ERG3/ERG11 Mutation And Drug Resistance Of Recurrent Vulvovaginal Candidiasis

Objective: ①To compare the differences of drugs susceptibilities between vulvovaginal candidiasis (VVC) and Recurrent Vulvovaginal Candidiasis (RVVC) patients and the resistance situation of each drug by detecting the susceptibilities of isolated Candida to eight kinds of antifungal drugs in vitro. ② To analyze the relationship between gene mutation and drug resistance by ERG 11 and ERG3 gene sequencing to samples of sensitive,dose dependent sensitive and resistant straints. Meanwhile, try to explore the resistant mechanism of RWC.Methods and material: ①A total of 406 Candida isolates,which were taken from WC and RVVC non-pregant patients, were identified with chromogenic medium CHROMagar and the chromogenic medium of Autobio and the VITEK 2. ②A modifying broth microdilution antifungal susceptibility test was preformed with the Clinical And Laboratory Standards Institute(CLSI) document M27-A3 guidelines. According to the minimum inhibitory concentration (MIC) of the drugs, the suscepitibility of drug was determined. ③ extracted DNA from samples, amplified gene ERG3/ERG11 and obtain DNA sequencing, then compared with standard sequence respectively and found out the point mutations.Results:Candida albicans is dominant isolate of VVC and RVVC. Candida glatraba was dominant isolate in non-albicans. For Candida albicans, drug susceptibility of fluconazole was highest sensitive while of itraconazole was worst. For order of sensitive in two groups as follows,VVC:fluconazole>5-fluorocytosine> amphotericin B> clotrimazole> nystatin> econazole> miconazole>itraconazole; RVVC:amphotericinB>fluconazole/5-fluorocytosine>clotrimazole>miconazole> econazole> nystatin> itraconazole. Amphotericin B and miconazole were more sensitive in RVVC group than in VVC group (p<0.05). For non-albicans, drug susceptibility of 5-fluorocytosine was highest sensitive and stable while of itraconazole was worst. For order of sensitive in two groups as follows,VVC: amphotericinB>5-fluorocytosine>miconazole>fluconazole>nystatin>clotrimazole>ec onazole>itraconazole;RVVC:5-fluorocytosine/amphotericinB>fluconazole>miconazo le/clotrimazole> nystatin> econazole> itraconazole. There were no differences of drug susceptibility between RVVC and VVC group. Candida glatraba was less sensitive to nystatin and clotrimazole and fluconazole than Candida tropical is (P<0.05).For missense mutations of ERG3 in Candida albicans, there were three strains in RVVC group:the strains with T59T/G and C82C/T missense mutations were no drug resistance, and the strain with C223T missense mutation was resistant to econazole and miconazole. There was one strain in VVC group:the strain with G880G/A missense mutation was resistant to clotrimazole and itraconazole. There was no difference for percentage of missense mutations between two groups (P>0.05). For missense mutations of ERG3 in non-albicans, there was one strain with A334A/G missense mutation in each group, and both of them were resistant to various azoles. There was no difference for percentage of missense mutations between two groups (P>0.05).For missense mutations of ERG 11 in non-albicans, there were three strains in RVVC group and eleven strains in VVC group. There were twelve missense mutations which might be related to drug resistance:T394C、A383、C982C/G、 G340T、A1025A/G、T26T/A、T1021T/G、T26T/G、C166T、T395A、G1193T、 A569A/G. There was no difference for percentage of missense mutations between two groups (P>0.05). Only VVC group had multi-missense mutations. For missense mutations of ERG 11 in Candida albicans, there were seventy-four strains in RVVC group and seventy-three strains in VVC group. There were fifteen missense mutations which might be related to drug resistance:A383A/C、G340T、T394C、C368T、T769C、 G1393A、A1444A/C、A1025A/G、A620A/G、G640T、C341T、T433C、A1303A/C、 A1007A/G、T1346A. G1354G/A and G1343A missense mutations may be not associated with azoles resistance.Percentage of missense mutations in RVVC group was higher than that in WC group (49.3% VS 35.6%), P=0.01. Except the percentage of resistance of miconazole in VVC group was higher than that in RVVC group (P<0.05), there were no statistically significance for the rest of azoles (P>0.05). For multi-missense mutations of ERG11 in Candida albicans, there were twenty-eight strains in RWC group and thirteen in VVC group, and there was significant difference between two groups, P=0.007.Conclusion: ①Candida albicans is dominant isolate of VVC and RWC. For albicans, the priority of antifungal drug for WC is fluconazole, and the second one is 5-fluorocytosine; the priority of antifungal drug for RWC treatment is amphotericinB, and the second one is 5-fluorocytosine or fluconazole. For non-albicans, the priority of antifungal drug for VVC is 5-fluorocytosine or amphotericinB, and the second one is fluconazole or miconazole; the priority of antifungal drug for RWC treatment is 5-fluorocytosine or amphotericinB, and the second one is fluconazole.Itraconazole is not recommended in all of them.② T59T/G and C82C/T missense mutations of ERG3 may be not associated with azoles resistance. C223T, G880G/A and A334A/G missense mutations of ERG3 might were related to azoles resistance. ③ It was found out twenty-seven missense mutations of ERG 11 which might be related to azoles resistance in this study. In non-albicans, only WC group had multi-missense mutations. In Candida albicans, Percentage of missense mutations and multi- missense mutations in RVVC group were higher than that in VVC group.