Tracing Transmission of Tuberculosis By Random Amplified Polymorphic DNA (RAPD) Analysis Within Same

Oct 04, 04:13 AM

Current Headlines: By Singh, H B Chauhan, D S; Faujdar, J; Upadhyay, P; Et al

Sir, Molecular epidemiology that integrates molecular approaches into the convention epidemiologic investigations has proved quite useful in tracing the transmission of drug resistant Mycobacterium tuberculosis in USA1. Molecular epidemiological tools for tuberculosis include both restriction fragment length polymorphism (RFLP) as well as PCR methods2,3. The most useful methods are the IS 6110 RFLP analysis and the two PCR-based methods-spoligolytyping and mycobacterial interspersed repetitive unit (MIRU) analysis4-6. In our previous report, the typing of Indian isolates of Mycobacterium tuberculosis by random amplified polymorphic DNA (RAPD) analysis using an arbitrary primer 986 FP7, was found to be reproducible and promising to establish similarity in profiles, and clustering was found among isolates from Ranchi, New Delhi and Chandigarh8. In this report we have focused on the application of this system on analysis of M. tuberculosis isolates from a small group of patients living in two closely located slum areas of Agra city in north India to find out its utility in rapid discrimination of isolates.

A total of 16 coded isolates of M. tuberculosis from 16 patients (2 from the same family and 14 from neighbourhood) stored in the Mycobacterial Repository Centre of National JALMA Institute for Leprosy & other mycobacterial Diseases, Agra, obtained from TB sputum samples of Meena Charitable Hospital Lohamandi, Agra, were included. Isolates obtained from the sputa received from TB patients living in two closely located slum areas of Agra city viz., Jagdeeshpura and Lohamandi during April 2004 to September 2004, were studied. The isolates used in the study were obtained as a part of surveillance study on drug resistance of Government of India which was duly approved by the Ethical Committee of the Institute. DNAs from these isolates were isolated4 and were analyzed RAPD technique as described earlier7,8 taking the usual precautions.

There was a clear cluster of isolates from patients from same family (DAU-166, DAU-168) and neighbourhood (DAU-171, DAU-180, DAU- 165, DAU-162, DAU-156, DAU-40, DAU-16 (Figs. 1 and 2). These observations indicate utility of RAPD analysis in rapid discrimination of isolates from cases within a family and also from those who were living in these slum areas. Similar findings of showing evidence of transmission have been reported from Taiwan city using this system9. RAPD reproducibility is known to be affected by several factors such as the thermal cycler used10, the enzyme involved in the amplification11 and the age of the strain studied12. If the effect of these factors is not properly controlled, the RAPD analysis may not be suitable to be implemented as a molecular epidemiological tool. According to some earlier studies3,13, the RAPD analysis, although using different primer compared to the present study, showed a low sensitivity, reproducibility and discriminatory power in comparison with most prominent genotyping methods like IS 6110. MIRU- VNTR and spoligotyping. Unlike these studies, our RAPD analysis using primers described earlier did not produce different pattern for duplicate DNA sample8. Reproducibility was also reported to be good in other studies7,9. Though RAPD analysis has the advantage of being a simple, easy, fast and relatively low cost molecular typing method, important aspects like reproducibility, comparability and complimentary role with other discriminatory method like IS 6110(4), MIRU- VNTR6. spoligotyping5 and amplified fragment length polymorphism (AFLP)14 need to studied and are to be taken into consideration. There is an intense need to conduct well planned studies specially in densely populated areas such as slum areas of major cities of India and to find complementary role of RAPD with other discriminatory methods for studying dynamics of transmission, especially after introduction of DOTS. Our study indicates that RAPD analysis seems to have potential in tracing transmission of tuberculosis within same family and neighbourhood.

Acknowledgment Authors thank all technical colleagues for support provided in carrying out this study. Gift of reagents/plastic ware by LEPRA (UK) is gratefully acknowledged. Authors acknowledge the Central TB Division, New Delhi, Government of India for providing financial support.

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H.B. Singh, D.S. Chauhan, J. Faujdar P. Upadhyay, N. Saxena, V.S. Yadav, A. Kumar* I. Ahmed**, V.M. Katoch & V.D. Sharma[dagger]

Departments of Microbiology & Molecular Biology & * Biostatistics, National JALMA Institute for Leprosy (ICMR) & ** Meena Charitable Hospital

Agra 282001, India

[dagger] For corresponding e-mail: jalma@sancharnet.in

Copyright Indian Council of Medical Research Jul 2007

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