M.J. Najafzadeh

Waterborne Exophiala species causing disease in cold-blooded animals

The majority of mesophilic waterborne species of the black yeast genus Exophiala (Chaetothyriales) belong to a single clade judging from SSU rDNA data. Most taxa are also found to cause cutaneous or disseminated infections in cold-blooded, water animals, occasionally reaching epidemic proportions. Hosts are mainly fish, frogs, toads, turtles or crabs, all sharing smooth, moist or mucous skins and waterborne or amphibian lifestyles; occasionally superficial infections in humans are noted. Cold-blooded animals with strictly terrestrial life styles, such as reptiles and birds are missing. It is concluded that animals with moist skins, i.e. those being waterborne and those possessing sweat glands, are more susceptible to black yeast infection. Melanin and the ability to assimilate alkylbenzenes are purported general virulence factors. Thermotolerance influences the choice of host. Exophiala species in ocean water mostly have maximum growth temperatures below 30 °C, whereas those able to grow until 33(−36) °C are found in shallow waters and occasionally on humans. Tissue responses vary with the phylogenetic position of the host, the lower animals showing poor granulome formation. Species circumscriptions have been determined by multilocus analyses involving partial ITS, TEF1, BT2 and ACT1.

Fonsecaea nubica sp. nov, a new agent of human chromoblastomycosis revealed using molecular data

A new species of Fonsecaea, Fonsecaea nubica, morphologically similar to F. pedrosoi and F. monophora, is described using multilocus molecular data including AFLP profiles, sequences of the ribosomal internal transcribed spacers (ITS), and partial sequences of the cell division cycle (cdc42), beta-tubulin (tub1) and actin (act1) genes. A phylogenetic approach was used to evaluate species delimitation. Topologies of the trees were concordant. Fonsecaea strains could be classified into three major entities, i.e., one representing Fonsecaea pedrosoi isolates, another consisting of strains of F. monophora, and a third, unnamed group comprising isolates mostly recovered from cases of chromoblastomycosis in South America and China. F. nubica is part of this latter group. Based on strains analyzed thus far, we have found that the pathologies of these three Fonsecaea species are somewhat different in that F. pedrosoi and F. nubica are preponderantly associated with chromoblastomycosis, while F. monophora may also act as a systemic opportunist in cases involving brain infections. The latter species is also the most frequently recovered of the three from environmental samples.

Molecular Epidemiology of Fonsecaea Species

These fungi disperse slowly, leading to changes in structure at different geographic locations.

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

Exophiala jeanselmei is clinically redefined 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 finds tumefaction, abscess formation and draining sinuses. The species has been described as being common in the environment, but molecular methods have only confirmed 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 reflects the taxonomy of this group. The first purpose of this study was the re-identification 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 of Exophiala infections remains to be determined.

Molecular techniques for pathogen identification and fungus detection in the environment.

Many species of fungi can cause disease in plants, animals and humans. Accurate and robust detection and quantification of fungi is essential for diagnosis, modeling and surveillance. Also direct detection of fungi enables a deeper understanding of natural microbial communities, particularly as a great many fungi are difficult or impossible to cultivate. In the last decade, effective amplification platforms, probe development and various quantitative PCR technologies have revolutionized research on fungal detection and identification. Examples of the latest technology in fungal detection and differentiation are discussed here.

In Vitro Activities of Eight Antifungal Drugs against 55 Clinical Isolates of Fonsecaea spp.

ABSTRACT The in vitro activities of eight antifungal drugs against clinical isolates of Fonsecaea pedrosoi (n = 21), Fonsecaea monophora (n = 25), and Fonsecaea nubica (n = 9) were tested. The resulting MIC90s for all strains (n = 55) were as follows, in increasing order: posaconazole, 0.063 μg/ml; itraconazole, 0.125 μg/ml; isavuconazole, 0.25 μg/ml; voriconazole, 0.5 μg/ml; amphotericin B, 2 μg/ml; caspofungin, 2 μg/ml; anidulafungin, 2 μg/ml; and fluconazole, 32 μg/ml.

Cyphellophora and its relatives in Phialophora: biodiversity and possible role in human infection

Cyphellophora is a genus of black yeast-like fungi characterised by having simple phialides with multiseptate, curved conidia. Judging from SSU and LSU data, Cyphellophora was found to be located in a well-supported clade within the Chaetothyriales comprising a number of species occurring on human skin and nail. Cyphellophora is phylogenetically close to Phialophora europaea, P. reptans and P. oxyspora, though morphologically these species produce single-celled phialoconidia rather than multiseptate ones. Pseudomicrodochium suttonii and P. fusarioides have dark colonies and phylogenetically fit in with Cyphellophora; the type species of Pseudomicrodochium, P. aciculare, has similar, septate conidia but has a hyaline thallus. In the present study, multilocus phylogenetic analyses were combined with morphology and physiology. Sequences of the internal transcribed spacer region, the DNA dependent RNA polymerase II largest subunit and the partial beta tubulin gene were analysed for a set of 30 strains. Two novel species, Cyphellophora pauciseptata and Phialophora ambigua were discovered. Cyphellophora eucalypti was reduced to synonymy of C. guyanensis. The role of the studied fungi between colonization and infection of human skin was discussed. Putative virulence factors for these black yeast-like fungi were hypothesized to be the ability to assimilate monoaromatic hydrocarbons, to produce melanin pigments, and to tolerate the temperature of epidermal human skin.

Identification of Pseudallescheria and Scedosporium Species by Three Molecular Methods

ABSTRACT The major clinically relevant species in Scedosporium (teleomorph Pseudallescheria) are Pseudallescheria boydii, Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium prolificans, while Pseudallescheria minutispora, Petriellopsis desertorum, and Scedosporium dehoogii are exceptional agents of disease. Three molecular methods targeting the partial β-tubulin gene were developed and evaluated to identify six closely related species of the S. apiospermum complex using quantitative real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), and loop-mediated isothermal amplification (LAMP). qPCR was not specific enough for the identification of all species but had the highest sensitivity. The PCR-RLB assay was efficient for the identification of five species. LAMP distinguished all six species unambiguously. The analytical sensitivities of qPCR, PCR-RLB, and LAMP combined with MagNAPure, CTAB (cetyltrimethylammonium bromide), and FTA filter (Whatman) extraction were 50, 5 × 103, and 5 × 102 cells/μl, respectively. When LAMP was combined with a simplified DNA extraction method using an FTA filter, identification to the species level was achieved within 2 h, including DNA extraction. The FTA-LAMP assay is therefore recommended as a cost-effective, simple, and rapid method for the identification of Scedosporium species.

Successful treatment of chromoblastomycosis of 36 years duration caused by Fonsecaea monophora

We report a case of chromoblastomycosis in a 67-year-old female farmer, which involved a large (20 x 30 cm) cicatricial erythematous plaque on the inner side of her right thigh. The lesion was initially a small nodule which gradually extended over 36 years. Direct microscopic examination revealed a granulomatous lesion with muriform cells surrounded by giant cells. The mould recovered in cultures was dark olivaceous and identified as Fonsecaea monophora by ribosomal internal transcribe spacer (ITS) sequence data. The lesion was successfully cured after 4 months treatment with itraconazole, but there was a relapse.

Rapid identification of fungal pathogens by rolling circle amplification using Fonsecaea as a model

We aimed to describe a rapid and sensitive assay for identification of pathogenic fungi without sequencing. The method of rolling circle amplification (RCA) is presented with species of Fonsecaea, agents of human chromoblastomycosis, as a model. The internal transcribed spacer (ITS) rDNA region of 103 Fonsecaea strains was sequenced and aligned in view of designing three specific padlock probes to be used for the detection of single nucleotide polymorphisms in three Fonsecaea species. The 38 strains included for testing the specificity of RCA comprised 17 isolates of Fonsecaea pedrosoi, 13 of Fonsecaea monophora and eight of Fonsecaea nubica. The assay successfully amplified DNA of the target fungi at the level of species, while no cross reactivity was observed. The amplification product was visualised on a 1% agarose gel to verify the specificity of probe–template binding. Amounts of reagents were minimised to avoid the generation of false‐positive results. The simplicity, sensitivity, robustness and low costs provide RCA a distinct position among isothermal techniques for DNA diagnostics as a very practical identification method.