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Background: The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues. Methodology/Principle Findings: Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations. Conclusions: hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications. [ABSTRACT FROM AUTHOR]

Cytokine-activated macrophages (MΦ) employ reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to combat pathogens. The requirement for ROI for an effective host response to experimental leprosy using mice which have a disruption in the 91-kD subunit of the NAPDH oxidase cytochrome b ( phox91−/−) was examined. Mycobacterium leprae multiplication in phox91−/− foot pads (FP) was elevated early in infection but subsequently arrested similarly to control mice within a noninvasive granuloma. Using a modified lepromin test model, a similar cellular composition in the M. leprae-induced FP granuloma in both strains with lymphocyte infiltration consisting primarily of CD4+CD44hiCD62Llo effector cells was found. Of great interest was the disparity in the T cell population between the granuloma and the draining lymph node which contained predominantly naïve CD4+CD44loCD62Lhi cells and was, therefore, not representative of the infection site. TH1 cytokines, chemokines and inducible nitric oxide synthase were comparably expressed in the FP of both strains. When infected in vitro, normal MΦ from B6 and phox91−/− mice supported bacterial viability, whereas IFNγ-activated MΦ killed M. leprae in a RNI-dependent manner, emphasizing that ROI was dispensable. These data show that phox91−/− mice generate a strong adaptive immune response and control long-term infection with M. leprae. [ABSTRACT FROM AUTHOR]