Effect of Mycobacterium leprae on neurotrophins expression in human Schwann cells and mouse sciatic nerves

BACKGROUND Although Mycobacterium leprae (ML) is well characterised as the causative agent of leprosy, the pathophysiological mechanisms underlying peripheral nerve damage still need further understanding. In vitro and in vivo studies have yielded insights into molecular mechanisms of ML interaction with Schwann cells (SC), indicating the regulation of genes and proteins crucial to neural plasticity. OBJECTIVES We aimed to investigate the effect of ML on neurotrophins expression in human SC (hSC) and mice sciatic nerves to better understand their role in leprosy neuropathy, and aiming to contribute to future therapeutic approaches. METHODS We evaluated mRNA and protein expression of BDNF, NGF, NT-3, NT-4 in hSC from amputation nerve fragments, as well as in athymic nude mice, infected by ML for eight months. FINDINGS and MAIN CONCLUSIONS Our in vitro results showed a trend to decline in NGF and BDNF mRNA in ML-treated hSC, compared to controls. The immunodetection of BDNF and NT-4 was significantly downregulated in ML-treated hSC. Conversely, ML-infected mice demonstrated upregulation of NT-3, compared to non-infected animals. Our findings indicate that ML may be involved in neurotrophins regulation, suggesting that a pathogen-related imbalance of these growth factors may have a role in the neural impairment of leprosy.

Epidemiological and geographical characterization of leprosy in a Brazilian hyperendemic municipality / Caracterização epidemiológica e geográfica da hanseníase em um município brasileiro hiperendêmico / Caracterización epidemiológica y geográfica de lepra en un municipio brasileño hiperendémico

Abstract: This study aimed to identify the distribution pattern of leprosy in a hyperendemic municipality in Brazil and determine its relationship with the clinico-epidemiological situation over 11 years. The geographic information system, MapInfo, spatial scan statistics and the Moran I index were used to analyze new cases. The digital cartographic base was used to map clusters of new paucibacillary and multibacillary cases and cases in minors under 15 years old. Socioeconomic indicators are shown using the choropleth mapping technique. A reduction in the detection coefficient, increases in high-risk spatial clusters, marked changes in the distribution of high-risk and low-risk clusters, and high-risk clusters of minors under 15 years old were observed from 2006 to 2010, showing recent illness, the presence of active foci, and overlapping of high-risk clusters of multibacillary infection in minors under 15 years old. Leprosy remains a public health problem in Rondonópolis, Mato Grosso State; the high-risk areas require an intensification of control measures and active search strategies to detect new cases.

Resumo: O estudo teve como objetivos identificar o padrão de distribuição da hanseníase em um município brasileiro hiperendêmico e determinar a relação com o quadro clínico-epidemiológico ao longo de 11 anos. Os casos novos foram analisados com o sistema de informação geográfica, MapInfo, estatística scan espacial e índice Moran I. A base cartográfica digital foi usada para mapear os clusters de casos paucibacilares e multibacilares novos e casos em menores de 15 anos. Os indicadores socioeconômicos são mostrados através da técnica de mapeamento coroplético. Entre 2006 e 2010, foram observados uma redução no coeficiente de detecção, aumento no clusters espaciais de alto risco, mudanças marcantes na distribuição de clusters de alto e baixo risco e clusters de alto risco em menores de 15 anos, sugerindo doença recente, a presença de focos ativos e a sobreposição de clusters de alto risco para infecção multibacilar em menores de 15 anos. A hanseníase persiste enquanto problema de saúde pública em Rondonópolis, Mato Grosso; as áreas de alto risco exigem a intensificação de medidas de controle, além de estratégias de busca ativa para detectar casos novos.

Resumen: Este estudio tuvo como objetivo identificar la distribución de los patrones de lepra en una municipalidad hiperendémica en Brasil y determinar su relación con la situación clínico-epidemiológica durante 11 años. El sistema de información geográfica, MapInfo, estadísticas de escaneo espacial y el índice de Moran se usaron para analizar nuevos casos. La base cartográfica digital se usó para mapear clústeres de nuevos casos multibacilares y paucibacilares, así como casos en menores por debajo de 15 años de edad. Los indicadores socioeconómicos se presentan usando la técnica de mapeo de coropletas. La reducción en la detección del coeficiente, se incrementa en los clústeres de alto riesgo espaciales, asimismo, se observaron de 2006 a 2010 cambios considerables en la distribución de los clústeres de alto riesgo y bajo riesgo, así como en clústeres de alto riesgo con menores con menos de 15 años de edad, mostrando los casos de enfermedad reciente la presencia de focos activos, así como solapando clústeres de alto riesgo de infección multibacilar en menores por debajo de los 15 años de edad. La lepra continúa siendo un problema de salud pública en Rondonópolis, Mato Grosso; las áreas de alto riesgo necesitan una intensificación de las medidas de control y una búsqueda activa de estrategias, con el fin de detectar nuevos casos.

qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms

Abstract Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

High rates of undiagnosed leprosy and subclinical infection amongst school children in the Amazon Region

Leprosy in children is correlated with community-level factors, including the recent presence of disease and active foci of transmission in the community. We performed clinical and serological examinations of 1,592 randomly selected school children (SC) in a cross-sectional study of eight hyperendemic municipalities in the Brazilian Amazon Region. Sixty-three (4%) SC, with a mean age of 13.3 years (standard deviation = 2.6), were diagnosed with leprosy and 777 (48.8%) were seropositive for anti-phenolic glycolipid-I (PGL-I). Additionally, we evaluated 256 house-hold contacts (HHCs) of the students diagnosed with leprosy; 24 (9.4%) HHC were also diagnosed with leprosy and 107 (41.8%) were seropositive. The seroprevalence of anti-PGL-I was significantly higher amongst girls, students from urban areas and students from public schools (p < 0.0001). Forty-five (71.4%) new cases detected amongst SC were classified as paucibacillary and 59 (93.6%) patients did not demonstrate any degree of physical disability at diagnosis. The results of this study suggest that there is a high rate of undiagnosed leprosy and subclinical infection amongst children in the Amazon Region. The advantages of school surveys in hyperendemic areas include identifying leprosy patients at an early stage when they show no physical disabilities, preventing the spread of the infection in the community and breaking the chain of transmission.

Ten years experience with Jorge Lobo's disease in the state of Acre, Amazon region, Brazil / Dez anos de experiência com a Doença de Jorge Lobo no estado do Acre, região amazônica, Brasil

Jorge Lobo's disease is a cutaneous and subcutaneous mycosis that affects patients in the Amazon region. The number of patients is relatively small, but the real situation of the disease as public health problem is not known, because Jorge Lobo's disease is not a notifiable disease. This study aims to report the clinical evolution in patients affected and to determine the prevalence and areas of occurrence of the disease. A retrospective study was carried out based on the analysis of the clinical records, which included a collection of photographs of patients in the Department of Sanitary Dermatology, in Rio Branco, and patients seen in the interior of the state. In a decade, in Rio Branco, 249 cases of the disease were reported, 30 were females and 219 males. Of these patients, 153 had localized lesions, 94 of them were on one ear, 55 had multifocal lesions and 41 had disseminated lesions. The average time between the onset of symptoms and diagnosis was 19 years. The average age at the time of diagnosis was 53 years, and ages ranged from 14 to 96 years.

A doença de Jorge Lobo é micose cutânea e subcutânea que afeta pessoas na região Amazônica. O número de pacientes é relativamente pequeno, no entanto, a real prevalência da doença como problema de saúde pública é pouco conhecida. A doença de Jorge Lobo não é de notificação compulsória. Este estudo teve como objetivo determinar a prevalência, as áreas de ocorrência da doença de Jorge Lobo, além de sua evolução clínica. Um estudo retrospectivo foi desenvolvido com base na análise de prontuários de pacientes, incluindo documentação fotográfica dos mesmos, que foram atendidos no Departamento de Dermatologia Sanitária em Rio Branco e no interior do Estado. Foram registrados 249 casos em uma década em Rio Branco, 30 mulheres e 219 homens. Do total 153 apresentavam lesões localizadas, 94 lesões em apenas uma orelha, 55 lesões multifocais e 41 lesões disseminadas. A média de tempo entre o início dos sintomas e o diagnóstico foi de 19 anos. A média de idade no momento do diagnóstico foi de 53 anos, e as idades variaram de 14 a 96 anos.

Search for Mycobacterium leprae in wild mammals

Leprosy is still a worldwide public health problem. Brazil and India show the highest prevalence rates of the disease. Natural infection of armadillos Dasypus novemcinctus with Mycobacterium leprae has been reported in some regions of the United States. Identification of bacilli is difficult, particularly due to its inability to grow in vitro. The use of molecular tools represents a fast and sensitive alternative method for diagnosis of mycobacteriosis. In the present study, the diagnostic methods used were bacilloscopy, histopathology, microbiology, and PCR using specific primers for M. leprae repetitive sequences. PCR were performed using genomic DNA extracted from 138 samples of liver, spleen, lymph nodes, and skin of 44 D. novemcinctus, Euphractus sexcinctus, Cabassous unicinctus, and C. tatouay armadillos from the Middle Western region of the state of São Paulo and from the experimental station of Embrapa Pantanal, located in Pantanal da Nhecolândia of Mato Grosso do Sul state. Also, the molecular analysis of 19 samples from internal organs of other road killed species of wild animals, such as Nasua nasua (ring-tailed coati), Procyon cancrivoros (hand-skinned), Cerdocyon thous (dog-pity-bush), Cavia aperea (restless cavy), Didelphis albiventris (skunk), Sphigurrus spinosus (hedgehog), and Gallictis vittata (ferret) showed PCR negative data. None of the 157 analyzed samples had shown natural mycobacterial infection. Only the armadillo inoculated with material collected from untreated multibacillary leprosy patient presented PCR positive and its genomic sequencing revealed 100 percent identity with M. leprae. According to these preliminary studies, based on the used methodology, it is possible to conclude that wild mammals seem not to play an important role in the epidemiology of leprosy in the Middle Western region of the São Paulo state and in the Pantanal of Mato Grosso do Sul state.

The effect of iron supplementation in the diet of Dasypus novemcinctus (Linnaeus, 1758) armadillos in captivity / O efeito da suplementação com ferro na dieta de tatus Dasypus novemcinctus (Linnaeus, 1758) em cativeiro

Armadillos of the species Dasypus novemcinctus have been used as an experimental model of leprosy. Besides non-human primates, they are the only species naturally infected with Mycobacterium leprae and when experimentally inoculated, reproduce the lepromatous form of the disease producing large quantities of bacilli. This species has been maintained in captivity by numerous researchers and specific housing and feeding requirements have been developed to guarantee their survival during long experimental periods. In the "Lauro de Souza Lima" Institute, armadillos receive dog food, ground beef, boiled eggs and vitamin C. However, despite the balanced diet, anemia has been observed in some captive animals, especially in armadillos inoculated with M. leprae in advanced stages of infection. Thus, the objective of the present study was to evaluate the effect of iron sulfate supplementation in the feed provided for armadillos, both inoculated and non-inoculated with M. leprae, by means of the evaluation of their hematological profile. Fourteen armadillos received 10 mg/animal of iron sulfate (Hematofer®) diluted in sterile water mixed with their daily feed for 50 days. Hemograms and serum iron dosages for each armadillo were performed before and after supplementation. The hematocrit values increased significantly after iron supplementation, both in armadillos inoculated and non-inoculated with M. leprae. It is possible that the amount of iron in the feed is insufficient for the formation of hemoglobin, leading to microcytic anemia. Dietary supplementation with iron sulfate reversed this state, showing the importance of understanding the metabolism of exotic species for their maintenance in captivity, and thus ensuring their well-being.

O tatu da espécie Dasypus novemcinctus tem sido utilizado como modelo experimental para a hanseníase. Eles são a única espécie, além de primatas não humanos, que se apresentam naturalmente infectados pelo Mycobacterium leprae. Estes tatus, quando experimentalmente inoculados, reproduzem a forma virchoviana da hanseníase produzindo grandes quantidades de bacilos. Esta espécie tem sido mantida em cativeiro por vários pesquisadores, no entanto, alguns cuidados são necessários para garantir a sobrevivência destes por longos períodos experimentais, tais como alojamento e alimentação. No Instituto Lauro de Souza Lima, os animais em cativeiro recebem ração canina, carne bovina, ovos cozidos e vitamina C, mesmo assim, tem-se observado o desenvolvimento de quadros de anemia nestes animais, principalmente nos inoculados com o M. leprae, em estados avançados da infecção. Sendo assim, o objetivo deste estudo foi avaliar o efeito da suplementação com sulfato ferroso na alimentação fornecida aos tatus, inoculados e não inoculados com M. leprae, por meio de avaliação de seu perfil hematológico. Foram utilizados 14 animais que receberam 10 mg/animal de sulfato ferroso (Hematofer®) diluídos em água estéril e misturados diariamente à ração por 50 dias. Foram feitos hemograma e dosagem de ferro sérico de cada animal antes e após a suplementação. Os valores de hematócrito aumentaram significativamente após a suplementação, tanto nos animais inoculados como nos não inoculados com M. leprae. É possível que a quantidade de ferro na alimentação seja insuficiente para a formação da hemoglobina, levando a um quadro de anemia microcítica. A suplementação da dieta com sulfato ferroso reverteu este quadro, mostrando a importância de se conhecer o metabolismo de espécies exóticas para manutenção destas em cativeiro, assegurando seu bem estar.

Recidivas associadas à resistência a drogas na hanseníase / Relapse related to drug resistance in leprosy

A poliquimioterapia/Organização Mundial da Saúde foi implantada efetivamente no Brasil em 1991, contribuindo drasticamente para redução da taxa de prevalência e cura da hanseníase. No entanto, a sua comprovada eficácia não tem impedido a ocorrência de recidiva da doença. Falha no tratamento, persistência bacilar ou resistência a drogas são fatores que podem ou não estarem associados a ela. O objetivo deste estudo foi verificar a ocorrência de recidiva e associá-la com a presença de cepas resistentes do Mycobacterium leprae entre 28 indivíduos que apresentaram suspeita clínica de recidiva após tratamento por monoterapia sulfônica, esquema da Divisão Nacional de Dermatologia Sanitária ou poliquimioterapia. Biópsias das lesões de pacientes multibacilares, com diagnóstico clínico de recidiva, atendidos por demanda espontânea, foram coletadas para avaliar resistência a drogas por meio da técnica de inoculação em pata de camundongo. Dentre as amostras avaliadas 42,8% apresentaram bacilos sensíveis à dapsona e rifampicina e 10,7% apresentaram resistência à dapsona; não foram isolados bacilos resistentes à rifampicina. A emergência de bacilos resistentes, especialmente à rifampicina, é um alerta para os programas de controle da hanseníase. Monitorar a disseminação destas cepas é importante, pois elas apresentam um sério obstáculo para a eliminação da doença, principalmente em países onde a hanseníase ainda é endêmica.

The multidrugtherapy proposed by the World Health Organization has been effectively implemented in Brazil in 1991. It helped reduce the prevalence and achieve the cure of leprosy. However, its proven efficacy has not prevented the occurrence of relapses in some leprosy patients. Irregular treatment, bacillary persistence or resistance of Mycobacterium leprae to drugs are factors that may be associated with relapse. The objective of this study was assess the occurrence of relapse and associate it with the presence of Mycobacterium leprae resistant strains. In order to do that, 28 individuals who were clinically diagnosed as relapse after treatment with sulphone monotherapy, the National Division of Sanitary Dermatology scheme or multidrugtherapy. Biopsies from lesions of multibacillary patients attended by spontaneous demand were collected to verify resistance to drugs through the mouse foot pad inoculation technique. Among the samples evaluated 42.8% had bacilli susceptible to dapsone and rifampicin and 10.7% showed resistance to dapsone. No rifampicin resistant bacilli were isolated. The emergence of resistant strains, especially to rifampicin, is a threat to leprosy control programs, therefore, monitoring the spread of these strains is important because resistance pose a serious obstacle to the elimination of disease, particularly in countries where the disease is endemic.

Distribuição fenotípica e a freqüência genotípica do sistema sangüíneo Duffy em pacientes com a doença de Jorge Lobo / Phenotypic distribution and genotypic frequency of the Duffy blood group system in Jorge Lobo's disease patients

A doença de Jorge Lobo é uma micose cutânea e subcutânea de evolução crônica, causada pelo Lacazia loboi. Os mecanismos envolvidos na suscetibilidade/resistência do hospedeiro frente ao agente não estão ainda elucidados, dentre eles encontra-se a constituição genética. Portanto, o objetivo deste trabalho é avaliar a distribuição fenotípica do grupo sangüíneo Duffy em pacientes com doença de Jorge Lobo e calcular suas freqüências gênicas. Foram analisados 21 pacientes, 13 caucasóides e oito mestiços (tipo moreno claro), provenientes do estado do Acre, e 44 indivíduos, não acometidos por esta doença, habitantes da cidade de Rio Branco (AC), como grupo controle. O fenótipo Duffy foi definido pela presença dos antígenos nas hemácias pela reação de aglutinação dos eritrócitos com anti-soros anti-Fya e anti-Fyb incubando-se a 37ºC e aplicando-se o teste da antiglobulina humana. Os resultados mostram que a distribuição fenotípica do sistema Duffy nos pacientes difere significativamente daquela observada nos controles, pela alta freqüência de indivíduos com fenótipo Fy(a-b-). Observa-se claramente que os pacientes apresentam maior freqüência do alelo FY e menor do alelo FYA do que os controles. O alelo FYB (não inclui os alelos que não são expressos) tem praticamente a mesma freqüência nos dois grupos. Com relação à comparação entre distribuição do grupo sangüíneo Duffy com as formas clínicas, sugere maior número de pacientes com forma localizada da doença de Jorge Lobo apresentando um predomínio do fenótipo Fy(a-b+) seguido do fenótipo Fy(a-b-) e inexistência do fenótipo Fy(a+b-). Por outro lado, temos a forma disseminada com maior incidência de fenótipo Fy(a+b-). A forma multifocal foi menos expressiva. Desse modo, podemos inferir uma provável prevalência de fenótipo Duffy negativo na região por nós estudada e os poucos dados disponíveis até o momento tornam clara a necessidade de elaborar tabelas de freqüências fenotípicas para a população...

Jorge Lobo's disease is a chronic cutaneous-subcutaneous mycosis caused by the fungus Lacazia loboi. The mechanisms involved in susceptibility and resistance to infection by this pathogen, especially genetic background, are yet to be understood. The objective of the present study was to evaluate the phenotypic distribution of the Duffy blood group in Jorge Lobo's disease patients and calculate its gene frequencies. Twenty one patients were analyzed, among them 13 Caucasoid and 8 from mixed ethnical backgrounds (Afro-Brazilians) originating from the State of Acre. The control group consisted of 44 healthy individuals from the city of Rio Branco (AC). The Duffy phenotype was defined by the presence of antigens in erythrocytes as visualized by the agglutination reaction of erythrocytes in the presence of anti-Fya and anti-Fyb antisera incubated at 37ºC by the human antiglobulin test. Results showed that the phenotypic distribution of the Duffy blood group system in patients was significantly different from controls, with the frequency of individuals with phenotype Fy(a-b-) being very high. Patients presented remarkably higher frequencies of the allele FY and ower frequencies of the allele FYA compared to controls. Frequencies of the allele FYB (not including non-expressed alleles) were similar in both groups. The comparison between Duffy blood group distribution with clinical forms of the disease suggests predominance of the phenotype Fy(a-b+) in patients with the localized form of Jorge Lobo's disease, followed by phenotype Fy(a-b-) and absence of phenotype (a+b-). On the other hand, we observed a higher incidence of phenotype Fy(a+b-) in patients with the disseminated form of the disease. The multifocal forms were less expressive. Therefore, we may infer that the negative Duffy phenotype is probably more prevalent in the region studied. There is scarce data available on phenotype distributions, it is necessary to develop similar studies...

Mitsuda reaction in armadillos Dasypus novemcinctus using human and armadillo derived antigens

The armadillo has been an important experimental model for leprosy, besides it is still an important resource for bacilli. Despite the innumerous studies about armadillos of the Genus Dasypus, little is known about the real susceptibility of this species to the Hansen’s bacillus after experimental infection with M. leprae. Many authors have reported that 80 per cent of the inoculated animal will develop the disease. In Brazil, positive inoculation of this species was obtained only twice, being raised the hypothesis that these animals are more resistant to experimental infection. In the present study the response to the Mitsuda antigen was used as an indicative of cellular immune response to M. leprae in armadillos. Twenty one animals were tested with two Mitsuda antigen preparations, human derived (4,2 x 109 bacilli/ml) and armadillo derived (1,6 x 108 bacilli/ml) antigens. Response after 28 days of intradermal testing showed that most of the animals presented an infiltrate composed by grouped macrophages with vacuolated cytoplasm and rare lymphocytes. This response resembles lepromatous leprosy in humans and suggests that these animals would be susceptible to development of disseminated leprosy when successfully inoculated. Bacilloscopy in these animals varied from 3+ to 4+ according to Ridley’s scale (1966). Two animals developed a granulomatous reaction with borderline pattern and bacilloscopy varying from 1+ to 3+.