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Two new species in the order Mucorales, Mucor velutinosus and Mucor ellipsoideus, isolated from human clinical specimens in the USA, are described and illustrated. The former species is similar to Mucor ramosissimus, from which it can be differentiated by its ability to grow at 37°C and produce verrucose sporangiospores. Mucor ellipsoideus is also able to grow and sporulate at 37°C like M. indicus, the nearest phylogenetic species in this study, however, the former has narrow ellipsoidal sporangiospores in contrast to the subglobose to ellipsoidal sporangiospores of M. indicus. Analysis of the sequences of the ITS and the D1-D2 regions of the rRNA genes confirmed the novelty of these species. The in vitro antifungal susceptibility of the new species showed that amphotericin B was active against all isolates and posaconazole and itraconazole showed low activity.

We examined the utility of agar dilution to screen yeasts for reduced susceptibility to several newer antifungal drugs including echinocandins and azoles. We compared agar dilution susceptibility screening with the Clinical and Laboratory Standards Institute (CLSI) method for Candida isolates. We added echinocandins and azoles to CHROMagar Candida medium prior to its solidifi cation. Assessment of resistance was based on growth characteristics, wherein decreased colony size in the presence of antifungal drugs was used as an indicator of susceptibility. Clinical Candida isolates of C. albicans , C. glabrata , C. parapsilosis , C. tropicalis , C. guilliermondii , C. lusitaniae , C. rugosa and C. dubliniensis were screened for drug susceptibility. Overall, antifungal susceptibility of the yeasts to anidulafungin, caspofungin, micafungin, posaconazole and voriconazole, determined using CHROMagar agar dilution, were shown to be 96, 80, 94, 90 and 97% as accurate, respectively, as those determined by the CLSI method, i.e., within one tube dilution of CLSI MICs. Categorical errors by percentage had a broader range. Major errors noted with anidulafungin, caspofungin and micafungin were 3, 6 and 0%, respectively, while very major errors were 15, 55 and 38%, respectively. Major errors with posaconazole and voriconazole were 12 and 0%, respectively, while very major errors were 0 and 22%, respectively, compared to CLSI standards. Most of the assessment errors were found with C. glabrata and C. parapsilosis . Agar dilution screening for drug susceptibility with the current panel of antifungal drugs is rapid, accurate and effective. However, the determination of resistance or non-susceptibility in yeasts may be more problematic, and may be species dependent.

We report a case of a life-threatening, recurrent, and azole-resistant pulmonary coccidioidomycosis in a patient receiving long-term fluconazole therapy for a history of coccidioidal meningitis. Since this diagnosis, the patient has received weekly amphotericin B for more than four years and remains in remission with a stable serum Coccidioides complement fixation antibody titer.

MALDI-TOF MS can rapidly identify microorganisms to the species level and may be able to detect antimicrobial resistance. We evaluated the ability of this technology to detect triazole resistance in Candida species.35 C. albicans, 35 C. glabrata, and 37 C. tropicalis strains were exposed to fluconazole, voriconazole, or posaconazole at two different concentrations plus a drug-free control: a midrange concentration (CLSI clinical breakpoint or epidemiologic cut-off value), and a high concentration (fluconazole 64 μg/ml, voriconazole & posaconazole 16 μg/ml). The MALDI-TOF MS spectra at these concentrations were used to create the individual composite correlation index (CCI) matrices for each isolate. When the CCI of the midrange/highest concentration was lower than that of the midrange/null concentration, the strain was classified as resistant. These results were then compared to the classifications for susceptible or resistant obtained by measuring the MICs according to the CLSI M27-A3 antifungal susceptibility testing (AFST) method.The MALDI-TOF MS assay was able to classify triazole susceptibility against all strains. Overall, essential agreement between MALDI-TOF MS and AFST varied between 54% and 97%, and was highest for posaconazole against C. glabrata. The reproducibility of the MALDI-TOF MS assay varied between 54.3 and 82.9% and was best for fluconazole against C. albicans and posaconazole against C. glabrata. Reproducibility was also higher for C. glabrata isolates compared to C. albicans and C. tropicalis.These results demonstrate that MALDI-TOF MS may be used to simultaneously determine the Candida species and classification as susceptible or resistant to triazole antifungals. Further studies are needed to refine the methodology and improve the reproducibility of this assay.

The in vitro activities of three new triazoles were determined and compared to those of itraconazole and fluconazole against 306 clinical isolates of Blastomyces dermatitidis, Cladophialophora carrionii, Coccidioides immitis, Fonsecaea pedrosoi, Fusarium spp., Histoplasma capsulatum, Paecilomyces lilacinus, Pseudallescheria boydii and Sporothrix schenckii. Minimum inhibitory concentrations (MIC) were determined by a broth macrodilution method of the National Committee for Clinical Laboratory Standards M38-A procedure. Itraconazole (geometric mean MIC, 0.16-0.65 microg/ml), voriconazole (geometric mean MIC, 0.18-1.44 microg/ml), ravuconazole (geometric mean MIC, 0.18-1.09 microg/ml), and posaconazole (geometric mean MIC, 0.18-1.38 microg/ml), had relatively uniform values showing potent in vitro inhibitory activity against B. dermatitidis, C. carrionii, C. immitis, F. pedrosoi, H. capsulatum, and S. schenckii. The in vitro activity was variable with strains of P. boydii, P. lilacinus and Fusarium spp.

Cerebral phaeohyphomycosis caused by Rhinocladiella mackenziei (formerly Ramichlo-ridium mackenziei) is extremely rare, and geographically limited to the Middle East. The fungus exclusively targets the brain and infections have a grave prognosis. Eighteen cases have been reported in the literature from 1983 to 2004 with almost 100% mortality. Our patient presented in February 2008 with a brain abscess while receiving chemotherapy for carcinoma of the breast. Diagnosis was by craniotomy and aspiration of the brain abscess. Direct microscopy showed dematiaceous fungal hyphae. R. mackenziei was recovered in culture and this identification was confirmed by molecular analysis. Examination of histopathological sections of tissue from the brain biopsy revealed moniliform hyphae characteristic for phaeohyphomycosis. The patient failed to respond to antifungal therapy with amphotericin B and voriconazole or amphotericin B and posaconazole and finally expired 64 days after diagnosis. In vitro antifungal susceptibility testing showed this isolate to be resistant to amphotericin B while susceptible to itraconazole, voriconazole, and posaconazole. Previously published antifungal susceptibility data indicate that although strains show variable susceptibility to amphotericin B, the organism is generally refractory to treatment with this agent. Similar outcomes are seen with the azole agents used alone or in combination with other drugs. Although no specific risk factors have been identified, the majority of cases have occurred in immunocompromised individuals. R. mackenziei is a highly virulent agent of serious cerebral phaeohyphomycosis, and should be considered in the differential diagnosis of central nervous system disease in the Middle East.

Frequently, diseases caused by black yeasts are chronic in nature with a high morbidity. In addition, these infections are often fatal and relapse is common, even after prolonged treatment. Although the CLSI Document M38-A outlines methods for antifungal susceptibility testing of moulds, Exophiala spp. are not directly discussed. In an effort to determine the antifungal susceptibility patterns of Exophiala spp. we tested 160 clinical isolates against amphotericin B, itraconazole, posaconazole, and voriconazole in a head-to-head comparison. Posaconazole and itraconazole were the most active in vitro with MICs falling well below the achievable serum levels typically observed with standard dosing regimens.