Leprosy is a chronic human disease caused by the yet-uncultured pathogen Mycobacterium leprae. Although readily curable with multidrug therapy (MDT), over 200,000 new cases are still reported annually. Here, we obtain M. leprae genome sequences from DNA extracted directly from patients’ skin biopsies using a customized protocol. Comparative and phylogenetic analysis of 154 genomes from 25 countries provides insight into evolution and antimicrobial resistance, uncovering lineages and phylogeographic trends, with the most ancestral strains linked to the Far East. In addition to known MDT-resistance mutations, we detect other mutations associated with antibiotic resistance, and retrace a potential stepwise emergence of extensive drug resistance in the pre-MDT era. Some of the previously undescribed mutations occur in genes that are apparently subject to positive selection, and two of these (ribD, fadD9) are restricted to drug-resistant strains. Finally, nonsense mutations in the nth excision repair gene are associated with greater sequence diversity and drug resistance.

The chronic course of lepromatous leprosy may be interrupted by acute inflammatory episodes known as erythema nodosum leprosum (ENL). Despite its being a major cause of peripheral nerve damage in leprosy patients, the immunopathogenesis of ENL remains ill-defined. Recognized by distinct families of germline-encoded pattern recognition receptors, endogenous and pathogen-derived nucleic acids are highly immunostimulatory molecules that play a major role in the host defense against infections, autoimmunity, and autoinflammation. The aim of this work was to investigate whether DNA sensing via TLR-9 constitutes a major inflammatory pathway during ENL. Flow cytometry and immunohistochemistry analysis showed significantly higher TLR-9 expression in ENL when compared with nonreactional lepromatous patients, both locally in the skin lesions and in circulating mononuclear cells. The levels of endogenous and pathogen-derived TLR-9 ligands in the circulation of ENL patients were also higher. Furthermore, PBMCs isolated from the ENL patients secreted higher levels of TNF, IL-6, and IL-1β in response to a TLR-9 agonist than those of the nonreactional patients and healthy individuals. Finally, E6446, a TLR-9 synthetic antagonist, was able to significantly inhibit the secretion of proinflammatory cytokines by ENL PBMCs in response to Mycobacterium leprae lysate. Our data strongly indicate that DNA sensing via TLR-9 constitutes a major innate immunity pathway involved in the pathogenesis and evolution of ENL. Thus, the use of TLR-9 antagonists emerges as a potential alternative to more effectively treat ENL aiming to prevent the development of nerve injuries and deformities in leprosy.

Leprosy is an infectious disease caused by Mycobacterium leprae and frequently resulting in irreversible deformities and disabilities. Ticks play an important role in infectious disease transmission due to their low host specificity, worldwide distribution, and the biological ability to support transovarial transmission of a wide spectrum of pathogens, including viruses, bacteria and protozoa. To investigate a possible role for ticks as vectors of leprosy, we assessed transovarial transmission of M. leprae in artificially-fed adult female Amblyomma sculptum ticks, and infection and growth of M. leprae in tick cell lines. Our results revealed M. leprae RNA and antigens persisting in the midgut and present in the ovaries of adult female A. sculptum at least 2 days after oral infection, and present in their progeny (eggs and larvae), which demonstrates the occurrence of transovarial transmission of this pathogen. Infected tick larvae were able to inoculate viable bacilli during blood-feeding on a rabbit. Moreover, following inoculation with M. leprae, the Ixodes scapularis embryo-derived tick cell line IDE8 supported a detectable increase in the number of bacilli for at least 20 days, presenting a doubling time of approximately 12 days. As far as we know, this is the first in vitro cellular system able to promote growth of M. leprae. Finally, we successfully transformed a clinical M. leprae isolate by inserting the reporter plasmid pCHERRY3; transformed bacteria infected and grew in IDE8 cells over a 2-month period. Taken together, our data not only support the hypothesis that ticks may have the potential to act as a reservoir and/or vector of leprosy, but also suggest the feasibility of technological development of tick cell lines as a tool for large-scale production of M. leprae bacteria, as well as describing for the first time a method for their transformation.

Background Leprosy has been treated with multidrug therapy, which has been distributed for free across the globe and regarded as highly efficient. However, the impossibility of growing Mycobacterium leprae in axenic media has historically impaired assessments of M. leprae resistance, a parameter only recently detectable through molecular methods. Methods A systematic, population-based search for M. leprae resistance in suspected leprosy relapse cases and contacts was performed in Prata Village, an isolated, hyperendemic, former leprosy colony located in the Brazilian Amazon. Results led to an extended active search involving the entire Prata population. Confirmed leprosy cases were investigated for bacterial resistance using a combination of in vivo testing and direct sequencing of resistance genes folP1, rpoB, and gyrA. A molecular epidemiology analysis was performed using data from 17 variable number tandem repeats (VNTR). Results Mycobacterium leprae was obtained from biopsies of 37 leprosy cases (18 relapses and 19 new cases): 16 (43.24%) displayed drug-resistance variants. Multidrug resistance to rifampicin and dapsone was observed in 8 relapses and 4 new cases. Single resistance to rifampicin was detected in 1 new case. Resistance to dapsone was present in 2 relapses and 1 new case. Combined molecular resistance and VNTR data revealed evidence of intra-familial primary transmission of resistant M. leprae. Conclusions A comprehensive, population-based systematic approach to investigate M. leprae resistance in a unique population revealed an alarming scenario of the emergence and transmission of resistant strains. These findings may be used for the development of new strategies for surveillance of drug resistance in other populations.

We performed genotyping of Mycobacterium leprae present in skin biopsy samples that were collected during the first and the second disease occurrences from eight leprosy patients, seven of whom were diagnosed as suffering from disease relapse. Sequence analysis of part of the M. leprae rpoB, folP1, gyrB and gyrA genes did not show genetic change that supported the presence of drug-resistant bacilli. However, we observed a synonymous nucleotide change at position 297 of gyrA among five of these patients, one presenting C to T (CgyrAT) and four presenting T to C (TgyrAC) at this position. Additional genotyping by analysis of the four short tandem repeats GAA, GTA9, AT17 and TA18 showed that the gyrA single nucleotide polymorphism change was accompanied by a change in short tandem repeat genotype. Our data suggest that leprosy relapse in these patients, living in an area endemic for leprosy, could be caused by M. leprae with a genotype different from the one that caused initial disease.