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Journal of Clinical Microbiology, July 2009, p. 2124-2130, Vol. 47, No. 7
0095-1137/09/$08.00+0     doi:10.1128/JCM.00512-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Molecular Determination of Mycobacterium leprae Viability by Use of Real-Time PCR{triangledown}

Alejandra N. Martinez,1,2 Ramanuj Lahiri,2 Tana L. Pittman,2 David Scollard,2 Richard Truman,2 Milton O. Moraes,1* and Diana L. Williams2*

Laboratório de Hanseníase, Instituto Oswaldo Cruz—Fiocruz, Rio de Janeiro, Brazil,1 HRSA, BPHC, Division of National Hansen's Disease Programs, Laboratory Research Branch at the School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana2

Received 12 March 2009/ Accepted 1 May 2009

Mycobacterium leprae, the etiological agent of leprosy, is noncultivable on axenic media. Therefore, the viability of M. leprae for clinical or experimental applications is often unknown. To provide new tools for M. leprae viability determination, two quantitative reverse transcriptase PCR (RT-PCR) assays were developed and characterized. M. leprae sodA mRNA and 16S rRNA were used as RNA targets, and M. leprae repetitive element (RLEP) DNA was used to determine relative bacterial numbers in the same purified bacterial preparations or from crude biological specimens. Results demonstrated that both assays were good predictors of M. leprae viability during short-term experiments (48 h) involving rifampin (rifampicin) treatment in axenic medium, within rifampin-treated murine macrophages (M{Phi}), or within immune-activated M{Phi}. Moreover, these results strongly correlated those of other M. leprae viability assays, including radiorespirometry-based and Live/Dead BacLight viability assays. The 16S rRNA/RLEP assay consistently identified the presence of M. leprae in eight multibacillary leprosy patient biopsy specimens prior to multidrug therapy (MDT) and demonstrated a decline in viability during the course of MDT. In contrast, the sodA/RLEP assay was able to detect the presence of M. leprae in only 25% of pretreatment biopsy specimens. In conclusion, new tools for M. leprae viability determination were developed. The 16S rRNA/RLEP RT-PCR M. leprae viability assay should be useful both for short-term experimental purposes and for predicting M. leprae viability in biopsy specimens to monitor treatment efficacy, whereas the sodA/RLEP RT-PCR M. leprae viability assay should be limited to short-term experimental research purposes.

* Corresponding author. Mailing address for Milton O. Moraes: Departamento de Hanseníase, IOC—Fiocruz, Avenida Brasil, 4365 Manguinhos, 21040-900 Rio de Janeiro, Brazil. Phone: 055 (21) 2598-4467. Fax: 055 (21) 2270-9997. E-mail: mmoraes{at}fiocruz.br. Mailing address for Diana L. Williams: Molecular Biology Research Dept., Laboratory Research Branch, National Hansen's Disease Programs at SVM, LSU, Rm. 3517W, Skip Bertman Dr., Baton Rouge, LA. Phone: (225) 578-9839. Fax: (225) 578-9856. E-mail: dwill21{at}lsu.edu

{triangledown} Published ahead of print on 13 May 2009.

Journal of Clinical Microbiology, July 2009, p. 2124-2130, Vol. 47, No. 7
0095-1137/09/$08.00+0     doi:10.1128/JCM.00512-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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