Performance of optimized McRAPD in identification of 9 yeast species frequently isolated from patient samples: potential for automation

<p>Abstract</p> <p>Background</p> <p>Rapid, easy, economical and accurate species identification of yeasts isolated from clinical samples remains an important challenge for routine microbiological laboratories, because susceptibility to antifungal agents, probability to develop resistance and ability to cause disease vary in different species. To overcome the drawbacks of the currently available techniques we have recently proposed an innovative approach to yeast species identification based on RAPD genotyping and termed McRAPD (Melting curve of RAPD). Here we have evaluated its performance on a broader spectrum of clinically relevant yeast species and also examined the potential of automated and semi-automated interpretation of McRAPD data for yeast species identification.</p> <p>Results</p> <p>A simple fully automated algorithm based on normalized melting data identified 80% of the isolates correctly. When this algorithm was supplemented by semi-automated matching of decisive peaks in first derivative plots, 87% of the isolates were identified correctly. However, a computer-aided visual matching of derivative plots showed the best performance with average 98.3% of the accurately identified isolates, almost matching the 99.4% performance of traditional RAPD fingerprinting.</p> <p>Conclusion</p> <p>Since McRAPD technique omits gel electrophoresis and can be performed in a rapid, economical and convenient way, we believe that it can find its place in routine identification of medically important yeasts in advanced diagnostic laboratories that are able to adopt this technique. It can also serve as a broad-range high-throughput technique for epidemiological surveillance.</p

Evaluation multicentrique d'une méthode EUCAST pour tester la sensibilité antifongique des dermatophytes produisant des spores

Background: Terbinafine resistance is increasingly reported in Trichophyton rubrum and Trichophyton interdigitale rendering susceptibility testing important particularly in non-responding cases. We performed a multicentre evaluation of a recently proposed modified EUCAST method implementing medium supplemented with chloramphenicol and cycloheximide (CC) to avoid contamination. Materials/methods: A blinded panel of wild-type and squalene epoxidase (SQLE) target gene mutant T. rubrum and T. interdigitale strains were distributed to 10 European laboratories. Susceptibility to terbinafine, itraconazole, voriconazole and amorolfine) were performed according to the E.Def 9.3.1 method with and without addition of chloramphenicol and cycloheximide (final concentrations 50 mg/L and 300 mg/L, respectively). Plates were incubated at 25 °C (one laboratory used 30 °C) for 5-7 days until sufficient growth. MICs were determined visually (ignoring trailing growth for itraconazole) and spectrophotometrically with 90% and 50% endpoints yielding a total of 7,829 MICs. A. flavus ATCC 204304 and A. flavus CNM-CM1813 were included as controls. Results: 100%/96% (voriconazole) and 84%/84% (itraconazole) MIC determinations fell within the QC ranges for the two QC strains, respectively, and 96%/92% terbinafine MICs fell in a 0.25-1 mg/L 3 two-fold-dilution range suggesting a high interlaboratory reproducibility. Across the six methods, the number of terbinafine MEs varied from 2 (2.6%) to 5 (6.6%) for T. rubrum and between 0 and 2 (2.0%) for T. interdigitale (lowest for the CC-method (2.6%-4.4%/ 0-1% for T. rubrum/T. interdigitale). The difference between the modes for the wt and mutant population were ≥7 two-fold-dilutions in all cases (Table). If excluding a I121M/V237I T. rubrum mutant, and two mixed T. interdigitale strains, the number of VMEs were CC visual: T. rubrum: 1/77 (1.3%), CC spec-90%: 3/68 (4.4%) and CC spec-50%: 1/76 (1.3%), and none for T. interdigitale. The activity of voriconazole, itraconazole and amorolfine were quite uniform against T. rubrum and T. interdigitale, but unacceptably wide MIC ranges were found for the visual and spec-90% inhibition methods for itraconazole (data not shown). Conclusions: Although none of the laboratories perform dermatophyte testing at a regular basis an acceptable interlaboratory agreement and good separation between SQLE wt and mutants were found, suggesting a robust performance of the proposed method

MixInYeast: A Multicenter Study on Mixed Yeast Infections

Invasive candidiasis remains one of the most prevalent systemic mycoses, and several studies have documented the presence of mixed yeast (MY) infections. Here, we describe the epidemiology, clinical, and microbiological characteristics of MY infections causing invasive candidiasis in a multicenter prospective study. Thirty-four centers from 14 countries participated. Samples were collected in each center between April to September 2018, and they were sent to a reference center to confirm identification by sequencing methods and to perform antifungal susceptibility testing, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). A total of 6895 yeast cultures were identified and MY occurred in 150 cases (2.2%). Europe accounted for the highest number of centers, with an overall MY rate of 4.2% (118 out of 2840 yeast cultures). Of 122 MY cases, the most frequent combinations were Candida albicans/C. glabrata (42, 34.4%), C. albicans/C. parapsilosis (17, 14%), and C. glabrata/C. tropicalis (8, 6.5%). All Candida isolates were susceptible to amphotericin B, 6.4% were fluconazole-resistant, and two isolates (1.6%) were echinocandin-resistant. Accurate identification of the species involved in MY infections is essential to guide treatment decisions

Azole-Resistance in Aspergillus terreus and Related Species: An Emerging Problem or a Rare Phenomenon?

Raquel Sabino was not included as an author in the published article. It was corrected a posteriori.Erratum in - Corrigendum: Azole-Resistance in Aspergillus terreus and Related Species: An Emerging Problem or a Rare Phenomenon? [Front Microbiol. 2018] Front Microbiol. 2019 Jan 14;9:3245. doi: 10.3389/fmicb.2018.03245. eCollection 2018.Disponível em: https://www.frontiersin.org/articles/10.3389/fmicb.2018.03245/fullFree PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882871/ | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340063/Objectives: Invasive mold infections associated with Aspergillus species are a significant cause of mortality in immunocompromised patients. The most frequently occurring aetiological pathogens are members of the Aspergillus section Fumigati followed by members of the section Terrei. The frequency of Aspergillus terreus and related (cryptic) species in clinical specimens, as well as the percentage of azole-resistant strains remains to be studied. Methods: A global set (n = 498) of A. terreus and phenotypically related isolates was molecularly identified (beta-tubulin), tested for antifungal susceptibility against posaconazole, voriconazole, and itraconazole, and resistant phenotypes were correlated with point mutations in the cyp51A gene. Results: The majority of isolates was identified as A. terreus (86.8%), followed by A. citrinoterreus (8.4%), A. hortai (2.6%), A. alabamensis (1.6%), A. neoafricanus (0.2%), and A. floccosus (0.2%). One isolate failed to match a known Aspergillus sp., but was found most closely related to A. alabamensis. According to EUCAST clinical breakpoints azole resistance was detected in 5.4% of all tested isolates, 6.2% of A. terreus sensu stricto (s.s.) were posaconazole-resistant. Posaconazole resistance differed geographically and ranged from 0% in the Czech Republic, Greece, and Turkey to 13.7% in Germany. In contrast, azole resistance among cryptic species was rare 2 out of 66 isolates and was observed only in one A. citrinoterreus and one A. alabamensis isolate. The most affected amino acid position of the Cyp51A gene correlating with the posaconazole resistant phenotype was M217, which was found in the variation M217T and M217V. Conclusions:Aspergillus terreus was most prevalent, followed by A. citrinoterreus. Posaconazole was the most potent drug against A. terreus, but 5.4% of A. terreus sensu stricto showed resistance against this azole. In Austria, Germany, and the United Kingdom posaconazole-resistance in all A. terreus isolates was higher than 10%, resistance against voriconazole was rare and absent for itraconazole.This work was supported by ECMM, ISHAM, and EFISG and in part by an unrestricted research grant through the Investigator Initiated Studies Programof Astellas, MSD, and Pfizer. This study was fundet by the Christian Doppler Laboratory for invasive fungal infections.info:eu-repo/semantics/publishedVersio