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.
Highlights
In spite of the implementation of the Revised National Tuberculosis Control Program (RNTCP), the burden of tuberculosis (TB) in India is still the highest which is an important public health concern [1]
44 (23.28%) Mycobacterium tuberculosis complex (MTBC) isolates had 43 unknown spoligopatterns
The Beijing lineage (SIT1) representing 35.45% (n = 189) of the total MTBC isolates was found to be the most dominant spoligotype followed by Central Asian-Delhi (CAS1_Delhi) (11.64%)
Summary
In spite of the implementation of the Revised National Tuberculosis Control Program (RNTCP), the burden of tuberculosis (TB) in India is still the highest which is an important public health concern [1]. MIRU-VNTR typing method is more discriminatory and is less prone to homoplasy and has been increasingly used along with spoligotyping as a better tool for assessment of tuberculosis progression among infected associates [22]. We combined these two typing methods in the present study. Till date there is no published report of molecular genotyping study of MTBC based on combined analysis of spoligotyping and MIRU-VNTR typing from the state of Assam located in the northeastern (NE) region of India. In this study 189 MTBC isolates from the state of Assam were studied using spoligotyping along with 24-loci MIRU-VNTR typing to better understand the diversity of MTBC isolates and to find the value of different MIRU-VNTR loci for their ability and usefulness to characterize MTBC isolates from this region
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