SUMMARY Leprosy is best understood as two conjoined diseases. The first is a chronic mycobacterial infection that elicits an extraordinary range of cellular immune responses in humans. The second is a peripheral neuropathy that is initiated by the infection and the accompanying immunological events. The infection is curable but not preventable, and leprosy remains a major global health problem, especially in the developing world, publicity to the contrary notwithstanding. Mycobacterium leprae remains noncultivable, and for over a century leprosy has presented major challenges in the fields of microbiology, pathology, immunology, and genetics; it continues to do so today. This review focuses on recent advances in our understanding of M. leprae and the host response to it, especially concerning molecular identification of M. leprae, knowledge of its genome, transcriptome, and proteome, its mechanisms of microbial resistance, and recognition of strains by variable-number tandem repeat analysis. Advances in experimental models include studies in gene knockout mice and the development of molecular techniques to explore the armadillo model. In clinical studies, notable progress has been made concerning the immunology and immunopathology of leprosy, the genetics of human resistance, mechanisms of nerve injury, and chemotherapy. In nearly all of these areas, however, leprosy remains poorly understood compared to other major bacterial diseases.

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Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae–infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories.

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In a bioassay-guided search for antimycobacterial compounds from higher plants of the southeastern United States, we have chemically investigated the sea daisy (Borrichia frutescens) from coastal marshes of Louisiana for their active constituents. Bioactive chromatographic fractions provided two new triterpenes, (24R)-24,25-epoxycycloartan-3-one (1) and (23R)-3-oxolanosta-8,24-dien-23-ol (4), and (3 alpha H, 24R)-24,25-epoxycycloartan-3-ol (3a). Compound 3a had been previously isolated as a mixture of C-24 epimers. The structures of 1, 3a, and 4 were established by spectroscopic methods and chemical transformations, and the molecular structures of 1 and 4 were determined by single-crystal X-ray diffraction. In a radiorespirometric bioassay against Mycobacterium tuberculosis, the epoxycycloartanes 1 and 3a exhibited minimum inhibitory concentrations of 8 micrograms/mL. In contrast, the lanostadiene-type triterpene 4 showed no significant inhibition at 128 micrograms/mL, as did the acetate 3b. Cytotoxicity for Vero cells gave IC50 values of 71.8, 39.8, and 103.6 micrograms/mL for triterpenes 1, 3a, and 4, respectively.

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.

Tumor necrosis factor (TNF) plays a pivotal role in inflammatory phenomena that culminate in either pathogenesis or resistance in mycobacterial disease. The regulatory role of TNF in murine tuberculosis was examined by administering a recombinant adenovirus encoding a fusion protein consisting of the human 55-kDa TNF receptor extracellular domain and the mouse IgG heavy chain domain (AdTNFR). During acute infections with Mycobacterium tuberculosis, AdTNFR pretreatment induced elevated mycobacterial burdens of 1 log10 in the tissues of H37Ra-infected mice and 2 log10 (spleen and liver) and 4 log10 (lungs) in H37Rv-infected mice. In mice infected chronically with H37Rv, AdTNFR treatment induced a 3-log10 increase of M. tuberculosis in the lungs, in which a tuberculous bronchopneumonia developed with numerous acid-fast bacilli visible in alveoli and bronchi. Administration of AdTNFR may serve as a useful model for studying the pathogenesis and chemotherapy of progressive primary tuberculosis.

Type-1 reactions (T1R) are pathological inflammatory episodes and main contributors to nerve damage in leprosy. Here, we evaluate the genewise enrichment of rare protein-altering variants in 7 genes where common variants were previously associated with T1R. We selected 474 Vietnamese leprosy patients of which 237 were T1R-affected and 237 were T1R-free matched controls. Genewise enrichment of nonsynonymous variants was tested with both kernel-based (sequence kernel association test [SKAT]) and burden methods. Of the 7 genes tested 2 showed statistical evidence of association with T1R. For the LRRK2 gene an enrichment of nonsynonymous variants was observed in T1R-free controls (PSKAT-O = 1.6 × 10−4). This genewise association was driven almost entirely by the gain-of-function variant R1628P (P = 0.004; odds ratio = 0.29). The second genewise association was found for the Parkin coding gene PRKN (formerly PARK2) where 7 rare variants were enriched in T1R-affected cases (PSKAT-O = 7.4 × 10−5). Mutations in both PRKN and LRRK2 are known causes of Parkinson’s disease (PD). Hence, we evaluated to what extent such rare amino acid changes observed in T1R are shared with PD. We observed that amino acids in Parkin targeted by nonsynonymous T1R-risk mutations were also enriched for mutations implicated in PD (P = 1.5 × 10−4). Hence, neuroinflammation in PD and peripheral nerve damage due to inflammation in T1R share overlapping genetic control of pathogenicity.