BackgroundThe 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 FindingsTwo 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.
Mycobacterium leprae infection was evaluated in interferon-gamma knockout (GKO) mice. At 4 months, growth of the bacilli in the footpads of GKO mice plateaued a log(10) higher than that in control mice. Control mice exhibited mild lymphocytic and histiocytic infiltrates, whereas GKO mice developed large, unorganized infiltrates of epithelioid macrophages and scattered CD4 and CD8 T cells. Flow cytometric analysis of popliteal lymph node cells demonstrated similar profiles of T cells; however, GKO cells exhibited an elevated proliferative response to M. leprae antigen. Expression of inducible nitric oxide synthase mRNA was decreased in GKO mice, whereas macrophage inflammatory protein-1alpha and interleukin-4 and -10 mRNA expression were augmented. Control and GKO activated macrophages inhibited bacterial metabolism and produced nitrite. Thus, although deficient in an important Th1 cytokine, GKO mice possess compensatory mechanisms to control M. leprae growth and feature elements resembling mid-borderline leprosy in humans.
Recent studies identified an association between genetic variants in the lymphotoxin-␣ (LT␣) gene and leprosy.To study the influence of LT␣ on the control of experimental leprosy, both low-and high-dose Mycobacterium leprae foot pad (FP) infections were evaluated in LT␣-deficient chimeric (cLT␣ On infection with many intracellular organisms, especially mycobacterial pathogens, a granulomatous response ensues. This complex process involves the participation of cell-mediated immunity (CMI), activation of endothelial cells, enhancement of adhesion molecule expression, management of macrophage and lymphocyte infiltration, and induction of the microbiostatic and microbicidal effects of macrophages.1 In addition, extensive intercellular communication, chiefly through cytokine and chemokine signals, is required to orchestrate this cellular accumulation and results in a three-dimensional structure that limits or prevents dissemination of the pathogen and is largely protective.The contributions of both soluble and membranebound tumor necrosis factor (TNF) to granuloma development have been established.2-5 Lymphotoxin (LT)-␣ is also a member of the TNF superfamily, but as compared with TNF, much less is known about its function. LT␣ is required for the development of secondary lymphoid tissue 6 through its association with LT, to form heterotrimeric LT␣ 1  2 and LT␣ 2  1 , which enable interaction between lymphocytes and surrounding fibroblasts, and epithelial and myeloid cells that express the LT receptor. Current evidence also suggests that LT␣ plays a regulatory role in CMI 7 and serves as an initiating stimuSupported by National Institutes of Health (NIH) grant AI-50027 (L. Adams) and the NHMRC of Australia (B. Saunders and W. Britton).
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