In Vitro Antifungal Susceptibility And Molecular Identification Of Aspergillus Spp. Commonly Encountered In Clinical Setting

【Objective】To investigate the in vitro antifungal susceptibility of classic and emerging antifungal agents such as voriconazole, itroconazole, amphotericin B, posaconazole and caspofungin to Aspergillus spp. commonly encountered in clinical setting and to establish and evaluate a real-time PCR assay for identification of Aspergillus spp. to species level.【Methods】Fungi were identified by morphology and molecular biology before in vitro antifungal susceptibility testing. E-test method was employed for MIC determination of the 5 antifungal agents including voriconazole, itroconazole, amphotericin B, posaconazole and caspofungin. Consequently, ITS regions in ribosome DNA (rDNA) of the 5 Aspergillus spp. (47 strains) were amplified by common PCR assay. Species-specific probes were then confirmed and modified according to sequences acquired by sequencing of ITS regions. After that, a real-time PCR assay was established for identification of Aspergillus spp. to species level by the probes'specific melting temperatures (Tm). Finally, the sensitivity, specificity and reproducity of the real-time PCR assay were evaluated.【Results】The 5 Aspergillus spp. can basically be identified by morphology, however, it's difficult to identify other fungi to species levels by this method. The MIC90 of caspofungin against A.fumigatus, A.flavus and A.nidulans was 0.094μg/ml whereas the MIC90 against A.niger was 0.19μg/ml. The MIC90 of caspofungin against these 4 species was the lowest among the 5 antifungal agents. For A.niger, the MIC90 of posaconazole was the lowest. The MIC90 against A.fumigatus, A.flavus, and A.niger in order of increasing was caspofungin, posaconazole, voriconazole, itraconazole, amphotericin B. The MIC of amphotericin B against A.terrus was higher than 32μg/ml in all 7 strains tested. The concentration of Aspergillus DNA extracted by MasterPureTM Yeast DNA Purification Kit is low. The OD260/OD280 of DNA extracted by solid culture with liquid nitrogen grinding method, liquid culture with liquid nitrogen grinding method, and solid culture with liquid nitrogen grinding method and purification is 1.73,1.96 and 1.80, respectively. The Tm value of A.terrus specific probe was 64.5℃and 55.2℃, and that of A.fumigatus, A.flavus, A.niger and A.nidulans specific probe was 61.4℃,57.4℃,67.7℃and 65.8℃, respectively. The detection limit of A.fumigatus A.flavus, A.niger, A.terrus and A.nidulans was 56.8fg, 1110fg,13.7fg,123fg,780fg, respectively. Phialophora verrucosa and Rhizopus microspores had cross reactions with A.fumigatus specific probe. The Tm value fluctuation was within 0.5℃in repeated reactions.[Conclusion] Morphology combined with sequencing of ITS region was helpful for identification of Aspergillus spp. less commonly encountered. The in vitro antifungal susceptibility of Etest method showed the new drug caspofungin, which is a kind of echinocandins, had good activity to the five species of Aspergillus spp. and all the tested triazoles had better in vitro activity than traditional amphotericin B. High quality of Aspergillus DNA can be acquired by solid culture with liquid nitrogen grinding and purification. The five species of Aspergillus spp. can be clearly distinguished by melting curve analysis in a single reaction. The real-time PCR is time-saving and with high sensitivity, specificity and reproducity. Therefore, it is promising for diagnosis of invasive aspergillosis.