Rapid detection of rifampin resistance mutations in clinical isolates of Mycobacterium tuberculosis by Dot-Blot hybridisation assay

Abstract number: 1732_316

Varma-Basil M., Pathak R., Ahmed S., Bhatnagar A., Bose M.

Objectives: A Dot-Blot hybridisation assay that detects all mutations occurring in the M. tuberculosis rpoB hot-spot region is being developed. The assay uses five probes, capable of binding to a different target segment within the rpoB hot-spot region of the wild-type M. tuberculosis genome. The present study is a preliminary investigation to assess the suitability of the assay for detection of resistance mutations in rpoB in clinical isolates of M. tuberculosis from Delhi, India.

Methods: Susceptibility testing of 142 isolates of M. tuberculosis was carried out by proportion method and confirmed by BACTEC 460TB system. Dot-Blot assay was performed on 106 isolates with two of the probes hybridising to the wild-type sequence of M. tuberculosis, from codons 522 to 527 (Probe D) and 528 to 533 (Probe E). Absence of hybridisation with any of the probes in the assay when a mutation was present indicated Rifampicin resistance, a surrogate marker for Multidrug-resistant M. tuberculosis.

Results: Susceptibility testing of 142 isolates revealed isoniazid resistance in 53.5%, rifampicin resistance in 41%, streptomycin resistance in 53% and ethambutol resistance in 37% of strains. Forty-five strains (32%) were multidrug resistant. Further analysis of the data showed that 96.4% of rifampicin-resistant strains and 100% of ethambutol-resistant strains had co resistance to one or the other antituberculous drug. It was also interesting to note that 77% of the rifampicin-resistant strains were multidrug resistant, while the corresponding figure for ethambutol-resistant strains was 87%.

Dot-Blot hybridisation assay with probes D and E was carried out on 106 isolates of M. tuberculosis, of which 43 were resistant to rifampicin. Of the rifampicin-resistant isolates tested, 15 did not hybridise with probe E, indicating a mutation at this site. The results of 10 strains were confirmed by sequencing when a mutation was detected in 9 strains at codon 531 and in one of them at codon 533. The remaining 28 rifampicin-resistant strains, which hybridised with probe E, may have a mutation at a site other than that complimentary to probe E. Of these, 10 hybridised with probe D, indicating a mutation at the site complimentary to probe D (codons 522 to 527). Of the 63 rifampicin-susceptible isolates, 62 (98%) hybridised with probes D and E, indicating a wild-type sequence.

Conclusions: The Dot-Blot assay was found to be a sensitive and specific assay.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Location: ICC, Munich, Germany
Presentation type:
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