1887

Abstract

Rifampicin-resistant is regularly reported and drug resistance is a major threat for the elimination of leprosy. There is an urgent need for a simple method that can detect rifampicin resistance in clinical isolates. This study developed a multiple-primer PCR amplification refractory mutation system, a simple, reliable and economical method for clinical specimens that allowed the rapid detection of mutations in the nucleotides of the codon for Ser425 of the gene, mutation of which to Leu, Met or Phe is associated with rifampicin resistance. The approach involved a multiple-primer PCR in which both mutant-specific and normal sets of primers were included in the reaction. The mutant-specific primer was complementary to the corresponding sequence of the wild-type gene except for one additional deliberate mismatch at the fourth nucleotide from the 3′-OH terminus. A single mismatch has little influence on the yield of PCR products, but if there are two mismatches as a result of mutation at the position being tested, the mutant-specific primer will not function in PCR under appropriate conditions, leading to no yield of PCR product from the mutant allele. The assay was evaluated successfully using a panel of plasmids and reference strains carrying the wild-type or known mutations. The assay was subsequently applied to DNA extracts from skin biopsies taken from patients. In all biopsy samples, the wild-type allele was detected for Ser425. The PCR results correlated with rifampicin susceptibility, as also measured by the traditional mouse footpad technique.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.47534-0
2008-02-01
2019-10-21
Loading full text...

Full text loading...

/deliver/fulltext/jmm/57/2/179.html?itemId=/content/journal/jmm/10.1099/jmm.0.47534-0&mimeType=html&fmt=ahah

References

  1. Cambau, E., Bonnafous, P., Perani, E., Sougakoff, W., Ji, B. & Jarlier, V. ( 2002; ). Molecular detection of rifampin and ofloxacin resistance for patients who experience relapse of multibacillary leprosy. Clin Infect Dis 34, 39–45.[CrossRef]
    [Google Scholar]
  2. Ebenezer, G. J., Norman, G., Joseph, G. A., Daniel, S. & Job, C. K. ( 2002; ). Drug resistant Mycobacterium leprae – results of mouse footpad studies from a laboratory in south India. Indian J Lepr 74, 301–312.
    [Google Scholar]
  3. Fan, X. Y., Hu, Z. Y., Xu, F. H., Yan, Z. Q., Guo, S. Q. & Li, Z. M. ( 2003; ). Rapid detection of rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates in Shanghai by using the amplification refractory mutation system. J Clin Microbiol 41, 993–997.[CrossRef]
    [Google Scholar]
  4. Grosset, J. H., Guelpa-Lauras, C. C., Bobin, P., Brucker, G., Cartel, J. L., Constant-Desportes, M., Flageul, B., Frederic, M., Guillaume, J. C. & Millan, J. ( 1989; ). Study of 39 documented relapses of multibacillary leprosy after treatment with rifampin. Int J Lepr Other Mycobact Dis 57, 607–614.
    [Google Scholar]
  5. Honoré, N. & Cole, S. T. ( 1993; ). Molecular basis of rifampin resistance in Mycobacterium leprae. Antimicrob Agents Chemother 37, 414–418.[CrossRef]
    [Google Scholar]
  6. Honoré, N., Perrani, E. & Telenti, A. ( 1993; ). A simple and rapid technique for the detection of rifampin resistance in Mycobacterium leprae. Int J Lepr Other Mycobact Dis 61, 600–604.
    [Google Scholar]
  7. Honoré, N., Roche, P. W., Grosset, J. H. & Cole, S. T. ( 2001; ). A method for rapid detection of rifampicin-resistant isolates of Mycobacterium leprae. Lepr Rev 72, 441–448.
    [Google Scholar]
  8. Ji, B. ( 1987; ). Drug susceptibility testing of Mycobacterium leprae. Int J Lepr Other Mycobact Dis 55, S830–S835Medline
    [Google Scholar]
  9. Ji, B. ( 2002; ). Rifampicin resistant leprosy: a review and a research proposal of a pilot study. Lepr Rev 73, 2–8.
    [Google Scholar]
  10. Levy, L. & Ji, B. ( 2006; ). The mouse foot-pad technique for cultivation of Mycobacterium leprae. Lepr Rev 77, 5–24.
    [Google Scholar]
  11. Maeda, S. ( 2004; ). Multi-drug resistant Mycobacterium leprae from patients with leprosy. Nihon Hansenbyo Gakkai Zasshi 73, 227–233.[CrossRef]
    [Google Scholar]
  12. Maeda, S., Matsuoka, M., Nakata, N., Kai, M., Maeda, Y., Hashimoto, K., Kimura, H., Kobayashi, K. & Kashiwabara, Y. ( 2001; ). Multidrug resistant Mycobacterium leprae from patients with leprosy. Antimicrob Agents Chemother 45, 3635–3639.[CrossRef]
    [Google Scholar]
  13. Matsuoka, M., Kashiwabara, Y. & Namisato, M. ( 2000; ). A Mycobacterium leprae isolate resistant to dapsone, rifampin, ofloxacin and sparfloxacin. Int J Lepr Other Mycobact Dis 68, 452–455.
    [Google Scholar]
  14. Newton, C. R., Graham, A., Heptinstall, L. E., Powell, S. J., Summers, C., Kalsheker, N., Smith, J. C. & Markham, A. F. ( 1989; ). Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 17, 2503–2516.[CrossRef]
    [Google Scholar]
  15. Norman, G., Joseph, G., Ebenezer, G., Rao, S. P. & Job, C. K. ( 2003; ). Secondary rifampin resistance following multi-drug therapy – a case report. Int J Lepr Other Mycobact Dis 71, 18–21.[CrossRef]
    [Google Scholar]
  16. Sapkota, B. R., Ranjit, C. & Macdonald, M. ( 2006; ). Reverse line probe assay for the rapid detection of rifampicin resistance in Mycobacterium leprae. Nepal Med Coll J 8, 122–127.
    [Google Scholar]
  17. Scollard, D. M., Adams, L. B., Gillis, T. P., Truman, J. L. R. W. & Williams, D. L. ( 2006; ). The continuing challenges of leprosy. Clin Microbiol Rev 19, 338–381.[CrossRef]
    [Google Scholar]
  18. Shepard, C. ( 1960; ). The experimental disease that follows the injection of human leprosy bacillus into footpads of mice. J Exp Med 112, 445–454.[CrossRef]
    [Google Scholar]
  19. Shepard, C. C. & McRae, D. H. ( 1968; ). A method for counting acid-fast bacteria. Int J Lepr Other Mycobact Dis 36, 78–82.
    [Google Scholar]
  20. Suzuki, Y. & Matsuoka, M. ( 2006; ). DNA microarray based rapid drug susceptibility test for Mycobacterium leprae. Nihon Hansenbyo Gakkai Zasshi 75, 271–277 (in Japanese).[CrossRef]
    [Google Scholar]
  21. Williams, D. L. & Gillis, T. P. ( 2004; ). Molecular detection of drug resistance in Mycobacterium leprae. Lepr Rev 75, 118–130.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.47534-0
Loading
/content/journal/jmm/10.1099/jmm.0.47534-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error