Late leprosy reaction presenting as erythema multiforme-like erythema nodosum leprosum with underlying rifampicin resistance and its potential implications.

Erythema multiforme (EM)-like erythema nodosum leprosum (ENL) is a rare atypical presentation, and its late appearance after the completion of multidrug therapy (MDT) is unusual. We describe the case of a lepromatous leprosy patient who after the completion of MDT presented to us with late EM-like ENL and was found to be resistant to rifampicin. We discuss the implications of this finding and the potential role of resistant bacilli in causing reactions with atypical presentations.

Insights from the Genome Sequence of Mycobacterium lepraemurium: Massive Gene Decay and Reductive Evolution.

Mycobacterium lepraemurium is the causative agent of murine leprosy, a chronic, granulomatous disease similar to human leprosy. Due to the similar clinical manifestations of human and murine leprosy and the difficulty of growing both bacilli axenically, Mycobacterium leprae and M. lepraemurium were once thought to be closely related, although it was later suggested that M. lepraemurium might be related to Mycobacterium avium In this study, the complete genome of M. lepraemurium was sequenced using a combination of PacBio and Illumina sequencing. Phylogenomic analyses confirmed that M. lepraemurium is a distinct species within the M. avium complex (MAC). The M. lepraemurium genome is 4.05 Mb in length, which is considerably smaller than other MAC genomes, and it comprises 2,682 functional genes and 1,139 pseudogenes, which indicates that M. lepraemurium has undergone genome reduction. An error-prone repair homologue of the DNA polymerase III α-subunit was found to be nonfunctional in M. lepraemurium, which might contribute to pseudogene formation due to the accumulation of mutations in nonessential genes. M. lepraemurium has retained the functionality of several genes thought to influence virulence among members of the MAC.IMPORTANCEMycobacterium lepraemurium seems to be evolving toward a minimal set of genes required for an obligatory intracellular lifestyle within its host, a niche seldom adopted by most mycobacteria, as they are free-living. M. lepraemurium could be used as a model to elucidate functions of genes shared with other members of the MAC. Its reduced gene set can be exploited for studying the essentiality of genes in related pathogenic species, which might lead to discovery of common virulence factors or clarify host-pathogen interactions. M. lepraemurium can be cultivated in vitro only under specific conditions and even then with difficulty. Elucidating the metabolic (in)capabilities of M. lepraemurium will help develop suitable axenic media and facilitate genetic studies.

PCR studies of feline leprosy cases.

16S rRNA gene sequence analysis provided evidence for two different mycobacterial species, Mycobacterium lepraemurium and a potentially novel species, as causative agents of 'feline leprosy'. Comparison of 16S rRNA gene sequence data obtained for M. lepraemurium and the potentially novel species indicated 12 nucleotide differences over a 446 bp region encompassing the V2 and V3 hypervariable regions. From available 16S rRNA gene sequence data, M. lepraemurium shared greatest nucleotide identity with M. avium subsp paratuberculosis and M. avium. The novel species had a long helix 18 in the V3 region and shared greatest nucleotide identity with M. leprae, M. haemophilum and M. malmoense. The novel species had an additional 'A' nucleotide at position 105 of the aligned 16S rRNA gene sequence, the only other mycobacterial database sequence having this same extra nucleotide being M. leprae. This nucleotide variation was exploited to develop specific PCR assays for the two species. These were found to be effective and specific when tested against a panel of mycobacteria including species found in feline leprosy lesions and closely related mycobacteria and also when applied directly to formalin-fixed, paraffin-embedded tissues from feline leprosy cases.

Mycobacterium leprae and Mycobacterium lepraemurium infections in domestic and wild animals.

Mycobacterium leprae, the aetiological agent of leprosy in humans, gives rise to a chronic granulomatous disease that affects primarily the skin and peripheral nerves, and secondarily some internal organs such as the testis and the eye; viscera are seldom involved. Depending on host resistance, leprosy may present as a benign disease (tuberculoid leprosy) or as a malignant disease (lepromatous leprosy), with a spectrum of intermediate stages appearing between the two. Immunity against leprosy depends on the cell-mediated immunity of the host, and this is severely compromised in the malignant (lepromatous) form of leprosy. Although culture of M. leprae has never been achieved in artificial media, the bacterium may be grown in several experimental animals, including the armadillo, non-human primates, and to a certain extent, rodents. Naturally acquired leprosy has been reported in wild nine-banded armadillos (Dasypus novemcinctus) and in three species of non-human primates (chimpanzees [Pan troglodytes], sooty mangabey monkeys [Cercocebus atys] and cynomolgus macaques [Macaca fascicularis]), thus qualifying leprosy as a zoonosis. Murine leprosy is a leprosy-like disease of rats and mice, caused by Mycobacterium lepraemurium. The disease affects primarily viscera and the skin, and very rarely peripheral nerves. Depending on the host strain, rodent leprosy may also evolve as 'lepromatous' or 'tuberculoid' leprosy, and strains of mouse that develop intermediate forms of the disease may exist. Growth of M. lepraemurium on conventional media for mycobacteria is not successful, but the bacterium has been cultured on an egg yolk-based medium. Naturally acquired murine leprosy has been observed in rats, mice and cats, but not in humans or any other species. Thus, in contrast to human leprosy, murine leprosy is not a zoonosis.

Determination of the etiology of presumptive feline leprosy by 16S rRNA gene analysis.

PCR-amplified 16S rRNA gene sequences were obtained directly from tissue specimens from eight cats with presumptive feline leprosy. Acid-fast bacilli were observed in sections from all eight specimens, but culture for mycobacteria was successful for one specimen only. Analysis of the V2 variable region of each 16S rRNA PCR product identified a sequence with 100% nucleotide identity to the sequences of Mycobacterium lepraemurium, Mycobacterium avium, and Mycobacterium paratuberculosis in four of the specimens from cats with feline leprosy. Separate M. paratuberculosis- and M. avium-specific PCR amplifications of the four specimens were negative, thus substantiating the identification of M. lepraemurium in these specimens from cats with feline leprosy. Further sequence analysis of the V3 variable region of one of the four specimens provided conclusive evidence of the presence of M. lepraemurium. This is the first report of the definitive identification of M. lepraemurium in cats with feline leprosy by molecular biology-based analyses. M. avium, which is rarely reported in cats, and Mycobacterium chitae, a reported nonpathogenic, rapidly growing mycobacterial species found in the environment, were identified in the specimen from which acid-fast bacilli were cultured. Two of the specimens from cats were infected with a potentially novel species of mycobacteria which had a 16S rRNA gene sequence sharing the closest nucleotide sequence identity with that of Mycobacterium malmoense. Molecular biology-based analyses provided for the accurate and rapid diagnosis of mycobacterial infections in cats and circumvented the problems of culture and misdiagnosis of feline leprosy associated with traditional methods.

Evaluation of methods for isolation of DNA from slowly and rapidly growing mycobacteria.

Mycobacteria generally have thick cell walls and contain large amounts of lipid, making them resistant to DNA extraction. Five methods, namely, extensive enzymic digestion method (M1), 2-min mechanical glass-bead disruption method (M2), thermal shock method (M3), modified conventional enzymic digestion method (M4), and manual disruption with modified conventional enzymic digestion method (M5), were used to compare their effectiveness and simplicity in extracting DNA from slowly growing mycobacteria (Mycobacterium leprae, M. lepraemurium and M. bovis BCG), and a rapidly growing mycobacterium (M. phlei). The highest DNA yield was obtained by M2 from M. lepraemurium which produced 2.82 micrograms DNA/mg wet weight of cells, representing a theoretical yield of 78%. M3 gave the lowest DNA yield; 0.01 microgram DNA/mg wet weight of cells of M. lepraemurium was obtained. M4, in which proteinase K was used, is more effective than M1, in which subtilisin and pronase were used. M5 yielded a higher amount of DNA, but it required more manipulations to extract DNA as compared to M4. Extraction of DNA of M. leprae from nude mice is more difficult than that of M. leprae from armadillos by all of the methods used. These results suggest that the biosynthetic capabilities of these two forms of M. leprae may vary, depending on their cultural conditions and/or strain differences. Our results have shown that both M2 and M4 are the simplest, most effective and time-saving methods which are suitable for every routine laboratory to extract DNA from slowly and rapidly growing mycobacteria.

The use of DNA probes identifying restriction-fragment-length polymorphisms to examine the Mycobacterium avium complex.

DNA probes were used to identify restriction-fragment-length polymorphisms (RFLPs) in DNA samples, demonstrating that the Mycobacterium avium complex could be clearly divided into M. avium and Mycobacterium intracellulare strains. Less than 2% DNA base substitution was found between M. avium strains, whereas the M. intracellulare strains had greater than 15% base substitution. The Johne's disease bacillus, Mycobacterium paratuberculosis (American type strain), was found to be distinguishable from the M. avium complex serotypes examined. Strain 18 was found to be identical to M. avium. The rat leprosy bacillus, Mycobacterium lepraemurium, was found to be very closely related, but not identical, to M. avium.

Ação da hialoronidase testicular sôbre a evolução da lepra murina

The effect of testicular hyaluronidasis on the evolution of murine leprosy was studied in rats, inoculated with M. lepraemurium by the intraperitoneal route. The results, which were based on the statistical analysis of the survical time and on teh evolution of the leprosy lesions show, that the hyaluronidasis, administred by the subcutaneous route (dose: 25 U each two days), does not modify the evolutive rate of the disease. This enzyme has neither determined more rapid bacillary dissemination, nor has influenced the leprous lesions or mycobacteria growth rate in the rat tissues. There is note interaction between hyaluronidasis and DDS treatment.

Relação entre a dose de M. lepraemurium experimentalmente inoculada e a sobrevivência de ratos tratados e não tratados pela 4-4 diaminodifenilsulfona

Grupos de ratos com 90-120 g de pêso foram inoculados, por via intraperitoneal, com suspensão de M. lepraemurium contendo 5,7 mg, 2,85 mg ou 0,57 mg de bacilos. Uma parte dos animais de cada grupo foi tratada co DDS (tratamento iniciado 7 dias após a inoculação) permanecendo os animais restantes sem tratamento. Baseado no estudo histológico da evolução das lesões e na sobrevivência foram analisados estat¡sticamente (análise de variância e linha de regressão de sobrevivência), tendo-se verificado: a) o tratamento sulfônico aumenta a sobrevivência dos animais; b) o aumento da sobrevivência devido ao tratamento ‚ independente da dose de bacilos inoculada; c) a sobrevivência dos animais nâo tratados depende da dose de bacilos inoculada, sendo tanto maior quanto menor a dose e vice-versa, d) nos animais não tratados a relação entre a sobrevivência e o log. da dose de bacilos ‚ linear, dentro dos limites usados, o que permite efetuar dosagem biológica do inóculo, conhecendo-se a sobrevivência dos animais; e) os resultados de tratamento sulfônico são tanto mais evidentes quanto maior a dose do inóculo. Sendo linear a regressão dose do inóculo-sobrevivência, nos animais não tratados, calculou-se, com base nesses resultados, o ritmo de multiplicação do M. lepraemurium, obtendo-se o seguinte valor médio: 55,1 dias (+/- 4,1 para intervalos de confiança de 5%). O valor obtido mostra que o ritmo de reprodução do bacilo ‚ lento. Os resultados foram discutidos com relação ao mecanismo de ação de DDS na lepra murina.

Efeito da vacinação pelo BCG sôbre a evolução da lepra murina; observação em ratos inoculados, por via peritoneal, com pequena dose de M. lepraemurium / Effect of BCG vaccination on the evolution of murine leprosy; observation in mice inoculated by peritoneal route, with a small dose of M. lepraemurium

The peritoneal inoculation of M. lepraemurium in the rat produces generalized lesions and a disease which shows uniforms evolution. The evolutive speed depends on the weight of inoculated bacilli. The incoculations of nearly o.03 mg of M. lepraemurium provokes only histological lesions, which progress very slowly. This slow evolution permits the observation of the influence of previous mycobacterial vaccination upon the progress and the influence of the citology of the lesions, allowing the study of the acquired resistance. Fifty young rats were divided in 4 lots: 1) 13 rats vaccinated with 25 mg of BCG by oral route; 2) 12 rats vaccinated with 5 mg of BCG, by oral route; 3) 12 rats vaccinated with 5 mg of BCG, by intramuscular route; 4) 13 non vaccinated rats (controls). All animals were inoculated 90 days after vaccination with a suspension containing approximately 0.03 mg of M. lepraemurium (by peritoneal route) and observed 450 days. The gain of weight and the histological picture observed in vaccinated and control animals are identicals; the disease shows the same evolution. The BCG vaccination does not suscitate, in the rat, acquired resistance against M. lepraemurium demonstrable by histological methods. The lesions in the vaccinated and control groups reach the same intensity in the lymph nodes, liver and apleen, and show the same slow evolution. In the animals of both groups the lesions remain almost stationary (during the all observed time), or grow slowly and progressively, or more rarely undergo involution (without complete disappearing). The histological method also shows that macrophages of the normal rat have no ability to destroy the M. lepraemurium, M. leprae and M. tuberculosis; the BCG vaccination does not induce at the macrophages the aptitude to lyse these acid-fast bacilli. On the other hand, the macrophages of normal guinea pigs have the ability to lyse these mycobacteria; the BCG vaccination provokes in the guinea pigs this physiologic...

O comportamento do Mycobacterium lepraemurium nos testes de redução do azul de metileno e do tetrazólio / The behavior of \"Mycobacterium lepraemurium in tests to reduce the methylene blue and tetrazolium

Using M. lepraemurium suspensions obtained from leproma of rats inoculated by subcutaneous route or from nodules of the peritoneal cavity, the AA. have tested the capacity of these bacilli in reduce the methylene blue and formation of formazan from the tetrazolium. The time or intensity of reduction of methylene blue or formation of formazan are proportional to the concentration of bacilli in the suspensions and is increased with the storage in the refrigerator at 1øC. In comparison with tests perfomed upon other acid fast bacilli the ones with M. lepraemurium are developed very slowly.

Estudo histológico das lesões de lepra murina em involução / Histological study of lesions of murine leprosy in involution

The cytological and histological aspects showed by the involutive stage of the murine leprosy lesions has been considered and analised, in rats inoculated with M. leparemurium by the peritonial route and treated by some chemiotherapeutic agents. Under this condition there are alterations in the leprous cells and consequently in the general structure of the lesions. The cytological alterations has degenerative character (cytoplasmic micro-vacuolization; cromatolysis, cariolysis and picnosis of nucleous) and at the end of the process we can see frequently cellular necrosis. The alterations of general structure are determined by the cytological degenerative process which constitute less compact and disorganized lesions. The involution of lesions is continous; but in the scope to simplify the understanding, it has been considered theree different degrees of involutive stages: inicial (early), medial and final. At first (inicial degree) the involutive alteration of lesions are observed in the marginal area only; in the medial degree the central area is compromised too and in the final degree there are evidents structural alterations and the lesions diminuish to little sizes. At the end of involutive process the lesions disappear without sign of cicatricial residue; only in rare cases a little fibroblastic cicatricial nodule remain. In connexion with the histological alterations of involutive character there are numerical and morphologic alterations of bacilli. The microscopical aspects of involutive murine leprosy lesions are very resembling to the human leprosy beings in the same condition. In the involutive stage the most part of leprous cells become degenerated and disappear (celular necrosis); a few number of then, in another side, there are not distroyed by necrosis but show cytologic alterations taking the characters of R.E.S. cells. The observation of the records show all intermediated degrees between the typical leprous cells at one side and the normal...