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  Table of Contents  
Year : 2012  |  Volume : 30  |  Issue : 4  |  Page : 470-473

A pilot study to determine genetic polymorphism in Mycobacterium tuberculosis isolates in Central India

1 Department of Microbiology, Bhopal Memorial Hospital and Research Centre, Raisen Bypass Road, Karond, Bhopal, India
2 Department of Microbiology and Molecular Biology, National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR), Tajganj, Agra, India
3 Indian Council of Medical Research, Ansari Nagar, New Delhi, India

Date of Submission27-Mar-2012
Date of Acceptance20-Jun-2012
Date of Web Publication24-Nov-2012

Correspondence Address:
P Desikan
Department of Microbiology, Bhopal Memorial Hospital and Research Centre, Raisen Bypass Road, Karond, Bhopal
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.103774

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 ~ Abstract 

This study was carried out to identify predominant spoligotypes responsible for transmission and prevalence of tuberculosis in central India since there is no data available about the genetic biodiversity of Mycobacterium tuberculosis isolates from patients with tuberculosis in this region. 35 strains of Mycobacterium tuberculosis were subjected to spoligotyping according to the standard protocol. A total of 25 strains out of the 35 (71.42%) could be grouped in to 6 clusters. The largest cluster comprised 8 isolates. Unique (Non-clustered) spoligotypes were seen in 10 isolates, Nine strains did not match the data base (Spol DB-4 data base). The results indicate that there may be a number of orphan strains unique to this geographical area. Further studies on a larger sample size derived from this area would help us delineate the epidemiology of Mycobacterium tuberculosis infection in this area.

Keywords: Genetic polymorphism, Mycobacterium tuberculosis, spoligotyping

How to cite this article:
Desikan P, Chauhan D S, Sharma P, Panwalkar N, Gautam S, Katoch V M. A pilot study to determine genetic polymorphism in Mycobacterium tuberculosis isolates in Central India. Indian J Med Microbiol 2012;30:470-3

How to cite this URL:
Desikan P, Chauhan D S, Sharma P, Panwalkar N, Gautam S, Katoch V M. A pilot study to determine genetic polymorphism in Mycobacterium tuberculosis isolates in Central India. Indian J Med Microbiol [serial online] 2012 [cited 2021 Mar 5];30:470-3. Available from:

 ~ Introduction Top

India is the second-most populous country in the world, but has more new TB cases annually than any other country. In 2009, out of the estimated global annual incidence of 9.4 million TB cases, 2 million were estimated to have occurred in India. India, therefore, has one-fifth of the global burden of TB. Also, it is estimated that about 40% of the Indian population is infected with Mycobacterium tuberculosis. [1] Given this background, it has become increasingly important to understand the epidemiology and transmission dynamics of M. tuberculosis in India, to formulate policies for prevention and control of the disease. Molecular typing of M. tuberculosis strains has now become a valuable tool for identification of circulating strains in any geographical region. There has been no study on the genetic polymorphism of M. tuberculosis strains in Central India till date. This pilot study attempts to identify the genetic biodiversity of strains of M. tuberculosis in Central India, using spoligotyping as a method of molecular typing of M. tuberculosis strains in this region.

 ~ Materials and Methods Top

Mycobacterium tuberculosis strains were isolated from patients attending a tertiary care hospital and district microscopy centres in Central India. A total of 35 strains of M. tuberculosis were isolated from 28 sputa, 4 bronchial aspirates, and 1 each from pleural fluid, pus and CSF. The strains were identified as M. tuberculosis, based on a positive niacin accumulation test, positive nitrate reduction test, negative catalase test, absence of growth on Para-nitro benzoic acid incorporated Lowenstein Jensen media, and an rRNA-directed DNA hybridization assay for M. tuberculosis using the AccuProbe system (GenPprobe Inc., San Diego, CA, USA).

Genomic DNA was extracted from the strains of M. tuberculosis using a commercial kit, QIAamp DNA Mini Kit (Qiagen, Dusseldorf, Germany) as per the manufacturer's protocol. All the strains were subjected to spoligotyping using a commercially available kit (Isogen Biosciences BV, Maarsen, the Netherlands) in order to detect 43 known spacers in the direct locus according to the standard protocol provided by the manufacturer. Spoligotyping was carried out at the National JALMA Institute for Leprosy and other Mycobacterial Diseases, ICMR, Agra.H37Rv was used as a positive control and double-distilled water as a negative control. The DRa (5′ biotinylated) and DRb primers were used for amplification of DR regions. Forty-three oligonucleotides directed toward unique spacer sequence within the DR locus were hybridised by Enhanced Chemiluminesence (Amersham, Buckinghamshire, UK) followed by exposure to X-ray film (Hyperfilm ECL, Amersham), according to the manufacturer's instructions. The films were analysed and results were recorded in the form of binary codes for each spacer oligonucleotide probe. Presence of spacer was observed as a dark spot on the 'X' ray film which was recorded as 'one' whereas absence of dark spot was indicated as 'zero' representing absence of spacer oligonucleotide. The binary codes were converted into octal codes. The octal codes were analysed using the international spolDB4 database in order to locate the families of spoligotypes. A cluster was defined as two or more isolates with similar spoligotype patterns. However, if the spoligotype pattern in the spolDB4 database corresponded to only one isolate, then the pattern was called 'unique'. The spoligotypes that did not match any pattern in the database were defined as 'orphan'.

 ~ Results Top

Spoligotyping of the 35 strains of M. tuberculosis showed that 19 strains were clustered in three families. The biggest cluster consisted of nine strains, which constituted 25.71% of all strains. These strains belonged to the shared type ST11/EAI3_IND. Six strains of shared type ST26/CAS1_DEL formed a second cluster which constituted 17.14 % of all strains. A third cluster of four strains (11.42% of all strains), of shared type ST288/CAS2, was detected. There were seven unique (non-clustered) strains, as detailed in [Table 1]. Nine strains were orphan strains, which could not fit into the spolDB4 database. Orphan strains were analysed using SPOTCLUST database in order to find out their most probable families. Out of nine orphan isolates, three were identified as CAS family, two isolates as Family 33, one each as Family 34 and 36, and one each as EAI-3 and EAI-5.
Table 1: Details of mycobacterial strains

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 ~ Discussion Top

The present pilot study is an attempt to detect predominant shared types/families of strains of M. tuberculosis circulating in Madhya Pradesh, Central India. Genetic polymorphism of strains of M. tuberculosis in this region has not been characterised till date. Madhya Pradesh is a vast state in the heartland of India, with an area of 308,000 sq. km, and a population of approximately 726 lakhs, as per the 2011 census. [2] Out of 52,451 smear-positive cases diagnosed in the state between the fourth quarter of 2009 to the third quarter of 2010, 3242 were from Bhopal District. [1] This underscores the need to identify the genetic polymorphism of strains of M. tuberculosis in this region, in order to understand the origins and transmission dynamics of the strains.

The predominant Spoligo-International Type (SIT) in this area was found to be SIT11, followed by SIT26, which belong to the East African-Indian (EAI) and Central Asian (CAS) clades respectively. This finding conforms to a previous indication that most of the TB burden in India is caused by strains derived from a few ancestral, well-established clones. [3],[4] A previous study [3] had found a clear difference between the strains circulating between North and South India. It was found that SIT26 strains were predominant in the north, and SIT11 strains were predominant in the South. Bhopal is midway between the two areas; hence it is possible that these predominant local strains are derived from both areas. A third cluster of four strains (11.4% of all strains), of shared type ST288/CAS2 was found, which has also previously been isolated from India. [5],[6] Of the seven unique strains, three belonged to the CAS clade, two to the EAI clade, and one each to T3 and U clades. There has been no previous isolation of strains of the T3 clade from India reported in the spolDB4 database as yet. [5] This strain was isolated from the sputum of a 60-year-old female patient with pulmonary tuberculosis. Quite surprisingly, we did not find any isolate of type ST1 (Beijing type). Clusters of the Beijing type have frequently been reported from other parts of India. [4],[7],[8] In addition, though a study from Maharashtra reported Manu 1 as one of the predominant spoligotypes, [9] we did not find any strain of this spoligotype. There were nine orphan strains (21.7% of all strains) of which three were isolated from extra-pulmonary samples and these were only the extra-pulmonary samples included in the study. The significant numbers of orphan strains suggest that there may have been various evolutionary pressures that may have played a role in the development of the existing genetic topography of strains of M. tuberculosis in this region. It is important to assess the spatial and temporal evolution of the genetic landscape of M. tuberculosis strains isolated from our area, particularly in order to find a clinical correlation of the pattern of disease progression with specific spoligotypes, and also to understand the transmission dynamics of these strains. Further studies, with a greater number of strains are needed to understand this pattern.

 ~ References Top

1.TB India 2011: RNTCP Annual Report, 2011. Available from: [Last accessed 2012 March 06].  Back to cited text no. 1
2.Provisional Population Totals: Census of India, 2011. Available from: [Last accessed 2012 March 06].  Back to cited text no. 2
3.Singh UB, Arora J, Suresh N, Pant H, Rana T, Sola C, et al. Genetic biodiversity of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in India. Infect Genet Evol 2007;7:441-8.  Back to cited text no. 3
4.Gutierrez MC, Ahmed N, Willery E, Narayanan S, Hasnain SE, Chauhan DS, et al. Predominance of ancestral lineages of Mycobacterium tuberculosis in India. Emerg Infect Dis 2006;12:1367-74.  Back to cited text no. 4
5.Spoldb4, sorted by increasing spoligo-international-type number. Available from: [Last accessed 2012 March 06]  Back to cited text no. 5
6.Brudey K, Driscoll JR, Rigouts L, Prodinger WM, Gori A, Al-Hajoj SA, et al. Mycobacterium tuberculosis complex genetic diversity: Mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiol 2006;6:23.  Back to cited text no. 6
7.Singh UB, Suresh N, Bhanu VN, Arora J, Pant H, Sinha S, et al. Predominant spoligotypes, Delhi, India. Emerg Infect Dis 2004;10:1138-42.  Back to cited text no. 7
8.Sharma P, Chauhan DS, Upadhyay P, Faujdar J, Lavania M, Sachan S, et al. Molecular typing of Mycobacterium tuberculosis isolates from a rural area of Kanpur by spoligotyping and mycobacterial interspersed repetitive units (MIRUs) typing. Infect Genet Evol 2008;8:621-6.  Back to cited text no. 8
9.Chatterjee A, D'Souza D, Vira T, Bamne A, Ambe GT, Nicol MP, et al. Strains of Mycobacterium tuberculosis from western Maharashtra, India, exhibit a high degree of diversity and strain-specific associations with drug resistance, cavitary disease, and treatment failure. J Clin Microbiol 2010;48:3593-9.  Back to cited text no. 9


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