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

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。     

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。     

细菌性阴道病的菌群亚型与治疗结局的相关性研究

目的探讨细菌性阴道病的菌群亚型与不同治疗结局的相关性,为细菌性阴道病分型诊断及个体化治疗提供理论依据。方法选取2012年9月至2013年7月在北京大学第一医院妇产科门诊诊断为细菌性阴道病的育龄期女性为研究对象,规范使用甲硝唑凝胶治疗,分别采集患者用药前、用药后6~8d、用药后1个月的阴道分泌物,提取细菌组DNA,进行454焦磷酸测序,分析阴道微生物菌群的多样性和丰度差异,探讨细菌性阴道病患者阴道菌群的构成差异与预后之间的相关性。结果共有109例细菌性阴道病患者,其中61名患者完成了2次随访,共计183份阴道分泌物样品。对所有采集的标本进行454焦磷酸测序分析,共发现192种细菌。根据高通量测序结果分析,可将细菌性阴道病的菌群结构分为3种亚型:普雷沃菌型(Prevotella,I型)、阿托波菌型(Atopobium,II型)和乳杆菌型(Lactobacillus,III型)。细菌性阴道病不同的菌群亚型,其治愈率是不同的。乳杆菌型的治愈率最高,约为76.5%,阿托波菌型的治愈率最低,约为42.8%。结论细菌性阴道病的菌群存在不同亚型,且与治疗结局存在相关性,乳杆菌型菌群结构的患者对甲硝唑的治愈率最高。     

阴道常见乳杆菌及相关研究进展

近年来,随着全基因组测序等分子生物技术的应用,人们对阴道微生态系统的结构和功能的认识逐渐清晰。研究发现很多妇科疾病的出现伴随着阴道菌群失调,因此研究正常的阴道菌群结构和功能对预防和治疗菌群失调引起的疾病有重要意义,这也是目前人体微生态研究领域的热点。本文总结了近年关于健康女性阴道菌群的组成和类型的研究,阐述了阴道中常见乳杆菌如卷曲乳杆菌、惰性乳杆菌、格氏乳杆菌、詹氏乳杆菌等在功能研究方面的最新进展,以期为乳杆菌在防治妇科疾病如细菌性阴道病(BV)、外阴假丝酵母菌病(VVC)并推进其产品开发及临床应用等方面提供一些理论参考。     

宫颈上皮内瘤变与宫颈微生物群落结构的相关性

目的分析宫颈上皮内瘤变与宫颈微生物群落结构的相关性。方法选取2017年1月至2018年10月于我院就诊的253例女性进行回顾性分析,根据是否患有宫颈上皮内瘤变分为CIN组(86例)及对照组(167例)。采集纳入对象的宫颈菌群并提取细菌基因组DNA,对细菌16S rRNA V3、V4区片段进行PCR扩增,并采用Illuminate Miseq测序平台对PCR产物进行测序,分析两组对象宫颈微生物群落结构,并分析患者宫颈微生物群落结构与宫颈上皮内瘤变的相关性。结果两组对象宫颈微生物群落的Simpson指数及Shannon指数比较差异无统计学意义(t=1.474、1.636,P0.05),而CIN组的Chao指数及ACE指数均高于对照组(t=9.213、10.420,P0.05)。16S rRNA分析显示放线菌是宫颈部位的主要菌群。CIN组女性宫颈微生物群落中放线菌、奇异菌属、放线菌门、阴道奇异菌及奇异变形菌占主要优势(LDA4log10),对照组中杆菌、厚壁菌门等占主要优势。Spearman相关分析示卷曲乳杆菌、惰性乳杆菌、格氏乳杆菌、詹氏乳杆菌与宫颈上皮内瘤变呈负相关,而加特纳菌、阴道奇异菌、普雷沃氏菌、粪球菌与宫颈上皮内瘤变呈正相关(均P0.05)。结论 CIN女性宫颈菌群与健康女性存在明显差异,其中卷曲乳杆菌、惰性乳杆菌、格氏乳杆菌、詹氏乳杆菌与宫颈上皮内瘤变呈负相关,而加特纳菌、阴道奇异菌、普雷沃氏菌、粪球菌与宫颈上皮内瘤变呈正相关。     

不同生理阶段女性阴道微生态状况分析

目的 通过对女性阴道微生态的检查,全面评估不同生理阶段的女性阴道微生态状况,为临床对女性阴道感染性疾病的诊断和治疗提供较全面客观的实验室依据。方法 选取2017年1月1日至2017年6月30日在山东省立医院妇产科门诊就诊的女性患者2311例,分别采集患者的阴道分泌物,通过相差显微镜、革兰染色和干化学检测等方法,分别对患者阴道分泌物的清洁度、菌群密集度、多样性,pH值、AV评分、Nugent评分和功能评价等进行分析。结果 （1）2311例患者中育龄期患者组1806例,围绝经期患者组380例,老年绝经期患者组125例,其中育龄期患者组阴道优势菌为G+大杆菌（26.57%,480/1806）,菌群密集度（+++/++++,76.97%,1390/1806）、菌群多样性（++/+++,27.96%,505/1806）与围绝经期患者组和老年绝经期患者组比较,差异均有统计学意义（Ps<0.01）。围绝经期患者组阴道清洁度（III/IV度,50.26%,191/380）,pH值（pH≥4.6,72.89%,277/380）,菌群密集度（未见,3.42%,13/380）,过氧化氢（+,84.74%,322/380）,唾液酸苷酶（+‒,14.21%,54/380;+,27.89%,106/380）,与育龄期患者组比较,差异均有统计学意义（Ps<0.01）;围绝经期患者组阴道优势菌为G‒小杆菌（20.26%,77/380）,革兰染色不定球杆菌（27.10%,103/380）、菌群多样性（+,78.16%,297/380）与育龄期患者组和老年绝经期患者组比较,差异均有统计学意义（Ps<0.01）。老年绝经期患者组阴道pH值（pH≥4.6,85.60%,107/125）,无优势菌（25.60%,32/125）,优势菌为G+球菌（39.20%,49/125）,菌群密集度（未见,20.80%,26/125;+/++,48.80%,61/125）、菌群多样性（未见,20.80%,26/125）、白细胞酯酶（+,77.60%,97/125）,与育龄期患者组和围绝经期患者组比较,差异均有统计学意义（Ps<0.01）。（2）老年绝经期患者组AV（中度,17.6%,22/125;重度,5.6%,7/125）、BV（中介,56%,70/125）均显著高于育龄期患者组和围绝经期患者组;育龄期患者组VVC（23.20%,419/1806）和CV（8.47%,153/1806）的发病率均显著高于围绝经期患者组和老年绝经期患者组;围绝经期患者组TV（7.10%,27/380）的发病率显著高于育龄期患者组和老年绝经期患者组。结论 通过分析2311例女性患者阴道微生态状况,发现女性不同生理阶段阴道的微生态情况和各种阴道炎的发病率有差异,因而阴道微生态评价能够为临床的诊断和及时治疗提供依据,更快速地解决患者的病痛,提高患者的生活质量。     

外阴阴道假丝酵母菌病患者阴道菌群多样性研究

目的探讨外阴阴道假丝酵母菌病患者阴道菌群多样性的变化。方法采集育龄期女性健康受试者(control,CN,n=50)、细菌性阴道病患者(Bacterial vaginosis,BV,n=50)、外阴阴道假丝酵母菌病患者(Vulvovaginal candidiasis,VVC,n=48)、细菌性阴道病合并外阴阴道假丝酵母菌病患者(Bacterial vaginosis in combination of vulvovaginal candidiasis,BVC,n=32)的阴道拭子,提取细菌基因组DNA,而后应用25%~55%变性梯度的PCR-DGGE进行指纹图谱分析。结果阴道菌群指纹图谱结果显示,VVC组中阴道优势菌群与CN组基本一致,但与BV组和BVC组比较差异均有统计学意义。结论外阴阴道假丝酵母菌病阴道菌群多样性无明显变化。     

阴道微生态失调与早产相关性研究进展

健康育龄妇女的阴道微生态环境呈现多样性,阴道内微生物菌群以乳杆菌为主。乳杆菌可维持妇女阴道微环境平衡和保持阴道酸性环境以抑制致病性病原体生长。当乳杆菌比例低至致病阈值以下或无乳杆菌存在时,阴道微环境的菌群组成转变成以兼性需氧及厌氧菌为主。越来越多的证据表明,怀孕期间阴道菌群的异常与早产、及因早产和围产期结局不佳导致的潜在新生儿后遗症有关。本文综述了妊娠期阴道菌群的变化、细菌性阴道炎与早产发生风险等方面的问题,目的是帮助临床医生确定怀孕期间有阴道炎症状女性是否可接受治疗,从而降低早产、早产胎膜早破、绒毛膜羊膜炎和母胎传染病的发病率。     

4 630例绝经后妇女阴道微生态状况分析

目的 分析绝经后妇女阴道微生态状况,为临床诊疗提供实验室依据。 方法 以2018年1月至2019年9月在我院妇科门诊就诊的4 630例绝经后妇女作为研究对象,对其阴道微生态检查结果进行回顾性分析。 结果 4 630例患者中阴道微生态正常者409例(8.83%),阴道微生态异常者4 221例(91.17%)。阴道微生态异常者中不能通过镜检明确病原菌者1 758例(37.97%),包含优势菌异常1 406例(30.37%)、菌群抑制351例(7.58%)、菌群增殖过度1例(0.02%);通过镜检明确病原菌者2 463例(53.20%),包含细菌性阴道病(BV)1 237例(26.72%)、需氧菌性阴道炎(AV)1 001例(21.62%)、外阴阴道假丝酵母菌病(VVC)159例(3.43%)、滴虫性阴道炎(TV)66例(1.43%)。不同年龄段绝经后妇女阴道微生态构成存在差异,其阴道清洁度(Ⅲ/Ⅳ)、菌群密集度(未见/Ⅰ)、菌群密集度(Ⅳ)、菌群多样性(未见/Ⅰ)、菌群多样性(Ⅳ)、优势菌(无)、优势菌(G+大杆菌)、优势菌(G-弧形菌)、优势菌(G+球菌)、VVC、TV、β 氨基半乳糖苷酶(+)、唾液酸苷酶(+)、过氧化氢(+)、阴道pH>4.5、微生态异常发生率在不同年龄段之间的分布差异均有统计学意义(均P结论 阴道微生态异常在妇科门诊绝经后妇女中发生率较高,且不同年龄段妇女阴道微生态异常构成存在差异,提示应结合年龄治疗阴道疾病,同时积极恢复阴道微生态环境。     

Real-time PCR分析滴虫性阴道炎患者微生物组成的变化

目的 探寻滴虫性阴道炎患者阴道微生物组成的特点,为临床诊治及减少疾病的复发提供参考依据。方法 选取2016年5‒6月复旦大学附属华东医院妇科门诊45岁以下确诊为滴虫性阴道炎的育龄期患者16例,以同期18例45岁以下育龄期健康女性为对照,采集患者和健康人上阴道壁1/3处的分泌物,进行常规临床分析和革兰染色,并以21种常见的阴道细菌的特异引物进行Real-time PCR检测,分析并比较这些细菌在滴虫性阴道炎患者和健康人阴道内的组成及变化。结果 在18例健康人中,其中13例的阴道微生物组成以乳酸杆菌（Lactobacillus）为优势菌,3例的阴道微生物组成无明显优势菌群或者优势菌不为检测的21种常见阴道微生物,2例的阴道微生物组成以加德纳菌为优势菌;在16例滴虫性阴道炎患者中,只有2例患者的阴道微生物菌群以乳酸杆菌为优势菌,14例以厌氧菌如普雷沃属（Prevotella）、纤毛菌（Leptotrichia）和斯尼思菌（Sneathia）为优势菌。经Metastats进行组间差异分析,结果表明Lactobacillus crispatus、Leptotrichia amnionii、Eggerthella sp.和Peptostreptococcus sp.在两组之间差异有统计学意义（P≤0.05）。PCoA主成分析结果亦显示滴虫性阴道炎患者的阴道常见21种微生物组成与健康人有显著差别。结论 滴虫性阴道炎患者阴道内微生物组成的多样性增加,普雷沃属、纤毛菌及斯尼思菌的明显增高可能滴虫性阴道炎的炎症发生有关,此结果对于临床治疗滴虫性阴道炎的及减少复发提供新的方向,具有一定的指导意义。     

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.     

Normal vaginal flora in chimpanzees (Pan troglodytes): qualitative and quantitative study

Lactobacilli are the predominant microorganisms in the vaginal flora of human beings, and are known to play an important role in protecting them from genital infections. On the other hand, the composition of the vaginal flora differs among laboratory animal species, and lactobacilli are not the predominant vaginal microorganism in many laboratory animals. We speculated that the vaginal flora of chimpanzees would be more similar to those of human beings than to those of other animal species, because chimpanzees are phylogenetically close to human beings, and their reproductive physiology is similar to that of human beings. To clarify our speculation, we examined the development of the vaginal flora in chimpanzees (Pan troglodytes). Streptococci, lactobacilli, and members of the family Bacteroidaceae were the most predominant bacteria in the vagina of mature chimpanzees (9 to 22 years old). During development of the vaginal flora of chimpanzees, the total number of bacteria increased with age and reached a plateau just before sexual maturity (5 to 7 years of age; juvenile period). Lactobacilli were already one of the predominant bacteria before sexual maturity. In mature chimpanzees, the total number of bacteria (aerobes and anaerobes) in the vagina was highest during the swelling phase of the menstrual cycle. During the swelling phase in mature chimpanzees, streptococci, lactobacilli, and Bacteroidaceae were the most frequently isolated (100%) organisms, and the total number of organisms recovered from vaginal specimens from these three groups was the highest. In mature chimpanzees in which the number of bacteria was the highest, lactobacilli were the predominant bacteria. Taken together, these results suggest that these three bacterial groups (streptococci, lactobacilli, and Bacteroidaceae) are indigenous to the vagina of chimpanzees, and chimpanzees would be the most suitable laboratory animals for studying the role of lactobacilli in the vagina of human beings.     

Obstetric and Gynecological Diseases and Complications Resulting from Vaginal Dysbacteriosis

Accurate knowledge of the composition and ecology of vaginal microbial environment of a healthy woman is necessary for the understanding of normal flora and how to reduce the risk for diseases. Vagina and its microflora form a balanced ecosystem in which dominated bacteria are vaginal lactobacilli. There are dynamic changes in this ecosystem having structure and composition depending on many factors. The term dysbacteriosis defines any movement outside the normal range for the given biotope of obligate and/or facultative microflora. Such a change in the quantity and quality of the respective microbial balance is fraught with danger and requires correction and recovery. The purpose of this overview is to examine obstetric and gynecological diseases that can cause vaginal impaired microbial balance. Vaginal dysbacteriosis is a cause, predecessor, and often also consequence of vaginal infections. In essence, any vaginal infection can be seen as dysbacteriosis, developed to the most severe extent. Here, there is a dominant microorganism other than lactic acid bacteria in the vagina (clinically manifested or not, respectively), depletion of defense mechanisms of the vagina associated with the shift of lactobacilli from their dominant role in the vaginal balance, decrease in their number and species diversity, and a resulting change in the healthy status of the vagina. Vaginal dysbacteriosis can be found in pathogenetic mechanism, whereby many obstetric and gynecological diseases develop. Most of these diseases lead directly to increased maternal and infant morbidity and mortality, so it is important to understand the reasons for them and the arrangements for their prevention.     

B族链球菌感染及微量元素营养干预对产妇阴道微生态的影响

分析产妇阴道微生态失衡的发生情况和B族链球菌（GBS）感染及营养相关因素在正常与微生态失衡产妇中的差异,同时检验不同干预的效果。

选取2020年1月—2021年6月本院就诊的105例产妇作为研究对象,采用随机数字表法将其分为观察组（51例）和对照组（54例）。对照组患者接受常规医疗照护干预,观察组在对照组基础上接受个体化营养干预（视产妇具体血清微量元素水平情况）,比较两组产妇阴道菌群分布、GBS感染、血清微量元素分布和新生儿情况。

全部受试对象阴道内优势菌群前5位依次是菌群抑制无优势菌、非典型乳杆菌类革兰阳性杆菌、革兰阳性短杆菌、革兰阳性大杆菌、革兰阳性球菌。阴道微生态指标检测结果显示,阴道微生态失衡产妇共49例,阴道微生态正常产妇共56例。阴道微生态失衡产妇的GBS感染率更高（30.61% vs 5.36%）,且微量元素锌和硒缺乏情况更为严重（P＜0.05）。营养干预后,观察组阴道菌群的分布异常情况改善优于对照组,且菌群多样性正常比例、锌和维生素D水平高于对照组,而GBS感染率低于对照组（P＜0.05）。

GBS感染可加重产妇阴道微生态紊乱,增加不良妊娠结局发生风险,而营养相关干预能够减低GBS感染率并提高微量元素的血清水平,有利于产妇健康,值得临床推广。

     

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。     

Methods for quantitative and qualitative evaluation of vaginal microflora during menstruation.

The quantitative and qualitative changes in the bacterial flora of the vagina during menstruation have received inadequate study. Similarly, the effect of vaginal tampons on the microbial flora as well as the relationship between the microbial flora of the vagina and that of the tampon has not been adequately evaluated. The purposes of the present study were (i) to develop quantitative methods for studying the vaginal flora and the flora of tampons obtained during menstruation and (ii) to determine whether there were differences between the microflora of the tampon and that of the vaginal vault. Tampon and swab samples were obtained at various times from eight young healthy volunteers for 8 to 10 menstrual cycles. Samples consisted of swabs from women wearing menstrual pads compared with swab and tampon samples taken at various times during the menstrual cycle. Samples were analyzed for total facultative and anaerobic bacterial counts, and the six dominant bacterial species in each culture were identified. Statistical evaluation of the results indicates that total bacterial counts decreased during menstruation and that swab and tampon samples yielded similar total counts per unit weight of sample. The numbers of bacteria in tampons tended to be lower than in swabs taken at the same time. Overall, during menstruation, the concentrations of lactobacilli declined, but otherwise there was little difference among the species found during menstruation compared with those found in intermenstrual samples. Cotton tampons had little discernible effect on the microbial flora.     

Methods for quantitative and qualitative evaluation of vaginal microflora during menstruation

The quantitative and qualitative changes in the bacterial flora of the vagina during menstruation have received inadequate study. Similarly, the effect of vaginal tampons on the microbial flora as well as the relationship between the microbial flora of the vagina and that of the tampon has not been adequately evaluated. The purposes of the present study were (i) to develop quantitative methods for studying the vaginal flora and the flora of tampons obtained during menstruation and (ii) to determine whether there were differences between the microflora of the tampon and that of the vaginal vault. Tampon and swab samples were obtained at various times from eight young healthy volunteers for 8 to 10 menstrual cycles. Samples consisted of swabs from women wearing menstrual pads compared with swab and tampon samples taken at various times during the menstrual cycle. Samples were analyzed for total facultative and anaerobic bacterial counts, and the six dominant bacterial species in each culture were identified. Statistical evaluation of the results indicates that total bacterial counts decreased during menstruation and that swab and tampon samples yielded similar total counts per unit weight of sample. The numbers of bacteria in tampons tended to be lower than in swabs taken at the same time. Overall, during menstruation, the concentrations of lactobacilli declined, but otherwise there was little difference among the species found during menstruation compared with those found in intermenstrual samples. Cotton tampons had little discernible effect on the microbial flora.