1887

Abstract

, the causative agent of leprosy, is uncultivable in defined media. Development of new diagnostic tools which do not depend on growth of bacteria is needed for the early detection of and for monitoring the effectiveness of chemotherapy. We used a real-time PCR-based assay to quantify the copy number of bacterial DNA and mRNA from 47 leprosy patients using paraffin-embedded biopsy samples. The assay used was specific, sensitive and reproducible. The applicability of this approach in monitoring the chemotherapy of leprosy was examined. A reduction in DNA and mRNA during chemotherapy was observed and mRNA could not be detected in patients who underwent 2 years of multidrug therapy (MDT). However, a considerable amount of DNA could be detected even after 2 years of MDT. A significant amount of mRNA was found in reactional cases as well. This raises important questions regarding the role of bacterial antigens in leprosy reactions and the rationale of omitting antibiotics in the treatment of reactional cases. Results in this study show that real-time PCR could be a better tool for the careful monitoring of bacillary DNA and mRNA in lesions, which will help to improve diagnosis, disease progression and the treatment regimen.

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2009-06-01
2019-10-15
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References

  1. Britton, W. J. & Lockwood, D. N. ( 2004; ). Leprosy. Lancet 363, 1209–1219.[CrossRef]
    [Google Scholar]
  2. Chae, G. T., Kim, M. J., Kang, T. J., Lee, S. B., Shin, H. K., Kim, J. P., Ko, Y. H., Kim, S. H. & Kim, N. H. ( 2002; ). DNA-PCR and RT-PCR for the 18-kDa gene of Mycobacterium leprae to assess the efficacy of multi-drug therapy for leprosy. J Med Microbiol 51, 417–422.
    [Google Scholar]
  3. Clark-Curtiss, J. E., Jacobs, W. R., Docherty, M. A., Ritchie, L. R. & Curtiss, R. ( 1985; ). Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae. J Bacteriol 161, 1093–1102.
    [Google Scholar]
  4. Cole, S. T., Eiglmeier, K., Parkhill, J., James, K. D., Thomson, N. R., Wheeler, P. R., Honoré, N., Garnier, T., Churcher, C. & other authors ( 2001; ). Massive gene decay in the leprosy bacillus. Nature 409, 1007–1011.[CrossRef]
    [Google Scholar]
  5. Cox, R. A., Kempsell, K., Fairclough, L. & Colston, M. J. ( 1991; ). The 16S ribosomal RNA of Mycobacterium leprae contains a unique sequence which can be used for identification by the polymerase chain reaction. J Med Microbiol 35, 284–290.[CrossRef]
    [Google Scholar]
  6. Donoghue, H. D., Holton, J. & Spigelman, M. ( 2001; ). PCR primers that can detect low levels of Mycobacterium leprae DNA. J Med Microbiol 50, 177–182.
    [Google Scholar]
  7. Giulietti, A., Overbergh, L., Valckx, D., Decallonne, B., Bouillon, R. & Mathieu, C. ( 2001; ). An overview of real time quantitative PCR: applications to quantify cytokine gene expression. Methods 25, 386–401.[CrossRef]
    [Google Scholar]
  8. Godfrey, T. E., Kim, S. H., Chavira, M., Ruff, D. W., Warren, R. S., Gray, J. W. & Jensen, R. H. ( 2000; ). Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitative reverse transcription-polymerase chain reaction. J Mol Diagn 2, 84–91.[CrossRef]
    [Google Scholar]
  9. Gupta, U. D., Katoch, K., Singh, H. B., Natrajan, M., Sharma, V. D. & Katoch, V. M. ( 1999; ). Detection of viable organisms in leprosy patients treated with multidrug therapy. Acta Leprol 11, 89–92.
    [Google Scholar]
  10. Gupta, U. D., Katoch, K., Singh, H. B., Natrajan, M. & Katoch, V. M. ( 2005; ). Persister studies in leprosy patients after multi-drug treatment. Int J Lepr Other Mycobact Dis 73, 100–104.
    [Google Scholar]
  11. Hu, Y., Mangan, J. A., Dhillon, J., Sole, K. M., Mitchison, D. A., Butcher, P. D. & Coates, A. R. ( 2000; ). Detection of mRNA transcripts and active transcription in persistent Mycobacterium tuberculosis induced by exposure to rifampin or pyrazinamide. J Bacteriol 182, 6358–6365.[CrossRef]
    [Google Scholar]
  12. Jamil, S., Keer, J. T., Lucas, S. B., Dockrell, H. M., Chiang, T. J., Hussain, R. & Stoker, N. G. ( 1993; ). Use of polymerase chain reaction to assess efficacy of leprosy chemotherapy. Lancet 342, 264–268.[CrossRef]
    [Google Scholar]
  13. Job, C. K., Drain, V., Williams, D. L., Gillis, T. P., Truman, R. W., Sanchez, R. M., Deming, A. T. & Hastings, R. C. ( 1991; ). Comparison of polymerase chain reaction technique with other methods for detection of Mycobacterium leprae in tissues of wild nine-banded armadillos. Lepr Rev 62, 362–373.
    [Google Scholar]
  14. Katoch, V. M. ( 2002; ). Advances in the diagnosis and treatment of leprosy. Expert Rev Mol Med 4, 1–14.
    [Google Scholar]
  15. Klee, S. R., Tyczka, J., Ellerbrok, H., Franz, T., Linke, S., Baljer, G. & Appel, B. ( 2006; ). Highly sensitive real-time PCR for specific detection and quantification of Coxiella burnetii. BMC Microbiol 6, 2 [CrossRef]
    [Google Scholar]
  16. Kramme, S., Bretzel, G., Panning, M., Kawuma, J. & Drosten, C. ( 2004; ). Detection and quantification of Mycobacterium leprae in tissue samples by real-time PCR. Med Microbiol Immunol 193, 189–193.[CrossRef]
    [Google Scholar]
  17. Kumar, B., Dogra, S. & Kaur, I. ( 2004; ). Epidemiological characteristics of leprosy reactions: 15 years experience from north India. Int J Lepr Other Mycobact Dis 72, 125–133.[CrossRef]
    [Google Scholar]
  18. Kurabachew, M., Wondimu, A. & Ryon, J. J. ( 1998; ). Reverse transcription-PCR detection of Mycobacterium leprae in clinical specimens. J Clin Microbiol 36, 1352–1356.
    [Google Scholar]
  19. Lehmann, U. & Kreipe, H. ( 2001; ). Real time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods 25, 409–418.[CrossRef]
    [Google Scholar]
  20. Lienhardt, C. & Fine, P. E. ( 1994; ). Type 1 reaction, neuritis and disability in leprosy. What is the current epidemiological situation? Lepr Rev 65, 9–33.
    [Google Scholar]
  21. Lini, N., Rehna, E. A., Shiburaj, S., Maheshwari, J. J., Shankernarayan, N. P. & Dharmalingam, K. ( 2008; ). Functional characterization of a small heat shock protein from Mycobacterium leprae. BMC Microbiol 8, 208 [CrossRef]
    [Google Scholar]
  22. Lockwood, D. N. ( 1996; ). The management of erythema nodosum leprosum: current and future options. Lepr Rev 67, 253–259.
    [Google Scholar]
  23. Martinez, A. N., Britto, C. F., Nery, J. A., Sampaio, E. P., Jardim, M. R., Sarno, E. N. & Moraes, M. O. ( 2006; ). Evaluation of real time and conventional PCR targeting complex 85 genes for detection of Mycobacterium leprae DNA in skin biopsy samples from patients diagnosed with leprosy. J Clin Microbiol 44, 3154–3159.[CrossRef]
    [Google Scholar]
  24. Naafs, B. ( 2000; ). Viewpoint: leprosy after the year 2000. Trop Med Int Health 5, 400–403.[CrossRef]
    [Google Scholar]
  25. Ohno, H., Zhu, G., Mohan, V. P., Chu, D., Kohno, S., Jacobs, W. R. J. & Chan, J. ( 2003; ). The effects of reactive nitrogen intermediates on gene expression in Mycobacterium tuberculosis. Cell Microbiol 5, 637–648.[CrossRef]
    [Google Scholar]
  26. Plikaytis, B. B., Gelber, R. H. & Shinnick, T. M. ( 1990; ). Rapid and sensitive detection of Mycobacterium leprae using a nested-primer gene amplification assay. J Clin Microbiol 28, 1913–1917.
    [Google Scholar]
  27. Rastogi, N., Goh, K. S. & Berchel, M. ( 1999; ). Species-specific identification of Mycobacterium leprae by PCR-restriction fragment length polymorphism analysis of the hsp65 gene. J Clin Microbiol 37, 2016–2019.
    [Google Scholar]
  28. Ridley, D. S. & Jopling, W. H. ( 1966; ). Classification of leprosy according to immunity. A five-group system. Int J Lepr Other Mycobact Dis 34, 255–273.
    [Google Scholar]
  29. Santhosh, R. S., Pandian, S. K., Lini, N., Shabaana, A. K., Nagavardhini, A. & Dharmalingam, K. ( 2005; ). Cloning of mce1 locus of Mycobacterium leprae in Mycobacterium smegmatis mc2 155 SMR5 and evaluation of expression of mce1 genes in M. smegmatis and M. leprae. FEMS Immunol Med Microbiol 45, 291–302.[CrossRef]
    [Google Scholar]
  30. Scollard, D. M., Adams, L. B., Gillis, T. P., Krahenbuhl, J. L., Truman, R. W. & Williams, D. L. ( 2006; ). The continuing challenges of leprosy. Clin Microbiol Rev 19, 338–381.[CrossRef]
    [Google Scholar]
  31. Shabaana, A. K., Venkatasubramani, R., Narayan, N. S., Hoessli, D. C. & Dharmalingam, K. ( 2001; ). Cytokine profiles in paraffin-embedded biopsy samples of lepromatous leprosy patients: semi-quantitative measure of cytokine mRNA using RT-PCR. Int J Lepr Other Mycobact Dis 69, 204–214.
    [Google Scholar]
  32. Shabaana, A. K., Shankernarayan, N. P. & Dharmalingam, K. ( 2003; ). Mycobacterium leprae 18-kDa heat shock protein gene is polymorphic. Curr Sci 84, 64–70.
    [Google Scholar]
  33. Sharma, A., Sharma, V. K., Rajwanshi, A., Das, A., Kaur, I. & Kumar, B. ( 1999; ). Presence of M. leprae in tissues in slit skin smear negative multibacillary (MB) patients after WHO-MBR. Lepr Rev 70, 281–286.
    [Google Scholar]
  34. Tadesse, A., Abebe, M., Bizuneh, E., Mulugeta, W., Aseffa, A. & Shannon, E. J. ( 2006; ). Effect of thalidomide on the expression of TNF-alpha m-RNA and synthesis of TNF-alpha in cells from leprosy patients with reversal reaction. Immunopharmacol Immunotoxicol 28, 431–441.[CrossRef]
    [Google Scholar]
  35. Van Brakel, W. H., Khawas, I. B. & Lucas, S. B. ( 1994; ). Reactions in leprosy: an epidemiological study of 386 patients in west Nepal. Lepr Rev 65, 190–203.
    [Google Scholar]
  36. Wakade, A. V. & Shetty, V. P. ( 2006; ). Isolation of Mycobacterium leprae from untreated borderline tuberculoid, mid-borderline and indeterminate cases using the mouse foot pad technique – a study of 209 cases. Lepr Rev 77, 366–370.
    [Google Scholar]
  37. Williams, D. L., Gillis, T. P., Booth, R. J., Looker, D. & Watson, J. D. ( 1990; ). The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae. J Infect Dis 162, 193–200.[CrossRef]
    [Google Scholar]
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vol. , part 6, pp. 753-759

Clinical features of the leprosy patients in this study. Absence of mRNA in skin lesions of psoriasis patients. [PDF](119 KB)



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