Molecular typing of Mycobacterium tuberculosis isolates from a rural area of Kanpur by spoligotyping and mycobacterial interspersed repetitive units (MIRUs) typing Pragya Sharma a , Devendra Singh Chauhan a , Prashant Upadhyay a , Jaya Faujdar a , Mallika Lavania a , Shailender Sachan c , Kiran Katoch b , Vishwa Mohan Katoch a, * a Department of Microbiology and Molecular Biology, National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR), Tajganj, Agra 282001, India b Medical Unit-1, National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR), Tajganj, Agra 282001, India c Model Rural Health Research Unit, Ghatampur, National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR), Tajganj, Agra 282001, India 1. Introduction Tuberculosis remains one of the leading causes of death worldwide with an estimated 8.8 million new TB cases. The majority of patients with tuberculosis live in the most populous countries of Asia; Bangladesh, China, India, Indonesia, and Pakistan together account for half (48%) the new cases that arise every year. India accounts for a fifth of the worlds’ new TB cases and 2/3rd of the cases in South-East Asia. This makes India the highest TB burden country in the world. It has been estimated for the year 2000, that there were about 3.8 million bacteriologically positive TB cases in the country (RNTCP Status report, 2007). The introduction of molecular techniques in the field of tuberculosis has opened new avenues in studying transmission dynamics and epidemiology in conjunction with classical epide- miological approaches. These techniques usually exploit various repetitive DNA elements as markers for strain identification of Mycobacterium tuberculosis. Most widely used and internationally accepted method is based on transposable element IS6110 (van Embden et al., 1993). Since its introduction it has been widely used worldwide in various molecular epidemiological studies including outbreak investigations, transmission in the community and dissemination of multi drug resistant (MDR) clones (Goyal et al., 1994; Alland et al., 1994; Edlin et al., 1992). It has also been used to detect suspected laboratory cross contamination (Small et al., 1993). Requirement of good quality of DNA and absence or low copy number of IS6110 element in the some strains of M. tuberculosis hinders full use of this technique in certain parts of the world especially in Southeast Asia (Das et al., 1995; Radhakrishnan et al., 2001; Gillespie et al., 1995; Chauhan et al., 2007). These drawbacks can be overcome with the help of some of the PCR based methods by targeting other gene/region. Spoligotyping and methods based on various tandem repeats are the second most widely used methods after IS6110 based fingerprinting (Goyal et al., 1999; Allix et al., 2004). Spoligotyping relies on polymorph- ism in the direct repeat (DR) locus of M. tuberculosis complex organism. The DR locus contains multiple direct variable repeats (DVRs) that consist of well conserved 36 bp DRs interspersed with Infection, Genetics and Evolution 8 (2008) 621–626 ARTICLE INFO Article history: Received 29 November 2007 Received in revised form 31 March 2008 Accepted 5 May 2008 Available online 9 May 2008 Keywords: Spoligotyping MIRU-VNTR Shared type Loci ABSTRACT Molecular typing of Mycobacterium tuberculosis isolates has greatly facilitated the understanding of epidemiology of tuberculosis (TB). This study was done to characterize prevalent genotypes of M. tuberculosis on a collection of 97 isolates based on spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing in rural area of Kanpur, North India. In this area different types of interventions are being undertaken and follow-up studies are progressing. Predominant spoligotypes prevalent in this region belonged to Central Asian–Delhi family (CAS1_Del) (37%), East African–Indian family (11%), T1 family (8%) and Beijing (4%) family. Highly distinct MIRU-VNTR genotypes were obtained. Significant spoligotypes such as Beijing and CAS1_Del type were further divided into subtypes with MIRU-VNTR. This preliminary study reveals that CAS is the most predominant family in this rural area of Kanpur. If confirmed in other areas, this combined approach of molecular typing can be preferably be used as first line tool for studying linkage and transmission dynamics of TB in India. ß 2008 Elsevier B.V. All rights reserved. * Corresponding author. Tel.: +91 562 2331756/2333595/2232222; fax: +91 562 2331755. E-mail addresses: vishwamohan_katoch@yahoo.co.in, jalma@sancharnet.in (V.M. Katoch). Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid 1567-1348/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2008.05.002