Study On The Bacterial Communities In Vagina And Genetic Polymorphisms Of Candida Albicans Associated With Vulvovaginal Candidiasis

Chapter1:Study on the vaginal microbiome in healthy women by using454pyrosequencingObjective To understand the composition of bacterial communities in healthy Chinese women. Methods Genome DNA were extracted directly from vaginal fluids of20healthy women. the primers target V1-V3region of bacterial16S rRNA were used to amplify the DNA. The purified PCR products were subjected to pyrosequencing. Valid sequences were calculated and richness and diversity of bacterial communities among the20subjects were also assessed. Taxonomical analysis was done on six levels including phylum, class, family, order, genus and species. Results A total of115329sequences were finally analyzed. The average number of sequences is5766, with an average length of477bp. After clustering and alignment,2363operational taxonomy unites were recognized. A total of6phylum were detected predominated by Firmacutes. was the most prevalent genus in vaginal tract, composing over90%of the total bacterial communities. Conclusions Lactobacillus was the largest composition of normal micorbiome. L.crispatus was the most common species detected.Chapter2Characterization of Candida albians associated with vulvovaginal candidiasis using microsatellite length polymorphismsObjective:To characterize the genotype distribution pattern of Candida albicans associated with vulvovaginal candidiasis in Nanjing, China. Methods:A molecular epidemiological study was conducted to explore the genotypes of C.albicans associated with VVC in Nanjing. Generally, microscopical examination of a wet smear combined with culture was used for the diagnosis of vulvovaginal candidiasis (VVC). A questionnaire about information associated with VVC was obtained from each participant. The genotypes of clinical isolates were determined by the analysis of PCR products amplified by microsatellite primers of CAI. Chi-square test was used to compare the genotype distribution of strains isolated from pregnant women with non-pregnant ones. Comparison among different age groups was also performed by using Chi-square test. Difference of genotype distribution of VVC with different clinical conditions was calculated by Chi-square test. Results:PCR fragments containing30:45,21:21and32:46alleles were the three major genotypes among the51genotypes detected, accounting for29.3%,13.0%and12.0%of206clinical isolates respectively. The21:21genotype was more prevalent in younger groups compared with other groups. The46:46,32:46and 21:21were important genotypes causing VVC in pregnant women. The30:45was rare in pregnant women, only one strain demonstrated this genotype. Patients with different clinical conditions had similar genotype distribution.Conclusions:A distinctive genotype distribution of C. albicans was detected in Nanjing, China, compared to previous reports, with21:21being the major genotype besides previously reported30:45and32:46. A different distribution pattern was also observed in pregnant women compared with non-conceived ones. No difference distribution of C.albicans was found among different age groups.Chapter3Genetic diversity of Candida albicans associated with vulvovaginal candidiasis in pregnant womenObjective To study the genetic diversity of C.albicans associated VVC in pregnant women in Nanjing. Methods Mid-term pregnant women diagnosed as VVC were collected for fungal detection and clinical information. Strains isolated from the discharge were subjected to morphologic and molecular identification on the genus level. Strains confirmed as Candida albicans were then conducted to perform multi locus sequence typing using the seven house-keeping genes. The sequence type of each strain was determined by comparison with database. Phylogenic analysis was done with the Mega5.1software. EBURST V3.0software was used to check the clonal complex of the sequence types. Results A total of84Candida strains were obtained from81pregnant women diagnosed as VVC. Among the84strains,77strains were finally identified as Canida albicans while the rest7belong the species Candida glabrata. Seven genes including TTla, ACC1, ADP1, MPIb, SYA1, VPS13and ZWFlb were successfully amplied. All the strains were nominated to60independent sequence types and all of them had not beed recognized before. Phylogenic analysis using the UPMGMA algorithm divived the sequence types into5groups and most of them were include in clade one.The largest clonal complex was consisted with23unique sequence types and the ancenstor was the sequence type drived from strain57. Conclusions The Candida albcians associated with VVC during pregnancy showed highly discrimative diversity by using MLST. However, the phylogenic analysis exhibited genetic similarity among the strains studied.