Comparative Study Of Vaginal Microflora Of VVC And RVVC Patients

BackgroundVVC (Vulvovaginal candidiasis) is a common vulva vagina inflammation. VVC occupies about 17%-39% patients who have symptoms of infection, about 25%-30%of microorganisms caused vaginitis,5%-8% of the VVC patients develop into recurrent RVVC (Vulvovaginal candidiasis), namely the VVC attack 4 times a year or more. Vulva vagina of Candida pathogens to C.albicans (Candida albicans) primarily, accounts for about 60%, other Candida also quite shares. Candida sojourn in vaginal mucous membrane. Together with lactobacillus bacteria, escherichia coli, staphylococcus epidermis, they engraftment vaginal normal flora. They have a relationship of a dynamic balance. The growth of the bacterial flora resists Candida. Vaginal mucous membrane prevents fungus invading. Parasitic vaginal anaerobic bacteria and lactic acid bacteria have protective effect on vaginal epithelial cells. Under normal circumstances, engraftment of Candida doesn’t cause any discomfort, but when the female reproductive tract microecological bacteria by environment change, drugs to stimulate, low levels of the hormone and immune status, VVC happened. The change of vagina micro ecological balance state is one of the important reasons of VVC. Candida is a kind of yeast who has two states. It is vulnerable to external environment influence and change. Therefore, it has highly vaginal fungal flora diversity. The changes in the structure have close relationship with RVVC development. Early studies mainly focused on the bacteria culture and drug susceptibility of Candida, less analysis of vaginal microecological bacteria and its elements in the whole in the happening of the disease development and outcome of the changes. Therefore, understanding of VVC and RVVC vaginal microecological bacteria in patients with structure and the dynamic change, is helpful for us to understand reproductive tract Candida disease occurrence, development process, further guide clinical treatment and prevention of recurrence.Vaginal flora is mainly for the anaerobic bacterium. The use of traditional culture and separated identification method is difficult to a comprehensive understanding of the vaginal flora diversity. With the rapid development of molecular biology techniques and the mature technology of cultivate dependency, the researchers can take advantage of the 16 s rRNA genes PCR (Polymerase chain reaction) amplification of the vaginal microbes culture method, by Illumina high-throughput Sequencing platform of Solexa Sequencing, Combined with labels of double end sequencing based on fluorescence sequencing platform BIPES (Barcoded Paired-End Illumina Sequencing) for a quick and comprehensive understanding of vaginal microbial community structure. There are reports clarified the microbial community composition and diversity of vaginal flora on healthy subjects and patients suffering from VVC. However, there is still no in-depth research on the overall structure, composition and change of the microbial community of the vaginal flora of Candida patients before and after active treatment.Research purposes1. Through study the vaginal microbial flora structure of VVC an RVVC patients before and after treatment by Illumina high-throughput sequencing technology and BIPES series research methods, we analyze the vaginal flora diversity change, understand the difference changes of the vaginal flora in patients with VVC and RWC by drug treatment in a attack period, investigate the relationship between the changes and treatment effect of bacteria.2.Tracking observation of vaginal flora structure information of VVC and RVVC patients in the second watch in the menstrual cycle (for non-invasive cycle) with the original corresponding to the days before and after the treatment, we understand the correlation between the occurrence of Candida, vaginal flora balance and dominant fungi.Methods1.The experimental object:Experiment 1:In April 2013-December 2013,6 outpatients from the department of gynaecology in the NO.1 people’s hospital, shunde district, foshan city, verage age (30.16±6.66). Patients were signed informed consent.VVC group (3 cases) inclusion criteria:(1) symptoms:pruritus vulvae, burning pain, accompanied by urinary symptoms such as pain and pain during sex, vaginal secretions increase; (2) signs:vulva erythema, edema, with scratches or skin chapping, exfoliation, flush on vaginal mucous membrane, the inside of nympha and vaginal mucosa membrane attached to white, white can be seen in the vagina viscous show latex or bean curd secretion; (3) laboratory examination:wet smear (with normal saline and 10% KOH preparation) confirmed the presence of Candida of budding spores or pseudohyphae. Or fungi cultivation culture has nearly smooth white candida, candida, such as tropical candida yeast growth.RWC group (3 cases) inclusion criteria:(1) conformed to the VVC diagnostic criteria, and in a year have symptoms and confirmed by mycology WC attack 4 times or more. (2) laboratory examination:wet smear (with normal saline and 10% KOH preparation) confirmed the presence of Candida of budding spores or pseudohyphae. Or fungi cultivation culture has nearly smooth white Candida, Candida, such as tropical Candida yeast growth.Two groups of patients diagnosed with Terbinafine Hydrochloride Vaginal Effervescent tablets (vagina medicine,50 mg/d, a total of 7 days), after treatment, the patients were consciously pruritus vulvae symptom relief, leucorrhea reduced, laboratory wet smears fungi negative.Experiment 2:6 cases of Experiment 1 selected observation object, respectively VVC Ⅱ group and RVVC Ⅱ group, with the onset of menstrual cycle for the second observation cycle. Inclusion criteria is the same with the experimental 1. Patients were signed informed consent.Exclusion criteria:The objects of study have regular menstruation, rule out during pregnancy and lactation; eliminate systemic diseases such as diabetes, and hormone treatment disease, such as acute or chronic nephritis, etc.; Rule out other vaginitis, such as:trichomonad sex, bacterial vaginal disease etc. One month no systemic antibiotics, immunosuppressants, vaginal medication history, asexual life history and vaginal lavage within three days.2. Specimen collection:The object of study, for the first observation, before treatment and in the 8th day after drug treatment, with sterile vaginal swabs from the vagina fornix vaginal wall take secretion. Puts vaginal swabs sterile into dry tubes and used to extract total DNA bacteria. Specimens collected in 4℃ specimen box save after inspection. Sampling cycle time for the second observation in experiment 2, a corresponding day of the menstrual cycle in experiment 1.3. The extract and detection of bacterial total DNA:the bacterial total DNA was extracted from the specimen by bacterial genomic DNA extraction kit, and the specific steps were referred manual. The purity and concentration of DNA was tested by ultraviolet spectrophotometer (the wavelength is 570). The products of PCR, after which were tested by 1% agarose gel electrophoresis, AGE, were keeping in refrigerator.4.16s rRNA V4 area gene segment amplification and Illumina sequencing: Base on the sample of the total DNA template,the primer (Shanghai sangon biological engineering co., LTD)of 514F和805R were used to amplify 16s rRNA V4 area gene segment. After recovery and purification, the products of PCR, whose output and character of were tested by 1% AGE, were sequencing its double terminal 100 bp through Illumina Hiseq 2000 platform.5. Bioinformatic analysis:after the tested gene order was preliminarily dispose by BIPES operation, its effective sequencing data was trimmed. And V4 sequence was fitting by the reverse complement. And the twostage-clustering was clustered. After every representative of operational taxonomy units was extract, the species were classify referring GAST. Those belong to lactic acid bacteria of the genus OTU were further assigned to the level by Blast. Unifrac distance of bacterial community is analyzed based on evolutionary relationship, and principal component analysis of vaginal flora was done by QIIME.6. Statistical processing:SPSS 13.0 software was used to statistical analysis, with the proportion or rate counting data. Use Chi-square test(x2) analysis of variance to compare the Measurement data set within or between groups, use mean ± standard deviation (x ± s) for Measurement data. P< 0.05 for the difference is statistically significant.ResultsExperiment 1:1. Sequencing BIPES flow used in the data analysis, adopting UCHIME remove chimeric sequences, there are 12 samples to sequence, eventually received 928028 sequences, each sample an average of 77340.2. Application Complete linkage clustering method of vaginal flora of VVC and RVVC group clustering analysis. VVC group and RVVC vaginal micro ecological diversity advantage bacterium group failed to for different disease group significantly, but is characterized by individual flora clustering. The remarkable differences of vaginal flora apparent after treatment in VVC group, while RWC group differences apparent vaginal flora structure is more similar. Account for that the vaginal flora is characterized by individual clustering,and the flora similarity by treatment is greater than the disease.3. Through the Euclidean distance to compare the differences of VVC and RVVC group samples, according to the varied range of vaginal flora is highest which of VVC group after treatment, while it is smaller or the same of RVVC, the differences between the two groups have statistical significance (P< 0.05).4. To compare the bacteria of the genus level, Bifidobacterium、L.crispatus and Gardnerella are the mainly predominant vaginal spcies in VVC group before treatment, accounted fo 80%.55% and 45%, while reduce about 15%～15% after treatment. Lactobacillus become the mainly vaginal flora by increasing its richness significantly.The differences have statistical significance by treatment in VVC group (P< 0.05).The differences have statistical smaller by treatment in RVVC group (P >0.05),while the species composition ratio of vaginal flora in RVVC group is basically identical after treatment.5. Through Observed-species (a)、Chaol (b)、PD-whole-tree (c) and Shannon Index(d) to compare the Alpha diversity of two group samples, according to the differences of vaginal flora Alpha diversity is statistical smaller of VVC group and RVVC group (P> 0.05).6. Through LEfSe statistics analyse the species differences in three groups,we found that no bacterial genus exist in VVC and RVVC group after treatment.Experiment 2:1. Sequencing BIPES flow used in the data analysis, adopting UCHIME remove chimeric sequences, there are 12 samples to sequence, eventually received 929032 clean sequences, each sample an average of 77420.2. Application Complete linkage clustering method of vaginal flora of VVC Ⅱ and RVVC Ⅱ group clustering analysis. VVC Ⅱ group and RVVC Ⅱ vaginal micro ecological diversity advantage bacterium group failed to for different disease group significantly, but is characterized by individual flora clustering. The remarkable differences of vaginal flora apparent after treatment in VVC Ⅱ group, while RVVC group differences apparent vaginal flora structure is more similar. The differences between the two groups have statistical non-significance (P=0.465).3.Analyse the differences in species composition ratio between the VVC Ⅱ group and RVVC Ⅱ group. Specimen Were collected from each patient before treatment and after treatment. Analyzed the vaginal bacteria species in the VVC Ⅱ group and RVVC Ⅱ before and after the treatment during the unaffected period and found there is statistical non-significance (P=0.465). This showed similarity bacterial flora was exsited in two groups of patients, and the vaginal flora ecology at a similar stable state.4. Analyse the differences advantage flora in VVC group and VVC Ⅱ group, the RVVC group and RVVC Ⅱ group respectively. Samples were collected from every patient before and after using the drug, medication during the same period before and after the same period. the vaginal flora of bacteria genera species composition ratio difference between the VVC and the RVVC group is analyzed both in the onset period and the non-onset period respectively. The dominant bacteria were significantly different between the VVCⅡ group and VVC group(P= 0.012), while the difference between the RVVCⅡ group and RVVC group were small (P= 0.541). This showed that the balance of vaginal flora may interfere by incidental candidiasis; when recurrent, correlation between the candidiasis and vaginal dominant bacteria were significantly reduced.Conclusion1.Antimicrobial killing Candida While reducing the Candia’s antagonism to Lactobacillus, therefore adjusted the structure of the vaginal flora and made the flora structure gradually tend to be normal. Antifungal drug treatment is not prompted the emergence of new species in patients, but to adjust the composition of the vaginal flora and restore the flora balance to achieve therapeutic effect.2.Vaginal micro ecological advantage bacterium group diversity of VVC group and RVVC group is based on individual flora feature clustering. The patients in VVC group were more sensitive to drug treatment. While the treatment to patients in RVVC group did not cause significant vaginal microecological changes. The vaginal microbes advantage bacterium for the non-onset period compared with the onset period, VVC group have significant difference, while RVVC group have no significant difference. This showed that the balance of vaginal flora may interfere by incidental candidiasis; when recurrent, correlation between the candidiasis and vaginal dominant bacteria were significantly reduced.