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Journal of Clinical Microbiology, July 2002, p. 2677-2680, Vol. 40, No. 7
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.7.2677-2680.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Spoligotyping of Mycobacterium tuberculosis Isolates from Multiple-Drug-Resistant Tuberculosis Patients from Bombay, India

Nerges F. Mistry,1* Anand M. Iyer,1 Desirée T. B. D'souza,1 G. Michael Taylor,2 Douglas B. Young,2 and Noshir H. Antia1

The Foundation for Medical Research, Bombay 400018, India,1 Department of Infectious Disease and Microbiology, Centre for Molecular Microbiology and Infection, Faculty of Medicine, Imperial College School of Science, Technology, and Medicine, London SW7 2AZ, United Kingdom2

Received 10 December 2001/ Returned for modification 12 March 2002/ Accepted 7 April 2002


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ABSTRACT
 
Spoligotyping was undertaken in 65 multiple-drug-resistant Mycobacterium tuberculosis isolates from Bombay, India. The spoligotype patterns showed seven closely related clusters, a cluster with 2 Beijing-like isolates, and unique spoligotypes (43%). Of the clusters, one with 29% of all the isolates suggested transmission of a dominant resistant clone.


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TEXT
 
The increasing worldwide prevalence of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis represents a major threat to tuberculosis (TB) control programs (8). Although data on drug-resistant TB are lacking in India due to the absence of a reliable surveillance network, a World Health Organization survey in 1997 in the state of Tamil Nadu (28) and a limited study in an urban tertiary care center in Bombay (25) reported figures of 7.1 and 58% prevalence of multidrug resistance, respectively, indicating that MDR TB may pose significant problems. The design of strategies for the management of MDR TB depends on an understanding of the development and spread of resistant isolates. Well-documented outbreaks in settings of low endemicity demonstrate the efficacy of MDR TB isolates in generating new incident cases (4), but less is known about the ability of resistant isolates to compete with other strains of M. tuberculosis in areas of high endemicity such as Bombay.

The objective of this study was therefore to obtain an initial assessment of the extent to which the transmission of dominant clones of M. tuberculosis contributes to MDR TB in Bombay. The few studies on the molecular epidemiology of Indian M. tuberculosis isolates show low copy numbers of the IS6110 insertion element, making them refractory to typing by the standard restriction fragment length polymorphism (RFLP) system (19, 20). The present study used spoligotyping, a PCR technique based on DNA polymorphism at the direct repeat locus of the genome of the M. tuberculosis complex (11, 13), as an alternative to IS6110 RFLP. Although the overall discriminatory power of spoligotyping is lower than that of IS6110 typing (11), it has the specific advantage of higher discrimination of strains with low copy numbers of IS6110 (3).

We describe a cross-sectional analysis of a panel of 65 MDR TB isolates from Bombay. The patients were referred to the Foundation for Medical Research by local public and private providers for refractoriness of their infections to standard antituberculous therapy. Demographic details of the patients are presented in Table 1.


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  		TABLE 1. Study population

Patient specimens, including early morning sputum samples (n = 53) or blood, in the absence of productive cough (in 12 human immunodeficiency virus [HIV]-positive patients), were collected. The sputum and blood samples were processed by the modified Petroff's method (2) and the lysis-centrifugation method (15), respectively, cultured in Dubos' liquid medium and Löwenstein-Jensen slants and tested for niacin and pyrazinamidase production to confirm their identity as M. tuberculosis (14).

Drug sensitivity testing was performed in 53 of the 65 isolates by the radiorespirometric Buddemeyer technique (5). A growth index of >=20% of the positive control for any drug was considered to indicate resistance to that drug. Multiple-drug resistance, as opposed to the classical definition, was defined as resistance to any two or more anti-TB drugs.

The genomic DNA from the isolates was extracted by the cetyltrimethylammonium bromide-phenol-chloroform method (22) and spoligotyped according to the method described previously (13). The spoligotype patterns were analyzed and corroborated manually by two independent observers. A cluster was defined as two or more isolates from different patients with identical spoligotype patterns, whereas nonclustered patterns were referred to as unique.

A total of 37 (57%) strains could be grouped into 8 different clusters (Fig. 1). The largest cluster (cluster 1) comprised 19 isolates, whereas the remaining seven clusters consisted of 2 to 4 isolates. Unique (nonclustered) spoligotype patterns were seen in 28 (43%) isolates. Of the 14 isolates from HIV-seropositive individuals, 9 belonged to the single largest cluster of 19 isolates (cluster 1).



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  		FIG. 1. Spoligotype patterns of clustered isolates. The filled boxes represent the presence of spacers, and the periods represent the absence of spacers. The numbers along the left of the figure are cluster numbers.

The spoligotype pattern of the 2 isolates in cluster 8 (Fig. 1), with hybridization only to the 3'-terminal spacers 35 to 43, is characteristic of the Beijing genotype, a dominant strain in many Asian countries (1, 26). IS6110 RFLP of the isolates showed identical patterns of 15 bands (data not shown), again consistent with the Beijing genotype. There was no obvious epidemiological link between the two patients infected with Beijing strains with respect to the locations of their residences, their occupations, or the commonality of referring health centers. Both isolates were resistant to all individual first- and second-line drugs. A similar frequency of the Beijing genotype (3%) was previously reported among a panel of strains from India (26) and is in marked contrast to the dominance of this genotype in other parts of Asia (1, 26). The detection of the Beijing genotype is important, however, because of its reported association with drug resistance (1, 7) and with outbreaks of MDR TB (4, 7).

Two nonclustered isolates (Fig. 2, E7 and E8) had spoligotype patterns lacking terminal spacers 39 to 43, a profile generally associated with Mycobacterium bovis (6, 13, 16). Although both isolates were positive for niacin production, like M. bovis, they showed a negative pyrazinamidase test. However, further PCR-based genotyping of these isolates with previously described methods for RD5 and RD7 deletions (21), and commonly associated with M. bovis strains (10, 17, 21, 27), showed an absence of these deletion events, as did a novel method employing flanking primers for the RD8 region. The sequences of the RD8 primers were 5'-GAGTCTATATAGTGTGCTCATGGGGCTAGC-3' (forward) and 5'-GCTTGCTGGCGATCATTGGTCT-3' (reverse). These amplify a 178-bp product in strains which have undergone this deletion event. PCR conditions for the RD8 PCR were identical to those for the RD7 PCR. Moreover, amplification and sequencing of the oxyR285 polymorphic site, as described previously (21, 24), showed nucleotide G, which is typical of M. tuberculosis isolates (23). The balance of evidence thus suggests the amplification failure of one or more terminal direct repeat spacers in M. tuberculosis, possibly due to the rearrangement or deletion of this region. Such intermediate profiles (12) may represent an ongoing evolution among these strains for broadening host tropism.



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  		FIG. 2. Spoligotype patterns of unique isolates. The filled boxes represent the presence of spacers, and the periods represent the absence of spacers. The codes along the left of the figure are patient codes.

The chi-square test with Yates' correction was used to compare the association of the clustering of spoligotypes with HIV serostatus, age, and gender of patients. Overall, there was no significant difference in the clustering of spoligotypes between HIV-seropositive and -seronegative groups (Table 1). Similarly, there was no association of clustering with age, gender, history of previous treatment (Table 1), or specific drug susceptibility patterns (Table 2).


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  		TABLE 2. Drug sensitivity testing of the isolates by using the radiorespirometric Buddemeyer assay

The detection of 36 different spoligotypes among the panel of drug-resistant isolates is consistent with a high rate of secondary, or acquired, resistance in this population, reflecting the problems of efficient TB control in a metropolis with overburdened health facilities (D. D'souza, N. F. Mistry, B. A. Rajgor, and N. H. Antia, submitted for publication). However, the sharing of a single spoligotype by 29% (19 of 65) of the isolates (cluster 1) suggests an important role for the transmission of a dominant resistant clone. While alternative genotypic techniques (9, 16, 18) are required to determine whether cluster 1 indeed represents a clonal population, this study may provide the basis for a systematic and extended study of MDR TB strains in India.


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ACKNOWLEDGMENTS
 
N.M. was supported by travel grant no. 062287/Z/00/Z from the Wellcome Trust, United Kingdom, for carrying out the spoligotyping of the M. tuberculosis isolates in London. The financial support received by the FMR from the Bombay Community Public Trust and ICICI Ltd. is gratefully acknowledged.

We are grateful to Sven Hoffner, The Swedish Institute for Infectious Disease Control, Stockholm, Sweden, for carrying out the IS6110 RFLP of the Beijing isolates. We thank Yatin Dholakia and Meena Hingorani for referring some of the patients included in the study and Bhakti Oza for technical assistance. We also thank Sheela Rangan and Tannaz Birdi for helpful discussions and inputs.


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FOOTNOTES
 
* Corresponding author. Mailing address: The Foundation for Medical Research, 84-A, R.G. Thadani Marg, Worli, Bombay 400018, India. Phone: 91 22 4934989. Fax: 91 22 4932876. E-mail: frchbom{at}bom2.vsnl.net.in. Back


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REFERENCES
 
    1
  1. Anh, D. D., M. W. Borgdoff, L. N. Van, N. T. N. Lan, T. van Gorkom, K. Kremer, and D. van Soolingen. 2000. Mycobacterium tuberculosis Beijing genotype emerging in Vietnam. Emerg. Infect. Dis. 6:302-305.[Medline]
    2. 2
  2. Baker, J. F., and R. E. Silverton. 1978. Introduction to medical laboratory technology, 5th ed., p. 528-530. Butterworths, London, England.
    3. 3
  3. Bauer, J., Å. B. Andersen, K. Kremer, and H. Miörner. 1999. Usefulness of spoligotyping to discriminate IS6110 low-copy-number Mycobacterium tuberculosis complex strains cultured in Denmark. J. Clin. Microbiol. 37:2602-2606.[Abstract/Free Full Text]
    4. 4
  4. Bifani, P. J., B. Mathema, Z. Lin, S. I. Mogazeh, B. Shopai, B. Tempalaki, J. Driacoll, R. Frothingham, J. M. Musser, P. Aleabea, and B. N. Kreiswirth. 1999. Identification of a W variant outbreak of Mycobacterium tuberculosis via population based molecular epidemiology. JAMA 282:2321-2327.[Abstract/Free Full Text]
    5. 5
  5. Boonkitticharoen, V., J. C. Ehrhardt, and P. T. Kirchner. 1987. Radiometric assay of bacterial growth: analysis of factors determining system performance and optimisation of assay technique. J. Nucl. Med. 28:209-217.[Abstract/Free Full Text]
    6. 6
  6. Costello, E., D. O'Grady, O. Flynn, R. O'Brien, M. Rogers, F. Quigley, J. Egan, and J. Griffin. 1999. Study of restriction fragment length polymorphism analysis and spoligotyping for epidemiological investigation of Mycobacterium bovis infection. J. Clin. Microbiol. 37:3217-3222.[Abstract/Free Full Text]
    7. 7
  7. Diaz, R., K. Kremer, P. E. W. de Haas, R. I. Gomez, A. Marrero, J. A. Valdivia, J. D. A. van Embden, and D. van Soolingen. 1998. Molecular epidemiology of tuberculosis in Cuba outside of Havana, July 1994-June 1994: utility of spoligotyping versus IS6110 restricted fragment length polymorphism. Int. J. Tuberc. Lung. Dis. 2:743-750.[Medline]
    8. 8
  8. Espinal, M., A. Laszlo, L. Simonsen, F. Boulahbal, S. J. Kim, A. Reniero, S. Hoffner, H. L. Rieder, N. Binkin, C. Dye, R. Williams, and M. C. L. Raviglione. 2001. Global trends in resistance to antituberculosis drugs. N. Engl. J. Med. 344:1294-1303.[Abstract/Free Full Text]
    9. 9
  9. Frothingham, R., and W. A. Meeker-O'Connell. 1998. Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats. Microbiology 144:1189-1196.[Abstract/Free Full Text]
    10. 10
  10. Gordon, S. V., R. Brosch, A. Billault, T. Garnier, K. Eiglemeier, and S. T. Cole. 1999. Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Mol. Microbiol. 32:643-655.[CrossRef][Medline]
    11. 11
  11. Goyal, M., N. A. Saunders, J. D. A. van Embden, D. B. Young, and R. J. Shaw. 1997. Differentiation of Mycobacterium tuberculosis isolates by spoligotyping and IS6110 restriction fragment length polymorphism. J. Clin. Microbiol. 35:647-651.[Abstract/Free Full Text]
    12. 12
  12. Kallenius, G., T. Koivula, S. Ghebremichael, S. E. Hoffner, R. Norberg, E. Svensson, F. Dias, B.-I. Marklund, and S. B. Svenson. 1999. Evolution and clonal traits of Mycobacterium tuberculosis complex in Guinea-Bissau. J. Clin. Microbiol. 37:3872-3878.[Abstract/Free Full Text]
    13. 13
  13. Kamerbeek, J., L. Schouls, A. Kolk, M. van Agterveld, D. van Soolingen, S. Kuijper, A. Bunschoten, H. Molhuizen, R. Shaw, M. Goyal, and J. D. A. van Embden. 1997. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35:907-914.[Abstract/Free Full Text]
    14. 14
  14. Kent, P. T., and G. P. Kubica. 1985. Public health mycobacteriology: a guide for the level III laboratory. Centers for Disease Control, U.S. Department of Health and Human Services, Atlanta, Ga.
    15. 15
  15. Kiehn, T. E., F. F. Edwards, P. Brannon, A. Y. Tsang, M. Maio, J. W. M. Gold, E. Whimbey, B. Wong, J. K. McClatchy, and D. Armstrong. 1985. Infections caused by Mycobacterium avium complex in immunocompromised patients. Diagnosis by blood culture and fecal contamination, antimicrobial susceptibility tests and morphological and seroagglutination characteristics. J. Clin. Microbiol. 21:168-173.[Abstract/Free Full Text]
    16. 16
  16. Kremer, K., D. van Soolingen, R. Frothingham, W. H. Haas, P. W. N. Hermans, C. Martin, P. Palittapongarnpim, B. B. Plikaytis, L. W. Riley, M. A. Yarkus, J. M. Musser, and J. D. A. van Embden. 1999. Comparison of methods based on different molecular epidemiological markers of typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility. J. Clin. Microbiol. 37:2607-2618.[Abstract/Free Full Text]
    17. 17
  17. Liebana, E., A. Aranaz, B. Francis, and D. Cousins. 1996. Assessment of genetic markers for species differentiation within the Mycobacterium tuberculosis complex. J. Clin. Microbiol. 34:933-938.[Abstract/Free Full Text]
    18. 18
  18. Mazars, E., S. Lesjean, A.-L. Banuls, M. Gilbert, V. Vincent, B. Gicquel, M. Tibayrenc, C. Locht, and P. Supply. 2001. High resolution minisatellite based typing of Mycobacterium tuberculosis molecular epidemiology. Proc. Natl. Acad. Sci. USA 98:1901-1906.[Abstract/Free Full Text]
    19. 19
  19. Radhakrishnan, I., Y. K. Manju, R. A. Kumar, and S. Mundayoor. 2001. Implications of low frequency of IS6110 in fingerprinting field isolates of Mycobacterium tuberculosis from Kerala, India. J. Clin. Microbiol. 39:1683.[Free Full Text]
    20. 20
  20. Sahadevan, R., S. Narayanan, C. N. Paramasivan, R. Prabhakar, and P. R. Narayanan. 1995. Restriction fragment length polymorphism typing of clinical isolates of Mycobacterium tuberculosis from patients with pulmonary tuberculosis in Madras, India, by use of direct-repeat probe. J. Clin. Microbiol. 33:3037-3039.[Abstract/Free Full Text]
    21. 21
  21. Sales, M. P. U., G. M. Taylor, S. Hughes, M. Yates, G. Hewinson, D. B. Young, and R. J. Shaw. 2001. Genetic diversity among Mycobacterium bovis isolates: a preliminary study of strains from animal and human sources. J. Clin. Microbiol. 39:4558-4562.[Abstract/Free Full Text]
    22. 22
  22. Small, P. M., R. W. Shafer, P. C. Hopewell, S. P. Singh, M. J. Murphy, E. Desmond, M. F. Sierra, and G. K. Schoolnik. 1993. Exogenous reinfection with multidrug-resistant Mycobacterium tuberculosis in patients with advanced HIV infection. N. Engl. J. Med. 328:1137-1143.[Abstract/Free Full Text]
    23. 23
  23. Sreevatsan, S., P. Escalante, X. Pan, D. A. Gillies II, S. Siddiqui, C. N. Khalaf, B. N. Kreiswirth, P. Bifani, L. G. Adams, T. Ficht, V. S. Perumaalla, M. D. Cave, J. D. A. van Embden, and J. M. Musser. 1996. Identification of a polymorphic nucleotide in oxyR specific for Mycobacterium bovis. J. Clin. Microbiol. 34:2007-2010.[Abstract/Free Full Text]
    24. 24
  24. Taylor, G. M., M. Goyal, A. J. Legge, R. J. Shaw, and D. Young. 1999. Genotypic analysis of Mycobacterium tuberculosis from medieval human remains. Microbiology 145:899-904.[Abstract/Free Full Text]
    25. 25
  25. Udwadia, Z. F., A. Hakimiyan, and C. Rodrigues. 1996. A profile of drug-resistant tuberculosis in Bombay. Chest 110:228S.
    26. 26
  26. van Soolingen, D., L. Qian, P. E. W. de Haas, J. T. Douglas, H. Traore, F. Portaels, H. Z. Qing, D. Enkhsaikan, P. Nyamadawa, and J. D. A. van Embden. 1995. Predominance of a single genotype of Mycobacterium tuberculosis in countries of East Asia. J. Clin. Microbiol. 33:3234-3238.[Abstract/Free Full Text]
    27. 27
  27. Weil, A., B. B. Plikaytis, W. R. Butler, C. L. Woodley, and T. M. Shinnick. 1996. The mtp40 gene is not present in all strains of Mycobacterium tuberculosis. J. Clin. Microbiol. 34:2309-2311.[Abstract/Free Full Text]
    28. 28
  28. World Health Organization. 2000. Antituberculosis drug resistance in the world. Report no. 2: prevalence and trends. W. H. O./CDS/TB/2000.278. World Health Organization, Geneva, Switzerland.

Journal of Clinical Microbiology, July 2002, p. 2677-2680, Vol. 40, No. 7
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.7.2677-2680.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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