两种不同方法对阴道感染的评价

目的探讨需氧菌阴道炎／细菌性阴道病五项联合测定试剂对阴道感染的临床应用价值。方法将380例就诊女性采用需氧菌阴道炎／细菌性阴道病五项联合测定试剂方法,同时采刖自带常规镜检＋PH值方法进行检测。结果380例阴道分泌物标本经五项联合测定试剂方法检出阴道微生态失衡345例（其中包括AV、BV、CV、VVT等感染）,阳性率为90．79％;采用白带常规镜检＋pH值方法检出阴道微生态失衡328例（其中包括AV、BV、CV、VVT等感染）,阳性率为86．32％,两种检查方法结果差异明显。结论需氧菌阴道炎／细菌性阴道病五项联合测定试剂法准确,快速,高效,能及时评价阴道微生态状况,提高临床合理用药程度,值得临床推广应用。     

基于实时定量PCR技术的育龄期细菌性阴道病患者 阴道菌群结构分析及其临床应用

摘要：目的 探讨育龄期女性细菌性阴道病（bacterial vaginosis,BV）患者阴道优势菌群变化及其与临床指标的相关性。方法 选择本院门诊确诊的育龄期BV患者35例和同期入院体检的健康体检女性37例,无菌拭子采集阴道中段壁分泌物,提取细菌基因组DNA,采用实时定量PCR技术（real-time quantitative PCR,qPCR）进行阴道优势细菌检测,并进行其与临床指标如阴道pH和Nugent评分相关性分析。结果 乳杆菌属细菌及其种水平的细菌如惰性乳杆菌、卷曲乳杆菌、詹氏乳杆菌等在BV患者中均显著下降,BV相关阴道致病细菌如加德纳菌属、奇异菌属、埃格特菌属、巨型球菌I型菌属、纤毛菌属和普氏菌属显著升高（P<0.05）。阴道致病菌群与阴道pH和Nugent评分呈显著正相关,而阴道卷曲乳杆菌和惰性乳杆菌与其呈负相关。结论 育龄期BV患者阴道优势菌群显著失衡,并与阴道pH和Nugent评分显著相关,提示阴道优势菌群改变参与BV发生发展。     

1260例阴道分泌物检测分析

目的调查海宁地区女性阴道感染情况。方法采用白带常规镜检联合需氧菌阴道炎／细菌性阴道病联合测定技术对1260例阴道分泌物进行检测。结果在1260例就诊患者中,清洁度I～II度526例（41．75％）,清洁度III～IV度734例（58．25％）,检出阴道感染患者912例,单一性感染487例（占患病人数的53．4％）,混合感染患者425例（占患病人数的46．6％）。在487例单一性感染患者中,需氧菌阴道炎（AV）患者123例,细菌性阴道病（BV）患者178例,胞溶性阴道病（CV）患者8例,外阴阴道假丝酵母菌病（VVC）患者146例,滴虫性阴道炎（TV）患者32例;在425例混合感染患者中,AV＋BV患者136例,AV＋VVC患者56例,AV＋TV患者62例,BV＋VVC患者103例,BV＋TV患者48例,AV＋BV＋VVC患者13例,AV＋BV＋TV患者7例。结论阴道混合感染及AV发病率较高,值得临床高度关注。     

需氧菌阴道炎／细菌性阴道病联合测定技术诊断需氧菌性阴道炎的临床价值

目的探讨需氧菌阴道炎／细菌性阴道病联合测定技术诊断需氧菌性阴道炎（AV）的临床价值。方法分别采用镜检法和需氧菌阴道炎／细菌性阴道病联合测定技术对200例疑似AV患者进行检测。结果在200例疑似AV患者中,镜检法检出阳性患者174例,需氧菌阴道炎／细菌性阴道病联合测定法检出阳性患者169例。以镜检法为诊断标准,需氧菌阴道炎／细菌性阴道病联合测定技术的敏感性为94．83％、特异性为84．62％。两种方法的符合率为93．50％[（165＋22）／200],两者呈中度一致性（Kappa=0．735,P〉0．05）。结论需氧菌阴道炎／细菌性阴道病联合测定技术诊断AV具有很高的敏感度和特异度,与目前临床使用的常规镜检法符合率高,且该方法操作简便、快速,值得临床推广使用。     

2 368株自动培养临床分离菌细菌谱分析

目的：了解本地区血(体)液中临床感染细菌的分布情况。方法：采用Mini VITAL全自动荧光血培养仪和VITEK32自动细菌鉴定仪对我院1999年1月～2002年12月临床送检的8362人份16395瓶血(体)液标本的培养结果作回顾性分析。结果：16395瓶临床血(体)液标本需氧和厌氧培养,3894瓶细菌培养阳性,总阳性率23．8％(血液23．2％,体液28．6％),专性厌氧菌占除甲型副伤寒沙门菌外的其他感染菌的3．7％;共检出各种感染菌41属96种2368株,革兰阴性杆菌：革兰阳性球菌：酵母样真菌=86．0：11．8：0．6;有77．3％的兼性厌氧菌同时在需氧和厌氧环境都生长,10．3％和12．4％只在需氧或厌氧培养中生长。结论：除传染病因素外,血(体)液中的感染菌群组成以革兰阳性球菌为主;同时做血(体液)标本的需氧和厌氧培养,可以提高细菌培养阳性率。     

鲤肠道正常菌群的研究

本实验用稀释滴种的定量方法,对淡水养殖地中健康鲤肠道内的１０种菌群进行了定性、定量分析,并对结果进行统计学处理,得到鲤肠菌群的一些生理值。结果表明鲤肠道中需氧、兼性厌氧优势菌是气单胞菌和酵母菌,厌氧优势菌是拟杆菌。对不同时间、不同温度条件下同一养殖池中鲤肠道菌群的测定结果比较表明：葡萄球菌、假单胞菌差异显著（ｐ＜０．０５）,气单胞菌、大肠杆菌、需氧芽胞杆菌、酵母菌、乳酸杆菌、双歧杆菌、拟杆菌、梭状芽胞杆菌差异不显著（ｐ＞０．０５）,表明该８种菌群在鲤肠道中的菌数处于相对稳定状态,属于肠道正常菌群。     

一次性肠道菌群分析培养瓶的研制和应用

目前国内外进行肠道菌群分析多一般使标本称重连续１０倍稀释后用选择性培养基和非选择性培养基,选择一定稀释度滴种（接种）于厌氧菌和需氧菌的有关培养基上分别进行培养,最后以活菌计数方法分析肠道菌群基本情况。这种常规的肠道菌群分析方法,不仅操作繁锁,且每次菌...     

惰性乳杆菌对女性生殖道健康影响的研究进展

乳杆菌是维持女性阴道环境的重要菌群。惰性乳杆菌是发现最晚、基因组最小的一种乳杆菌,也是健康女性阴道内众多乳杆菌中的一种。惰性乳杆菌表现出的特性与以往认识的乳杆菌不同,其存在往往与阴道微生态紊乱有关。由于常能在细菌性阴道病（bacterial vaginosis,BV）或BV中间型患者阴道中检测到该菌,因此有研究认为惰性乳杆菌通过某种机制促进阴道内致病菌的定植和侵袭,从而间接参与BV的发病。以惰性乳杆菌为优势菌的阴道环境明显比以卷曲乳杆菌为优势菌的阴道环境更容易定植假丝酵母。复发性外阴阴道假丝酵母菌病（vulvovaginal candidiasis,VVC）患者阴道菌群丰富度明显降低,并以惰性乳杆菌为优势菌,而卷曲乳杆菌含量明显减少。惰性乳杆菌在HIV感染、HPV感染及沙眼衣原体感染女性的阴道中具有较高的定植率,有研究表明其存在率也与宫颈上皮内瘤变有关。     

儿童乳牙根管感染的细菌学分析

对18例3～8岁儿童乳牙的根管感染以无菌技术进行定量取样,按种于12种选择性培养基和2种非选择性培养基上,进行需氧、微需氧和厌氧培养,并对细菌菌落计数。对牙髓拟杆菌和牙龈拟杆菌作半定量免疫荧光染色计数;并对其中9例病牙进行了菌相分析。检出的所有细菌中,厌氧菌占绝对优势;其中检出率较高的菌为:产黑色素拟杆菌属,厌氧革兰氏阳性球菌,微需氧革兰氏阳性球菌等.本试验证明,儿童乳牙根管感染是以厌氧菌为主的混合感染,其中以产黑色素拟杆菌属等最常见.     

利用X-Neu5Ac为底物测定唾液酸酶活性在诊断细菌性阴道病的价值

目的利用5溴-4氯-3吲哚乙酰基神经氨酸盐（X-Neu5Ac）为底物测定阴道唾液酸酶活性诊断细菌性阴道病（bacterial vaginosis,BV）的价值.方法健康妇女30例,临床Amsel法诊断为BV的患者45例,共计75例进行了阴道分泌物分析和检测,并与唾液酸酶活性法诊断作了对比研究.取阴道分泌物作为标本分别进行唾液酸酶活性和阴道菌群定量分析,检测细菌包括乳酸杆菌、类杆菌、肠杆菌、葡萄球菌、肠球菌和阴道加德纳菌.唾液酸酶活性测定利用的底物为X-Neu5Ac,特异活性用其产物 ——甲氧基苯酚的纳摩尔数来表示.结果阴道液唾液酸酶活性测定诊断细菌性阴道病的敏感性、特异性、阳性预期值和阴性预期值分别为88.9%、90%、93%和84.3%.唾液酸酶法在检测细菌性阴道病上和传统的Amsel法比较,差异无显著性（P＞0.05）.唾液酸酶阳性组Gv活菌数（6.96 log CFU/g）明显高于唾液酸酶阴性组（2.05 log CFU/g）（P＜0.01）.唾液酸酶阳性组产H2O2阴道乳杆菌（LB＋）活菌数（4.26 Log CFU/g）明显低于唾液酸酶阴性组（8.66 Log CFU/g）（P＜0.01）.唾液酸酶阳性组与唾液酸酶阴性组两组的阴道液中需氧菌活菌数差异无显著性（P＞0.05）,主要包括金黄色葡萄球菌、肠球菌和肠杆菌.结论利用X-Neu5Ac作为唾液酸酶的底物测定唾液酸酶活性的方法是诊断细菌性阴道病的有效检测方法.阴道内唾液酸酶活性增强,厌氧菌数量增加,LB＋数量减少,提示BV发生恶化.     

需氧菌阴道炎、细菌性阴道病阴道菌群分析

用阴道分泌物直接涂片、革兰染色、选择性培养分离和细菌预成酶谱分析等微生物学方法和生物化学方法,分析了正常女性、AV、BV患者的阴道菌群。正常女性阴道中以乳酸杆菌为主,产H2O2乳酸杆菌的检出率89%,AV的致病菌主要是革兰阳性率需氧菌、检出率可达80%,其中金黄色葡萄球菌,粪肠球菌和埃希氏大肠菌较为常见,B族链球菌的检出率较低;BV的致病菌主要是厌氧菌,以革兰阴性厌氧菌的检出率最高,加德纳菌的检出率不足40%。48例BV患者中有8例合并AV感染,31例AV患者中有6例合并BV感染。细菌谱分析发现,AV患者易感染消化链球菌、普雷沃菌等厌氧菌和加德纳菌,BV患者易感染粪肠球菌、大肠埃希菌等需氧菌。     

乳杆菌泡腾片治疗细菌性阴道病

目的观察10例BV患者采用了信谊制药总厂生产的阴道嗜酸乳杆菌泡腾片1个疗程(7 d)后阴道菌群的变化。方法采用7种选择或非选择培养基对阴道分泌物的细菌总数、乳杆菌等7个指标进行检测。结果治疗后阴道中乳杆菌显著增多,厌氧革兰阴性杆菌显著减少(两者P<0.001)。细菌总数、肠杆菌、酵母菌和阴道加德纳菌均减少(P<0.01)。结论信谊阴道嗜酸乳杆菌泡腾片具有较好的调整阴道微生态平衡的作用。     

Dynamics of vaginal bacterial communities in women developing bacterial vaginosis, candidiasis, or no infection, analyzed by PCR-denaturing gradient gel electrophoresis and real-time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

Clinical Features of Bacterial Vaginosis in a Murine Model of Vaginal Infection with Gardnerella vaginalis

Bacterial vaginosis (BV) is a dysbiosis of the vaginal flora characterized by a shift from a Lactobacillus-dominant environment to a polymicrobial mixture including Actinobacteria and Gram-negative bacilli. BV is a common vaginal condition in women and is associated with increased risk of sexually transmitted infection and adverse pregnancy outcomes such as preterm birth. Gardnerella vaginalis is one of the most frequently isolated bacterial species in BV. However, there has been much debate in the literature concerning the contribution of G. vaginalis to the etiology of BV, since it is also present in a significant proportion of healthy women. Here we present a new murine vaginal infection model with a clinical isolate of G. vaginalis. Our data demonstrate that this model displays key features used clinically to diagnose BV, including the presence of sialidase activity and exfoliated epithelial cells with adherent bacteria (reminiscent of clue cells). G. vaginalis was capable of ascending uterine infection, which correlated with the degree of vaginal infection and level of vaginal sialidase activity. The host response to G. vaginalis infection was characterized by robust vaginal epithelial cell exfoliation in the absence of histological inflammation. Our analyses of clinical specimens from women with BV revealed a measureable epithelial exfoliation response compared to women with normal flora, a phenotype that, to our knowledge, is measured here for the first time. The results of this study demonstrate that G. vaginalis is sufficient to cause BV phenotypes and suggest that this organism may contribute to BV etiology and associated complications. This is the first time vaginal infection by a BV associated bacterium in an animal has been shown to parallel the human disease with regard to clinical diagnostic features. Future studies with this model should facilitate investigation of important questions regarding BV etiology, pathogenesis and associated complications.     

4019例妇科门诊不同症状患者阴道微生态状况分析

目的探讨妇科门诊不同症状患者的阴道微生态状况。方法回顾性分析2011年11月至2012年9月西安交通大学医学院第一附属医院妇科门诊因不同症状就诊患者的阴道微生态评价（菌群的密集度、多样性、优势菌、病原菌、AV评分、Nugent评分及五项菌群功能及炎症反应指标）检测结果。结果4019例被检查者平均年龄为33．56岁。其中阴道微生态正常占n70％（28／4019）;阴道微生态失调者占99．30％（3991／4019）;后者包括未明确感染的阴道微生态失调患者占66．36％（2667／4019）;明确感染的阴道微生态失调患者占32．94％（1324／4019）;其中外阴阴道假丝酵母菌病（vulvovaginal candidiasis,VVC）患者占24．33％（978／4019）;需氧菌陛阴道炎（aerobic vaginitis,AV）患者占3．11％（125／4019）;细菌性阴道病（bacterial vaginosis,BV）患者占3．14％（126／4019）;滴虫阴道炎（trichomonas vaginitis,TV）患者占5．15％（207／4019）;混合感染患者占8．31％（110／1324）。优势菌群异常患者占54．32％（2183／4019）。结论妇科门诊中未明确感染的阴道微生态失调患者构成比最高;在明确感染的患者中外阴阴道假丝酵母菌病构成比最高;因此阴道微生态评价成为阴道感染性疾病诊断首选检测方法,在临床诊疗实践中具有重要意义。     

通过特异PCR扩增和16SrDNA序列分析检测动弯杆菌

细菌性阴道病(Bacterial Vaginosis,BV)是由于细菌过度生长所致阴道微生态非正常改变,从而导致的一类多微生物病(Polymicrobial Diseases)。动弯杆菌(Mobiluncus sp．)与BV发生有密切关系,但该菌为厌氧菌,营养要求苛刻,很难进行纯培养,国内鲜有研究报道。本文先对BV动物模型恒河猴阴道分泌物进行厌氧菌混合培养,抽提混合物染色体DNA,之后设计了一对动弯杆菌16SrRNA基因的特异性引物,用PCR的方法扩增出了特异片段。通过对扩增产物进行测序分析,确定检测出的为动弯杆菌,并且与羞怯动弯杆菌极为相似。     

通过特异PCR扩增和16S rDNA序列分析检测动弯杆菌

细菌性阴道病 (BacterialVaginosis,BV)是由于细菌过度生长所致阴道微生态非正常改变 ,从而导致的一类多微生物病 (PolymicrobialDiseases)。动弯杆菌 (Mobiluncussp .)与BV发生有密切关系 ,但该菌为厌氧菌 ,营养要求苛刻 ,很难进行纯培养 ,国内鲜有研究报道。本文先对BV动物模型恒河猴阴道分泌物进行厌氧菌混合培养 ,抽提混合物染色体DNA ,之后设计了一对动弯杆菌 16SrRNA基因的特异性引物 ,用PCR的方法扩增出了特异片段。通过对扩增产物进行测序分析 ,确定检测出的为动弯杆菌 ,并且与羞怯动弯杆菌极为相似     

Composition of the Vaginal Microbiota in Women of Reproductive Age – Sensitive and Specific Molecular Diagnosis of Bacterial Vaginosis Is Possible?

Background and Objective

Bacterial vaginosis (BV) is the most common vaginal disorder, characterized by depletion of the normal lactobacillus-dominant microbiota and overgrowth of commensal anaerobic bacteria. This study aimed to investigate the composition of the vaginal microbiota in women of reproductive age (healthy women and women with BV), with the view of developing molecular criteria for BV diagnosis.

Materials and Methods

Vaginal samples from 163 women (79 control, 73 BV and 11 intermediate (Lactobacillary grade II flora) cases) were analyzed using 454 pyrosequencing of the hypervariable regions V3–V4 of the 16S rRNA gene and 16 quantitative bacterial species/genus-specific real-time PCR assays. Sensitivities and specificities of potential BV markers were computed using the Amsel criteria as reference standard for BV. The use of quantitative thresholds for prediction of BV, determined for both relative abundance measured with 454 pyrosequencing and bacterial load measured with qPCR, was evaluated.

Results

Relative to the healthy women, the BV patients had in their vaginal microbiota significantly higher prevalence, loads and relative abundances of the majority of BV associated bacteria. However, only Gardnerella vaginalis, Atopobium vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 detected at or above optimal thresholds were highly predictable for BV, with the best diagnostic accuracy shown for A. vaginae. The depletion of Lactobacillus species combined with the presence of either G. vaginalis or A. vaginae at diagnostic levels was a highly accurate BV predictor.

Conclusions

Quantitative determination of the presence of G. vaginalis, A. vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Measurements of abundance of normal and BV microbiota relative to total bacteria in vaginal fluid may provide more accurate BV diagnosis, and be used for test-of-cure, rather than qualitative detection or absolute counts of BV related microorganisms.     

The complex vaginal flora of West African women with bacterial vaginosis

Background

The spectrum of bacteria associated with bacterial vaginosis (BV) has recently expanded through taxonomic changes and the use of molecular methods. These methods have yet to be used in large-scale epidemiological studies in Africa where BV is highly prevalent.

Methods

An analysis of samples obtained during a clinical trial of the management of vaginal discharge in four West African countries. Samples were available from 1555 participants; 843 (54%) had BV. Nucleic acids of 13 bacterial genera or species potentially associated with BV were detected through the polymerase chain reaction.

Results

The associations between various components of the vaginal flora were complex. Excluding Lactobacillus, the other 12 micro-organisms were all associated with each other at the p≤0.001 level. The prevalence of various bacterial genera or species varied according to age, sexual activity and HIV status. In multivariate analysis, the presence of Gardnerella vaginalis, Bifidobacterium, Megasphaera elsdenii, Dialister, Mycoplasma hominis, Leptotrichia, and Prevotella were independently associated with BV as was the absence of Lactobacillus and Peptoniphilus. However, Mobiluncus, Atopobium vaginae, Anaerococcus, and Eggerthella were not independently associated with BV. Unexpectedly, after treatment with a regimen that included either metronidazole or tinidazole, the proportion of patients with a complete resolution of symptoms by day 14 increased with the number of bacterial genera or species present at enrolment.

Conclusions

Numerous bacterial genera or species were strongly associated with each other in a pattern that suggested a symbiotic relationship. BV cases with a simpler flora were less likely to respond to treatment. Overall, the vaginal flora of West African women with BV was reminiscent of that of their counterparts in industrialized countries.