本文選題：泌尿生殖道　切入點：衣原體　出處：《北京協(xié)和醫(yī)學院》2016年博士論文　論文類型：學位論文

【摘要】：第一部分江蘇和廣西性病門診患者的泌尿生殖道沙眼衣原體高分辨多位點測序分型研究生殖道沙眼衣原體是全球最為常見的性傳播疾病之一,2008年WHO估計成人新發(fā)感染生殖道沙眼衣原體的人數(shù)達1億左右。傳統(tǒng)的基因分型方法分型能力有限,而多位點序列分型(multi-locus sequence typing, MLST)是一種基于核苷酸序列測定的細菌分型方法,該方法具有較高的分辨能力,既適于分子流行病學研究,也可用于分子進化學的研究。為了豐富我國沙眼衣原體分子生物學的遺傳背景信息,了解流行的分子學特征,本研究在廣西和江蘇地區(qū)收集了99例沙眼衣原體感染患者和16例隨訪患者的生殖道分泌物標本以及其相關流行病學資料。利用QIAxtractor全自動核酸純化儀對收集的標本進行DNA提取和沙眼衣原體淋球菌的實時定量PCR的檢測,沙眼衣原體核酸檢測的標本經(jīng)巢式PCR法擴增沙眼衣原體主要外膜蛋白(outer membrane protien, omp) ompA基因和MLST的CT046 (hctB)、CT058、CT144、CT172和CT682 (pbpB)五個基因。擴增產(chǎn)物經(jīng)正反雙向測序后,通過序列比對和型別分析獲得菌株的ompA基因型別及MLST型別。利用BioNumerics7軟件對菌株MLST的信息進行最小生成樹分析,比較來源于不同地區(qū)的菌株的差異。同時利用卡方檢驗探討菌株型別與其臨床表現(xiàn)的關系、菌株型別與淋球菌共感染的關系。結果顯示：90例成功分型的菌株中共含7種ompA基因型和26種MLST型別,其中5種序列型別(Sequence type, ST)型別為首次報道。江蘇和廣西兩個地區(qū)沙眼衣原體的ST差異較小；但與意大利的菌株的差異卻較大。6例隨訪陽性患者的標本經(jīng)MLST分型后,3例被鑒定為的新發(fā)感染。統(tǒng)計分析后顯示沙眼衣原體菌株型別與淋球菌共感染情況存在相關性、不同菌株型別與不同臨床癥狀間存在相關性：J型的沙眼衣原體合并淋球菌感染的比率顯著低于其他型別,合并淋球菌感染的比率較高的分別為：E-3和F-110；去除淋球菌感染對臨床癥狀的影響后,引起分泌物異常的菌株主要為J和E,引起尿道不適癥狀則主要是E和F,而J型菌株引起尿道不適癥狀比例顯著低于其他型別菌株。此外易引起分泌異常型別是E-3和J-264；易引起尿道不適癥狀的ompA-MLST型別是：D-35,F-110及E-3,但這還需要大量的數(shù)據(jù)進行驗證,從而更好的指導臨床。我國流行的沙眼衣原體ST型別與意大利流行的不同,隨訪陽性患者的標本中絕大多數(shù)為新發(fā)感染。不同ST型別的沙眼衣原體菌株與淋球菌共感染及其引起的臨床癥狀不同。第二部分沙眼衣原體新型變種在我國華南地區(qū)暗娼人群中的流行現(xiàn)狀調查及Abbott m2000 CT/NG檢測性能的評估2006年,瑞典科學家發(fā)現(xiàn)了可引起生殖道感染的沙眼衣原體新變種(new variant of chlamydia trachomatis, nvCT),該變種菌株的隱蔽質粒中缺失了377bp長片段,而當時的羅氏和雅培診斷試劑選擇的目標片段正是這一缺失的片段,從而導致了nvCT能夠在瑞典迅速傳播。我國使用核酸試劑一直是羅氏的診斷試劑,因此國內(nèi)nvCT的流行情況及其對我國公共衛(wèi)生的影響也尚不清楚。本研究旨在了解我國nvCT在女性性工作者(Female sexual workers, FSWs)人群中流行現(xiàn)狀,同時了解Abbott m2000 CT/NG核酸檢測試對該人群生殖道感染沙眼衣原體和淋球菌的檢測能力。在廣東和海南地區(qū)收集997例FSWs的宮頸拭子,同時利用Abbott m2000實時定量CT/NG全自動檢測平臺和羅氏Amplicor CT/NG檢測試劑進行沙眼衣原體和淋球菌的檢測,不一致的結果利用Qiagen care CT PCR試劑進行確認。羅氏檢測沙眼衣原體為陰性,而雅培檢測為陽性的標本,則懷疑為nvCT,并將該菌株的DNA標本進行測序鑒定,以確定其是否為nvCT。結果顯示：在該人群中沒有發(fā)現(xiàn)可能為沙眼衣原體新型變種的菌株存在。共有25份標本的沙眼衣原體和26份淋球菌的結果需要進行Qiagen試劑的確證。Abbott m2000實時定量PCR試劑對沙眼衣原體的敏感性和特異性分別為92.59%和100%,淋球菌的則為95.45%和99.90%。在暗娼人群中對沙眼衣原體的陽性預測值和陰性預測值分別為100%和98.52%,淋球菌的則為95.5%和99.90%。因此目前尚沒有證據(jù)顯示在中國廣東和海南地區(qū)的暗娼人群中有nvCT的流行。.Abbott m2000實時定量PCR試劑對沙眼衣原體和淋球菌的檢測特異性高,但是靈敏度低于Roche Cobas Amplicor核酸試劑。Abbott m2000實時定量PCR試劑在暗娼人群中對淋球菌檢測有著較高的陽性預測值,這提示該試劑能夠適用在淋球菌流行率較低的人群中。第三部分我國生殖道沙眼衣原體檢測現(xiàn)狀分析為了了解我國沙眼衣原體的檢測能力及其檢測情況,本研究通過對2015年度270家參評單位的沙眼衣原體質控的結果進行分析,評價我國檢測沙眼衣原體的能力；并選擇5個省共36家醫(yī)療單位進行沙眼衣原體檢測現(xiàn)狀調查,了解我國檢測沙眼衣原體的情況。結果顯示：共214家單位使用了快速免疫色譜法(Immunochromatographic test, ICT)進行沙眼衣原體的檢測,占反饋結果單位的79.3%；36家調研的醫(yī)療單位中共有4家單位沒有開展沙眼衣原體的檢測,共27家開展的是沙眼衣原體快速免疫色譜法,占84.4%；僅有5家使用的是核酸檢測。ICT的方法在進行弱陽性標本檢測26.64%,6家醫(yī)療單位ICT檢測陽性率(1.22%)明顯低于核酸檢測的陽性率(6.16%)。23家綜合醫(yī)院1-6月份沙眼衣原體的檢測量為34462例,9家婦幼保健醫(yī)院48219例。婦幼系統(tǒng)對目標人群的篩查高于綜合性醫(yī)院。我國檢測生殖道沙眼衣原體感染的檢測手段目前仍主要是ICT檢測,但該方法的靈敏性太低,不足以發(fā)現(xiàn)所有待檢標本中陽性感染,因此我國實驗室所采用的實驗方法對沙眼衣原體的檢測的能力有限。此外婦幼系統(tǒng)對目標人群沙眼衣原體篩查力度大于綜合性醫(yī)療機構,在綜合性醫(yī)療機構篩查力度有待進一步的加強。我國沙眼衣原體真實的流行態(tài)勢不容樂觀。
[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.

【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R691.3

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2 王千秋;泌尿生殖道沙眼衣原體感染的某些血清學和分子生物學研究[D];中國協(xié)和醫(yī)科大學;1997年

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7 王蕊;HaCaT細胞培養(yǎng)沙眼衣原體及體外藥敏試驗的探索[D];天津醫(yī)科大學;2012年

8 白光春;沙眼衣原體重組口服活疫苗的構建及其免疫學特性的初步研究[D];第四軍醫(yī)大學;1999年

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1 汪咪珍;呼吸道感染患兒沙眼衣原體檢測與基因分型[D];浙江大學;2015年

2 高磊瓊;沙眼衣原體質粒致病機制分析[D];重慶醫(yī)科大學;2015年

3 尤聰;沙眼衣原體培養(yǎng)陽性率提高及臨床株熱休克蛋白60的轉錄隨傳代的變化[D];天津醫(yī)科大學;2012年

4 邵麗麗;沙眼衣原體抗菌素耐藥及治療失敗的評估[D];天津醫(yī)科大學;2010年

5 朱輝;大環(huán)內(nèi)酯類藥物次抑菌濃度誘導沙眼衣原體耐藥的實驗研究[D];天津醫(yī)科大學;2010年

6 侯淑萍;天津市耐藥沙眼衣原體野生株的細胞學及基因學研究[D];天津醫(yī)科大學;2004年

7 姜慧芬;沙眼衣原體主要外膜蛋白重組質粒的構建、表達和活性鑒定[D];浙江大學;2010年

8 陳偉;沙眼衣原體泌尿生殖道感染遷移性臨床研究[D];天津醫(yī)科大學;2007年

9 展小飛;泌尿生殖道沙眼衣原體的隨訪和治療方法的評估及病程與維生素D3水平的聯(lián)系[D];天津醫(yī)科大學;2012年

10 布曉坤;中性粒細胞在沙眼衣原體小鼠肺炎易感性差異免疫學機制的研究[D];天津醫(yī)科大學;2008年

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