Genotyping and drug-resistance epidemiology of mycobacterium tuberculosis in Xuzhou, China.
ABSTRACT: BACKGROUND:To explore the genetic diversity and drug resistance status of MTB in Xuzhou, China. METHODS:A total of 325 clinical MTB strains were genotyped by spacer-oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive unit variable number of tandem repeats (MIRU-VNTR). Phenotypic resistance was assessed by drug susceptibility testing (DST). RESULT:Based on the spoligotyping method, 325 MTB isolates were classified into 5 known genotypes and 12 unknown genotypes, and the largest branch comprised 268 strains belonging to the Beijing family. Based on the 15-loci VNTR typing method, 325 MTB isolates were divided into 35 clusters and 220 unique patterns. Compared to the low discriminatory power of spoligotyping genotyping (HGDI = 0.3444), 15-loci VNTR genotyping had a significantly higher discriminatory power for all strains (HGDI = 0.9980), particularly for the Beijing family strains (HGDI = 0.9892). When spoligotyping and 15-loci VNTR methods were used together, the discriminatory power increased to 0.9991. The Beijing family strain presented increased risks for developing multi-drug resistance TB (P < 0.05). CONCLUSION:The Beijing family isolates is the most prevalent strains in Xuzhou. Spoligotyping, in combination with 15-loci MIRU-VNTR, is useful for epidemiological analysis of MTB transmission in Xuzhou.
Project description:<h4>Background</h4>Globally, China is the second place with high burden of tuberculosis (TB). To explore the characteristics of the pathogens of Mycobacterium tuberculosis (MTB) circulating in this area is helpful for understanding and controlling the spread of the strains. Recent developments in molecular biology have allowed prompt identification and tracking specific strains of MTB spreading through the population.<h4>Methods</h4>Spacer-oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) were performed in combination to yield specific genetic profiles of 260 MTB strains isolated from 30 counties of Jiangsu province in China between June and July 2010. The spoligotyping results were in comparison to the world Spoligotyping Database of Institute Pasteur de Guadeloupe (SpolDB4). Drug susceptibility test (DST) was performed on all strains by proportion method on Lowenstein-Jensen (LJ) culture media.<h4>Results</h4>Based on the spoligotyping method, 246 strains displayed known patterns and 14 were absent in the database. Predominant spoligotypes belonged to the Beijing family (80.4%). By using the 24-loci VNTR typing scheme, 224 different patterns were identified, including 20 clusters and 204 unique patterns. The largest clade comprised 195 strains belonging to the Beijing family. The combination of spoligotyping and 24-loci MIRU-VNTR demonstrated maximal discriminatory power. Furthermore, we observed a significant association between Beijing family strains and drug-resistant phenotypes. The Beijing family strains presented increased risks for developing multi-drug resistant TB, with the OR (95% CI) of 11.07(1.45-84.50).<h4>Conclusions</h4>The present study demonstrated that Beijing family isolates were the most prevalent strains circulating in Jiangsu province of China. The utility of spoligotyping in combination with 24-loci MIRU-VNTR might be a useful tool for epidemiological analysis of MTB transmission.
Project description:Bovine tuberculosis (bTB) caused by Mycobacterium (M.) bovis and M. caprae is a transmissible disease of livestock, notifiable to the World Organization for Animal Health (OIE). BTB particularly affects cattle and small ruminants and can be transmitted to humans thereby posing a significant threat to veterinary and public health worldwide. M. bovis is the principal cause of bTB in Algeria. In order to better understand the route of spreading and elaborate an eradication program, isolation and characterization of mycobacteria from Algerian cattle was performed. Sixty strains belonging to the M. tuberculosis complex were analyzed by spoligotyping, thereof 42 by 19-locus-MIRU-VNTR-typing. Spoligotyping revealed 16 distinguishable patterns (Hunter-Gaston discriminatory index [HGDI] of 0.8294), with types SB0120 (n = 20) and SB0121 (n = 13) being the most frequent patterns, representing 55% of the strains. Analyses based on 19-locus-MIRU-VNTR yielded 32 different profiles, five clusters and one orphan pattern, showing higher discriminatory power (HGDI = 0.9779) than spoligotyping. Seven VNTR-loci [VNTR 577 (alias ETR C), 2163b (QU11b), 2165 (ETR A), 2461 (ETR B), 3007 (MIRU 27), 2163a (QUB11a) and 3232 (QUB 3232)] were the most discriminative loci (HGDI ˃ 0.50). In conclusion, 19-locus-MIRU-VNTR yielded more information than spoligotyping concerning molecular differentiation of strains and better supports the elucidation of transmission routes of M. bovis between Algerian cattle herds.
Project description:The Mycobacterium tuberculosis (MTB) Beijing strain is highly virulent, drug resistant, and endemic over Asia. To explore the genetic diversity of this family in several different regions of eastern Asia, 338 Beijing strains collected in Taiwan (Republic of China) were analyzed by mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and compared with published MIRU-VNTR profiles and by the Hunter-Gaston diversity index (HGDI) of Beijing strains from Japan and South Korea. The results revealed that VNTR2163b (HGDI>0.6) and five other loci (VNTR424, VNTR4052, VNTR1955, VNTR4156 and VNTR 2996; HGDI>0.3) could be used to discriminate the Beijing strains in a given geographic region. Analysis based on the number of VNTR repeats showed three VNTRs (VNTR424, 3192, and 1955) to be phylogenetically informative loci. In addition, to determine the geographic variation of sequence types in MTB populations, we also compared sequence type (ST) data of our strains with published ST profiles of Beijing strains from Japan and Thailand. ST10, ST22, and ST19 were found to be prevalent in Taiwan (82%) and Thailand (92%). Furthermore, classification of Beijing sublineages as ancient or modern in Taiwan was found to depend on the repeat number of VNTR424. Finally, phylogenetic relationships of MTB isolates in Taiwan, South Korea, and Japan were revealed by a minimum spanning tree based on MIRU-VNTR genotyping. In this topology, the MIRU-VNTR genotypes of the respective clusters were tightly correlated to other genotypic characters. These results are consistent with the hypothesis that clonal evolution of these MTB lineages has occurred.
Project description:Tuberculosis (TB) is one of the major public health concerns in Assam, a remote state located in the northeastern (NE) region of India. The present study was undertaken to explore the circulating genotypes of Mycobacterium tuberculosis complex (MTBC) in this region. A total of 189 MTBC strains were collected from smear positive pulmonary tuberculosis cases from different designated microscopy centres (DMC) from various localities of Assam. All MTBC isolates were cultured on Lowenstein-Jensen (LJ) media and subsequently genotyped using spoligotyping and 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing. Spoligotyping of MTBC isolates revealed 89 distinct spoligo patterns. The most dominant MTBC strain belonged to Beijing lineage and was represented by 35.45% (n = 67) of total isolates, followed by MTBC strains belonging to Central Asian-Delhi (CAS/Delhi) lineage and East African Indian (EAI5) lineage. In addition, in the present study 43 unknown spoligo patterns were detected. The discriminatory power of spoligotyping was found to be 0.8637 based on Hunter Gaston Discriminatory Index (HGDI). On the other hand, 24-loci MIRU-VNTR typing revealed that out of total 189 MTBC isolates from Assam 185 (97.9%) isolates had unique MIRU-VNTR profiles and 4 isolates grouped into 2 clusters. Phylogenetic analysis of 67 Beijing isolates based on 24-loci MIRU-VNTR typing revealed that Beijing isolates from Assam represent two major groups, each comprising of several subgroups. Neighbour-Joining (NJ) phylogenetic tree analysis based on combined spoligotyping and 24-loci MIRU-VNTR data of 78 Non-Beijing isolates was carried out for strain lineage identification as implemented by MIRU-VNTRplus database. The important lineages of MTBC identified were CAS/CAS1_Delhi (41.02%, n = 78) and East-African-Indian (EAI, 33.33%). Interestingly, phylogenetic analysis of orphan (23.28%) MTBC spoligotypes revealed that majority of these orphan isolates from Assam represent two new sub-clades Assam/EAI and Assam/CAS. The prevalence of multidrug resistance (MDR) in Beijing and Non-Beijing strains was found to be 10.44% and 9.01% respectively. In conclusion, the present study has shown the predominance of Beijing isolates in Assam which is a matter of great concern because Beijing strains are considered to be ecologically more fit enabling wider dissemination of M. tuberculosis. Other interesting finding of the present study is the discovery of two new clades of MTBC isolates circulating in Assam. More elaborate longitudinal studies are required to be undertaken in this region to understand the transmission dynamics of MTBC.
Project description:<h4>Background</h4>Tuberculosis remains a serious threat to human health as an infectious disease in China. Henan, a most populated province in China, has a high incidence of tuberculosis (TB). Though the genetic diversity of Mycobacterium tuberculosis (MTB) has been investigated in many regions, there have been only a few studies on the molecular characteristics and drug resistance phenotypes in Henan. This is the first study on the genetic profile of MTB from Henan.<h4>Methods</h4>A total of 668 MTB isolates from various areas were genotyped with spoligotyping and 26-locus MIRU-VNTR (classical 24-locus MIRU-VNTR and 2 other loci). The association between TB spoligotype signatures and drug-resistant profiles was analysed.<h4>Results</h4>Our data revealed that MTB isolates circulating in Henan had a high degree of genetic variation. The Beijing family was the most predominant genotype (83.53%,n?=?558), and the typical Beijing type(ST1) was the major sublineage (81.73%,n?=?546). In total,668 isolates were divided into 567 different types, forming 38 clusters (2-15 isolates per cluster), and 529 unique types by 26-locus MIRU-VNTR analysis. There was no correlation between the Beijing family and gender, age at diagnosis or treatment history, whereas the Beijing family was significantly associated with all four first-line drug resistance and multidrug-resistant phenotypes. For these samples, 15 of 26 MIRU-VNTR loci had high or moderate discriminatory power according to the Hunter-Gaston discriminatory index. A combination of the 10 most polymorphic loci had similar discriminatory power as the 26-locus set.<h4>Conclusion</h4>The Beijing genotype is the most prevalent family. Ten-locus MIRU-VNTR in combination with spoligotyping can efficiently classify the molecular type of MTB in Henan Province.
Project description:<i>Objectives.</i> We studied the genetic diversity of clinical isolates from patients with tuberculosis in the multiethnic area of Xinjiang autonomous region in China. A total of 311 clinical <i>M. tuberculosis</i> isolates were collected in 2006 and 2011 and genotyped by two genotyping methods. All isolates were grouped into 68 distinct spoligotypes using the spoligotyping method. The Beijing family was dominant, followed by T1 and CAS. MIRU-VNTR results showed that a total of 195 different VNTR types were identified. Ten of the 15 loci were highly or moderately discriminant according to their HGDI scores, and 13 loci had good discriminatory power in non-Beijing family strains, whereas only two loci had good discriminatory power in Beijing family strains. Chi-square tests demonstrated that there were no correlations between four characteristics (sex, age, type of case, and treatment history) and the Beijing family. In summary, Beijing family strains were predominant in Xinjiang, and the VNTR-15<sub>China</sub> locus-set was suitable for genotyping all Xinjiang strains, but not for the Beijing family strains. Thus, these data suggested that different genotype distributions may exist in different regions; MLVA locus-sets should be adjusted accordingly, with newly added loci to increase resolution if necessary.
Project description:BACKGROUND: The Central Asian Strain 1 (CAS1) genogroup of Mycobacterium tuberculosis (MTB) is the most prevalent in Pakistan, India and Bangladesh. Mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) typing is a reliable and reproducible method for differentiation of MTB isolates. However, information of its utility in determining the diversity of CAS1 strain is limited. We performed standard 12 loci based MIRU-VNTR typing on previously spoligotyped CAS1 strains and 'unique' strains in order to evaluate its discriminatory power for these isolates. METHODS: Twelve loci based MIRU- VNTR typing was used to type 178 CAS1 and 189 'unique' MTB strains. The discriminatory index for each of the loci was calculated using the Hunter Gaston Discriminatory Index (HGDI). A subset of these strains (n = 78) were typed using IS6110 restriction fragment length polymorphism (RFLP). MIRU-VNTR profiles were studied together with their drug susceptibility patterns. RESULTS: A total of 349 MIRU patterns were obtained for the 367 strains tested. The CAS1 strains were subdivided into 160 distinct patterns; 15 clusters of 2 strains each, 1 cluster of four strains and 144 unique patterns. Using HGDI, seven MIRU loci, (numbers 26, 31, 27, 16, 10, 39, and 40) were found to be "highly discriminatory" (DI: >or=0.6), four MIRU loci (numbers 20, 24, 23, and 4) were "moderately discriminatory" (DI: 0.3-0.59), and one locus (number 2) was "poorly discriminatory" (DI< 0.3). Loci 26 and 31 were the most discriminatory for the CAS1 isolates. Amongst 'unique' strains in addition to loci 26, 31, 27, 16, 10, 39, and 40, locus 23 was highly discriminatory, while no locus was poorly discriminating. DI values for loci 4, 10 and 26 were significantly lower (P-value < .01) in CAS1 strains than in 'unique' strains. The association between CAS1 strains and MDR was not found to be significant (p value = 0.21). CONCLUSION: We propose that MIRU typing could be used to estimate the phylogenetic relatedness amongst prevalent CAS1 strains, for which MIRU loci 26, 31, 16, 10, 27, 39 and 40 were found to be the most discriminatory.
Project description:Today, significant attention is directed towards the global lineages and sublineages of Mycobacterium tuberculosis (Mtb). NEW-1 (SIT 127) and CAS1-Delhi (SIT 26) strains are recognized as growing and circulating Mtb genotypes, especially in Asian countries. It is crucial to develop or enhance Mtb genotyping methods for a more accurate and simple differentiation of these families. We used 24-loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing for genotyping 217 Mtb isolates. To select the optimal MIRU-VNTR loci, we calculated the Hunter-Gaston discriminatory index (HGDI), allelic diversity, and accumulation of percentage differences (APDs) between the strains among different groups of genotypes (NEW-1 and non-NEW-1; CAS1-Delhi and non-CAS). Finally, the minimum spanning tree was constructed for clustering analysis. In the NEW-1 population, loci with APD?>?60% were found to have a high discriminatory power. VNTR loci with APD?>?50% showed high discrimination power for the CAS population. Our findings suggest that APDs, which are valuable for the selection of VNTR loci sets, may improve the discriminatory power of MIRU-VNTR typing for identification of Mtb genotypes in specific regions.
Project description:<h4>Background</h4>The control of tuberculosis in densely populated cities is complicated by close human-to-human contacts and potential transmission of pathogens from multiple sources. We conducted a molecular epidemiologic analysis of 356 Mycobacterium tuberculosis (MTB) isolates from patients presenting pulmonary tuberculosis in metropolitan Taipei. Classical antibiogram studies and genetic characterization, using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing and spoligotyping, were applied after culture.<h4>Methods</h4>A total of 356 isolates were genotyped by standard spoligotyping and the strains were compared with in the international spoligotyping database (SpolDB4). All isolates were also categorized using the 15 loci MIRU-VNTR typing method and combin with NTF locus and RD deletion analyses.<h4>Results</h4>Of 356 isolates spoligotyped, 290 (81.4%) displayed known spoligotypes and 66 were not identified in the database. Major spoligotypes found were Beijing lineages (52.5%), followed by Haarlem lineages (13.5%) and EAI plus EAI-like lineages (11%). When MIRU-VNTR was employed, 140 patterns were identified, including 36 clusters by 252 isolates and 104 unique patterns, and the largest cluster comprised 95 isolates from the Beijing family. The combination of spoligotyping and MIRU-VNTR revealed that 236 (67%) of the 356 isolates were clustered in 43 genotypes. Strains of the Beijing family was more likely to be of modern strain and a higher percentage of multiple drug resistance than other families combined (P = 0.08). Patients infected with Beijing strains were younger than those with other strains (mean 58.7 vs. 64.2, p = 0.02). Moreover, 85.3% of infected persons younger than 25 years had Beijing modern strain, suggesting a possible recent spread in the young population by this family of TB strain in Taipei.<h4>Conclusion</h4>Our data on MTB genotype in Taipei suggest that MTB infection has not been optimally controlled. Control efforts should be reinforced in view of the high prevalence of the Beijing strain in young population and association with drug resistance.
Project description:In Morocco, the prevalence of multidrug resistant tuberculosis (MDR-TB) continues to increase especially within previously treated cases; these MDR cases may evolve to extensively drug resistant tuberculosis (XDR-TB) raising major concern to TB control programs. From an epidemiological window, scarce informations are available about the genetic diversity of Mycobacterium tuberculosis (MTB) strains fueling these forms of resistance. The aim of this study was to assess to genetic diversity of MDR-MTB strains. Hence, this prospective study was conducted on patients diagnosed with MDR-TB at Pasteur Institute of Casablanca from 2010 to 2013. A total of 70 MDR-MTB isolates were genotyped by spoligotyping and 15-loci MIRU-VNTR methods. Spoligotyping generated four orphan patterns, five unique profiles whereas 61 strains were grouped in nine clusters (2 to 25 strains per cluster), the clustering rates being 87.1%. Subtyping by 15 loci MIRU-VNTR splitted all clusters already established by spoligotyping and generated 70 unique profiles not recognized in SITVIT2 database; clustering rate was equal to zero. HGDI analysis of 15 loci MIRU demonstrated that eight out of 15 loci were highly discriminant. Of note, all pre-XDR strains belongs to many clades, meaning that there no association between gyrA mutants and particular clade. Overall, the data generated by this study (i) describe the population structure of MDR MTBC in Morocco which is highly homogenous, (ii) confirm that TB in Morocco is almost exclusively transmitted by modern and evolutionary lineages with high level of biodiversity seen by MIRU, and (iii) validate the use of optimized 15-loci MIRU-VNTR format for future investigations in Morocco.