Correlación clínico-histológica de lepra en Guantánamo / Clinicopathological correlation of leprosy in Guantanamo

Se realizó un estudio para determinar la correlación clínica e histológica de la lepra en el Hospital General Docente de Guantánamo Dr Agostinho Neto, durante el año 2011. El universo estuvo constituido por 23 pacientes diagnosticados con enfermedad de Hansen en la provincia. Se describieron variables como: sexo, grupos de edades, áreas de salud, formas clínicas, manifestaciones clínicas más frecuentes, así como la correlación clínica e histológica de los casos estudiados. Predominaron los pacientes masculinos, los de 55 y más años de edad. Las áreas del 4 de Abril y Asdrúbal López presentaron mayor incidencia, la forma clínica más frecuente encontrada fue la lepromatosa y en la clínica predominaron las máculas anestésicas y la infiltración difusa. Se llegó a la conclusión de que aunque existe buena correlación clínico histológica en la mayoría de los casos estudiados, dentro de los factores que dificultan esta correlación se encontró la mala selección de la muestra biopsiada, una técnica mal empleada, deficiencia de los reactivos empleados, entre otros (AU)

A study was conducted to determine the clinical and histological correlation of leprosy in Guantanamo at the General Teaching Hospital Dr Agostinho Neto , in 2011. The universe was composed of 23 patients diagnosed with Hansen's disease in the province. Sex, age groups, and areas of health, clinical forms, most frequent clinical manifestations, clinical and histological correlation of the studied cases: variables are described: Predominance of male patients, 55 and more years old; areas of the South and 4 de Abril had a higher incidence, the most common clinical form was found lepromatous and clinical predominant anesthetic macules and diffuse infiltration. It was concluded that although there is good histologic clinical correlation in most cases studied, among the factors that hinder this correlation poor selection of the biopsied sample, a misused technical deficiency of the reagents was found, among others(AU)

Optimized protocols for Mycobacterium leprae strain management: frozen stock preservation and maintenance in athymic nude mice.

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1(nu)). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Optimized protocols for Mycobacterium leprae strain management: frozen stock preservation and maintenance in athymic nude mice

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1(nu)). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Estudo do papel do Fator de Crescimento Semelhante à Insulina-I (IGF-I) na modulação funcional de macrófagos infectados pelo Mycobacterium leprae / Study of the role of growth factor insulin-like (IGF-I) in the functional modulation of macrophages infected with Mycobacterium leprae

Fagócitos mononucleares são as células-alvo para micobactérias patogênicas que geralmente requerem um ambiente intracelular adequado para sua sobrevivência e replicação. Mycobacterium leprae, o agente etiológico da Hanseníase, é capaz de subverter mecanismos microbicidas de macrófagos e sobreviver e replicar no interior dessas células. Contudo o mecanismo molecular envolvido na modulação da célula hospedeira, não étotalmente compreendido. O presente estudo mostrou o potencial papel exercido pelo IGF-I na patogênese causada pelo M. leprae. Foi demonstrado que o M. leprae induz a expressão do fator de crescimento semelhante a insulina (IGF-I), em macrófagos RAW 264.7. Curiosamente, apenas quando as células foram tratadas com anticorpo neutralizante para o receptor do tipo I de IGF-I (IGF-1R) o M. leprae foi capaz de regular positivamente a expressão da enzima óxido nítrico sintase induzível (iNOS) em macrófagos RAW 264.7 ou ativar o promotor de iNOS em células transfectadas com a construção contendo gene repórter da luciferase sob o controle do promotor de iNOSO bloqueio da sinalização de IGF-I reduziu a viabilidade intracelular do M. leprae determinada por qPCR. As células RAW 264.7 pré-tratadas com IGF-I apresentaram uma redução significativa da atividade do promotor iNOS em resposta ao Mycobacterium bovis BCG, Mycobacterium smegmatis e consequentemente um aumento na viabilidade intracelular monitorada através de contagem de Unidades Formadoras de Colônia (CFU). Outro fato é que IGF-I foi capaz de atenuar a ativação de macrófagos mediada por IFN-gama medida através da expressão de iNOS e da quantificação da fosforilação de Ativador de transcrição e de transdução de sinal (pSTAT1). Além disso, o IGF-I foi capaz de induzir aumento de PGE2 em macrófagos, um eicosanoide previamente implicado na persistência micobacteriana no hospedeiro...

Mononuclear phagocytes are targeted cells for pathogenic mycobacteria thatgenerally require afavorable intracellular environment in wich they survive and replicate. Mycobacterium leprae, the etiologic agent of leprosy, is able to subvert macrophage microbicidal mechanisms and survive and replicate within these cells. However, the molecular mechanism involved in pathogen driven host cellmodulation is not fully understood. The present study investigated the potential role of insulin-like growth factor-I (IGF-I) in M. leprae pathogenesis. We showed that M.leprae induces IGF-I expression in RAW 264.7 macrophages in a dose-dependent manner. Notably, only when cells were treated with a neutralizing antibody to IGFtype 1 receptor (IGF-1R), M. leprae infection was able to upregulate inducible nitricoxide sintase (iNOS) expression in RAW cells or to activate the iNOS promoter incells transfected with the iNOS-luciferase reporter construct. . The blockage ofIGF-I signaling also decreased the intracellular M. leprae viability as measured byqPCR. Moreover, RAW cells pre-treated with IGF-I showed a significant reduction in iNOS promoter activity in response to Mycobacterium bovis BCG and Mycobacterium smegmatis with subsequent increase in bacterial intracellular survival as accessed by colony-forming unit counts (CFU). Of note, IGF-I was able to attenuate interferon gamma (IFN-gama ) activating effects on macrophages asacessed by iNOS expression and quantification of phosphorylated SignalTransducers and Activators of Transcription 1 (p-STAT1). Additionally, IGF-I was able to induce PGE2 secretion in murine macrophages, an eicosanoid previously implicated in mycobacterial persistence in the host. Finally, immunohistochemicalanalysis of skin lesions of lepromatous leprosy (LL) patients revealed an abundantexpression of IGF-I by highly infected foamy macrophages...

Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice.

BACKGROUND: The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues. METHODOLOGY/PRINCIPLE FINDINGS: Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations. CONCLUSIONS: hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications.

Interação patógeno-hospedeiro na hanseníase: indução da via de interferon tipo I como potencial mecanismo de sobrevivência do Mycobacterium leprae em macrófagos humanos / Host-pathogen interaction in leprosy: induction of type I IFN pathway as a potential mechanism for survival of mycobacterium leprae in human macrophages

A indução da via de interferon (IFN) do tipo I (IFN-alfa e -beta) é crucial para uma resposta protetora contra infecções virais. Entretanto, estudos recentes demonstraram que esta classe de IFNs tem a ativação mediada por receptores citoplasmáticos de DNA e regula negativamente a resposta protetora contra a infecção por bactérias intracelulares, com destaque para o Mycobacterium tuberculosis, criando um nicho favorável para a replicação micobacteriana. Um estudo anterior do nosso grupo, utilizando uma abordagem de expressão gênica global por microarranjos, demonstrou que a infecção de células de Schwann com Mycobacterium leprae induz genes ativados por IFN do tipo I, com destaque para o gene OASL, o qual codifica a proteína 2’ 5’ oligoadenilato sintetase like. No presente estudo, esses resultados foram estendidos e validados utilizando o modelo de macrófagos derivados de células THP-1. Nossos dados demonstram que a OASL foi induzida por M. leprae vivo, mas não M. bovis BCG ou M. leprae morto. Adicionalmente, a transfecção de DNA de M. leprae, mas não de RNA, induziu a produção de OASL. Em nosso modelo, a infecção por M. leprae não foi capaz de induzir a produção de IFN-alfa, assim como o tratamento CpG não induziu OASL, excluindo assim a sinalização de DNA mediada por TLR9. Além disso, mostramos evidências da permeabilização fagossomal mediada pelo fator de virulência micobacteriano ESAT-6, a qual permite acesso do conteúdo do fagossomo ao citoplasma e, consequente, produção de IFN-beta. O bloqueio farmacológico de TBK1 foi capaz de inibir a produção de OASL mediada pela infecção com M. leprae, indicando, dessa forma, que a indução de IFN-beta e seus genes a jusante, como o OASL, ocorre através do eixo de sinalização STING/TBK1/IRF3...

The induction of interferon pathway (IFN) type I (IFN-alfa and-beta) is crucial for a protective response against viral infections. However, emergent research has shown that type I IFNs and cytoplasmic DNA signaling are negative regulators of the protective response against infection by intracellular bacteria, such as Mycobacterium tuberculosis, and provide a favorable niche for mycobacterial replication. Recently, using an approach of global geneexpression by microarray, our group showed that Schwann cells infection with Mycobacterium leprae induced genes activated by IFN type I, highlighting the OASL gene,which encodes the 2 '5' oligoadenylate synthetase like protein. In the present work, these results were extended and validated using the THP-1-derived macrophages model. Our datademonstrate that OASL and type I IFN pathway was upregulated by M. leprae, but not M. bovis BCG or dead M. leprae, in macrophage-like THP-1 infected cells. In addition, M. leprae DNA transfection but not RNA, was able to induce OASL production. In our model, M. leprae infection was not able to induce IFN-alfa and OASL was not induced by CpG, therefore excluding a TLR9 dependent pathway. Furthermore, we show evidence of fagossomal permeabilization mediated by mycobacterial virulence factor ESAT-6, which enables accessof contents of the phagosome to host cytoplasm and subsequent induction of IFN-beta. Pharmacological blockage of TBK1 was able to inhibit M. leprae-induced OASL mRNA expression, indicating thereby that the IFN-beta induction and its downstream genes, such as OASL occur through the STING/TBK1/IRF3 pathway...

Current status of leprosy: epidemiology, basic science and clinical perspectives.

Leprosy has affected humans for millennia and remains an important health problem worldwide, as evidenced by nearly 250 000 new cases detected every year. It is a chronic infectious disorder, caused by Mycobacterium leprae, that primarily affects the skin and peripheral nerves. Recent advances in basic science have improved our knowledge of the disease. Variation in the cellular immune response is the basis of a range of clinical manifestations. The introduction of multidrug therapy has significantly contributed to a decrease in the prevalence of the disease. However, leprosy control activities, including monitoring and prevention programs, must be maintained.

Deciphering the proteomic profile of Mycobacterium leprae cell envelope.

The complete sequence of the Mycobacterium leprae genome, an obligate intracellular pathogen, shows a dramatic reduction of functional genes, with a coding capacity of less than 50%. Despite this massive gene decay, the leprosy bacillus has managed to preserve a minimal gene set, most of it shared with Mycobacterium tuberculosis, allowing its survival in the host with ensuing pathological manifestations. Thus, the identification of proteins that are actually expressed in vivo by M. leprae is of high significance in understanding obligate, intracellular mycobacterial pathogenesis. In this study, a high-throughput proteomic approach was undertaken resulting in the identification of 218 new M. leprae proteins. Of these, 60 were in the soluble/cytosol fraction, 98 in the membrane and 104 in the cell wall. Although several proteins were identified in more than one subcellular fraction, the majority were unique to one. As expected, a high percentage of these included enzymes responsible for lipid biosynthesis and degradation, biosynthesis of the major components of the mycobacterial cell envelope, proteins involved in transportation across lipid barriers, and lipoproteins and transmembrane proteins with unknown functions. The data presented in this study contribute to our understanding of the in vivo composition and physiology of the mycobacterial cell envelope, a compartment known to play a major role in bacterial pathogenesis.

Anti-neutrophil cytoplasmic antibodies in leprosy.

INTRODUCTION: Anti-Neutrophil Cytoplasmic Antibodies (ANCA) are auto-antibodies directed to intracellular components of neutrophils and used to be considered as present almost exclusively in granulomatous vasculitis. Recently, these auto-antibodies have been found in other autoimmune disorders as well as infectious diseases. MATERIALS AND METHODS: We studied patients with leprosy confirmed by bacilloscopy and/or skin biopsy, in reaction phase from the Ambulatório de Hanseníase do Hospital Universitário Professor Edgar Santos. ANCA and Antinuclear antibodies (ANA) were determined by indirect immunofluorescence using commercially available kits. RESULTS: Twenty patients were enrolled in our study, nine males and 11 females. The mean age was 36.9+/-18.2 years. ANCA were present only in one patient, with a perinuclear staining pattern (p-ANCA), and no patient tested positive for ANA. DISCUSSION: Although other studies have shown the presence of ANCA in leprosy, the low frequency of these antibodies in leprosy sera demonstrated in the present study illustrates the high specificity of ANCA for the diagnosis of Wegener granulomatosis.

Técnica de la inoculación en Almohadilla plantar para cultivo del Mycobacterium Leprae / No disponible

Aunque la multiplicación del M. leprae en las almohadillas plantares de ratones inmuno-competentes es limitada y no se originan lesiones típicas de la enfermedad, este método representa el primer sistema útil y modelo animal reproducible de la infección por M.leprae. Su uso ha permitido establecer investigaciones sobre temas básicos referentes a la enfermedad y sobre la microbiología del M. leprae y la epidemiología, tratamiento y control de la lepra. Esta técnica es muy labroiosa y cara en cuanto a la compra y mantenimiento de los animales. Además es imprecisa e insensible comparada con las técnicas utilizadas con microorganismos cultivables. Por estas razones y también por el éxito de la multiterapia, ha sido abandonada por muchos centros. Sin embargo, hasta que se disponga de una técnica más sensible y simple para demostrar la viabilidad del M.leprae, sigue siendo un instrumento esencial para la investigación en este campo. En este trabajo, se revisa la técnica de la almohadilla en detalle, se analiza su precisión y limitaciones, sus importantes aplicaciones y se describe el método que puede reemplazar a este en el futuro

Although multiplication of Mycobacterium leprae in the foot pads of immune-competent mice is limited, and no leprosy-like lesions are produced in these animals, the Mouse foot-pad system represents the first truly useful and reproducible animal model of M.leprae infection. Its employments has enabled research into basic questions with respect to the microbiology of M.leprae and the epidemiology, treatment and control of leprosy. The muse foot-pad technique is labour-intensive and time-consuming, and is expensive in terms of the costs of animal purchase and maintenance. In addition, the technique employed in working with cultivable micro-organisms. For these reasons, and also as by-product of the success of multi-drug therapy, the technique has been abandoned in many research centres. Nevertheless, until a more simple and sensitive technique for demonstrating the viability of M. leprae is developed, the mouse foot-pad system remains an essential tool for leprosy research. In this review, we discuss the mouse foot-pad technique in detail, analyse its precision, point ourt its shortcomings, describe its most important applications, and prescribe a method by witch to assess the ability of an alternative technique to serve in place of this established technique

Concentracao serica da lectina ligante de manose (MBL) em pacientes portadores de hanseniase / Serum concentration of binder of mannose lectin (MBL) in patients with leprosy

Alectina ligante de manose (MBL) e uma proteina com importantepapel na primeira linha de defesa do sistema imune inato, cujos valores sericos sao determinados geneticamente. A MBL ativa a via da lectina do complemento, alem de mediar a opsonisaçao e fagocitose de microorganismos. A hanseniase e uma doença infecciosa cronica causada pelo Mycobacterium leprae, bacteria intracelular obrigatoria, que infecta fagocitos mononucleares do ospedeiro...

Fnac study of histoid nodule: an early tool for diagnosis.

Histoid lesion, a variety of lepromatous leprosy, is due to alteration in the growth pattern of Mycobacterium leprae, possibly due to loss of immunity in localized areas. The distinction is based on cellular morphology by demonstrating pallisading arrangement of multi-layered spindle-shaped histocytes. Cytodiagnosis by fine needle aspiration cytology is therefore an early tool to recognize the histoid variety, differentiating it from a conventional LL module, as it is a simple and less traumatic procedure.

Soroprevalencia para hanseniase em areas endemicas do estado do Para / Soroprevalence for leprosy in endemic areas of the state of Para

Analisou-se a prevalencia de anticoepos IgM utilizando teste sorologico e antigeno especifico Glicolipidio Fenolico I (PGL-I) do Mycobacterium leprae, por meio do teste rapido ML Dipstick em 1611 individuos sadios categorizados em contatos de pacientes hansenicos intradomiciliares e comunitarios, residentes em cinco municipios endemicos para hanseniase. Realizou-se estudo tranversal pela analise do banco de dados do laboratorio de hanseniase, Instituto Evandro Chagas. O estudo estendeu-se de março a dezembro de 1998, com reavaliaçoes anuais ate dezembro de 2002, no qual, os municipios selecionados-Curinopolis, Eldorado do Carajas, Xinguara, Rondon do Para e Dom Eliseu - apresentaram taxas de prevalencia em hanseniase maiores que 20 em cada 10.000 hab. e detecçao de casos novos maiores que quatro em cada 10.000 hab. A idade variou de 2 a 90 anos, devido ao fato de que a doença em areas de alta endemicidade possibilita o diagnostico de crianças. Estudou-se 957 mulheres (957/1611-59.4 por cento), 654 homens (654/1611-40.6 por cento) e observou-se predominancia de soropositividade entre as mulheres na faixa etaria entre 21 e 50 anos, 62.81 por cento contra 53.97 por cento nos homens. A analise foi realizada nos bancos de dados D-BASE, EPI-INFO 6.4 e 2002, BIOESTAT 3.0 e SINAM, existentes no Instituto Evandro Chagas e Secretaria Estadual de Saude do Para...

Leprosy

Leprosy remains an important health problem wordwide. The disease in caused by a chronic granulomatous infection of the skin and peripheral nerves with Mycobacterium leprae. The clinical range from tuberculoid to lepromatous leprosy is a result of variation in the cellular immune response to the mycobacterium. The resulting impairment of nerve fuinction causes be disabilities associated with leprosy. This review summarises recent advances in understanding of the biology of leprosy, clinical features of the disease, the current diagnostic criteria, and the new approaches to treatment of the infection and the immune-mediated complications. Supervesed multi-drug therapy (MDT) for fixed durations is highly effective for all forms of the disease. The widespread implemantation of MDT has been associated with a fall in the prevalence of the leprosy but as yet no reduction in the case-detection rate globally. Thus, leprosy control activities must be maintained for decades to interrupt transmission of infection

Comparative studies of the cell structures of Mycobacterium leprae and M. tuberculosis using the electron microscopy freeze-substitution technique.

The cell envelope and cytoplasmic architecture of the Mycobacterium leprae Thai-53 strain were examined using the freeze-substitution technique of electron microscopy and compared with those of the M. tuberculosis H37Rv strain. Both strains had similarly multilayered envelope architectures composed of an electron-translucent layer, a peptidoglycan layer and the plasma membrane, from outside to inside. A comparison of the structures of these two mycobacteria revealed that the M. leprae cell was smaller in size and had a thinner peptidoglycan layer than the M. tuberculosis cell. The cell widths measured on electron micrographs were 0.44 microm for M. tuberculosis and 0.38 microm for M. leprae. The peptidoglycan layer of M. leprae was 4-5 nm, while the corresponding layer of M. tuberculosis was 10-15 nm.

Histoid leprosy: diagnosis by fine needle aspiration cytology

Histoid leprosy is a distinct and rare form of multibacillary leprosy that presents clinically as well-demarcated cutaneous and subcutaneous nodules similar to dermatofibroma.....