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In vitro activity of Syn-2869, a novel triazole agent, against emerging and less common mold pathogens.

The in vitro activity of Syn-2869 was compared with that of amphotericin B and itraconazole. MICs for 100 isolates of pathogenic molds belonging to 12 species were determined by a broth microdilution adaptation of the method recommended by the National Committee for Clinical Laboratory Standards. Syn-2869 and itraconazole showed comparable, good activity against the dematiaceous molds Cladophialophora bantiana, Cladophialophora carrionii, Exophiala dermatitidis, Fonsecaea pedrosoi, Phialophora parasitica, and Ramichloridium mackenziei. Neither of the azole agents was active against the hyaline molds Fusarium solani, Scedosporium prolificans, and Scopulariopsis brevicaulis, but both were more active than amphotericin B against Scedosporium apiospermum. The MICs of the three agents were comparable for the mucoraceous mold Absidia corymbifera, but Syn-2869 appeared to be the least active against the dimorphic mold Sporothrix schenckii. Our results suggest that Syn-2869 could be effective against a range of mold infections in humans.

Antifungal Agents↗

In vitro activity of anidulafungin against selected clinically important mold isolates.

In this study, we evaluated the in vitro activity of anidulafungin against selected mold isolates. Anidulafungin showed promising activity against Bipolaris spicifera, Exophiala jeanselmei, Fonsecaea pedrosoi, Madurella mycetomatis, Penicillium marneffei, Phialophora verrucosa, Pseudallescheria boydii, Sporothrix schenckii, and Wangiella dermatitidis.

Amphotericin B↗

New method for susceptibility testing with antifungal agents.

A new method for the determination of minimal inhibitory and fungicidal concentrations of antifungal agents for filamentous fungi is described. 5-Fluorocytosine (5-FC) was used as a representative agent and was tested against the following genera: Allescheria, Aspergillus, Cladosporium, Fonsecaea, and Phialophora. 5-FC was found to be inhibitory but not fungicidal for many of the fungi tested, including eight of clinical origin.

Antifungal Agents↗

Relationships among non-Acremonium sp. fungal endophytes in five grass species.

Many cool-season grasses (subfamily Pooideae) possess maternally transmitted fungal symbionts which cause no known pathology and often enhance the ecological fitness and biochemical capabilities of the grass hosts. The most commonly described endophytes are the Acremonium section Albo-lanosa spp. (Acremonium endophytes), which are conidial anamorphs (strictly asexual forms) of Epichloë typhina. Other endophytes which have been noted are a Gliocladium-like fungus in perennial ryegrass (Lolium perenne L.) and a Phialophora-like fungus in tall fescue (Festuca arundinacea Schreb.). Here, we report the identification of additional non-Acremonium sp. endophytes (herein designated p-endophytes) in three more grass species: Festuca gigantea, Festuca arizonica, and Festuca pratensis. In each grass species, the p-endophyte was cosymbiotic with an Acremonium endophyte. Serological analysis and sequence determinations of variable portions of their rRNA genes indicated that the two previously identified non-Acremonium endophytes are closely related to each other and to the newly identified p-endophytes. Therefore, the p-endophytes represent a second group of widely distributed grass symbionts.

Base Sequence↗

Identification of volatile metabolites from five fungal species cultivated on two media.

Five fungal species, Aspergillus versicolor, Penicillium commune, Cladosporium cladosporioides, Paecilomyces variotii, and Phialophora fastigiata, were cultivated on two media, malt extract agar and dichloran glycerol agar. Culture flasks provided with inlet and outlet tubes were used and purified, and humidified air was constantly led through the flasks. Air samples from the cultures were sorbed on Tenax GR and analyzed by thermal desorption-gas chromatography. The produced volatile metabolites were analyzed by mass spectrometry. Various hydrocarbons, alcohols, ketones, ethers, esters, sulfur-containing compounds, and terpenes were identified. The most commonly produced substances were 2-methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 3-methylfuran, and dimethyl disulfide. The production was highly dependent on both medium and species.

Journal Article↗

Misdiagnosis caused by fungal contaminant in a histological stain solution.

Silver stains on tissue and cytology specimens are important in the evaluation of patients with suspected fungal infections. Care must be taken, however, to prevent misinterpretation of contamination artifacts. Two cases presenting such a problem are reported. The first patient had granulomatous leg lesions that microscopically showed characteristics of erythema induratum but with budding yeastlike organisms demonstrated by Grocott methenamine silver stain. Cultures and subsequent biopsies were negative for fungi. The second patient had a steroid-dependent chronic obstructive lung disease, and during evaluation for possible Pneumocystis carinii pneumonia, the Grocott methenamine silver stain on expectorated sputum showed budding yeastlike organisms. Sputum cultures were negative for fungi. Examination of the two Grocott light-green counterstain solutions demonstrated black, budding yeast cells similar to those seen in the specimens from the patients. Culture of the counterstain grew Exophiala (Phialophora) jeanselmei. Further studies revealed that this cause of misdiagnosis could be prevented by either filtering or adding thymol to the counterstain solution. Care regarding contamination of histological stain solutions is emphasized.

Granuloma↗

Exoantigen test for differentiation of Exophiala jeanselmei and Wangiella dermatitidis isolates from other dematiaceous fungi.

Concentrated (25X) exoantigens of 105 isolates of pathogenic and saprophytic dematiaceous fungi and 3 isolates of Sporothrix schenckii were analyzed by the microimmunodiffusion method. The reagents used were nonadsorbed and adsorbed sera produced in New Zealand rabbits. One set of rabbits was immunized with soluble antigens of a 1-month-old culture of Exophiala jeanselmei (ATCC 34123), and the other set was immunized with soluble antigens from a culture of Wangiella dermatitidis (ATCC 28869). The reference antigens were 25X-concentrated exoantigens of the above cultures. This exoantigen test permitted the differentiation of E. jeanselmei and W. dermatitidis from one another as well as from other Exophiala species, Fonsecaea species, Phialophora species, Cladosporium species, Rhinocladiella species, and Sporothrix schenckii by presence or absence of lines of identity or of partial identity, or lines of nonidentity. Using adsorbed serum eliminated the problems with cross-reactivity seen with nonadsorbed serum. Thus, with an adsorbed serum as the reagent, it was possible to presumptively differentiate E. jeanselmei and W. dermatitidis from one another and from other dematiaceous fungi.

Antigens, Fungal↗

Mycetoma of the foot caused by Cylindrocarpon destructans.

A 39-year-old male, originally from Antigua, West Indies, presented with a 12-year history of swelling of the left foot. A pathogen could not be recovered in cultures of three surgical biopsy specimens. During follow-up, pus and grains were expressed from a draining sinus tract and Cylindrocarpon destructans grew in pure culture. Retrospective examination of histologic sections of tissue removed during the third biopsy demonstrated a grain characteristic of eumycotic mycetoma. Although the fungus was susceptible to amphotericin B and ketoconazole in vitro, the patient refused treatment, and the clinical course over almost 19 years has been one of slow but progressive bone destruction. The fungus was identified by its microconidial morphology, the presence of chlamydospores, and an intense brown diffusible pigment. It was compared with another poorly known agent of white grain mycetoma, Phialophora cyanescens, characterized by phialidic conidia, chlamydospores in aggregations, and an intense diffusing pigment. The new combination Cylindrocarpon cyanescens (de Vries et al.) Sigler comb. nov. is proposed.

Adult↗

A phaeohyphomycotic cyst and peritonitis caused by Phialemonium species and a reevaluation of its taxonomy.

Two cases of human fungal infections caused by members of the genus Phialemonium, a genus proposed by Gams and McGinnis (1983) for fungi intermediate between the genera Acremonium and Phialophora, are presented. The first case was a phaeohyphomycotic cyst on the foot of a renal transplant recipient. The fungus was detected by direct examination and histopathology and was recovered by several procedures over 4 months. It was flat, glabrous, and white becoming yellow with the production of a diffusible yellow pigment; it had conidiophores that were mostly solitary and lateral and terminal phialides and adelophialides with distinct collarettes producing cylindrical to curved conidia. The isolate resembled both Phialemonium dimorphosporum and Phialemonium curvatum, although its characteristics were more consistent with those of the latter. The second case was peritonitis in a renal transplant recipient. The fungus was white-to-cream colored and yeast like, but later became black with a green diffusible pigment, and produced obovoid conidia; it was easily identified as Phialemonium obovatum. Difficulties encountered in the identification and taxonomy of members of this genus highlight the need for standardized conditions, e.g., potato dextrose agar culture incubated at 24 to 25 degrees C for morphologic comparisons, to control significant variations due to culture conditions.

Child, Preschool↗

Analysis of genes coding for small-subunit rRNA sequences in studying phylogenetics of dematiaceous fungal pathogens.

Because of their ability to display yeast-like growth forms in various environmental conditions, dematiaceous (melanized) hyphomycetes of the form-genera Exophiala, Rhinocladiella, and Wangiella have been informally termed "black yeasts." Cladistic analysis of 1,050 bp of the genes coding for small-subunit rRNA (SSU rDNA) supported a close relationship among species of these black yeasts with other dematiaceous hyphomycetes in the form-genera Fonsecaea, Phialophora, and Ramichloridium. The conventional categories of these fungi based on asexual states are not supported by phylogenetic analysis of SSU rDNA sequences. Isolates exhibiting annellidic modes of blastic conidiogenesis (e.g., Exophiala spp.) were not monophyletic and were placed as sister taxa to isolates that produce phialides or sympodulae. The results indicated very close relationships between isolates of Wangiella dermatitidis and Exophiala mansonii and between Rhinocladiella aquaspersa and Exophiala jeanselmei. This clade of dematiaceous hyphomycetes was a sister group to a clade comprising members of two orders of cleistothecial ascomycetes, Eurotiales and Onygenales. The etiological agents of chromoblastomycosis were found to be a closely related group (clade), while the agents of phaeohyphomycosis displayed a broader distribution on the SSU rDNA tree.

Ascomycota↗

Comparison of In vitro activities of the new triazole SCH56592 and the echinocandins MK-0991 (L-743,872) and LY303366 against opportunistic filamentous and dimorphic fungi and yeasts.

The in vitro antifungal activities of SCH56592, MK-0991, and LY303366 against 83 isolates of Acremonium strictum, Aspergillus flavus, Aspergillus fumigatus, Aspergillus terreus, Bipolaris spp., Blastomyces dermatitidis, Cladophialophora bantiana, Fusarium oxysporum, Fusarium solani, Histoplasma capsulatum, Phialophora spp., Pseudallescheria boydii, Rhizopus arrhizus, Scedosporium prolificans, and Sporothrix schenckii were compared. The in vitro activities of these agents against 104 isolates of yeast pathogens of Candida spp., Cryptococcus neoformans, and Trichosporon beigelii were also compared. MICs were determined by following a procedure under evaluation by the National Committee for Clinical Laboratory Standards (NCCLS) for broth microdilution testing of the filamentous fungi (visual MICs) and the NCCLS M27-A broth microdilution method for yeasts (both visual and turbidimetric MICs). The in vitro fungicidal activity of SCH56592 was superior (minimum fungicidal concentrations [MFCs], 0.25 to 4 microgram/ml for 7 of 18 species tested) to those of MK-0991 and LY303366 (MFCs, 8 to >16 microgram/ml for all species tested) for the molds tested, but the echinocandins had a broader spectrum of fungicidal activity (MFCs at which 90% of strains are inhibited [MFC90s], 0.5 to 4 microgram/ml for 6 of 9 species tested) than SCH56592 (MFC90s, 0.25 to 8 microgram/ml for 4 of 9 species tested) against most of the yeasts tested. Neither echinocandin had in vitro activity (MICs, >16 microgram/ml) against C. neoformans and T. beigelii, while the SCH56592 MICs ranged from 0.12 to 1.0 microgram/ml for these two species. The MICs of the three agents for the other species ranged from <0.03 to 4 microgram/ml. These results suggest that these new agents have broad-spectrum activities in vitro; their effectiveness in the treatment of human mycoses is to be determined.

Anidulafungin↗

Specific oligonucleotide primers for identification of Cladophialophora carrionii, a causative agent of chromoblastomycosis.

Cladophialophora carrionii is one of the relatively common causative agents of chromoblastomycosis. We have developed the specific oligonucleotide primer set based on the internal transcribed spacer regions of ribosomal DNA for the rapid identification of this pathogen. PCR with this primer set amplified a 362-bp amplicon from C. carrionii strains. From other relevant dematiaceous species, including medically important dematiaceous fungi, such as Fonsecaea pedrosoi, Phialophora verrucosa, and Exophiala dermatitidis, and eight species of medically important yeasts, such as Candida albicans and Cryptococcus neoformans var. neoformans, the primer set did not produce any amplicon. PCR with this primer set may be a useful tool for the identification of C. carrionii.

Chromoblastomycosis↗

Species of Phaeoacremonium associated with infections in humans and environmental reservoirs in infected woody plants.

To date, three species of Phaeoacremonium have been associated with phaeohyphomycosis. These are P. parasiticum (formerly Phialophora parasitica), P. inflatipes, and P. rubrigenum. Numerous unknown isolates resembling Phaeoacremonium spp. have in recent years been isolated from human patients as well as from woody plants that appear to be the main environmental source of these fungi. Nine new Phaeoacremonium species, of which six were obtained as etiologic agents of human opportunistic infection, are reported. They can be identified based on their cultural and morphological characters, and the identifications are strongly supported in phylogenetic analyses of partial sequences of the actin, beta-tubulin, and calmodulin genes. A multiple-entry electronic key based on morphological, cultural, and beta-tubulin sequence data was developed to facilitate routine species identification. Reexamination of all isolates of P. inflatipes associated with human disease showed them to be misidentified and to belong to the new taxa described here.

Actinidia↗

Internal transcribed spacer rRNA gene-based phylogenetic reconstruction using algorithms with local and global sequence alignment for black yeasts and their relatives.

Sequences of rRNA gene internal transcribed spacer (ITS) of a standard set of black yeast-like fungal pathogens were compared using two methods: local and global alignments. The latter is based on DNA-walk divergence analysis. This method has become recently available as an algorithm (DNAWD program) which converts sequences into three-dimensional walks. The walks are compared with, or fit to, each other generating global alignments. The DNA-walk geometry defines a proper metric used to create a distance matrix appropriated for phylogenetic reconstruction. In this work, the analyses were carried out for species currently classified in Capronia, Cladophialophora, Exophiala, Fonsecaea, Phialophora, and Ramichloridium. Main groups were verified by small-subunit rRNA gene data. DNAWD applied to ITS2 alone enabled species recognition as well as phylogenetic reconstruction reflecting clades discriminated in small-subunit rRNA gene phylogeny, which was not possible with any other algorithm using local alignment for the same data set. It is concluded that DNAWD provides rapid insight into broader relationships between groups using genes that otherwise would be hardly usable for this purpose.

Algorithms↗

Chromoblastomycosis simulating rhinosporidiosis in a patient from Ceylon.

A case of chromoblastomycosis confined to the mucous membrane of one side of the nasal septum is reported. The organism was not cultivated, but its characteristics in histological preparations were typical of those of the organisms in sections of cutaneous lesions known to be caused by Phialophora pedrosoi and related fungi. The diagnosis is considered to have been justified in spite of the great rarity of mucosal involvement in chromoblastomycosis and of the complete absence of lesions in the skin. The patient was a Sinhalese student working in London. He had first noticed the lesion before he left Ceylon, but the symptoms of nasal obstruction and bleeding were not sufficient to make him seek medical advice until two years later. If it is correct to assume that he contracted the infection in Ceylon his case is only the second on record in which there has been reason to suggest that Ceylon has been the geographical source of chromoblastomycosis. The lesion was excised and its site cauterized. There has been no sign of recurrence of the infection during the two years that have passed since the operation.

Chromoblastomycosis↗

THE DARK SIDE OF THE MYCELIUM: Melanins of Phytopathogenic Fungi.

Melanins are darkly pigmented polymers that protect organisms against environmental stress. Even when not directly involved in pathogenesis, fungal melanin is likely required by melanizing phytopathogens for survival in the environment. However, some phytopathogenic fungi that produce melanized appressoria for host invasion require appressorial melanogenesis for pathogenicity. Much less is known about the role melanins play in pathogenesis during infection by other phytopathogens that do not rely on appressoria for host penetration. Here we focus on one such phytopathogenic fungus, Gaeumannomyces graminis var. tritici, the etiologic agent of the devastating root disease of cereals, take-all. This fungus is lightly pigmented in culture, but requires melanin biosynthesis for pathogenesis, perhaps to produce melanized, ectotrophic macrohyphae on roots. However, the constitutively melanized, asexual Phialophora anamorph of G. graminis var. tritici is nonpathogenic. In addition, melanization of G. graminis var. graminis is not required to produce root disease on its rice host. Explanations for these apparent contradictions are suggested, as are other functions for the melanins of phytopathogenic fungi.

Journal Article↗

In vitro comparison of the antifungal activities of R34,000, miconazole and amphotericin B.

In vitro susceptibilities of 78 isolates of pathogenic filamentous fungi to the imidazole compounds R34,000 and miconazole and to amphotericin B were determined using an agar dilution technique. Allescheria boydii, Sporothrix schenckii and the dematiaceous fungi (Cladosporium, Fonsecaea and Phialophora spp.) were most susceptible to miconazole with minimum inhibitory concentration (MIC) values ranging from less than or equal to 0.25 to 32 microgram/ml and with geometric mean MIC (G-MIC) values of 0.56-1.24 microgram/ml. Isolates of Aspergillus fumigatus were more susceptible to amphotericin B and miconazole than to R34,000 with G-MIC values of 1.20 and 3.48 microgram/ml, respectively. Coccidioides immitis, Histoplasma capsulatum and Blastomyces dermatitidis were highly susceptible to all three drugs (G-MICs greater than 1 microgram/ml); R34,000 was the most active of the three compounds against C. immitis (G-MIC = 0.44 microgram/ml).

Amphotericin B↗

Epidemiology, clinical manifestations, and therapy of infections caused by dematiaceous fungi.

The dematiaceous (brown-pigmented) fungi are a large and heterogenous group of moulds that cause a wide range of diseases including phaeohyphomycosis, chromoblastomycosis, and eumycotic mycetoma. Among the more important human pathogens are Alternaria species, Bipolaris species, Cladophialophora bantiana, Curvularia species, Exophiala species, Fonsecaea pedrosoi, Madurella species, Phialophora species, Scedosporium prolificans, Scytalidium dimidiatum, and Wangiella dermatitidis. These organisms are widespread in the environment, being found in soil, wood, and decomposing plant debris. Cutaneous, subcutaneous, and corneal infections with dematiaceous fungi occur worldwide, but are more common in tropical and subtropical climates. Infection results from traumatic implantation. Most cases occur in immunocompetent individuals. Dematiaceous moulds are also important causes of invasive sinusitis and allergic fungal sinusitis. Infection is thought to follow inhalation. Although cerebral infection is the commonest form of systemic phaeohyphomycosis, other localized deep forms of the disease, such as arthritis, and endocarditis, have been reported. Disseminated infection is uncommon, but its incidence is increasing, particularly among immunocompromised individuals. Scedosporium prolificans is the most frequent cause. A number of dematiaceous fungi are neurotropic, including Cladophialophora bantiana, Ramichloridium mackenziei, and Wangiella dermatitidis. Although cases have occurred in immunocompromised persons, cerebral phaeohyphomycosis is most common in immunocompetent individuals with no obvious risk factors. Most forms of disease caused by dematiaceous fungi require both surgical and medical treatment. Itraconazole is currently the most effective antifungal agent for chromoblastomycosis and subcutaneous phaeohyphomycosis, while ketoconazole remains useful for mycetoma. Extensive surgical debridement combined with amphotericin B treatment is recommended for chronic invasive sinusitis. Long-term treatment with itraconazole has led to improvement or remission in some patients that had failed to respond to amphotericin B. Allergic fungal sinusitis requires surgical removal of impacted mucin combined with postoperative oral corticosteroids. Antifungal treatment is not usually of benefit, but post-operative itraconazole may reduce the need for reoperation. The clinical outcome of cerebral and other deep-seated forms of phaeohyphomycosis is dismal, with long-term survival being reported only when complete surgical resection of discrete lesions is possible. The development of new antifungal agents and combination treatment may help to improve the management of these infections.

Antifungal Agents↗