Study On The Correlation Of Invasiveness,genotype And Drug Sensitivity In Vitro Of RVVC Pathogens

Objectives:1.To identify the strain of the pathogenic Candida of vulvovaginal candidiasis(VVC)and recurrent vulvovaginal candidiasis(RVVC)patients,and analyze the strain distribution.2.To analyze genotype of Candida distribution and its relationship with its source by genotyping the pathogenic Candida.3.Through the in vitro susceptibility test of three antifungal drugs(fluconazole,amphotericin B,itraconazole),to compare drug sensitivity differences and analyze the relationship between drug sensitivity of different Candida and their sources and genotypes.4.Through determining the activity of secreted aspartic protease and extracellular phospholipase and the expression level of Sap-2 and PLB-1 of the pathogenic Candida,to analyze the relationship between the invasiveness-related factors of pathogenic Candida and its source,genotype and drug sensitivity.Methods:1.All pathogenic strains were collected from patients with vulvovaginal candidiasis in gynecology and obstetrics clinic of our hospital.Among them,95 strains were RVVC pathogenic strains and 223 strains were VVC pathogenic strains.Then all samples were isolated and purified and were initially identified with chromogenic medium CHROMagar and chromogenic medium of Autobio and finally identified with the VITEK-2 system.2.The genomic DNA of the sample was extracted,and according to the presence or size of transposable type I intron in the 25 SrDNA gene coding region of the sample,genotyping 270 strains of Candida albicans by PCR.3.Drug susceptibility test in vitro was preformed with the microdilution method recommended by the Clinical And Laboratory Standards Institute(CLSI)document M27-A3 guidelines.Drug susceptibility was judged by the breakpoint value provided by CLSI combined with the minimum inhibitory concentration(MIC)obtained from the experiment.4.The secreted aspartic protease and extracellular phospholipase activity of the sample were determined by using milk medium and egg yolk medium respectively.5.The expression levels of Sap-2 and PLB-1 in the sample were determined by ELISA.6.Statistical analysis of experimental data using statistical software.Results:1.A total of 318 pathogenic bacteria of vulvovaginal candidiasis were collected,including 196 non-pregnant VVC pathogenic bacteria,186 of which were Candida albicans(94.9%)and 75 non-pregnant RVVC pathogenic bacteria,69 of which were Candida albicans(92.0%)and 27 of which were pregnant VVC pathogenic bacteria,all of which were identified as Candida albicans(100%)and 20 of which were RVVC pathogenic bacteria during pregnancy,19 of which were Candida albicans(95%);suggesting that Candida albicans was the predominant pathogenic strain of VVC and RVVC in pregnancy and non-pregnancy.A total of 17 non-Candida albicans were detected in all samples,of which Candida glabrata was the predominant strain(52.9%).2.270 strains of Candida albicans were divided into three genotypes,of which 237 strains(87.8%)were intron-free strains,23 strains(8.5%)of type B and 10 strains(3.7%)of type C were intron-containing strains,and there was no significant difference in the proportion of intron-free group(type A)and intron-containing group(type B+C)in the pathogenic strains of pregnant and non-pregnant VVC and RVVC patients,P>0.05.Genotype A was the most common strain.3.The drug sensitivity of Candida albicans in non-pregnant RVVC group and VVC group was the highest and itraconazole was the worst;among the different genotypes of non-pregnant VVC patients,the sensitivity rates of fluconazole,amphotericin B and itraconazole for 24 hours in intron-free group(type A)and intron-containing group(type B+C)had no statistical difference,P>0.05;the sensitivity rates of fluconazole for 48 hours had no statistical significance.The sensitivity rate of amphotericin B in intron-free group(type A)was higher than that in intron-containing group(48h),while the sensitivity rate of itraconazole was lower than that in intron-containing group(P<0.05).4.The MIC values of different genotypes of non-pregnant VVC patients in vitro were significantly different,P<0.05.The MIC values of fluconazole and itraconazole without intron group(A)were higher than those of intron-containing(B+C)at 24 h and 48 h,while the MIC values of amphotericin B at 24 h and 48 h were lower than those of intron-containing group.5.In non-pregnant RVVC strains,the positive rate of extracellular phospholipase and enzyme activity of Candida albicans with intron group were higher than those without intron group,with statistical difference(P<0.05).6.The aspartate protease activity of itraconazole-sensitive strains was stronger than that of non-sensitive strains in non-pregnant RVVC pathogenic Candida albicans(P<0.05).7.The expression levels of Sap-2 and PLB-1 of Candida albicans in patients with RVVC were significantly higher than those of VVC patients,and the expression of PLB-1 was statistically significant(P<0.05).Conclusions:1.Candida albicans is still the main pathogenic strain of pregnant and non-pregnant VVC and RVVC.2.Fluconazole has the highest sensitivity among the three antifungal drugs,with a sensitivity rate of more than 95%,and can be the first choice for clinical treatment.3.The presence of transposable intron I in the coding region of 25SrDNA gene may affect the drug sensitivity of vaginal pathogenic Candida albicans to fluconazole,amphotericin B and itraconazole,and it may enhance the extracellular phospholipase activity of Candida albicans,while the aspartate protease activity of pathogenic Candida albicans may be related to the drug sensitivity of the strain to itraconazole.4.Recurrence of vulvovaginal candidiasis may be related to increased expression of Sap-2 and PLB-1 in pathogenic Candida albicans.