Multicenter Study Of Identification,Antifungal Susceptibility And Resistance Mechanism To Triazoles Of Aspergillus In China

[Objective]To evaluate the performance of gene-level and protein-level identification methods in Aspergillus identification.To analyze the in vitro antifungal susceptibility profiles of clinical Aspergillus isolates collected from multiple centers in China,and agreement between the commercial Sensititre YeastOne system and the reference broth microdilution method in in vitro antifungal susceptibility testing of Aspergillus was also assessed.To investigate the potential mechanism underlying triazole resistance of Aspergillus which is a hot topic in the global at present.[Methods]Gene sequencing analysis was carried out with DNA markers of the internal transcribed spacer(ITS),?-tubulin gene(BenA)and calmodulin gene(CaM)to identify 332 isolates of Aspergillus collected from 13 centers in China,and the most effective gene marker in Aspergillus identification will be selected.Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS)identification was performed and the optimized measures in the identification procedures and the application of the in-house database were also employed to improve the capacity of MALDI-TOF MS in Aspergillus identification.The in vitro antifungal susceptibility testing was performed according to the reference CLSI M38-A2 broth microdilution method to evaluate the antifungal susceptibility profiles and resistance rates of the tested multicenter Aspergillus isolates against the clinical used antifungal agents.Sensititre YeastOne antifungal susceptibility testing system was also employed in parallel and the agreement between those two methods in determination of in vitro antifungal susceptibility of Aspergillus was evaluated.The mutations in CYP51A gene of triazole-resistant Aspergillus were detected through molecular sequencing analysis and the expression of azole-resistant relevant genes including CYP51A,CYP51B and Cdr1B,etc.,were evaluated by real-time quantitative PCR.In vitro induction of triazole resistance was carried out on a triazole-susceptible Aspergillus fumigatus isolate to obtain the homologous triazole-resistant Aspergillus fumigatus,and the mutations and expression levels of genes involving in triazole resistance of Aspergillus during this induction process were analyzed.[Results]Gene sequencing analysis revealed that 332 Aspergillus isolates belonging to 15 species,and CaMgene had the highest species level identification capacity with rate of 97.9%to Aspergillus.Only 27.4%(91/332)isolates could be identified to species level and 56.3%(187/332)to genus level by MALDI-TOF MS with the Bruker recommended detection parameters.The species level identification rate of MALDI-TOF MS to Aspergillus could be increased significantly from 27.4%to 83.7%(278/332)with no increase of the false identification rate after the species level identification cut-off value of MALDI-TOF MS was properly adjusted from>2.0 to>1.7.With the combination of the in-house MS database,94.3%(313/332)Aspergillus isolates could be identified correctly at species level by MALDI-TOF MS.96.7%(321/332)Aspergillus isolates in our study were completely susceptible to the tested six antifungl agents.The voriconazole(VOR)resistance rate was the highest one of 2.4%(8/332)among Aspergillus isolates in this study.The essential agreement rate and the categorized agreement rate between Sensititre YeastOne and CLSI M38-A2 in antifungal susceptibility testing of Aspergillus could be reached to 91.2%and 98.8%,respectively.Two triazole-resistant Aspegillus fumigatus isolates were detected in this study and one was resistant to itraconazole(ITR)and VOR dually with mutation of TR34/L98H in CYP51A gene,the other one was only resistant to VOR with CYP51A gene mutation of TR46/Y121F/T289A.Both the triazole-resistant isolates presented significantly increased expression of CYP51A gene.Homologous triazole-resistant Aspegillus fumigatus isolates from a triazole-susceptible Aspergillus fumigatus were obtained through the in vitro induction assay with triazoles,and only single-site amino acid changes in CYP51A gene were detected including G448S and P216L;the expression of CYP51A gene in induced triazole-resistant Aspergillus fumigatus isolates were also increased remarkably compared to the homologous triazole-susceptible isolate.The in vitro induction with the combination of ITR and VOR could generate multiply triazole-resistant isolates and also significantly increased expression of Cdr1B gene.[Conclusions]Calmodulin gene(CaM)could be recommended as the first-line DNA marker for molecular sequencing identification of Aspergillus.MALDI-TOF MS is a preferable and practical method in identification of clinical Aspergillus isolates,especially when the proper species level identification cut-off value and the in-house MS database were employed together.The in vitro susceptibility profiles of clinical Aspergillus isolates from multiple centers in this study are pretty well,and the rate of triazole-resistant Aspergillus fumigatus in the clinical setting is still keeping in a low level,but the following-up surveillance will be advisable and necessary.The commercially available Sensititre YeastOne antifungal susceptibility testing system is an alternative method for in vitro antifungal susceptibility testing of Aspergillus.Mutations and highly increased expression of CYP51A gene are highly relevant to the resistance mechanism of triazole-resistant Aspergillus fumigatus in this study.In vitro induction of triazole resistance could cause single-site amino acid changes and moderate increase of CYP51A gene expression in Aspergillus fumigatus.In addition,the remarkably increased expression of Cdr1B gene might participate in the defensive response of Aspergillus fumigatus to the stress of higher concentration of antifungals surrounding.