Peiying Feng

Global ITS diversity in the Sporothrix schenckii complex

Molecular phylogeny has revealed that sporotrichosis is caused by several Sporothrix species which differ in clinical behavior. The complex is embedded within Ophiostoma, a genus mainly comprising fungi that live in association with bark beetles, but differs by a high virulence towards humans and other mammals. The different ecology is corroborated by phylogenetic separation. The aim of the present study was to determine the validity of the rDNA internal transcribed spacer (ITS) region as a marker for diagnostics of species in the clinical group, using beta-tubulin sequences to calibrate species delimitations. The topology of the two gene trees was concordant, and all clinically relevant Sporothrix species could easily be recognized by means of the ITS region. An increased geographic sampling did not affected delimitation success in the clinical clade of the S. schenckii complex.

Phylogeography and evolutionary patterns in Sporothrix spanning more than 14 000 human and animal case reports.

Pathology to vertebrate hosts has emerged repeatedly in the order Ophiostomatales. Occasional infections have been observed in Sporothrix mexicana at a low level of virulence, while the main pathogenic species cluster in a derived clade around S. schenckii s.str. In this paper, phylogeny and epidemiology of the members of this clade were investigated for 99 clinical and 36 environmental strains using four genetic loci, viz. rDNA ITS and partial CAL, TEF1, and TEF3; data are compared with amplified fragment length polymorphism (AFLP) genotyping. The four main species of the pathogenic clade were recognised. The species proved to show high degrees of endemicity, which enabled interpretation of literature data where live material or genetic information is lacking. The clade of four species comprised nine subclusters, which often had limited geographic distribution and were separate from each other in all partitions, suggesting low degrees of interbreeding between populations. In contrast, S. globosa exhibited consistent global distribution of identical AFLP types, suggesting another type of dispersal. Sporothrix brasiliensis is known to be involved in an expanding zoonosis and transmitted by cats, whereas S. globosa infections originated from putrid plant material, causing a sapronosis. Sporothrix schenckii s.str., the most variable species within the clade, also had a plant origin, with ecological similarities to that of S. globosa. A hypothesis was put forward that highly specific conditions in the plant material are required to promote the growth of Sporothrix. Fermented, self-heated plant debris may stimulate the thermodependent yeast-like invasive form of the fungus, which facilitates repeated infection of mammals.

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.

Novel taxa of thermally dimorphic systemic pathogens in the Ajellomycetaceae (Onygenales).

Recent discoveries of novel systemic fungal pathogens with thermally dimorphic yeast‐like phases have challenged the current taxonomy of the Ajellomycetaceae, a family currently comprising the genera Blastomyces, Emmonsia, Emmonsiellopsis, Helicocarpus, Histoplasma, Lacazia and Paracoccidioides. Our morphological, phylogenetic and phylogenomic analyses demonstrated species relationships and their specific phenotypes, clarified generic boundaries and provided the first annotated genome assemblies to support the description of two new species. A new genus, Emergomyces, accommodates Emmonsia pasteuriana as type species, and the new species Emergomyces africanus, the aetiological agent of case series of disseminated infections in South Africa. Both species produce small yeast cells that bud at a narrow base at 37°C and lack adiaspores, classically associated with the genus Emmonsia. Another novel dimorphic pathogen, producing broad‐based budding cells at 37°C and occurring outside North America, proved to belong to the genus Blastomyces, and is described as Blastomyces percursus.

50 Years of Emmonsia Disease in Humans: The Dramatic Emergence of a Cluster of Novel Fungal Pathogens

CITATION: Schwartz, I. S. et al. 2015. 50 years of Emmonsia disease in humans : the dramatic emergence of a cluster of novel fungal pathogens. PLoS Pathogens, 11(11): e1005198, doi:10.1371/journal.ppat.1005198.

Detection and identification of opportunistic Exophiala species using the rolling circle amplification of ribosomal internal transcribed spacers.

Deep infections by melanized fungi deserve special attention because of a potentially fatal, cerebral or disseminated course of disease in otherwise healthy patients. Timely diagnostics are a major problem with these infections. Rolling circle amplification (RCA) is a sensitive, specific and reproducible isothermal DNA amplification technique for rapid molecular identification of microorganisms. RCA-based diagnostics are characterized by good reproducibility, with few amplification errors compared to PCR. The method is applied here to species of Exophiala known to cause systemic infections in humans. The ITS rDNA region of five Exophiala species (E. dermatitidis, E. oligosperma, E. spinifera, E. xenobiotica, and E. jeanselmei) was sequenced and aligned in view of designing specific padlock probes to be used for the detection of single nucleotide polymorphisms (SNPs) of the Exophiala species concerned. The assay proved to successfully amplify DNA of the target fungi at the level of species; while no cross-reactivity was observed. Amplification products were visualized on 1% agarose gels to verify the specificity of probe-template binding. Amounts of reagents were minimized to avoid the generation of false positive results. The sensitivity of RCA may help to improve early diagnostics of these difficult to diagnose infections.

Spectrum of Fusarium infections in tropical dermatology evidenced by multilocus sequencing typing diagnostics.

Fusarium species are emerging causative agents of superficial, cutaneous and systemic human infections. In a study of the prevalence and genetic diversity of 464 fungal isolates from a dermatological ward in Thailand, 44 strains (9.5%) proved to belong to the genus Fusarium. Species identification was based on sequencing a portion of translation elongation factor 1‐alpha (tef1‐α), rDNA internal transcribed spacer and RNA‐dependent polymerase subunit II (rpb2). Our results revealed that 37 isolates (84%) belonged to the Fusarium solani species complex (FSSC), one strain matched with Fusarium oxysporum (FOSC) complex 33, while six others belonged to the Fusarium incarnatum‐equiseti species complex. Within the FSSC two predominant clusters represented Fusarium falciforme and recently described F. keratoplasticum. No gender differences in susceptibility to Fusarium were noted, but infections on the right side of the body prevailed. Eighty‐nine per cent of the Fusarium isolates were involved in onychomycosis, while the remaining ones caused paronychia or severe tinea pedis. Comparing literature data, superficial infections by FSSC appear to be prevalent in Asia and Latin America, whereas FOSC is more common in Europe. The available data suggest that Fusarium is a common opportunistic human pathogens in tropical areas and has significant genetic variation worldwide.

Molecular Characterization of Pathogenic Members of the Genus Fonsecaea Using Multilocus Analysis

Members of the fungal genus Fonsecaea causing human chromoblastomycosis show substantial geographic structuring. Genetic identity of clinical and environmental strains suggests transmission from plant debris, while the evolutionary processes that have led to spatially separated populations have remained unexplained. Sequences of ITS, BT2, ACT1, Cdc42, Lac and HmgA were analyzed, either by direct sequencing or by cloning. Thirty-seven clinical and environmental Fonsecaea strains from Central and South America, Asia, Africa and Europe were sequenced and possible recombination events were calculated. Phylogenetic trees of Cdc42, Lac and HmgA were statistically supported, but ITS, BT2 and ACT1 trees were not. The Standardized Index of Association (IA S) did not detect recombination (IA S = 0.4778), neither did the Phi-test for separate genes. In Fonsecaea nubica non-synonymous mutations causing functional changes were observed in Lac gene, even though no selection pressures were detected with the neutrality test (Tajima D test, p>0.05). Genetic differentiation of populations for each gene showed separation of American, African and Asian populations. Strains of clinical vs. environmental origin showed genetic distances that were comparable or lower than found in geographic differentiation. In conclusion, here we demonstrated clonality of sibling species using multilocus data, geographic structuring of populations, and a low functional and structural selective constraint during evolution of the genus Fonsecaea.

Disseminated infection caused by Emmonsia pasteuriana in a renal transplant recipient.

Emmonsia pasteuriana is a thermally dimorphic fungus identified in very few human cases. Here, we report a case of a 43‐year‐old male renal transplant patient from China presenting with multiple painful skin eruptions on his head, nose and left thigh, later accompanied by respiratory failure. Histopathology of the biopsy collected from the left thigh upper ulcer and occipital nodule both demonstrated chronic inflammation with granuloma formation and yeast‐like elements. Emmonsia pasteuriana was cultured from two biopsy specimens and their identity was confirmed by sequencing of the rDNA internal transcribed spacer. The patient in intensive care showed marked clinical improvement with antifungal treatment.

Identification and Typing of Isolates of Cyphellophora and Relatives by Use of Amplified Fragment Length Polymorphism and Rolling Circle Amplification

ABSTRACT The species diversity and identification of black fungi belonging to Cyphellophora and Phialophora, which colonize and infect human skin and nails, were studied using amplified fragment length polymorphism (AFLP). A total of 76 Cyphellophora and Phialophora isolates were evaluated, and their delimitation was compared to earlier studies using multilocus sequencing. The results of the AFLP analysis and sequencing were in complete agreement with each other. Seven species-specific padlock probes for the most prevalent species were designed on the basis of the ribosomal DNA internal transcribed spacer region, and identification of the respective species could easily be achieved with the aid of rolling circle amplification.