipres portal v.1.10, was used for the tree search and the bootstrap analysis (GTRMIX model of molecular evolution and 500 bootstrap replicates). Bootstrap values equal or greater than 70 % were considered significant (Hillis et al. 1993). Newly generated sequences were subjected to a BLAST search of the NCBI databases, sequences with high similarity (≥98%) were downloaded from GenBank and sequences were compared based on the alignment using a black yeast molecular database maintained at the CBS- KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands. If the similarity of sequences of the ITS region was more than 99% between a studied strain and its nearest neighbor the strain is concerned as belonging to the same species as its nearest neighbor.

In vitro susceptibility MICs (minimum inhibitory concentration) and MECs (minimum effective concentration, echinocandins only) were determined as described in the Clinical and laboratory Standards Institute (CLSI; formerly NCCLS) documents M38-A2. The eight antifungal agents amphotericin B (AMB, Bristol-Myers Squib, Woerden, The Netherlands), fluconazole (FLU, Pfizer Central Research Sandwich, U.K.), itraconazole (ITC, Janssen Research Foundation, Beerse, Belgium),

96 The clinical spectrum ofExophiala jeanselmei voriconazole (VOR, Pfizer), posaconazole (POS, Schering-Plough, Kenilworth, U.S.A.), isavuconazole (ISA, Basilea, Basel, Switzerland), caspofungin (CAS, Merck Sharp & Dohme, Haarlem, The Netherlands) and anidulafungin (ANI, Pfizer) were provided by the manufacturers as reagent-grade powders. As mentioned in the CLSI guidelines the stock solutions of the drugs were prepared in the appropriate solvent. The drugs were diluted in the standard RPMI-1640 medium (Sigma Chemical Co.) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma) with L-glutamine without bicarbonate to yield two times at their concentrations were as follows: Amphotericin B, itraconazole, voriconazole, posaconazole and caspofungin 0.016–16 μg/mL; fluconazole 0.063–64 μg/mL; isavuconazole 0.002–2 μg/mL and anidulafungin 0.008–8 μg/mL. Plates were stored at –70 °C until they were used. Briefly, Inoculum suspensions were prepared from 7 to 14 days cultures on Potato Dextrose Agar (PDA, Difco); by adding sterile saline solution with Tween 40 (0.05%) and slightly scarping the surface of mature colonies with sterile cotton swab. If large aggregates existed, they were allowed to settle for several minutes, then transferred the homogenous conidial suspensions to sterile tube and the supernatants were performed spectrophotometrically at 530 nm wavelength to optical density (OD) that ranged from 0.17 to 0.15 (68 to 71 T %). Therefore, the final size of the stock inoculum suspensions of the isolates tested ranged from 0.4×104–3.1×104 CFU/ml as performed by quantitative colony count on Sabouraud Glucose Agar (SGA, Difco) to determined the viable number of colony forming units per milliliter. The inoculum suspension including mostly non-germinated conidial were diluted 1:50 in RPMI 1640 medium. Microdilultion plates were incubated at 35 °C and examined optically and spectrophotometrically at 420 nm after 72 h (insufficient growths were incubated longer, until 96 h) for MICs and MECs determinations. Paecilomyces variotii (ATCC 22319), Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258) strains were used for quality control. The MICs endpoints for amphotericin B, itraconazole, voriconazole, posaconazole and isavuconazole were determined with the aid of a reading mirror as the lowest concentration of drug that prevent any recognizable growth (100 % 6 inhibition) and for fluconazole a prominent reduction of growth was ≥50 % inhibition compared to drug-free growth control. Regarding echinocandins (caspofungin and anidulafungin) MECs was defined microscopically as the lowest concentration of drug that leads to the growth of small, rounded, compact hyphal forms as compared with the long, unbranched hyphal clusters that were seen in the growth control.

Case report A 43-year-old Thai man was admitted in 2004 to the Institute of Tropical Medicine, Antwerp, Belgium, due to a complaint of a tumefaction that localized and developed on the left leg at the dorsum of the foot affecting the fourth and fifth toe (Fig. 1A-B). The patient did not remember any history of trauma or puncture at the site of the lesion. First surgical resection biopsy was performed in 2005 and initial clinical diagnosis was that of chromoblastomycosis. Histopathologically there was no evidence of presence of muriform cells. Subsequently the patient underwent surgical excision of the medial mass with skin graft closure and no antifungal drugs were prescribed. Two years later he was referred again to the Institute of Tropical Medicine, presenting with a relapse of the lesion which consisted of a deformed tumorous area, with nodules and multiple draining sinuses. Sinuses discharged small amounts of sanguineous fluid without visible grains. The patient limped due to discomfort at the site of infection. Radiography of the foot was normal with no visible bone destruction or osteomyelitis, and no significant regional lymphadenopathy was noted. Results of bacteriological (Syphilis) and parasitological (Leishmaniosis) evaluations, and serological test for human immunodeficiency virus (HIV) were all negative. Laboratory investigations including full

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Table 2. In vitro susceptibility of Exophiala jeanselmei and Exophiala oligosperma for eight antifungal drugs

expressed in μg/ml, demonstrating range MIC50. Source (No. strains) and drugs MIC range MIC50

Exophiala jeanselmei (9)

amphotericin B 0.25–2 1

fluconazole 8–32 16

itraconazole 0.031–0.25 0.125

voriconazole 0.25–2 1

posaconazole 0.016–0.063 0.031

isavuconazole 0.25–>2 2

caspofungin 2–8 4

anidulafungin 0.063–4 0.5

Exophiala oligosperma (5)

amphotericin B 1–2 1

fluconazole 16–32 32

itraconazole 0.031–0.25 0.063

voriconazole 1–4 1

posaconazole 0.016–0.125 0.031

isavuconazole 0.5–2 1

caspofungin 0.25–4 4

anidulafungin 0.016–2 0.5

Note. MIC90 not determined due to insufficient numbers of isolate tested. blood count, blood chemistry and renal tests were within normal limits, whereas liver functions were slightly disturbed: SGOT 46 (normal <59), SGPT 82 (normal <72), GGT 166 (normal <73). Therapy with oral itraconazole 400 mg/day for 6 months yielded good clinical response. The slight liver tests abnormalities were considered no contraindication for itraconazole treatment, but patient was requested to avoid alcoholic beverages. Additional local heat therapy (hot footbath, local heated dry pillow, heat bag and/or infrared lamp) was suggested, but it remained unclear how far this was implemented, since verbal communication was rather difficult. On January 2008, there was a clear improvement of the wound, but no complete healing. Liver tests had normalized since he stopped alcoholic drinking. Afterwards, the patient did not return for follow up.

Mycological identification The entire biopsied tissue was adjusted for mycological and histopathological investigation. Direct examination with KOH (10 %) revealed black granules and hyphal elements but were not obviously clear. Microsopic examination of biopsy sections stained with hematoxylin and eosin (H&E) revealed pigmented granules, some sickle-shaped, and surrounded by a dens inflammatory infiltrate consisting of numerous neutrophils and to the periphery chronic inflammation with fibrosis (Fig. 1C-D). The histopathological observations led to the diagnosis of a eumycotic mycetoma caused by an Exophiala species. Clinical specimens were cultured on Sabouraud glucose agar (SGA, Difco) and SGA supplemented with chloramphenicol (0.5 μg/ ml) and incubated at 27–30 °C for 7 days. Growth of dematiaceous fungi was observed and this were morphologically classified as Exophiala species. Stock cultures were maintained on slants of 2% MEA and OA at 24 °C (Gams et al. 2007), and a voucher strain was deposited in the CBS culture collection as CBS 122339. Microscopic studies using the slide culture techniques PDA or OA wher conducted.

98 The clinical spectrum ofExophiala jeanselmei

These media were selected becouse they readily induce sporulation and suppress growth of aerial hyphae (de Hoog et al. 2000). After 2 weeks slides were prepared frome these cultures in lactic acid or lactophenol cotton blue and light micrographs were taken using Nikon Eclipse 80i microscope with a Nikon digital sight DS-Fi1 camera. Colonies were moderately expanding and initially moist (yeast-like), forming velvety, olivaceous-green aerial hyphae; colony reverse was olivaceous-black (Fig. 2A). Numerous sub-spherical to ellipsoidal budding cells were present in young cultures, giving rise to short torulose hyphae that gradually change over into unswollen hyphae. Conidia were often cohering in long chains and changed over into hyphae. Conidiogenous cells were intercalary or lateral, then rocket-shaped, brown, with inconspicuous, slightly tapering annellated zones, producing smooth, narrow, thin-walled, broadly ellipsoidal conidia 2.6–5.9×1.2–2.5 µm (Fig. 2B-C). Cardinal growth temperatures of strain CBS 122339: growth between 9–37 °C, optimum at 27 °C, no growth was observed at 40 °C.

Results Seventeen strains from different geographical locations maintained as Exophiala jeanselmei in the CBS culture collection were sequenced using the ITS region (542 characters) and compared with alignable members of genus Exophiala. The ex-type strains ofE. jeanselmei (AY156963), E. spinifera (AY156976), E. exophialae (AY156973), E. dermatitis (FJ974060), E. oligosperma (AY163551), E. xenobiotica (DQ182587), E. bergeri (EF551462) and E. lecanii-corni (FJ974061) were used. The species E. jeanselmei was unambiguously distinguished in a robust branch with >98 % bootstrap support (Fig. 3). Nine out of 17 strains showed 98 % with the E. jeanselmei ex-type strain (CBS 507.90) whith had origanaly been isolated from a true mycetoma-like infection. Five strains were re-identified as E. oligosperma, and one stain each was confirmed as E. bergeri (CBS 102241), E.xenobiotica (CBS 527.76) and Capronia pilosella (CBS 663.76) (Table 1). Strains confirmed to be E. jeanselmei all originated from subcutaneous infections in human patients (current case, Naka et al. 1986, de Hoog et al. 2003, Langeron et al. 1928, Murray et al. 1963, Kawachi et al. 1995, 6 Yamada et al. 1989), whereas strains whith originated from environmental samples like soil, wood and plant or from clinical relevant eye or disseminated infections were considered to belong to E. oligosperma and E. xenobiotica (Bossler et al. 2003). Table 2 summarizes the results of in vitro antifungal susceptibility testing of eight antifungal drugs against E. jeanselmei (n=9) and E. oligosperma (n=5) employing the methods descriped in the CLSI guideline (M38-A2). The MIC90 was not determined due to insufficient numbers of isolates. Amphotericin B MICs for most non-dermatophyte opportunistic filamentous fungi isolates are clustered between 0.5–2 μg/ml. However, ther is very little data available regarding the correlation between MIC and outcome of treatment with amphotericin B for the filamentous fungi. Results have shown that amphotericin B MICs ranged from 0.25–2.0 and 1–2 μg/ml for E. jeanselmei and E. oligosperma, respectively. Itraconazole and posaconazole showed potent activity against all E. jeanselmei and E. oligosperma isolates, interestingly in terms of MIC50 (0.031 μg/ml) posaconazole was more active against E. jeanselmei than itraconazole (MIC50 0.125). Filamentous fungi are usually not susceptible to fluconazole and most MICs are >64 μg/ml for these isolates. In our study we found that fluconazole had the widest range and the highest MICs against E. jeanselmei and E. oligosperma ranging between 8–32 and 16–32 μg/ml, respectively. Voriconazole and isavuconazole had highest MICs with complete inhibition end points with MIC50 (1 μg/ ml and 2) against E. jeanselmei and MIC50 (both 1μg/ml) against E. oligosperma. Posaconazole demonstrated the lowest MIC50 (0.031 μg/ml) of all azoles. Concerning the echinocandin drugs caspofungin and anidulafungin, both exhibited low in vitro activity against E. jeanselmei and

E. oligosperma. Exophiala jeanselmei showed higher MEC50 (4 μg/ml) with caspofungin than to

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Fig. 1. (A–B) Clinical signs of mycetoma caused by Exophiala jeanselmei, deformed tumorous area of the foot, with nodules, draining sinuses with discharging and ulcers. (C–D) Section of the biopsy material stained with hematoxylin and eosin (H&E) exhibited pigmented granules; some were sickle-shaped, and surrounded by a dense inflammatory infiltrate consisting of numerous neutrophils (H&E stain ×400). anidulafungin (MEC50 0.5 μg/ml). Overall, in terms of MEC50 caspofungin had no activity against E. jeanselmei and E. oligosperma. There was no statistically significant difference when comparing the susceptibilities of E. jeanselmei and E. oligosperma isolates for all antifungal drugs (P< 0.05).

Discussion In our previous studies, phylogenetic analysis of nucSSU, nucLSU and RPB1 genes have shown that the ex-type strain of E. jeanselmei (CBS 507.96) is a member of the order Chaetothyriales, within the family Herpotrichiellaceae (Badali et al. 2008, Badali et al. 2009). Identification of this and related fungi with standard mycological procedures is difficult because of their high number of budding cells and poorly differentiated conidial structures. Nishimura et al. (1983) studied the conidiogenesis of E. dermatitidis, E. jeanselmei, and E. spinifera and reported that the conidial ontogenesis of these three species is annellidic and may sometimes be difficult to differentiate. Masuda et al. (1989) and Dixon et al. (1991) showed that the morphological variants of E. jeanselmei distinguished earlier by de Hoog (1977) are quite distinct on the molecular level and subsequently segregated into a number of individual species. Masuda et al. used DNA-DNA hybridization to classify E. jeanselmei into six groups. They suggest that DNA similarities between one group and another seem too low for these groups to comprise a single species (Masuda et al.1989). Kawasaki et al. (1999) classified strains of E. jeanselmei into 18 types based on mitochondrial restriction profiles and believed that the organism constitutes a complex. Wang et al. (2001) used the DNA and amino acid types of the mitochondrial cytochrome b. They proposed that on the

100 The clinical spectrum ofExophiala jeanselmei

Fig. 2. A. Culture on Malt extract agar (MEA, Difco) produced dark, moist, olivaceous-black yeast-like colony at room temperature. B–C. Conidia clustered at the apices of the tapered annellides and long, thick-walled, septate conidiophores. Scale bar = 10 μm bases of mtDNA RFLP typing and similarities of nucleotide and amino acid sequences, that some E. jeanselmei strains should be re-identified. Moreover, Haaseet al. (1999) suggested E. jeanselmei var. lecanii-cornii should be a distinct species, based on investigation of ribosomal DNA. In the present study, considerable sequence differences were found in nine out of 17 strains in the CBS culture collection. The isolate from the present case report exhibited ≥98 % ITS similarity to the ex-type strain of E. jeanselmei (CBS 507.90) and formed a single cluster of E. jeanselmei and consequently should be regarded as identical to this species. The intraspecific variability within two strains ITS1 region was 2 bp (in the nucleotide position of 82 and 107) and 4 bp in 6 ITS2 region (in the nucleotide position of 492, 519, 528 and 531), positions of ITS1 and 2 are counted after ATCATT. The ex-type strain originated from a case of mycetoma in a patient in France who was an imigrand from Martinique (Langeron 1928). Strain CBS 116.86 represents the etiologic agent of chromoblastomycosis-like infection (Naka et al. 1986) with muriform cells in tissue, however the clinical manifestation and the tissue form are different from that of the grains described by Langeron (1928). CBS 677.76 originated from a black-grain mycetoma in a patient from Pakistan, Murray (1963) and formed grains in vivo that were morphologically identical to those formed by the ex-type strain that also originated from mycetoma. Sequence data of CBS 677.76 proved it to be close to E. jeanselmei. CBS 148.97 caused phaeohyphomycotic cyst in a 59-year-old Japanese male with enlarging nodules on his finger and forearm associated with wooden splinters (Kawachi et al. 1995); it was identified as E. jeanselmei by morphological and molecular analysis. In contrast, the remaining strains (CBS 119095, CBS 109635, CBS 528.76 and CBS 664.76) from unpublished cases were maintained as originating from cutaneous infections (arm, hand or foot). Environmental strains formerly identified as E. jeanselmei were considered to be distinct from E. jeanselmei s. str. They were within 1 % differences with ex-type strains of E. oligosperma (CBS 725.88), E. xenobiotica (CBS 118157) and E. bergeri (CBS 353.52) (Fig. 3). Although E. oligosperma is phenotypically similar to E. jeanselmei and has mostly been confused with that taxon, genetically the species are clearly distinct. In our case report on CBS 122339, granules, the hallmark of mycetoma, were seen histopathologically in tissue (Fig. 1C- D). Parts of the grains were reniform, and clinical features of mycetoma, such as tumefaction, abscess formation, and fistulae were observed. In summary, nine strains confirmed asE. jeanselmei

101 Chapter 6

Fig. 3. Consensus tree of ITS rDNA obtained from a ML analysis using RAxML. Bootstrap support values were estimated based on 500 replicates, and are shown above the branches. Exophiala dermatitidis Clade was taken as outgroup.

102 The clinical spectrum ofExophiala jeanselmei originated from mycetoma or subcutaneous phaeohyphomycosis, as well as in one instance from chromoblastomycosis according to histopathological examination. Strains with identity (100 %) or near-identity (approximately 99 %) with the ex-type strain of E. jeanselmei appeared to be very consistent in clinical behavior. The case of chromoblastomycosis is remarkable. The switch between grain and muriform cell as a response to host factors needs to be studied.Over the past two decades, more serious black yeast infections have been recognized. Different antifungal agents have been used in the treatment of mycetoma, such as amphotericin B, fluorocytosine, itraconazole, fluconazole, and voriconazole (Ameen et al. 2008). In the past, amphotericin B was the most potent antifungal drug for severe invasive fungal infections, but its use is associated with severe side effects, particularly nephrotoxicity and low efficacy and its use has been replaced by newer azoles and echinocanains. Unfortunately, little data are available on the in vitro antifungal susceptibility of conventional and new antifungal agents against confirmed strains ofE. jeanselmei. In a comparative overview of in vitro activities of posaconazole, itraconazole, voriconazole, and amphotericin B against over 19,000 clinically yeasts and moulds, just fourteen Exophiala species were included, without molecular identification (Sabatelli et al. 2006). Data have shown that, posaconazole has good activity against agents that cause chromoblastomycosis, mycetoma, and phaeohyphomycosis, including Exophiala species and posaconazole was generally more active than itraconazole and amphotericin B against these organisms (Sabatelli et al. 2006, Zeng et al. 2007). Two recent studies reported in vitro antifungal data on amphotericin B, itraconazole, posaconazole and voriconazole against moleculary confirmed Exophiala species. In the first, 188 clinical strains were analyzed and most appeared to be susceptible to the four widely used antifungal agents, except Exophiala attenuata which was resistant to amphotericin B. MIC50 of all E. jeanselmei for amphotericin B, itraconazole, voriconazole and posaconazole were 0.5, 0.03, 0.125 and ≤ 0.015

μg/ml, respectively, whereas for E. oligosperma MIC50 were 0.25, 0.06, 0.25, and 0.03, respectively (Zeng et al. 2007). Fothergill et al. (2009) studied amphotericin B, itraconazole, posaconazole and voriconazole for 160 Exophiala strains including E. jeanselmei (n=8, originating from 6 subcutaneous and deep infections) and E. oligosperma (n=40, from cutaneous, subcutaneous and deep infection). Concerning E. jeanselmei MIC50 were determined for amphotericin B (0.5 μg/ ml), itraconazole (0.03 μg/ml), posaconazole (<0.015 μg/ml) and voriconazole (0.125 μg/ml) bud posaconazole was more active than others. Similar results were found in our series, posaconazole showing high activity against E. jeanselmei and E. oligosperma in terms MIC50. In vitro testing of isavuconazole and voriconazole exhibited broad-spectrum activity against the majority of the opportunistic and pathogenic fungi (Guinea et al. 2008). In the present study, low in vitro activities isavuconazole (MIC50 2 μg/ml) and caspogungin (MIC50 4 μg/ml) were found with E. jeanselmei suggesting that these drugs might not be the optimal choice for treatment. In contrast, posaconazole demonstrated potent in vitro activity (MIC50 0.031 μg/ml) against E. jeanselmei that caused mycetoma. Itraconazole has been used clinically in cases of mycetoma due to E. jeanselmei, with successful outcome (Al-Tawfiq et al. 2009). Voriconazole has only been employed in some cases of cutaneous infections (Loulergue et al. 2006). Isavuconazole until now has not been used clinically for Exophiala infections, but itraconazole was the only clinically active antifungal drug in the treatment of mycetoma due to E. jeanselmei. Infections by Exophiala species may require a combination of surgical and medical treatment. Although amphotericin B and itraconazole, with or without additional flucytosine, are currently regarded to be efficacious against cutaneous and subcutaneous lesions, the newer triazole agents, itraconazole and posaconazole, expand the therapeutic options for these mycoses. In conclusion, posaconazole and itraconazole demonstrated the highest in vitro antifungal activity against E. jeanselmei and E. oligosperma that had high MICs for caspofungin. Therefore posaconazole and itraconazole seem to be the most active drugs

103 Chapter 6 for treating Exophiala infections. We did not investigate the relation between MIC and clinical response to treatment their clinical effectiveness in the treatment of Exophiala infections remains to be determined.

Acknowledgments This study and the work of Hamid Badali were financially funded by a grant (No. 13081) from Ministry of Health and Medical Education of Islamic Republic of Iran and the School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. As well as a part of this study was supported by a grant from Basilea Pharmaceutica, Basel, Switzerland. We thank I. Curfs- Breuker for help in building up part of the test.

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Langeron M (1928). Mycétome à Torula jeanselmei Langeron, 1928. Nouveau type de mycétome à grains noirs. Ann Parasitol Hum Comp 6: 385-403. Loulergue P, Hot A, Dannaoui E, et al (2006). Successful treatment of black grain mycetoma with voriconazole. Am J Trop Med Hyge 75: 1106-1107. Masuda M, Naka W, Tajima S, et al (1989). Deoxyribonucleic acid hybridization studies of Exophiala dermatitidis and Exophiala jeanselmei. Microbiol Immunol 33: 631-639. McGinnis MR (1996). Mycetoma. Dermatol Clin 14: 97-104. Murray IG, Dunkerley GE, Hughes KEA (1963). A case of Madura foot caused by Phialophora jeanselmei. Sabouraudia 3:175-177 Naka W, Harada T, Nishikawa T. Fukushiro R (1986). A case of chromoblastomycosis with special reference to the mycology of the isolated Exophiala jeanselmei. Mykosen 29: 445-452 Nishimura K, Miyaji M (1983). Studies on the phylogenesis of pathogenic ‘black yeasts.’ Mycopathologia 81: 135-144. Nucci M, Akiti T, Barreiros G, et al (2001). Nosocomial fungemia due to Exophiala jeanselmei var. jeanselmei and a Rhinocladiella species: newly described causes of bloodstream infection. J Clin Microbiol 39: 514-518. Stamatakis A, Hoover P, Rougemont J (2008). A rapid bootstrap algorithm for the RAxML Web-Servers. Syste Biol 57: 758-771. Sabatelli F, Patel R, Mann PA, et al (2006). In vitro activities of posaconazole, fluconazole, itraconazole, voriconazole, and amphotericin B against a large collection of clinically important molds and yeasts. Antimicrob Agents Chemother 50: 2009-2015. Satow MM, Attili-Angelis D, De Hoog GS, et al (2008). Selective factors involved in oil flotation isolation of black yeasts from the environment. Stud Mycol 61: 157-163 Werlinger KD, Yen Moore A (2005). Eumycotic mycetoma caused by Cladophialophora bantiana in a patient with systemic lupus erythematous. J Am Acad Dermatology 52: 114–7. Wang L, Yokoyama K, Miyaji M, and Nishimura K (2001). Identification, classification, and phylogeny of the pathogenic species Exophiala jeanselmei and related species by mitochondrial cytochrome b gene analysis. J Clin Microbiol 39: 4462-4467. White TJ, Bruns T, Lee S, Taylor J (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DA, Sninsky JJ, White TJ (eds). PCR Protocols: A Guide to Methods and Applications. San Diego: Academic Press: 315-322. Yamada Y, sugihara K, Van Eijk GW, et al (1989). Coenzyme Q system in ascomycetous black yeasts. Antonie van Leeuwenhoek 56: 349-356. Zeng JS, Sutton DA, Fothergill AW, et al (2007). Spectrum of clinically relevant Exophiala species in the U.S.A. J Clin Microbiol 45: 3713-3720. 6

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Rhinocladiella aquaspersa, proven agent of verrucous skin infection and a novel type of chromoblastomycosis

H. Badali 1, 2, 3, A. Bonifaz 4, T. Barron-Tapia 5, D. Vazquez-Gonzalez 4, L. Estrada-Aguilar 5, N.M. Cavalcante Oliveiar 6, J.F. Sobralfilho7 , J. Guarro 8, J.F.G.M. Meis 9, G.S. de Hoog 1, 2, 10

1CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands, 2Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands, 3Department of Medical Mycology and Parasitology, School of Medicine /Molecular and Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran. 4Mycology Department, Dermatology Service. General Hospital of Mexico, 5Dermatology Service, Regional Hospital Lic. Adolfo López Mateos, Mexico, 6 Laboratório de Micologia, Departamento de Ciências Farmacêuticas, 7 Serviço de Dermatologia, Hospital Universitário Lauro Wanderley. Universidade Federal da Paraíba, Joao Pessoa, Brazil. 8Mycology Unit, Medical School, IISPV, Rovira i Virgili University, Reus, Spain,9Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands, 10Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China.

Published: Medical Mycology 2010 Jan 29, DOI:10.3109/13693780903471073 7107 Chapter 7

Abstract We report a case of chromoblastomycosis, which resembled sporotrichosis due to the presence of warty nodules and lymphatic distribution on the forearm in a 56-year old male. Mycological and histopathological investigation of exudates and biopsy tissue revealed a granulomatous lesion with muriform cells, the hallmark of chromoblastomycosis. The infection showed only localised expansion with verrucous plaques, thus presenting a new clinical type of the disease. The causative agent was identified as Rhinocladiella aquaspersa. This case prompted a study of the clinical spectrum of R. aquaspersa, towards which end we identified in a 62-year old Brazilian female a second case due to R. aquaspersa exhibiting hyphae rather than muriform cells in tissue. Given the difficulties treating chromoblastomycosis and other infections by melanized fungi, we evaluated the in vitro activities of extended-spectrum triazoles, amphotericin B, and echinocandins towards these clinical isolates of R. aquaspersa. Itraconazole (MIC, 0.063 μg/ml) and posaconazole (MIC, 0.125 μg/ml) had the highest in vitro activities towards this species, while voriconazole and isavuconazole had somewhat lower activities (MICs, 2 μg/ml). Amphotericin B and anidulafungin each had an MIC of 1 μg/ml, whereas the MIC of caspofungin was 8 μg/ml.

Key words: Chromoblastomycosis, Rhinocladiella aquaspersa, Rhinocladiella phaeophora, ITS rDNA, black yeasts.

Introduction Chromoblastomycosis is a chronic, progressive cutaneous and subcutaneous disorder histologically characterised by nodular and verrucous skin lesions. Tissue proliferation usually occurs around the area of inoculation, producing crusted, verrucose, wart-like lesions eventually leading to emerging, cauliflower-like forms (Queiroz-Telleset al. 2009). Infections occur primarily in immunocompetent individuals and are supposed to originate by traumatic implantation of fungal elements into the skin. The fungus multiplies in the tissue producing muriform cells, which represent the invasive form of fungi causing chromoblastomycosis (Mendoza et al. 1993). The mostcommon etiological agents in chromoblastomycosis are the melanized fungi namely Fonsecaea pedrosoi, F. monophora, Cladophialophora carrionii and Phialophora verrucosa, all members of the ascomycete order Chaetothyriales in the family Herpotrichiellaceae (Badali et al. 2008), and occasional reports of chromoblastomycosis involving other members of the order Chaetothyriales, such as Exophiala jeanselmei, E. spinifera, E. dermatitidis (Badali et al. 2009, Queiroz-Telles et al. 2003, Padhye et al. 1987) and Cladophialophora samoënsis (Badali et al. 2008) have appeared. A few species outside the order Chaetothyriales have been implicated in chromoblastomycosis-like infections, such as Chaetomium funicola (order Sordariales, family Chaetomiaceae) (Piepenbring et al. 2007), Catenulostroma chromoblastomycosum (order Capnodiales, family Teratosphaeriaceae (Crous et al. 2007), and an unidentified capnodialean fungus (Campolina et al. 2009). However, these mostly concerned infections with hyphae and chlamydospore-like cells in tissue which does not fit with the concept of chromoblastomycosis. Chromoblastomycosis is currently classified in five clinical typesQueiroz-Telles ( et al. 2009): nodular, plaque, tumoral, cicatricial and verrucous, based on a subdivision made by Carrión in 1950. Queiroz-Telles et al. (2009) graded lesions according to their expansion, viz. mild, moderate and severe, and noted that in the most severe cases, chronic lymphedema and ankylosis develop. However, some atypical cases of chromoblastomycosis show lymphatic dissemination, a trait usually associated with sporotrichosis (Ogawa et al. 2003, Takase et al. 1988). For example, Muhammed et al. (2006) reported a case of lymphangitic chromoblastomycosis due to F. pedrosoi in a 40-year-old male who presented with a non-healing ulcer on the right big toe and multiple

108 Chromoblastomycosis and phaeohyphomycosis due to R. aquaspersa nodules over the anterior aspect of the right leg and foot. Interestingly, Ogawa et al. (2003) used lymphoscintigraphy as an objective method to show the morphology of the lymph vessels and to assess lymphedema secondary to chromoblastomycosis. Lymphatic dissemination leads to a different appearance of chromoblastomycosis, as illustrated in the present case reports of a Mexican male and a Brazilian female infected by Rhinocladiella aquaspersa (Borelli 1972, Arango et al. 1998, Marques et al. 2004). The chaetothyrialean fungus R. aquaspersa is a rare cause of chromoblastomycosis, cases of which are confined largely to Latin America. The infection caused by species of the genus Rhinocladiella is clinically diverse: while R. aquaspersa gives rise to skin infections, Rhinocladiella mackenziei causes brain infections in otherwise healthy individuals and is associated with high mortality (Sutton et al. 1998, Campbell et al. 1993). The aforementioned cases of R. aquaspersa chromoblastomycosis prompted a study on the clinical spectrum of R. aquaspersa, based on strains verified by sequencing and with analysis of related species and led us to evaluate the in vitro activities of amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole, caspofungin, and anidulafungin against this fungal pathogen.

Case report 1 A 56 year-old male rural worker, professional plumber of drainage systems (residual water handling), native and resident of Alpuyeca, Morelos (120 km south of Mexico City), presented with a localized dermatosis affecting the dorsum of the left hand and the ipsilateral forearm. The lesions consisted of multiple, well-defined verrucous plaques with the same color as the skin or slightly greyish, and following the trajectory of lymphatic vessels of the arm, with mild pruritus (Fig. 1A-B). The dermatosis reportedly had begun fifteen years ago. The patient referred to several work-related traumata. Prior treatment included topical ketoconazole. The entire biopsied tissue was subjected to mycological and histopathological investigation. Direct examination of scales of the verrucous plaques in KOH (20%) demonstrated multiple dark brown muriform cells, approximately 6 µm in diameter. Section of the biopsied material stained with hematoxylin and eosin (H&E) revealed pseudoepitheliomatous hyperplasia and a granulomatous inflammatory infiltrate with lymphocytes, multinucleated giant cells (Langhan’s type) and muriform cells (Fig. 1D). Clinical specimens were cultured on Sabouraud’s glucose agar (SGA, Difco) and on SGA supplemented with chloramphenicol (0.5 μg/ml) at 27–30°C for two weeks. Growth of a melanized 7 fungus was observed repeatedly. The fungus was provisionally identified as a Rhinocladiella sp. by morphological criteria, and a voucher strain was deposited in the CBS-KNAW culture collection (accession number CBS 122635). The patient was treated successfully with oral terbinafine (500 mg/day), with good clinical response (significant reduction of the affected area) after six months of therapy, then put on maintenance therapy with oral itraconazole solution (200 mg/day) for three months. The patient also had bimonthly cryosurgery sessions during one year. Follow-up showed improvement of the dermatosis, with residual hypochromic areas and recovery of terminal hair in the previously verrucous plaques, though a 5-mm lesion remained in the distal third of the forearm (Fig. 1C).

Case report 2 A 62 year-old black female agricultural worker, from Solânea in the state of Paraiba in the North East of Brazil with a history of puncture with an unidentified cactus approximately 20 years earlier, consulted the hospital in 2001 because of the presence of multiple verrucose lesions measuring 2 to 8 cm in diameter, forming plaques on the left foot (Fig.2A). Mycological and histopathological

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Fig. 1. Case-1 (A–B) multiple and well-defined verrucous plaques due to Rhinocladiella aquaspersa concolorous with the skin or slightly greyish, and following the trajectory of lymphatic vessels of the arm. (C) Improvement of the dermatosis, residual hypochromic areas with recovering of the terminal hair in the previously verrucous plaques. (D) Histopathological examination showing muriform cells stained with hematoxylin & eosin (H&E ×400). investigations of the biopsied verrucous lesion by direct examination in KOH (20 %) and KOH supplemented with lactophenol revealed dark brown septate hyphae (Fig. 2B). A section of the biopsied material stained with H&E showed no muriform cells. Clinical specimens were cultured on SGA with and without chloramphenicol (0.5 μg/ml) and incubated at 27–30 °C for two weeks and growth of a melanized fungus was observed repeatedly. The fungus was provisionally identified as a Rhinocladiella sp. by morphological criteria, and a voucher strain was deposited in the culture collection of the Universitat Rovira i Virgili School of Medicine in Reus, Spain (accession number FMR 7699). The patient was treated with antiseptics, antibiotics, and corticoids without improvement, followed by electrocauterization and oral itraconazole (200 mg/day). The lesions improved progressively, and were successfully cured.

Materials and Methods

Mycology Stock cultures of both representative strains (Table.1) were maintained on slants of 2% malt extract agar (MEA, Difco) and oatmeal agar (OA, Difco) at 24°C (Gams et al. 2007). Microscopy was performed by the slide culture technique using potato dextrose agar (PDA, Difco) or OA which readily induce sporulation and suppress aerial hyphae growth (de Hoog et al. 2000).Each slide culture was prepared in culture plates containing sterile water into which a U-shaped glass rod

110 Chromoblastomycosis and phaeohyphomycosis due to R. aquaspersa

Fig. 2. Case-2 (A) verrucose lesions due to Rhinocladiella aquaspersa. (B) Direct examination of verrucous lesion in KOH (20%) supplemented with lactophenol demonstrating dark brown septate hyphae. was placed, extending above the water surface. A block of freshly-grown fungal colony (ca. 1cm2) was placed onto a sterile microscope slide, covered with a somewhat larger, sterile glass cover slip, and incubated in the moist chamber. After two weeks slides were stained with both lactic acid and lactophenol cotton blue and light micrographs recorded using a Nikon Eclipse 80i microscope fitted with a Nikon digital sight DS-Fi1 camera. For DNA extraction, mycelia were grown for 2 weeks at 24 °C on plates of 2 % MEA. DNA was extracted using an Ultra Clean Microbial DNA Isolation Kit (Mobio, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. Methods for PCR amplification and sequencing were those of Badali et al. (2008). DNA from both strains (strains from case-1 and case-2) including the ex-type strains of R. aquaspersa (CBS 313.73) and R. phaeophora (CBS 496.78) were subjected to DNA sequencing of internal transcribed spacer region (ITS rDNA), the partial β-tubulin gene (TUB) and the partial gene and introns of actin (ACT1). In addition, the nuclear ribosomal small subunit (nucSSU) gene of the ex-type strains also were sequenced (Table 1). Obtained sequences were compared with selected sequences once in GenBank (http://www.ncbi.nlm.nih. gov) and using a black yeast database maintained for research purposes at the CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands. 7 Antifungal susceptibility testing Minimal inhibitory concentrations (MICs) and minimum effective concentrations (MECs) for the three available clinical isolates of R. aquaspersa and environmental isolate of R. phaeophora towards eight antifungal agents were determined according to Clinical and Laboratory Standards Institute guidelines. Amphotericin B (Bristol-Myers-Squib, Woerden, The Netherlands), fluconazole (Pfizer Central Research, Sandwich, U.K.), itraconazole (Janssen Research Foundation, Beerse, Belgium), voriconazole (Pfizer), posaconazole (Schering-Plough, Kenilworth, U.S.A.), isavuconazole (Basilea Pharmaceutica International AG, Basel, Switzerland), caspofungin (Merck Sharp & Dohme, Haarlem, The Netherlands) and anidulafungin (Pfizer) were obtained as reagent-grade powders from their respective manufacturers. Antifungal agents were dissolved as prescribed by the CLSI; stock solutions of the experimental triazole isavuconazole were prepared in DMSO. Antimycotics were diluted in RPMI 1640 medium (GIBCO BRL, Life Technologies, Woerden, The Netherlands) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) (Sigma- Aldrich Chemie GmbH, Steinheim, Germany) and dispensed into 96-well microdilution trays at the following concentration ranges: amphotericin B, itraconazole, voriconazole, posaconazole,

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Fig. 3. (A-B) Colonies of Rhinocladiella aquaspersa on malt extract agar (MEA, Difco) and potato dextrose agar (PDA, Difco) moderately expanding, velvety, elevated, olivaceous-black. (C-E) Conidiophores straight, upright, unbranched, thick-walled, dark brown. (F) Conidia subhyaline, smooth- and thin-walled, one- or occasionally two-celled, ellipsoidal to clavate. Scale bar = 10 μm. caspofungin: 0.016-16 μg/ml; fluconazole: 0.063-64 μg/ml; isavuconazole, anidulafungin: 0.008-8 μg/ml. Microdilution trays were stored at -70 °C prior to use. Conidial suspensions in physiological saline containing Tween 40 (0.05 %) were adjusted spectrophotometrically (530 nm) to optical densities in the range between 0.15-0.17, then diluted 1:50 in buffered RPMI 1640 medium; final inoculum suspensions contained 0.5 × 104 - 4 × 104 CFU/ml, as verified by colony counts on SGA. Microtiter plates inoculated with R. aquaspersa were incubated at 35 °C, whereas microtiter plates inoculated with R. phaeophora were incubated at 30 °C; after 96 h the plates were examined visually and MICs and MECs determined. Candida parapsilosis (ATCC 22019) (21), Candida krusei (ATCC 6258), and Paecilomyces variotii (ATCC 22319) were used for quality control.

Results Colonies of R. aquaspersa were moderately expanding; upper surfaces were velvety, elevated, and olivaceous-black; reverses were dark olivaceous (Fig. 3A-B). Hyphae were pale olivaceous, smooth- or slightly rough-walled. Conidiophores were straight, upright, unbranched, thick-

112 Chromoblastomycosis and phaeohyphomycosis due to R. aquaspersa

Table. 1. Isolation data of examined strains.

Name Reference Gene Bank Source Origin number ITS, TUB, ACT, SSU

Rhinocladiella CBS 313.73 (T) GU017733, GU079660, GU079656, RAU20512 Chromoblastomycosis, male Mexico aquaspersa

Rhinocladiella CBS 122635 GU017732, GU079659, GU079658, GU079655 Chromoblastomycosis, male Mexico aquaspersa

Rhinocladiella FMR 7699 GU053606, -, -, - Phaeohyphomycosis, male Brazil aquaspersa

Rhinocladiella CBS 496.78 (T) EU041811, GU079661, GU079657, EF137366 maize-field soil Colombia phaeophora

Abbrevations used: CBS = CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; ITS rDNA = Internal Transcribed Spacer ribosomal DNA, TUB = the partial β-tubulin gene, ACT1 = the partial gene and introns of actin and nucSSU= the nuclear ribosomal small subunit. T = ex-type culture walled, dark brown. Conidiogenous cells were usually terminal, cylindrical, 9 −23 μm long, with crowded, slightly prominent denticles with dark scars with hyaline centre (Fig. 3 C-E). Conidia were subhyaline, smooth- and thin-walled, one- or occasionally two-celled, ellipsoidal to clavate, 4.5−7.5 × 1.8–2.4 μm (Fig. 2F). Presence of annellides are occasionally described for this fungus but were not noticed in our isolates. The teleomorph is unknown. Growth occurred between 9-37°C (optimum, 27°C), with no growth at 40 °C. The strains isolated from chromoblastomycosis (case-1, CBS 122635) and phaeohyphomycosis (case-2, FMR 7699) were identified as R. aquaspersa by 100 % identity by multilocus sequence typing to ex-type strain of R. aquaspersa (CBS 313.73) originated from human chromoblastomycosis, Mexico). The related species Rhinocladiella phaeophora (ex-type strain CBS 496.78) was unambiguously distinguishable from R. aquaspersa by all genes sequenced (97 %, 95 % and 97.8 % similarity in ITS, TUB and ACT1 genes, respectively) forming a robust branch with 100% bootstrap support. Morphologically R. phaeophora had sympodial, brown conidiophores which were arranged in a more profusely branched conidial apparatus than R. aquaspersa. MICs for the three clinical isolates of R. aquaspersa towards eight antifungal drugs surveyed were: amphotericin B, 1-2 μg/ml; fluconazole, 32-64 μg/ml, itraconazole and posaconazole, 0.063- 0.125 μg/ml, isavuconazole and voriconazole, 2 μg/ml; caspofungin, 8 μg/ml; anidulafungin, 1 μg/ml. For the environmental isolate of R. phaeophora examined, itraconazole (MIC, 0.5 μg/ml) 7 and posaconazole (MIC, 0.25 μg/ml) were the most active drugs, followed by amphotericin B, voriconazole, and isavuconazole (2 μg/ml). MICs for caspofungin and anidulafungin towards R. phaeophora were each 8 μg/ml, whereas fluconazole had an MIC >64μ g/ml.

Discussion Our previous study (Badali et al. 2008), based on multigene phylogeny analysis, showed that Rhinocladiella is a genus of black yeast-like fungi closely related to other members of the order Chaetothyriales with sympodial conidiogenesis (Arzanlou et al. 2007) consistently associated with opportunistic infections, mostly species belonging to the family Herpotrichiellaceae (e.g., Cladophialophora, Exophiala and Fonsecaea). The initial R. aquaspersa isolate (strain CBS 313.73) was obtained by Borelli as “Acrotheca aquaspersa Borelli” in 1972 from a chromoblastomycosis patient in Mexico; the species was later renamed “R. aquaspersa” by Schell et al. (1983) following isolation of the organism from a patient in Brazil. Padhye et al. (1987) re-described these two earlier cases and presented other reports of chromoblastomycosis from Colombia, Brazil, and Mexico ascribed to this fungus. Arango et al. (1998) reported a further case in a 60 year-old male with auricular localization and an infiltrative, dark-colored encrusted, scaly lesion. This patient

113 Chapter 7 suffered from the infection for 5 years, but was successfully treated with itraconazole. Marques et al, (2004) reported an unusual case of R. aquaspersa chromoblastomycosis in a 52-year-old male farm worker from Brazil, with extensive lesions in scaly plaques erupting in three different body sites over two years. Jae et al. (2004) published a further case of chromoblastomycosis due to this fungus in a 52-year-old female from Korea in which the lesion, an erythematous, verrucous abdominal plaque 2 cm × 1.3 cm, was cured with oral itraconazole solution (200 mg/ day administered over 4 months) together with surgical excision. All cases of chromoblastomycosis due to R. aquaspersa were diagnosed on the basis of clinical and pathological data and confirmed by conventional examination of lesion biopsies showing numerous muriform cells. Misidentification of R. aquaspersa is likely using conventional morphological methods, especially as the literature is replete with incorrectly-named organisms. It may be noted that the species has dark conidial denticles with hyaline scars, resembling the poroconidia of dematiaceous fungi (de Hoog et al. 2000). Over the past two decades chromoblastomycosis and phaeohyphomycosis have been attributed principally to C. carrionii, F. pedrosoi, F. monophora and P. verrucosa (Queiroz-Telles et al 2009, Mendoza et al. 1993, Badali et al. 2008); our data confirm that R. aquaspersa also can cause chromoblastomycosis. Our case report from Mexico clinically resembled sporotrichosis. The dry encrusted verrucous violaceous lesions and chronic lymphedema were characteristic of chromoblastomycosis, but the infection usually shows only localized expansion with verrucous plaques (Queiroz-Telles et al. 2009, de Hoog et al. 2000), and thus our cases represent a new clinical presentation of the disease that also may occur in patients infected with F. pedrosoi (Ogawa et al. 2003, Takase et al. 1988, Muhammed et al. 2006), and lymphatic distribution in chromoblastomycosis may appear after cryosurgical treatment (R. Isa, Dominican Republic, personal communication). d’Ávila et al. (2002) characterised cell-mediated tissue reactions in chromoblastomycotic skin lesions and showed that distinct immunohistopathological alterations correlated with clinical aspects of the lesions: patients with verrucous plaques had a type Th2 immunological response, whereas patients with erythematous atrophic plaques had a type Th1 response. Corbellini et al. (2006) studied delayed-type hypersensitivity responses to crude and fractionated antigens from F. pedrosoi cultivated in different media, and suggested that a delayed- type skin test using antigens produced in synthetic media may be useful for assessing primary exposure to chromoblastomycosis. Iwatsu et al. (1982) observed cutaneous delayed hypersensitivity in animal experiments, and Ahrens et al. (1989) reported enlargement and metastasis of lesions infected by F. pedrosoi in immunosuppressed but not in immunocompetent mice. Esterre et al. (2006) suggested that, relative to cell-mediated immunity, humoral immunity does not seem to offer much protection against chromoblastomycosis. Cardona-Castroet al. (1999) detected chronic infection after healthy mice were inoculated intraperitoneally with F. pedrosoi. An unambiguous connection between host defense mechanisms and clinical aspects of chromoblastomycosis remains to be proven. Most countries in which cases of R. aquaspersa have been reported have subtropical climates with alternating well-defined dry and humid seasons. The related fungus R. phaeophora originally was obtained from maize field soil from Colombia, but R. aquaspersa thus far has been found only in humans. For infections caused by black yeast-like fungi, the mammalian host seems to select a small number of opportunistic species. De Hoog et al. (2007) found that a C. carrionii- positive patient was repeatedly pricked by cacti which carried Cladophialophora yegresii rather than C. carrionii on their spines; possibly the patient became infected by C. carrionii present at sub- detectable levels on the cactus spines, or repeated exposure to Cladophialophora yegresii sensitized the patient to subsequent exposure through a different venue to C. carrionii. Harutyunyan et al.

114 Chromoblastomycosis and phaeohyphomycosis due to R. aquaspersa

2008 reported the presence of distantly related Rhinocladiella species in lichens from arid habitats devoid of pathogenity towards mammals. Treatment of chromoblastomycosis is difficult, since prolonged therapy is needed and relapses are frequent. Itraconazole and terbinafine are the most frequently applied antifungals (Queiroz-Telles et al. 2009, Bonifaz et al. 2004, Bonifaz et al. 2005), but no standard therapy is has been identified, and there are limited data on in vitro antifungal activity against R. aquaspersa. MICs for amphotericin B towards most non-dermatophyte opportunistic filamentous fungi cluster in the 0.5-2 μg/ml range, but correlations between MIC and outcome of treatments with amphotericin B for filamentous fungi are poorly documented. Vanden Bossche et al. (1998) reported that for black fungi amphotericin B MIC values were mostly >4 μg/ml. In contrast, Marques et al. (2004), using a broth macrodilution method, obtained amphotericin B MICs of 0.8 μg/ml for R. aquaspersa. Our results corroborate previous studies (Gonzalez et al. 2005) that itraconazole (MICs, 0.063 μg/ml) and posaconazole (MICs, 0.125 μg/ml) have potent activity against R. aquaspersa, whereas voriconazole and isavuconazole have lower activities. Cuenca-Estrella et al. (2006) likewise noted that posaconazole was a superior drug against Rhinocladiella strains while caspofungin had poor activity; however, we also found that R. aquaspersa was more sensitive to anidulafungin (MICs, 1 μg/ml) than was R. phaeophora (MICs, 8 μg/ml). Odabasi et al. (2004) showed that anidulafungin is a potent drug against P. verrucosa, and Badali et al. (2009) demonstrated that, although anidulafungin had potent in vitro activity against Alternaria clinical isolates (Alternaria infectoria, A. alternata), it had poor activity against Alternaria environmental isolates (A. malorum). This is concordant with the finding presented here, that anidulafungin has higher in vitro activity against clinical isolates of Rhinocladiella (R. aquaspersa) than against environmental isolates of this genus (R. phaeophora). We conclude that itraconazole and posaconazole may become therapeutic options and alternatives to amphotericin B for treating infections due to the rare etiologic agent R. aquaspersa, although in vitro studies using considerably larger numbers of strains, as well as animal models of infection and relevant clinical trials will be required these before these azoles receive an official imprimatur from the medical community for treatment of R. aquaspersa chromoblastomycosis.

Acknowledgments This work was financially supported by a grant from the Ministry of Health and Medical Education of Islamic Republic of Iran (No. 13081) and the School of Medicine, Mazandaran University of 7 Medical Sciences, Sari, Iran, which we gratefully acknowledge. The authors would like to thank Walter Gams and Dr. Mark Jones (Basilea Pharmaceutica International AG, Basel, Switzerland) for critically reviewing the manuscript.

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Corbellini VA, Scroferneker ML, Carissimi M, Santolin LD (2006). Delayed-type hypersensitivity response to crude and fractionated antigens from Fonsecaea pedrosoi CMMI 1 grown in different culture media.Mycopathologia 162: 51-55. Crous PW, Braun U, Groenewald JZ (2007). Mycosphaerella is polyphyletic. Stud Mycol 58: 1-32. Cuenca-Estrella M, Gomez-Lopez A, Mellado E, et al (2006). Head-to-head comparison of the activities of currently available antifungal agents against 3378 Spanish clinical isolates of yeasts and filamentous fungi. Antimicrob Agents Chemother 50: 917-921. De Hoog GS, Guarro J, Gené J, Figueras MJ (2000). Atlas of Clinical Fungi, 2nd ed. Utrecht, Reus: CBS-KNAW Fungal Biodiversity Centre, Universitat Rovira i Virgili. De Hoog GS, Nishikaku AS, Fernández Zeppenfeldt G, et al (2007). Molecular analysis and pathogenicity of the Cladophialophora carrionii complex, with the description of a novel species. Stud Mycol 58: 219-234. Esterre P, Queiroz-Telles F (2006). Managements of chromoblastomycosis: novel perspectives. Curr Opin Infect Dis 19: 148-152. Gams W, Verkley GJM, Crous PW (2007). CBS course of mycology, 5th ed. CBS-KNAW Fungal Biodiversity Centre, Utrecht. Gonzalez GM, Fothergill AW, Sutton DA, et al (2005). In vitro activities of new and established triazoles against opportunistic filamentous and dimorphic fungi.Med Mycol 43: 281-284. Harutyunyan S, Muggia L Grube M (2008). Black fungi in lichens from seasonally arid habitats. Stud Mycol 61: 83-90. Iwatsu T, Miyaji M, Taguchi H, Okamoto S (1982). Evaluation of skin test for chromoblastomycosis using antigens prepared from culture filtrates of Fonsecaea pedrosoi, Phialophora verrucosa, Wangiella dermatitidis and Exophiala jeanselmei. Mycopathologia 77: 59-64. Jae BJ, Park YJ, Kim DW, Suh SB (2004). Chromoblastomycosis caused by Rhinocladiella aquaspersa. Kor J Med Mycol 9: 117-122. Marques S G, Pedrozo Silva CM, Resende MA, et al 2004. Chromoblastomycosis caused by Rhinocladiella aquaspersa. Med Mycol; 42: 261-265. Mendoza L, Karuppayil SM, Szaniszlo PJ (1993). Calcium regulates in vitro dimorphism in chromoblastomycotic fungi. Mycoses 36:157-164. Muhammed K, Nandakumar G, Asokan KK, Vimi P (2006). Lymphangitic chromoblastomycosis. Indian J Dermatol Venereol Leprol 72: 443-445. Odabasi Z, Paetznick VL, RodriguezJR , et al (2004). In vitro activity of anidulafungin against selected clinically important mold isolates, Antimicrob Agents Chemother 48: 1912-1915. Ogawa MM, Alchorne MM, Barbieri A, et al (2003). Lymphoscintigraphic analysis in chromoblastomycosis. Int J Dermatol 42: 622-625. Padhye AA, Ajello L (1987). A case of chromoblastomycosis with special reference to the mycology of the isolated Exophiala jeanselmei. Mykosen 30: 134. Piepenbring M, Cáceres Mendez OA, Espino Espinoza A, et al (2007). Chromoblastomycosis caused by Chaetomium funicola: a case report from Western Panama. Brit J Derm 157: 1025-1029. Pires d’Avila SCG, Pagliari C, Duarte MIS (2002). The cell-mediated immune reaction in the cutaneous lesion of chromoblastomycosis and their correlation with different clinical forms of the disease.Mycopathologia 156: 51-60. Queiroz-Telles F, Esterre P, Perez-Blanco M, et al (2009). Chromoblastomycosis: an overview of clinical manifestations, diagnosis and treatment. Med Mycol 47: 3-15 Queiroz-Telles F, McGinnis MR, Salkin I, et al (2003). Subcutaneous mycoses. Infect Dis Clin North Am 17: 59-85. Sutton DA, Slifkin M, Yakulis R, et al (1998). Case report of cerebral phaeohyphomycosis caused by Ramichloridium obovoideum (R. mackenziei): criteria for identification, therapy, and review of other known dematiaceous neurotropic taxa. J Clin Microbiol 36: 708-715. Schell WA, McGinnis MR, Borelli D (1983). Rhinocladiella aquaspersa a new combination for Acrotheca aquaspersa. Mycotaxon 17: 341-348. Takase T, Baba T, Uyeno K. Chromomycosis (1988). A case with a widespread rash, lymph node metastasis and multiple subcutaneous nodules. Mycoses 31: 343-352. Vanden Bossche H, Dromer F, Improvisi I, et al (1998). Antifungal drug resistance in pathogenic fungi. Med Mycol 38: 116119-128. 8 Chapter 8

First autochthonous case of Rhinocladiella mackenziei cerebral abscess outside the Middle East

H. Badali 1, 2, 3, J. Chander 4, S. Bansal 5, A. Aher 6, S. S. Borkar 7, J.F.G.M. Meis 8, G.S. de Hoog 1, 2, 9

1CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands, 2Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands, 3Department of Medical Mycology and Parasitology, School of Medicine/Molecular and Cell Biology Research Centre, Mazandaran University of Medical Sciences, Sari, Iran, 4Department of Microbiology, Government Medical College Hospital, Chandigarh, India,5 Department of Medicine, 6Microbiology and 7Pathology, People’s College of Medical Sciences & Research Centre, Bhopal, Madhya Pradesh, 8Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands, 9Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China.

Published: Journal of Clinical Microbiology 2010; 48(2): 646-9. 8117 Chapter 8

Abstract Cerebral phaeohyphomycosis due to Rhinocladiella mackenziei is a severe infection in the Middle East, with nearly 100 % mortality despite the application of combined surgical and antifungal therapy and occasionally occurring in otherwise healthy patients. We report the first case of brain infection in an elderly male in India, where R. mackenziei is not endemic.

Key words: Cerebral phaeohyphomycosis, R. mackenziei, ITS rDNA.

Case report A 50-year-old Indian male who had type two diabetes mellitus for the last four years and suffered from psychiatric illness was admitted to the Department of Neurology, Peoples College of Medical Sciences, Bhopal, India, and presented with a one-day history of frontal headache, dizziness, slurring speech and weakness over the left half of his body. Ten days previously he had become inattentive due to his uncontrolled diet and irregular anti-diabetic treatment. First computed tomography (CT) scan of the brain demonstrated a mass lesion and patient underwent indigenous (Ayurvedic) treatment for 5-6 days. The chest X-ray was normal. Laboratory investigations of full blood analysis revealed a hemoglobin level of 16.8 g/dl; white blood cells, serum electrolytes and liver function tests were within normal range. Blood cultures were sterile and urine cultures became positive with Escherichia coli. Later, a second CT scan revealed a large (around 5-6 cm in diam), discrete, irregular, peripheral ring-enhancing necrotic lesion in the right frontoparietal region causing significant mass effects and midline shift with perifocal oedema (Fig. 1A-B). Neurosurgical intervention consisted of a right frontoparietal craniotomy for decompression of the space-occupying lesion. The necrotic material was encapsulated and approximately 8 ml thick viscous black fluid, was aspirated which was predominately caseous. The material was analyzed by two mycological and histopathological laboratories involved. The lesions were multiple ring- enhancing, greyish-white, soft, measuring 1.5 × 0.4 cm. Microscopic examination of biopsy sections (pus or necrotic tissue) stained with hematoxylin and eosin revealed necrosis with dense and diffuse mixed inflammatory infiltrates. There were several foreign bodies and Langhans’ type of giant cells with granuloma formation and the presence of numerous septate hyphae surrounded by a dense inflammatory response (Fig. 2A-B). Following this the biopsy specimens were also stained with Ziehl- Neelsen and revealed many moniliform septated hyphal elements (Fig 2C). The tentative diagnosis of chronic granulomatous inflammation with fungal infection was made. The clinical specimens were cultured on Sabouraud dextrose agar (SDA, Difco) and SDA supplemented with chloramphenicol (0.5 mg/ ml) and incubated at 30 –35 °C for up to 10 days, as well as on brain-heart infusion agar with 5 % sheep blood at 37 °C (Oxoid LTD., Basingstoke, Hampshire, England). Growth of melanized fungi after one week was observed and these were morphologically classified asRhinocladiella mackenziei (formerly, Ramichloridium mackenziei). Stock cultures were maintained on slants of 2 % Malt extract agar (MEA, Difco) and Oat meal agar (OA, Difco) at 30 °C, and a voucher strain was deposited into the CBS-KNAW culture collection and preserved as CBS 125089. Microscopic studies using slide culture techniques with potato dextrose agar (PDA) were conducted. This medium was selected because it readily induces sporulation and suppresses growth of aerial hyphae (de Hoog et al. 2000). After two weeks, slides were prepared from these cultures in lactic acid or lactophenol cotton blue under BSL-3 safety regulations and light micrographs were taken using Nikon Eclipse 80i microscope with a Nikon digital sight DS-Fi1 camera. After 2 weeks growth at 27 °C on MEA in darkness the moderately rapidly growing colonies, were velvety and olivaceous-

118 Rhinocladiella mackenziei cerebral abscess outside the Middle East brown; colony reverse was olivaceous-black (Fig. 3A). Conidiophores arose at right angles from creeping hyphae, were stout, thick-walled, brown, 3.0–4.5 μm wide, cells 10–25 μm long, apically with short cylindrical denticles and brown conidia which were ellipsoidal, 8.5–12.0 × 4–5 μm, with prominent, 1 μm wide basal scars (Fig. 3B-C). Cardinal growth temperatures of strain CBS 125089 were between 9 and >40 °C, with an optimum at 30 °C and still some growth at 40 °C. Morphologically R. mackenziei resembles Pleurothecium obovoideum (Mats.) Arzanlou & Crous from dead wood, but P. obovoideum has distinct conidiophores, and the ascending hyphae are thick-walled with cylindrical denticles up to 1.5 μm long. In contrast, R. mackenziei has only slightly prominent denticles. Pleurothecium obovoideum clusters in the order Chaetosphaeriales (Arzanlou et al. 2007). DNA was extracted using an Ultra Clean Microbial DNA Isolation Kit (Mobio, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. ITS rDNA was amplified using primers V9G (5’-TTA CGT CCC TGC CCT TTG TA-3’) and LS266 (5’-GCAT TCC CAAACA ACT CGA CTC-3’) and sequenced with the internal primers ITS1 (5’-TCC GTA GGT GAA CCT GCG G-3’) and ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’). PCR amplification and sequencing were according to Badali et al. (2009). Sequences were compared with GenBank and through local blast with a molecular database maintained for research purposes at the CBS- KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands. The isolate (CBS 125089 = GQ863214) was identified as R. mackenziei having 99.5 % sequence identity with the ex-type isolate of that species (CBS 650.93 = AY857540) which had originally been isolated from a case of cerebral phaeohyphomycosis from Saudi Arabia. The molecular results confirmed the mycological diagnosis, and histopathological observation led to the diagnosis of a cerebral phaeohyphomycosis due to Rhinocladiella mackenziei. For the management of the case insulin infusion was given along with intravenous amphotericin B deoxycholate (0.6 mg/kg/day). Simultaneously empiric anti-tuberculous and antibacterial therapy was started, consisting of sequential betalactam antibiotics (ceftriaxone, piperacillin, cefoperazone) combined with metronidazole and amikacin. During follow-up there was no clinical improvement. Therefore, successive CT scans after 8 days were obtained, showing decreases in size of the lesions (around 4 cm); however, an increase in parietal edema, enlargement of mass effects and multiple, coalescing ring-enhancing, cerebral lesions were seen (Fig 1C). Despite amphotericin B therapy the patient’s condition continued to deteriorate and eventually he expired two weeks after diagnosis of the disease. Thein vitro antifungal susceptibilities of this strain (CBS 125089) were determined by microbroth dilution according to the Clinical and Laboratory Standard Institute document M38-A2. Methods for sporulation and preparation of suspensions were according to Badali et al. 2009. Paecilomyces variotii (ATCC 22319), Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258) were used as quality control (13). The MICs of eight antifungal drugs were as 8 follows: amphotericin B, 16 μg/ml; fluconazole 32μ g/ml, itraconazole 0.063 μg/ml, voriconazole 0.5 μg/ml, posaconazole 0.031 μg/ml and isavuconazole 0.5 μg/ml. The two echinocandin agents yielded MECs of 8 μg/ml and 4 μg/ml for caspofungin and anidulafungin, respectively.

Discussion Cerebral phaeohyphomycosis by melanized fungi is a rare, but highly significant disease due to the regional prevalence and high mortality of up to 70 % despite combined surgical and antifungal therapy (Horre et al. 1999). The majority of reported central nervous system infections caused by dematiaceous fungi were found to be brain abscesses in patients with no predisposing factors or immunodeficiency; symptoms included headache, seizures cerebral irritation, fever and neurological deficits (Li et al. 2009). Binford et al. (1952) described one of the first cases of brain

119 Chapter 8

FIG. 1. A–B. Initial computed tomography (CT) showing a mass lesion, discrete, large (around 5-6 cm in diam), irregular, peripheral ring-enhancing necrotic lesion in the right frontoparietal region with mass effects and midline shift with perifocal oedema. C. Third CT showing decreases in size of lesions (around 4 cm), however increasing of parietal edema, enlargement in mass effects and multiple, coalescing ring-enhancing, cerebral lesions were seen.

FIG. 2. A–B. Hematoxylin-eosin stain (H&E) revealed necrosis with dense and diffuse mixed inflammatory infiltrates with granuloma formation and the presence of numerous septate hyphae (arrows) surrounded by a dense inflammatory response. C. Biopsy specimens were also stained with Ziehl- Neelsen and revealed many moniliform branched septated hyphal elements.

FIG. 3. Rhinocladiella mackenziei (CBS 125089). A. Colony on malt extract agar (MEA, Difco) at 30 °C after 2 weeks in darkness. B–C. Semi-micronematous conidiophores and sympodially proliferating conidiogenous cells (arrows). Scale bar =10 μm

120 Rhinocladiella mackenziei cerebral abscess outside the Middle East abscess due to Cladosporium trichoides (= Cladophialophora bantiana) and Campbell & Al-Hedaithy (1993) reported on a similar case by Ramichloridium mackenziei (= Rhinocladiella mackenziei). The latter review listed 8 cases of this infection, all occurring in countries of the Middle East. Exophiala dermatitidis (Chang et al. 2000), Cladophialophora bantiana (Levin et al. 2004) and Rhinocladiella mackenziei (Campbell et al. 1993, Sutton et al. 1998), all members of the order Chaetothyriales, are notorious agents of cerebral infections. One of the most striking features of these organisms is their neurotropism in humans. Arzanlou et al. (2007) studied the phylogenetic and morphotaxonomic revision of Ramichloridium and allied genera, because Rhinocladiella was in the past frequently confused with the genus Ramichloridium. Rhinocladiella species were shown to cluster in the Chaetothyriales, while Ramichloridium clustered in the order of Capnodiales. Rhinocladiella mackenziei affects only the central nervous system and the integuments, with nearly 100 % mortality in both immunocompetent and immunocompromised individuals. This species is the most common agent of brain infection and was thought to be restricted to the Middle East and the Persian Gulf region (Campbell et al. 1993). Cases diagnosed in the U.K. or U.S.A. concerned immigrants from Saudi Arabia and Kuwait (Sutton et al. 1998). Until now R. mackenziei has never been isolated from the environment. The natural niche of this organism thus remains unknown. To the best of our knowledge this patient represents the first case of R. mackenziei outside the arid climate zone of the Middle East. In contrast to all cases reported thus far, this strain (CBS 125089) originated from a humid sub-tropical climate, infecting a patient who claimed he had never travelled outside of India. Cerebral infections due to R. mackenziei have thus far been diagnosed after CT-guided needle aspiration and were proven by positive histopathology and cultures. The mortality is almost 100% for all reported cases of infection despite surgical resection and antifungal therapy. There is no standard therapy for this disorder. Treatment as presented in the literature mostly involved (high-dose lipid) amphotericin B, itraconazole and 5 flucytosine, or a combination of these drugs (Revankar et al. 2004). However strains of R. mackenziei isolated from infected patients are in vitro resistant to amphotericin B, which is often used as the gold standard of treatment. Only a single patient is reported to have survived an R. mackenziei cerebral infection, with pronounced radiological and clinical improvement after switching therapy from a combination of liposomal amphotericin B, 5-flucytosine, and itraconazole to posaconazole (Al- Abdely et al. 2005). This is supported by in vitro results and data from a murine infection model (Abdely et al. 2000), in which posaconazole prolonged the survival of mice and reduced the brain fungal burden compared to itraconazole and amphotericin B. Previous in vitro antifungal susceptibility testing of 10 strains of R. mackenziei (Badali et al. 2009) has shown that the widest range (2 to 16 μg/ml) and the highest MICs were recorded 8 for amphotericin B (MIC50 8 μg/ml and MIC90 16 μg/ml). In contrast, quite uniform patterns were obtained for susceptibility to itraconazole, posaconazole, and isavuconazole. Our results were in line with animal test data, but clinical experience and animal experiments have not been reported for the newer antifungal drugs. Animal studies have suggested that no benefit is achieved with amphotericin B in experimental infection due to poor penetration into the central nervous system (CNS) (Abdely et al. 2000). Although liposomal amphotericin B likely has a better CNS penetration the only patient surviving a R. mackenziei cerebral infection (Abdely et al. 2005) failed liposomal amphotericin and only improved on posaconazole an agent with good CNS penetration (Ruping et al. 2008) and a low MIC for the pathogen (Abdely et al. 2005) Delay in diagnosis, misidentification, poor standard therapy and limited data on effective alternative drugs are the main factors promoting the development of cerebral phaeohyphomycosis. With early diagnosis,

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when the lesion is singular, and with effective therapy with complete surgical excision of brain abscesses, patient’s outcome may be improved. Brain abscesses incited by R. mackenziei in patients from non-endemic areas have not been previously reported. Now that the fungus has been observed outside its endemic area, R. mackenziei, along with other neurotropic agents, should always be considered as a potential etiologic agent of cerebral phaeohyphomycosis in all patients, regardless of their area of residence.

Acknowledgments This work was supported by a grant (No. 13081) to H. Badali from the Ministry of Health and Medical Education of the Islamic Republic of Iran and the School of Medicine, Mazandaran University of Medical Sciences, Sari-Iran. The authors report no conflicts of interest. We thank Dr. Vichal Rastogi and Dr. V. K. Ramnani, Department of Microbiology, Dr. Sushil Jindal, Department of Medicine, Dr. Mridul Shai, Department of Surgery, People’s College of Medical Sciences & Research Centre, Bhopal, Madhya Pradesh, India for their contribution. As well as, Dr. Hena Rani, Department of Microbiology, Government Medical College Hospital, Chandigarh, India is acknowledged for help in building up part of the microbiological work.

References Al-Abdel HM, Najvar L, Bocanegra R, et al (2000). SCH 56592, amphotericin B, or itraconazole therapy of experimental murine cerebral phaeohyphomycosis due to Ramichloridium obovoideum (“Ramichloridium mackenziei”). Antimicrob Agents Chemother 44: 1159-1162. Al-Abdely HM, Alkhunaizi AM, Al-Tawfiq JA, et al (2005). Successful therapy of cerebral phaeohyphomycosis due to Ramichloridium mackenziei with the new triazole posaconazole. Med Mycol 43: 91-95. Arzanlou M, Groenewald JZ, Gams W, et al (2007). Phylogenetic and morphotaxonomic revision of Ramichloridium and allied genera. Stud Mycol 58: 57-93. Badali H, Carvalho VO, Vicente V, et al (2009). Cladophialophora saturnica sp. nov., a new opportunistic species of Chaetothyriales revealed using molecular data. Med Mycol 47: 51-62. Badali H, De Hoog GS, Curfs-Breuker I, Meis JF (2009). In vitro activities of antifungal drugs against Rhinocladiella mackenziei, an agent of fatal brain infection. J Antimicrob Chemother 65: 175-7 Binford CH, Thompson RK, and Gorhan ME (1952). Mycotic brain abscess due toCladosporium trichoides, a new species; report of a case. Am J Clin Pathol 22: 535-542. Campbell CL, Al-Hedaithy SS (1993). Phaeohyphomycosis of the brain caused by Ramichloridium mackenziei sp. nov. in Middle Eastern countries. J Med Vet Mycol 31: 325-332. Chang CL, Kim DS, Park DJ, et al. (2000). Acute cerebral phaeohyphomycosis due to Wangiella dermatitidis accompanied by cerebrospinal fluid eosinophilia. J Clin Microbiol 38: 1965-1966. De Hoog GS, Guarro J, Gené J, Figueras MJ (2000). Atlas of clinical fungi, 2nd ed. CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands. Clinical and Laboratory Standards Institute (2008). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved Standard; 2nd Ed; Document M38-A2. Clinical and Laboratory Standards Institute, Wayne, PA. Horré R, and De Hoog GS (1999). Primary cerebral infections by melanized fungi: a review. Stud Mycol 43: 176-193. Levin TP, Baty DE, Fekete T, et al (2004). Cladophialophora bantiana brain abscess in a solid-organ transplant recipient: case report and review of the literature. J Clin Microbiol 42: 4374-4378. Li DM, and De Hoog GS (2009). Cerebral phaeohyphomycosis – a cure at what lengths? Lancet Infect Dis 9: 376-383. Revankar SG, Sutton DA, Rinaldi MG (2004). Primary central nervous system phaeohyphomycosis: A review of 101 cases. Clin Infect Dis 38: 206-216. Ruping MJGT, Albermannn N, Ebinger F, et al (2008). Posaconazole concentrations in the central nervous system. J Antimicrob Chemother 62: 1468-1470. Sutton DA, Slifkin M, Yakulis R, Rinaldi MG (1998). U.S. case report of cerebral phaeohyphomycosis caused by Ramichloridium obovoideum (R. mackenziei): criteria for identification, therapy, and review of other known dematiaceous neurotropic taxa. J Clin Microbiol 36: 708-715. 122 9 Chapter 9

In vitro activities of antifungal drugs against Rhinocladiella mackenziei, an agent of fatal brain infection

H. Badali 1, 2, 3, G.S. de Hoog 1, 2, I. Curfs-Breuker 4 and J.F.G.M. Meis 4

1CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; 2Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands, 3Department of Medical Mycology and Parasitology, School of Medicine/Molecular and Cell Biology Research Centre, Mazandaran University of Medical Sciences, Sari, Iran, 4Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands.

Published: Journal of Antimacrobial Chemotherapy 2010; 65: 175-177 9123 Chapter 9

Cerebral phaeohyphomycosis is a rare disease with a mortality of up to 70 % despite combinations of surgical and antifungal therapy (Li et al. 2009). This infection is predominantly caused by Exophiala dermatitidis, Cladophialophora bantiana, and Rhinocladiella mackenziei (order Chaetothyriales, family Herpotrichiellaceae); though a few etiological agents are distributed among the orders Pleosporales, Sordariales, Xylariales, and Helotiales. Rhinocladiella is a genus of black yeast-like fungi which are encountered in human infections ranging from mild cutaneous lesions to fatal brain infections (Kantarcioglu et al. 2004). Rhinocladiella mackenziei (formerly Ramichloridium mackenziei) is an extremely neurotropic fungus, infections by which lead to almost 100 % mortality (Li et al. 2009, Kantarcioglu et al. 2004). The infection is restricted largely to the Middle East, especially Saudi Arabia and Kuwait, although sporadic cases involving visitors or immigrants from endemic areas have been diagnosed in the U.K. and U.S.A. No clear epidemiologic factors other than area of residence link these patients. Most R. mackenziei infections are brain abscesses in patients with no predisposing factors or immunodeficiency (Li et al. 2009, Kantarcioglu et al. 2004). Symptoms included headache, neurological deficits, and seizures. Pathogenesis may be haematogenous from a subclinical pulmonary focus, though it remains unclear why this fungus preferentially causes central nervous system disease in immunocompetent individuals (Kantarcioglu et al. 2004). Treatment regimens, which involve mostly amphotericin B, itraconazole and 5-flucytosine, are associated with a poor outcome in both animal and clinical studies (Al-Abdely et al. 2000). In vitro data for R. mackenziei indicate resistance to amphotericin B but susceptibility to triazoles such as itraconazole (Al-Abdely et al. 2000, Sutton et al. 1998, Al-Abdely et al. 2005) though experimental R. mackenziei cerebral infection in mice indicate that posaconazole clearly is superior to either amphotericin B or itraconazole for treatment of this condition (Al-Abdely et al. 2000). Here we describe the in vitro activity of a new triazole antimycotic, isavuconazole, and 7 comparators against 10 clinical isolates of R. mackenziei. Strains of R. mackenziei (9 from cerebral abscesses, 1 from a liver abscess) were obtained from the CBS-KNAW Fungal Biodiversity Centre (Utrecht, The Netherlands) and identified as R. mackenziei by sequencing of the internal transcribed spacer regions of rDNA. Antifungal susceptibility testing was performed according to CLSI guidelines. Amphotericin B (Bristol–Myers Squibb, Woerden, The Netherlands), fluconazole (Pfizer Central Research, Sandwich, Kent, U.K.), voriconazole (Pfizer), anidulafungin (Pfizer), itraconazole (Janssen Research Foundation, Beerse, Belgium), posaconazole (Schering- Plough, Kenilworth, N.J., U.S.A.), isavuconazole (Basilea Pharmaceutica Ltd, Basel, Switzerland) and caspofungin (Merck Sharp & Dohme BV, Haarlem, The Netherlands) were obtained as reagent grade powders. The antifungal agents were dispensed into microdilution trays at a final concentration of 0.016-16 μg/ml for amphotericin B, itraconazole, voriconazole, posaconazole, and caspofungin, 0.063-64 μg/ml for fluconazole, and 0.008-8 μg/ml for isavuconazole and anidulafungin. Conidial inocula were prepared from ≤2-week potato dextrose agar cultures by gently scraping the surface of mature colonies with a sterile cotton swab moistened with sterile physiological saline containing Tween 40 (0.05 %) under BSL-3 safety regulations. Supernatants were adjusted spectrophotometrically (530 nm) to a percentage transmission in the range 68- 71 (corresponding to 1.5-4 × 104 cfu/mL). Quality control strains Paecilomyces variotii ATCC 22319, Candida krusei ATCC 6258, and Candida parapsilosis ATCC 22019, obtained from the American Type Culture Collection (Manassas, VA), were included in each assay run. Microdilution plates were incubated at 35 °C for 96 h. The MIC endpoint for amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole, determined with the aid of a magnifying mirror, corresponded to the lowest drug concentration resulting in wells without any recognizable growth; the MIC endpoint for fluconazole was defined as the minimum drug concentration resulting

124 In vitro activities of antifungal drugs against Rhinocladiella mackenziei

Table 1. Source of Rhinocladiella mackenziei strains (n = 10)

CBS No Other collection No Clinical aspect Sex Age Therapy Out come Country

CBS 650.93 NCPF 2808 brain abscess F 55 AmB,5FC, KET Died Saudi Arabia

CBS 367.92 NCPF 2738 brain abscess F 60 -- -- Israel

CBS 368.92 UTMB 3170 brain abscess M -- -- Died Israel

CBS 102587 NCPF 2810 brain abscess M 70 AmB,5FC Died Saudi Arabia

CBS 102588 NCPF 2780 hepatic abscess M 55 AmB Died Qatar, UK

CBS 102590 NCPF 2853 brain abscess M -- -- Died UAE

CBS 102591 NCPF 7123 brain abscess, F 67 AmB Died Saudi Arabia

CBS 102592 NCPF 7460 brain abscess M 65 AmB Died Saudi Arabia

CBS 109634 -- brain abscess M 56 AmB Died Egypt, Kuwait

CBS 102589 NCPF 2779 brain abscess F 32 -- -- Oman

Table. 2 MIC ranges, MIC50 and MIC90 for 8 antifungal drugs against Rhinocladiella mackenziei.

Antifungal drugs MIC (mg/L) MIC (mg/L)

Range MIC50 MIC90

Amphotericin B 2 – 16 8 16

Fluconazole 16 – 64 32 64

Itraconazole 0.063 – 0.25 0.125 0.25

Voriconazole 0.25 – 2 1 2

Posaconazole 0.016 – 0.063 0.031 0.063

Isavuconazole 0.25 – 1 0.5 1

Caspofungin 4 – 8 8 8

Anidulafungin 1 – 8 2 8 in a prominent reduction (≥50 %) of growth. For the echinocandins MICs were determined microscopically as the lowest concentration of caspofungin or anidulafungin leading to formation of small, rounded, compact hyphal forms rather than the long unbranched hyphal clusters seen in growth controls devoid of antimycotic. The origin, CBS identification numbers, and MIC values for theR. mackenziei isolates surveyed are summarized in Table 1-2. MIC ranges for amphotericin B, voriconazole, and anidulafungin ranged over 3 log2 dilution steps, whereas MIC ranges for the remaining azoles ranged over 2 log2 dilution steps, and MIC range for caspofungin ranged over 1 log2 dilution step. Fluconazole had high MICs, indicating poor activity towards this pathogen, while lowest MIC values were seen for posaconazole. Drug treatment of human R. mackenziei cerebral phaeohyphomycosis, according to several case reports, has been unsuccessful despite aggressive therapy with amphotericin B and/or itraconazole, with or without 5-flucytosine and irrespective of surgical intervention (Liet al. 2009, Kantarcioglu et al. 2004, Al- Abdely et al. 200) only a single patient is reported to have survived an R. mackenziei cerebral infection, with pronounced radiological and clinical improvement after 9 switching therapy from a combination of liposomal amphotericin B, 5-flucytosine, and itraconazole to posaconazole (Al-Abdely et al. 2005) This sole surviving patient relapsed on itraconazole and displayed symptoms of progressive disease after several months of treatment with voriconazole, though when posaconazole therapy was resumed his condition improved significantly (Al- Abdely et al. 2005). Treatment of R. mackenziei cerebral infection with posaconazole is supported by in vitro results and data from a murine infection model (Al-Abdely et al. 2000), in which posaconazole prolonged the survival of mice and reduced the brain fungal burden compared

125 Chapter 9 to itraconazole and amphotericin B. Posaconazole apparently good penetration into the central nervous system (Ruping et al. 2008), combined with excellent in vitro data and activity in animal models, supports use of posaconazole for this difficult-to-treat infection. Most melanized fungi appear to be non-susceptible to echinocandins, probably due to a reduced presence of β-glucan in their cell walls (Odabasi et al. 2004) and we found poor activity of caspofungin and anidulafungin against R. mackenziei (MIC90, 8 μg/ml). Posaconazole (MIC90 0.063 μg/ml) and itraconazole

(MIC90 0.25 μg/ml) demonstrated the best in vitro activities, followed by isavuconazole (MIC90 1 μg/ml), though clinical experience strongly suggests that itraconazole is not a suitable choice for treating an R. mackenziei cerebral infection (Sutton et al. 1998, Al-Abdely et al. 2005). The new drug isavuconazole may be a promising alternative for this indication, but its activity against R. mackenziei needs to be confirmed in animal models.

Acknowledgments These data were presented as a poster presentation at the International Society of Human and Animal Mycology, Tokyo, Japan, 2009 (Abstract PP-03-23). The authors thank Dr. Stuart Shapiro (Basilea Pharmaceutica, Basel, Switzerland) for critically reviewing this manuscript prior to submission. The work of Hamid Badali was supported by the Ministry of Health and Medical Education of the Islamic Republic of Iran (no. 13081). This study was partially funded by an unrestricted grant from Basilea Pharmaceutica Ltd, Basel, Switzerland.

References Al-Abdely HM, Najvar L, Bocanegra R, et al (2000). SCH 56592, amphotericin B, or itraconazole therapy of experimental murine cerebral phaeohyphomycosis due to Ramichloridium obovoideum (“R. mackenziei”). Antimicrob Agents Chemother 44: 1159-1162. Al-Abdely HM, Alkhunaizi AM, Al-Tawfiq JA, et al (2005). Successful therapy of cerebral phaeohyphomycosis due to Ramichloridium mackenziei with the new triazole posaconazole. Med Mycol 43: 91-95. Clinical Laboratory Standards Institute (2008). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved Standard; 2nd Ed; Document M38-A2 Clinical Laboratory Standards Institute. Wayne, PA, USA. Kantarcioglu AS, de Hoog GS (2004). Infections of the central nervous system by melanized fungi: a review of cases presented between 1999 and 2004. Mycoses 47: 4-13. Li DM, de Hoog GS (2009). Cerebral phaeohyphomycosis - a cure at what lengths? Lancet Infect Dis 9: 376-83. Odabasi Z, Paetznick VL, Rodiguez JR, et al (2004). In vitro activity of anidulafungin against selected clinically important mold isolates. Antimicrob Agents Chemother 48: 1912-15. Ruping MJGT, Albermannn N, Ebinger F et al (2008). Posaconazole concentrations in the central nervous system. J Antimicrob Chemother 62: 1468-1470 Sutton DA, Slifkin M, Yakulis R, Rinaldi MG (1998). US case report of cerebral phaeohyphomycosis caused by Ramichloridium obovoideum (R. mackenziei): criteria for identification, therapy, and review of other known dematiaceous neurotropic taxa. J Clin Microbiol 36: 708-715. 126 10 Chapter 10

Discussion Conclusion & Summary Conclusies en samenvatting (Dutch) Acknowledgements About the author List of publications List of abstracts 10127 Chapter 10

Discussion

Ecology and pathogenicity of black yeasts and relatives The order Chaetothyriales includes non-lichenised ascomycetes, which are characterised by a dark mycelium, and are divided in two families, the Chaetothyriaceae and the Herpotrichiellaceae (Geiser et al. 2006). The Chaetothyriaceae are epiphytes, colonizing the leaves and the bark of trees in the tropics (Batista & Ciferri 1962). For many of these species of Chaetothyriaceae, it is still unclear whether they are saprophytic or biotrophic (Barr 1987). Species of the Herpotrichiellaceae are known either from their teleomorph growing as saprotrophes on dead plant material (Barr 1987, Untereiner & Naveau 1999b), from their anamorphs often occurring as opportunistic pathogens in humans and animals (de Hoog et al. 2000, Horré and de Hoog 1999), or from both. A new area of biodiversity is emerging from the environment due to the use of isolation by enrichment methods (Prenafeta-Boldú et al. 2006, Prenafeta-Boldú et al. 2001a, Zhao et al. 2010). Morphologically, many species are poorly differentiated, and can be identified reliably only since the development of molecular methods. These methods also revealed some connections between anamorphs and teleomorphs (Untereiner 1997, 2000, Untereiner & Naveau 1999a), but for the great majority of clinically relevant species teleomorphs are unknown. Thus far the teleomorphic genus Capronia appears to be polyphyletic (Untereiner 2000). Little is known of the natural ecology of black yeast-like fungi. The study of life cycles and the isolation of strains in their putative primary habitats are key issues to understanding the evolution in this group (de Hoog et al. 2000). Recently, Ruibal (2004) showed that some slow-growing melanized fungi inhabiting rocks in harsh environments also belong to the Chaetothyriales. Molecular data suggest that they form novel lineages within Chaetothyriales and indicate that more diversity is yet to be discovered for these rock-inhabiting species (Ruibal 2004). Rock fungi were qualified as extremotolerant, because they are able to tolerate very high or low temperatures (Sterflinger 1998a), repeated freezing and thawing cycles (Selbmannet al. 2002), low water activity (Sterflinger 1998b, Zalar et al. 1999), meristematic growth expressed at low pH (Mendoza et al. 1993, Badali et al. 2008), nutrient deficiency (Sterflinger et al. 1999), osmotic stress, desiccation, and UV radiation. Among the adaptations are melanization, meristematic growth, oligotrophism and slow, inexpensive metabolism. Meristematic and yeast-like growth is widely distributed in the Herpotrichiellaceae. The mycelium is obligatorily darkened due to the production of DHN- melanin derived from the precursor molecule 1,8-dihidroxynaphthalene (DHN) via the polyketide pathway. Melanin is frequently viewed as a prime virulence factor in black fungi, playing an essential role in pathogenicity to humans (Wheeler, Bell 1988, Schnitzler et al. 1999, Paolo et al. 2006). Pathogenicity and virulence are enhanced by melanin which protects fungal cells against oxidants, by impairing the development of cell-mediated responses, interfere with complement activation and reduce the susceptibility of cells to antifungal agents. The organism is also protected against environmental factors (Butler et al. 1998, Jacobson 2000). However, the presence of melanin is not sufficient to explain pathogenicity, and additional factors must be involved to explain the virulence of these fungi (de Hoog et al. 2000, Badali et al. 2008). In this thesis we observed recurrent pathogenicity all over the Chaetothyriales. Sometimes virulence was expressed in a sibling of a species with another type of ecology. This was first noted in the species pair Cladophialophora carrionii / C. yegresii (de Hoog et al. 2007). Cladophialophora carrionii is the agent of the skin disorder chromoblastomycosis, which occurs in semiarid climates, and is supposed to be inoculated via e.g. pricks of cactus spines. However, the species that occurs on live cactus, expressing the same morphology in spines as in human skin, is C. yegresii,

128 Disscusion which has never been found on humans. De Hoog et al. (2005) noticed that at the ordinal level, human pathogenicity is associated with stress-factors like osmotolerance, but the two factors are nearly mutually exclusive at the species level. It was hypothesized that extremotolerance may facilitate pathogenic evolution, but extremotolerance appears to be required, but is not sufficient. Closely related species may evolve towards two different types of ecology that share a number of basic factors, a condition termed ‘dual ecology’ by Prenafeta-Boldú et al. (2006). In the case of the Chaetothyriales we observe pathogenicity next to the ability to assimilate monoaromatic hydrocarbons, both in polluted and in natural environments such as cactus spines. Consequently, several organisms are of interest for the bioremediation of hydrocarbon pollution, while their sister species are significant in human disease. In contrast, several plant-pathogenic Cladophialophora species specific for particular host plants (Crous et al. 2007, Davey et al. 2007) are only distantly related and are unlikely to belong to the Chaetothyriales. No hydrocarbon association or human pathogenicity has ever been reported in these species.

Cladophialophora-like anamorphs are polyphyletic The family Chaetothyriaceae is poorly known and molecular data are lacking for most species. Relationships with other members of the Chaetothyriales are still unclear. At this moment we suppose that the Chaetothyriaceae comprise the ancestral lineages 2, 3 and 4 of the Chaetothyriales, paraphyletic to the Herpotrichiellaceae forming a derived lineage (Fig. 1, Chapter 2). Within the Herpotrichiellaceae, generic delimitations are problematic due to the fact that the numerous anamorphic genera (e.g., Cladophialophora, Rhinocladiella and Exophiala) appear to be polyphyletic (Haase et al. 1999, Badali et al. 2008). The genusCladophialophora is morphologically characterised by poorly or profusely branched chains of dry, rather strongly coherent, moderately melanized conidia. This simple type of propagation is found all over the Chaetothyriales. In our multilocus phylogeny of the Chaetothyriales, a number of plant-associated species (C. hostae, C. scillae, C. proteae, C. sylvestris, C. minutissima, and C. humicola) appear to be rather closely related to Ceramothyrium carniolicum, a putative member of lineage 2 of the Chaetothyriaceae, but only distantly related to the type species of Cladophialophora, i.e. C. bantiana. Most of the Cladophialophora species consistently associated with pathology to humans, nearly all belong to the Herpotrichiellaceae, a family containing many opportunistic fungi, which are classified in the genera Exophiala, Fonsecaea, Phialophora and Rhinocladiella. The only agent of a severe mycosis in the ancestral lineages is Cladophialophora modesta, causing brain infections after traumatic inoculation. Some very mild, often asymptomatic species on skin and in nails in lineages 2 and 3 are Phialophora europaea, Coniosporium epidermidis and Cyphellophora laciniata. The more severe, non-traumatic cases are all located in the derived lineage. Cladophialophora-like morphologies have been observed in several unrelated environmental fungi, particularly in Cladosporium, Pseudocladosporium, Fusicladium, Devriesia, Capnobotryella and Mycosphaerella (Braun et al. 2003, Seifert et al. 2004, Crous et al. 2007). These genera are assigned to different families within the Dothideales and Capnodiales, two ascomycete orders for which species are only exceptionally encountered in a clinical setting. Conidial scars in these genera have a pale pigmentation in Cladophialophora, which is in contrast to members of the common saprobes and hospital contaminants of Cladosporium, where pronounced, dark conidial scars are present. Conidiophores in most Cladophialophora species are poorly differentiated, while those of Cladosporium species are usually erect and significantly darker than the rest of the mycelium. A further distinctive characteristic is found in the conidial chains of Cladophialophora species which 10 are coherent, while those of Cladosporium detach very easily. Remaining Cladophialophora look- alikes have never been encountered in clinical samples.

129 Chapter 10

Opportunistic species of Cladophialophora are able to tolerate high temperatures. Tolerance of human body temperature is an essential requirement for pathogenicity, but this trait may have incidentally been acquired via adaptation to warm environments, such as hot surfaces in semiarid climates, or as a side effect of osmotolerance, noted in several species occurring in food stuffs and juices. The capacity of some species of Cladophialophora to grow in a meristematic form both in human hosts and under extreme environmental conditions supports the suggestion that the muriform cells may be a virulence factor in the development of the disease. The conversion of hyphae to muriform cells can be induced in vitro under acidic conditions and low concentration of calcium (Chapter 2). In Chapter 4 the new species Cladophialophora psammophila is described. (Badali et al. 2010a) It morphologically resembles C. bantiana, a virulent neurotropic agent of fatal cerebral phaeohyphomycosis in humans causing granolumatous abscesses in the brain. Members of C. bantiana are unique by consistently containing a distinctive 558-bp intron beginning at position 1768 in the small subunit of the ribosomal DNA gene (Gerrits van den Ende et al. 1999). Interestingly, this intron is also present in C. psammophila, underlining the close kinship of the two species. The multiple gene sequences and AFLP data generated in our study deviate sufficiently from those of C. bantiana to be sure that the two species be considered separate though highly related species. Cladophialophora bantiana has a maximum growth temperature of 42 °C and kills mice within 2 weeks at experimental inoculation, whereas C. psammophila grows up to 37 °C and is non-virulent to mice (Badali et al. 2010a). Cladophialophora bantiana could be isolated from the environment only once by using a mammal vector (Conti-Diaz et al. 1977, Dixon et al. 1980a , 1980b). In contrast, C. psammophila was isolated by hydrocarbon enrichment from a location where hydrocarbon pollution was prevalent. Both Cladophialophora species, although at short phylogenetic distance, thus exhibit striking ecological differences. Future genome comparisons could reveal the essential virulence factors involved in the pathogenicity of the Bio-Safety Level-3 species C. bantiana. Enrichment on toluene as the sole sources of carbon and energy is a key factor in the isolation of black yeast like fungi. The physiological and metabolic characterization of axenic cultures demonstrated that assimilation of aromatic hydrocarbons was limited to certain alkylbenzenes, such as toluene and ethylbenzene (Prenafeta-Boldú et al. 2001b). This biodegradation pattern was confirmed in assays of complex BTEX mixtures simulating gasoline pollution, in which only toluene and ethylbenzene were mineralized, while the xylene isomers were co-metabolized and benzene remained undegraded throughout the experiment (Prenafeta Boldú et al. 2002). Alkylbenzene degradation proceeded through initial oxidation of the alkyl side chain, rather than the aromatic ring (Prenafeta Boldú et al. 2001). Nevertheless, Rustler et al. (2008) suggested that E. oligosperma could further metabolize 2-hydroxyphenylacetate and finally cleave the aromatic ring (Rustler and Stolz 2007). Therefore, E. oligosperma was grown with phenylacetonitrile as sole source of carbon and energy. Degradation of phenylacetonitrile presumably proceeds via phenylacetic acid, 2-hydroxyphenylacetic acid, 2, 5-dihydroxyphenylacetic acid, maleylacetoacetate and fumarylacetoacetate (Rustler et al. 2008) The exceptional metabolic and physiological capacities of C. psammophila have been investigated in relation to the biofiltration of waste gases polluted with volatile aromatic hydrocarbons and for the bioremediation of BTEX pollution in soil microcosms (Prenafeta-Boldu et al. 2004, 2008). When metabolism of aromatic hydrocarbons becomes associated with pathogenicity in the same species, infections are nearly asymptomatic. Occurrence on skin and nails has been observed in Cladophialophora immunda (Chapter 2) and in C. saturnica (Chapter 3), as well as in the related genus Exophiala, particularly in E. oligosperma and E. xenobiotica (Badali et al. 2008; de Hoog et al. 2003; Vicente et al. 2008). Infection patterns of these species tend to be coincidental,

130 Disscusion and are mostly limited to skin and nails, and are similar to infections found in members of ancestral chaetothyrialean lineages, such as Phialophora europaea, Cyphellophora laciniata and Coniosporium epidermidis.

In vitro antifungal susceptibility of Cladophialophora bantiana In case of a cerebral infection, it is crucial to correctly identify the causative organism to the species level because of possible differences in susceptibility to antifungal drugs among closely related, sibling species. Several novel species of Cladophialophora causing infections in humans have recently been described. Diagnostics and identification with obsolete mycological procedures is insufficient due to the high degree of phenotypic similarity between these fungi. In contrast, genetic diversity allows unambiguous identification of the species (Zeng et al. 2007). In Chapter 5 the genetic diversity in a large collection of C. bantiana strains was monitored (Badali et al. 2010b). Previous studies had shown that ITS sequencing indicates a low degree of variability of C. bantiana. Strains of the species differ in less than 6 bp in the ITS1 region. Gerrits and de Hoog (1999) used SSU and ITS RFLP to assign 31 strains to this species. RFLP proved to be a reliable diagnostic technique with sufficient discriminative power with the set of restriction enzymes used. In addition, AFLP not only has higher reproducibility, resolution, and sensitivity at the whole genome level compared to other techniques, but it also has the capability to amplify between 50 and 100 fragments at one time, moreover no prior sequence information is needed for amplification (Walzet al. 1997). AFLP was expected to reveal the existence of sub populations within C. bantiana, as was found for Exophiala dermatitidis by Sudhadham et al. (2010). In the AFLP data presented in Chapter 5, nearly all strains proved to be identical, suggesting that the species practically consists of a single, recently emerged clone. Based on the AFLP patterns we did not see any misidentification for those taxa. All strains in this study were originally identified as C. bantiana, C. emmonsii, C. arxii, C. devriesii and C. modesta based on sequencing, phenotypic and physiological criteria which is in the line with the cluster analysis demonstrating a clear separation of C. bantiana as a rather homogeneous group from C. modesta, C. emmonsii, C. arxii and C. devriesii (Badali et al. 2010b). Very little data are available in the literature regarding the susceptibility of C. bantiana isolates to antifungals (Radford et al. 1997). In vitro activity of all isolates in our study had a reduced susceptibility to fluconazole. However, treatment with itraconazole and voriconazole (Fica et al. 2003) against cerebral abscess due to C. bantiana were less successful, although itraconazole had low MICs (MIC90 0.125 μg/ml) in our study. A likely explanation is that these last-mentioned antifungals do not penetrate well into the CNS. Whereas voriconazole has a good penetration into the CNS the MICs of C. bantiana were in the higher ranges (MIC90 2 μg/ml). Some authors report successful treatment of C. bantiana brain abscess with voriconazole (Lyons et al. 2005) while others report clinical failure (Fica et al. 2003). While an excellent drug for treatment of cerebral aspergillosis, voriconazole might not be a first choice forC. bantiana. In contrast to another study in which only seven strains of C. bantiana were investigated (Radford et al. 1997), giving a MIC range 0.12–1.0 μg/ml, we found a large range of activity (MIC 0.125-4 μg/ml). Thereforein vitro susceptibility testing may be warranted before using voriconazole. Posaconazole and itraconazole

(MIC90 0.125 μg/ml) demonstrated the best in vitro activities, followed by isavuconazole (MIC90 0.5 μg/ml), although clinical experience suggests that monotherapy with itraconazole is not a suitable choice for treating a C. bantiana cerebral infection. Treatment with posaconazole is supported by our in vitro results and data from murine infection models in which posaconazole 10 prolonged the survival of mice and reduced the brain fungal burden compared to itraconazole and amphotericin B (Al-Abdely et al. 2005a; Mariné et al. 2009). Moreover, its apparent good

131 Chapter 10 penetration into the CNS (Rüping et al. 2008) supports use of posaconazole for this difficult to treat infection. Most melanized fungi appear to be resistant to echinocandins, probably due to reduced β-glucan in their cell walls (Odabasi et al. 2004). We found poor activity of caspofungin but anidulafungin had activity for C. bantiana showing a geometric mean MIC of only (0.073 μg/ ml). In conclusion, itraconazole, posaconazole and isavuconazole demonstrated in vitro antifungal activity against neurotropic isolates of C. bantiana, although in case of isavuconazole clinical value needs to be confirmed in animal models.

Mycetoma in Exophiala jeanselmei Phylogenetic analyses have shown that the ex-type strain of E. jeanselmei (CBS 507.96) is a member of the order Chaetothyriales and is located within the family Herpotrichiellaceae (Badali et al. 2008, 2009). For this and other related species, identification with standard mycological procedures is difficult because of the preponderance of either budding cells or poorly differentiated conidial structures. In Chapter 6 we sequenced 17 strains from the CBS collection maintained as E. jeanselmei. Considerable sequence diversity was found in eight of these strains, which were therefore attributed to other black yeast species. The remaining strains, including one from a recent subcutaneous infection, exhibited ≥98 % ITS similarity to the ex-type strain of E. jeanselmei (CBS 507.90) and formed a single cluster of E. jeanselmei. In our case, as well as in several published cases, granules, the hallmark of mycetoma, were seen histopathologically in tissue (Badali et al. 2010c). Grains were often reniform and clinical features of mycetoma, such as tumefaction, abscess formation, and fistulae were observed. Some cases were subcutaneous but with hyphae rather than granules in tissue. Subcutaneous infection is regularly observed in E. jeanselmei, while it is hardly ever seen in other black yeast-like fungi. Exophiala jeanselmei appears to be very consistent in its clinical behavior. Only exceptionally cases were interpreted as chromoblastomycosis, judging from the presence of muriform cells in tissue. The switch between granules and muriform cell as a response to host factors needs to be further studied. Since at least 100 years, very serious black yeast infections are known to occur. The frequency of infections seems to be stable, and is not even changing as a result of growing populations of hospitalized patients with impaired immunity. In fact, the majority of infections, be it cutaneous, mycetoma, chromoblastomycosis, systemic and disseminated, occurs in otherwise healthy individuals. A large diversity of therapeutic regimens has been applied for treatments. In the past, amphotericin B was the most potent antifungal drug for severe invasive fungal infections, but with low efficacy, and its use is associated with severe side effects, particularly nephrotoxicity. The newer azoles and echinocandins have now replaced amphotericin B. As yet there are only few data available on susceptibilities of conventional and new antifungal agents against confirmed strains of black fungi, such as E. jeanselmei. In a comparative overview of in vitro activities of posaconazole, itraconazole, voriconazole, and amphotericin B against over 19,000 clinical yeasts and moulds, just 14 Exophiala strains were included, without molecular identification (Sabatelli et al. 2009). Data published by these authors (Sabatelli et al. 2009) and by others (e.g. Zeng et al. 2007) have shown that posaconazole has good activity against agents that cause chromoblastomycosis, mycetoma, and phaeohyphomycosis, including Exophiala species. Posaconazole was frequently found to be the most active drug against these organisms (Sabatelli et al. 2006, Zeng et al. 2007, Fothergill et al. 2009). Similar results were found in our series, posaconazole showing highest activity against E. jeanselmei and E. oligosperma in terms of MIC50. In vitro testing of isavuconazole and voriconazole exhibited broad-spectrum activity against the majority of the opportunistic and pathogenic fungi (Guinea et al. 2008). In the present study with E. jeanselmei (Chapter 6) low in vitro activities for isavuconazole and voriconazole were found, suggesting that these drugs might

132 Disscusion not be the optimal choice for treatment. In contrast, posaconazole demonstrated potent in vitro activity against strains of E. jeanselmei causing mycetoma. Itraconazole has been successfully used clinically in cases of mycetoma due to E. jeanselmei (Al-Tawfiqet al. 2009). Voriconazole has been used only in some cases of cutaneous infections (Loulergue et al. 2006). Isavuconazole until now has not been used clinically for Exophiala infections. Infections by Exophiala species may require a combination of surgical and medical treatment. Although amphotericin B and itraconazole, with or without additional flucytosine, are currently regarded to be effective against cutaneous and subcutaneous lesions, the newer triazole agents, voriconazole and posaconazole, expand the therapeutic options for treatments of these mycoses. In conclusion, posaconazole and itraconazole seem to be the most active drugs for treating Exophiala infections, but their clinical effectiveness in the treatment of Exophiala infections remains to be determined.

Clinical types of chromoblastomycosis Chromoblastomycosis is a chronic, cutaneous and subcutaneous infection characterised by slowly expanding skin lesions with muriform cells in tissue, provoking a granulomatous immune response. The infection usually shows only localized expansion with verrucous plaques (Queiroz- Telles et al. 2009, de Hoog et al. 2000). All prevalent agents (Cladophialophora carrionii and several Fonsecaea species) are members of the Chaetothyriales. That also holds true for the more rare agent Rhinocladiella aquaspersa (Marques et al. 2004, Jae et al. 2004), which is characterised by sympodial conidiogenesis (Arzanlou et al. 2007). Published cases of chromoblastomycosis due to R. aquaspersa have been diagnosed on the basis of clinical and pathological data with biopsies showing numerous muriform cells and complied with the clinical criteria known for the disease. Our case report (Chapter 7) from Mexico, however, had dry, encrusted, verrucous violaceous lesions and chronic lymphedema as in chromoblastomycosis, but clinically resembled sporotrichosis by lymphatic expansion. Thus our case showed a new clinical presentation of the disease (Badali et al. 2010d). Retrospective analysis demonstrated that this type may also occur in patients infected with F. pedrosoi (Ogawa et al. 2003, Takase et al. 1988). Lymphatic dissemination in chromoblastomycosis may also appear after cryosurgical treatment (R. Isa, Dominican Republic, pers. comm.). An analysis of all infections caused by R. aquaspersa revealed that the species, in addition to causing chromoblastomycosis, may also present with hyphae in tissue. The related fungusRhinocladiella phaeophora originally was obtained from maize field soil from Colombia, but R. aquaspersa thus far has been found only in humans. Harutyunyan et al. (2008) reported on the distantly related Rhinocladiella species located in the lower, ancestral lineages of Chaetothyriales. These species inhabit lichens in arid climates and are devoid of pathogenicity towards mammals. Pathogenicity to vertebrates seems to have evolved in the derived lineages of the Chaetothyriales. Treatment of chromoblastomycosis is difficult, because prolonged therapy is needed and relapses are frequent. Itraconazole and terbinafine are the most frequently applied antifungals (Queiroz-Telles et al. 2009, Bonifaz et al. 2004, 2005), but no standard therapy has been determined. In cases caused by C. carrionii and Phialophora verrucosa, patients generally respond well to relatively low doses of most antimycotics. Thein vitro susceptibilities of C. carrionii strains to antifungal drugs (Vital et al, 2009) were similar to those of the Fonsecaea spp. Najafzadeh et al. (2010) have tested in vitro activities of eight antifungal drugs against clinical isolates of F. pedrosoi, F. monophora and F. nubica and the resulting MIC90 for all strains were as follows, in increasing order: posaconazole, 0.063 μg/ml; itraconazole, 0.125 μg/ml; isavuconazole, 0.25 μg/ 10 ml; voriconazole, 0.5 μg/ml; amphotericin B, 2 μg/ml; caspofungin, 2 μg/ml; anidulafungin, 2 μg/ml; and fluconazole, 32 μg/ml. Vanden Bossche et al. (1998) reported that for black fungi

133 Chapter 10 amphotericin B MIC values were mostly >4 μg/ml. In contrast, Marques et al. (2004), using a broth macrodilution method, obtained an amphotericin B MIC of 0.8 mg/L for R. aquaspersa. Our results corroborate previous studies (Gonzalez et al. 2005) that itraconazole (MIC 0.063 μg/ ml) and posaconazole (MIC 0.125 μg/ml) have potent activity against R. aquaspersa, whereas voriconazole and isavuconazole have lower activities. Cuenca-Estrella et al. (2006) likewise noted that posaconazole was a superior drug against Rhinocladiella strains while caspofungin had poor activity. We also found that R. aquaspersa was more sensitive to anidulafungin (MIC 1 μg/ml) than was R. phaeophora (MIC 8 μg/ml). This is concordant with the finding presented here, that anidulafungin has higher in vitro activity against clinical isolates of Rhinocladiella (R. aquaspersa) than against environmental isolates of this genus (R. phaeophora). We conclude that itraconazole and posaconazole may become therapeutic options and alternatives to amphotericin B for treating infections due to the rare etiologic agent R. aquaspersa.

Rhinocladiella mackenziei brain infection Rhinocladiella mackenziei is an extremely neurotropic fungus, and infections that invade the CNS cause death in most instances. Thus far the infection was restricted to the Middle East and the Persian Gulf region (Campbell et al. 1993), especially Saudi Arabia and Kuwait. Sporadic cases outside these regions concerned visitors or immigrants from endemic areas diagnosed in the U.K. and the U.S.A. No clear epidemiological factors other than area of residence link these patients. Cerebral phaeohyphomycosis caused by R. mackenziei is a rare but highly significant disease due to the regional prevalence and high mortality nearly 100 % despite combined surgical and antifungal therapy (Horré & de Hoog 1999) in both immunocompetent and immunocompromised individuals. Previously, the majority of reported central nervous system (CNS) infections caused by dematiaceous fungi were found to be brain abscesses in patients with no predisposing factors or immunodeficiency. Symptoms included headache, neurological deficits and seizures. Pathogenesis may be haematogenous from a subclinical pulmonary focus, although it remains unclear why this fungus preferentially causes CNS disease in immunocompetent individuals. Until now R. mackenziei has never been isolated from the environment. The natural niche of this organism thus remains unknown. In Chapter 8 we present the first case ofR. mackenziei outside the arid climate zone of the Middle East. In contrast to all reported cases, this strain (CBS 125089) originated from a humid sub-tropical climate, infecting a patient who claimed he had never travelled outside of India (Badali et al. 2010e). Cerebral infections due to R. mackenziei have thus far been diagnosed after CT-guided needle aspiration and were proven by positive histopathology and cultures. Treatment mostly involved (high-dose lipid) amphotericin B, itraconazole and 5 flucytosine, or a combination of these drugs (Revankar et al. 2004). However, strains of R. mackenziei isolated from infected patients are in vitro resistant to amphotericin B, which is often used as the gold standard of treatment. Only a single patient was reported to have survived an R. mackenziei cerebral infection, with pronounced radiological and clinical improvement after switching therapy from a combination of liposomal amphotericin B, 5-flucytosine, and itraconazole to posaconazole (Al-Abdely et al. 2005b). This is supported by in vitro data from a murine infection model (Abdely et al. 2000), in which posaconazole prolonged the survival of mice and reduced the brain fungal burden compared to itraconazole and amphotericin B. The apparently good penetration of posaconazole into the central nervous system (Ruping et al. 2008), combined with excellent activity in animal models, makes a good case for posaconazole for this difficult-to-treat infection. Delay in diagnosis and limited data on effective treatment are main factors the development of cerebral abscess. Establishment of in vitro activities (Chapter 9) demonstrated that R. mackenziei is susceptible to itraconazole,

134 Disscusion posaconazole, and isavuconazole. The latter drug may be a promising alternative for this indication (Badali et al. 2010f).

Identification of human-associated black yeasts and relatives In this thesis, I have demonstrated that human infections by members of the Chaetothyriales are a very consistent phenomenon. Infectious species show quite specific behavior in the human host, and therefore clinical data should play a role in identification of the causative agent, but molecular identification is indispensible. As representatives of the black yeast and allies, diagnostics of the various species of Cladophialophora (Chapters 2−5), Exophiala (Chapter 6) and Rhinocladiella (Chapter 7−9) were developed using different molecular and mycological techniques. Only a limited number of taxa can be recognized by morphological features. Available physiological data are insufficient to separate and distinguished all species from each other, particularly in members of Chaetothyriales. For example, C. carrionii and C. samoensis, the true agents of chromoblastomycosis, share major phenotypic characters with the environmental only species C. yegresii, Exophiala jeanselmei, E. oligosperma and E. xenobiotica are morphologically nearly indistinguishable, but cause different diseases. Molecular data thus are indispensible to come to a final diagnosis. A large diversity of molecular biological data has been applied to build up a modern classification of black yeasts and relatives and to understand patterns of epidemiology (Badali et al. 2008, Wang et al. 2001, Najafzadeh et al. 2009, Arzanlou et al. 2007). These studies uncovered genetic variation of taxa at different levels of diversity and provide clues for species diagnoses. Sequencing the ITS rDNA region has been most frequently applied. In Chapter 2, it was noted that human-associated Cladophialophora species are divided over two main clades when applying ITS sequences: the carrionii clade and the bantiana clade. This separation was supported by robust data in conserved (nucSSU, nucLSU and RPB1) and well as in variable genes (ITS, TUB and EF1-α). Topological conflicts were detected for the variable genes only below the species level relationships, and therefore – in agreement with the genealogical recognition of the species concept (Taylor et al. 2000) – the conflicts were ignored and the loci combined. Thus, within the order Chaetothyriales the agents of chromoblastomycosis are polyphyletic.

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Conclusion & Summary The black yeast like fungi and relatives in the Chaetothyriales comprise a large number of species occurring as opportunists in cold-blooded animals and humans. Diseases caused are highly diverse, but characteristic within each species, and range from mild cutaneous infections to fatal encephalitis. Particularly species of Cladophialophora are well-known for their high virulence. They may infect their hosts by inhalation, ingestion or traumatic inoculation, provoking a variety of disorders. Most infections are found in otherwise healthy individuals. Because of diagnostic problems the Chaetothyriales have received limited attention in the past,

138 Disscusion but recent developments in molecular techniques have made them a very interesting study object. Understanding of pathogenicity and evolution is now within reach. Given the phylogenetic kinship of etiologic agents, it is likely that species share a number of essential virulence factors, which must have come about by shared processes of evolution. Chaetothyriales comprise teleomorphs that occur as saprophytes on decaying wood and mushrooms (Untereiner et al. 1995, 1999b, Untereiner 1997, 2000). Previous studies of the evolution of lichenisation have shown that the ancestor of this order was likely lichenised, and that opportunistic pathogens and saprophytes within Chaetothyriales probably derived from this ancestor by a loss of lichenisation (Lutzoni et al. 2001, James et al. 2006). Gueidan et al. (2008) showed that lineages diverging early in the evolution of the Chaetothyriales comprise a high diversity of rock-inhabiting fungi, and reconstructed that the ancestor of this order was a rock-inhabitant. Several authors (e.g. Schnitzler et al. 1999, Feng et al. 2001) believed that among the basic set of virulence factors conferring invasive capability are melanin formation and meristematic growth. These factors have primarily evolved in response to extreme environments (de Hoog 1993, Haase et al. 1999, Prenafeta-Boldú et al. 2006). Success rates of isolation of Chaetothyriales from the environment vary greatly. Often enrichment methods are necessary, e.g. using toxic hydrocarbons (Prenafeta-Boldú et al. 2006). Some species can be found on plant debris where they reside in passive (meristematic) form and then are supposed to be traumatically inoculated into humans. However, this relationship is not straightforward, because the plant-inhabiting species are frequently non-pathogenic siblings of the invasive species. Until now numerous species of Cladophialophora and Fonsecaea have been identified from environments which have not been isolated from mammalian tissue, andvice versa. For example, C. yegresii was isolated from living, asymptomatic Stenocereus plants surrounding the cabin of a symptomatic patient with C. carrionii. Although some publications suggested that infections originated from inoculation by plant material (e.g., Salgado et al. 2004), it now becomes clear that the environmental look-alikes of clinical strains do not necessarily belong to the same species. An unambiguous connection between a clinical and any environmental strain still has to be proven for many species, such as Cladophialophora bantiana. We hypothesize that there is an adaptive step between the plant species and its pathogenic sibling. Specific characters evolved in a rock-inhabiting ancestor for life in extreme habitats and potentially shared by all descendants within this lineage, may explain the numerous independent shifts to pathogenicity that have occurred in more derived clades of the Chaetothyriales. Because clinical strains have to grow and multiply at body temperature, tolerance of high temperatures is a prerequisite for the evolution of pathogens on animals and humans (Prenafeta-Boldú et al. 2006). Evolution of the Chaetothyriales underlines the role of extremotolerance in lifestyle transitions in black fungi. De Hoog et al. (2007) performed inoculation experiments with Cladophialophora carrionii (from humans) and C. yegresii (from cactus) and showed that both species were able to cause infection in cactus tissue with very low degrees of tissue necrosis. Cactus tissue is rich in carbohydrates, vitamins and minerals (Vélez & Chávez 1980) which may promote endophytic growth. The two species differed in the degree of scarring after traumatic inoculation into mature plants. It was concluded that clinical C. carrionii was consistently more virulent than C. yegresii that originated from the same host plant. The difference in virulence may be explained by C. carrionii, which lives as a saprobe on dead cactus debris for part of its life cycle and is less adapted to an endophytic life style. In nature, the fungi are likely to invade only when the integrity of the epidermis is broken, such as by goat feeding or transmission by sap-sucking birds or piercing insects. The two species also show the same transformation to meristematic morphology (González 10 et al. 1990) when entering hard, lignin-rich spine tissue. Lignin is a complex chemical compound which is relatively hydrophobic and aromatic in nature, most commonly derived from wood,

139 Chapter 10 and is an integral part of the secondary cell walls of plants. It plays a crucial part in conducting water in plant stems and might be a trigger for fungal meristematic conversion in the spines. Spines play a role in mechanic dispersal of the fungi by traumatic inoculation into living tissue of humans or animals, where the same muriform cells are formed, leading to the skin disease chromoblastomycosis. An interesting question is whether animal/human inoculation plays a role in the evolution of the fungus. Cladophialophora carrionii as a member of the Chaetothyriales is known to occur on lignified materials like wood chips or dead plant debris (de Hoog et al. 2007). Also Cryptococcus neoformans is a pathogen known to have an essential part of its life cycle in hollows of Eucalyptus trees. Cryptococcus neoformans produces diphenol oxidase to degrade lignin, an aromatic polymer in the cell wall of plants and a component of wood (Cabral 1999). Similar degradation pathways are present in Cladophialophora carrionii (Prenafeta- Boldú et al. 2006). Tolerance of high and low temperature, acid tolerance, meristematic growth, melanin protection and the use of aromatic hydrocarbons all contribute to the ability to cause recurrent infections, and to the high diversity of clinical syndromes in the derived lineages of Chaetothyriales. Corbellini et al. (2006) studied delayed-type hypersensitivity responses to crude and fractionated antigens from Fonsecaea pedrosoi and suggested that skin test using antigens produced in synthetic media may be useful for assessing primary exposure to chromoblastomycosis. The presence of memory suggests regular confrontation of host and fungus in history. d’Ávila et al. (2002) demonstrated cell-mediated tissue reactions in chromoblastomycotic skin lesions. They believed that patients with verrucous plaques had a Th2 response, whereas patients with erythematous atrophic plaques had a Th1 response. Iwatsu et al. (1982) observed cutaneous delayed hypersensitivity in animal experiments. Esterre et al. (2006) suggested that, relative to cell-mediated immunity, humoral immunity does not seem to offer much protection against chromoblastomycosis. Therefore, cell-mediated immunity seems to play a consistent role in the disease. This suggests that there is a long-standing relationship between fungus and host,the fungus having evolved from opportunism to pathogenicity. This matches with the observation that in endemic areas humans nearly always carry C. carrionii, F. pedrosoi or F. monophora rather than other, common environmental Cladophialophora and Fonsecaea species (de Hoog et al. 2007, Vicente et al. 2008). Central nervous system infection due to C. bantiana is reported worldwide, though a general preference for warmer climates with high humidity is apparent (Horré et al. 1999). The mode of infection is supposed to be either by haematogenous spread from an unrecognized pulmonary focus, and not through direct extension from adjacent paranasal sinuses, although the majority had had no recent evidence of pulmonary or sinus infections (Horré et al. 1999). Cladophialophora bantiana is able to pass the blood brain barrier of laboratory animals (Al-Abdely et al. 2005a). The narrow ecological amplitude of C. bantiana seems to match with factors provided by the hosts. The species shows very constant AFLP profiles, having the appearance of a recently evolved single clone. Etiological agents of some human disorders may have their natural niche in the environment, where they may be relatively harmless. The recurrent opportunism in Chaetothyriales seems only occasionally to have lead to some degree of pathogenicity. Most members of the order are potentially able to cause severe disease in healthy people, but seem to lack mechanisms to efficiently enter the human body. In order to better understand their evolution, ancestral state reconstructions methods have been used to characterize these ecological shifts (Hibbett et al. 2000, Lutzoni et al. 2001). The essential factors may however be unexpected and difficult to determine, such as the ability to assimilate monoaromatic hydrocarbons. More knowledge on primary niches, ecological

140 Disscusion preferences and factors involved in pathogenicity are needed to fully understand the ecology of Chaetothyriales. Opportunistic fungal pathogens have become a major concern in public health, further work on their ecology and evolution is crucially important.

Conclusies en samenvatting (Conclusion & Summary in Dutch) De groep van de zwarte gisten en verwanten in de ascomyceten orde Chaetothyriales bevat een groot aantal soorten welke infecties in vooral de mens en koudbloedige dieren kunnen veroorzaken. De ziektebeelden zijn uitermate divers, maar binnen elke soort zijn ze karakteristiek. Het totale spectrum varieert van milde cutane infecties tot levensbedreigende hersenabscessen. Vooral soorten van het geslacht Cladophialophora zijn berucht vanwege hun pathogeniteit en hoge virulentie. Infectie kan via verschillende routes plaatsvinden: inhalatie, ingestie en traumatische inoculatie, leidend tot heel verschillende ziektebeelden. Bijna alle infecties komen voor in patiënten met een actief, functioneel immuunsysteem. Veel soorten in de Chaetothyriales zijn erg moeilijk morfologisch te determineren, en dat heeft in het verleden de belangstelling voor deze groep getemperd. Maar dankzij ontwikkeling van moderne, moleculaire methoden realiseren we ons nu dat ze en buitengewoon interessant studieobject vormen. Inzicht in pathogeniteit en evolutie is nu binnen handbereik. Veel pathogene soorten zijn fylogenetisch verwant, hebben gedeelde voorouders, en delen derhalve naar alle waarschijnlijkheid ook een aantal essentiële virulentie-factoren. Van enkele leden van de Chaetothyriales zijn teleomorfen (sexuele stadia) bekend die saprofytair voorkomen op houtige planten en op paddestoelen (Untereiner et al. 1995, 1999b, Untereiner 1997, 2000). Het wordt aangenomen dat in de vroegste evolutie van de Chaetothyriales gelicheniseerde soorten voorkwamen, en dat de opportunisten en saprofieten hiervan afstammen na verlies van lichenisatie (Lutzoni et al. 2001, James et al. 2006). Gueidan et al. (2008) toonden aan dat de deze groepen veel species bevatten die ook zonder lichenisatie op gesteente kunnen leven. Ook de oudste directe voorouder van de Chaetothyriales was een fungus die rotsen koloniseerde. Daarvan is extremotolerantie als belangrijke eigenschap overgebleven (de Hoog 1993, Haase et al. 1999, Prenafeta-Boldú et al. 2006). Verschillende auteurs (o.a. Schnitzler et al. 1999, Feng et al. 2001) veronderstelden daarom dat aanwezigheid van melanine in de celwand en meristematische groei de potentie tot invasieve groei in mensenlijk weefsel hebben bevorderd. Het is ook niet altijd eenvoudig zwarte gisten en verwanten in de Chaetothyriales uit de omgeving te isoleren. Dat lukt vaak alleen indien ophopings-methoden worden toegepast, zoals met gebruikmaking van giftige monoaromaten als selectieve stof (Prenafeta-Boldú et al. 2006), of inoculatie in een proefdier. Sommige soorten komen in passieve, zgn. meristematische stadia voor op resten van moeilijk afbreekbare planten. Het werd altijd aangenomen dat ze het menselijk lichaam binnenkomen via trauma, bij voorbeeld na een prik van een plantenstekel. Maar we hebben gemerkt dat deze relatie niet zo eenduidig is, want de stammen uit de omgeving zijn vaak nauw verwant, maar niet identiek met de stam op de plant. Meestal levert isolatie van monsters die verondersteld worden de bron van de infectie te zijn zwarte gisten op die wel lijken op de invasieve stammen, maar tot andere soorten van hetzelfde genus behoren. Cladophialophora yegresii bij voorbeeld komt voor als endofiet in stekels van levende Stenocereus cactusplanten. In een onderzoek (Zeppenfeldt et al. 1994) werd deze soort meermaals geïsoleerd van een cactus-haag rond de woning van een patiënt met chromoblastomycose veroorzaakt door C. carrionii. Hoewel sommige publicaties (b.v. Salgado et al. 2004) suggereren dat de infectieuze organismen direct uit de omgeving geïsoleerd kunnen worden, blijkt deze relatie toch ingewikkelder. Voor veel soorten, 10 zoals voor de neurotrope species Cladophialophora bantiana, wordt nog steeds gezocht naar de omgevings niche. Onze hypothese is dat er een evolutionaire adaptatie stap zit tussen de direct

141 Chapter 10 isoleerbare soort, en z’n invasieve afgeleide. De vele gastheerwisselingen in vele takken van de Chaetothyriales kunnen deels worden verklaard door kenmerken met een lange evolutionaire geschiedenis, die geëvolueerd zijn in voorouders met aanpassingen aan extreme condities, bijvoorbeeld op kaal gesteente. Omdat klinische stamen moeten kunnen groeien bij de lichaamstemperatuur van zoogdieren, is thermotolerantie een eerste vereiste voor het ontstaan van pathogenen op mens en dier (Prenafeta-Boldú et al. 2006). Maar andere eigenschappen zijn wellicht belangrijker. De Hoog et al. (2007) voerden inoculatie experimenten uit met levende cactusplanten, waarin Cladophialophora carrionii (een stam van een menselijke patiënt) en C. yegresii (een stam van cactus) werden geïnoculeerd. De auteurs konden aantonen dat beide soorten in de levende plant konden groeien, met geinge weefselschade. Cactus weefsel bevat veel koolstof, vitaminen en mineralen (Vélez & Chávez 1980), welke de endofytische groei bevorderen. De twee soorten verschilden in de mate waarin bij inoculatie in volwassen planten wonden werden veroorzaakt. De conclusie was dat de klinische soort C. carrionii in de plant virulenter was dan C. yegresii van dezelfde plantaardige gastheer. Het verschil in virulentie werd verklaard door aan te nemen dat C. carrionii, die ook is gevonden al seen saprofiet op dode cactus, minder is aangepast aan een endofytische levensstijl. In de natuur komen deze fungi waarschijnlijk alleen de plant binnen via bestaande wonden, bij voorbeeld bij geitenvraat of via vogels of insecten op zoek naar cactus sap. De twee soorten vertonen vergelijkbare transitie naar meristematische groei (González et al. 1990) wanneer ze hard stekel weefsel tegenkomen dat rijk is aan lignine. Lignine is een complexe chemische verbinding die relatief hydrofoob is en benzeen-eenheden bevat. De stof is een hoofdbestanddeel van hout, en komt voor in de celwand van veel planten. Het speelt een belangrijke rol in de geleiding van water door plantenstelen, en is anderzijds mogelijk betrokken in de meristematische conversie in b.v. cactus-stekels. Deze stekels spelen niet alleen een rol bij de passieve verspreiding van de schimmels, maar kunnen ook in de huid van mens en dier worden geïnoculeerd. Daar worden vergelijkbare muriforme cellen gevormd, die nu verantwoordelijk zijn voor de huidziekte chromoblastomycose. Een interessante vraag is of pathogeniteit op dier en mens vervolgens ook een rol speelt in de evolutie van Chaetothyriales. Ook de pathogene gist Cryptococcus neoformans brengt een deel van zijn levenscyclus door in holle Eucalyptus bomen. De gist produceert diphenol oxidasen om lignine aft e breken. Vergelijkbare enzymen komen voor in Cladophialophora carrionii (Prenafeta- Boldú et al. 2006). Tolerantie van extreme temperaturen, meristematische groei, melaninevorming en de assimilatie van aromatische componenten dragen allemaal bij aan de potentie infecties te veroorzaken, waardoor we zo’n veelheid aan klinische syndromen kennen in de verschillende afstammingslijnen der Chaetothyriales. Corbellini et al. (2006) bestudeerde vertraagde overgevoeligheid tegen ruwe en gezuiverde antigenen van Fonsecaea pedrosoi en bewezen dat huidtesten tegen antigenen in synthetische media kunnen worden gebruikt om vroege infecties door veroorzakers van chromoblastomycose op te sporen. De aanwezigheid van geheugen suggereert dat de gastheer in de loop van de evolutie veelvuldig met infecties in aanraking is geweest. d’Ávila et al. (2002) demonstreerde cellulaire weefsel reacties in chromoblastomycose. Zij concludeerden dat patiënten met ruwe lesies een T-helper cel-2 antwoord hadden, terwijl patiënten met atrofiërende lesies een Th-1 antwoord hadden. Iwatsu et al. (1982) demonsteerden het bestaan van cutane vertraagde overgevoeligheid via dierexperimenten. Esterre et al. (2006) meenden dat humorale immuniteit vergeleken met specifieke cellulaire immuniteit geen grote rol speelt in de bescherming tegen chromoblastomycose. Alles tesamen nemend kan worden geconcludeerd dat cellulaire adaptieve immuniteit erg belangrijk is in chromoblastomycose. Hoogst waarschijnlijk is er dus een langdurige relatie tussen gastheer en pathogen, en is er pathogeniteit ontstaan waar voordien slechts sprake was van opportunisme. Dit

142 Disscusion komt overeen met de constatering dat in endemische gebieden mensen heel frequent geïnfecteerd zijn met C. carrionii, F. pedrosoi of F. monophora, en dat andere, nauw verwante Cladophialophora en Fonsecaea soorten geen chromoblastomycose kunnen veroorzaken (de Hoog et al. 2007, Vicente et al. 2008). Infecties van het centrale zenuwstelsel door C. bantiana komen wereldwijd voor, maar vooral in warmere en vochtige klimaat zones (Horré et al. 1999). De infectieroute is vermoedelijk hematogene verspreiding vanuit een voordien niet opgemerkte haard in de longen, en niet door directe uitbreiding van lesies vanuit de paranasale holten. Verreweg de meeste patiëten met cerebrale abscessen hadden geen van beide infectiehaarden (Horré et al. 1999). Cladophialophora bantiana kan de bloed-hersenbarrière van laboratoriumdieren passeren (Al-Abdely et al. 2005a). Het nauwe ecologische spectrum van C. bantiana (bijna alleen in menselijke hersenen) lijkt op een bestaan als een echt pathogeen in de menselijke / dierlijke gastheer te wijzen. AFLP profielen van stammen van deze soort lijken erg veel op elkaar, en maken de indruk dat C. bantiana bestaat uit een enkele, recent ontstane kloon. Veroorzakers van sommige humane ziektebeelden hebben hun natuurlijke habitat in de omgeving, en ze hun infecties zijn erg zeldzaam. Maar het voortdurend weerkerende opportunism van leden van de orde Chaetothyriales lijkt in een aantal gevallen geleid te hebben tot een bepaalde graad van pathogeniteit. De meeste leden van de groep kunnen infecties veroorzaken in voor het overige gezonde personen, maar lijken geen efficiënte mechanismen te hebben om de gastheer binnen te dringen. Om hun evolutie beter te kunnen begrijpen, is het nodig evolutionaire voorourders te reconstrueren om gastheerwisselingen te localiseren (Hibbett et al. 2000, Lutzoni et al. 2001). De doorslaggevende factoren bij zulke adaptaties zouden echter weleens ongewoon kunnen zijn en daardoor moeilijk te vinden, zoals het vermogen om benzeen-achtige stoffen te assimileren. Meer kennis over primaire niches, ecologische voorkeuren en factoren die bij pathogeniteit betrokken zijn zijn nodig om de ecologie van de Chaetothyriales te begrijpen. In sommige delen van de wereld vormen opportunistische schimmels een ernstige bedreiging voor de volksgezondheid, en verder onderzoek naar hun ecologie en evolutie is noodzakelijk. Hoofstuk een is een overzicht van de gemelaniseerde fungi (zwarte gisten en verwanten) in de orde Chaetothyriales, voornamelijk in de familie Herpotrichiellaceae, en met accent op het geslacht Cladophialophora. De huidige taxonomie en ecologie van de zwarte gisten wordt besproken. Hoofdstuk twee is is het belangrijkste hoofdstuk van dit proefschrift en vergelijkt ecologische informatie met phylogenetische and taxonomische data van ziekteverwekkende Cladophialophora soorten. De resultaten worden geïnterpreteerd in het licht van de mogelijke gezondheidsrisico’s van ogenschijnlijk saprofytaire schimmels in voedingsmiddelen. Een multilocus studie werd verricht met behulp van de internal transcribed spacer region (ITS rDNA), de translation elongation factor 1-α (EF1-α), een deel van het beta tubulin gen (TUB), en de kleine subeenheid van het ribosomale kern RNA (nucSSU). Vier nieuwe soorten werden ontdekt in soft drinks, in grond verontreinigd met alkylbenzenen, en in menselijke patiënten. Het behoren tot de carrionii of bantiana clades geeft een potentiële virulente potentie aan. De evolutie van pathogeniteit in het geslacht Cladophialophora wordt bestudeerd in het licht van aanpassingen aan extreme milieu’s, met factoren als melanine productie, meristematische groei, groei bij lage pH, en thermotolerantie. Hoofdstuk drie beschrijft een nieuwe soort: Cladophialophora saturnica, een opportunistist in de Chaetothyriales van een interdigitale tinea nigra-achtige lesie in een HIV-positief Braziliaans kind. De soort verschilt van bekende Cladophialophora species door verschillen in ITS rDNA en het partiële EF1-α gen, maar ook door een andere ecologie. Dezelfde soort werd uit de natuur 10 geïsoleerd met behulp van selectieve isolatie-technieken. Hoofdstuk vier beschrijft een nieuwe soort van Cladophialophora uit grond vervuild met

143 benzeen, tolueen, ethylbenzeen en xyleen (BTEX). De soort bleek te kunnen groeien met tolueen en andere alkylbenzenen als koolstof- en energiebron. De fungus is belangrijk vanwege de complete afbraak van tolueen en andere xenobiotica onder groeibeperkende factoren, en is toepasbaar in het bijzonder in lucht biofilters die opereren onder droge en/of zure condities. Een eerste MLST en AFLP analyse toonde aan dat deze schimmel nauw verwant is met de neutrotrope fungus C. bantiana; zelfs een C. bantiana-specifiek SSU intron bleek identiek. Maar de soort kon niet groeien bij 40 °C en bleek in een muis-experiment niet virulent te zijn. Een goede fylogenetische en ecofysiologische afgrenzing van deze soort is essentieel met het oog op het voorkomen van gezondheidsrisico’s bij toepassingen in de bioremediatie. Hoofdstuk vijf evaluateert de inter- en intraspecifieke genomische variatie van 42 Cladophialophora isolaten van ziektegevallen met o.a. cerebrale phaeohyphomycose. Stammen werden vergeleken met behulp van de ‘vingerafdruk’ techniek AFLP. Duidelijke soort-specifieke predilecties konden worden vastgesteld. Vervolgens werden de gevoeligheid bepaald tegen acht conventionele en moderne middelen tegen C. bantiana, om de antifungale therapie in patiënten te verbeteren. Hoofdstuk zes betreft de herdeterminatie van alle klinische en omgevingsisolaten die in de CBS collectie zijn bewaard onder de naam Exophiala jeanselmei. Daartoe wordt het ITS gesequenced en de klinische manifestaties geanalyseerd. Een nieuw ziektegeval van een mycetoma door deze schimmel wordt beschreven. De soort blijkt betrokken in diverse typen cutane en vooral subcutane infecties. Vanwege de hoge incidentie van eumycetoma in endemische gebieden wordt ook de in vitro gevoeligheid van E. jeanselmei voor verschillende antimycotica bepaald, om een betere therapie te ontwikkelen. Hoofdstuk zeven behandelt de indeling van chromoblastomycose door Carrión in 1950 in vijf verschillende typen (nodulair, plaque type, tumoraal, cicatriciaal, hyperkeratotisch). We beschrijven een nieuw type chromoblastomycose, nl. met lymphatische verspreiding. We tonen aan dat Rhinocladiella aquaspersa chromoblastomycose en phaeohyphomycose kan veroorzaken. Vanwege de moeilijke behandeling van chromoblastomycose en andere door R. aquaspersa veroorzaakte infecties evalueren we in vitro gevoeligheden tegen een uitgebreid panel triazoles, amphotericin B, en echinocandins. Hoofdstuk acht behandelt het eerste geval van een cerebrale infectie door Rhinocladiella mackenziei buiten de endemische area. Patiënt was een man van middelbare leeftijd in India. Dit ziektebeeld komt veel voor in het Midden-Oosten, en leidt dan tot praktisch 100 % mortaliteit, zelfs met gecombineerde chirurgie en antimycotische therapie. De soort komt ook voor in ogenschijnlijk gezonde patiënten. Hoofdstuk tien evalueert de in vitro activiteit van een nieuw triazol antimycoticum, isavuconazol, en zeven ander middelen (amphotericine B, fluconazol, itraconazol, voriconazol, posaconazol, caspofungine and anidulafungine) tegen hoogvirulente isolaten van Rhinocladiella mackenziei. Sommige R. mackenziei infecties leiden tot hersenabscessen in patiënten zonder bijzondere predispositie of immunodeficiëntie. Behandeling is meestal met amphotericine B, itraconazol en 5-flucytosine, maar meestal met matig resultaat, in dierexperimenten zowel als in klinische studies. De nieuwe stoffen zijn veelbelovend, hoewel voor vaststelling van de effectiviteit van isavuconazol tegen R. mackenziei nog verdere dier-experimenten nodig zijn.

144 Disscusion

Acknowledgements My dream started since 2004 when I passed the exam and received a scholarship from the Ministry of Health and Medical Education of Iran. It was a great challenge for me and finally my dream became true when Prof. Dr. de Hoog accepted me as a PhD student in the Netherlands. I had a wonderful and pleasend time during this period of my life. Working in several laboratories gave me an opportunity to meet different persons and to make very good friends. Therefore, in general I would like to acknowledge all who supported me during my PhD period, because without their help this would not have been possible. Those people and institutes involved I would like to thank wholeheartedly here. I would like to thank Ministry of Health and Medical Education of Iran for the financial support (grant No, 13081), also on behalf of my family. I would like to express my appreciation to my promoter Sybren de Hoog for giving me thies opportunity to carry out my PhD in his group at CBS Fungal Biodiversity Centre (CBS-KNAW). Thank you for your kind invitation, excellent supervision, patient guidance, encouragement, advice, valuable discussions and critical reading of manuscripts. I really enjoyed working in your group as a team working and appreciated your optimistic words and flexibility. Also I would like to thank you for all the time that you spent for me to discuss both scientific and personal problems. I would like to also express my deepest gratitude and appreciation to my second supervisor Prof. Dr. Steph Menken for his helpful advice, useful comments, invaluable guidance, and continuous encouragements throughout the time of my studies. I am deeply indebted to Dr. Jacques Meis for giving me the opportunity to dedicate time to my studies in the Canisius Wilhelmina Hospital (CWZ) in Nijmegen. Dear Dr. Jacques, you guided me how to become an objective scientist and I appreciate your sharp and useful comments and suggestions on the obtained results. Thank you also for introducing another style in my work. You always sharing with me your scientific experience and motivating me in goal oriented approach. Finally, I thank Labland BV, Wijchen for additional financial support. I am really indebted to Prof. dr. Walter Gams and I express my special thanks to him. Dear Walter, I appreciate your sharp and useful comments and suggestions with critical review of my manuscripts. Thank you also of Sophia, for all the warm receptions and hospitalities at your house in Baarn. I would like to express my deepest gratitude and appreciation to Dr. Grit Walther and Dr. Cecile Gueidan who were joined to our group at CBS since 2007 until 2009. I really enjoyed working with both of you and I learned scientific goal orientation. Dear Grit and Cecile thank you for useful comments, invaluable guidance, and continuous encouragements throughout the time of my studies. Thank you so much for everything, your teaching, your flexibility. I really appreciate your friendship. I am very grateful to Kasper Luijsterburg and Bert Gerrits van den Ende, my daily supervisors in our group for their helpful technical assistance during my experiments. I really enjoyed working in the BYP group, thank you so much for everything. I wish to express my appreciation to Dr. Corné. H. Klaassen, Ilse. Breuker-Curfs and Erik Geertsen of the Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands for their technical help in molecular and mycological analysis and their professional comments to direct me through this part of my research. Many thanks to all the staff members of CBS-KNAW fungal biodiversity Centre, Utrecht, The Netherlands for your great support during my study; I would like to express my deepest gratitude 10 and appreciation to all staff members of the departments of culture collection, because without you not only my project, but also all research at CBS would not have been possible. Special thanks

145 Chapter 10 to Marlene Setropawiro for your entire predisposition to help me with anything that I asked. My colleagues from the Department of Microbiology, Government Medical College Hospital, Chandigarh, India, especially Prof. Dr. Chander and his team from whom I received good clinical samples in the field of medical mycology, are thanked for their kind cooparation I would like to thank my internal promoter in Iran, Prof. Dr. Shokohi for excellent collaboration and helpful discussions scientifically and personallity. Thanks to all my Iranian friends in the Netherlands because I had a good time with all of you: Farid, Atosa, Ramin, Maryam, Esmael, Fahimeh, Ramin (Rezapour), Mahnaz, Mohammad, Zohreh, Hadi, Mehdi, Reza, Mehdi (rad), Fatemeh, Sadegh, Javad, Amir, Haleh. I would like to express my especial appreciation to my father, my dear mother and my family for their warm feelings and moral support. They always encouraged me to study continuously. Thank you for your concern, support and strength during all these years. My deepest gratitude to wife, Sahar for your understanding, patience, flexibility and support.I do not know how to thank. My dear and little son, Ilya, I am very happy and glad with every progress you make.

About the Author Hamid Badali was born on 15th of April 1977 in Iran, Zanjan in the North West of Iran, where he did his studies from primary to high school. Subsequently he carried on his education in the Azad University in field of Molecular and Cellular Microbiology (BSc). Since 2000 he moved to North East of Iran to do his Master of Science at Mazandaran University of Medical Sciences, Sari, in the field of Medical Mycology. His MSs project was entitled ‘Immunoblotting analysis of sera from patients with acute and chronic vaginitis for IgE and IgG antibodies against Candida albicans’. In 2005 he was supported by a grant (No. 13081) from the Ministry of Health and Medical Education of the Islamic Republic of Iran and the School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran for a PhD degree. Thereafter, he joined the Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam and the CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands where he started his PhD program. His research project focused on biodiversity, pathogenicity and antifungal susceptibility of black yeast and relatives, particularly on Cladophialophora species, from March 2006 until June 2010. In the meantime, as a researcher internship he had collaboration with at the department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands. The results he obtained are published in this book and related journals. During this time he was carried out his project under the supervision of Prof. Dr. G.S. de Hoog and Prof. Dr. S.B.J Menken. Finally, after defence on 25th of June 2010 he will join to the department of Medical Mycology/Molecular and Cell Biology Research Center, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.

List of Publications Badali H, de Hoog GS, Breuker-Curfs I, Klaassen W CH, Meis JF (2010). Use of Amplified Fragment Length Polymorphism to identify 42 Cladophialophora strains related to cerebral phaeohyphomycosis with in vitro antifungal susceptibility. J Clin Microbiol (in press). Badali H, Bonifaz A, Barrón-Tapia T, Vázquez-González D, Estrada-Aguilar L, Cavalcante Oliveira NM, Sobral Filho JF, Guarro J, Meis JF, de Hoog GS (2010). Rhinocladiella aquaspersa, proven agent of verrucous skin infection and a novel type of chromoblastomycosis. Med Mycol Jan 29. Najafzadeh MJ, Badali H, Illnait-Zaragozi MT, de Hoog GS, Meis JF (2010). In vitro activities of eight antifungal drugs against 55 clinical isolates of Fonsecaea spp. Antimicrob Agents Chemother 54:1636-1638. Badali H, Chander J, Gulati N, Attri A, Chopra R, Najafzadeh MJ, Chhabra S, Meis JF, de Hoog GS (2010). Subcutaneous phaeohyphomycotic cyst caused by Pyrenochaeta romeroi. Med Mycol Jan 8. Badali H, Chander J, Bansal S, Aher A, Borkar SS, Meis JF, de Hoog GS (2010). First autochthonous case of Rhinocladiella

146 Disscusion

mackenziei cerebral abscess outside the Middle East. J Clin Microbiol 48: 646-649. Badali H, de Hoog GS, Curfs-Breuker I, Meis JF. (2010). In vitro activities of antifungal drugs against Rhinocladiella mackenziei, an agent of fatal brain infection. J Antimicrob Chemother 65(1): 175-177. Badali H, Najafzadeh MJ, van Esbroeck M, van den Enden E, Tarazooie B, Meis JF, de Hoog GS (2010). The clinical spectrum of Exophiala jeanselmei, with a case report and in vitro antifungal susceptibility of the species. Med Mycol (in press). Najafzadeh MJ, Rezusta A, Cameo MI, Zubiri ML, Yus MC, Badali H, Revillo MJ, de Hoog GS (2010). Successful treatment of chromoblastomycosis of 36 years duration caused by Fonsecaea monophora. Med Mycol (in press). Badali H, de Hoog GS, Curfs-Breuker I, Andersen B, Meis JF (2009). In vitro activities of eight antifungal drugs against 70 clinical and environmental isolates of Alternaria species.J Antimicrob Chemother 63: 1295-1297. Najafzadeh MJ, Gueidan C, Badali H, van den Ende AH, Xi L, de Hoog GS (2009).Genetic diversity and species delimitation in the opportunistic genus Fonsecaea. Med Mycol 47: 17-25. Nourian AA, Amiri M, Ataeian A, Haniloo A, Mosavinasab SN, Badali H (2008). Seroepidemiological study for toxocariasis among children in Zanjan-northwest of Iran. Pak J Biol Sci 11: 1844-1847. Badali H, Carvalho VO, Vicente V, Attili-Angelis D, Kwiatkowski IB, Gerrits van den ende AH, de Hoog GS (2009). Cladophialophora saturnica sp. nov., a new opportunistic species of Chaetothyriales revealed using molecular data. Med Mycol 47: 51-62. de Hoog GS, Nishikaku AS, Fernandez-Zeppenfeldt G, Padín-González C, Burger E, Badali H, Richard-Yegres N, van den Ende AH (2007). Molecular analysis and pathogenicity of the Cladophialophora carrionii complex, with the description of a novel species. Stud Mycol 58: 219-234. Badali H, Gueidan C, Najafzadeh MJ, Bonifaz A, van den Ende AH, de Hoog GS (2008). Biodiversity of the genus Cladophialophora. Stud Mycol 61: 175-191. Bonifaz A, Badali H, de Hoog GS, Cruz M, Araiza J, Cruz MA, Fierro L, Ponce RM (2008). Tinea nigra by Hortaea werneckii, a report of 22 cases from Mexico. Stud Mycol 61: 77-82.

List of Abstracts Badali H, de Hoog GS, Curfs-Breuker I, Klaassen CH, Meis JF. Use of Amplified Fragment Length Polymorphism for identification of 29 neurotropicCladophialophora isolates with in vitro activities of eight antifungal drugs. Poster at the 4th Trends in Medical Mycology, Athens, Greece, October 18-21, 2009. Badali H, Najafzadeh MJ, de Hoog GS, Curfs-Breuker I, Meis JF. Microdilution in vitro susceptibility of agents of chromoblastomycosis, Fonsecaea pedrosoi and Fonsecaea monophora, for 8 antifungal agents. Poster at the 4th Trends in Medical Mycology, Athens, Greece, October 18-21, 2009 . Badali H, Najafzadeh MJ, Vanesbroeck M, Van den Enden E, Meis JF de Hoog GS. The clinically important black yeast, Exophiala jeanselmei and its in vitro susceptibility to antifungal agents. Poster at the 4th Trends in Medical Mycology, Athens, Greece, October 18-21, 2009 . Badali H, de Hoog GS, Curfs-Breuker I, Meis JF. Antifungal activity of isavuconazole and comparators against chromoblastomycotic Cladophialophora spp. Poster at the 4th Trends in Medical Mycology, Athens, Greece, October 18-21, 2009 . Badali H, de Hoog GS, Breuker-Curfs I, Andersen B, Meis JF. In vitro activities of eight antifungal drugs against 70 clinical and environmental isolates of Alternaria species. Oral presentation at the 17th congress of International Society for Human and Animal Mycology 2009, May 25-29 Tokyo, Japan. Badali H, de Hoog GS, Breuker-Curfs I, Heep M, Meis JF. In vitro activities of conventional and new antifungal drugs against Rhinocladiella mackenziei an agent of cerebral phaeohyphomycosis. Oral presentation at the 17th congress of International Society for Human and Animal Mycology 2009, May 25-29 Tokyo, Japan. Najafzadeh MJ, Gueidan C, Badali H, Gerrits van den Ende AHG, de Hoog GS. Genetic diversity and species delimitation in the opportunistic genus Fonsecaea. 17th congress of International Society for Human and Animal Mycology 2009, May 25-29 Tokyo, Japan. Badali H, Gueidan C, Gerrits van den Ende AHG, de Hoog GS. Cladophialophora with an infectious potential occurring in soft drinks. International congress of mycology, IUMS 2008, August 5-9, Istanbul, Turkey. Badali H, de Hoog GS, Gerrits van den Ende AHG, Najafzadeh MJ, Biodiversity of the genus Cladophialophora, agent of human infection, 3rd trend Medical Mycology congress 2007, October 27- 31 Torino, Italy. Najafzadeh MJ, Badali H, Gerrits van den Ende AHG, de Hoog GS. Multilocus Sequence Typing (MLST) of the pathogenic genus Fonsecaea. 3rd Trends in Medical Mycology congress 2007, October 27- 31 Torino, Italy. Badali H, Vicente V, Attili-Angelis D, Kwiatkowski IB, Gerrits van den Ende AHG, de Hoog GS, A new, opportunistic species of Cladophialophora. 12th Netherlands Society for Medical Mycology 2007, December. 10 Badali H, Nourian AA, Mohseni S, Haniloo A, Khodaverdi M, Hamzehei H, Airborne fungi isolated from Zanjan– Iran.12th International Congress on Infection Disease, 2006, June Portugal, Lisbon. Hedayati MT, Badali H. Mycoflora of stored wheat in warehouses of Mazandaran Province, Iran. 13t VAM congress 2001,

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August 27-30, Kuala Lumpur, Malaysia. Hedayati MT, Badali H, Vasheghani F, Aghili R, Mohammad Pour RA. Immunoblotting analysis of sera from patients with acute and chronic candidal and noncandidal vaginitis for IgE and IgG antibodies to Candida albicans. 13th European Congress of Clinical Microbiology and Infectious Diseases, 2003 May 10 -13, Glasgow, UK.

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Scholarships, Awards and Honors: 1. Scholarship for PhD degree in field of Medical Mycology from the Ministry of Health and Medical Education of Islamic Republic of Iran from 2006 to 2010. 2. ISHAM young investigators travel award worth 1500 $ for an outstanding presentation at the 17th Congress of The International Society for Human and Animal Mycology, Tokyo, Japan, May 2009 3. ECMM young investigators travel award worth 1000 Euro for an outstanding presentation at the 4th Trends in Medical Mycology, Athens, Greece, October 18-21, 2009

Professional memberships: 1. Member of the International Society of Human and Animal Mycology (ISHAM). 2. Member of the Young International Society of Human and Animal Mycology (ISHAM). 3. Member of the European Society of Clinical Microbiology and Infection Disease (ESCMID). 4. The Young ISHAM’ representative for Iran, Turkey and Middle East [email protected]

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Biodiversity, pathogenicity and antifungal susceptibility of

Biodiversity, pathogenicity and antifungal susceptibility of Cladophialophora and relatives Cladophialophora and relatives

Hamid Badali Hamid Badali

2010