Research Of The Relation Among Mrr2?CDR1 And Fluconazole Resistance In VVC Patients' Candida Albicans

BackgroundVulvovaginal Candidasis(VVC)is a kind of vulva and vagina inflammatory disease mainly caused by Candida spp.VVC is characterized by vulva edema,erosion and increased residue-like leucorrhea,they often accompany pruritus vulvae,burning-sensitive pain,frequency,urgency,dysuria,algopareunia,and even restless sleep and sitting,which could threaten women's physical and mental health,it's the second largest common Vulvovaginitis after the bacterial vaginosis.VVC has a high morbidity and recurrence rate,so the prevention and treatment of VVC is particularly important.Candida albicans is the most common pathogen of VVC.Oral azole drugs are mainly used in the treatment of VVC now.Fluconazole(FCA)is one of the most commonly used azole drugs in the treatment of VVC,but long-term non-standard treatment,pregnancy,low immunity and many other reasons have made FCA drug-resistant strains more common,which straightly lead to failure of VVC treatment.Thus,the research of FCA resistance mechanism of Candida albicans in VVC patients is imminent.ObjectiveTo investigate the distribution of Candida albicans isolated from local VVC patients and its in vitroresistance to FCA.To investigate the relationship between Mrr2 mutation and FCA resistance in Candida albicans isolated from VVC patients.To investigate the relationship between the mRNA expression of Mrr2 and FCA resistance in Candida albicans isolated from VVC patients.To investigate the relationship between the expression of CDR1 and FCA resistance in Candida albicans isolated from VVC patients.Further investigate the interaction between Mrr2 expression and CDR1 expression in FCA resistance.To investigate the interaction between Mrr2 mutation and Mrr2 expression in Candida albicans isolated from VVC patients in FCA resistance.Methods:Collected 155 vaginal secretion samples from suspected VVC patients from November 2015 to May 2016 in the Second Hospital of Shanxi Medical University dermatological department.Strain identification was performed by Fungal microscopy,CHROMagar Candida culture medium and API 20 C AUX Candida identification system,and 102 samples were positive.M27-A3 method was used in FCA susceptibility test of 80 Candida albicans.Preliminary extracted DNA from 80 Candida albicans,amplified the Mrr2 gene fragment by Polymerase Chain Reaction(PCR)and sequenced to find missense mutations.Extracted the total RNA,and detected the expression of Mrr2 and CDR1 by Real-time Fluorescence Quantitative PCR(FQ-RT-PCR)with the help of reverse transcription cDNA,then do statistical analysis.Results:(1)A total of 102 Candida spp.strains were obtained from 155 samples of vaginal secretions.There were 80 Candida albicans(78.4%),followed by 15 Candida glabrata(14.7%),4 Candida tropicalis(3.9%),2 Candida parapsilosis(2.0%),1 Candidakrusei(1.0%).(2)FCA susceptibility test was performed by M27-A3 method,40 Candida albicans were susceptible to FCA,10 dose-dependent and 30 resistant,the resistance rate was 37.5%.(3)Sequenced the Mrr2 gene obtained from 80 Candida albicans.The mutation rate of Mrr2 gene was 56.67% in FCA-resistant group,and 26.08% in FCA-sensitive group.The Mrr2 gene mutation and FCA resistance of Candida albicans were tested by the four-cell chi-square test,P<0.05,the difference was statistically significant.The mutation rate of Mrr2 gene in the resistant group was higher than sensitive group.(4)Detected Mrr2 and CDR1 mRNA expression levels by statistical analysis:(1)The mRNA expression of Mrr2 in FCA-resistant group was higher than that in sensitive group(1.10 ± 0.50 vs 0.52 ± 0.45),P < 0.05,the difference was statistically significant.(2)The mRNA expression level of CDR1 in FCA-resistant group was higher than that in sensitive group(0.42 ± 0.294 vs 0.25 ± 0.289),P < 0.05,the difference was statistically significant.(3)CDR1 was positively correlated with Mrr2,r = 37.6%.(4)The FCA resistance in Mrr2 gene mutationand overexpression groupwas 47.50 times higher than that of the none-mutation and low expression group,there might be a synergistic effect between Mrr2 gene mutation and high expression in the production of FCA resistance.Conclusion(1)The main pathogen of local VVC patients is Candida albicans,and it has a higher FCA resistance rate.Based on drug sensitivity results,rational antifungal drugs should be selected for precise VVC treatment.(2)The FCA resistance of Candida albicans isolated from local VVC patients may be related to Mrr2 mutation.(3)Overexpression of CDR1 may directly lead to Candida albicans resistant to FCA.Mrr2 overexpression may directly lead to Candida albicans resistant to FCA.Moreover,Mrr2 overexpression also promotes the high expression of CDR1,which mediates the resistance of Candida albicans to FCA.(4)Mrr2 gene mutation and overexpression may have a synergistic effect on FCA resistance.