JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 3593–3599 Vol. 48, No. 10 0095-1137/10/$12.00 doi:10.1128/JCM.00430-10 Copyright ? 2010, American Society for Microbiology. All Rights Reserved.
Strains of Mycobacterium tuberculosis from Western Maharashtra,
India, Exhibit a High Degree of Diversity and Strain-Speci，c
Associations with Drug Resistance, Cavitary Disease, and
Treatment Failure †
111342Anirvan Chatterjee,Desiree D’Souza,Tina Vira,Arun Bamne,Gurish T. Ambe,Mark P. Nicol, 1Robert J. Wilkinson,5,6,7 and Nerges Mistry*
1Foundation for Medical Research, 84-A, R. G. Thadani Marg, Worli, Mumbai 400018, India; Division of Medical Microbiology, 2Medical School, University of Cape Town, Observatory, Cape Town 7925, South Africa; Mumbai District TB Control Society 111, 3New Municipal Building, Dr. E. Moses Road, Worli, Mumbai 400018, India; Joint Executive Health Of;cer, F(S) Ward Building, 4Brihanmumbai Municipal Corporation (BMC), Mumbai 400012, India; Division of Medicine, Imperial College London, 5London W2 1PG, United Kingdom; Institute of Infectious Diseases and Molecular Medicine and Department of 6Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa; and 7MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom
Received 2 March 2010/Returned for modi，cation 21 June 2010/Accepted 10 August 2010
We performed spoligotyping of Mycobacterium tuberculosis isolates from 833 systematically sampled pulmo- nary tuberculosis (TB) patients in urban Mumbai, India (723 patients), and adjacent rural areas in western India (110 patients). The urban cohort consisted of two groups of patients, new cases (646 patients) and ；rst-time treatment failures (77 patients), while only new cases were recruited in the rural areas. The isolates from urban new cases showed 71% clustering, with 168 Manu1, 62 CAS, 22 Beijing, and 30 EAI-5 isolates. The isolates from ；rst-time treatment failures were 69% clustered, with 14 Manu1, 8 CAS, 8 Beijing, and 6 EAI-5 isolates. The proportion of Beijing strains was higher in this group than in urban new cases (odds ratio [OR], 3.29; 95% con；dence limit [95% CL], 1.29 to 8.14; P 0.003). The isolates from rural new cases showed 69% clustering, with 38 Manu1, 7 CAS, and 1 EAI-5 isolate. Beijing was absent in the rural cohort. Manu1 was found to be more common in the rural cohort (OR, 0.67; 95% CL, 0.42 to 1.05; P 0.06). In total, 71% of isolates were clustered into 58 spoligotypes with 4 predominant strains, Manu1 (26%), CAS (9%), EAI-5 (4%), and Beijing (4%), along with 246 unique spoligotypes. In the isolates from urban new cases, we found Beijing to be associated with multidrug resistance (MDR) (OR, 3.40; 95% CL, 1.20 to 9.62; P 0.02). CAS was found to be associated with pansensitivity (OR, 1.83; 95% CL, 1.03 to 3.24; P 0.03) and cavities as seen on chest radiographs (OR, 2.72; 95% CL, 1.34 to 5.53; P 0.006). We recorded 239 new spoligotypes yet unreported in the global databases, suggesting that the local TB strains exhibit a high degree of diversity.
The resurgence of tuberculosis (TB) fuelled by multidrug simultaneously for long durations, resulting in a set of varied resistance (MDR) and extensive drug resistance has caused strain types (6, 20, 21). signi，cant concern among health care practitioners (36, 37). Studies from India show differential strain predominance There have been renewed efforts to understand the biology of between the southern and northern regions of the country. the pathogen alongside its epidemiology. Such data have come While the central Asian strain (CAS) is dominant in the north, mostly from regions of sporadic incidence or from populations East African Indian strains (EAI) are observed more fre- where the disease is driven by high HIV prevalence (8). Data quently in the southern regions (29). Most studies from India from some of the highest-disease-burden regions, where tuber- as well as speci，cally from Mumbai, India, showed CAS and culosis has remained endemic, are scarce. Manu1 as the predominant spoligotypes along with EAI as a India is one such region where Mycobacterium tuberculosis third large strain lineage (3, 16, 18, 22, 28, 29, 30). Another has remained in equilibrium with the population, resulting in study from a tertiary care center in Mumbai reported a high an area of tuberculosis endemicity (6, 27). Under such condi- proportion of Beijing strains (23%) in a cohort associated with tions, the strain diversity is expected to be different compared a high proportion of MDR (1). Interestingly, TbD1-positive to that for epidemic or sporadic incidents, where a few speci，c strains of tuberculosis (such as EAI) predominate in India,
strain types dominate (8, 23); in a setting where tuberculosis is whereas TbD1-negative strains of M. tuberculosis are more
endemic, the pathogen and the host are expected to evolve common in the rest of the world (11). Although previous studies provided preliminary data from various sites in India, they did not re！ect strain variability from * Corresponding author. Mailing address: Foundation for Medical a single cosmopolitan region. Additionally, studies from Mum- Research, 84-A, R. G. Thadani Marg, Worli, Mumbai 400018, India. bai were biased toward MDR cases (1), had small sample sizes Phone: 91 22 4934989. Fax: 91 22 4932876. E-mail: email@example.com. (16), or were derived from a cohort of chronic (re-treated) TB † Supplemental material for this article may be found at http://jcm cases accessing tertiary care hospitals (22). .asm.org/. Epidemiological studies of M. tuberculosis have been facili- Published ahead of print on 18 August 2010.
3594 CHATTERJEE ET AL. J. CLIN. MICROBIOL.
tated by a variety of genotyping tools. IS6110 restriction frag- ment length polymorphism (RFLP) remains the gold standard due to its high level of discrimination (15) but is time-consum- ing and less suitable in populations with low copy numbers (7). Mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing (10, 19, 31) is a high- throughput and discriminatory method, but the best combina- tion of MIRU loci is yet to be achieved, and combinations may differ between populations (33). We used spoligotyping as a primary ，ngerprint method, due to its relatively high through- put nature. Spoligotyping has a lower discriminatory power than MIRU-VNTR typing, making it less suitable for deter- mining strain transmission. However, spoligotyping served as a useful primary ，ngerprinting tool allowing comparisons of strain types from strains around the world through updated global databases (5, 17, 35). In this study, we describe the distribution of strain genotypes from a systematic collection of strains from urban Mumbai and two neighboring rural areas. Mumbai is a location where a con！uence of people from all parts of the country live in poor, congested neighborhoods with high population densities, up to 64,168 people per square kilometer in one of the city wards as
per the 2001 census (26). These conditions, coupled with a high
proportion of MDR cases in the region (2, 9), were cause for
concern and underlined the need for more information on FIG. 1. Geographical demarcations of India. (The map was created local circulating strains. We wished to determine the distribu- using MapXL Maps of India, v.6 [Compare Infobase Limited].) tion of strains among newly diagnosed TB patients in the region to extend previous observations (16, 22). This study describes the spoligotypes present in the cohort All rural patients were new cases sampled at onset of therapy. Patients were and the extent of their clustering. We further analyzed the recruited from April 2004 to September 2007 at RNTCP DOTS centers. association of spoligotypes with other parameters, namely, age, Inclusion criteria for patients were (i) smear positivity, (ii) age from 15 to 70 gender, geographical origin of the host, radiology, and multi- years, (iii) residency in Mumbai for at least 3 years immediately prior to diag- drug resistance, to obtain deeper insights into strain behavior. nosis, and (iv) residence in the same area as the health posts where treatment was sought. Sputum-negative cases were excluded, as we were unable to perform sputum MATERIALS AND METHODS culture on all patients, due to study constraints. Patients who had not resided in Location of study. The Revised National Tuberculosis Control Programme the area for at least 3 years were excluded, as we were interested primarily in (RNTCP) in Mumbai is implemented in individual wards of the city. Since this strains causing disease in the resident population of Mumbai. Since the study study was part of a larger epidemiological project assessing transmission of MDR aimed to identify strains circulating in the region during the study period, pre- TB in a setting of TB endemicity, 4 centrally located wards (F/N, G/N, H/E, and viously treated patients were excluded to eliminate relapse cases, which are less K/E) characterized by a high sputum-positive case load, with moderately subop- likely to relate to a current transmission event. Patients with a history of TB or timal cure rates ranging between 78 and 81%, were selected (RNTCP quarterly antituberculosis therapy were determined through interview and scrutiny of reports 2001; can be sourced from firstname.lastname@example.org). As far as could be district TB registers and patients defaulting during therapy (in the case of ascertained, there was no apparent deviation in the RNTCP functioning in these