| Objective:1.To understand the distribution of Candida strains and the sensitivity to azole drugs in VVC patients in this region.2.To investigate the relationship between ERG3,Efg1 mutation and expression level of Candida albicans in patients with VVC and the drug resistance of azole drugs.3.To investigate the relationship between Efg1 and ERG3 of VVC in resistance to azole drugs.Methods:Collected from November 2015 to May 2016 in Shanxi Province Maternal and Child Health Hospital gynecological clinic VVC patients with vaginal discharge specimens of 184 cases,Through culture,CHROMagar Candida culture,API20 CAUX identification system and molecular biology method for isolation and identification.Fifty Candida albicans were tested by M27-A3 micro-liquid-based dilution method.They were divided into sensitive group and resistant group.The genomic DNA of the two strains was extracted,and the specific primers of ERG3 and Efg1 were amplified and sequenced.The mutations were found by comparison with the standard sequences in genbank.The relationship between mutation and drug resistance was studied.The total mRNA of the strain was analyzed by fluorescence quantitative PCR and its expression was correlated with drug resistance.Results:1.A total of 184 vaginal secretions of VVC patients were collected,The species distribution shows: Candida albicans 146(79.35%),Candida glabrata 22(22.0%),Candida tropicalis 6(3.62%),Candida parapsilosis 5(2.72%),Candida albicans 2(1.01%),the other 3(1.63%).2.In vitro drug susceptibility tests showed that the resistance rates of 50 Candida albicans to fluconazole(FCA),itraconazole(ITR)and voriconazole(VRC)were 44%,56% and 50%,There is cross-resistance phenomenon.3.Gene sequencing showed that:(1)ERG3 sequencing results: the R352 H missense mutation occurred in one strain,the W219 C missense mutation occurred in the two strains,and the three strains were resistant to FCA,ITR and VRC;(2)Efg1 sequencing results: 6 strains of V86 I missense mutation occurred,of which 1 strains were resistant to ITR alone,more than 5 strains were sensitive strains.4.The expression of ERG3 and Efg1 mRNA in drug-resistant group(FCA,ITR,VRC and cross-resistance)and corresponding sensitive group were statistically analyzed:(1)ERG3 mRNA expression analysis: 8 groups of data are in line with the normal distribution,using t test,the results with the mean±standard deviation(?x±s)said.FCA-sensitive group(2.01±0.60)was higher than that in resistant group(0.59±0.36),t=10.29,P<0.001,the difference was statistically significant;ITR-sensitive group(2.21±0.52)was higher than that in the resistant group(0.73±0.43),t=10.97,P<0.001,the difference was statistically significant;VRC-sensitive group(2.10±0.57)was higher than that in the resistant group(0.66±0.40),t=10.42,P<0.001,the difference was statistically significant;the sensitive group(2.54±0.39)was higher than that in the cross-resistant group(0.31±0.22),t=17.99,P<0.001,the difference was statistically significant.(2)Efg1 mRNA expression analysis:Four groups of drug-resistant groups were not subject to normal distribution,using rank sum test,the results with the median±four quartile spacing(M±QR)said.FCA-resistant group(2.02±2.78)was higher than that in the sensitive group(0.55±0.65),the average rankings are 39.5 and 14.5,P<0.001,the difference was statistically significant;ITR-resistant group(1.74±1.75)was higher than that in the sensitive group(0.45±0.34),the average rankings are 36.48 and 11.52,P<0.001,the difference was statistically significant;VRC-resistant group(1.88±2.22)was higher than that in sensitive group(0.48±0.50),the average rankings are 38 and 13,P<0.001,the difference was statistically significant;the cross-resistant group(3.68±5.88)was higher than that in the sensitive group(0.34±0.11),the average rankings are 19.5 and 7.0,P<0.001,the difference was statistically significant.(3)The correlation between ERG3 and Efg1 was analyzed by Spearman correlation analysis.The results showed that the correlation coefficient r =-0.614,P <0.001,which was negatively correlated.Conclusion:1.Candida albicans in the region is still dominant in VVC infection,Candida albicans have different degrees of resistance to azole drugs,so the treatment of VVC should be identified as bacteria and drug sensitivity analysis as a guide.2.ERG3 high expression may increase the sensitivity of Candida albicans to azole drugs;ERG3 mutations can make Candida albicans resistant to azole drugs,W219 C,R352H is involved in azole drug resistance still need to be further confirmed.3.Efg1 overexpression may increase the resistance of azole drugs,but the relationship between Efg1 mutations and drug resistance remains to be further studied.4.Candida albicans Efg1 and ERG3 were negatively correlated with azole drug resistance and Efg1 negatively regulated ERG3. |
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