早期梅毒的分子流行病学研究：我国多地区性病门诊的横断面调查
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摘要
梅毒是世界范围内常见的性传播疾病之一,据WHO (World Health Organization,世界卫生组织)最新估计全球每年新发梅毒患者1060万例。梅毒对人类的危害和致病性一直备受关注,其病原体T.pallidum (Treponema pallidum,梅毒螺旋体)可侵犯皮肤黏膜、心血管、神经、骨骼等造成多系统损害；梅毒能促进HIV的感染和传播,加速艾滋病的病程；感染梅毒的妊娠期妇女可发生流产、死胎、早产,以及分娩低出生体重儿和先天梅毒儿等。我国于1964年基本消灭了梅毒,但自从20世纪70年代末期实行对外开放政策和经济体制改革以来,由于大量农村人口向城市流动,以及卖淫嫖娼等社会现象的出现,各种性病特别是梅毒又死灰复燃并迅速蔓延。据全国性病疫情监测系统统计,我国梅毒发病率自2003年至今呈显著增长趋势,2011年的梅毒报告发病率高达32.04/10万,比2010年增长10.89%,梅毒发病人数居全国乙类传染病第三位。开展梅毒发病、流行和预防控制相关领域的研究已经成为我国性病防治研究的重要内容。
T. pallidum无法在体外成功培养为梅毒的研究带来一定的困难。20世纪90年代,Nichols株基因组全部碱基对的成功破解使得从基因分子生物学的角度研究梅毒成为可能。分子流行病学是传统流行病学与分子生物学交叉融合的产物,是研究传染性疾病流行特征的有力工具。其应用先进的技术测量人群中特异性分子生物学标志的分布情况,并结合流行病学的现场研究方法探讨疾病的流行特征。基因分型系统是开展梅毒分子流行病学研究的重要方法之一,能从微观层面区分不同的菌株,从而有助于探讨传播的网络关系、追溯传染源、区分复发和再感染,同时可以研究基因型别与临床特征之间的联系,还有助于更好的研究梅毒在不同的时间、空间和人群中的流行特点。1998年,美国CDC (Center for Disease Control and Prevention,疾病预防控制中心)建立了基于arp和tprⅡ亚家族的双基因分型系统。2010年,美国华盛顿大学的梅毒研究团队又在此基础上引入tp0548基因,进一步提高了分型的区分度。
梅毒分子流行病学的另一个重要方面是通过对基因突变的检测监测T. pallidum对大环内酯类药物(如阿奇霉素)耐药的状况。由于阿奇霉素具有单剂量口服治疗梅毒且不会导致青霉素所致的过敏反应,现场防治和性伴治疗时方便快捷、依从性好等特点,同时在部分国家开展的随机对照临床试验证实其疗效和青霉素相当,因此单剂量阿奇霉素在梅毒治疗中的应用受到广泛关注。然而,近年来在美国等发达国家不断有临床使用阿奇霉素治疗梅毒失败(耐药)的报道,并且这种耐药性正在一定的性网络中传播,有逐年上升的趋势。已有分子生物学研究证实耐阿奇霉素菌株的23SrRNA基因第2058位点的碱基发生A至G的点突变。在特定的时间、区域和人群中用该分子生物学标志监测耐药菌株的流行情况,不仅可以了解阿奇霉素耐药的状况,而且有助于指导区域特异性的梅毒治疗,特别是梅毒防控的现场治疗。
目前,我国湖南、广州、江门和上海有关于双基因定位分型的研究报道；同时上海有关于阿奇霉素耐药性突变监测的报道,但均是小范围研究且样本量小。我国是世界上梅毒疫情严重的国家之一,尚未有多地区较大规模的梅毒分子流行病学研究,尚不了解T. pallidum基因型别的分布特点、阿奇霉素耐药性突变的总体流行情况以及对防治实践的影响。
本研究分为三个部分。第一部分,世界范围内梅毒分子流行病学的系统综述和meta分析：本部分在5个数据库中全面检索自1998年10月17日首次报道梅毒基因分型的研究以来截至2010年12月31日所有已发表的梅毒分子流行病学研究,共16篇,并给予系统评价。结果表明早期梅毒的皮损标本,如硬下疳、扁平湿疣、湿丘疹或粘膜斑,最适合用于分子诊断和分子流行病学调查。耳垂刮出血有望成为研究无明显皮损的梅毒病例的首选标本类型,但尚需大样本研究加以证实。世界范围内T. pallidum的基因型别存在高度的多样性,其中14d、14f、14a、13d和15d最为流行。基因型别和阿奇霉素耐药性突变以及神经梅毒之间联系的相关资料比较有限。第二部分,梅毒的分子诊断、基因分型和阿奇霉素耐药性突变检测的实验室方法学建立：本部分在第一部分系统比较国外各个研究的实验室方法的基础上,结合中国医学科学院皮肤病研究所性病控制中心STD (Sexually transmitted diseases,性传播疾病)参比实验室的科研条件,建立基于polA基因的梅毒分子诊断的实验室方法。并在此基础上,建立基于arp、tprⅡ和tp0548基因的分型系统,以及基于23SrRNA基因的阿奇霉素耐药性突变的检测方法。第三部分,我国多地区STD门诊早期梅毒分子流行病学的初步研究：本部分将第二部分建立的实验室方法初步用于我国梅毒分子流行病学研究。在2008至2011年间,我们在我国东部、南部和北部三大地区的8个STD门诊共招募391例早期梅毒患者,在知情同意的前提下每人完成一份结构式问卷调查表,并采集一份皮损标本。其中211例标本能进行三基因完全分型和阿奇霉素耐药性突变的检测。总计发现27种基因型别,其中14d/f占39.8%,具有绝对的流行优势。三大地区基因型别分布存在显著性差异(χ2=18.1,p=0.021)。91.9%的临床菌株发生阿奇霉素耐药性突变,并且与大环内酯类抗生素用药史密切相关(χ2=37.5,p<0.001)
Syphilis is one of the common sexually transmitted diseases in the world. The World Health Organization recently estimated10.6million new cases of infectious syphilis each year. Treponema pallidum (T. pallidum), the pathogen of syphilis, can enter skin mucosa, cardiovascular system, nerve and bone. Syphilis infection can facilitate the transmission of HIV infection and the adverse development of AIDS. Syphilis can also increase the risk of adverse pregnancy outcomes, including abortion, stillbirth, premature delivery, low birth weight and congenital syphilis. China eliminated syphilis in1964, but syphilis resurged in late1970s after implementation of reform and opening policies because of rural-to-urban migration, higher-risk behaviors, and sexual networks of heterosexual, homosexual and bisexual contacts. According to surveillance results from the National Center for STD Control and Prevention, syphilis incidence has been increasing sharply since2003. In2011, syphilis incidence was as high as32.04per100,000persons, which was10.89%higher than that in2010. The annual number of new syphilis cases ranks third out of all categories B infectious diseases nationally, following tuberculosis and hepatitis B. It is very important to study the epidemiology, control and prevention of syphilis to decrease the transmission of syphilis in China.
T. pallidum cannot be cultured in vitro, which makes syphilis research difficult. Since the1990s, it has been possible to investigate the molecular biology of syphilis due to the complete genome sequence determination of the Nichols strain. Molecular epidemiology is the integration of molecular biology with traditional epidemiologic research and a powerful tool for determining diversity and epidemiology of infectious diseases using molecular biomarkers. Molecular typing of T. pallidum clincal strains is an important aspect of the molecular epidemiology of syphilis. It helps to characterize syphilis transmission networks, trace infectious sources, differentiate between relapse and re-infection episodes, evaluate subtypes associated with clinical characteristics, and better understand the spatial, temporal and population distributions of T. pallidum. In1998, the first molecular typing method was introduced by the United States Centers for Disease Control and Prevention and is based on the interstrain variability of the acidic repeat protein gene and the T. pallidum repeat gene subfamily Ⅱ. Moreover, a recent study developed a third gene named tp0548with a better discriminatory typing power in2010at the University of Washington.
Another important aspect of the molecular epidemiology of syphilis is monitoring macrolide antibiotic resistance, such as azithromycin. The use of azithromycin for the treatment of early syphilis offers certain advantages over conventional intramuscular therapy with benzathine penicillin G, including oral route of delivery with single-dose, no risk of anaphylactic shock, the ability to treat in the field and the convenience of partner-delivery therapy. The efficacy, safety and ease of azithromycin usage were verified by randomized controlled clinical trials. However, azithromycin treatment failures for early syphilis have been documented in multiple geographic locations in developed countries. Moreover, azithromycin resistance has been increasing each year and transmitting in certain sexual networks in these developed countries. Biological research has demonstrated that a single mutation from adenine to guanine in locus2058of the23SrRNA gene confers azithromycin resistance. Monitoring resistant strains helps to guide syphilis treatment in a defined time, space and population using the23SrRNA gene as a molecular biomarker.
In China, four small-scale molecular typing studies, which were based on the arp and tprⅡ genes, have described the subtype distributions of T. pallidum in South Hunan, Guangzhou, Jiangmen and Shanghai. Shanghai has also reported the epidemiology of azithromycin resistant strains. China is a huge country with the severe resurgence of syphilis. The strain type distribution of T. pallidum and epidemiology of azithromycin resistance are still not well known due to the absence of molecular epidemiology data across multiple geographic areas.
There are three sections for this study. Section one is a systematic review and meta-analysis of the molecular epidemiology of syphilis globally. A total of16published studies were included in five databases from Oct.17th1998to Dec.31st2010. The main results based on systematic analyses were as following:(1) primary lesions, such as genital ulcer, condyloma lata, papulae and mucosal patches, were better specimens for the molecular diagnosis and epidemiologic study of syphilis;(2) ear lobe scraping was a promising specimen type when no visible skin lesions existed, but it needs to be validated by more research with larger sample sizes;(3) there was substantial genetic diversity of T. pallidum globally and14d,14f,14a,13d and15d were the most prevalent subtypes in descending order;(4) subtype data associated with azithromycin resistance and neurosyphilis were limited.
Section two discusses the establishment of laboratory methods for molecular diagnosis, genotyping and azithromycin-resistant mutant identification for syphilis. This section described a detailed methodology established for syphilis molecular epidemiology research in the STD reference laboratory of the National Center for STD Control and Prevention based on the systematic review of laboratory methods in the first section. Positive DNA was first screened by a diagnostic PCR assay of polA gene, which is unique to T. pallidum. Molecular typing was based on the interstrain variability of the arp, tpr subfamily Ⅱ and tp0548genes. Azithromycin resistance identification was based on a single mutation from adenine to guanine in locus2058of23SrRNA gene.
Section three is a preliminary molecular epidemiology study of early syphilis in STD clinics in multiple geographic areas of China. We conducted a cross-sectional study in STD clinics from eight cities located in East, South and North China during the years2008to2011. A total of391outpatients with early syphilis were recruited based on an oral informed consent. Each subject was interviewed using a short structured questionnaire and encouraged to provide a moist skin lesion for specimen collection.211clinical specimens could be fully molecular typed and identified azithromycin-resistant mutations. Overall,27strain types were identified.14d/f accounted for39.8%prevalence and was the predominant strain type across China. Strain type distribution was significantly different across geographic areas (χ2=18.1,p=0.021).91.9%clinical strains were azithromycin resistant, and resistant mutations were significantly associated with history of macrolide antibiotics use (χ2=37.5,p<0.001).
T. pallidum无法在体外成功培养为梅毒的研究带来一定的困难。20世纪90年代,Nichols株基因组全部碱基对的成功破解使得从基因分子生物学的角度研究梅毒成为可能。分子流行病学是传统流行病学与分子生物学交叉融合的产物,是研究传染性疾病流行特征的有力工具。其应用先进的技术测量人群中特异性分子生物学标志的分布情况,并结合流行病学的现场研究方法探讨疾病的流行特征。基因分型系统是开展梅毒分子流行病学研究的重要方法之一,能从微观层面区分不同的菌株,从而有助于探讨传播的网络关系、追溯传染源、区分复发和再感染,同时可以研究基因型别与临床特征之间的联系,还有助于更好的研究梅毒在不同的时间、空间和人群中的流行特点。1998年,美国CDC (Center for Disease Control and Prevention,疾病预防控制中心)建立了基于arp和tprⅡ亚家族的双基因分型系统。2010年,美国华盛顿大学的梅毒研究团队又在此基础上引入tp0548基因,进一步提高了分型的区分度。
梅毒分子流行病学的另一个重要方面是通过对基因突变的检测监测T. pallidum对大环内酯类药物(如阿奇霉素)耐药的状况。由于阿奇霉素具有单剂量口服治疗梅毒且不会导致青霉素所致的过敏反应,现场防治和性伴治疗时方便快捷、依从性好等特点,同时在部分国家开展的随机对照临床试验证实其疗效和青霉素相当,因此单剂量阿奇霉素在梅毒治疗中的应用受到广泛关注。然而,近年来在美国等发达国家不断有临床使用阿奇霉素治疗梅毒失败(耐药)的报道,并且这种耐药性正在一定的性网络中传播,有逐年上升的趋势。已有分子生物学研究证实耐阿奇霉素菌株的23SrRNA基因第2058位点的碱基发生A至G的点突变。在特定的时间、区域和人群中用该分子生物学标志监测耐药菌株的流行情况,不仅可以了解阿奇霉素耐药的状况,而且有助于指导区域特异性的梅毒治疗,特别是梅毒防控的现场治疗。
目前,我国湖南、广州、江门和上海有关于双基因定位分型的研究报道；同时上海有关于阿奇霉素耐药性突变监测的报道,但均是小范围研究且样本量小。我国是世界上梅毒疫情严重的国家之一,尚未有多地区较大规模的梅毒分子流行病学研究,尚不了解T. pallidum基因型别的分布特点、阿奇霉素耐药性突变的总体流行情况以及对防治实践的影响。
本研究分为三个部分。第一部分,世界范围内梅毒分子流行病学的系统综述和meta分析：本部分在5个数据库中全面检索自1998年10月17日首次报道梅毒基因分型的研究以来截至2010年12月31日所有已发表的梅毒分子流行病学研究,共16篇,并给予系统评价。结果表明早期梅毒的皮损标本,如硬下疳、扁平湿疣、湿丘疹或粘膜斑,最适合用于分子诊断和分子流行病学调查。耳垂刮出血有望成为研究无明显皮损的梅毒病例的首选标本类型,但尚需大样本研究加以证实。世界范围内T. pallidum的基因型别存在高度的多样性,其中14d、14f、14a、13d和15d最为流行。基因型别和阿奇霉素耐药性突变以及神经梅毒之间联系的相关资料比较有限。第二部分,梅毒的分子诊断、基因分型和阿奇霉素耐药性突变检测的实验室方法学建立：本部分在第一部分系统比较国外各个研究的实验室方法的基础上,结合中国医学科学院皮肤病研究所性病控制中心STD (Sexually transmitted diseases,性传播疾病)参比实验室的科研条件,建立基于polA基因的梅毒分子诊断的实验室方法。并在此基础上,建立基于arp、tprⅡ和tp0548基因的分型系统,以及基于23SrRNA基因的阿奇霉素耐药性突变的检测方法。第三部分,我国多地区STD门诊早期梅毒分子流行病学的初步研究：本部分将第二部分建立的实验室方法初步用于我国梅毒分子流行病学研究。在2008至2011年间,我们在我国东部、南部和北部三大地区的8个STD门诊共招募391例早期梅毒患者,在知情同意的前提下每人完成一份结构式问卷调查表,并采集一份皮损标本。其中211例标本能进行三基因完全分型和阿奇霉素耐药性突变的检测。总计发现27种基因型别,其中14d/f占39.8%,具有绝对的流行优势。三大地区基因型别分布存在显著性差异(χ2=18.1,p=0.021)。91.9%的临床菌株发生阿奇霉素耐药性突变,并且与大环内酯类抗生素用药史密切相关(χ2=37.5,p<0.001)
Syphilis is one of the common sexually transmitted diseases in the world. The World Health Organization recently estimated10.6million new cases of infectious syphilis each year. Treponema pallidum (T. pallidum), the pathogen of syphilis, can enter skin mucosa, cardiovascular system, nerve and bone. Syphilis infection can facilitate the transmission of HIV infection and the adverse development of AIDS. Syphilis can also increase the risk of adverse pregnancy outcomes, including abortion, stillbirth, premature delivery, low birth weight and congenital syphilis. China eliminated syphilis in1964, but syphilis resurged in late1970s after implementation of reform and opening policies because of rural-to-urban migration, higher-risk behaviors, and sexual networks of heterosexual, homosexual and bisexual contacts. According to surveillance results from the National Center for STD Control and Prevention, syphilis incidence has been increasing sharply since2003. In2011, syphilis incidence was as high as32.04per100,000persons, which was10.89%higher than that in2010. The annual number of new syphilis cases ranks third out of all categories B infectious diseases nationally, following tuberculosis and hepatitis B. It is very important to study the epidemiology, control and prevention of syphilis to decrease the transmission of syphilis in China.
T. pallidum cannot be cultured in vitro, which makes syphilis research difficult. Since the1990s, it has been possible to investigate the molecular biology of syphilis due to the complete genome sequence determination of the Nichols strain. Molecular epidemiology is the integration of molecular biology with traditional epidemiologic research and a powerful tool for determining diversity and epidemiology of infectious diseases using molecular biomarkers. Molecular typing of T. pallidum clincal strains is an important aspect of the molecular epidemiology of syphilis. It helps to characterize syphilis transmission networks, trace infectious sources, differentiate between relapse and re-infection episodes, evaluate subtypes associated with clinical characteristics, and better understand the spatial, temporal and population distributions of T. pallidum. In1998, the first molecular typing method was introduced by the United States Centers for Disease Control and Prevention and is based on the interstrain variability of the acidic repeat protein gene and the T. pallidum repeat gene subfamily Ⅱ. Moreover, a recent study developed a third gene named tp0548with a better discriminatory typing power in2010at the University of Washington.
Another important aspect of the molecular epidemiology of syphilis is monitoring macrolide antibiotic resistance, such as azithromycin. The use of azithromycin for the treatment of early syphilis offers certain advantages over conventional intramuscular therapy with benzathine penicillin G, including oral route of delivery with single-dose, no risk of anaphylactic shock, the ability to treat in the field and the convenience of partner-delivery therapy. The efficacy, safety and ease of azithromycin usage were verified by randomized controlled clinical trials. However, azithromycin treatment failures for early syphilis have been documented in multiple geographic locations in developed countries. Moreover, azithromycin resistance has been increasing each year and transmitting in certain sexual networks in these developed countries. Biological research has demonstrated that a single mutation from adenine to guanine in locus2058of the23SrRNA gene confers azithromycin resistance. Monitoring resistant strains helps to guide syphilis treatment in a defined time, space and population using the23SrRNA gene as a molecular biomarker.
In China, four small-scale molecular typing studies, which were based on the arp and tprⅡ genes, have described the subtype distributions of T. pallidum in South Hunan, Guangzhou, Jiangmen and Shanghai. Shanghai has also reported the epidemiology of azithromycin resistant strains. China is a huge country with the severe resurgence of syphilis. The strain type distribution of T. pallidum and epidemiology of azithromycin resistance are still not well known due to the absence of molecular epidemiology data across multiple geographic areas.
There are three sections for this study. Section one is a systematic review and meta-analysis of the molecular epidemiology of syphilis globally. A total of16published studies were included in five databases from Oct.17th1998to Dec.31st2010. The main results based on systematic analyses were as following:(1) primary lesions, such as genital ulcer, condyloma lata, papulae and mucosal patches, were better specimens for the molecular diagnosis and epidemiologic study of syphilis;(2) ear lobe scraping was a promising specimen type when no visible skin lesions existed, but it needs to be validated by more research with larger sample sizes;(3) there was substantial genetic diversity of T. pallidum globally and14d,14f,14a,13d and15d were the most prevalent subtypes in descending order;(4) subtype data associated with azithromycin resistance and neurosyphilis were limited.
Section two discusses the establishment of laboratory methods for molecular diagnosis, genotyping and azithromycin-resistant mutant identification for syphilis. This section described a detailed methodology established for syphilis molecular epidemiology research in the STD reference laboratory of the National Center for STD Control and Prevention based on the systematic review of laboratory methods in the first section. Positive DNA was first screened by a diagnostic PCR assay of polA gene, which is unique to T. pallidum. Molecular typing was based on the interstrain variability of the arp, tpr subfamily Ⅱ and tp0548genes. Azithromycin resistance identification was based on a single mutation from adenine to guanine in locus2058of23SrRNA gene.
Section three is a preliminary molecular epidemiology study of early syphilis in STD clinics in multiple geographic areas of China. We conducted a cross-sectional study in STD clinics from eight cities located in East, South and North China during the years2008to2011. A total of391outpatients with early syphilis were recruited based on an oral informed consent. Each subject was interviewed using a short structured questionnaire and encouraged to provide a moist skin lesion for specimen collection.211clinical specimens could be fully molecular typed and identified azithromycin-resistant mutations. Overall,27strain types were identified.14d/f accounted for39.8%prevalence and was the predominant strain type across China. Strain type distribution was significantly different across geographic areas (χ2=18.1,p=0.021).91.9%clinical strains were azithromycin resistant, and resistant mutations were significantly associated with history of macrolide antibiotics use (χ2=37.5,p<0.001).
引文
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