2.1 Subjects and study design
A cross-sectional study was conducted in Gynecology Outpatient Clinic of the First Affiliated Hospital of Xi'an Jiaotong University between November 2016 and November 2020. Prior to data collection, participants ≥ 16 years signed written informed consent, and those < 16 years provided assent and parental consent from their parents/guardians. The study protocol was approved by the ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University. Collected information including age, status of their pregnancy, main symptoms (color and amount of discharge, itching, burning pain, urodynia, etc), medical history before gynecological examination. All had simultaneous STIs test(including CT, MG and NG)using the RNA-based SAT and vaginal microecology evaluation based on VMES. The exclusion criteria were: (1) Women during menstrual period; (2) women received hormones, antibiotic or immunosuppressive agents within 2 weeks; (3) women had a history of vulva or vaginal medication in the last 7 days. Eligible patients were required to complete a comprehensive gynecologic examination after collecting the basic information. Samples of vaginal fluid from the lateral vaginal walls were obtained using two sterile cotton swabs, one was used for the functional detection of vaginal microecology evaluation, another vaginal swab was streaked on a glass slide and air dried to Gram stain for morphological interpretations. Additionally, a long sterile cotton swab was used to collect the endocervical samples for the test of STIs. All samples were taken to the laboratory for testing immediately.
2.2 Vaginal microecology evaluation system
(1) Microscopic evaluation on Gram-stained vaginal smears:
(A) Bacterial density: it refers to the average number of bacteria in the microscopic field under oil immersion lens. The results were classified into four levels(denoted as + to ++++): grade I (+): 1–9; grade II (++): 10–99; grade III (+++): 100 and above; and grade IV: (++++): bacteria clustered full of vision. Both “++” and “+++” mean normal.
(B) Flora diversity: it refers to the number of distinguishable bacterial species under oil immersion lens. The results were also divided into four levels (denoted as + to ++++): grade I (+): 1–3 kinds of flora; grade II (++): 4–6 kinds of flora; grade III (+++): 7–10 kinds of flora; and grade IV (++++): more than 10 kinds of flora. “++” and “+++” were viewed as normal.
(C) Predominant flora: it refers to the maximum number of microorganism observed in the microscopic field under oil immersion lens. Only Large Gram-positive rods (Lactobacillus) was determined as a normal flora.
(D) Vaginal cleanliness evaluation: It was graded by the density of Lactobacillus, epithelial cells, white blood cells, other flora into 4 levels, with Grade I-II as normal and grades III-IV as abnormal usually accompanied by inflammation. Specifically, the judgment of vaginal cleanliness were as follows (Table S1) : Grade I was a large number of large Gram-positive rods (indicative of Lactobacillus spp.), vaginal epithelial cells, and no other bacteria observed with WBC 0–5/HP under microscopy. Grade II was some Lactobacillus spp. and vaginal epithelial cells, some pus cells, and other bacteria observed under microscopy with WBC 10–15/HP. Grade III was a small amount of Lactobacillus spp., a large number of pus cells, and other bacteria observed under microscopy with WBC 15–30/HP. Grade IV was no Lactobacillus spp. but pus cells and other bacteria observed under microscopy with WBC more than 30/HP(Table S1).
(E) Bacterial vaginosis (BV) was assessed by Nugent score and a score of 7 to 10 was defined as BV(Table S2). Aerobic vaginitis (AV) were assessed by VMES according to the AV score[9, 10](Table S3).
(F) Pathogen: TV (Trichomonas vaginosis) was diagnosed by motile flagellates under a microscope and VVC (vulvovaginal candidiasis) was confirmed when the yeast and(or) pseudohyphae were observed through a microscope.
(2) Functional detection of vaginal microecology evaluation:
(A) Vaginal preformed enzymes: H2O2, sialidase, β-glucuronidase, leukocyte esterase, and acetylglucosaminidase were detected following manufacturer's directions (Chaoshi-Bio, Jiangsu China). All these enzymes were classified as positive or negative result.
(B) Vaginal pH: pH paper strips ranging from 3.8 to 5.4 were used to measure the vaginal pH. The normal vaginal pH values range from 3.8 to 4.5.
2.4 Statistical analyses
Differences in study population characteristics were tested using the χ2, Fisher exact or Mann-Whitney U test, accordingly. The unadjusted odds ratios (ORs) were obtained from an unconditional, binary logistic model with only one dichotomous covariate of interest in the model. All variables associated with the outcome (STIs) at P < 0.1 were entered into the logistic regression model. Then stepwise backward elimination logistic regression was performed to determine which factors were independently associated with STIs, and the final reduced model was constructed. P values < 0.05 were considered significant. Finally, we also presented the results in the subset of women without BV using the stepwise backward elimination logistic regression. We obtained the unadjusted and adjusted log odds using computer software (IBM SPSS version 25).