北京地区结核分枝杆菌基因分型及与耐药性关系的研究
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摘要
目的了解北京地区679株分枝杆菌临床分离株的基因型分布、主要流行特点,比较465株分枝杆菌间隔区寡核苷酸分型(Spoligotyping)和散在分布重复单位-数目可变串联重复序列(MIRU-VNTR)两种基因型方法试验结果的异同,分析北京基因型与耐药性的相互关系,从而探讨北京地区结核分枝杆菌的分子流行特点,分析结核分枝杆菌的耐药性特点及其同分子流行病学的联系,并评价两种基因分型方法。方法对收集的北京市679株分枝杆菌临床分离株进行Spoligotyping基因分型研究,其中的465株分枝杆菌同时运用MIRU-VNTR方法进行基因分型研究。用NatureEdge软件和BioNumerics软件将分型结果进行聚类分析。应用比例法药物敏感试验对268株结核分枝杆菌进行检测,最后将药敏结果结合流行病学资料进行分析。采用χ2检验比较不同组间结果的差别。结果在分离培养的679株菌株中,具有特异Spoligotyping指纹图谱的北京基因型菌株在北京地区分布达82.6%(561/679),为北京地区2008年主要流行的基因型菌株。未接种过卡介苗(BCG)的患者中北京基因型菌株占83.5%(240/269),接种过BCG的患者中北京基因型菌株占81.7%(384/470),两者差异无统计学意义。十种药物耐药性由高到低依次为丁胺卡那霉素(61.76%)、利福平(54.65%)、乙硫异烟胺(44.16%)、利福布汀(39.71%)、异烟肼(23.37%)、链霉素(22.22%)、左氧氟沙星(18.87%)、对氨基水杨酸(8.70%)、卷曲霉素(6.52%)、乙胺丁醇(4.71%)。北京基因型菌株耐药率为86.11%(186/216),低于非北京基因型菌株的耐药率93.18%(41/44),但二者差异也无统计学意义。Spoligotyping分型方法显示成簇菌株占所有菌株的93.93%(437/465),分73个基因型,MIRU-VNTR分型方法显示成簇菌株占所有菌株的22.37%(104/465),398个基因型。本地户籍成为菌株成簇的一个危险因素,OR值为1.90。VNTR方法对465株分枝杆菌的分辨率指数为0.9576。对北京基因型的分辨率指数为0.9547,同样显示了很高的分辨率指数。而Spoligotyping分型方法对465株分枝杆菌的分辨率指数0.4131,对北京基因型的分辨率指数为0.1430,低于MIRU-VNTR方法。不同的菌株和同一菌株在不同位点的MIRU-VNTR DNA指纹图谱呈现出多态性,各位点呈现的等位基因多态性有差异,多态性最高的位点是QUB-11b,多态性最低的位点为QUB-4156c。对北京基因型位点多态性分析也显示了相同的最高位点和最低位点。结论北京基因型菌株2008年在北京地区有广泛分布,为占绝对优势的菌株；北京基因型菌株与BCG接种无关,与一线、二线抗结核药物耐药无关；结核病患者中有部分是由于近期传播而引起的。MIRU-VNTR基因分型方法能够对Spoligotyping分型结果继续分型,分辨率指数要高于Spoligotyping分型方法。MIRU-VNTR分型方法对所有基因型和对北京基因型的分辨指数差别不大。Spoligotyping分型方法对北京基因型的分辨率指数较低。
Objective To find out the distribution of M.tb genotyping,leading epidemic type of679clinial isolates of TB in Beijing to compare the similarities and differences of two genotyping methods-Spoligotyping and MIRU-VNTR. Also to analyze the correlation between different genotypes and drug resistance. So as to explore the molecular-epidemiology of Mycobacterium tuberculosis in Beijing and analyze the two genotyping methods. Methods679M.tuberculosis strains were collected from20TB dispensaries or hospitals in Beijing in2008. The465clinical isolates were genotyped by mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and Spoligotyping methods respectively and the results of two genotypes were compared. The data of the tests were dealt with cluster analysis by means of the NatureEdge and the main epidemic strains and genotype were obtained. The genotyping results analyzed and combined with DST (appropriate concentrations)as well as epidemiological data. Chi-square was used to compare the difference between different groups. Results The Spoligotyping results demonstrated that82.6%(561/679) of the strains belonged to the Beijing genotype which is the domain genotype in Beijing in2008.Of the patients who had received BCG-vaccination,81.7%(384/470) infectied with strains of Beijing genotype and83.5%(240/269) of the patients were not BCG-vaccinated.However,the difference was not statistically significant.In the study,drug resistance rates to Km, RFB, Pto, RFT, INH, S, LFX, PAS, Cm, EMB were61.76%,54.65%,44.16%,39.71%,23.37%,22.22%,18.87%,8.70%,6.52%,4.71%.Drug resistant rate from those strains of Beijing genotype was86.11%(186/216), lower than that of non-Beijing genotype93.93%(437/465).The Spoligotyping showed93.93%(437/465) were strains of cluster and73genotypes,while22.37%(104/465) that of MIRU-VNTR and398genotypes. HGI of MIRU-VNTR465isolates and Beijing genotype isolates are0.9576which is higher than that of Spoligotyping (0.9547).DNA fingerprint of MIRU -VNTR presented polymorphism to different strain or different locus, there was different allelic diversity at each MIRU-VNTR locus,the QUB-1lblocus had the highest diversity and the QUB-4156c locus has the lowest diversity.And this is the same results to Beijing genotype. Conclusions The Beijing genotype of M.tuberculosis were found to be the dominant strains in Beijing in2008.The association between Beijing genotype of M.tuberculosis were vaccination or drug-resistant were not found.Results from cluster analysis suggested that some cases might belong to the newly developed cases. MIRU-VNTR has more diversified results and its HGI is higher than that of Spoligotyping.HGI difference between all isolates and Beijing genotype isolates is very slight by MIRU-VNTR, whereas high by Spoligotyping.
Objective To find out the distribution of M.tb genotyping,leading epidemic type of679clinial isolates of TB in Beijing to compare the similarities and differences of two genotyping methods-Spoligotyping and MIRU-VNTR. Also to analyze the correlation between different genotypes and drug resistance. So as to explore the molecular-epidemiology of Mycobacterium tuberculosis in Beijing and analyze the two genotyping methods. Methods679M.tuberculosis strains were collected from20TB dispensaries or hospitals in Beijing in2008. The465clinical isolates were genotyped by mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and Spoligotyping methods respectively and the results of two genotypes were compared. The data of the tests were dealt with cluster analysis by means of the NatureEdge and the main epidemic strains and genotype were obtained. The genotyping results analyzed and combined with DST (appropriate concentrations)as well as epidemiological data. Chi-square was used to compare the difference between different groups. Results The Spoligotyping results demonstrated that82.6%(561/679) of the strains belonged to the Beijing genotype which is the domain genotype in Beijing in2008.Of the patients who had received BCG-vaccination,81.7%(384/470) infectied with strains of Beijing genotype and83.5%(240/269) of the patients were not BCG-vaccinated.However,the difference was not statistically significant.In the study,drug resistance rates to Km, RFB, Pto, RFT, INH, S, LFX, PAS, Cm, EMB were61.76%,54.65%,44.16%,39.71%,23.37%,22.22%,18.87%,8.70%,6.52%,4.71%.Drug resistant rate from those strains of Beijing genotype was86.11%(186/216), lower than that of non-Beijing genotype93.93%(437/465).The Spoligotyping showed93.93%(437/465) were strains of cluster and73genotypes,while22.37%(104/465) that of MIRU-VNTR and398genotypes. HGI of MIRU-VNTR465isolates and Beijing genotype isolates are0.9576which is higher than that of Spoligotyping (0.9547).DNA fingerprint of MIRU -VNTR presented polymorphism to different strain or different locus, there was different allelic diversity at each MIRU-VNTR locus,the QUB-1lblocus had the highest diversity and the QUB-4156c locus has the lowest diversity.And this is the same results to Beijing genotype. Conclusions The Beijing genotype of M.tuberculosis were found to be the dominant strains in Beijing in2008.The association between Beijing genotype of M.tuberculosis were vaccination or drug-resistant were not found.Results from cluster analysis suggested that some cases might belong to the newly developed cases. MIRU-VNTR has more diversified results and its HGI is higher than that of Spoligotyping.HGI difference between all isolates and Beijing genotype isolates is very slight by MIRU-VNTR, whereas high by Spoligotyping.
引文
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