Received 2 March 2010/Returned for modification 21 June 2010/Accepted 10 August 2010

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p Vol. 48, No /10/$12.00 doi: /jcm Copyright 2010, American Society for Microbiology. All Rights Reserved. Strains of Mycobacterium tuberculosis from Western Maharashtra,, Exhibit a High Degree of Diversity and Strain-Specific Associations with Drug Resistance, Cavitary Disease, and Treatment Failure Anirvan Chatterjee, 1 Desiree D Souza, 1 Tina Vira, 1 Arun Bamne, 3 Gurish T. Ambe, 4 Mark P. Nicol, 2 Robert J. Wilkinson, 5,6,7 and Nerges Mistry 1 * Foundation for Medical Research, 84-A, R. G. Thadani Marg, Worli, Mumbai , 1 ; Division of Medical Microbiology, Medical School, University of Cape Town, Observatory, Cape Town 7925, South Africa 2 ; Mumbai District TB Control Society 111, New Municipal Building, Dr. E. Moses Road, Worli, Mumbai , 3 ; Joint Executive Health Officer, F(S) Ward Building, Brihanmumbai Municipal Corporation (BMC), Mumbai , 4 ; Division of Medicine, Imperial College London, London W2 1PG, United Kingdom 5 ; Institute of Infectious Diseases and Molecular Medicine and Department of Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa 6 ; and MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom 7 Received 2 March 2010/Returned for modification 21 June 2010/Accepted 10 August 2010 We performed spoligotyping of Mycobacterium tuberculosis isolates from 833 systematically sampled pulmonary tuberculosis (TB) patients in urban Mumbai, (723 patients), and adjacent rural areas in western (110 patients). The urban cohort consisted of two groups of patients, new cases (646 patients) and first-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 first-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% confidence 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 resistance (MDR) and extensive drug resistance has caused significant concern among health care practitioners (36, 37). There have been renewed efforts to understand the biology of the pathogen alongside its epidemiology. Such data have come mostly from regions of sporadic incidence or from populations where the disease is driven by high HIV prevalence (8). Data from some of the highest-disease-burden regions, where tuberculosis has remained endemic, are scarce. is one such region where Mycobacterium tuberculosis has remained in equilibrium with the population, resulting in an area of tuberculosis endemicity (6, 27). Under such conditions, the strain diversity is expected to be different compared to that for epidemic or sporadic incidents, where a few specific strain types dominate (8, 23); in a setting where tuberculosis is endemic, the pathogen and the host are expected to evolve * Corresponding author. Mailing address: Foundation for Medical Research, 84-A, R. G. Thadani Marg, Worli, Mumbai ,. Phone: Fax: fmr@fmrindia.org. Supplemental material for this article may be found at Published ahead of print on 18 August simultaneously for long durations, resulting in a set of varied strain types (6, 20, 21). Studies from show differential strain predominance between the southern and northern regions of the country. While the central Asian strain (CAS) is dominant in the north, East African n strains (EAI) are observed more frequently in the southern regions (29). Most studies from as well as specifically from Mumbai,, showed CAS and Manu1 as the predominant spoligotypes along with EAI as a third large strain lineage (3, 16, 18, 22, 28, 29, 30). Another study from a tertiary care center in Mumbai reported a high proportion of Beijing strains (23%) in a cohort associated with a high proportion of MDR (1). Interestingly, TbD1-positive strains of tuberculosis (such as EAI) predominate in, whereas TbD1-negative strains of M. tuberculosis are more common in the rest of the world (11). Although previous studies provided preliminary data from various sites in, they did not reflect strain variability from a single cosmopolitan region. Additionally, studies from Mumbai were biased toward MDR cases (1), had small sample sizes (16), or were derived from a cohort of chronic (re-treated) TB cases accessing tertiary care hospitals (22). Epidemiological studies of M. tuberculosis have been facili- 3593

2 3594 CHATTERJEE ET AL. J. CLIN. MICROBIOL. tated by a variety of genotyping tools. IS6110 restriction fragment length polymorphism (RFLP) remains the gold standard due to its high level of discrimination (15) but is time-consuming 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 highthroughput and discriminatory method, but the best combination of MIRU loci is yet to be achieved, and combinations may differ between populations (33). We used spoligotyping as a primary fingerprint method, due to its relatively high throughput nature. Spoligotyping has a lower discriminatory power than MIRU-VNTR typing, making it less suitable for determining strain transmission. However, spoligotyping served as a useful primary fingerprinting 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 confluence 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 local circulating strains. We wished to determine the distribution 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 and the extent of their clustering. We further analyzed the association of spoligotypes with other parameters, namely, age, gender, geographical origin of the host, radiology, and multidrug resistance, to obtain deeper insights into strain behavior. MATERIALS AND METHODS Location of study. The Revised National Tuberculosis Control Programme (RNTCP) in Mumbai is implemented in individual wards of the city. Since this study was part of a larger epidemiological project assessing transmission of MDR TB in a setting of TB endemicity, 4 centrally located wards (F/N, G/N, H/E, and K/E) characterized by a high sputum-positive case load, with moderately suboptimal cure rates ranging between 78 and 81%, were selected (RNTCP quarterly reports 2001; can be sourced from dtomhbmc@rntcp.org). As far as could be ascertained, there was no apparent deviation in the RNTCP functioning in these wards compared to that in the other wards in Mumbai. A significant proportion of the resident population of these 4 selected wards belonged to the middle or lower socioeconomic class and resided in informal housing in slums. Cumulatively, the 4 wards covered 38 DOTS (directly observed therapy short course) centers, with a population of 3 million. In addition, we sampled patients from rural TB units (located 250 and 280 km from Mumbai), covering a population of 860,000. This facilitated studying spoligotypes from a rural cohort which, as opposed to Mumbai, had a homogeneous population comprising an indigenous community with little or no migration from other parts of. Unlike the rural cohort, that from the Mumbai region included a large migrant population from other parts of (37%) (26). We could thus compare strain diversities in homogenous (rural) and cosmopolitan (Mumbai, urban) cohorts in the same geographical location. Additionally, there exists a movement of people from these rural areas to Mumbai, in the form of unskilled industrial labor, which provided the opportunity to study the overlap of TB strains between the two locations. Case definition. Sputum-positive new cases of TB were broadly classified into two groups: (i) new cases sampled at onset of CAT1 therapy, a regimen of 2(isoniazid [H]-ethambutol [E]-rifampin [R]-pyrazinamide [Z])3 4(H-R)3, comprising 2 months of H-E-R-Z thrice weekly followed by 4 months of H-R thrice weekly), and (ii) first-time treatment failures, or new cases who remained sputum smear positive at the fifth month after commencement of CAT1 therapy. FIG. 1. Geographical demarcations of. (The map was created using MapXL Maps of, v.6 [Compare Infobase Limited].) All rural patients were new cases sampled at onset of therapy. Patients were recruited from April 2004 to September 2007 at RNTCP DOTS centers. Inclusion criteria for patients were (i) smear positivity, (ii) age from 15 to 70 years, (iii) residency in Mumbai for at least 3 years immediately prior to diagnosis, 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 culture on all patients, due to study constraints. Patients who had not resided in the area for at least 3 years were excluded, as we were interested primarily in strains causing disease in the resident population of Mumbai. Since the study aimed to identify strains circulating in the region during the study period, previously treated patients were excluded to eliminate relapse cases, which are less likely to relate to a current transmission event. Patients with a history of TB or antituberculosis therapy were determined through interview and scrutiny of district TB registers and patients defaulting during therapy (in the case of first-time treatment failures). As far as could be ascertained, the first-time treatment failures had not received any antituberculosis therapy prior to the current episode. Patients were recruited after informed consent and referred for HIV counseling and testing. Clearance for this study was obtained from the Foundation for Medical Research (FMR) institutional ethics committee ( /01). Patient investigations. Patient demographic details, including age, gender, and geographical origin, were recorded. Since many patients were found to be migrants from other parts of the country, they were analyzed based on 3 groups: south, north, and Maharashtra (considered the native population) (Fig. 1). All patients recruited were subjected to radiological examination and were binomially classified into groups by presence or absence of cavities. Sample processing and culture. Sputum samples (one per patient) were collected in cetylpyridinium chloride (CPC)-NaCl vials as previously described (9). These samples were then concentrated by Petroff s method (4). The concentrated samples were inoculated onto solid Lowenstein-Jensen slants (Hi-Media, ) and incubated at 37 C until growth was observed. DNA extraction. A loopful of culture was used to extract DNA by a standard cetyltrimethylammonium bromide (CTAB) mycobacterial DNA extraction procedure, followed by phenol chloroform purification (13, 14). Spoligotyping. Spoligotyping was conducted as previously described (12). Clusters were defined as at least 2 isolates with identical spoligotypes.

3 VOL. 48, 2010 SPOLIGOTYPING M. TUBERCULOSIS STRAINS IN WESTERN INDIA 3595 Patient study group and cohort TABLE 1. Patient distribution in the cohort across the different locations and patient types Ward or TB unit No. (%) of clustered isolates Spoligotype clustering No. of unique isolates No. of isolates of major spoligotype Total Manu1 CAS Beijing EAI-5 Treatment failures Mumbai (urban) F/N 20 (67) G/N 4 (50) H/E 13 (65) K/E 16 (84) Total 53 (69) New cases Mumbai (urban) F/N 194 (72) G/N 78 (70) H/E 86 (74) K/E 100 (67) Total 458 (71) Rural Narayangaon 46 (70) Bhor 30 (68) Total 76 (69) Total 587 (71) A random selection of samples (DNA extracted from 80 isolates in Tris-EDTA [TE] in freezer packs) was sent to the Institute of Infectious Diseases and Molecular Medicine, University of Cape Town (IIDMM, UCT), for singleblinded quality control. Drug susceptibility testing. Drug susceptibility testing for the four first-line drugs rifampin (R), isoniazid (H), ethambutol (E), and pyrazinamide (Z) was performed by the radiorespirometric Buddemeyer technique (a manual modification of the BACTEC 460 technique) (9). Strains resistant to at least H and R were classified as multidrug resistant (MDR). Resistance to H, R, and an additional first-line drug was represented as MDR. Ten percent of the isolates were sent to the Swedish Institute for Infectious Disease Control (supranational reference laboratory), Stockholm, for external quality assurance by the BACTEC method. Kappa scores showed good agreement for H (0.76) and R (0.77) (9). Statistical analysis. Multivariate analysis (binary logistic regression) was performed with the spoligotype as the dependent variable and MDR, susceptibility, HIV status, and cavitation in the new cases (first-time treatment failures excluded) as covariates. A chi-square test was performed to find associations between spoligotype and (i) patient subgroup (first-time treatment failures, urban new cases, and rural cases) or (ii) region of origin. All patient data were entered and maintained using SPSS v10.0 (SPSS, Inc.). Analyses were done with SPSS and Microsoft Office Excel 1997 (Microsoft Corporation). Spoligotype comparison to SpoldB3 (35) ( bennek/spotclust.html) and SpoldB4 (17) ( -guadeloupe.fr:8081/sitvitdemo/) was done using tools available on the respective websites. RESULTS Patient selection. (i) Urban. We screened 1,136/2,184 (52%) new cases who presented to the RNTCP for diagnosis between April 2004 and September We included 646 new cases in the urban cohort from the 1,136 screened, based on our inclusion criteria. The 490 patients excluded from the study consisted of 135 (28%) with prior antituberculosis treatment, 222 (45%) who had taken more than 5 doses of antituberculosis therapy before sampling, and 24 (5%) who resided outside the study area. One hundred nine patients (22%) refused to participate in the study. During the same period, we screened 318 treatment failures, of which 77 were included. The 241 excluded patients consisted of 86 (36%) with prior antituberculosis therapy, 114 (47%) with an interruption in treatment of more than 2 weeks, and 14 (6%) who resided outside the study area. Twenty-seven patients (11%) refused to participate in the study. (ii) Rural. The rural cohort consisted of 110 patients out of 262 screened (42%).Thus, a total of 833 isolates from urban (n 723) and rural (n 110) patients were spoligotyped (Tables 1, 2, 3, and 4). Spoligotyping. The four major clusters, designated C1, C2, C15, and C24, contained 77, 30, 220, and 37 isolates, respectively (Fig. 2). The clusters were labeled C1 through C58 sequentially as they were formed during the study period. When all of the spoligotypes were compared to the international spoligotype database SpoldB4 (5), the shared types (ST) and labels were C1, CAS1_Delhi (ST26); C2, Beijing (ST1); C15, Manu1 (ST100); and C24, EAI-5 (ST236). Therefore, Manu1 emerged as the largest strain type, infecting 26.4% of the urban and rural cohorts (see Table S1 in the supplemental material). New cases (urban). The urban cohort consisted of 646 isolates from new cases sampled at onset of therapy. Seventyone percent (458/646) of these were found to be clustered into 56 spoligotypes (Table 3). Manu1 was the largest cluster, with 168 (26%) isolates (Table 1). We found 62 (9.5%) CAS isolates, 22 (3.4%) Beijing isolates, and 30 (4.6%) EAI-5 isolates in the cohort (Table 1). Although this was the largest subgroup TABLE 2. Distribution of clustered strains in the total cohort No. of isolates within cluster No. of clusters with this characteristic No. of strains with this characteristic/total no. of isolates (%) /833 (51) /833 (20) /833 (13) /833 (0.06)

4 3596 CHATTERJEE ET AL. J. CLIN. MICROBIOL. TABLE 3. All spoligotypes in different patient types Cluster or unique spoligotype No. of isolates in patient study group Urban treatment failures Urban new cases Rural new cases Total C C C C C5 8 8 C C C8 5 5 C9 2 2 C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C Unique spoligotype Total of patients, it did not contain 2 spoligotypes (C9 and C6) (Table 3). First-time treatment failures. Seventy-seven treatment failures sampled at the end of 5 months of treatment yielded 24 unique spoligotypes and a clustering of 69% (Tables 1 and 3). The 53 clustered strains were divided into 18 spoligotypes. Manu1 was the largest cluster, with 14 isolates (18%), followed by CAS and Beijing, with 8 isolates each (10%). EAI-5 was the fourth largest cluster, with 6 isolates (8%) (Table 1). The proportion of Beijing isolates in the treatment failures was found to be significantly higher than that in the urban new cases (odds ratio [OR], 3.29; 95% confidence limit [95% CL], TABLE 4. Distribution of major spoligotypes based on geographical origin of patient Spoligotype category and parameter Maharashtra Result for patients with GO a of: Northern Eastern Western Southern Total Manu1 No. of isolates % within cluster % within GO % of total CAS No. of isolates % within cluster % within GO % of total EAI-5 No. of isolates % within cluster % within GO % of total Beijing No. of isolates % within cluster % within GO % of total Other clusters No. of isolates % within cluster % within GO % of total Unique spoligotype No. of isolates % within cluster % within GO % of total Total No. of isolates % within cluster % within GO % of total a GO, geographical origin to 8.14; P 0.003) (Table 5). This subgroup consisted of both the isolates that formed the C9 cluster not found in new cases and 1 isolate of the C6 cluster (Table 3). New cases (rural). The 110 isolates in the rural cohort were divided into 21 clusters and 34 unique spoligotypes. The 21 clusters comprised 69% (76/110) of the rural isolates and were also seen in the urban cohort. Manu1 was the largest cluster, with 38 (34.5%) of the isolates, followed by 7 CAS isolates (6.3%) and 1 EAI-5 isolate. Additionally, we found no Beijing strains among the 110 isolates (Table 1). In total, we found 833 spoligotypes, of which 246 were unique, while the remaining were distributed across 58 spoligotype clusters. A total of 70.5% of strains were clustered. The proportions of clustered strains were similar in the urban and rural cohorts (70.6% and 69.1%, respectively). Although 71% of the isolates were present in clusters, a large number of these clusters had few isolates in them. Of the 587 clustered strains, 421 (72%) were in clusters with greater than 10 isolates in them. Fifty-one percent of the isolates formed only 8 clusters. Twenty-three clusters had only 2 isolates in them (Table 2). Additionally, we have reported 239/304 (79%) new spoligo-

5 VOL. 48, 2010 SPOLIGOTYPING M. TUBERCULOSIS STRAINS IN WESTERN INDIA 3597 types (22 clustered and 217 unique) which may be added to existing databases to provide a better representation of strains from a high-burden region (see Table S1 in the supplemental material). A concordance of 97% was found between spoligotyping results from our laboratory and IIDMM, UCT. Spoligotype associations (new cases). (i) Multidrug resistance. We did not observe any significant difference in the clustering percentages between treatment failures and new cases (68.8% and 70.6%, respectively) (data not shown) or across different drug susceptibility profiles (sensitive, 68%; MDR, 68%; single-drug resistant, 75%; other resistance, 73%) (data not shown). Analyses of associations between spoligotype and drug resistance showed a significantly higher proportion of Beijing strains than other strain types among the MDR isolates (OR, 3.40; 95% CL, 1.20 to 9.62; P 0.02). We also found a significantly higher proportion of CAS strains than other strain types in the pansensitive group (OR, 1.83; 95% CL, 1.03 to 3.24; P 0.03) (Table 5). Beijing strains were also found to be significantly associated with treatment failures (OR, 3.29; 95% CL, 1.29 to 8.14; P 0.003), which was independent of MDR, as found by multivariate analysis. (ii) Extent of radiographic disease. We further observed that infection with CAS was significantly associated with the presence of cavities on chest radiographs, compared to results FIG. 2. Spoligotype proportions. for the rest of the cohort (OR, 2.72; 95% CL, 1.34 to 5.53; P 0.006) (Table 5). (iii) Geographical origin of patient. On analyzing the spoligotypes for association with specific host background (Table 4), we found a significantly high number of CAS strains in isolates from people originating from the northern regions of compared to the numbers in the rest of the cohort (OR, 1.84; 95% CL, 1.08 to 2.99; P 0.02) (Table 5) and in the indigenous population (OR, 2.05; 95% CL, 1.18 to 3.56; P 0.006). We also found a higher proportion of Manu1 strains in the rural population than in the urban new cases (OR, 0.67; 95% CL, 0.42 to 1.05; P 0.06) (data not shown) and a significantly higher proportion than in the urban cohort (OR, 1.57; 95% CL, 0.41 to 1.00; P 0.04). No significant associations between spoligotype and age or gender were seen. (iv) HIV status. Of the 833 patients, HIV status was available for 722 individuals. Of these, 32 (4.5%) had tested positive. No association between HIV status and spoligotype was found. DISCUSSION This to the best of our knowledge is the largest communitybased molecular fingerprinting study of M. tuberculosis with a well-characterized cohort of new pulmonary tuberculosis cases TABLE 5. Significant associations between spoligotype and biological parameter Parameter No. of isolates of spoligotype c OR 95% CL P value Manu1 CAS Beijing EAI-5 Rural cohort a 38* Treatment failures b * % of isolates MDR within a cluster (urban new cases) b * % of isolates pansensitive within a cluster (urban new cases) b 33 53* % of patients with lung cavities within a cluster a 54 83* % of patients from north within a cluster b 27 40* a Chi-square test. b Binary logistic regression. c *, proportion is significantly high compared to those of other spoligotypes.

6 3598 CHATTERJEE ET AL. J. CLIN. MICROBIOL. from (1, 2, 3, 11, 16, 22, 28, 29, 30). We included only sputum-positive patients to enhance the possibility of typing actively transmitting strains. Although the exclusion of sputum-negative cases could potentially have introduced a bias in the strain composition, we were unable to perform sputum culture on all patients due to study constraints. We demonstrated a predominance of Manu1 along with a high proportion of CAS strains in our cohort, versus the predominance of Harlem and LAM strains in Africa and South America, Beijing and T strains in the Americas, the Beijing strain in East Asia, and the T strain in Europe (5, 32). Concurrent with earlier reports from (28) and specifically Mumbai (16), we found a low proportion of the Beijing strain in our cohort. This is different from the 23% proportion of Beijing reported by a tertiary care center in Mumbai catering to a more affluent population (1). The high incidence of Beijing in that study may be attributed to the selection bias toward MDR TB patients and also to a significantly higher proportion of migrant Tibetan populations accessing the tertiary care center (personal communication from Camilla Rodriguez, Hinduja Hospital, Mumbai). Additionally, we found a high strain diversity, with a large number of small clusters, as well as a significant proportion (79%) of strains hitherto unreported in the global databases. In comparing the spoligotype proportions reported from other studies in (3, 28, 29) to our observations, we found that our cohort had a higher proportion of Manu1 (26% versus 7%), a lower proportion of EAI-5 (4% versus 20 to 45%), and a comparable proportion of CAS (9% versus 5 to 43%). These results may indicate that different strains predominate in different regions of the country. The low proportion of Beijing in the urban cohort and its absence in the rural cohort suggest that this strain remains uncommon among the general population in this part of. The independent association of Beijing with MDR and treatment failure cases (as brought out by multivariate analysis) supports earlier findings (23, 25). In contrast, CAS was found to be associated with drug sensitivity and with cavitation. These observations suggest that modern strains like Beijing may have evolved more under drug pressures, leading to an accumulation of drug-resistant mutations, while ancestral strains, like CAS, may have evolved to cause cavitation and hence increase the opportunity for transmission (24, 34). Conclusion. Results from this study raise issues that may be typical for a cosmopolitan population in which TB is endemic. The constant exposure of the host to the pathogen (as in a setting of disease endemicity) has probably resulted in the high strain diversity. In contrast to the variability, the predominance of Manu1 (highest proportion ever reported) is indicative of a persistence of local strains. Interestingly, the high proportion of Manu1 in the urban cohort was significantly lower than that in the more homogenous rural cohort. Concurrently, Beijing (considered to be an imported strain), displaying a low proportion in the urban cohort, was absent in the more homogeneous rural cohort. The increasing predominance of one strain (Manu1) along with the decreasing penetrance of another (Beijing) as the homogeneity of the population increased from urban to rural probably reflects the effect of migration. This is further substantiated by the significant presence of strains predominant elsewhere in (CAS and EAI-5) in the cosmopolitan urban cohort. The preliminary inferences from this study plead for a more extensive analysis of the data to study the variability of M. tuberculosis strains and their transmission dynamics. 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