Evaluation Of Antifungal Susceptibility Of Candida Auris To Ravuconazole And Preliminary Study Of Resistance Mechanism

Part ?In vitro activity of ravuconazole against vaginal Candida isolates Objective:To investigate the activity of ravuconazole against vaginal Candida isolates of China and assess the feasibility of ravuconazole in the treatment of vulvovaginal candidiasis.Method:We determined the MICs of ravuconazole and fluconazole on 525 vaginal Candida isolates(Candida albicans,Candida tropicalis,Candida glabrata and Candida parapsilosis)from 9 provinces of China by Clinical and Laboratory Standards Institute(CLSI)methodology.Result:For vaginal Candida isolates,the proportion of ravuconazole-resistant isolates is 0.19%(1/525),the proportion of fluconazole-resistant isolates is 5.52%(29/525).Conclusion:Ravuconazole was in good active against vaginal Candida isolates,which suggested ravuconazole could be used in the treatment of vulvovaginal candidiasis.Part ?Evaluation of drug sensitivity of Candida auris to ravuconazole and preliminary study on resistance mechanismObjectives:To investigate the in vitro and in vivo activity of ravuconazole and fluconazole against C.auris and explore the expression of virulence and drug-resistance related gene of C.auris.Methods:1.We determined the in vitro activity of ravuconazole and 9 comparators against 15 C.auris isolates by CLSI M27-E4 methodology.2.Galleria mellonella was the infection model.We investigated the in vivo activity of ravuconazole and fluconazole at different concentrations against C.auris by measuring the survival rate and fungal load and observing the pathological sections of infected larvae.3.The expression of adhesion related genes(ALS5,HYR3),hydrolase related genes(SAP5,PLB1),fungal cell wall,membrane and extracellular matrix related genes(ERG2?KRE6?EXG?ENG1)of C.auris were detected by real-time fluorescence quantitative PCR.Observing the protein expression of C.auris by MALDI-TOF MS.Results:1.The MICs of fluconazole and amphotericin B on C.auris were much higher than second-generation azoles and echinocandins.Ravuconazole was active against all the C.auris isolates and as effective as isavuconazole,posaconazole and echinocandins while showed a better antifungal activity than itraconazole,voriconazole to C.auris.2.The survival rate and fungal load of infected models suggested that antifungal drugs could significantly elevate the survival rate and reduce the fungal load compared to the control.In the tissue section,the fungus clump was the least in the high concentration ravuconazole group.3.The effect of ravuconazole on the virulence and drug-resistance related gene expression of C.auris.MALDI-TOF MS:the mass spectra of C.auris treated with ravuconazole and fluconazole showed that the peaks of protein in different daltons changed.Quantitative RT-PCR:the expression of virulence and drug-resistance related genes in CBS 12766 group was higher than that in CBS 10913 group.Compared with fluconazole group,the gene expression was significantly up-regulated in the ravuconazole group.Conclusion:Ravuconazole was in good active against C.auris in vitro and vivo.The virulence of CBS 12766 was higher than CBS 10193.By comparing the gene expression status,we found that C,auris would up-regulated the drug-resistance and virulence related genes to cope with the external environmental pressure.Compared with fluconazole,the external environmental pressure caused by ravuconazole was more significant.Part ?In vitro activity of itraconazole combined with terbinafine against Candida aurisObjective:To determine the potential antifungal effect of itraconazole combined with terbinafine against C.auris in vitro.Method:We determined the in vitro activity of itraconazole and terbinafine in combination against 15 C.auris isolates by the checkerboard method according to CLSI M27-E4 documents.The fractional inhibitory concentration index(FICI)model was used to evaluate the in vitro antifungal effect of itraconazole combined with terbinafine.Result:The FICIs of 15 C.auris isolates ranged from 0.75 to 2,indicating that itraconazole combined with terbinafine has indifference effect against C.auris.Terbinafine combined with itraconazole can reduce the "tailing phenomenon" of itraconazole in vitro susceptibility test.Conclusion:Itraconazole combined with terbinafine against C.auris showed indifference effect.By naked eye observation,the combination of the two drugs can inhibit the growth of C.auris,which may be used in the treatment of C.auris infection.