Preliminary Study On Genotype And Evolution Of Beijing Family Of Mycobacterium Tuberculosis In Beijing, China

OBJECTIVE To understand the genotype features、main prevalent genotype and evolution of Beijing family of Mycobacterium tuberculosis (M. tuberculosis) in Beijing, China; to provide scientific evidence for tracing the source of the infection, finding the way of transmission and strengthening the prevention and control of tuberculosis (TB).METHODS Chaoyang districts of Beijing were selected as the survey point using the random cluster sampling method.From March2009to December2010, all patients suspectedwith TB coming in the center for TB control and prevention were investigated, whose sputum sample and clinical background material were collected. M. tuberculosis in the sputum sample was isolated by means of the Lowenstein-Jenson medium. The cultures were identified by biochemical tests, then the genomic DNA were extracting by boiling method. Molecular typing identification of the strains was done by means of Spoligotyping with the DR region amplified by PCR, MLVA with15VNTR loci and LSP with RD105, RD181and NTF loci. Cluster analysis was done by BioNumerics software5.0and HGI, Chi-square test. The multivariate logistic regression analysis were done using excel, SPSS, SAS software.RESULTS360Mycobacterium strain were isolated, of which354strains were identified as M. tuberculosis by biochemical testing with PNB/TCH media.1. Spacer oligonucleotide typing (Spoligotyping)354M. tuberculosis strains were classified into40genotypes (Shared international types, the STs), representing12clusters and28single isolates (also called unique pattern), of which, the predominant STs were Beijing family (1-9STs)(n=292,82.5%), while non-Beijing family represented high polymorphism,30STs including25single isolates. The main genotypes of non-Beijing family were T1(n=31,8.8%) and T2(n=5,1.4%) family. The results of multivariate logistic regression analysis showed that:the proportion of M tuberculosis Beijing family in TB patients whose registered residence were in the north was4.624times of that in the south in China,95%CI was2.517to8.497.2. multiple locus VNTR analysis (MLVA)Among354M tuberculosis strains being genotyped,228different VNTR genotypes were detected, of which189(53.4%) genotypes were unique pattern, while Beijing family were classified into172genotypes, of which138strains (80.2%) were unique pattern; the cluster rate of Beijing family and non-Beijing family was52.4%and20.3%respectively. For TB patients whose registered residence in the north and the south, the cluster rate of M. tuberculosis was50.6%and25.7%respectively, the difference between them has statistically significance (P﹤0.05); the results of multivariate logistic regression analysis showed that registered residence in the north of TB patients could positive predict the cluster rate of M. tuberculosis (OR=2.207,95%CI:1.201to4.057), the Beijing family of M. tuberculosis could positive predicted the cluster rate (OR=4.240,95%CI:2.0228.891); The HGI of15VNTR loci for all strains and Beijing family was0.978and0.967respectively. Each VNTR locus had different discriminatory power for all M. tuberculosis strains and Beijing family.3. Preliminary exploration research of M. tuberculosis Beijing family stainsAmong290Beijing family strains, the proportion of the ancient and the modern was53.1%and46.2%respectively; Beijing family strains mainly represented four subgroups, of which, the predominant subgroup belonging to the modern accounted for49.0%showing the RD105, RD181deletion and IS6110insertion in the NTF region. The Kappa value representing the degree of consistency of RD105and Spoligotyping identifying Beijing family was0.77. Four loci including ETRD, MIRU26, Mtub21and Mtub30can provide signals for Beijing family phylogeny evolution.CONCLUSION1. In Beijing area clinical M. tuberculosis strains have strong allelic diversity and Beijing family is the predominat prevalence genotype. The epidemic of Beijing family strains is related to the registered residence of TB patients.The recent spread of tuberculosis is related to the registered residence of TB patients.2. Among Beijing clinical strains of M. tuberculosis in Beijing area, ancient type and modern type had the similar proportion, the former was little higher than the latter; Beijing family of M. tuberculosis mainly represented four subgroups, the characteristic of the predominant subgroup belonging to modern type represented RD105and RD181deletion and IS6110insertion in the NTF region. It was probably a important branch that leading the evolution process of Beijing family.3. RD105, as a molecular marker in great worth to identify Beijing family, can’t completely replace the gold standard, Spoligotyping. So it should be carefully used.4. MLVA also has a certain marker effect in exploring the phylogeny evolution of Beijing family. For Beijing family strains in Beijing area, ETRD, MIRU26, Mtub21and Mtub30could provide the signal for the systemic evolution of Beijing family. Referring to different regions, different strains, appropriate VNTR locus should be selected to carry out the study of microevolution of Beijing family strains, which provide assistance to establishment of molecular epidemiological framework of Beijing family strains, the study of systemic evolution and the analysis of the genotype of clinical strains.