泌尿生殖道沙眼衣原体多位点测序分型研究和新型变种流行现状研究

发布时间：2018-03-17 00:17

本文选题：泌尿生殖道　切入点：衣原体　出处：《北京协和医学院》2016年博士论文　论文类型：学位论文

【摘要】：第一部分江苏和广西性病门诊患者的泌尿生殖道沙眼衣原体高分辨多位点测序分型研究生殖道沙眼衣原体是全球最为常见的性传播疾病之一,2008年WHO估计成人新发感染生殖道沙眼衣原体的人数达1亿左右。传统的基因分型方法分型能力有限,而多位点序列分型(multi-locus sequence typing, MLST)是一种基于核苷酸序列测定的细菌分型方法,该方法具有较高的分辨能力,既适于分子流行病学研究,也可用于分子进化学的研究。为了丰富我国沙眼衣原体分子生物学的遗传背景信息,了解流行的分子学特征,本研究在广西和江苏地区收集了99例沙眼衣原体感染患者和16例随访患者的生殖道分泌物标本以及其相关流行病学资料。利用QIAxtractor全自动核酸纯化仪对收集的标本进行DNA提取和沙眼衣原体淋球菌的实时定量PCR的检测,沙眼衣原体核酸检测的标本经巢式PCR法扩增沙眼衣原体主要外膜蛋白(outer membrane protien, omp) ompA基因和MLST的CT046 (hctB)、CT058、CT144、CT172和CT682 (pbpB)五个基因。扩增产物经正反双向测序后,通过序列比对和型别分析获得菌株的ompA基因型别及MLST型别。利用BioNumerics7软件对菌株MLST的信息进行最小生成树分析,比较来源于不同地区的菌株的差异。同时利用卡方检验探讨菌株型别与其临床表现的关系、菌株型别与淋球菌共感染的关系。结果显示：90例成功分型的菌株中共含7种ompA基因型和26种MLST型别,其中5种序列型别(Sequence type, ST)型别为首次报道。江苏和广西两个地区沙眼衣原体的ST差异较小；但与意大利的菌株的差异却较大。6例随访阳性患者的标本经MLST分型后,3例被鉴定为的新发感染。统计分析后显示沙眼衣原体菌株型别与淋球菌共感染情况存在相关性、不同菌株型别与不同临床症状间存在相关性：J型的沙眼衣原体合并淋球菌感染的比率显著低于其他型别,合并淋球菌感染的比率较高的分别为：E-3和F-110；去除淋球菌感染对临床症状的影响后,引起分泌物异常的菌株主要为J和E,引起尿道不适症状则主要是E和F,而J型菌株引起尿道不适症状比例显著低于其他型别菌株。此外易引起分泌异常型别是E-3和J-264；易引起尿道不适症状的ompA-MLST型别是：D-35,F-110及E-3,但这还需要大量的数据进行验证,从而更好的指导临床。我国流行的沙眼衣原体ST型别与意大利流行的不同,随访阳性患者的标本中绝大多数为新发感染。不同ST型别的沙眼衣原体菌株与淋球菌共感染及其引起的临床症状不同。第二部分沙眼衣原体新型变种在我国华南地区暗娼人群中的流行现状调查及Abbott m2000 CT/NG检测性能的评估2006年,瑞典科学家发现了可引起生殖道感染的沙眼衣原体新变种(new variant of chlamydia trachomatis, nvCT),该变种菌株的隐蔽质粒中缺失了377bp长片段,而当时的罗氏和雅培诊断试剂选择的目标片段正是这一缺失的片段,从而导致了nvCT能够在瑞典迅速传播。我国使用核酸试剂一直是罗氏的诊断试剂,因此国内nvCT的流行情况及其对我国公共卫生的影响也尚不清楚。本研究旨在了解我国nvCT在女性性工作者(Female sexual workers, FSWs)人群中流行现状,同时了解Abbott m2000 CT/NG核酸检测试对该人群生殖道感染沙眼衣原体和淋球菌的检测能力。在广东和海南地区收集997例FSWs的宫颈拭子,同时利用Abbott m2000实时定量CT/NG全自动检测平台和罗氏Amplicor CT/NG检测试剂进行沙眼衣原体和淋球菌的检测,不一致的结果利用Qiagen care CT PCR试剂进行确认。罗氏检测沙眼衣原体为阴性,而雅培检测为阳性的标本,则怀疑为nvCT,并将该菌株的DNA标本进行测序鉴定,以确定其是否为nvCT。结果显示：在该人群中没有发现可能为沙眼衣原体新型变种的菌株存在。共有25份标本的沙眼衣原体和26份淋球菌的结果需要进行Qiagen试剂的确证。Abbott m2000实时定量PCR试剂对沙眼衣原体的敏感性和特异性分别为92.59%和100%,淋球菌的则为95.45%和99.90%。在暗娼人群中对沙眼衣原体的阳性预测值和阴性预测值分别为100%和98.52%,淋球菌的则为95.5%和99.90%。因此目前尚没有证据显示在中国广东和海南地区的暗娼人群中有nvCT的流行。.Abbott m2000实时定量PCR试剂对沙眼衣原体和淋球菌的检测特异性高,但是灵敏度低于Roche Cobas Amplicor核酸试剂。Abbott m2000实时定量PCR试剂在暗娼人群中对淋球菌检测有着较高的阳性预测值,这提示该试剂能够适用在淋球菌流行率较低的人群中。第三部分我国生殖道沙眼衣原体检测现状分析为了了解我国沙眼衣原体的检测能力及其检测情况,本研究通过对2015年度270家参评单位的沙眼衣原体质控的结果进行分析,评价我国检测沙眼衣原体的能力；并选择5个省共36家医疗单位进行沙眼衣原体检测现状调查,了解我国检测沙眼衣原体的情况。结果显示：共214家单位使用了快速免疫色谱法(Immunochromatographic test, ICT)进行沙眼衣原体的检测,占反馈结果单位的79.3%；36家调研的医疗单位中共有4家单位没有开展沙眼衣原体的检测,共27家开展的是沙眼衣原体快速免疫色谱法,占84.4%；仅有5家使用的是核酸检测。ICT的方法在进行弱阳性标本检测26.64%,6家医疗单位ICT检测阳性率(1.22%)明显低于核酸检测的阳性率(6.16%)。23家综合医院1-6月份沙眼衣原体的检测量为34462例,9家妇幼保健医院48219例。妇幼系统对目标人群的筛查高于综合性医院。我国检测生殖道沙眼衣原体感染的检测手段目前仍主要是ICT检测,但该方法的灵敏性太低,不足以发现所有待检标本中阳性感染,因此我国实验室所采用的实验方法对沙眼衣原体的检测的能力有限。此外妇幼系统对目标人群沙眼衣原体筛查力度大于综合性医疗机构,在综合性医疗机构筛查力度有待进一步的加强。我国沙眼衣原体真实的流行态势不容乐观。
[Abstract]:The first part of Jiangsu and Guangxi among STD clinic patients of urogenital Chlamydia trachomatis high-resolution multilocus sequence typing study of Chlamydia trachomatis is one of the most common sexually transmitted disease worldwide, 2008 WHO estimates the number of adult new infections of Chlamydia trachomatis was about 100 million. The traditional gene typing method limited however, multilocus sequence typing (multi-locus sequence, typing, MLST) is a bacterial nucleotide sequencing based typing methods, this method has high resolution and is suitable for the study of molecular epidemiology, can also be used to study the molecular evolutionary genetic background information. In order to enrich our understanding of the molecular biology of Chlamydia trachomatis. The molecular characteristics of popular, this study in Guangxi and Jiangsu area collected 99 cases of Chlamydia trachomatis infection patients and 16 patients with genital tract secretion Samples and related epidemiological data. By using real time quantitative PCR detection of QIAxtractor automatic nucleic acid purification samples for DNA extraction and Chlamydia trachomatis, Neisseria gonorrhoeae, Chlamydia trachomatis nucleic acid detection specimens by nested PCR amplification of Chlamydia trachomatis major outer membrane protein (outer membrane protien, OMP ompA and MLST CT046 gene) (hctB), CT058, CT144, CT172 and CT682 (pbpB) five gene. PCR products by bidirectional sequencing, sequence alignment and analysis by type strains of ompA genotype and MLST type analysis. The minimum spanning tree using BioNumerics7 software information of strain MLST, differences between the strains from different areas at the same time. The relationship between strain types and clinical manifestations of the chi square test, the relationship between strain types and gonococcal infection. The results showed that 90 cases of success The type of strains containing 7 ompA genotypes and 26 MLST genotypes, including 5 kinds of sequence types (Sequence type, ST) were reported for the first time. The smaller the ST differences between Jiangsu and Guangxi two regions of Chlamydia trachomatis; but the difference with Italy strains were larger in.6 cases with positive specimens of MLST type, 3 cases were identified as new infections. Statistical analysis shows that Chlamydia trachomatis strains of Neisseria gonorrhoeae infection type and the correlation between the correlation between different strains and different types of clinical symptoms: ratio of type J of Chlamydia trachomatis with gonococcal infection was significantly lower than other type, with gonococcal infection rate high respectively: E-3 and F-110; the removal of gonococcal infection on clinical symptoms, caused by abnormal secretion was mainly J and E, cause urethral discomfort symptoms are mainly E and F, and J strains Urinary tract symptoms was significantly lower than other type strains. In addition can cause abnormal secretion of type is E-3 and J-264; easy to cause urethral discomfort symptoms of type ompA-MLST are: D-35, F-110 and E-3, but it also requires a large amount of data verification, and guide clinical better. China's popular Chlamydia trachomatis ST type with the Italy epidemic of different follow-up patients with positive specimens for the vast majority of new infections. Different type ST strains of Chlamydia trachomatis and Neisseria gonorrhoeae infection and the clinical symptoms caused by different model variants. The second part of Chlamydia trachomatis in China among the Southern China area in the investigation on the epidemic situation of Abbott and M2000 CT/NG detection performance evaluation in 2006, Swedish scientists found new variants of Chlamydia trachomatis can cause reproductive tract infection (new variant of Chlamydia trachomatis, nvCT), the variant strain The cryptic plasmid 377bp deletion in the long fragment, and the choice of Roche and Abbott diagnostics was the target fragment is the deletion fragment, resulting in nvCT can spread rapidly in Sweden. China's use of nucleic acid reagents have been Roche diagnostic reagents, the epidemic situation of domestic nvCT and its influence on public health the country is also unclear. The purpose of this study is to understand China's nvCT among female sex workers (Female sexual, workers, FSWs) the current population, and understand the Abbott M2000 CT/NG nucleic acid detection testing ability of the population trachoma chlamydial and gonococcal genitourinary tract infection. 997 cases of FSWs patients were collected in Guangdong and Hainan area cervical swab test, using Abbott M2000 automatic detection platform for real-time CT/NG and Roche Amplicor CT/NG kit for Chlamydia trachomatis and Neisseria gonorrhoeae, the result is not consistent Care CT PCR was confirmed by Qiagen reagent. Roche detection of Chlamydia trachomatis was negative, while Abbott test positive specimens, is suspected to be nvCT, and the sequencing of the strain DNA were to determine whether the nvCT. results show that did not find the existence of possible for new varieties of Chlamydia trachomatis strains in the crowd the sensitivity and specificity. A total of 25 specimens of Chlamydia trachomatis and Neisseria gonorrhoeae 26 results need Qiagen reagent confirmed.Abbott M2000 real-time quantitative PCR reagent for Chlamydia trachomatis were 92.59% and 100%, gonococcus is 95.45% and 99.90%. in FSWs positive predictive value and negative for Chlamydia trachomatis the prediction values were 100% and 98.52%, Neisseria gonorrhoeae was 95.5% and 99.90%. so there is no evidence of a nvCT in Guangdong and Hainan area Chinese FSWs pop ..Abbott M2000 real time quantitative PCR reagent for Chlamydia trachomatis and Neisseria gonorrhoeae detection with high specificity, but the sensitivity is lower than the Roche Cobas Amplicor.Abbott M2000 PCR real time quantitative nucleic acid reagents reagents in FSWs for detection of Neisseria gonorrhoeae has a high positive predictive value, suggesting that this reagent can be used in the low prevalence of Neisseria gonorrhoeae in the crowd the third part detection. The status quo of China's genital Chlamydia trachomatis detection analysis in order to understand our ability of Chlamydia trachomatis and detection, the results of this study by Chlamydia trachomatis quality control for the year 2015 270 participating units were analyzed to evaluate the ability of detection of Chlamydia trachomatis in China; and a total of 5 provinces, 36 medical units survey the present condition of the detection of Chlamydia trachomatis, understand the detection of Chlamydia trachomatis in China. Results: a total of 214 units using a rapid immune color Spectrum method (Immunochromatographic test, ICT) for detection of Chlamydia trachomatis, accounting for 79.3% of 36 units of feedback; research the medical units in a total of 4 units did not carry out the detection of Chlamydia trachomatis, a total of 27 to carry out the Chlamydia rapid immunochromatographic assay, accounting for 84.4%; only 5 are using the method of nucleic acid detection.ICT in weak positive samples 26.64%, the positive rate of medical unit 6 ICT detection (1.22%) was significantly lower than the positive rate of nucleic acid detection (6.16%) detected.23 in 1-6 general hospitals in Chlamydia trachomatis in 34462 cases, 9 cases of maternal and child health hospital 48219. Maternal and child screening system of the target population is higher than that of our hospital. Detection of genital Chlamydia trachomatis infection is still the main means of detection is ICT detection, but the sensitivity of the method is too low, not enough to find all the detected specimen in the positive, so I The ability of detection of Chlamydia trachomatis in the laboratory experiment method is limited. In addition the system is greater than the maternal and child comprehensive medical institutions for the target population of chlamydia screening efforts in comprehensive medical screening efforts need to be further strengthened. The epidemic situation of China's real Chlamydia trachomatis is not optimistic.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R691.3

【相似文献】