健康妇女和外阴阴道假丝酵母菌病患者阴道乳杆菌对阴道假丝酵母菌抑制作用的实验研究

目的:探讨正常健康和外阴阴道假丝酵母菌病(VVC)妇女阴道分泌物中分离的乳杆菌对假丝酵母菌生长的抑制。方法:健康正常(正常组)和VVC初发(VVC组)妇女各30名,通过定量采集阴道分泌物培养乳杆菌,观察阴道乳杆菌数量及菌种分布情况,并将获得的乳杆菌及VVC患者阴道分泌物中假丝酵母菌分离纯化及鉴定后,采用牛津杯法观察乳杆菌对假丝酵母菌生长的抑制作用,同时比较VVC组和正常组乳杆菌产H2O2能力的差异。结果:①乳杆菌检出率及菌种分布组间无统计学差异(P>0.05);②VVC组阴道乳杆菌数量的平均对数值与正常组相比显著减少(P<0.01);③阴道内乳杆菌BV试剂盒检测为阴性者,正常组显著高于VVC组(P<0.01);④正常组阴道分离的乳杆菌与VVC组比较,对白色和热带假丝酵母菌的生长抑制有统计学差异(P<0.05),对克柔假丝酵母菌的抑制无统计学差异(P>0.05);正常组分离的嗜酸乳杆菌与VVC组的比较,对白色和热带假丝酵母菌的生长抑制均有统计学差异(P<0.01,P<0.05),对克柔假丝酵母菌的生长抑制无统计学差异(P>0.05);但是正常组和VVC组分离的詹氏乳杆菌对上述3种假丝酵母菌的生长抑制均无统计学差异(P>0.05)。结论:①正常妇女与VVC患者阴道内乳杆菌检出率、菌种分布相似,均以嗜酸、詹氏乳杆菌为优势菌种;②VVC患者阴道乳杆菌的数量显著减少;③VVC组产生H2O2的能力下降,可能与VVC发生相关;④正常状态下阴道乳杆菌对假丝酵母菌抑制作用优于VVC感染状态,其中以嗜酸乳杆菌的抑菌作用最为明显。     

内蒙古牧区育龄期健康及细菌性阴道病妇女阴道乳杆菌种的鉴定与分析

目的 对内蒙古牧区育龄期健康妇女及细菌性阴道病(BV)患者的阴道乳杆菌种进行鉴定与分析,为阴道微生态治疗提供依据.方法 2008年6至12月,选取内蒙古自治区3个牧区旗县(正镶白旗、达尔罕茂明安联合旗、百灵庙镇)育龄期蒙古族健康妇女203例、汉族健康妇女74例,蒙古族BV妇女102例,采用改良乳杆菌培养基(MRS)对阴道侧壁分泌物中乳杆菌进行分离、培养,提取细菌总DNA,进行核糖体脱氧核糖核酸(16S rDNA)基因序列鉴定,并采用四甲基联苯胺-辣根过氧化物酶-乳杆菌培养基(TMB-HRP-MRS)对乳杆菌进行产H2O2分析.结果 (1)检出率:203例蒙古族健康妇女乳杆菌检出率为76.8%(156/203),蒙古族BV妇女乳杆菌检出率为21.6%(22/102),两者比较,差异有统计学意义(P＜0.01);汉族健康妇女乳杆菌检出率为82.4%(61/74),与蒙古族健康妇女比较,差异无统计学意义(P＞0.05).(2)阴道乳杆菌鉴定:蒙古族健康妇女阴道分泌物中共检出193株乳杆菌株,共11种,蒙古族BV妇女阴道分泌物中检出22株乳杆菌株,共4种.(3)H2O2阳性乳杆菌的鉴定:对分离出乳杆菌的74例蒙古族健康妇女及22例蒙古族BV妇女进行阴道乳杆菌H2O2实验,其中蒙古族健康妇女阴道H2O2阳性乳杆菌的检出率为75.7%(56/74),蒙古族BV妇女阴道H2O2阳性乳杆菌的检出率为27.3%(6/22),两者比较,差异有统计学意义(P＜0.05).结论 内蒙古牧区育龄期健康妇女阴道乳杆菌检出率与种族无明显相关性;BV妇女的阴道乳杆菌及H2O2阳性乳杆菌检出率均较健康妇女明显降低.

Abstract：

Objective To identify and analyze the species of vaginal lactobacilli between patients with bacterial vaginosis (BV) and healthy women at childbearing age in Inner Mongolia. Methods From Jun. 2008 to Dec. 2008, 203 Mongolian healthy women, 74 Han healthy women and 102 Mongolian patients with BV from 3 pastoral areas were enrolled in this study. Isolation and culture of lactobacilli from vaginal wall were performed by modified culture medium. DNA of lactobacilli were extracted and sequenced. H2O2 were detected by TMB-HRP-MRS. Results(1)The rate of lactobacilli identification were 76.8%(156/203) in Mongolian healthy women and 21.6% (22/102) in Mongolian patients with BV, which reached statistical difference(P＜0.01).Lactobacilli identification in Han healthy women [82.4%(61/74)] did not show significant difference with that of Mongolian healthy women (P＞0.05). (2) The total of 193 strains and 11 species of Lactobacillus were detected in 203 Mongolian healthy women. Meanwhile,22 strains and 4 species of Lactobacillus were found in 102 Mongolian BV cases.(3)The rate of H2O2 generating Lactobacilli was 27.3% (6/22) in Mongolian BV patients and 75.7% (56/74)in Mongolian healthy women, which showed statistical difference(P＜0.05). Conclusions The rate of Lactobacillus was not related with the race of women in pastoral area in Inner Mongolian. The amount of lactobacilli and H2O2 generating Lactobacilli in the vagina of BV patients was remarkably lower than those of healthy women at childbearing age.     

阴道乳杆菌对常见阴道假丝酵母菌抑制作用的体外试验研究

目的:观察阴道乳杆菌菌株在体外实验中对假丝酵母菌生长的抑制作用。方法:分离健康妇女阴道中常见的乳杆菌和外阴、阴道假丝酵母菌病(vulvovaginal candidiasis,VVC)患者阴道中的常见假丝酵母菌,采用96孔板混合生长抑制法观察处于生长对数期的乳杆菌对假丝酵母菌生长的抑制作用。结果:23例健康妇女阴道分泌物中分离出3种乳杆菌:嗜酸乳杆菌(La)、干酪乳杆菌(Lca)和卷曲乳杆菌(Lcr),15例VVC初发患者阴道分泌物中分离出3种假丝酵母菌:白色假丝酵母菌(Ca)、热带假丝酵母菌(Ct)和克柔假丝酵母菌(Ck)。各种乳杆菌均表现出对假丝酵母菌生长的抑制能力,La对各种假丝酵母菌的抑制能力均优于Lcr;与Lca相比,La对Ct的抑制更为明显;产H2O2的Lcr与不产H2O2的Lca对假丝酵母菌的抑制能力无显著差异。结论:各种乳杆菌表现出对假丝酵母菌生长的不同抑制能力,La对研究的3种假丝酵母菌的抑制能力均优于Lcr;与Lca相比,La的唯一优势体现在对Ct的抑制上;乳杆菌对假丝酵母菌的抑制可能不仅仅与产生H2O2能力有关。     

重组人干扰素α-2b阴道泡腾胶囊对细菌性阴道病患者阴道内环境影响的临床研究

目的:评价重组人干扰素α-2b阴道泡腾胶囊对细菌性阴道病患者阴道内环境紊乱的治疗效果.方法:选择2008年10月至2009年5月就诊的诊断为细菌性阴道病的患者58例.采用每晚阴道内放置重组人干扰素α-2b阴道泡腾胶囊,连用10天,对其治疗前后阴道内环境情况进行对照研究.结果:治疗前后患者的乳杆菌检出率、pH和线索细胞检出率指标均有明显改善.治疗前后比较,差异有高度统计学意义(P<0.01).结论:重组人干扰素α-2b阴道泡腾胶囊能有效改善阴道内环境紊乱,疗效确定,值得推广.     

阴道微生态的临床研究及意义

目的:研究人群的阴道微生态状况,为阴道炎症的防治提供新思路。方法:采用整群二级抽样方式,随机抽取313例符合条件的育龄妇女进行调查及阴道微生态检测(其中阴道炎症74例),采用成组病例对照法进行分析。结果:阴道微生态失调者阴道pH值4.4,明显高于阴道微生态正常者4.0(t=6.3581,P<0.01),但并未超出阴道正常酸性环境范围;各类阴道炎症患者的阴道pH均值增高更为明显(P均<0.01),且均超出了阴道正常的酸性环境范围。微生态失调者阴道乳杆菌和H2O2(+)检出率均明显低于微生态正常者(P均<0.01),而阴道炎症者此两项指标降低更为明显(P均<0.01),且阴道炎症患者乳杆菌检出率明显低于无阴道炎症妇女(27.0%vs74.9%,P<0.01)。阴道乳杆菌检出率降低幅度越大,患常见阴道炎症的相对危险度就会越高(P均<0.01)。结论:阴道pH值升高可作为阴道微生态异常状况的预警信号;阴道乳杆菌检出率可作为阴道健康状况的监测指标或阴道炎症治疗后的康复指标。     

常用抗假丝酵母菌药物对阴道常见乳杆菌生长影响的研究

目的:探讨治疗外阴阴道假丝酵母菌病(VVC)的药物对阴道常见乳杆菌生长的影响。方法:VVC患者随机分为3组,分别应用咪康唑(咪康唑组)、伊曲康唑(伊曲康唑组)、制霉菌素栓(制霉菌素组)进行治疗,治疗前、后分别进行乳杆菌Nugent评分,治疗后观察患者症状缓解时间、阴道pH值、乳杆菌数量及H2O2阳性乳杆菌的检出率等。结果:患者症状缓解时间、总有效率3组间差异无统计学意义(P>0.05)。3组乳杆菌Nugent评分均较治疗前有所降低,且伊曲康唑组乳杆菌Nugent评分显著高于咪康唑组和制霉菌素组,差异有统计学意义(P<0.05)。咪康唑组、伊曲康唑组、制霉菌素组中H2O2阳性的乳杆菌分别占30.0%、60.0%和25.0%,差异有统计学意义(P<0.05),伊曲康唑组H2O2阳性的乳杆菌的检出率显著高于其余2组,差异有统计学意义(P<0.05)。结论:伊曲康唑治疗VVC疗效确切,对阴道乳杆菌生长的影响较小,有助于维护阴道正常菌群,是一种治疗VVC较理想的抗真菌药物。     

复发性细菌性阴道病的发病机制和治疗

细菌性阴道病(bacterial vaginosis,BV)是生育年龄妇女最常见的阴道感染.系由于阴道内产过氧化氢(H_2O_2)的乳杆菌减少,而阴道加德纳菌、各种厌氧菌、动弯杆菌及支原体等大量繁殖引起混合感染,导致阴道微生态平衡失调的临床症候群~([1～3]).     

三种人工阴道成形术后阴道乳杆菌定植情况研究

目的探讨结肠代阴道、皮肤代阴道及猪小肠黏膜下层生物补片代阴道术后阴道乳杆菌定植情况。方法回顾性分析2012年7月至2014年7月在上海交通大学医学院附属第九人民医院行人工阴道成形术的患者资料,根据所用术式分为:结肠代阴道组(23例)、皮肤代阴道组(18例)、补片代阴道组(20例),分别在术后1周、1个月、6个月进行阴道分泌物乳杆菌培养检测;对照组为同期就诊的健康女性(30例)。比较各组阴道乳杆菌的检出情况。结果术后1周的阴道乳杆菌检出率,结肠代阴道组(17.39%)、皮肤代阴道组(16.67%)及补片代阴道组(20%)与对照组(60%)相比,差异均有统计学意义(P0.05);术后1个月的阴道乳杆菌检出率,结肠代阴道组(34.78%)、补片代阴道组(35.00%)与对照组相比差异均无统计学意义(P0.05),皮肤代阴道组(22.22%)与对照组相比差异有统计学意义(P0.05);术后6个月的阴道乳杆菌检出率,结肠代阴道组(47.83%)、补片代阴道组(40.00%)与对照组相比差异均无统计学意义(P0.05),皮肤代阴道组(27.78%)与对照组相比差异有统计学意义(P0.05)。结论术后1周内乳杆菌即可在人工阴道内定植;术后1、6个月时结肠代阴道和补片代阴道患者阴道内乳杆菌检出情况与健康妇女水平相近,皮肤代阴道乳杆菌检出率较低,有必要进一步研究。     

黄体酮阴道环在家兔的药代动力学研究

目的:提供研制可供哺乳期妇女避孕的黄体酮阴道环的药理学依据。方法:将18只去卵巢后2周的新西兰雌兔随机分为3组:阴道环低剂量组(175mg,A组)、高剂量组(350mg,B组)及肌注组(C组),分别在放置阴道环及肌注黄体酮前后不同时点取静脉血,用磁性分离酶联免疫法测定兔血清中黄体酮的浓度,用PK-Graph程序计算药代动力学参数。结果:黄体酮阴道环和黄体酮注射剂在兔体内的药代动力学符合二室开放模型。主要药代动力学参数分别是A组:Tmax:2.23±1.3h,Cmax:47.64±23.58ng/ml,T1/2:818.08±511.77h,AUC:16115.11±8398.88ng·ml·h-1;B组:Tmax:2.03±1.33h,Cmax:74.04±24.57ng/ml,T1/2:730.31±306.49h,AUC:28751.95±7151.95ml·h-1;C组:Tmax:1.54±0.77h,Cmax:138.88±60.96ng/ml,T1/2:2.55±0.89h。其中A组的AUC(56d)明显低于B组,二者有显著性差异(P<0.05);A组和B组的Cmax均低于C组,差异显著(P<0.05);A组和B组的T1/2均明显高于C组(P<0.01),但A、B组间无差异。结论:与传统的黄体酮注射剂相比,黄体酮阴道环的药代动力学参数呈现明显的长效缓释特征。应可成为安全长效且使用方便的哺乳期避孕药具。     

妇女阴道微生态状态研究

目的:研究中小学女教师群体阴道微生态状态及年龄、绝经与否、避孕方式对阴道pH和阴道乳杆菌分布的影响.方法:对1000名贵阳市中小学女教师,采用问卷调查、病史询问、妇科检查及阴道微生态检查方法,将被检者进行分组比较.结果:①阴道微生态异常率较高(67.20%);②在30～60岁年龄段,随着年龄的增长,阴道平均pH值升高,阴道乳杆菌检出率下降;③与未绝经者相比,绝经者阴道平均pH值升高,阴道乳杆菌检出率下降;④使用避孕套者细菌性阴道病的检出率明显低于其他避孕方式;⑤患细菌性阴道病者,阴道pH值升高,乳杆菌检出率降低.结论:女性阴道微生态变化受年龄、绝经、避孕方式及细菌性阴道病的影响.     

Hydrogen peroxide-producing Lactobacillus species in healthy women and in women with bacterial vaginosis

We compare the prevalence of H2O2--producing Lactobacillus species in women with or without bacterial vaginosis. H2O2--producing facultative Lactobacillus species were found in the vaginas of 37 (92.5%) of 40 normal women and 3 (4.1%) of 72 women with BV. Anaerobic Lactobacillus species (which do not produce H2O2) were isolated from 12 (16.6%) of 72 women with BV and 1 (2.5%) of 40 normal women. The production of H2O2 by Lactobacillus species may represent a nonspecific antimicrobial defence mechanism of the normal vaginal ecosystem.     

In vitro antagonistic effect of Lactobacillus on organisms associated with bacterial vaginosis

OBJECTIVE: To assess antagonistic properties of Lactobacillus strains isolated from the vaginas of healthy women as compared to the most common bacterial agents related to vaginosis. STUDY DESIGN: Antagonistic activity of different Lactobacillus strains isolated from the vaginas of healthy women not treated for infections with an antibiotic for the previous three months was screened using an agar slab method. The activity was tested against test organisms associated with bacterial vaginosis and/or urinary tract infections: Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, Escherichia coli, Gardnerella vaginalis, Peptostreptococcus anaerobius and Prevotella bivia. RESULTS: Many of the 146 Lactobacillus strains tested exerted apparent antagonistic activities against gram-positive aerobic cocci and gram-negative rods, such as S aureus and E coli, and a marked number of Lactobacillus strains inhibited facultative bacteria, such as Gardnerella vaginalis and the anaerobes P anaerobius and P bivia. Only a few lactobacilli were able to inhibit growth of E faecalis and S agalactiae. Indicator bacteria growth inhibition probably relies upon several different complementary mechanisms. The specific indicator bacteria species determines which mechanism predominates. CONCLUSION: Lactobacillus strains taken from normal vaginal flora demonstrated antagonistic activity against a variety of bacteria related to vaginal and urinary tract infections. The specific occurrence rates of active Lactobacillus strains are different, and this difference is dependent on the indicator bacteria species.     

Hydrogen peroxide producing lactobacilli in women with vaginal infections

OBJECTIVE: Hydrogen peroxide producing lactobacilli are isolated from the vaginas of a majority of healthy reproductive age women. Their toxic and inhibitory effect against the overgrowth of pathogens in the vagina is documented by in vitro studies. Clinical studies concerning the role of hydrogen peroxide producing lactobacilli in vaginal infections are controversial. STUDY DESIGN: The aim of this study was to isolate lactobacilli from women with major vaginal infections: bacterial vaginosis (BV), vulvovaginal candidiasis (VVC) and trichomoniasis (TV), and from women with normal flora (NF), to test their ability to produce hydrogen peroxide, to quantitate lactobacilli and to evaluate factors related to the colonization of vagina with hydrogen peroxide producing lactobacilli. The study group comprised 108 women: 27 with VVC, 26 with BV, 20 with TV and 35 with NF. Data was analysed with Fisher exact test, chi2-test, and ANOVA. RESULTS: Lactobacilli were isolated from 81.5% of women with VVC, 58% of women with BV (p=0019), 55% of women with TV (p=0.015) and from 86% of women with NF. The majority of isolates of lactobacilli from women with VVC, TV and NF produced hydrogen peroxide (77%, 63.5% and 80%, respectively), compared with women with BV where only 26.5% of isolates produced hydrogen peroxide (p=0.01). Quantitative analysis showed that in an overwhelming majority of women colonized with lactobacilli, their count was >10(7) CFU/ml. Upon evaluation of behavioural and demographic characteristics, we found that smokers had a lower incidence of isolation of hydrogen peroxide producing lactobacilli (p=0.018). Our results showed that women with BV lack lactobacilli, especially hydrogen peroxide producing ones. Women with TV had a lower rate of lactobacilli. In women with VVC, there was neither significant difference in the isolation of lactobacilli, nor in their ability to produce hydrogen peroxide, compared to women with NF. CONCLUSION: Our results point out that hydrogen peroxide producing lactobacilli could protect against the development of BV, but not against VVC and TV.     

The relationship of hydrogen peroxide-producing lactobacilli to bacterial vaginosis and genital microflora in pregnant women.

Lactobacilli provide an important microbial defense against genital colonization by pathogens. The role of hydrogen peroxide (H2O2) in the control of genital microflora was explored in a cross-sectional study of 275 women in the second trimester of pregnancy. Vaginal cultures were obtained for detection of H2O2-positive and H2O2-negative lactobacilli and other members of the genital microflora. Compared with women with H2O2-negative lactobacilli, women colonized by H2O2-positive lactobacilli were less likely to have bacterial vaginosis, symptomatic candidiasis, and vaginal colonization by Gardnerella vaginalis, Bacteroides, Peptostreptococcus, Mycoplasma hominis, Ureaplasma urealyticum, and viridans streptococci (P less than or equal to .05 for each comparison). In addition to the above organisms, women without vaginal lactobacilli were more likely than those women with H2O2-positive lactobacilli to have Chlamydia trachomatis, and less likely to be colonized by Enterococcus or coagulase-negative staphylococci (P less than .05 for each comparison). Vaginal colonization by group B streptococci or Escherichia coli was not related to the presence of H2O2-positive lactobacilli. These data suggest that the presence of H2O2-positive lactobacilli in the vagina is inversely correlated with infection by some genital pathogens in pregnant women.     

A study of the prevalence of hydrogen peroxide generating Lactobacilli in bacterial vaginosis: the determination of H2O2 concentrations generated, in vitro , by isolated strains and the levels found in vaginal secretions of women with and without infection.

The concentration of hydrogen peroxide in the vaginal secretions of normal women were compared with levels found in women infected with bacterial vaginosis (BV). The mean concentration found in 90 normal samples was 0.171 microg per ml, whereas in women with BV, the mean level was 0.04 microg per ml (P < 0.001). In vitro culture experiments using TMB agar showed that in normal women, 75% of Lactobacillus strains produced H2O2 and 25% did not, whereas in women 'infected' with BV. 14% of the isolates produced H2O2 and 86% did not (P < 0.001). Quantitative culture tests showed that both anaerobic and facultative strains of Lactobacilli isolated from normal women generated significantly higher levels of H2O2 than strains cultured from women with BV. Anaerobic strains isolated from normal women produced a mean level of 6.8 microg per ml, compared with the mean level of 1.4 microg per ml from women with BV. Facultative strains from normal women produced mean levels of 18.3 microg per ml compared with 2.4 microg per ml produced by strains from BV-infected women, which had been incubated under the same conditions (P < 0.001). There results indicate that Lactobacilli which produce H2O2 probably have a significant role in protecting the vaginal ecosystem from BV infection.     

Treatment of bacterial vaginosis with an acid cream: a comparison between the effect of lactate-gel and metronidazole

Bacteriological isolation of anaerobes, Gardnerella and lactobacilli was carried out in a group of 62 women with the diagnosis bacterial vaginosis and 42 control women. Lactobacilli were the predominant organisms in the control group whereas anaerobes dominated the flora in bacterial vaginosis patients. Lactate-gel (pH 3.5, 5 ml) inserted into the vagina daily for 7 days is as effective as oral metronidazole, 500 mg twice daily for 7 days. The women in both groups became symptom-free and objectively improved. Anaerobes were significantly reduced (p less than 0.0001) in both groups after 1-week treatment but Gardnerella was not significantly reduced. As bacterial vaginosis is generally looked upon as a mild noninflammatory condition lactate-gel seems to be an ideal treatment for this disease.     

Could bacterial vaginosis be due to the competitive suppression of lactobacilli by aerobic microorganisms?

Bacterial vaginosis (BV) is characterized by lack of lactobacilli and predominance of anaerobic species. Ciprofloxacin is active against a great number of aerobic bacteria. The effect of ciprofloxacin on the vaginal microbial flora was studied both in healthy subjects and in patients with BV in order to test if BV may be due to competitive suppression of lactobacilli by aerobic species. About half of the patients were relieved of their symptoms. No adverse effects on the microbial profile or colonization by lactobacilli were produced by the drug. Lactobacilli known to be able to produce hydrogen peroxide were found to be significantly more common in healthy women than in women with BV. The species most frequently related to vaginal health were Lactobacillus jensenii and Lactobacillus rogosae.     

The in vitro activity of vaginal Lactobacillus with probiotic properties against Candida

Lactobacilli, the predominant vaginal microorganisms in healthy premenopausal women, control other members of the vaginal microflora and thus protect against bacterial vaginosis and urinary tract infections. It has been claimed that some lactobacilli are also protective against Candida vaginitis. Little is known, however, about the mechanisms by which these lactobacilli can control vaginal populations of Candida and prevent vaginitis. To address this question, vaginal Lactobacillus strains with known antagonistic properties against bacteria were tested for their cell surface properties, adhesion to vaginal cell lines in vitro and antagonistic activities against Candida. A small proportion of the lactobacilli tested adhered strongly to cultured vaginal epithelial cells and inhibited growth of Candida albicans but not of C. pseudotropicalis. This anticandidal activity was in some Lactobacillus strains related to hydrogen peroxide (H2O2) production, but catalase treatment did not suppress this activity in other Lactobacillus strains, suggesting alternative mechanism(s). Moreover, tested vaginal Candida strains were resistant to relatively high concentrations of H2O2 that markedly exceeded those produced by even the most active Lactobacillus strains.