扬州市结核分枝杆菌分子分型及耐药性监测

发布时间：2019-05-15 18:35

【摘要】：结核病(Tuberculosis, TB)是由结核分枝杆菌复合群(Mycobacterium tuberculosis complex, MTBC)引起的一种慢性传染病,主要感染人的呼吸道系统。据WHO估计,世界上近三分之一的人口已感染结核分枝杆菌。虽然TB流行水平呈下降趋势,但每年新发病例仍突破900万例,并且,随着耐药菌株的不断出现,对结核病的治疗和防控造成很大的挑战。因此,结核分枝杆菌菌种的鉴定分型以及耐药性监测对于该病的诊断、治疗以及预后都有着重要的意义。本研究针对结核分枝杆菌扬州临床分离株,运用传统细菌学方法和多位点PCR相结合,初步鉴定分枝杆菌的种属,再利用MIRU-VNTR和Spoligotyping分型方法研究其流行病学特征,最后通过比例法、MIC和耐药基因检测分析结核分枝杆菌耐药性情况。1扬州市结核分枝杆菌的分离培养与鉴定用抗酸染色实验确定抗酸杆菌阳性后,用PNB/TCH试验和多位点PCR从抗酸杆菌阳性菌株中鉴定出结核分枝杆菌(Mycobacterium tuberculosis, MTB),并比较两个方法之间的差异。结果显示,289株抗酸染色阳性临床分离株,经过PNB/TCH试验和多位点PCR进一步鉴定,其中270株为MTB,9株为非结核分枝杆菌(nontuberculous mycobacteria, NTM),2株为M. africanum,剩余8株需进一步鉴定。PNB/TCH试验结果与多位点PCR结果无统计学显著性差异。鉴定结果表明,扬州市结核病例绝大多数是由结核分枝杆菌所引起的,但也存在非结核分枝杆菌引起的结核病。2结核分枝杆菌扬州分离株的分子分型研究CTAB法提取到270株MTB分离株基因组DNA,用MIRU-VNTR和Spoligotyping进行基因分型。Spoligotyping方法共区分出8个家族,其中北京家族233株、T1家族15株、未知家族菌株11株、相似北京家族4株、U家族3株、H3家族2株、MANU2家族1株、T2家族1株。MIRU-VNTR方法共区分出154个基因型,其中144株组成28个簇,剩余126株均为独立基因型,该方法的分辨力为0.960。在15个VNTR位点中,不管是对全部菌株还是只对北京家族进行分析,Mtub21位点的HGDI值均为最高,分别为0.545和0.451。MIRU-VNTR分型方法的分辨率(0.960)明显高于Spoligotyping方法的分辨力(0.253),且两种分型方法相结合会使分辨力更高。通过分型研究,我们发现结核分枝杆菌扬州分离株具有一定的基因多态性,北京家族基因型菌株为该市的主要流行菌株,其流行趋势具有高度集中性,同时Spoligotyping方法也发现了11个新基因型,丰富了SpolDB4数据库。3结核分枝杆菌扬州分离株的耐药表型及耐药基因分析通过传统比例法和MIC方法对174株临床表现为耐药的分离株进行耐药性检测,并针对鉴定为耐药的菌株进行耐药基因的测序(主要针对四种一线药：异烟肼INH、利福平RFP、链霉素SM、乙胺丁醇EMB)。传统比例法耐药性结果显示174株MTB中有72株耐药(41.4%),多重耐药菌株有32株(18.4%)。对72株耐药菌株的耐药性检测方面,MIC法与传统比例法的结果之间无统计学显著性差异。耐药基因比对结果显示,INH耐药菌株中,katG突变率为72.0%(36/50),inhA突变率为6.0%(3/50)；RFP耐药菌株中,rpoB突变率为67.5%(27/40)；EMB耐药菌株中,embB突变率为52.6%(10/19)；SM耐药菌株中,rrs突变率为15.4%(6/39),rpsL突变率为71.8%(28/39)。通过比对分析,耐药基因的突变与结核分枝杆菌耐药表型密切相关,出现耐药表型的菌株不一定发生耐药基因突变,但是耐药基因突变的菌株全部耐药。本研究以扬州市2012-2015年结核分枝杆菌为对象,通过分离鉴定、分子分型、药物敏感性试验和耐药基因突变位点分析等检测,在分子水平分析扬州地区结核分枝杆菌的分布和传播特征,为该地区结核病的防治策略的订制提供科学参考依据,同时为结核病分子流行病学研究奠定了基础,并为结核病耐药监测提供了相关背景数据。
[Abstract]:Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis complex (MTBC). According to the WHO estimates, nearly one-third of the world's population has been infected with Mycobacterium tuberculosis. Although the prevalence of TB has a downward trend, new cases of TB continue to break through 9 million cases every year, and with the continuous emergence of drug-resistant strains, the treatment and prevention and control of tuberculosis pose a great challenge. Therefore, the identification and classification of Mycobacterium tuberculosis and the monitoring of drug resistance are of great significance to the diagnosis, treatment and prognosis of the disease. In this study, for the clinical isolates of M. tuberculosis in Yangzhou, the species of M. tuberculosis were identified by the combination of traditional bacteriological method and multi-site PCR. The epidemiological characteristics of M. tuberculosis were studied by using the methods of MIRU-VNTR and Spirigotyping. The drug resistance of Mycobacterium tuberculosis was analyzed by MIC and drug-resistant gene test. The isolated culture and identification of Mycobacterium tuberculosis in Yangzhou were determined by acid-fast staining. After the positive of acid-fast bacilli, Mycobacterium tuberculosis (M. tuberculosis) was identified from the positive strain of the acid-fast bacilli by using the PNB/ TCH test and the multi-site PCR. MTB) and comparing the differences between the two methods. The results showed that 289 strains of acid-fast stain positive clinical isolates were further identified by PNB/ TCH and multi-site PCR, of which 270 were MTB and 9 were non-tuberculous mycobacteria (NTM),2 were M. aficianum, and the remaining 8 strains were further identified. There was no statistically significant difference between the results of the PNB/ TCH test and the multi-site PCR. The results showed that the majority of the tuberculosis cases in Yangzhou were caused by Mycobacterium tuberculosis, but there were no M.tuberculosis caused by M.tuberculosis. The molecular typing of the isolated strains of M. tuberculosis was extracted by CTAB method to the genomic DNA of 270 MTB isolates. Genotyping was performed with MIRU-VNTR and Spirigotyping. A total of 8 families were identified, including 233 strains of the Beijing family,15 strains of the T1 family,11 strains of the unknown family strain,4 similar to the Beijing family,3 in the U family,2 in the H3 family,1 in the MANU2 family and 1 in the T2 family. A total of 154 genotypes were identified by the MIRU-VNTR method, of which 144 were 28 clusters, and the remaining 126 were independent. The resolution of the method was 0.960. In the 15 VNTR sites, the HGDI values of the Mtb21 site were the highest, 0.545 and 0.451.The resolution (0.960) of the Mtb21 locus was significantly higher than the resolution (0.253), and the combination of the two typing methods would make the resolution higher. By typing, we found that the isolated strain of M. tuberculosis has a certain gene polymorphism, and the Beijing family genotype strain is the main epidemic strain of the city, and the epidemic trend of the strain is highly concentrated, and 11 new genotypes are also found in the Spirigoinging method. The drug-resistant phenotype and the drug-resistance gene of the isolated strain of Mycobacterium tuberculosis were analyzed by the traditional method and the MIC method, and the drug resistance of the isolates was detected by the traditional method and the MIC method. And sequencing of the drug resistant genes for the strains identified as resistant (mainly aiming at the four first-line drugs: the heterosmotic INH, the rifampin RFP, the streptomycin SM, and the ethylamine-butanol EMB). The results showed that there were 72 drug-resistant strains (41.4%) in 174 strains of MTB and 32 (18.4%) of the multiple drug-resistant strains. There was no statistically significant difference between the results of the drug resistance of 72 strains of drug-resistant strains and the results of the method of MIC and the traditional method. The results showed that the mutation rate was 72.0% (36/50), the mutation rate of inhA was 6.0% (3/50), the mutation rate of rpoB in the resistant strain of RFP was 67.5% (27/40), and the mutation rate of rpoB in the drug-resistant strain was 52.6% (10/19). In the SM-resistant strain, the mutation rate of rrs was 15.4% (6/39), and the rpsL mutation rate was 71.8% (28/39). Compared with the analysis, the mutation of the drug-resistant gene is closely related to the drug-resistant phenotype of the mycobacterium tuberculosis, and the strain of the drug-resistant phenotype does not necessarily have the drug-resistant gene mutation, but the strain of the drug-resistant gene mutation is all resistant. In this study, the distribution and transmission characteristics of Mycobacterium tuberculosis in Yangzhou area were analyzed by means of isolation and identification, molecular typing, drug sensitivity test and resistance gene mutation site analysis in Yangzhou,2012-2015. It provides scientific reference for the customization of tuberculosis control strategy in the region, and lays a foundation for the epidemiological study of tuberculosis, and provides relevant background data for tuberculosis drug resistance monitoring.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R52;R446.5

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