妇科门诊育龄妇女与孕妇阴道菌群状态及常规感染的比较分析

目的比较妇科门诊育龄妇女与孕妇的阴道菌群状态及常规感染,分析阴道菌群改变对阴道常规感染影响。方法用革兰染色法分别检查普通育龄妇女和孕妇的阴道分泌物,并进行Nugent评分。结果妇科门诊育龄妇女和孕妇的假丝酵母菌阳性率分别为28．6％和9．2％,滴虫阳性率分别为2．4％和0．6％;细菌性阴道病（BV）阳性率分别为32．4％和9．6％;乳杆菌3＋到4＋的比例分别为74．4％和44．8％。2组之间比较差异均有显著性（P〈0．05）。结论相对于妇科门诊育龄妇女而言,孕妇的阴道微生态状况更健康,对各种病原微生物有更强的抵抗力,受假丝酵母菌、滴虫和细菌性阴道病等常规疾病感染的机会明显降低。     

Comparison between Gram stain and culture for the characterization of vaginal microflora: Definition of a distinct grade that resembles grade I microflora and revised categorization of grade I microflora

Background  

The microbiological diagnosis of bacterial vaginosis is usually made using Nugent's criteria, a useful but rather laborious scoring system based on counting bacterial cell types on Gram stained slides of vaginal smears. Ison and Hay have simplified the score system to three categories and added a fourth category for microflora with a predominance of the Streptococcus cell type. Because in the Nugent system several cell types are not taken into account for a final score, we carried out a detailed assessment of the composition of the vaginal microflora in relation to standard Gram stain in order the improve the diagnostic value of the Gram stain. To this purpose we compared Gram stain based categorization of vaginal smears with i) species specific PCR for the detection of Gardnerella vaginalis and Atopobium vaginae and with ii) tDNA-PCR for the identification of most cultivable species.     

Assessment of Lactobacillus species colonizing the vagina of apparently healthy Nigerian women, using PCR-DGGE and 16S rRNA gene sequencing

The identity of lactobacilli colonizing Africa-American women, have previously been based on culture-dependent methods. This led to some misleading speculations that black women lack lactobacilli in their vagina and are therefore highly susceptible to Bacterial vaginosis and STDs including HIV. In this study we used culture-independent procedures.About 241 vaginal swabs were obtained from ‘apparently’ healthy premenopausal women, between 18 and 48 years. Samples were Gram stained for the Nugent score evaluation. DNA was extracted from the bacteria on the vaginal swabs and amplified using polymerase chain reaction (PCR), with Lactobacillus primers. Samples with PCR products were separated with denaturing gradient gel electrophoresis (DGGE) and cut bands were re-amplified with Lactobacillus primers without the GC clamp. The re-amplified product was purified and sequenced with ABI PRISM Big-Dye Terminator. The sequence was identified with BLAST algorithm having the highest Gene Bank Accession number.Out of the 241 vaginal swab samples, 207 (85.8%) had PCR products, indicating the presence of lactobacilli, while 34 (14.2%) showed absence of lactobacilli and the Nugent scores were synonymous to either intermediate bacterial vaginosis or bacterial vaginosis (BV). Out of the 207 samples that had PCR product for Lactobacillus, 149 (72%) had sequence results as revealed by the BLAST algorithm. Most of the women (64%) were colonized by Lactobacillus iners as the predominant strain. Lactobacillus gasseri had 7.3%, followed by L. plantarum and L suntoryeus (6.0%) each. Others were colonized by Lactobacillus crispatus (3.0%), Lactobacillus rhamnosus (2.7%), Lactobacillus vaginalis (2.7%), Lactobacillus rennanqilfy (2.7%), followed by Lactobacillus fermentum (1.3%), Lactobacillus helveticus (1.3%), Lactobacillus johnsonii (1.3%) and Lactobacillus salivarus (1.3%). To our knowledge, this is the first report of Lactobacillus iners being the predominant species colonizing the vagina of the Nigerian women examined. This finding is in line with those from a recent study conducted among Canadian and Swedish women.     

Gram Stains: A Resource for Retrospective Analysis of Bacterial Pathogens in Clinical Studies

We demonstrate the feasibility of using qPCR on DNA extracted from vaginal Gram stain slides to estimate the presence and relative abundance of specific bacterial pathogens. We first tested Gram stained slides spiked with a mix of 10⁸ cfu/ml of Escherichia coli and 10⁵ cfu/ml of Lactobacillus acidophilus. Primers were designed for amplification of total and species-specific bacterial DNA based on 16S ribosomal gene regions. Sample DNA was pre-amplified with nearly full length 16S rDNA ribosomal gene fragment, followed by quantitative PCR with genera and species-specific 16S rDNA primers. Pre-amplification PCR increased the bacterial amounts; relative proportions of Escherichia coli and Lactobacillus recovered from spiked slides remained unchanged. We applied this method to forty two archived Gram stained slides available from a clinical trial of cerclage in pregnant women at high risk of preterm birth. We found a high correlation between Nugent scores based on bacterial morphology of Lactobacillus, Gardenerella and Mobiluncus and amounts of quantitative PCR estimated genus specific DNA (rrn copies) from Gram stained slides. Testing of a convenience sample of eight paired vaginal swabs and Gram stains freshly collected from healthy women found similar qPCR generated estimates of Lactobacillus proportions from Gram stained slides and vaginal swabs. Archived Gram stained slides collected from large scale epidemiologic and clinical studies represent a valuable, untapped resource for research on the composition of bacterial communities that colonize human mucosal surfaces.     

The evaluation of prevalence and the impact of pathological microflora of the lower genital tract among women at early pregnancy on the risk of preterm delivery

The main aim of this prospective study was to determine the prevalence and an association between pathological microflora of the lower genital tract diagnosed at early pregnancy and the risk of preterm delivery. The study group comprised 179 randomly selected pregnant women from Lodz region, between 8 and 16 week of pregnancy. For the qualitative and quantitative assessment of biocenosis of the lower genital tract vaginal and cervical swabs were collected from the pregnant women under study. The C. trachomatis antigen was detected by direct immunofluorescence assay. The vaginal swabs were tested for aerobic and anaerobic bacteria. Bacterial vaginosis was diagnosed by Gram stain according to Spiegel's criteria. To evaluate the risk factors odds ratios were calculated using EPI INFO software. 21 (11.7%) women delivered before 37th week of pregnancy. Bacterial vaginosis was diagnosed among 51 (28.5%) pregnant women while intermediate microflora was diagnosed by Gram stain in 62 (34.6%) women. The shortest mean gestational age at delivery was noted among women with BV. The rate of preterm delivery in BV group was 15.7% comparing to 9.1% among women with normal microflora. Among women with preterm delivery BV was diagnosed in 38.1% (OR = 1.86). Based on culture results only 84 (46.9%) women had normal microflora at early pregnancy. The pathological culture was associated with slightly increased preterm delivery rate (12.6%) as compare to 10.7% in control group. Positive culture for Bacteroides and Mobiluncus was connected with nonstatistical rise in the risk of preterm delivery. No association between C. trachomatis infection at early pregnancy and elevated risk of preterm delivery was found. Early pregnancy diagnosis of bacterial vaginosis and its treatment should lower the rate of prematurity in Poland.     

Response of Gardnerella vaginalis biofilm to 5 days of moxifloxacin treatment

Polymicrobial communities are often recalcitrant to antibiotics. We tested whether the polymicrobial Gardnerella vaginalis biofilm can be eradicated with moxifloxacin. Twenty women with bacterial vaginosis were treated with 400 mg moxifloxacin for 5 days. The changes in the occurrence and proportions of Gardnerella, Atopobium and Lactobacillus spp. were assessed using FISH. The bacterial biofilm was investigated using desquamated epithelial cells of spontaneously voided urine and sections of vaginal biopsies. Fifteen of 20 women showed a significant and sustained clinical response to moxifloxacin according to Amsel and Nugent criteria. The concentrations of adherent bacteria decreased significantly. The incidence and proportion of Atopobium declined sustainably. The proportions of Lactobacillus in the biofilm mass increased following therapy. Initially, Gardnerella was the main component of the polymicrobial biofilm. Following treatment, Gardnerella was not accessible to FISH in the urine and vaginal samples of 75% of all women. Ten to 12 weeks after the end of therapy, Gardnerella biofilm was cumulatively present in 40%. This was not due to newly acquired disease, but due to reactivation of the persisting, but biochemically inactive biofilm. Despite clear clinical efficacy, and initially definite suppression of the biofilm, moxifloxacin was, similar to metronidazole, not able to eradicate the Gardnerella vaginalis biofilm in all patients.     

Bacterial Vaginosis Is Associated with Loss of Gamma Delta T Cells in the Female Reproductive Tract in Women in the Miami Women Interagency HIV Study (WIHS): A Cross Sectional Study

Bacterial vaginosis (BV) is the most common female reproductive tract infection and is associated with an increased risk of acquiring and transmitting HIV by a mechanism that is not well understood. Gamma delta (GD) T cells are essential components of the adaptive and innate immune system, are present in the female reproductive tract, and play an important role in epithelial barrier protection. GD1 cells predominate in the mucosal tissue and are important in maintaining mucosal integrity. GD2 cells predominate in peripheral blood and play a role in humoral immunity and in the immune response to pathogens. HIV infection is associated with changes in GD T cells frequencies in the periphery and in the female reproductive tract. The objective of this study is to evaluate if changes in vaginal flora occurring with BV are associated with changes in endocervical GD T cell responses, which could account for increased susceptibility to HIV. Seventeen HIV-infected (HIV+) and 17 HIV-uninfected (HIV-) pre-menopausal women underwent collection of vaginal swabs and endocervical cytobrushes. Vaginal flora was assessed using the Nugent score. GD T cells were assessed in cytobrush samples by flow cytometry. Median Nugent score was 5.0 and 41% of women had abnormal vaginal flora. In HIV uninfected women there was a negative correlation between Nugent score and cervical GD1 T cells (b for interaction = - 0.176, p<0.01); cervical GD1 T cells were higher in women with normal vaginal flora than in those with abnormal flora (45.00% vs 9.95%, p = 0.005); and cervical GD2 T cells were higher in women with abnormal flora than in those with normal flora (1.70% vs 0.35%, p = 0.023). GD T cells in the genital tract are protective (GD1) and are targets for HIV entry (GD2). The decrease in cervical GD1 and increase in GD2 T cells among women with abnormal vaginal flora predisposes women with BV to HIV acquisition. We propose to use GD T cell as markers of female genital tract vulnerability to HIV.     

纵向研究妊娠期妇女的阴道微生态状况

目的分析妊娠期妇女阴道微生态状况的纵向变化。方法选择健康无症状单胎妊娠期妇女50例,分别于孕早期（10.2±2.1）周、孕中期（24.6±3.7）周、孕晚期（33.2±3.4）周,取其阴道分泌物,进行pH、H2O2含量检测及细菌培养,并经革兰染色后在油镜下观察,进行阴道微生态（阴道菌群的密集度、多样性、优势菌、炎症反应状况等）状况评价。结果 （1）50例孕妇中,孕早期38例（76%）阴道微生态正常,其中31例（81.58%）整个孕期维持阴道微生态正常,7例（18.42%）孕妇于孕中期或孕晚期转变为阴道微生态失调;12例（24%）孕早期阴道微生态失调,其中6例（50%）于孕中期或孕晚期转变为阴道微生态正常。（2）无症状性细菌性阴道病随着孕周增加部分可自然消退40%（2/5）。（3）H2O2阳性、阴道pH低者易从阴道微生态失调转为阴道微生态正常。结论妊娠期阴道微生态是动态变化的,部分细菌性阴道炎可自然消退,H2O2阳性及低阴道pH为维持正常阴道微生态的重要因素。     

800例妇科门诊妇女阴道感染及微生态状况分析

目的了解普通妇科门诊妇女阴道分泌物病原体感染及阴道微生态状况。方法用革兰染色法检查普通妇科门诊妇女阴道分泌物,并进行Nugent评分。结果800例普通妇科门诊妇女阴道分泌物中,假丝酵母菌阳性236例,阳性率29．5％;阴道毛滴虫阳性21例,阳性率2．63％;Nugent评分：正常（0-3分）248例（31．0％）,临界（4-6分）283例（35．4％）,细菌性阴道病（BV）（7-10分）269例（33．6％）。结论妇科门诊妇女对各种病原微生物抵抗力不足,阴道微生态状况有待改善。     

More Than Meets the Eye: Associations of Vaginal Bacteria with Gram Stain Morphotypes Using Molecular Phylogenetic Analysis

Bacterial vaginosis (BV) is a highly prevalent condition associated with adverse health outcomes. Gram stain analysis of vaginal fluid is the standard for confirming the diagnosis of BV, wherein abundances of key bacterial morphotypes are assessed. These Lactobacillus, Gardnerella, Bacteroides, and Mobiluncus morphotypes were originally linked to particular bacterial species through cultivation studies, but no studies have systematically investigated associations between uncultivated bacteria detected by molecular methods and Gram stain findings. In this study, 16S-rRNA PCR/pyrosequencing was used to examine associations between vaginal bacteria and bacterial morphotypes in 220 women with and without BV. Species-specific quantitative PCR (qPCR) and fluorescence in Situ hybridization (FISH) methods were used to document concentrations of two bacteria with curved rod morphologies: Mobiluncus and the fastidious BV-associated bacterium-1 (BVAB1). Rank abundance of vaginal bacteria in samples with evidence of curved gram-negative rods showed that BVAB1 was dominant (26.1%), while Mobiluncus was rare (0.2% of sequence reads). BVAB1 sequence reads were associated with Mobiluncus morphotypes (p<0.001). Among women with curved rods, mean concentration of BVAB1 DNA was 2 log units greater than Mobiluncus (p<0.001) using species-specific quantitative PCR. FISH analyses revealed that mean number of BVAB1 cells was 2 log units greater than Mobiluncus cells in women with highest Nugent score (p<0.001). Prevotella and Porphyromonas spp. were significantly associated with the “Bacteroides morphotype,” whereas Bacteroides species were rare. Gram-negative rods designated Mobiluncus morphotypes on Gram stain are more likely BVAB1. These findings provide a clearer picture of the bacteria associated with morphotypes on vaginal Gram stain.     

Vaginal microbial flora in normal young women.

Vaginal swabs were taken from 1498 women attending a family planning clinic. The flora was assessed in the absence of any information about the women to whom the swabs related. Yeasts and fungi were present in 311 women (21%) and were no more prevalent among "pill" users than others. Candida albicans was significantly associated with vulval itching and with a vaginal discharge described as heavier than normal or curdy on clinical examination, though these abnormalities were present in only a minority of women with the organism. Trichomonas vaginalis was found in 14 women (1%) and was associated with abnormalities of vaginal discharge in all but one. Gram-negative anaerobic bacilli were significantly more common in women with a troublesome vaginal discharge and those who used an intrauterine device than others. No associations were found between fungi other than C albicans or the other bacteria sought and either symptoms or clinical abnormalities of vaginal discharge.     

Impact of Bacterial Vaginosis on Perineal Tears during Delivery: A Prospective Cohort Study

Objective

Long term effects of perineal tears pose a major worldwide health issue for women during delivery. Since bacterial vaginosis is related to major obstacles in obstetrics the aim of this study was to determine the relationship between bacterial vaginosis and the occurrence of perineal tears during vaginal delivery.

Methods

Between June 2013 and December 2013 pregnant women delivering after 37 weeks were recruited at one University hospital / tertiary care referral center in the course of this single-center, prospective cohort study. Bacterial vaginosis was assessed according to Nugent score method. Logistic-regression model was used to estimate odds ratios, adjusted for other risk factors to test the relationship between bacterial vaginosis and the occurrence of 1st to 4th degree perineal tears in women undergoing vaginal delivery.

Results

A total of 728 woman were included, 662 analyzed with a complete Nugent Score of the vaginal swab. The prevalence of 1st to 4th degree perineal tears was 35.8% (95% Confidence Interval (95%CI) = [32.2; 39.6]). The presence of BV was not significantly associated to the incidence of perineal tears neither in the univariate analysis (crude Odds Ratio = 1.43; 95%CI = [0.79; 2.60]; p = 0.235) nor in the multivariate analysis (adjusted Odds Ratio = 1.65; 95%CI = [0.81; 3.36]; p = 0.167). Instrumental delivery was the most important risk factor for perineal lacerations.

Conclusions

There is no evidence that vaginosis is a risk factor for vaginal tears.

Trial Registration

ClinicalTrials.gov N° NCT01822782     

Diagnostic value of Gram stain for assessment of vaginal smears during pregnancy

The main aim of this study was to compare the diagnostic value of Giemsa stained method with Gram stained method for the evaluation of vaginal smears among pregnant women. A study population comprised 111 pregnant between 6 and 30 weeks of gestation. The vaginal smears from every subject was diagnosed according to Giemsa and Gram stained method and micro-organisms were isolated by culture. In 29.3% cases diagnosed as normal flora (2a) on the basis of Giemsa method bacterial vaginosis was detected in Gram stains according to Spiegel's criteria and pathological microflora in concentration > or = 10(5) CFU/ml was cultured among 75.9% of them. Among 31.7% women who had grade 3a (abnormal) in Giemsa stains method normal flora was diagnosed on the basis on Gram's method and from 17.1% pregnant women from this group we did not isolated any pathogens. For evaluation of vaginal smears during pregnancy the Giemsa method should be replaced by Gram stained method.     

Evidence for Gardnerella vaginalis uptake and internalization by squamous vaginal epithelial cells: implications for the pathogenesis of bacterial vaginosis

Bacterial vaginosis (BV), a common condition seen in premenopausal women, is associated with preterm labor, pelvic inflammatory disease, and delivery of low birth weight infants. Gardnerella vaginalis is the predominant bacterial species associated with BV, although its exact role in the pathology of BV is unknown. Using immunofluorescence, confocal and transmission electron microscopy, we found that VK2 vaginal epithelial cells take up G. vaginalis after exposure to the bacteria. Confocal microscopy also indicated the presence of internalized G. vaginalis within vaginal epithelial cells obtained from a subject with BV. Using VK2 cells and (35)S labeled bacteria in an invasion assay, we found that a 1 h uptake of G. vaginalis was 21.8-fold higher than heat-killed G. vaginalis, 84-fold compared to Lactobacillus acidophilus and 6.6-fold compared to Lactobacillus crispatus. Internalization was inhibited by pre-exposure of cells to cytochalasin-D. In addition, the cytoskeletal protein vimentin was upregulated in VK2 cells exposed to G. vaginalis, but there was no change in actin cytoskeletal polymerization/rearrangements or vimentin subcellular relocalization post exposure. Cytoskeletal protein modifications could represent a potential mechanism for G. vaginalis mediated internalization by vaginal epithelial cells. Finally, understanding vaginal bacteria/host interactions will allow us to better understand the underlying mechanisms of BV pathogenesis.     

A clinical study on the association of Trichomonas vaginalis and Mycoplasma hominis infections in women attending a sexually transmitted disease (STD) outpatient clinic

Swabs from the posterior vaginal fornix were obtained from 804 consecutive female patients visiting a large Dutch sexually transmitted diseases (STD) outpatient clinic. A detailed clinical history was obtained and complaints concerning the lower genital tract, such as vaginal discharge or vulval and vaginal irritation, were recorded. Patients were examined and the presence of non-physiological vaginal secretions was established by speculum examination. The swabs were monitored for bacterial vaginosis (BV) or Candida albicans infection. PCR diagnosis of Chlamydia trachomatis and Trichomonas vaginalis was performed as well. Four groups of patients (n=14-21) with BV or single infections caused by one of these three pathogens and a control group with no pathogens were selected and Mycoplasma hominis PCR was performed additionally. At clinical presentation, controls and single-infected patient groups were comparable with regard to complaints of the lower genital tract and sexual risk behavior defined as having prior STDs and/or admitted prostitution. Only in the T. vaginalis-positive group significantly more women reporting sexual risk behavior were found than in controls. In agreement with former in vitro observations, an in vivo association between the PCR-detected presence of M. hominis and T. vaginalis was established. In 79% of all samples positive for T. vaginalis, M. hominis could be detected, as compared to only 6% in control samples (P=0.0004). However, since single infections by either of the two pathogens were regularly observed, there does not seem to be an exclusive association between the species, as the bacterium is also more frequently found in cases of BV (P=0.026). Co-infection of M. hominis with C. albicans (11%) or C. trachomatis (0%) did not differ significantly from controls (6%). M. hominis did not associate with complaints of the lower genital tract. However, if all groups were combined there appears to be a very significant association between the presence of M. hominis and sexual risk behavior (P=0.0004). M. hominis and sexual risk behavior were more closely associated than M. hominis and T. vaginalis. No indications were found for an enhanced pathogenicity by either of the symbionts.     

细菌性阴道病微生态与益生菌

细菌性阴道病(BV)是育龄妇女阴道炎的最常见原因,其特征是栖居在阴道内的乳酸杆菌减少导致阴道菌群平衡失调的复杂变化。用乳酸杆菌替代抗生素是治疗BV的一种有效的治疗措施。乳酸杆菌产生过氧化氢、乳酸、杆菌素能抑制引起BV的细菌生长。多胺在BV的病理机制中有重要作用。本研究拟综述BV患者阴道菌群的变化,免疫防御反应,阴道乳酸杆菌对BV的影响,多胺的意义,益生菌在治疗选择中的作用。     

Oral probiotics can resolve urogenital infections

We report the first clinical evidence that probiotic lactobacilli can be delivered to the vagina following oral intake. In 10 women with a history of recurrent yeast vaginitis, bacterial vaginosis (BV) and urinary tract infections, strains Lactobacillus rhamnosus GR-1 and Lactobacillus fermentum RC-14 suspended in skim milk and given twice daily for 14 days, were recovered from the vagina and identified by morphology and molecular typing within 1 week of commencement of therapy. In six cases of asymptomatic BV or intermediate BV (based upon Nugent scoring) was resolved within 1 week of therapy.     

Augmentation of antimicrobial metronidazole therapy of bacterial vaginosis with oral probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14: randomized, double-blind, placebo controlled trial

This study enrolled 125 premenopausal women diagnosed with bacterial vaginosis (BV) by presence of vaginal irritation, discharge and 'fishy' odor, and Nugent criteria and detection of sialidase enzyme. The subjects were treated with oral metronidazole (500 mg) twice daily from days 1 to 7, and randomized to receive oral Lactobacillus rhamnosus GR-1 (1 x 10(9)) and Lactobacillus reuteri RC-14 (1 x 10(9)) or placebo twice daily from days 1 to 30. Primary outcome was cure of BV as determined by normal Nugent score, negative sialidase test and no symptoms or signs of BV at day 30. A total of 106 subjects returned for 30-day follow-up, of which 88% were cured in the antibiotic/probiotic group compared to 40% in the antibiotic/placebo group (p<0.001). Of the remaining subjects, 30% subjects in the placebo group and none in the probiotic group had BV, while 30% in the placebo and 12% in the probiotic group fell into the intermediate category based upon Nugent score, sialidase result and clinical findings. High counts of Lactobacillus sp. (>10(5) CFU/ml) were recovered from the vagina of 96% probiotic-treated subjects compared to 53% controls at day 30. In summary, this study showed efficacious use of lactobacilli and antibiotic in the eradication of BV in black African women.