Intergenic spacer (IGS-1) region sequence-based identification, genotypic analysis, and antifungal susceptibility of clinical Trichosporon species.

OBJECTIVES: Molecular genotyping of Trichosporon species using intergenic spacer region (IGS-1) sequencing and antifungal drug susceptibility testing of T. asahii clinical isolates from Indian patients. MATERIALS AND METHODS: Fifty-five Trichosporon strains were characterized using IGS-1 sequencing from 2006 to 2018 and tested against 5 antifungals using CLSI M27-A3 guidelines. RESULTS: In this study, broad-spectrum antibiotics with steroids, catheters, and ICU stays were major underlying risk factors. These cases were most commonly associated with diabetes (type-2), chronic obstructive pulmonary disease, and hypertension. Out of fifty-five isolates, 47 (85%) were identified as T. asahii, and the remaining 6 were T. inkin (11%) and 2 were Cutaneotrichosporon dermatis (3.6%). The most common genotype of T. asahii was G3 (22; 49%) subsequently G4 (12; 23%), G1 (8; 17%), and G7 (2; 4%). One new genotype of T asahii was found in addition to the fifteen already known genotypes. Indian T. asahii isolates showed a low level of amphotericin B (range 0.06-4 â€‹mg/l) resistance but relatively higher in fluconazole (range 0.25-64 â€‹mg/l). Although, comparatively low MIC ranges were found in the case of voriconazole (0.03-1 â€‹mg/l), posaconazole (0.06-1 â€‹mg/l) and itraconazole (0.06-1 â€‹mg/l). Voriconazole appeared to be the most active drug in T. asahii isolates. The MICs for all the drugs were comparatively lower in the case of non-Trichosporon asahii strains. CONCLUSION: T. asahii was the most common Trichosporon isolate. Speciation is necessary for optimal antifungal therapy. Voriconazole-based treatment, Steroids, removal of catheters and control of underlying conditions results in positive outcomes.

Faster and accurate identification of clinically important Trichosporon using MALDI TOF MS.

PURPOSE: Trichosporon species are emerging human pathogens, accounting for the second most common cause of non-candidal mycosis. Rapid and reliable identification of these agents allows a better understanding of their epidemiology and therapeutic management. The Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) technique has the potential to be precise, fast and cost-effective. However, the precision of identification totally depends upon the type of protein extraction method used and embedded database in the system. Our objectives were to standardize the protein extraction technique and expand the present Bruker database by creating an in-house database and validating it with diverse clinical Trichosporon species of Indian origin. METHODS: Two different protein extraction protocols (on-plate and off-plate) were evaluated. The off-plate protocol was finalized for the identification. MALDI TOF MS with the existing Bruker database was evaluated for its ability to identify a total of 79 intergenic spacer 1 (IGS1) gene sequence confirmed clinical isolates of 5 different Trichosporon species. RESULTS: As outcome, off plate protocol yielded higher accuracy (73% on the species level and 95% on the genus level) than on-plate (25% on the genus level) in terms of log scores. The existing database for Trichosporon species was enriched with 28 sequence confirmed isolates, which improved accuracy from 73% to 100% and were identified up to species level with a log score >2.3. CONCLUSIONS: Used with standardized protein-extraction protocol along with an expanded database, MALDI-TOF MS could be a rapid and reliable approach to identify clinical Trichosporon species routinely in the laboratory.