Comparison of the Vitek 2 yeast susceptibility system with CLSI microdilution for antifungal susceptibility testing of fluconazole and voriconazole against Candida spp., using new clinical breakpoints and epidemiological cutoff values.

A commercially available, fully automated yeast susceptibility test system (Vitek 2; bioMérieux, Marcy d'Etoile, France) was compared in 3 different laboratories with the Clinical and Laboratory Standards Institute (CLSI) reference microdilution (BMD) method by testing 2 quality control strains, 10 reproducibility strains, and 425 isolates of Candida spp. against fluconazole and voriconazole. Reference CLSI BMD MIC endpoints and Vitek 2 MIC endpoints were read after 24 hours and 9.1-27.1 hours incubation, respectively. Excellent essential agreement (within 2 dilutions) between the reference and Vitek 2 MICs was observed for fluconazole (97.9%) and voriconazole (96.7%). Categorical agreement (CA) between the 2 methods was assessed using the new species-specific clinical breakpoints (CBPs): susceptible (S) ≤2 µg/mL, susceptible dose-dependent (SDD) 4 µg/mL, and resistant (R) ≥8 µg/mL for fluconazole and Candida albicans, Candida tropicalis, and Candida parapsilosis and ≤32 µg/mL (SDD), ≥64 µg/mL (R) for Candida glabrata; S ≤0.12 µg/mL, SDD 0.25-0.5 µg/mL, R ≥1 µg/mL for voriconazole and C. albicans, C. tropicalis, and C. parapsilosis, and ≤0.5 µg/mL (S), 1 µg/mL (SDD), ≥2 µg/mL (R) for Candida krusei. The epidemiological cutoff value (ECV) of 0.5 µg/mL for voriconazole and C. glabrata was used to differentiate wild-type (WT; MIC ≤ ECV) from non-WT (MIC > ECV) strains of this species. Due to the lack of CBPs for the less common species, the ECVs for fluconazole and voriconazole, respectively, were used for Candida lusitaniae (2 µg/mL and 0.03 µg/mL), Candida dubliniensis (0.5 µg/mL and 0.03 µg/mL), Candida guilliermondii (8 µg/mL and 0.25 µg/mL), and Candida pelliculosa (4 µg/mL and 0.25 µg/mL) to categorize isolates of these species as WT and non-WT. CA between the 2 methods was 96.8% for fluconazole and 96.5% for voriconazole with less than 1% very major errors and 1.3-3.0% major errors. The Vitek 2 yeast susceptibility system remains comparable to the CLSI BMD reference method for testing the susceptibility of Candida spp. when using the new (lower) CBPs and ECVs.

Evaluation of the correlation of caspofungin MICs and treatment outcome in murine infections by wild type strains of Candida parapsilosis.

We have evaluated the in vitro activity of caspofungin against 36 wild-type strains of Candida parapsilosis sensu stricto using 3 techniques: broth microdilution, disk diffusion, and the determination of minimal fungicidal concentration (MFC). The first 2 methods showed a good in vitro activity of caspofungin, but the MFCs were ≥2 dilutions above their corresponding MICs. In a murine model of disseminated infection, we evaluated the efficacy of caspofungin at 5 mg/kg against 8 strains of C. parapsilosis representing different degrees of in vitro susceptibility (0.12-1 µg/mL). All the isolates responded to treatment and (1→3)-ß-D-glucan levels were reduced in all the cases; however, the study revealed differences among isolates, since caspofungin reduced the tissue burden of mice infected with isolates with MICs ≤0.5 µg/mL but was less effective against those with MICs of 1 µg/mL.

MIC values of voriconazole are predictive of treatment results in murine infections by Aspergillus terreus species complex.

We evaluated the efficacy of voriconazole against nine strains of Aspergillus terreus with different MICs (0.12 to 4 µg/ml) by using a murine model. Markers of efficacy included survival, tissue burden, galactomannan antigenemia, and drug serum levels. Voriconazole was especially effective in prolonging survival and reducing the fungal load in infections by strains that showed MICs that were less than or equal to the epidemiological cutoff value (1 µg/ml). In vitro data might be useful for predicting the outcome of A. terreus infections.

Are epidemiologic cut-off values predictors of the in vivo efficacy of azoles in experimental aspergillosis?

We tried to correlate the in vitro activity and the in vivo efficacy of voriconazole (VRC) and posaconazole (POS) against Aspergillus flavus and Aspergillus niger. The in vitro susceptibility of a large set of isolates was determined by broth microdilution and disk diffusion methods, while the in vivo efficacy was assessed in a murine model of disseminated infection. For A. flavus, VRC showed efficacy even for strains with MICs above the epidemiologic cutoff value (ECV) (1 µg/mL). For A. niger, VRC was ineffective against those strains for which the MIC was 1 dilution below the corresponding ECV (2 µg/mL). POS showed efficacy against all the strains of both species included in the study, although isolates with MICs > ECV were not tested.

In vitro and in vivo activities of posaconazole and amphotericin B in a murine invasive infection by Mucor circinelloides: poor efficacy of posaconazole.

The in vitro susceptibility of 17 strains of Mucor circinelloides to amphotericin B and posaconazole was ascertained by using broth microdilution and disk diffusion methods and by determining the minimal fungicidal concentration (MFC). We evaluated the efficacy of posaconazole at 40 mg/kg of body weight/day and amphotericin B at 0.8 mg/kg/day in a neutropenic murine model of disseminated infection by M. circinelloides by using 6 different strains tested previously in vitro. In general, most of the posaconazole MICs were within the range of susceptibility or intermediate susceptibility, while the small inhibition zone diameters (IZDs) were indicative of nonsusceptibility for all isolates tested. The MFCs were ≥ 3 dilutions higher than the corresponding MICs. In contrast, amphotericin B showed good activity against all of the strains tested regardless of the method used. The in vivo studies demonstrated that amphotericin B was effective in prolonging survival and reducing the fungal load. Posaconazole showed poor in vivo efficacy with no correlation with the MIC values. The results suggested that posaconazole should be used with caution in the treatment of infections caused by Mucor circinelloides or by strains of Mucor not identified to the species level.

Pharmacokinetics of anidulafungin in pleural fluid during the treatment of a patient with Candida empyema.

Candida empyema is a serious complication of disseminated candidiasis. However, little is known about the intrapleural pharmacokinetics of echinocandins. We report the penetration of anidulafungin into the pleural fluid of a patient with Candida tropicalis empyema. The anidulafungin ratio for the area under the concentration-time curve from 0 h to the last measurement between pleural fluid and serum values was only 0.125 (12.5%), with pleural fluid concentrations ranging between 0.67 and 0.88 µg/ml.

Multicenter comparison of the Vitek 2 antifungal susceptibility test with the CLSI broth microdilution reference method for testing caspofungin, micafungin, and posaconazole against Candida spp.

The performance of the automated Vitek 2 (bioMérieux, Inc., Marcy l'Etoile, France) antifungal susceptibility system was compared to that of broth microdilution (BMD) for the determination of MICs of various antifungal drugs. A total of 112 challenge strains and 755 clinical isolates of Candida spp. were tested against caspofungin and micafungin. An additional 452 clinical isolates of Candida albicans were tested against posaconazole. Reference BMD MIC endpoints were established after 24 h of incubation for caspofungin and micafungin and after 48 h of incubation for posaconazole. Essential agreements (EAs) between the Vitek 2 and BMD methods for caspofungin and micafungin were 99.5% and 98.6%, respectively. EA between the Vitek 2 and BMD methods was 95.6% for posaconazole. The overall categorical agreements (CAs) between the Vitek 2 system and BMD were 99.8% for caspofungin, 98.2% for micafungin, and 98.1% for posaconazole. The Vitek 2 system reliably determined caspofungin and micafungin MICs among Candida spp. and posaconazole MICs among C. albicans isolates and demonstrated excellent quantitative and qualitative agreement with the reference BMD method.

Two new species of Mucor from clinical samples.

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.

Internet-accessible DNA sequence database for identifying fusaria from human and animal infections.

Because less than one-third of clinically relevant fusaria can be accurately identified to species level using phenotypic data (i.e., morphological species recognition), we constructed a three-locus DNA sequence database to facilitate molecular identification of the 69 Fusarium species associated with human or animal mycoses encountered in clinical microbiology laboratories. The database comprises partial sequences from three nuclear genes: translation elongation factor 1α (EF-1α), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2). These three gene fragments can be amplified by PCR and sequenced using primers that are conserved across the phylogenetic breadth of Fusarium. Phylogenetic analyses of the combined data set reveal that, with the exception of two monotypic lineages, all clinically relevant fusaria are nested in one of eight variously sized and strongly supported species complexes. The monophyletic lineages have been named informally to facilitate communication of an isolate's clade membership and genetic diversity. To identify isolates to the species included within the database, partial DNA sequence data from one or more of the three genes can be used as a BLAST query against the database which is Web accessible at FUSARIUM-ID (http://isolate.fusariumdb.org) and the Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Center (http://www.cbs.knaw.nl/fusarium). Alternatively, isolates can be identified via phylogenetic analysis by adding sequences of unknowns to the DNA sequence alignment, which can be downloaded from the two aforementioned websites. The utility of this database should increase significantly as members of the clinical microbiology community deposit in internationally accessible culture collections (e.g., CBS-KNAW or the Fusarium Research Center) cultures of novel mycosis-associated fusaria, along with associated, corrected sequence chromatograms and data, so that the sequence results can be verified and isolates are made available for future study.

Molecular phylogenetic diversity of the emerging mucoralean fungus Apophysomyces: proposal of three new species

Antecedentes Apophysomyces es un género monoespécifico perteneciente al orden Mucorales. La especie Apophysomyces elegans, ha sido reportada como causante de infecciones severas en pacientes inmunocomprometidos e inmunocompetentes. En un estudio previo (Álvarez et al., J Clin Microbiol. 2009;47:1650–6 ), demostramos la elevada variabilidad dentro de las secuencias del gen 5.8S del ARNr en un grupo de cepas clínicas de A. elegans.Material y métodosHemos realizado un estudio polifásico basado en el análisis de las secuencias del gen de la histona 3, la región de los espaciadores internos del ADNr y los dominios D1 y D2 del gen 28S del ARNr, así como la evaluación de caracteres morfológicos y fisiológicos relevantes de un grupo de cepas clínicas y ambientales de A. elegans.Resultados y conclusionesHemos demostrado que A. elegans es un complejo de especies. Proponemos como nuevas especies para la ciencia Apophysomyces ossiformis, caracterizada por sus esporangiosporas con forma de hueso; Apophysomyces trapeziformis, con esporangiosporas trapezoidales; y Apophysomyces variabilis, con esporangiosporas de formas variables. Las nuevas especies no asimilan la esculina, en tanto que A. elegans fue capaz de asimilar dicho glicósido. La anfotericina B y el posaconazol fueron los antifúngicos más activos frente a Apophysomyces(AU)

Background Apophysomyces is a monotypic genus belonging to the order Mucorales. The species Apophysomyces elegans has been reported to cause severe infections in immunocompromised and immunocompetent people. In a previous study of Álvarez et al.3 [J Clin Microbiol 2009;47:1650–6], we demonstrated a high variability among the 5.8S rRNA gene sequences of clinical strains of A. elegans.Material and methodsWe performed a polyphasic study based on the analysis of the sequences of the histone 3 gene, the internal transcribed spacer region of the rDNA gene, and domains D1 and D2 of the 28S rRNA gene, as well as by evaluation of some relevant morphological and physiological characteristics of a set of clinical and environmental strains of A. elegans.Results and conclusionsWe have demonstrated that A. elegans is a complex of species. We propose as new species Apophysomyces ossiformis, characterised by bone-shaped sporangiospores, Apophysomyces trapeziformis, with trapezoid-shaped sporangiospores, and Apophysomyces variabilis, with variable-shaped sporangiospores. These species failed to assimilate esculin, whereas A. elegans was able to assimilate that glycoside. Amphotericin B and posaconazole are the most active in vitro drugs against Apophysomyces(AU)

Molecular phylogenetic diversity of the emerging mucoralean fungus Apophysomyces: proposal of three new species.

BACKGROUND: Apophysomyces is a monotypic genus belonging to the order Mucorales. The species Apophysomyces elegans has been reported to cause severe infections in immunocompromised and immunocompetent people. In a previous study of Alvarez et al.(3) [J Clin Microbiol 2009;47:1650-6], we demonstrated a high variability among the 5.8S rRNA gene sequences of clinical strains of A. elegans. MATERIAL AND METHODS: We performed a polyphasic study based on the analysis of the sequences of the histone 3 gene, the internal transcribed spacer region of the rDNA gene, and domains D1 and D2 of the 28S rRNA gene, as well as by evaluation of some relevant morphological and physiological characteristics of a set of clinical and environmental strains of A. elegans. RESULTS AND CONCLUSIONS: We have demonstrated that A. elegans is a complex of species. We propose as new species Apophysomyces ossiformis, characterised by bone-shaped sporangiospores, Apophysomyces trapeziformis, with trapezoid-shaped sporangiospores, and Apophysomyces variabilis, with variable-shaped sporangiospores. These species failed to assimilate esculin, whereas A. elegans was able to assimilate that glycoside. Amphotericin B and posaconazole are the most active in vitro drugs against Apophysomyces.

Correlation between in vitro activity of posaconazole and in vivo efficacy against Rhizopus oryzae infection in mice.

We have evaluated the in vitro activity of posaconazole (PSC) against 50 clinical strains of Rhizopus oryzae using a broth microdilution method, the Neo-Sensitabs tablet diffusion method, and minimal fungicidal concentration (MFC) determination. In general, PSC showed low MICs against this fungus, and the MICs correlated with the inhibition zone diameters. Most of the MFCs, however, were from 1 to 4 dilutions higher than their corresponding MICs. We then investigated the efficacies of several different doses of PSC in a murine model. All treatments began 24 h after challenge and lasted for 7 days. The drug was administered twice a day to mice infected with three strains that showed intermediate PSC susceptibility (MIC = 2 microg/ml) and three PSC-susceptible strains (MIC = 0.25 microg/ml). A dose of 10 mg/kg of body weight was ineffective, while doses of 20 and 30 mg/kg prolonged the survival of the mice. The 50 strains tested were segregated into two groups on the basis of the in vitro data. For the group with the most strains (85%), the strains had low PSC MICs, mice infected with the strains showed higher rates of survival (30 to 40%), and PSC was able to reduce the fungal load in the kidney and less regularly in the brain. For the second group (15% of the strains), the strains had intermediate PSC MICs, mice infected with the strains had lower survival rates (10 to 20%), and PSC treatment resulted in variable and no reductions in the fungal loads in the kidneys and brains, respectively.

Sequence-based identification of filamentous basidiomycetous fungi from clinical specimens: a cautionary note.

The species-level identification of sterile and/or arthroconidium-forming filamentous fungi presumed to be basidiomycetes based upon morphological or physiological features alone is usually not possible due to the limited amount of hyphal differentiation. Therefore, a reliable molecular approach capable of the unambiguous identification of clinical isolates is needed. One hundred sixty-eight presumptive basidiomycetes were screened by sequence analysis of the internal transcribed spacer (ITS) and D1/D2 ribosomal DNA regions in an effort to obtain a species identification. Through the use of this approach, identification of a basidiomycetous fungus to the species level was obtained for 167/168 of the isolates. However, comparison of the BLAST results for each isolate for both regions revealed that only 28.6% (48/168) of the isolates had the same species identification by use of both the ITS and the D1/D2 regions, regardless of the percent identity. At the less stringent genus-only level, the identities for only 48.8% (82/168) of the isolates agreed for both regions. Investigation of the causes for this low level of agreement revealed that 14% of the species lacked an ITS region deposit and 16% lacked a D1/D2 region deposit. Few GenBank deposits were found to be complete for either region, with only 8% of the isolates having a complete ITS region and 10% having a complete D1/D2 region. This study demonstrates that while sequence-based identification is a powerful tool for many fungi, sequence data derived from filamentous basidiomycetes should be interpreted carefully, particularly in the context of missing or incomplete GenBank data, and, whenever possible, should be evaluated in light of compatible morphological features.

Pharmacokinetics of liposomal amphotericin B in pleural fluid.

We report the penetration of liposomal amphotericin B into the pleural fluid of a patient with pulmonary zygomycosis and empyema. The ratio of area under the concentration-versus-time curve in pleural fluid (AUC(pleural fluid)) to that in serum (AUC(serum)) for liposomal amphotericin B over 24 h was 9.4%, with pleural fluid concentrations of 2.12 to 4.91 microg/ml. Given the relatively low level of intrapleural penetration of liposomal amphotericin B, chest tube drainage may be warranted for successful treatment of zygomycotic empyema.

Micafungin concentrations from brain tissue and pancreatic pseudocyst fluid.

We report the attainment of micafungin concentrations from brain tissue and pancreatic pseudocyst fluid from two patients with invasive candidiasis. Micafungin was present in low levels at both body sites, indicating limited penetration into central nervous system (CNS) tissue and pancreatic fluid. Further studies are needed to fully characterize its pharmacokinetics at these locations, as micafungin may potentially serve as an alternative antifungal therapy for CNS or pancreatic candidal infections for which the currently recommended first-line therapy fails.

Novel multilocus sequence typing scheme reveals high genetic diversity of human pathogenic members of the Fusarium incarnatum-F. equiseti and F. chlamydosporum species complexes within the United States.

Species limits within the clinically important Fusarium incarnatum-F. equiseti and F. chlamydosporum species complexes (FIESC and FCSC, respectively) were investigated using multilocus DNA sequence data. Maximum-parsimony and maximum-likelihood analyses of aligned DNA sequences from four loci resolved 28 species within the FIESC, within which the species were evenly divided among two clades designated Incarnatum and Equiseti, and four species within the FCSC. Sequence data from a fifth locus, beta-tubulin, was excluded from the study due to the presence of highly divergent paralogs or xenologs. The multilocus haplotype nomenclature adopted in a previous study (K. O'Donnell, D. A. Sutton, A. Fothergill, D. McCarthy, M. G. Rinaldi, M. E. Brandt, N. Zhang, and D. M. Geiser, J. Clin. Microbiol. 46:2477-2490, 2008) was expanded to all of the species within the FIESC and FCSC to provide the first DNA sequence-based typing schemes for these fusaria, thereby facilitating future epidemiological investigations. Multilocus DNA typing identified sixty-two sequence types (STs) among 88 FIESC isolates and 20 STs among 26 FCSC isolates. This result corresponds to indices of discrimination of 0.985 and 0.966, respectively, for the FIESC and FCSC four-locus typing scheme using Simpson's index of discrimination. Lastly, four human and two veterinary isolates, received as members of the FIESC or FCSC, were resolved as five phylogenetically distinct species nested outside these species complexes. To our knowledge, these five species heretofore have not been reported to cause mycotic infections (i.e., F. armeniacum, F. brachygibbosum, F. flocciferum, and two unnamed Fusarium species within the F. tricinctum species complex).

Cerebral aspergillosis caused by Aspergillus granulosus.

Disseminated disease by Aspergillus granulosus has been reported only once previously in a cardiac transplant recipient. We report a fatal central nervous system infection in a lung transplant recipient. Key features of this species in the section Usti include growth at 37 degrees C and large, randomly spaced aggregates of variably shaped Hülle cells.

Trichosporon mycotoxinivorans, a novel respiratory pathogen in patients with cystic fibrosis.

This report describes the molecular epidemiology, in vitro susceptibility, colonial and microscopic morphologies, and biochemical features of Trichosporon mycotoxinivorans, a newly recognized pathogen that appears to have a propensity for patients with cystic fibrosis. The index patient died with histologically documented Trichosporon pneumonia complicating cystic fibrosis. This is also the first report of disease caused by a Trichosporon species in a nontransplant patient with cystic fibrosis. As T. mycotoxinivorans has not previously been recognized as a respiratory pathogen, the significance of its recovery from sputum samples was not initially appreciated. Genetic analysis of archived clinical samples found three additional cases of T. mycotoxinivorans infection which had previously been identified as other members of the genus. An additional isolate of T. mycotoxinivorans was identified from a clinical sample on initial testing. Three of these four cases were also patients with cystic fibrosis. All isolates had MICs at 48 h of amphotericin B of > or = 1 microg/ml and of echinocandins of > or = 16 microg/ml, but they displayed various susceptibilities to the triazoles. In summary, Trichosporon mycotoxinivorans is a newly recognized human pathogen that is associated with cystic fibrosis.

Cutaneous infection caused by Macrophomina phaseolina in a child with acute myeloid leukemia.

We report a case of Macrophomina phaseolina skin infection in an immunocompromised child with acute myeloid leukemia, which was treated successfully with posaconazole without recurrence after a hematopoietic stem cell transplant. The fungus was identified by DNA sequencing using both the internal transcribed spacer and D1/D2 region of the 28S ribosomal DNA gene.