Phialophora section Catenulatae disassembled: New genera, species, and combinations and a new family encompassing taxa with cleistothecial ascomata and phialidic asexual states.

The Pleuroascaceae (Leotiomycetes) is introduced for Phialophora hyalina (section Catenulatae) and its closest relatives based on analyses of DNA sequences of five gene regions and the comparison of cultural and micromorphological characters. The family is resolved as a strongly supported clade that encompasses Pleuroascus and the new anamorph genera Entimomentora and Venustampulla. The latter includes V. parva, a species placed formerly in Scopulariopsis, and V. echinocandica, which is established for the echinocandin-producing isolate BP-5553. Entimomentora includes E. hyalina, a species based on the ex-type strain of Ph. hyalina. Additional isolates identified as Ph. hyalina are distantly related to the Pleuroacaceae and include Psychrophila antarctica (Arachnopezizaceae) and Cryonesomyces dreyfussii, the sole member of the new genus Cryonesomyces (incertae sedis). Isolates identified or deposited as Ph. alba are also not closely related; they include a species for which we propose the name Neobulgaria koningiana (Gelatinodiscaceae) and a second psychrophilic species that we describe as Psychrophila lagodekhiensis. Of the 13 isolates assessed for in vitro antifungal activity, only V. echinocandica inhibited the growth of Candida albicans.

In vitro activities of nine antifungal drugs and their combinations against Phialophora verrucosa.

The in vitro activities of nine antifungal drugs and their combinations against 31 clinical and 15 environmental Phialophora verrucosa strains were tested. The MIC90/90% minimum effective concentration (MIC/MEC90) values (µg/ml) across all strains were as follows: for terbinafine, 0.25; for posaconazole, 0.5; for voriconazole, 1; for itraconazole, 2; for amphotericin B, 4; for caspofungin and micafungin, 16; and for fluconazole and flucytosine, 64. The highest synergy was shown by the combination of itraconazole plus caspofungin (with synergy against 100% of the 31 clinical strains), followed by amphotericin B plus flucytosine (45.2%) and itraconazole plus terbinafine or micafungin (25.8% or 12.9%, respectively).

Identification and typing of isolates of Cyphellophora and relatives by use of amplified fragment length polymorphism and rolling circle amplification.

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.

Identification of pathogenic dematiaceous fungi and related taxa based on large subunit ribosomal DNA D1/D2 domain sequence analysis.

The nucleotide sequences of the D1/D2 domains of large subunit (26S) ribosomal DNA for 76 strains of 46 species of pathogenic dematiaceous fungi and related taxa were determined. Intra-species sequence diversity of medically important dematiaceous fungi including Phialophora verrucosa, Fonsecaea pedrosoi, Fonsecaea compacta, Cladophialophora carrionii, Cladophialophora bantiana, Exophiala dermatitidis, Exophiala jeanselmei, Exophiala spinifera, Exophiala moniliae, and Hortaea werneckii were extremely small; as few as 0 changes were detected in C. bantiana, Fonsecaea and Exophiala species, 1 bp in C. carrionii and H. werneckii, and 2 bp in P. verrucosa. Inter-species nucleotide diversity between most species was higher. These data suggested that the D1/D2 domain is sufficiently variable for identification of pathogenic dematiaceous fungi and relevant species. The phylogenetic trees constructed from the sequence data revealed that most human pathogenic species formed a single cluster and that Cladosporium and Phialophora species were distributed polyphyletically into several clusters.

A new species, Phialophora europaea, causing superficial infections in humans.

A new species, Phialophora europaea, member of the P. verrucosa complex, is introduced. It is distinguished from existing species by reduced, flaring phialidic collarettes and inability to assimilate melibiose as sole source of carbon. Analysis of ITS1 and 2 rDNA of six strains attributed to the species show it to be clearly individualized. All strains originated from cutaneous and nail infections of humans in North-western Europe. A key to morphologically similar taxa is provided.

Phaeohyphomycosis caused by Phaeoacremonium inflatipes.

Phaeoacremonium inflatipes, one of three species previously classified as strains of Phialophora parasitica, was identified as the causal agent of a subcutaneous infection of the left foot of an 83-year-old woman from South Carolina. The patient had a granulomatous growth over the anteromedial aspect of her left foot. It was surgically excised, which led to complete healing without complications. Tissue sections of the excised mass stained with hematoxylin and eosin and Gomori's methenamine silver strains showed many septate hyphal elements of various lengths, some exhibiting brownish pigment in the cell walls of the hyphae. Portions of the tissue, when cultured, yielded many colonies which were initially glabrous, off white becoming velvety, greyish brown on aging. Microscopically, their hyphae were septate, branched, and phaeoid and bore lateral and terminal, erect, septate conidiophores. The conidiogenous cells (phialides) were terminal or lateral, mostly monophialidic, subcylindrical to spinelike in shape, and constricted at their bases and bore funnel-shaped, inconspicuous collarettes at their tips. The conidia were subhyaline, oblong, and ellipsoid to allantoid.

Mitochondrial DNA analysis of Phialophora verrucosa.

Restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) was examined in 32 isolates of Phialophora verrucosa (eight isolates from Japan, 10 from China, four from the USA, six from Venezuela and four from Colombia) and in three of Phialophora americana using five restriction enzymes. P. verrucosa isolates were divided into 10 mtDNA types based on RFLP patterns. Phylogeny constructed on sequence divergence of mtDNA indicated that P. verrucosa is a single species and isolates are clustered into three groups. Japan and the USA contained Group A and Group B isolates, China Group B isolates and South America Group B and Group C isolates. RFLP patterns of P. americana mtDNA were identical to those of Type 1 or Type 4 of P. verrucosa mtDNA, suggesting that both are identical. RFLP patterns of P. verrucosa were distinct from those of other dematiaceous fungi including Exophiala jeanselmei, E. moniliae, E. dermatitidis, E. spinifera, Cladophialophora (Cladosporium) carrionii, Fonsecaea pedrosoi, and Hortaea werneckii. These results indicate that RFLP analysis of mtDNA is a useful method for the identification, taxonomy, typing, epidemiology and phylogeny of P. verrucosa.

Identification of dematiaceous fungi and their role in human disease.

Phaeohyphomycosis is the disease caused by the dematiaceous hyphomycetes or those fungi that are darkly pigmented because of the presence of melanin in their cell wall. Infections with these fungi may remain localized at the site of traumatic inoculation or within the sinuses or may become disseminated. As these fungi pose a significant problem to both the clinician who must treat the patient and the laboratorian faced with identification of the organism, a selection of representative organisms was presented at the meeting entitled "Focus on Fungus Infections." These fungi often infect patients who are not overtly immunocompromised and therefore may be encountered in a wide variety of cases.

Evaluation of the API 20C yeast identification system for the differentiation of some dematiaceous fungi.

Ninety-seven isolates of Cladosporium spp., Exophiala spp., Fonsecaea spp., Lecythophora hoffmannii, Phaeoannellomyces werneckii, Phialophora spp., Wangiella dermatitidis, and Xylohypha bantiana were used to evaluate the API 20C Yeast Identification System for the differentiation of dematiaceous fungi. Using the API 20C system, we were able to distinguish most species of Phialophora and Cladosporium and to separate L. hoffmannii from the species of Phialophora tested; X. bantiana from C. carrionii, C. resinae, and C. sphaerospermum; and W. dermatitidis from Exophiala jeanselmei and Exophiala spinifera. Ninety-two (60.1%) of 153 possible species-pair combinations were separated.

Evaluation of proteolytic activity to differentiate some dematiaceous fungi.

A total of 123 isolates of Cladosporium spp., Exophiala spp., Fonsecaea spp., Lecythophora hoffmannii, Phaeoannellomyces werneckii, Phialophora spp., Wangiella dermatitidis, and Xylohypha bantiana were tested for proteolytic activity by using 26 different formulations of gelatin, milk, casein, and Loeffler media. Other physiological properties examined included hydrolysis of tyrosine and xanthine, sodium nitrate utilization in Czapek Dox agar, and thermotolerance. Isolates of Exophiala jeanselmei, Fonsecaea compacta, Fonsecaea pedrosoi, W. dermatitidis, and X. bantiana lacked proteolytic activity. Proteolytic activity was variable among the remaining species, depending on the type of medium used. Thermotolerance had value in distinguishing some taxa.

Use of carbohydrate and nitrate assimilations in the identification of dematiaceous fungi.

A total of 61 isolates of dematiaceous fungi, including Exophiala jeanselmei, Wangiella dermatitidis, Fonsecaea pedrosoi, Phialophora verrucosa, and a few isolates of related organisms were evaluated for their ability to assimilate 13 carbohydrates and sodium nitrate. Results indicated that patterns of assimilations can facilitate specific identifications when used with microscopic morphologic features. Eleven isolates of W. dermatitidis demonstrated negative results for nitrate assimilation, although most of the other fungi tested had positive reactions. The tests did aid in separating this very complex group of fungi.

Exoantigen test for Cladosporium bantianum, Fonsecaea pedrosoi, and Phialophora verrucosa.

Exoantigens from 10-day-old cultures of 100 isolates of pathogenic and saprophytic dematiaceous fungi were analyzed by the exoantigen test. Antisera to Cladosporium bantianum ATCC 10958, Fonsecaea pedrosoi CDC AMO-B06, and Phialophora verrucosa CDC AMO-C12 were prepared in New Zealand rabbits immunized with soluble antigens from 1-month-old cultures. Absorbed and nonabsorbed antisera and exoantigens from the same organisms were used as reference reagents. Serologic reactions were analyzed in terms of the presence or absence of lines of identity or nonidentity. These reactions allowed presumptive differentiation of C. bantianum, F. pedrosoi, and Phialophora verrucosa from other dematiaceous fungi, including Cladosporium spp. (28 isolates), Exophiala spp. (18 isolates), Fonsecaea spp. (17 isolates). Lecythophora hoffmannii (4 isolates), Phaeoannellomyces werneckii (3 isolates), Phialophora spp. (17 isolates), Wangiella dermatitidis (9 isolates), and Rhinocladiella spp. (4 isolates).

The relationship of Phialophora verrucosa to Phialophora americana.

Phialophora verrucosa and P. americana, two dematiaceous hyphomycetes, are known to cause chromoblastomycosis. Even though most medical mycologists consider P. americana as synonymous with P. verrucosa, others maintain them as two distinct species on the basis that the phialides of P. americana have deeper collarettes than those of P. verrucosa. Thirty-two isolates, identified either as P. americana or P. verrucosa, were studied for their morphologic, physiologic, and antigenic characteristics to evaluate their taxonomic status. Collarette morphology was found to be a variable character in 12 of the 32 isolates. Those 12 produced phialides with both shallow and deep collarettes. All of the isolates hydrolysed urea within 7 days at 25 degrees C, and failed to liquefy gelatin after 3 weeks at 25 degrees C. None of the isolates decomposed casein, xanthine, or hypoxanthine at the end of 3 weeks at 25 degrees C. All decomposed tyrosine. They grew at 25 degrees C and 37 degrees C, but failed to grow at 40 degrees C. The antigenic relationship between the two species was studied by the exoantigen procedure. The 32 isolates showed close antigenic relatedness. Adsorptions of antisera with homologous and heterologous antigens rendered the antisera free of precipitin bands when studied by the microimmunodiffusion test. The depth of phialide collarettes produced by the two species, being found to be variable character, and the identical nature of the two species with respect to their physiologic and antigenic characteristics, led us to conclude that P. americana should be considered as a synonym of P. verrucosa.

Pathogenicity of Exophiala jeanselmei for ddY mice.

The pathogenicity of three cultures isolated as Phialophora jeanselmei was compared with that of three cultures of Phialophora gougerotii using ddY mice. One hundred and twenty mice were used. They were divided into 6 groups consisting of 20 each. Each culture was evaluated in 20 mice. Mice were inoculated intravenously with 0.2 ml of a 1% (wet weight/vol.) yeast-like cell suspension and sacrificed at adequate intervals until the 30th day. As results, 1) the virulence of the three cultures each of P. jeanselmei and P. gougerotii to ddY mice was mild. 2) These cultures were not neurotropic. 3) P. gougerotii survived longer in the mice than P. jeanselmei. 4) There were no major differences in histopathology of the lesions in the mice inoculated with the two taxa.

Studies on the phylogenesis of pathogenic 'black yeasts'.

This paper deals mainly with the conidium ontogenesis and phylogenesis of black yeasts such as E. jeanselmei, E. gougerotii, E. dermatitidis and E. spinifera. The conidium ontogenesis of E. jeanselmei, E. gougerotii and E. dermatitidis was almost the same. One to five annellated tips were observed through a scanning microscope at the apices of conidiogenous cells, which were bottle- or jar-shaped. Annellations on the tips looked like fringes and the conidiogenous cells of these three species were annellides. Annellated projections occurred on hyphae and annelloconidia were also produced from them. Occasionally, secondary annellides occurred from primary ones. They looked like moniliform hyphae. Daughter conidia sometimes budded directly from mother cells. The shapes and sizes of the conidia of these species were very similar to each other. The conidium ontogenesis of E. spinifera was annellidic as well. However, a single annellated tip usually occurred on an annellide. The annellated tips of the fungus were long and more than 20 annellations were observed on their walls. The conidiogenesis of the four species of Exophiala is only annellidic. There were no differences in the biological examinations except KNO3 assimilation among these four species. The growth of E. jeanselmei and E. gougerotii was poor at 37 degrees C. The GC contents of E. jeanselmei 1171, E. gougerotii B-1800, E. dermatitidis MM-7 and E. spinifera DU-3342 were 54.6, 54.6, 56.6 and 59.7%, respectively.

Chromohyphomycosis of the hand due to Exophiala jeanselmei (Phialophora jeanselmei, Phialophora gougerotii)--case report and review.

A case of an unusual cause of chromomycosis of the hand is described. Follow-up at 2 months revealed a complete recovery following removal of the mass, without treatment with antifungal therapy. A review of the different disease entities of the chromomycoses and a brief generic classification of the dematiacious fungi are presented.