Heterogeneous persistence of Mycobacterium leprae in oral and nasal mucosa of multibacillary patients during multidrug therapy.

BACKGROUND: Leprosy is curable by multidrug therapy (MDT) treatment regimen ranging from six to 12 months. The variable levels of tolerance and adherence among patients can, however, result in treatment failure and the emergence of drug-resistant strains. OBJECTIVES: Describe the impact of MDT over Mycobacterium leprae viability in patient's oral and nasal mucosa along treatment. METHODS: Mycobacterium leprae viability was monitored by quantitative polymerase chain reaction (qPCR) quantification of 16S rRNA in lateral and contralateral scrapings of oral and nasal mucosa of 10 multibacillary patients along the initial five months of treatment. FINDINGS: The results demonstrated high heterogenicity of M. leprae viability among patients and between nasal and oral samples. Of six patients who presented good adherence and tolerance to the treatment, only four displayed absence of M. leprae viability in both samples three months after the first MDT dose, while for the other two, the absence of M. leprae viability in the oral and nasal cavities was only detected five months after the first dose. MAIN CONCLUSIONS: We concluded that qPCR of 16S rRNA for the determination of M. leprae viability in nasal and oral scraping samples could represent an interesting approach to monitor treatment efficacy.

Heterogeneous persistence of Mycobacterium leprae in oral and nasal mucosa of multibacillary patients during multidrug therapy

BACKGROUND Leprosy is curable by multidrug therapy (MDT) treatment regimen ranging from six to 12 months. The variable levels of tolerance and adherence among patients can, however, result in treatment failure and the emergence of drug-resistant strains. OBJECTIVES Describe the impact of MDT over Mycobacterium leprae viability in patient's oral and nasal mucosa along treatment. METHODS Mycobacterium leprae viability was monitored by quantitative polymerase chain reaction (qPCR) quantification of 16S rRNA in lateral and contralateral scrapings of oral and nasal mucosa of 10 multibacillary patients along the initial five months of treatment. FINDINGS The results demonstrated high heterogenicity of M. leprae viability among patients and between nasal and oral samples. Of six patients who presented good adherence and tolerance to the treatment, only four displayed absence of M. leprae viability in both samples three months after the first MDT dose, while for the other two, the absence of M. leprae viability in the oral and nasal cavities was only detected five months after the first dose. MAIN CONCLUSIONS We concluded that qPCR of 16S rRNA for the determination of M. leprae viability in nasal and oral scraping samples could represent an interesting approach to monitor treatment efficacy.

Emergence and transmission of drug-/multidrug-resistant Mycobacterium leprae in a former leprosy colony in the Brazilian Amazon

BACKGROUND: Leprosy has been treated with multidrug therapy (MDT) distributed for free across the globe and regarded as highly efficient. However, the impossibility to grow M. leprae in axenic media has historically impaired assessment 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, hyper-endemic 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. Molecular epidemiology analysis was performed using data from 17 variable number tandem repeats (VNTR). RESULTS: M. leprae was obtained from biopsies of 37 leprosy cases (18 relapses and 19 new); 16 (43.24%) displayed drug-resistance variants. Multi-drug resistance to rifampicin and dapsone was observed in 8 relapses and 4 new cases. Single resistance to rifampicin was detected in one new case. Resistance to dapsone was present in two relapses and one 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 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.

Ticks as potential vectors of Mycobacterium leprae: Use of tick cell lines to culture the bacilli and generate transgenic strains.

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.

Genotyping comparison of Mycobacterium leprae isolates by VNTR analysis from nasal samples in a Brazilian endemic region.

This study analyzed the genetic diversity by MIRU-VNTR of Mycobacterium leprae isolates from nasal cavities and related to epidemiological and clinical data. The sample consisted of 48 newly diagnosed leprosy cases that tested positive for M. leprae PCR in nasal secretion (NS) attending to the National Reference Center of Dermatology Dona Libania (CDERM), Fortaleza, Brazil. Total DNA was extracted from NS of each patient and used for amplification of four M. leprae VNTR loci. Four clusters of M. leprae isolates were formed with identical genotypes. In the spatial analysis, 12 leprosy cases presented similar genotypes organized into 4 clusters. The most common genotypes in the current study was AC8b: 8, AC9: 7, AC8a: 8, GTA9: 10, which may represent a genotype of circulating strains most often in Ceará. A minimum set of four MIRU-VNTR loci was demonstrated to study the genetic diversity of M. leprae isolates from NS.

Ticks as potential vectors of Mycobacterium leprae: use of tick cell lines to culture the bacilli and generate transgenic strains

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.

Experimental Infection of Rhodnius prolixus (Hemiptera, Triatominae) with Mycobacterium leprae Indicates Potential for Leprosy Transmission.

Leprosy is a chronic dermato-neurological disease caused by infection with Mycobacterium leprae. In 2013 almost 200,000 new cases of leprosy were detected around the world. Since the first symptoms take from years to decades to appear, the total number of asymptomatic patients is impossible to predict. Although leprosy is one of the oldest records of human disease, the mechanisms involved with its transmission and epidemiology are still not completely understood. In the present work, we experimentally investigated the hypothesis that the mosquitoes Aedes aegypti and Culex quinquefasciatus and the hemiptera Rhodnius prolixus act as leprosy vectors. By means of real-time PCR quantification of M. leprae 16SrRNA, we found that M. leprae remained viable inside the digestive tract of Rhodnius prolixus for 20 days after oral infection. In contrast, in the gut of both mosquito species tested, we were not able to detect M. leprae RNA after a similar period of time. Inside the kissing bug Rhodnius prolixus digestive tract, M. leprae was initially restricted to the anterior midgut, but gradually moved towards the hindgut, in a time course reminiscent of the life cycle of Trypanosoma cruzi, a well-known pathogen transmitted by this insect. The maintenance of M. leprae infectivity inside the digestive tract of this kissing bug is further supported by successful mice footpad inoculation with feces collected 20 days after infection. We conclude that Rhodnius prolixus defecate infective M. leprae, justifying the evaluation of the presence of M. leprae among sylvatic and domestic kissing bugs in countries endemic for leprosy.

Experimental infection of Rhodnius prolixus (Hemiptera, Triatominae) with Mycobacterium leprae indicates potential for leprosy transmission

Leprosy is a chronic dermato-neurological disease caused by infection with Mycobacterium leprae. In 2013 almost 200,000 new cases of leprosy were detected around the world. Since the first symptoms take from years to decades to appear, the total number of asymptomatic patients is impossible to predict. Although leprosy is one of the oldest records of human disease, the mechanisms involved with its transmission and epidemiology are still not completely understood. In the present work, we experimentally investigated the hypothesis that the mosquitoes Aedes aegypti and Culex quinquefasciatus and the hemiptera Rhodnius prolixus act as leprosy vectors. By means of real-time PCR quantification of M. leprae 16SrRNA, we found that M. leprae remained viable inside the digestive tract of Rhodnius prolixus for 20 days after oral infection. In contrast, in the gut of both mosquito species tested, we were not able to detect M. leprae RNA after a similar period of time. Inside the kissing bug Rhodnius prolixus digestive tract, M. leprae was initially restricted to the anterior midgut, but gradually moved towards the hindgut, in a time course reminiscent of the life cycle of Trypanosoma cruzi, a well-known pathogen transmitted by this insect. The maintenance of M. leprae infectivity inside the digestive tract of this kissing bug is further supported by successful mice footpad inoculation with feces collected 20 days after infection. We conclude that Rhodnius prolixus defecate infective M. leprae, justifying the evaluation of the presence of M. leprae among sylvatic and domestic kissing bugs in countries endemic for leprosy.

Genotyping of Mycobacterium leprae present on Ziehl-Neelsen-stained microscopic slides and in skin biopsy samples from leprosy patients in different geographic regions of Brazil

We analysed 16 variable number tandem repeats (VNTR) and three single-nucleotide polymorphisms (SNP) in Mycobacterium leprae present on 115 Ziehl-Neelsen (Z-N)-stained slides and in 51 skin biopsy samples derived from leprosy patients from Ceará (n = 23), Pernambuco (n = 41), Rio de Janeiro (n = 22) and Rondônia (RO) (n = 78). All skin biopsies yielded SNP-based genotypes, while 48 of the samples (94.1%) yielded complete VNTR genotypes. We evaluated two procedures for extracting M. leprae DNA from Z-N-stained slides: the first including Chelex and the other combining proteinase and sodium dodecyl sulfate. Of the 76 samples processed using the first procedure, 30.2% were positive for 16 or 15 VNTRs, whereas of the 39 samples processed using the second procedure, 28.2% yielded genotypes defined by at least 10 VNTRs. Combined VNTR and SNP analysis revealed large variability in genotypes, but a high prevalence of SNP genotype 4 in the Northeast Region of Brazil. Our observation of two samples from RO with an identical genotype and seven groups with similar genotypes, including four derived from residents of the same state or region, suggest a tendency to form groups according to the origin of the isolates. This study demonstrates the existence of geographically related M. leprae genotypes and that Z-N-stained slides are an alternative source for M. leprae genotyping.

Genotyping of Mycobacterium leprae present on Ziehl-Neelsen-stained microscopic slides and in skin biopsy samples from leprosy patients in different geographic regions of Brazil.

We analysed 16 variable number tandem repeats (VNTR) and three single-nucleotide polymorphisms (SNP) in Mycobacterium leprae present on 115 Ziehl-Neelsen (Z-N)-stained slides and in 51 skin biopsy samples derived from leprosy patients from Ceará (n = 23), Pernambuco (n = 41), Rio de Janeiro (n = 22) and Rondônia (RO) (n = 78). All skin biopsies yielded SNP-based genotypes, while 48 of the samples (94.1%) yielded complete VNTR genotypes. We evaluated two procedures for extracting M. leprae DNA from Z-N-stained slides: the first including Chelex and the other combining proteinase and sodium dodecyl sulfate. Of the 76 samples processed using the first procedure, 30.2% were positive for 16 or 15 VNTRs, whereas of the 39 samples processed using the second procedure, 28.2% yielded genotypes defined by at least 10 VNTRs. Combined VNTR and SNP analysis revealed large variability in genotypes, but a high prevalence of SNP genotype 4 in the Northeast Region of Brazil. Our observation of two samples from RO with an identical genotype and seven groups with similar genotypes, including four derived from residents of the same state or region, suggest a tendency to form groups according to the origin of the isolates. This study demonstrates the existence of geographically related M. leprae genotypes and that Z-N-stained slides are an alternative source for M. leprae genotyping.

Genotipagem de isolados de mycobacterium leprae de pacientes hansenianos do Brasil / Genotyping of isolates of mycobacterium leprae from leprosy patients in Brazil

A hanseníase é uma doença infecto-contagiosa crônica, causada pelo Mycobacterium leprae. Após o sequenciamento do genoma deste patógeno, inúmeros esforços têm sido feitos na busca por seqüências repetitivas com potencial para diferenciar isolados de M. leprae. Atualmente VNTRs têm sido utilizados com o objetivo de compreender a diversidade genética deste patógeno em áreas com alta prevalência da doença. Além disso, SNPs também têm contribuído para elucidar aspectos referentes à disseminação da hanseníase pelo mundo. Neste estudo, a variabilidade genética de 292 isolados de M. leprae oriundos de amostras clínicas de pacientes hansenianos das regiões norte, nordeste e sudeste do país foi avaliada utilizando 16 VNTRs e três SNPs. O polimorfismo dos diferentes VNTRs foi determinado através de MVLA (Multiple-locus VNTR analysis) utilizando FLA (Fragment lenght analysis), enquanto que os SNPs foram analisados através de PCR-RFLP e, ou sequenciamento. O poder discriminatório dos 16 VNTRs foi de 0.999, sendo que as repetições de dinucleotídeos AT e o trinucleotídeo GAA21 apresentaram os maiores índices de discriminação alélica. Além da genotipagem de isolados de M. leprae de biópsias de pele, material de linfa fixado em lâmina de baciloscopia também foi utilizado como uma fonte alternativa para obtenção de DNA deste bacilo. Com relação à genotipagem por SNP, foi possível observar a predominância do genótipo 3, associado à população européia, em Rondônia, Rio de Janeiro e São Paulo. Já o genótipo 4, oriundo da África Ocidental, foi predominante em Pernambuco e Fortaleza. A presença dos diferentes genótipos e sua predominância em determinadas áreas corroboram com o processo de colonização do país que se reflete na atual composição étnica da população brasileira. Com base nos perfis obtidos através dos VNTR ser SNPs, foram identificados seis grupos geneticamente idênticos: quatro do Ceará, um de Rondônia e outro formado entre amostras de Minas Gerais e São Paulo. Nenhuma associação entre os pacientes enquadrados nos grupos da região norte e sudeste do país foi estabelecida. Todavia, foi possível observar que os grupos foram formados entre indivíduos oriundos de mesmo estado ou região geográfica. Com relação aos grupos formados entre as amostras do Ceará, associações entre o gênero masculino e o local de origem das amostras foram estabelecidas com base nos genótipos de M. leprae, sugerindo que estes seriam fatores de risco para a transmissão da ganseníase. Neste estudo, também foi possível avaliar amostras de pacientes com hanseníase recidiva para quatro VNTRs e os achados sugerem que estes pacientes foram re-infectados ou que houve mudança da população bacteriana durante a recidiva da doença. Os resultados comprovam que a genotipagem de M. leprae é uma ferramenta importande na elucidação de aspectos relativos à transmissão e disseminação da doença pelo mundo.

Genetic diversity of mycobacterium leprae isolates from Brazilian leprosy patients.

INTRODUCTION: Leprosy is a chronic disease caused by infection with Mycobacterium leprae, an obligate intracellular parasite. A problem in studying the transmission of leprosy is the small amount of variation in bacterial genomic DNA. The discovery of variable number of tandem repeats (VNTRs) allowed the detection of strain variation in areas with a high prevalence of leprosy. Four genotypes of M. leprae based on three single-nucleotide polymorphism (SNPs) were also discovered to be useful for analysis of the global spread of leprosy. METHODS: In this present study, we examined the allelic diversity of M. leprae at 16 select VNTR and three SNP loci using 89 clinical isolates obtained from patients mainly from the neighbouring states of São Paulo and Rio de Janeiro Brazil. RESULTS AND CONCLUSION: By use of a PCR-RFLP-based procedure that allows the recognition of SNP types 3 and 4 without the need for the more expensive DNA sequencing steps, characterisation of the main M. leprae genotypes was easy. When applied on the study population, it was found that the SNP type 3 is most frequent in these two states of Brazil, and that VNTRs provided further discrimination of the isolates. Two Short Tandem Repeats (STRs) were monomorphic, with the remaining 14 STRs represented by two to 18 alleles. Epidemiological associations with township or state were not evident in this random collection and require further investigations. In phylogenetic trees, branches formed by all 16 STRs clearly separated SNP type 3 organisms from the other types while the allelic patterns of two minisatellite loci 27-5 and 12-5 were highly correlated with SNP type 3. This strain typing study provide the basis for comparison of M. leprae strain types within Brazil and with those from other countries, and informed selection of genomic markers and methods for future studies.