华东部分地区W-北京基因型结核分枝杆菌传播特征研究

发布时间：2018-06-12 13:02

本文选题：结核分枝杆菌 + 北京家族株　；参考：《复旦大学》2013年硕士论文

【摘要】：结核病是一种对人类健康和社会稳定造成严重威胁的传染病,中国是结核病高负担国家之一,尽管近些年来结核患病率有所下降,但我国的结核病疾病负担依然很严重。W-北京基因型结核分枝杆菌是一类由共同祖先进化而来、具有相似遗传背景的M.TB菌株,在世界各地均有不同水平的流行。W-北京家族菌株可能与结核耐药有关,能够引起暴发,形成流行优势。追踪传染源、阐明传播途径是结核病流行病学研究的两大问题,结核具有潜伏感染的特点,传统的流行病学方法并不能很好地阐述其传播机制,而以基因型分型技术为基础的结核分子流行病学在这方面有不可替代的优势。如与传统流行病学相结合,分子流行病学能更准确地预测疾病的流行、暴发,掌握其流行特征和传播模式。因此,本研究选取华东地区的阜宁县(江苏省)、德清县(江苏省)和鄞州区(浙江省)为研究现场,采用横断面调查和分子分型技术相结合的方法,描述华东地区W-北京基因型结核分枝杆菌的流行状况,分析影响W-北京株流行的社会人口学特征、临床治疗史、既往治疗史及人口流动等因素；确定菌株的VNTR-MIRU基因型,评估结核病的近期传播比例,探讨华东地区结核病的传播模式,为控制结核在人群中的传播提供线索；比较不同位点VNTR-MIRU的分型效果及应用前景,为完善结核分枝杆菌基因信息数据库提供资料。主要研究方法和结果如下： 1、W-北京基因型结核分枝杆菌的流行及影响因素研究研究收集三个现场2009年6月/7月-2010年11月/12月在各区县疾控中心登记的痰培养阳性结核患者基本资料,收集患者的痰标本进行菌株培养、提取DNA,采用横断面研究结合DTM-PCR分型的方法,识别W-北京家族菌株及现代型W-北京株。研究共纳入346例结核患者的M.TB菌株,经RD105分型,90%(311株)属于北京家族,阜宁、德清和鄞州的W-北京株检出比例分别为91%(J64株)、96%(23株)和87%(124株),差别没有统计学意义。W-北京株经RD181进一步分型,分别获得现代型W-北京株294株(77%)、古典型W-北京株90株(23%)。分析年龄、健康状况(BMI)等社会人口学因素及抗结核治疗史等临床特征对W-北京家族株流行的影响,除鄞州地区男性比女性具有更高的感染风险(OR=3.415,95%CI:1.071-10.884)外,未发现能够明显影响W-北京株流行的因素。 2、结核分枝杆菌耐药基因型与传播关系采用直接测序法检测与异烟肼、利福平耐药有关的基因katG、rpoB。研究发现,309株获得katG基因的测序结果的M.TB菌株中,24株(7.77%)在katG基因315位存在突变,突变形式为AGC-ACC,其中23株属于W-北京家族菌株；有145株M.TB菌株获得rpoB基因测序结果,有7株(4.83%)发生531位突变,突变形式均为TCG-TTG,其中有6株(85.71%)为W-北京家族株。katG和rpoB基因在人群中的突变率差别没有统计学意义。仅有1株发生katG基因突变的M.TB菌株发生成簇,突变菌株的成簇比例为4%；发生rpoB基因突变的M.TB菌株的基因型均为“唯一”型,无菌株发生成簇。 3、W-北京基因型结核分枝杆菌的基因型成簇特征及影响因素分析采用7位点VNTR-MIRU分型方法对成功获得DNA的426株M.TB菌株进行基因分型,识别了409种基因型,包括395种“唯一”基因型和14个簇(包括31株分离株),每个簇内有2-3株菌株不等,成簇比例为7.28%,近期传播比例为3.99%。阜宁有29株形成13个簇,成簇比例为15%；德清有2株形成1个簇,成簇比例为2%；鄞州无菌株成簇,均为“唯一型”；三地区成簇比例存在显著差异(x2=30.435,P0.001)。成簇菌株均为W_北京家族菌株,现代北京株的成簇率为7%,古典北京株成簇率为10%,二者的差别没有统计学意义,并且发现现代型与古典型菌株之间发生成簇。分析成簇患者的社会人口学及临床特征发现,户籍是影响M.TB菌株成簇的因素之一,流动人口的成簇风险明显低于本地人口(aOR=0.107,95%CI:0.013-0.856)；未发现BCG接种史、结核治疗史及痰涂片结果对菌株成簇有影响。成簇患者中有6例为复治病人,均为其所在簇内最早出现结核症状的患者。在德清的成簇患者中,发现本地人口与流动人口之间存在近期传播。簇内各患者呈散在分布,相互间没有发现流行病学关联。 4、VNTR-MIRU分型方法比较对阜宁地区199株M.TB菌株采用国际通用的15位点VNTR-MIRU进行基因分型,比较15位点与7位点VNTR-MIRU的分型能力。7位点组合中,各位点分辨力较高,最高为VNTR3820(0.8597),最低为Mtub21(0.6050);而15位点组合中,各个VNTR-MIRU位点分辨力水平有较大差异,从0.0804(ETRC)到0.7753(MIRU26)不等。15位点组合对阜宁地区M.TB菌株及北京株的总分辨力(HGI值)均为0.9994,而7位点组合的分辨力分别为0.9990和0.9988,略低于15位点组合。MIRU-15将阜宁199株M.TB菌株分为190种基因型,16株菌株成7个簇,成簇比例为8%,显著低于MIRU-7的成簇率(8%vs.15%,x2=4.234,P=0.040)。位点联合使用可以提高VNTR-MIRU分型方法的分辨力,但过多增加位点数量并不能使总分辨力得到进一步提高。通过本研究,我们可以得出如下结论： 1.W-北京家族菌株在华东地区的流行水平很高(90%),且没有发现年龄、健康状况(BMI)、户籍等社会人口学因素与北京株的分布的关联,也没有发现BCG接种史、结核治疗史、痰涂片结果等临床特征与北京株的流行有关。 2.W-北京株中,现代北京株的比例(77%)高于古典北京株,且现代北京株的分布不因社会人口学和临床特征而不同,始终占有较高的流行比例,提示现代型可能具有较强的致病性。 3.研究地区M.TB菌株成簇比例较低,提示华东地区结核病的发生主要是由既往感染结核复发造成,存在较小比例的近期传播,而且未发现簇内患者之间存在流行病学关联,提示由偶然接触导致结核发生可能是华东地区结核病传播的主要模式。 4.户籍是影响M.TB菌株成簇的主要因素之一,本地人口的成簇风险显著高于流动人口,提示本地人口比流动人口更容易发生结核的近期传播,同时研究也发现本地人口与流动人口之间能发生结核的交叉传播。 5.推荐选择来自MIRU-7的VNTR3820、Qub11a、Qub18和来自MIRU-15的Qub11b、MIRU10、MIRU26、MIRU31位点进行华东地区M.TB菌株的基因分型研究。
[Abstract]:Tuberculosis is an infectious disease that poses a serious threat to human health and social stability. China is one of the countries with a high burden of tuberculosis. Despite the decline in the prevalence of tuberculosis in recent years, the burden of tuberculosis disease in China is still very serious..W- Beijing Mycobacterium tuberculosis is a kind of common ancestor evolved from a common ancestor. M.TB strains of genetic background, in all parts of the world, have different levels of epidemic.W- Beijing family strains, which may be related to tuberculosis resistance, which can cause outbreak and form epidemic advantages.
Tracing the source of infection and clarifying the way of transmission is the two major problem in the epidemiological study of tuberculosis. Tuberculosis has the characteristics of latent infection. The traditional epidemiological method can not explain its transmission mechanism well, and the molecular epidemiology of tuberculosis based on genotyping technology has an irreplaceable advantage in this respect. With the combination of diseases and epidemiology, molecular epidemiology can predict the epidemic and outbreak of disease more accurately, and grasp its epidemic characteristics and mode of transmission. Therefore, this study selects Funing county (Jiangsu province), Deqing County (Jiangsu province) and Yinzhou District (Zhejiang province) in East China as the research site, and uses a method of combining cross-sectional investigation and molecular typing to describe the method of combining cross-sectional investigation and molecular typing. The epidemic status of Mycobacterium tuberculosis in W- Beijing genotypes in East China was analyzed. The social demographic characteristics, history of clinical treatment, history of treatment and population flow and other factors affecting the epidemic of W- strains were analyzed. The VNTR-MIRU genotype of the strain was determined, the rate of tuberculosis was assessed in the near future, and the mode of tuberculosis transmission in East China was discussed. It provides clues for the spread of tuberculosis in the population, compares the typing effect of VNTR-MIRU with different loci and its application prospects, and provides information for improving the genetic information database of Mycobacterium tuberculosis.
The main research methods and results are as follows:
1, the prevalence and influencing factors of W- Mycobacterium tuberculosis in Beijing
The basic data of sputum positive tuberculosis patients registered in each district and county CDC in June 2009 /7 month -2010 and /12 month in November /12 month were collected and collected. The sputum specimens of the patients were collected for strain culture, and DNA was extracted. A cross-sectional study combined with DTM-PCR typing was used to identify the W- Beijing family strain and the modern W- Beijing strain. M.TB strains of 346 tuberculosis patients, RD105 classification, 90% (311 strains) belong to Beijing family, Funing, Deqing and Yinzhou W- Beijing strains were 91% (J64 strain), 96% (23 strains) and 87% (124 strains), the difference was not statistically significant.W- Beijing strains through the one step type, respectively, the modern W- Beijing strain 294 strains (77%), the classical W- Beijing, respectively. Strain 90 (23%). Analysis of the social demographic factors such as age, health status (BMI) and the history of anti tuberculosis treatment history and other clinical characteristics on the epidemic of W- Beijing family, except for the higher risk of infection (OR=3.415,95%CI:1.071-10.884) of men in Yinzhou than women (OR=3.415,95%CI:1.071-10.884), there is no obvious influence on the factors of the epidemic of the W- Beijing strain.
2, the relationship between drug-resistant genotypes and transmission of Mycobacterium tuberculosis
The direct sequencing method was used to detect the gene katG related to isoniazid and rifampicin resistance. RpoB. studies found that 24 (7.77%) of the M.TB strains obtained from the sequencing of katG gene were mutated in the katG gene, and the mutation was AGC-ACC, 23 of which belonged to the W- Beijing family strain; 145 M.TB strains obtained the rpoB gene sequencing node. 531 mutations were found in 7 (4.83%) strains, all of which were TCG-TTG, of which 6 (85.71%) were W- Beijing family.KatG and rpoB gene mutations in the population with no statistical significance. Only 1 strains of katG gene mutation were produced by M.TB strains, and the proportion of mutant strains was 4%; rpoB gene mutation of M.TB bacteria. The genotypes of the plants were all "only", and the aseptic plants were clustered.
3, genotypic clustering characteristics and influencing factors of W- Beijing Mycobacterium tuberculosis
The 7 loci VNTR-MIRU typing method was used to genotyping 426 strains of M.TB strains, which were successfully obtained. 409 genotypes were identified, including 395 "unique" genotypes and 14 clusters (including 31 isolates). There were 2-3 strains in each cluster, the cluster ratio was 7.28%, the proportion of the near phase transmission was 29 in 3.99%. Funing, and 13 clusters were formed. The proportion of Yinzhou was 15%, 1 clusters were formed in Deqing and 2% in cluster proportion; all the sterile plants in Yinzhou were "unique". There were significant differences in the cluster proportion of three regions (P0.001). The cluster strains were all W_ Beijing family strains, the clustering rate of modern Beijing strain was 7%, and the clustering rate of Beijing strain in ancient Scripture was 10%, and there was no statistical difference between two. Significance and discovery of clusters between modern and classical strains.
The social demography and clinical characteristics of the cluster patients showed that the household registration was one of the factors affecting the cluster of M.TB strains. The cluster risk of the floating population was significantly lower than that of the local population (aOR=0.107,95%CI:0.013-0.856); the history of BCG inoculation, the history of tuberculosis treatment and the sputum smear results affected the strain of the strain. 6 of the cluster patients were retreated. Patients were the earliest patients with tuberculosis symptoms in their cluster. In the cluster patients of Deqing, there was a recent spread between the local population and the floating population. The patients were scattered in clusters, and there was no epidemiological association between each other.
4, comparison of VNTR-MIRU typing methods
In Funing, 199 strains of M.TB strains were genotyping by international VNTR-MIRU loci VNTR-MIRU. In the combination of the 15 loci and the 7 locus VNTR-MIRU, the resolution of each point was higher, the highest was VNTR3820 (0.8597) and the lowest was Mtub21 (0.6050), while the resolution level of the VNTR-MIRU loci in the 15 bit combination had a great difference. The total resolution of M.TB strain and Beijing strain in Funing area from 0.0804 (ETRC) to 0.7753 (MIRU26) is 0.9994, and the resolution of the 7 loci combination is 0.9990 and 0.9988 respectively, and the 7 loci combination.MIRU-15 divides 199 strains of M.TB strains in Funing into 190 genotypes, 16 strains are divided into 7 clusters, and the cluster proportion is 8. %, significantly lower than the clustering rate of MIRU-7 (8%vs.15%, x2=4.234, P=0.040). The combined use of loci can improve the resolution of the VNTR-MIRU typing method, but the number of excessive increasing loci can not further improve the total resolution.
Through this study, we can draw the following conclusions:
The prevalence of 1.W- Beijing family strains in East China was very high (90%), and no age, health status (BMI), household registration and other social demographic factors were associated with the distribution of Beijing strains. There was no history of BCG inoculation, tuberculosis treatment history, sputum smear results and other clinical features related to the epidemic of Beijing strain.
In the 2.W- Beijing strain, the proportion of modern Beijing strains (77%) is higher than that of the classical Beijing strain, and the distribution of modern Beijing strains is not different from the social demography and clinical characteristics, and it always occupies a high prevalence rate, suggesting that modern type may have a strong pathogenicity.
3. the cluster proportion of M.TB strains in the study area is low, suggesting that the occurrence of tuberculosis in East China is mainly caused by the recurrence of tuberculosis, there is a small proportion of the recent transmission, and there is no epidemiological association between the patients in the cluster. It is suggested that the occurrence of tuberculosis from accidental contact may be the main transmission of tuberculosis in East China. Pattern.
The 4. domicile is one of the main factors affecting the cluster of M.TB strains. The local population has a higher risk of clustering than the floating population, suggesting that the local population is more likely to have the recent spread of tuberculosis than the floating population, and the study also finds that the local population and the floating population can cross the cross transmission of tuberculosis.
5. recommend VNTR3820, Qub11a, Qub18 and Qub11b, MIRU10, MIRU26, and MIRU31 from MIRU-7, MIRU10, MIRU26, and MIRU31 loci to study the genotyping of M.TB strains in East China.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R52;R181.3

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