In Vitro Antifungal Susceptibilities And Moleculer Identification Of Clinical Important Trichosporon Species

【Objective】To investigate the in vitro activities of five antifungal drugs against medically important Trichosporon species,and then evaluate a combined PCR-reverse line blot(RLB) hybridization assay to identify 48 Trichosporon isolates.【Method】Firstly,48 Trichosporon strains were charactered by morphology,API 20C AUX,Vitek 2 Compact initially and then we did accurate identification by rDNA ITS and 28S D1-D2 region sequencing.We determined the sequence of intergenic spacer (IGS) 1 region,which is located between the 28S and 5S rRNA genes,in 36 T.asahii isolates for genotyping.MICs of fluconazole,voriconazole,itraconazole,amphotericin B and caspofungin against 48 Trichosporon spp.were detected by E-test method.Probes targeting either ITS or D1-D2 polymorphism(s) between species were designed for evaluate the ability of reverse line blot(RLB) hybridization to detect Trichosporon species.【Result】Morphological characters were unable to differentiate Trichosporon species clearly.Biochemical methods,such as API 20C AUX or Vitek 2 Compact can only detect T.asahii,T.asteroids and T.inkin.Following analysis of both ITS and D1-D2 region, there were 8 Trichosporon species,including 36 T.asahii.1 T.inkin,1 T.dermatis,1 T.cutaneum,1 T.laibachii.2 T.japonicum,4 T.domesticum and 2 T.jirovecii.Comparative sequence analysis of IGS1 region revealed that all 35 T.asahii(excluding strain PUMCH30418) belong to genotype 1,3,4,6,which accounted for 28.6%,20%,48.5%, 2.9%,respectively.It is reported most of the isolates that originated in Japan were of genotype 1,whereas the American isolates were of genotype 3 or 5.The in vitro susceptibility result revealed that Amphotericin B and fluconzole have poor antifungal activities towards 36 Trichosporon asahii,MIC₅₀:24μg/mL and 2μg/mL, MIC₉₀:256μg/mL and 64μg/mL.Except for one T.dermatis isolate has high MIC to voriconazole,voriconazole and itraconazole has good activity against most Trichosporon species.Caspofungin has no in vitro activity against Trichosporon spp.(MIC＞32μg/mL), except for T.cutaneum.Based on rDNA ITS or D1-D2 polymorphism(s),we designed 34 probes for 8 Trichosporon species,which have similar physical characteristics,namely, length,18-30 bp;Tm(melting temperature),58-65℃;secondary structure-no more than moderate;no dimer formation.It costs about 10 hours through the whole procedure. Compared to sequencing results,we successfully applied the RLB assay to identify 48 Trichosporon isolates to species level with high specificity. 【Conclusion】Identification of Trichosporon spp.by conventional morphological methods is often difficult and inconclusive.Biochemical identification(e.g.Vitek-2 or API20C) has always been not suitable to uncommon species.DNA-based molecular procedures,especially ITS and D1-D2 region sequencing,can provide alternative and more useful methods for the characterization and identification of Trichosporon spp. Based on rDNA ITS or D1-D2 polymorphism(s),RLB assay identified 48 Trichosporon isolates to species level successfully,and demonstrated to be a practical,convenient molecular epidemiological and diagnostic tool.The susceptibility patterns of Trichosporon spp.among species varied greatly,and also differ largely to other common yeasts.Amphotericin B and fluconazole show poor antifungal activity against Trichosporon spp.Caspofungin has no in vitro activity.Voriconazole and itraconazole seems to be the most effective agents in vitro.