Genetic Diversity Of Mycobacterium Tuberculosis From 13 Provinces In China

The genotyping of Mycobacterium tuberculosis (M. tuberculosis) strains is important for TB control because it allows for the detection of suspected outbreaks and the tracing of transmission chains. It is also important to monitor species diversity as well as identify genetic relationship. Spacer oligonucleotide typing (Spoligotyping) and variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) are the common methods in the molecular epidemiological research in M. tuberculosis, and have been widely used in epidemiological research of tuberculosis around the world. The genotyping results of M. tuberculosis showed that the prevalence of tuberculosis in the world mainly caused by several special M. tuberculosis genetic lineage (family) strains. Each family strains have unique molecular characteristics, regional distribution and pathogenicity. So the genotyping of M. tuberculosis play an important role in the tuberculosis prevention and control.In order to understand the molecular epidemical characters of M. tuberculosis, such as genetic diversity, regional distribution and major epidemic strains, et al, a total of 2346 M. tuberculosis isolates from 13 provinces in China were genotyped by Spoligotyping. Two hundred seventy-eight spoligotypes were identified:2153 isolates were grouped into 85 clusters, and the remaining 193 isolates were orphans. Comparison with the SITVIT2 database revealed that 118 spoligotypes had shared international type numbers in the database and the other 160 spoligotypes were novel.Family assignment revealed that the most frequent strains were Beijing family strains (1738/2356,74.08%), including typical Beijing family strains and atypical Beijing family strains. In addition, the strains belonged to other family, such as T family, CAS family, EAI family, Haarlem family, S family, MANU2 family, X family, LAM family and U family, were also found in China.The percentage of Beijing family strains varied greatly among the 13 provinces (54.50～92.59%). The highest prevalence was found in Beijing and its surrounding areas, while a lower prevalence was found in Southern China. The geographical distribution of Beijing family strains provides evidence for the recent clonal expansion of this particular family strain. The low diversity and the highly conserved DR region of the highly prevalent Beijing family strains in this study might indicate that the Beijing family is spreading rapidly in China.One interesting finding in this study is that 12 strains tested belong to the CAS family, these strains were only found in Tibet and Xinjiang. Among the 12 CAS family strains,3 and 9 strains originated from patients of Tibetan and Uyghur ethnicity, respectively. This finding suggests that CAS family might have biogeographical specificity. Moreover, Tibet and Xinjiang share geographic borders with India where the CAS family is dominant. It has also been suggested that this family strains might be transported by trade, tourism, or migration from India.The TbDl analysis employed in this study showed that the majority of isolates (99.97%) belonged to the TbD1-/modern strains, whereas only seven strains (0.03%) were TbDl+/EAI isolates. These seven TbDl+/EAI strains were only found in Fujian province where located in Southeastern China. The M. tuberculosis strains that contained the TbD1 region were designated as ancestral M. tuberculosis strains, while the M. tuberculosis strains missing the TbD1 region were designated as modern M. tuberculosis strains. The predominance of modern M. tuberculosis strains in China supports the hypothesis that China has a relatively modern endemic focus of TB. Since the ancestral TbD1+/EAI strains were only found in Fujian province, this suggests that they are endemic there.Tuberculosis (TB) is one of the major public health problems in Tibet. However, information about lineages of M. tuberculosis isolates from Tibetan patients is unknown. In order to detect the genetic diversity of M. tuberculosis isolates from Tibetan patients in Tibet, compare techniques of MIRU-VNTR verus Spoligotyping for study of genetic pattern of M. tuberculosis strains, and evaluate the discriminatory power of each locus. Five hundred sevety-seven isolates from Tibetan TB patients throughout Tibet were genotyped by Spoligotyping and 24-locus MIRU-VNTR methods in this study.Based on Spoligotyping results, a total of 23 different spoligotypes were identified among 577 M. tuberculosis isolates. The majority (97.57%,563/577) of the isolates belonged to four known families:Beijing family, T family, CAS family and MANU2 family. While the other 14 isolates belonged to 11 novel spoligotypes. Classical Spoligotyping confirmed that Beijing family is highly prevalent in Tibet (90.47% in whole sample). A high rate of clustering (96.01%, nine clusters,2 to 512 strains/cluster) was observed upon Spoligotyping; most of it was due to the lower discriminatory power of this method for the ST1 genotype (n=512; 88.73% of the isolates). In this study, there is no relationship between Beijing family strains and age, sex, BCG vaccination and treatment history, respectively (p> 0.05)Using 24-locus MIRU-VNTR to genotype 577 M. tuberculosis isolates, three hundred forty-seven distinct MIRU-VNTR patterns were detected. Two hundred ninety-nine strains were grouped into 1 of 69 different MIRU-VNTR clusters and 278 isolates were unique. The Hunter-Gaston discriminatory index of the method was 0.991, indicating the higher power of discrimination of MIRU-VNTR than Spoligotyping (the Hunter-Gaston discriminatory index was 0.221). The discriminatory power of 7 loci (MIRU31, Qub11b2, Qub26, Qub4156c, Mtub21, MIRU20 and MIRU26) were moderately or highly discrimination accroding to their alllelic diversity, while the other 17 loci have poor discrimination. Especilly, the number repeats of these 577 isolates in MIRU24 are all 1, suggesting that these isolates are all modern M. tuberculosis strains.By direct comparison with previously typed MIRU-VNTR patterns and by genetic relationship analyses, we could identify and clearly define four epidemic groups of M. tuberculosis:Beijing family, T family, CAS family and LAM family. The family designation of most strains corresponded to the results as confirmed by Spoligotyping. However,1 isolates with a Spoligotyping profile corresponding to MANU2 was clustered with Beijing strains. This confusing finding was due to mixed infection by PCR analysis.In conclusion, the M. tuberculosis isolates from Tibetan patients in Tibet in this study exhibit low genetic polymorphism. Beijing family strains predominant and spread rapidly in Tibet. Majority of VNTR loci in this 24-locus MIRU-VNTR method is unfit for TB genityping in areas where Beijing family strains are dominant. If appropriate loci are added to the MIRU-VNTR analysis, MIRU-VNTR genotyping could be a valuable tool for strain typing and epidemiological research of M. tuberculosis. With this approach a more clear understanding about genetic pattern of M. tuberculosis can be achieved.