Genetic diversity of Mycobacterium leprae in the state of São Paulo, an area of low-leprosy incidence in Brazil.

BACKGROUND: Brazil has the second largest number of leprosy cases worldwide, and the state of São Paulo has been considered non-endemic since 2006. METHODS: We analyzed 16 variable number tandem repeats loci and three single nucleotide polymorphisms loci of Mycobacterium leprae (M. leprae) in 125 clinical isolates from patients in different municipalities in the state. RESULTS: The clustering pattern of M. leprae indicated that the transmission of leprosy persisted in the state and included scenarios of intra-extra-familial transmission in areas with low endemicity. CONCLUSIONS: A significantly active circulation of M. leprae was observed. Therefore, surveillance and control measures must be implemented.

Genetic diversity of Mycobacterium leprae in the state of São Paulo, an area of low-leprosy incidence in Brazil

ABSTRACT Background: Brazil has the second largest number of leprosy cases worldwide, and the state of São Paulo has been considered non-endemic since 2006. Methods: We analyzed 16 variable number tandem repeats loci and three single nucleotide polymorphisms loci of Mycobacterium leprae (M. leprae) in 125 clinical isolates from patients in different municipalities in the state. Results: The clustering pattern of M. leprae indicated that the transmission of leprosy persisted in the state and included scenarios of intra-extra-familial transmission in areas with low endemicity. Conclusions: A significantly active circulation of M. leprae was observed. Therefore, surveillance and control measures must be implemented.

Social school contacts of multibacillary leprosy cases in children living in the hyperendemic region of the Midwest of Brazil

Abstract Objective: To analyze the prevalence of Mycobacterium leprae detection and the associated factors among social contacts in the school environment of multibacillary cases living in a hyperendemic municipality of the state of Mato Grosso. Methods: Cross-sectional study with 236 social contacts of multibacillary leprosy from public schools and residents in Cuiabá (Mato Grosso) in 2018. The sources of information were interviews and nasal swab tests for molecular analysis by polymerase chain reaction - PCR. For the prevalence ratio estimates, crude and adjusted analyses were performed using robust Poisson regression and their respective confidence intervals (95% CI). The ArcGIS 9.1 software was used for the geographic distribution analyses. Results: The prevalence of detection of M. leprae in social contacts was 14%. A total of 63.6% of the schools surveyed had 5.1% to 50% of the social contacts of leprosy with positive PCR. The analysis of the geographic distribution in the neighborhoods showed a high prevalence of infection, being higher than 50% in some localities. The highest proportion of positive results occurred in the northern region of the city and from a precarious socioeconomic class. Conclusion: The results showed a high prevalence of detection of M. leprae among social contacts in areas with poor socioeconomic conditions. In these regions, there is a greater risk of

Social school contacts of multibacillary leprosy cases in children living in the hyperendemic region of the Midwest of Brazil.

OBJECTIVE: To analyze the prevalence of Mycobacterium leprae detection and the associated factors among social contacts in the school environment of multibacillary cases living in a hyperendemic municipality of the state of Mato Grosso. METHODS: Cross-sectional study with 236 social contacts of multibacillary leprosy from public schools and residents in Cuiabá (Mato Grosso) in 2018. The sources of information were interviews and nasal swab tests for molecular analysis by polymerase chain reaction - PCR. For the prevalence ratio estimates, crude and adjusted analyses were performed using robust Poisson regression and their respective confidence intervals (95% CI). The ArcGIS 9.1 software was used for the geographic distribution analyses. RESULTS: The prevalence of detection of M. leprae in social contacts was 14%. A total of 63.6% of the schools surveyed had 5.1% to 50% of the social contacts of leprosy with positive PCR. The analysis of the geographic distribution in the neighborhoods showed a high prevalence of infection, being higher than 50% in some localities. The highest proportion of positive results occurred in the northern region of the city and from a precarious socioeconomic class. CONCLUSION: The results showed a high prevalence of detection of M. leprae among social contacts in areas with poor socioeconomic conditions. In these regions, there is a greater risk of infection and of getting sick.

Perfil sociodemográfico, clínico e geoespacial de casos novos de hanseníase diagnosticados no Instituto Lauro de Souza Lima, Bauru, São Paulo, entre 2015 e 2019 / Sociodemographic, clinical and geospatial profile of new leprosy cases diagnosed at Institute Lauro de Souza Lima, Bauru, São Paulo, between 2015 and 2019

Embora o Brasil venha apresentando uma redução considerável no número de casos novos de hanseníase, alguns estudos têm demonstrado transmissão ativa mesmo em áreas não endêmicas, como o estado de São Paulo. Diante disso, investigamos o perfil sociodemográfico, clínico e geoespacial dos casos novos de hanseníase diagnosticados entre 2015 e 2019 no Instituto Lauro de Souza Lima (ILSL), um centro de referência localizado no município de Bauru, interior do estado de São Paulo. Foram diagnosticados 177 novos casos de hanseníase nesse período, sendo 61,6% dos pacientes naturais do estado. A maioria dos indivíduos era do sexo masculino (59,9%) e a faixa etária mais prevalente foi de 60 a 69 anos; 79,1% se autodeclaravam brancos e 65,6% possuíam pouca ou nenhuma escolaridade. A forma clínica dimorfa foi a mais frequente (42,4%), a baciloscopia foi positiva em 38,4% dos pacientes (49,0% entre o sexo masculino e 22,5% entre o sexo feminino) e 49,0% dos pacientes possuíam incapacidades no diagnóstico. O georreferenciamento, realizado para os casos oriundos do município de Bauru (n = 31), revelou que a maioria dos pacientes residia em regiões com elevado nível de vulnerabilidade social. O perfil dos pacientes atendidos no ILSL apontou para predominância de homens adultos ou idosos com baixa escolaridade, multibacilares, apresentando incapacidades físicas e longo tempo de sintomas. Em conjunto, nossos dados sugerem atraso no diagnóstico que pode contribuir para a manutenção da transmissão da hanseníase mesmo numa região não endêmica.(AU)

Although Brazil has shown a considerable reduction in the number of new cases of leprosy, some studies have shown active transmission even in non-endemic areas, such as São Paulo state. Considering this, we investigated the sociodemographic, clinical and geospatial profile of new cases of leprosy diagnosed between 2015 and 2019 at the Lauro de Souza Lima Institute (ILSL) a reference center localized in Bauru, a municipality in the interior of São Paulo state. A total of 177 new cases of leprosy were diagnosed in this period, with 61.6% of the patients born in the state. The majority of the individuals was male (59.9%), the most prevalent age group was 60 to 69 years old, 79.1% declared themselves white color/race and 65.6% had little or no schooling. The borderline form of leprosy was the most frequent (42.4%), slit skin smear was positive in 38.4% of patients (49.0% among men and 22.5% among women) and 49.0% had disabilities at diagnosis. Georeferencing, performed for cases from the municipality of Bauru (n = 31), revealed that most patients lived in regions with a high level of social vulnerability. The profile of leprosy new cases found at ILSL pointed to a predominance of adult or elderly men with low education, multibacillary, presenting physical disabilities and a long time of symptoms. Altogether, our data suggests a delay in diagnosis that may contribute to the maintenance of leprosy transmission even in a non-endemic region.(AU)

Phylogenomics and antimicrobial resistance of the leprosy bacillus Mycobacterium leprae

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.

Analysis of immune response markers in Jorge Lobo's disease lesions suggests the occurrence of mixed T Helper responses with the dominance of regulatory T cell activity

Jorge Lobo's disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-ß and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-ß1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-ß1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-ß1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis.

Pre-miR 146a (rs2910164 G>C) single nucleotide polymorphism is genetically and functionally associated with leprosy

Mycobacterium leprae infects macrophages and Schwann cells inducing a gene expression program to facilitate its replication and progression to disease. MicroRNAs (miRNAs) are key regulators of gene expression and could be involved during the infection. To address the genetic influence of miRNAs in leprosy, we enrolled 1,098 individuals and conducted a case-control analysis in order to study four miRNAs genes containing single nucleotide polymorphism (miRSNP). We tested miRSNP-125a (rs12975333 G>T), miRSNP-223 (rs34952329 *>T), miRSNP-196a-2 (rs11614913 C>T) and miRSNP-146a (rs2910164 G>C). Amongst them, miRSNP-146a was the unique gene associated with risk to leprosy per se (GC OR = 1.44, p = 0.04; CC OR = 2.18, p = 0.0091). We replicated this finding showing that the C-allele was over-transmitted (p = 0.003) using a transmission-disequilibrium test. A functional analysis revealed that live M. leprae (MOI 100:1) was able to induce miR-146a expression in THP-1 (p<0.05). Furthermore, pure neural leprosy biopsies expressed augmented levels of that miRNA as compared to biopsy samples from neuropathies not related with leprosy (p = 0.001). Interestingly, carriers of the risk variant (C-allele) produce higher levels of mature miR-146a in nerves (p = 0.04). From skin biopsies, although we observed augmented levels of miR-146a, we were not able to correlate it with a particular clinical form or neither host genotype. MiR-146a is known to modulate TNF levels, thus we assessed TNF expression (nerve biopsies) and released by peripheral blood mononuclear cells infected with BCG Moreau. In both cases lower TNF levels correlates with subjects carrying the risk C-allele, (p = 0.0453 and p = 0.0352; respectively), which is consistent with an immunomodulatory role of this miRNA in 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.

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.

Tipagem molecular de cepas de Mycobacterium tuberculosis isoladas de pacientes HIV soropositivos e soronegativos na regiao de Bauru - SP

O presente estudo avaliou cepas de Mycobacterium tuberculosis isoladas de pacientes do Hospital Manoel de Abreu e de pacientes atendidos nos ambulatorios do Instituto Lauro de Souza Lima e Instituto Adolfo Lutz, com sorologia positiva e negativa para o HIV, com objetivo de caracterizar o perfil molecular das cepas. Um total de 57 cepas foram submetidas ao teste de sensibilidade as drogas isoniazida, rifampicina, estreptomicina, etambutol e pirazinamida e a taxa total de resistencia a estas drogas foi de 42,6 por cento. A tipagem molecular das cepas de M. tuberculosis foi realizada por duas tecnicas diferentes: o Restriction Fragment Lenght Polymorphism (RFLP) usando a sequencia de insercao IS6110 e o Double Repetitive Element PCR (DRE-PCR)...

Hanseníase experimental murina: inoculaçäo do Mycobaterium leprae via intranasal / Murine experimental hansen's disease: intranasal inoculation of the mycobacterium leprae

Considerando as vias aéreas superiores como a mais provável porta de entrada do M.leprae no organismo, no presente estudo avaliou-se o comportamento do bacilo inoculado experimentalmente via intranasal. Assim, camondongos suiços foram inoculados por instilaçäo nasal com M.leprae e sacrificados em diferentes períodos de tempo. Em cada cada sacrifício os pulmöes foram removidos e submetidos ao lavado broncoalveolar (LBA) e análise histopatológica. O LBA foi avaliado quanto ao número de bacilos recuperados e número total e diferencial de células inflamatórias; foi ainda semeado em meios de cultivo Lowenstein-Jensen e Middlebrook 7H9 e 7H11. No LBA foram encontrados bacilos até o 6§ mês de inoculaçäo, näo houve crescimento de micobactérias nas culturas. O número total de células, linfócitos e neutrófilos do LBA obtido de camundongos infectados foi maior que o do grupo controle até o 6§ mês da experimentaçäo. A análise histopatológica revelou pequenos infiltrados celulares distribuídos pelo parênquima pulmonar e ao redor dos vasos até o 6§ mês de sacrifício; näo foram observadas lesöes de disseminaçäo. Os resultados obtidos revelam que a inoculaçäo de M. leprae, via intranasal, em camundongos Suíços, näo levou ao desenvolvimento da doença nem a multiplicaçäo dos bacilos; assim estudos aidcionais envolvendo outras linhagens de camundongos poderäo ser úteis para determinar a importância da via intranasal na infecçäo hansênica