Pathogenic Study On Candida Species Isolated Form Pregnant Women And The Susceptibility Test To Clotrimazole, Miconazole And Nystatin

Vulvovaginal candidiasis (WC) is an opportunistic fungal infection caused by Candida species. About 75% of all women will have at least one VVC infection during their lifetime. Candida albicans is the dominant species that causes vaginitis, while Candida glabrata is the second most common Candida pathogen. The prevalence rates of Candida among pregnant women are reported to be higher than those in nonpregnant women. C. albicans is by far the most common species during pregnancy. At the end of pregnancy, C. albicans is found in vaginal secretions in 25~30% of the women. About 70~85% of these women subpartally contaminate their infants with this yeast. Recurrent vaginal candidiasis is caused by the persistence of a single yeast genotype during different trimesters. The susceptibility of different isolates to the antifungal agents was variable.In the absence of epidemiologic data, restriction in treatment and susceptibility to VVC during pregnancy, cure VVC during pregnancy is difficult. In present study, pathogenic study on vaginal Candida species isolated form pregnant women and the susceptibility test were conducted on more than 600 pregnant women, the aims of this study were showed as follows: â‘  to determine the vaginal colonization by Candida species and the prevalence of vaginal candidiasis during pregnancy, identify Candida species isolates from pregnant women;â‘¡determine the genotype polymorphism of major Candida species and provide molecular epidemiologic data;â‘¢compare the susceptibility difference to antifungal drug between C. albicans and nonalbicans species, and find out the relationship between genotypes of Candida andantifungal susceptibility in order to provide appropriate advices to treatment on vaginal candidiasis.Part One Epidemiologic study on vaginal Candida colonization and Candida species identify during pregnancyMethods. Vaginal swabs from 624 pregnant women (628 person-time) with a gestation period of 5~40 weeks were isolated and cultured after direct mycologic examination. Candida species were confirmed by using the chromogenic medium CHROMagar (CHROMagar Co., France) and the API20Caux yeast identification kit (bioMerieux). The patients with clinical signs such as vulvovaginal pruritus, irritation, edema, erythema, or abnormal discharge were defined as symptomatic patients (vulvaginal Candidiasis, VVC). And the other patients were categorized into asymptomatic patients.Results. The numbers of women from the first, the second and the third trimesters were 224, 224 and 180. A positive culture was obtained in 31.37% patients, and this proportion was change slight among different trimesters CP>0.05) . The prevalence of vaginal candidiasis during whole gestation wasl9.43%, and this prevalence during the first trimester, the second trimester and the third trimester was 17.86%^ 19.20% and 21.67% in each. A total of nine Candida species were obtained in present study, and the species numbers obtained in the first, the second, and the third trimesters were 6, 5, 7. C. albicans was the predominant species (63.64%), followed by C. glabrata (25.25%) and C. krusi (4.04%) . The symptomatic patients caused by C. albicans decreased whereas those caused by nonalbicans species increased as pregnancy advanced.Part Two Genotypes study on C. albicans and C. glabrataMethods. A total of 176 Candida isolates, including 126 of C. albicans and 50 C. glabrata were genotyped by INT-PCR (Polymerase chain reaction with INT primers),randomly amplified polymorphic DNA (RAPD) analysis and restriction endonuclease analysis (REA) methods. The two genotypic methods of RAPD and REA were compared at the same time.Results. INT-PCR divided 126 C. albicans strains into three groups on the basis of the presence of a 450bp or 840bp product. Genotype A strains were the most frequently strains in each trimester. After digested with EcoR I , 119 C. albicans strains were separated into three major groups and nine subgroups based on the presence of 3.7kb or 4.2kb major bands. This result was very similar to that from INT-PCR. For RAPD method with primer Ml3, 35 polymorphic PCR fragments of differentsizes from C. albicans and 20 fragments from C. glabrata were detected and scored. In total, 34 polymorphic PCR fragments were detected and scored with primer PA03, from C. albicans and C. glabrata in half. A total of 24 Hint I -digested patterns were obtained from C. albicans isolates, while 19 patterns obtained from C. glabrata. The number of EcoR I digested patterns obtained from C. glabrata was 11. The Candida isolates of identical genotypes, highly similar genotypes and unrelated genotypes were all be identified in pregnant women. The genotypes of the C. albicans strains isolated from the same patient during different trimesters were identical for individual. The genotypic polymorphism of C. albicans obtained from symptomatic patients was more obvious than that from asymptomatic patients (P <0.05 ) , whereas this discrimination was not found among C. galbrata isolates. RAPD method divided all isolates into different genotypic groups successful whereas REA method failed to do so in few isolates.Part Three Susceptibility test to Clotrimazole, Miconazole and NystatinMethods. A total of 195 Candida isolates, including 126 of C. albicans and 69 of nonalbicans) were tested in this part. A modifying broth microdilution antifungal susceptibility test performed according to the National Committee for Clinical Laboratory Standards (NCCLS) document M27-A guidelines. Clotrimazole and clotrimazole vaginal tablet (class B), nystatin and mycostatin vaginal tablet (class B),and miconazole (class C) were chose for in vitro testing. The antifungal activity against isolates with different genotypes from C. albicans or C.glabrata was compared at the same time.Results. ? The minimum inhibitory concentration (MIC) was determined as the lowest drug that maintained a blue or a blue-pink hue. As a result, C. albicans isolates were more susceptible to miconazole, elotrimazole and elotrimazole vaginal tablet at 24h. Mycostatin vaginal tablets exhibited the highest antifungal activity at 48h, followed by miconazole. Except elotrimazole, the C. albicans from different trimesters had no susceptible differences to the antifungal agents (P >0.05) . For nonalbicans isolates, the antifungal agent with ideal antifungal activity at either 24h or 48h was miconazole, while the agent with the lowest antifungal activity was elotrimazole. The isolates from the third trimester were less susceptible to those from the other two trimesters (P <0.05 ) . Most C. albicans isolates from whole gestation and nonalbicans isolates from the first two trimesters were susceptible to nystatin and mycostatin vaginal tablet, though these two agents exhibited high MICs at 24h. The Candida isolates from all pregnant women and those from VVC patients had similar results of antifungal susceptibility test. ?With INT-PCR, C. albicans were devidede into three genotypes and the MICs for genotype A isolates of elotrimazole, miconazole, elotrimazole vaginal tablet at 24h and 48h were significant higher than those for genotype B and (or) C (.P<0.05) . The RAPD genotypes of C. albicans were related to the MICs of mycostatin vaginal tablet at 24h, and nystatin at 48h, and elotrimazole vaginal tablet at 24h or 48h (PO.05) . For C. glabrata, the MICs of mycostatin vaginal tablet at 48h for isolates with different RAPD genotypes were discriminating (P<0.05) .Conclusions1 Vaginal Candida isolates from pregnant women had high species diversity, which was more obvious during the third trimester.2 The C. albicans and C.glabrata strains isolated from separate pregnant womenduring different trimesters show highly genotypic polymorphic. The "replacement hypothesis" and the "persistent hypothesis" both were pathogenesis of VVC during pregnancy.3 C. albicans isolates were more susceptible to antifungal agents and mycostatin vaginal tablet was the first choice. The treatment in VVC caused by C. albicans during different trimesters was very similar.4 Cure of VVC caused by nonalbicans species was not so easy and clotrimazole should not be used. The first choice agent during the first and the second trimesters was mycostatin vaginal tablet. During the third trimester, miconazole was the first choice.5 Medicine used directly in susceptibility test should be more helpful to clinical treatment.6 The relation between the Candida genotypes and the antifungal susceptibilities might be useful to treatment in vaginal candidiasis during pregnancy.