High Incidence of the Beijing Genotype among Multidrug-Resistant Isolates of Mycobacterium tuberculosis in a Tertiary Care Center in Mumbai, India

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1 BRIEF REPORT High Incidence of the Beijing Genotype among Multidrug-Resistant Isolates of Mycobacterium tuberculosis in a Tertiary Care Center in Mumbai, India Deepak Almeida, 1 Camilla Rodrigues, 2 Tester F. Ashavaid, 3 Ajit Lalvani, 5 Zarir F. Udwadia, 4 and Ajita Mehta 2 1 Research Labs, and Departments of 2 Microbiology, 3 Biochemistry, and 4 Pulmonary Medicine, P. D. Hinduja National Hospital and Medical Research Centre, Mahim, Mumbai, India; and 4 Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom We report a high frequency (35%) of the Beijing genotype among multidrug-resistant isolates recovered in and around Mumbai, India. Further restriction fragment length polymorphism typing showed that these strains were closely related. We also report a high frequency of the Delhi genotype (31% of isolates). Our data indicate considerable ongoing transmission of multidrug-resistant Mycobacterium tuberculosis strains of the Beijing genotype in Mumbai. The emergence and spread of drug-resistant tuberculosis has exacerbated the global resurgence of tuberculosis [1, 2]. A recent study performed at our hospital (P. D. Hinduja National Hospital and Medical Research Centre), a tertiary health care center in Mumbai, India, found that 51% of the patients who requested susceptibilty testing had multidrug-resistant tuberculosis [3]. Sampling bias cannot be ruled out, but, considering the fact that Mumbai is one of the densely populated cities in India, the study suggested the possibility of clonal spread. Studies have associated Mycobacterium tuberculosis of the Beijing genotype with drug resistance [4 6] and efficient transmission [1, 7 10]. This genotype is known to be predominant in Southeast Asia [4, 7, 11 13]; however, no studies from India, thus far, have reported a significant presence of these strains. There are very few genotyping studies from South and North India Received 13 July 2004; accepted 22 October 2004; electronically published 18 February Reprints or correspondence: Dr. Ajita Mehta, Dept. of Microbiology, P. D. Hinduja National Hospital and Medical Research Centre, Veer Sawarkar Marg, Mahim, Mumbai , India (dr_amehta@hindujahospital.com). Clinical Infectious Diseases 2005; 40: by the Infectious Diseases Society of America. All rights reserved /2005/ $15.00 in the literature [14 19], and there is only 1 study from Mumbai [20]. We therefore performed fingerprinting analysis of our drug-susceptible and multidrug-resistant isolates to determine whether there is any significant clonal spread in our geographic region. Also, fingerprinting analysis would serve to answer the following questions: (1) Does the Beijing genotype contribute significantly to the burden of tuberculosis in India? (2) If so, is the Beijing genotype preferentially associated with multidrug resistance? Methods. Our hospital is a tertiary care center with a referral bias toward nonresponding cases; it is located in central Mumbai. Culture, identification, and susceptibility testing of M. tuberculosis isolates were done using TB-Bactec 460 (Becton Dickinson). Of the 4009 samples that were cultured for M. tuberculosis between January 2001 and July 2002, a total of 1192 were positive for M. tuberculosis complex. Susceptibility testing was requested for 582 isolates, of which 342 were found to be mul- Table 1. Demographic data and infection and treatment history for 196 patients with Mycobacterium tuberculosis infection in Mumbai, India. Characteristic Patients with multidrug-resistant isolates (n p 129) Patients with pansusceptible isolates (n p 67) Age, years 15 9 (7) 2 (3) (52) 22 (33) (24) 20 (30) (13) 16 (23) 61 5 (4) 7 (10) Range Mean SD Sex Male 69 (53) 31 (46) Female 60 (47) 36 (54) Type of infection Pulmonary 113 (88) 48 (72) Extrapulmonary 16 (12) 19 (28) Treatment history No treatment Previous and/or current therapy Not available 3 4 NOTE. Data are no. (%) of patients, unless otherwise indicated. BRIEF REPORT CID 2005:40 (15 March) 881

2 Table 2. Clusters and unique isolates identified by spoligotyping. Cluster Multidrugresistant (n p 129) Isolates Spacer sequence Pansusceptible (n p 67) (34.8) 0 (0) (4.6) 3 (4.5) X.. X X X X X X X X X X X X X X X X X 3 4 (3.1) 0 (0) X X X X X X X X X X X X X X X X X X X X 4 5 (3.8) 3 (4.5) X X X X X X X X X X X X X 5 7 (5.4) 0 (0) X X X X X X X X X X X X X X X X X X X X 6 15 (11.6) 16 (23.9) X X X.... X X X X X X X X X X X X X 7 2 (1.5) 1 (1.5) X X X X X X X X X X X X X X X X X. X X 8 1 (0.8) 3 (4.5) X X X.... X X X X X... X X X X X 9 6 (4.6) 2 (3.0) X X X X X X X X X X X X. X X X X X X X 10 2 (1.5) 0 (0) X.. X X X X X X X X X X X X X X X X X 11 3 (2.3) 0 (0) X X X.... X X X X X X X X X X X X X 12 1 (0.8) 2 (3.0) X X X.... X X X X X X X X X X X X X 13 2 (1.5) 0 (0) X.. X X X X X X X X X X X X X X X X X 14 1 (0.8) 3 (4.5) X X. X X X X X X X X X. X X X X X X X 15 0 (0) 2 (3.0) X X X X X X X (1.5) 0 (0) X X..... X X X X X X X X X X X X X 17 1 (0.8) 1 (1.5) X X X.... X X X X X X X X X X X X. None a 26 (20.1) 31 (46.3) NOTE. The largest cluster, cluster 1, consisted of 45 strains and was identified as Beijing genotype; the genotype was present only in multidrug-resistant isolates. The second-largest cluster, cluster 6, consisted of 31 strains; the genotype was present in multidrug-resistant isolates, as well as in pansusceptible isolates. a Refers to unique isolates with fingerprints that were not shared by any other isolate. tidrug resistant and 129 were found to be pansusceptible. Spoligotyping was performed for 129 multidrug-resistant and 67 pansusceptible isolates recovered from 196 patients for whom demographic data and residential details were available. DNA extraction was done according to the standard cetyltrimethylammonium bromide sodium chloride procedure, as described elsewhere [21]. Spoligotyping was performed as described by Kamerbeek et al. [22]. IS6110 restriction fragment length polymorphism (RFLP) typing was performed according to a standard procedure [23]; only 88 strains (55 multidrugresistant and 33 pansusceptible strains) could be typed, because of a lack of adequate or good-quality DNA from the other strains. Multidrug-resistant strains were defined as strains that were resistant to (at least) both isoniazid and rifampin. Pansusceptible strains were defined as strains that were susceptible to streptomycin, isoniazid, rifampin, ethambutol, and pyrazinamide. A cluster was defined as 2 or more M. tuberculosis isolates that showed identical fingerprints (as determined either by spoligotyping or RFLP typing). Unique isolates were defined as isolates with distinct fingerprints (as determined either by spoligotyping or RFLP typing) that were not shared by any other isolate. Primary resistance was defined as resistance that was seen in patients with no history of antituberculosis therapy, and secondary resistance was defined as resistance that was seen in patients with a history of antituberculosis therapy. Results. The details of the patient demographics are given in table 1. Spoligotyping of 129 multidrug-resistant and 67 pansusceptible isolates showed 26 and 31 unique fingerprints, respectively, and 16 and 11 clusters, respectively; some clusters were common to both of the groups. Details of the results with the representative patterns are shown in table 2. Two large clusters were seen: cluster S1 and cluster S6. The latter cluster, which consisted of 15 multidrug-resistant and 16 pansusceptible isolates, was characterized by the presence of spacer numbers 4 7 and Cluster S1, the largest cluster, comprised 45 isolates that were present exclusively in the multidrugresistant group. The isolates in this cluster were characterized by the presence of 9 terminal spacer sequences, which is typical of the Beijing genotype (table 2). 882 CID 2005:40 (15 March) BRIEF REPORT

3 Spacer sequence Octal code (family/clade) X X X X X X X X X (Beijing) X X X X X X X X.... X. X X... X X X X (EAI3) X X X X X X X X.... X. X X X X X. X X X (EAI1) X X X X X X X X X X X (CAS2) X X X X X X X X X X X X.... X X X X X X X (T1) X X X X X X X X X X X (cas1 [Delhi]) X X X X X X X X X X X X.... X X X X X X X (X1) X X X X X X X X X X X (cas1) X X X X X X X X X X X X.... X X X X X X X (T3) X X X X X X X X X X. X.... X X X X X X X X X X X X X.. X X X X X X X X X X X X X.... X. X X X X X X X X X X X X X X X X X.... X X X. X X X X X X X X X X X X X.. X X. X X X X X X X X X X X X X X X X X X X X X RFLP typing was performed for 88 isolates; these isolates included, in addition to other smaller clusters, 32 isolates from the Beijing genotype (cluster 1) and 18 isolates from cluster 6. RFLP typing revealed that 29 (33%) of the 88 strains were closely related and were characterized by the presence of kilobase (kb) and 10.1-kb bands (figure 1). These strains represented a distinct group, which has recently been designated as the Delhi genotype [18]; 17 (94%) of the 18 cluster S6 isolates showed this genotype. Of 55 multidrug-resistant isolates, 32 showed unique patterns, and 23 were divided into 8 clusters, which were named R1 R8. Clusters R1 R3, R5, and R7 consisted of 2 isolates each. Cluster R4 contained 3 isolates, and the largest cluster, R6, contained 6 isolates. All strains in clusters R1 R7 belonged to the Beijing genotype (as identified by spoligotyping). Cluster R8 consisted of 4 isolates with a single IS6110 copy. Three of these strains also had the same spoligotype (i.e., cluster S2). In the pansusceptible group, only 1 cluster which comprised 3 isolates was seen; the RFLP pattern was same as that of cluster R8. Clustering was seen in 85 (75%) of the 113 patients with acquired resistance in the multidrug-resistant group (n p 129); 51% of the isolates were of the Beijing genotype. Clustering was also seen in 8 (62%) of 13 patients with primary resistance, and 37% of the isolates recovered were of the Beijing genotype. Overall, multidrug-resistant strains were more likely to be clustered than were pansusceptible strains (as determined by both spoligotyping and RFLP typing). The number of unique strains seen in the multidrug-resistant group was significantly less than the number seen in the pansusceptible group; the percentage of unique isolates is shown in table 3. Most isolates showed a preponderance of high numbers of IS6110 copies: of 88 isolates, 15 (17%) had 5 copies, and 9 (10%) had a single copy. Detailed distribution of the IS6110 copies is shown in table 4. Discussion. The present study identified the Beijing genotype as a predominant group among the multidrug-resistant isolates in our geographical region, accounting for nearly onethird of the multidrug-resistant isolates. None of the pansusceptible isolates showed this genotype. In contrast to the findings of the present study, a previous study from Mumbai [20] in 1999 found that, of 65 multidrug-resistant isolates, only 2 belonged to the Beijing genotype family. Another study from Delhi, India [18], found that, of 86 isolates, only 1 isolate belonged to the Beijing genotype. A more recent study from Delhi found that 8% of the isolates recovered in the study BRIEF REPORT CID 2005:40 (15 March) 883

4 Figure 1. Autoradiograph showing typical restriction fragment length polymorphism patterns. The external marker is a Mycobacterium tuberculosis (Mt) reference strain showing 12 predetermined bands. Numbers on the left indicate the molecular weight in kilobases (Kb); numbers at the bottom indicate the spoligotype patterns of the strains. Arrows denote the characteristic 12.1-kb and 10.1-kb band of the Delhi genotype. Lanes labeled S1 are isolates of the Beijing genotype strain (cluster S1). Lanes labeled S6 are isolates of the Delhi genotype strain (cluster S6). Lanes labeled U are unique isolates. belonged to the Beijing genotype [19]. Additional RFLP typing of our strains of the Beijing genotype did not reveal any large clusters, except for 1 cluster that contained 6 strains (i.e., cluster R6). Most of the strains, although not identical, were very closely related (figure 2). This similarity among RFLP patterns is suggestive of recent transmission among the Beijing strains [24] (figure 2). In addition, analysis of mutations in the rpob region of 23 multidrug-resistant, non Beijing genotype strains (with distinct fingerprints) showed a predominance of S531L mutations (in 61% of strains), followed by H526D, D516G, and D516Y mutations in 22%, 1%, and 1% of strains, respectively. Of the 7 Beijing strains that were analyzed (all with distinct RFLP profiles), a majority of strains (6 strains [86%]) had a S531L mutation, and only 1 (14%) had a H526D mutation. However, because the S531L mutation is the most commonly encountered mutation, it may not necessarily indicate recent transmission [25]. The present study also found that the newly identified Delhi genotype was prevalent in both multidrug-resistant and pansusceptible isolates. This genotype was found to be predominant (75%) among North Indian isolates [18]. Other studies have shown a lower prevalence of this Delhi genotype in Iran (27%) and in Southern India (13%) [18]. The high prevalence of this genotype in the present study and in other studies from North India [18, 19] may indicate that the phenotypic properties of the Delhi genotype are similar to the phenotypic properties of the Beijing genotype in that they aid in rapid dissemination across large geographic regions. The Beijing genotype was found to be associated exclusively with multidrug resistance. Although most of the strains of the Beijing genotype reported in the literature are pansusceptible [1], all of the strains of the Beijing genotype that were identified in the present study were multidrug resistant. The spread of multidrug-resistant strains of the Beijing genotype also has been documented in different geographic areas of the world, including Estonia, New York City, Cuba, and Vietnam [6 8, 13]. The Delhi genotype, unlike the Beijing genotype, was not re- Table 3. Percentage of unique isolates determined by spoligotyping and restriction fragment length polymorphism (RFLP) typing. Method Multidrug-resistant isolates Pansusceptible isolates x 2 P Spoligotyping 20 (26/129) 46 (31/67) 14.58!.001 RFLP typing 58 (32/55) 90 (30/33) 10.6!.01 NOTE. Data are percentage of unique isolates identified (no. of unique isolates identified/no. of isolates typed), unless otherwise indicated. 884 CID 2005:40 (15 March) BRIEF REPORT

5 Table 4. Total number of insertion elements seen in strains. No. of insertion elements No. of strains Total 88 NOTE. The distribution of the IS6110 copies among the strains is shown. Most of the strains showed a high number of copies; however, 9isolates contained a single copy. Strains of the Beijing genotype showed 9 18 copies, with a maximum (21 strains) showing copies. Strains of the Delhi genotype showed 9 18 copies, with a maximum (21 strains) showing copies. stricted to multidrug-resistant isolates but was also found in pansusceptible isolates; this indicates that the Beijing genotype was more associated with drug resistance than was the Delhi genotype. RFLP typing showed, contrary to the findings of studies from South India [16, 26], that most of the isolates had high numbers of IS6110 copies. None of our strains lacked the IS6110 sequence. This finding indicates considerable heterogeneity among Indian isolates with respect to IS6110 copies. The strains belonging to the Beijing/W genotype family have been shown to be associated with drug resistance [1] and are suggested to be genotypes with the potential to spread faster than other genotypes [10 13]. The high incidence of the Beijing genotype in our geographic region could possibly explain the high incidence of multidrug-resistant tuberculosis [3] and is a cause for great concern. Studies have also linked the Beijing genotype with patients belonging to a younger age group [7], but we did not find any significant association. The present study highlights considerable ongoing transmission of multidrug-resistant M. tuberculosis strains of the Beijing genotype and a high prevalence of strains of the Delhi genotype in Mumbai. These findings could have important implications for the control and prevention of tuberculosis. However, the results of the present study should not be overinterpreted. Because our samples were obtained from a tertiary care center, sampling bias cannot be ruled out. Also, the fact that many of the Beijing strains were from patients with acquired resistance strongly suggests that a susceptible population could be present in Mumbai. Moreover, we have no information about epidemiological associations between the patients. We presume that there were no such associations, because the patients were not from the same household or work place; however, we cannot rule out other possible associations. Larger studies with representative sampling are needed to elucidate the role of these genotypes in the dissemination and transmission of tuberculosis in India. Acknowledgments We thank Dick Van Soolingen (National Institute of Public Health and the Environment [RIVM]; Bilthoven, The Netherlands), for providing us with the probe and the standard markers for RFLP typing, and Peter Godfrey-Faussett (London School of Hygiene and Tropical Medicine; London, United Kingdom) and Tanu Singhal (P.D. Hinduja National Hospital and Medical Research Centre; Mumbai, India), for helpful advice. Financial support. National Health and Education Society of the P. D. Hinduja Hospital and Medical Research Centre. Potential conflicts of interest. All authors: no conflicts. Figure 2. Lane maps of IS6110 Southern blot hybridization patterns on some of the strains of the Beijing genotype. R1 R7 denote clusters, and unique strains are numbered. BRIEF REPORT CID 2005:40 (15 March) 885

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