Genetic Diversity of Mycobacterium tuberculosis Isolates from Assam, India: Dominance of Beijing Family and Discovery of Two New Clades Related to CAS1_Delhi and EAI Family Based on Spoligotyping and MIRU-VNTR Typing

Abstract

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.

Evaluation of 24-locus MIRU-VNTR genotyping in Mycobacterium tuberculosis cluster investigations in four jurisdictions in the United States, 2006–2010

“…In particular, the five East Asian (L2) PCR00002 clusters had significantly decreased odds of epidemiologic linkages using GENType (OR=0.05, 95% CI 0.001, 0.95) and were heterogeneous both by birth country and MIRU24; only 26% (10/38) of patients clustered by GENType, which was significantly less than patients in other clusters (272/304, 90%, p<0.001). These findings appear to be consistent with the insufficient discrimination power of MIRU24 in isolates from the Beijing family that were reported by other authors [22–23]. …”

Genetic diversity and distribution dynamics of multidrug-resistant Mycobacterium tuberculosis isolates in Nepal

“…People from Dharana city in eastern Nepal used to frequently travel to Hong Kong, China to work as members of the British Gurkha Army 24 . There is still frequent movement of people between the eastern region of Nepal and north-eastern parts of India where the Beijing family is dominant 25 . Since, MTB is suggested to have co-expanded in line with human migration 26 , these phenomena might explain the establishment of the Beijing family in eastern and central region and might be one of reasons for the higher chance of isolating the Beijing family in eastern Nepal (Tables 4 – 5 ).…”

Genotypic diversity ofMycobacterium tuberculosisisolates from North-Central Indian population

“…Of these, Sahariya tribe has been most widely studied in context of TB prevalence, genetic susceptibility to TB and associated risk factors [41]. But, the [7,17] The present study revealed EAI3_IND/SIT11 as one of the major genotypes in Sahariya tribe followed by CAS1_Delhi/SIT26 while in non-tribes, EAI3_IND/SIT11 and CAS1_Delhi/SIT26 both genotypes were observed to a similar degree. A 3.04 fold higher risk of infection with EAI3_IND/SIT11 genotype was observed in Sahariya tribe as compared to non-tribes.…”

“…Molecular typing has always facilitated in population based monitoring of TB control programmes and understanding the TB epidemics [13]. The most commonly used tools for molecular epidemiological investigations are IS6110-RFLP typing, spoligotyping and MIRU-VNTR typing [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28]. These markers have been demonstrated successfully in studying micro-evolutionary changes and mixed strain infections in MTB strains [29,30].…”