生殖支原体Taqman MGB荧光PCR方法的建立及在高危人群流行病学调查中的应用
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摘要
生殖支原体(Mycoplasma genitalium, Mg)是近年来确认的性传播疾病(STD)病原体之一,在男性引起非淋球菌性尿道炎(NGU),在女性导致宫颈炎、子宫内膜炎及盆腔炎等,如不治疗可致异位妊娠、不孕等不良结局。
     NUG是常见的STD,世界范围而言,每年有8900万的新发病例,NGU的发病率呈逐年上升的趋势。在我国,自1988年起,NGU作为重点监测的性病在一些监测点对其流行情况进行监测,2002年以来NGU的病例数超过淋病,居所有规定上报的STD首位,在随后几年中均占全国STD病例报告数构成比的35%以上,然而自2008年起NGU的监测被沙眼衣原体(CT)感染所取代,最主要的原因是CT感染已被确认为NGU的主要病因之一,可在11%-43%的NGU患者中检出。
     研究表明Mg是NGU的另一个重要的常见病原之一,在9%--25%NGU患者中可检出,与CT合并感染少见,规范治疗后复发性及持续性NGU患者中Mg阳性多见,因此在CT阴性的NGU患者中Mg可能发挥重要作用。采用推荐治疗药物阿奇霉素或强力霉素规范治疗后,对Mg阳性的NGU治疗失败的病例陆续有报道,说明有耐药的产生。随着对Mg在NGU中致病作用的认识,Mg在国外日益得到重视,2009年的欧洲男性NGU诊疗指南中提到需要把Mg的检测纳入临床诊疗中,对慢性NGU患者的治疗应该覆盖治疗Mg的药物,2010年东欧国家确立了关于Mg感染的实验室诊断指南。
     对Mg在中国人群中流行情况的研究比较有限,既往研究发现在NGU患者中Mg的流行率为20.6%-25%,在CT阴性的NGU患者中可达44.19%,此发现与其他一些国家的研究类似,说明Mg在中国的流行应引起重视。
     女性性工作者(FSWs),性病门诊男性就诊者(MSP)及男男性行为者(MSM)是STD/HIV感染的高危人群,也是HIV哨点监测的重点人群,随着近年来HIV传播模式的转变,经性传播已成为HIV传播的主要模式,上述人群在HIV的传播起着重要作用。近来研究发现Mg与HIV密切相关,Mg感染可能促进HIV的感染及传播,一项对泌尿生殖道Mg感染与HIV阳性的相关性的系统性综述及Meta分析发现Mg感染与HIV阳性显著相关,Mg感染者中HIV阳性率是Mg未感染者的2倍,此外有研究报道Mg感染可能通过促进HIVDNA的脱落进而促进HIV的传播。因此了解上述高危人群中Mg的流行情况,及时干预,不仅有助于控制Mg的传播,预防不良结局的发生,而且可能对于HIV的防治也起着积极作用。
     尽管Mg在国际社会越来越受到重视,然而由于Mg体外培养极其困难,且目前国内没有商品化检测试剂盒等,因此大大限制了对Mg的流行病学研究,缺乏相应的流行病学资料。本研究旨在采用文献报道的简便快捷,敏感性高,特异性好的TaqmanMGB实时定量PCR方法对MSP中Mg的流行情况进行调查,并对相关危险因素进行分析,为高危人群中Mg的流行及监测提供基线资料,为制定防治策略提供参考。全文分为三部分,各部分主要内容分述如下:
     第一部分:Taqman MGB荧光PCR方法建立
     采用文献中报道特异性及敏感性较高的以Mg主要黏附素基因(MgPa基因)为靶基因的Taqman MGB荧光PCR方法,用Mg标准菌株G37制备浓度分别为1×101拷贝/gl、1×102拷贝/gl、1×103拷贝/μl、1×104拷贝/μl、1×10。拷贝/μl、1×106拷贝/μl的6个标准品,制备标准曲线,并对该方法的重复性、灵敏度及特异性进行检测,确定该方法的检测限(LOD)及定量限(LOQ)。结果显示Taqman MGB实时定量PCR方法检测的线性范围好(1×101拷贝/μl--l×106拷贝/μl),标准曲线回归方程y=-3.982x+40.80,R2=0.993,重复性好(批内变异系数=0.7%,批间变异系数=1.09%),敏感性高,检测限(LOD)为每个PCR反应10拷贝,定量限(LOQ)为10拷贝/μl,用该方法检测泌尿生殖道常见10种病原体均为阴性,说明该方法灵敏度高,特异性好,且有较好的重复性。我们认为针对MgPa基因的Taqman MGB实时PCR可以快速、灵敏的对Mg进行定性及定量的检测,为检测人群中Mg的感染情况提供了极大的便利。
     第二部分:广西女性性工作者及性病门诊男性就诊者中生殖支原体的流行病学调查及危险因素分析
     采用第一部分建立的Taqman MGB荧光PCR方法对广西FSWs及MSP中Mg的流行情况进行调查,采用基于场所的分层抽样方法,于2009年7月至9月对广西梧州、贺州的宾馆、酒店、夜总会、KTV、发廊、路边店、站街、公园等娱乐场所及室外场所的FSWs及贺州性病门诊就诊的男性进行调查。按工作场所及每次性交易收入将FSWs划分为3类,路边店、出租屋、站街及公园工作或每次性交易低于50元者为低档FSWs, KTV、发廊、美容美发等场所工作者为中档FSWs,宾馆、酒店、夜总会为高档FSWs,取得知情同意后对受试者进行问卷调查并采集FSWs的宫颈拭子及MSP的尿液标本,Taqman MGB荧光PCR检测Mg。结果共纳入810名FSWs及423名MSP,收集到810份宫颈拭子及407份尿液标本。FSWs中Mg的感染率为13.2%(95%CI=10.87%-15.52%),高中低档FSWs Mg感染率分别为15.7%(95%CI=13.12%-18.2%),10.8%(95%CI=8.67%-12.93%)及14.6%(95%CI=12.17%-17.03%),不同档次间无统计学差异,受教育程度低于初中(AOR=2.36,95%CI=1.15-4.87),未婚(AOR=2.27,95%CI=1.42-3.62),非当地居民(AOR=2.03,95%CI=1.29-3.20),过去1年无STIs相关症状(AOR=1.66,95%CI=1.09-2.52)与Mg感染显著相关。MSP中Mg感染率为28%(95%CI=25.50%-30.49%),独居(AOR=3.84,95%CI=1.58-9.33),过去1年无STIs相关症状(AOR=2.21,95%CI=1.35-3.62),最近3个月与FSWs发生性行为(AOR=2.27,95%CI=1.41-3.67)与Mg感染相关。以上研究结果表明在FSWs及MSP人群中Mg感染率高,且多为无症状感染,提示对上述高危人群中Mg感染进行常规筛查成为必要。
     第三部分:广州男男性行为者中生殖支原体的流行率调查及危险因素分析
     于2010年1月至5月采用滚雪球的方法对广州市同性恋酒吧、茶吧、桑拿浴池、夜总会、公园、公厕等场所的MSM进行问卷调查并采集尿液标本,Taqman MGB荧光PCR检测Mg。328名MSM中,97.6%自我认同为同性恋,其中41.8%虽自我认同为同性恋但也与女性发生性关系,87.8%半年内与男性发生过肛交,63.2%最近一次肛交中使用了安全套,16.8%半年内与女性发生过性行为,仅27.3%在最近一次与女性发生性行为时使用了安全套,4.6%参加过性交易,寻找性伴的方式以互联网为主,占79.0%。323份尿液标本中Mg感染率为4.3%(95%CI=3.1%-5.4%),未发现与Mg感染相关的因素。研究结果表明在MSM人群中Mg感染率低,提示Mg感染以异性性行为途径传播为主,MSM作为Mg感染的传染源意义更大。
Mycoplasma genitalium (Mg) was first isolated from the urethras of two men with nongonococcal urethritis (NGU) in1980, and it has been widely recognized as a sexually transmitted pathogen and one of the common causes of NGU. Mg is associated with cervicitis, endometritis, and pelvic inflammatory diseases (PID) in women, if left untreated, Mg infection can lead to tubal factor infertility.
     In China, NGU has been resurgent since1980s and used to be one of sexually transmitted infections (STIs) for case-reporting before2008. Since then NGU has been replaced by Chlamydia trachomatis (CT) for national sentinel surveillance programme. A few studies on Mg were conducted in China, showing a prevalence rate of20.6%to25%among NGU patients, and44.19%among non-chlamydial NGU patients. These findings indicate that Mg infection is prevalent in China.
     Even though the overall HIV prevalence in China is low, HIV infection through heterosexual transmission is increasing. Female sex workers, male STD clinic patients and men who have sex with men are high risk populations and potential bridge populations for STD and HIV infection and transmission. A significant association between Mg and HIV infection has been observed in many cross-sectional studies and is well-reviewed. In a systematic review and meta-analysis of19studies, Mg infection was associated with a2-fold increased risk of HIV infection. Although Mg prevalence in high-risk populations such as female sex workers and men who have sex with men is gaining increasing attention worldwide, only a few studies estimated Mg prevalence among small Chinese female sex workers, male STD clinic patients and men who have sex with men. This report is the first to determine the prevalence and risk factors for Mg among relative large sample size of these three high risk populations in southwest China.
     Sectionl:To establish a Taqman MGB real time PCR for detection of Mycoplasma genitalium
     To development a previously published and validated method of real-time PCR targeting the mgpA gene in our laboratory by using the standard Mg G37strain (ATCC33530) to build a standard curve, two important values:limit of detection as well as limit of quantification were estimated, the results indicated that the Taqman MGB Real time PCR had good linear range (1×101copies/μl---1×106copies/μL R2=0.993), great repeatability, and very sensitive with limit of detection was2copies/μl, limit of quantification was10copies/μl. We concluded that the Taqman MGB Real time PCR was a rapid and sensitive method which was available not only for detection but also for quantification, it may play an important role in detecting and monitoring Mg prevalence in high risk populations.
     Section2:High prevalence and risk factors for Mycoplasma genitalium among female sex workers and male STD clinic patients in Guangxi, China.
     The study populations included females who were older than18years and reported having provided commercial sex in the past year and male adults who attended in STD clinics. From July to September2009,810venue-based FSWs were recruited in two cities, Wuzhou and Hezhou,423male STD clinic patients were recruited in Hezhou of Guangxi Autonomous Region. After informed content was obtained, information regarding socio-demographic and behavioral characteristics was collected by a questionnaire-based interview by outreach workers. Cervical swab specimens and urine specimens were collected from female sex workers and male STD clinic patients respectively. Mg was detected by using a real time polymerase chain reaction assay targeting the mgpA gene. The overall prevalence of Mg infection among810FSWs was13.2%(95%CI=10.87%-15.52%). Mg infection rates among high-tier FSWs, middle-tier FSWs and low-tier FSWs were15.7%(95%CI=13.12%-18.2%),10.8%(95%CI=8.67%-12.93%), and14.6%(95%CI=12.17%-17.03%), respectively. Mg infection was only independently associated with less education (AOR=2.36,95% CI1.15-4.87), being single (AOR=2.27,95% CI=1.42-3.62), migrant backgrounds (AOR=2.03,95% CI=1.29-3.20), and absence of STIs symptoms in the previous year (AOR=1.66,95%CI=1.09-2.52). For MSP, Mg infection rate was28%(95%CI=25.50%-30.49%). Mg infection related to living alone (AOR=3.84,95%CI=1.58-9.33), ever had sex with FSWs within3months (AOR=2.27,95%CI=1.41-3.67), and absence of STIs symptoms in the previous year (AOR=2.21,95%CI=1.35-3.62) among MSP. In this study, the overall prevalence of Mg infection was found to be substantial among female sex workers as well as male STD clinic patients in study areas. These findings highlight the needs for increased attention towards prevention and intervention of Mg infection among these two high risk populations. Routine screening, timely diagnosis, and treatment of Mg infection should be considered in comprehensive HIV/STIs control program in China.
     Section3:The prevalence of urethral Mycoplasma genitalium and its associated factors among men who have sex with men in Guangzhou, China
     Men who have sex with men from different venues including:gar bars, sauna, night clubs and parks were recruited by snowball method in Guangzhou, China, during Jan to May2010. Socio-demographic and behavioral characteristics information was collected by a questionnaire-based interview by outreach workers. Urine specimens were collected. Mg was detected by a real time PCR targeting the MgPa gene. Among328MSM, majority of study subjects (97.6%) were self-identified as gay, however, among them41.8%also had sex with women,16.8%of study subjects reported had sex with women within the previous6months,27.3%used condom during the last intercourse with women. Over half (58.8%) of study subjects only had sex with men, majority (87.8%) of study subjects had sex with men within the previous6months, and63.2%used condom during the last anal sex. Out of all MSM,79.0%solicited sex partners by internet.323urine specimens were collected, Mg infection rate was4.3%(95%CI=3.1%-5.4%). These findings indicate that Mg is relatively low prevalent among MSM, which suggests that Mg infection mainly by heterosexual transmission other than homosexual transmission, however MSM as source of Mg infection should not be neglected.
引文
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