Hanseníase em gêmeos de 3 anos em Mato Grosso, Brasil: a importância da baciloscopia no diagnóstico / Leprosy in 3-year-old twin children in Mato Grosso, Brazil: the importance of bacilloscopy in the diagnosis

Introdução: o diagnóstico clínico da hanseníase em crianças é particularmente difícil. Relato de Caso: crianças gêmeas bivitelinas, com três anos de idade, eram contactantes de pai com hanseníase Virchowiana. Os dois pacientes têm lesões cutâneas bem definidas e irregulares, anteriormente tratadas como micoses e uma cicatriz de BCG. Foram confirmados positivos para Mycobacterium por análise histopatológica da pele. Discussão: especialmente, com menos de cinco anos, os diagnósticos de hanseníase são raros e difíceis porque simulam outras doenças. Esses diagnósticos são alarmes epidemiológicos para áreas endêmicas e mostram a importância dos sintomas em crianças e o rastreamento nos contactantes dos pacientes.

Introduction: the clinical diagnosis of leprosy in children is particularly difficult. Case Report: fraternal twins, three years old, were in contact with a father with Virchowian leprosy. Both patients have well-defined and irregular skin lesions previously treated as mycoses and a BCG scar. They were confirmed positive for Mycobacterium by histopathological analysis of the skin. Discussion:especially, with less than five years, leprosy diagnoses are rare and difficult because they simulate other diseases. These diagnoses are epidemiological alarms for endemic areas and show the importance of symptoms in children and tracking of patients' contacts.

Células gigantes en lepra lepromatosa: dermatitis difusa con células gigantes exuberantes de tipo cuerpo extraño en lepra lepromatosa tratada / Giant cells lepromatous leprosy: Diffuse dermatitis with exuberant foreign body giant cells in treated lepromatous leprosy

Resumen Los pacientes con lepra lepromatosa que han recibido tratamiento durante años, usualmente requieren seguimiento con biopsias de piel para detectar lesiones persistentes o si la baciloscopia es positiva, incluso si los valores son menores que los iniciales. Se presenta el caso de una mujer de 48 años de edad con lepra lepromatosa de 15 años de evolución, índice bacilar de 4 en el extendido directo y en la biopsia, que recibió tratamiento con múltiples medicamentos durante 32 meses, aunque lo recomendado por la Organización Mundial de la Salud (OMS) es una duración de 12 meses. Se tomó una biopsia de piel para determinar si la enfermedad estaba activa. Se observó inflamación dérmica difusa con numerosas células gigantes de tipo cuerpo extraño y macrófagos vacuolados (células de Virchow). Estas células, CD68 positivas, contenían material granular ácido-alcohol resistente positivo con inmunohistoquímica para BCG. Se encontraron bacilos fragmentados y el índice bacilar fue de 2. Se interpretó como una forma residual de lepra lepromatosa y se concluyó que la paciente no requería prolongar el tratamiento con múltiples medicamentos. Este perfil histológico se ha observado en casos similares, pero sin datos clínicos estas biopsias representan un reto diagnóstico. La acumulación de lípidos en estas células gigantes se debe a la destrucción bacilar y a la fusión de macrófagos vacuolados. Se revisó el papel de los lípidos del bacilo y del huésped en la patogenia de la lepra lepromatosa. En estos casos, no es necesario extender los 12 meses de tratamiento con múltiples medicamentos recomendados por la OMS. En el seguimiento de los pacientes, se recomienda contar con los hallazgos clínicos, la baciloscopia, la biopsia anual de piel y los títulos IgM antiglucolípido fenólico.

Abstract Patients with lepromatous leprosy that have received treatment for many years usually get follow up biopsies for persistent skin lesions or positive bacilloscopy even if the values are lower than in the initial bacilloscopy. We report the case of a 48-year old woman with long-standing lepromatous leprosy of 15 years of evolution, with a bacterial index of 4 in the direct smear and the initial skin biopsy. The patient was treated with multidrug therapy for 32 months although the treatment recommended by the World Health Organization (WHO) is only for 12 months. A skin biopsy was taken to determine if there was an active disease. We observed a diffuse dermal inflammation with numerous foreign body giant cells and vacuolated macrophages (Virchow´s cells). These cells contained granular acid-fast material that was also positive with immunohistochemistry for BCG. There were fragmented bacilli and the BI was 2. These cells were also strongly positive for CD68. The biopsy was interpreted as a residual form of lepromatous leprosy that did not require further multidrug therapy. We have observed similar histological profiles in several cases. The lack of clinical data makes it a histological challenge. The accumulation of lipids in these giant cells is due to bacillary destruction and fusion of vacuolated macrophages. We discuss here the role of bacillary and host lipids in the pathogenesis of lepromatous leprosy. We concluded that there was no need to extend the 12-month multidrug therapy recommended by WHO. Clinical findings, bacilloscopy, annual skin biopsy, and anti-phenolic glycolipid-I IgM titers are recommended procedures for the follow-up of these patients.

Performance of diagnostic tests for pulmonary tuberculosis in indigenous populations in Brazil: the contribution of Rapid Molecular Testing / Desempenho de testes para o diagnóstico de tuberculose pulmonar em populações indígenas no Brasil: a contribuição do Teste Rápido Molecular

ABSTRACT Objective: To evaluate the accuracy of rapid molecular testing as a diagnostic tool and estimate the incidence of smear-positive pulmonary tuberculosis among the indigenous population. Methods: This is an epidemiological study based on secondary data. We calculated the incidence of smear-positive pulmonary tuberculosis between January 1st, 2011 and December 31, 2016, and the performance of bacilloscopy and rapid molecular testing in diagnosing pulmonary tuberculosis compared to sputum culture (standard test). Results: We included 4,048 cases of indigenous people with respiratory symptoms who provided sputum samples for analysis. Among them, 3.7%, 6.7%, and 3.7% had positive results for bacilloscopy, sputum culture, and rapid molecular testing, respectively. The mean incidence of pulmonary tuberculosis was 269.3/100 thousand inhabitants. Rapid molecular testing had 93.1% sensitivity and 98.2% specificity, compared to sputum culture. Bacilloscopy showed 55.1% sensitivity and 99.6% specificity. Conclusions: Rapid molecular testing can be useful in remote areas with limited resources and a high incidence of tuberculosis, such as indigenous villages in rural regions of Brazil. In addition, the main advantages of rapid molecular testing are its easy handling, fast results, and the possibility of detecting rifampicin resistance. Together, these attributes enable the early start of treatment, contributing to reduce the transmission in communities recognized as vulnerable to infection and disease.

RESUMO Objetivo: Avaliar a acurácia do teste rápido molecular como ferramenta diagnóstica e estimar a incidência de casos pulmonares positivos entre a população indígena. Métodos: Estudo epidemiológico baseado em dados secundários. Foi calculada a incidência de casos de tuberculose pulmonar positiva entre 1° de janeiro de 2011 e 31 de dezembro de 2016, e o desempenho da baciloscopia e do teste rápido molecular no diagnóstico de tuberculose pulmonar, em comparação à cultura de escarro (teste padrão). Resultados: Foram incluídos 4.048 casos de indígenas considerados sintomáticos respiratórios, que forneceram amostras de escarro para análise. Destes, 3,7%, 6,7% e 3,7% apresentaram resultados positivos para baciloscopia, cultura e teste rápido molecular, respectivamente. A incidência média de tuberculose pulmonar foi de 269,3/100 mil habitantes. A sensibilidade do teste rápido molecular, em relação à cultura, foi 93,1% e a especificidade foi 98,2%. A baciloscopia apresentou sensibilidade 55,1% e especificidade 99,6%. Conclusões: O teste rápido molecular pode ser útil em áreas remotas, com recursos limitados e incidência de tuberculose elevada, como as aldeias indígenas nas áreas rurais do país. Ademais, o teste rápido molecular apresenta como principais vantagens o fácil manuseio, os resultados rápidos e a possibilidade de identificar a resistência à rifampicina. Em conjunto, esses atributos facilitam o início do tratamento precoce, contribuindo para reduzir a transmissão em comunidades reconhecidamente vulneráveis à infecção e à doença.

Relationship between bacilloscopy and operational classification of Hansen's disease in patients with reactions

Abstract: Leprosy can be classified according to its operational form as paucibacillary or multibacillary. Bacilloscopy integrates its diagnostic armamentarium. Patients with the disease may present leprosy reactions. This study describes the association of bacilloscopy results and the type of operational classification of leprosy in patients with leprosy reactions. Medical records were analyzed at a reference center between 2010 and 2015. Reactions occurred in almost half of the patients, making their identification important. The bacilloscopic and operational characterization indicates a greater occurrence of leprosy reactions in patients with positive bacilloscopy and also in multibacillary.

Natural environmental water sources in endemic regions of northeastern Brazil are potential reservoirs of viable Mycobacterium leprae

BACKGROUND The detection of live Mycobacterium leprae in soil and animals other than humans suggests that the environment plays a role in the transmission of leprosy. OBJECTIVE The objective of this study was to investigate the presence of viable M. leprae in natural water sources used by the local population in five municipalities in the state of Ceará, northeastern Brazil. METHODS Samples were collected from 30 different sources. Viable bacilli were identified by reverse transcriptase polymerase chain reaction (PCR) of the M. leprae gyrA gene and sequencing of the PCR products. Physicochemical properties of each water source were also assessed. FINDINGS M. leprae gyrA mRNA was found in 23 (76.7%) of the water sources. No association was found between depth of the water and sample positivity, nor was there any association between the type of water used by the population and sample positivity. An association between viable M. leprae and temperature and pH was found. Georeferencing showed a relation between the residences of leprosy cases and water source containing the bacterium. MAIN CONCLUSIONS The finding of viable M. leprae in natural water sources associated with human contact suggests that the environment plays an important role in maintaining endemic leprosy in the study region.

Presence of Mycobacterium leprae genotype 4 in environmental waters in Northeast Brazil

Abstract INTRODUCTION: This study quantified Mycobacterium leprae bacilli in environmental water samples from five municipalities in the State of Ceará by quantitative polymerase chain reaction (qPCR) and compared the identified genotypes with those obtained from leprosy patient biopsies. METHODS: We collected five replicas from each of the 30 selected reservoirs and skin lesion biopsies from 25 new leprosy cases treated at a reference center in Fortaleza, Ceará from 2010 to 2013. The 16S rRNA gene region of M. leprae was amplified by qPCR and a standard curve was created with the pIDTBlue 16SrRNAMlep plasmid. The Juazeiro do Norte water samples and the biopsies were genotyped (single nucleotide polymorphism [SNP] 1 to 4) and the SNP 4 genotypes were subtyped. RESULTS: Of the 149 water samples analyzed, 54.4% were positive for the M. leprae DNA. The M. leprae bacilli copy number ranged from 1.42 × 10 -1 to 1.44 × 10 + 2 . Most biopsies showed SNP type 4 (64%), while all samples from Juazeiro do Norte were SNP type 4, with subtype 4-N appearing at the highest frequency. CONCLUSIONS: We suggest that environmental waters containing M. leprae bacilli play an important role in disease transmission, justifying PGL-1 seropositivity in individuals living in areas where there is no reported case, and in leprosy cases individuals who report no previous contact with other case. Therefore, further investigation is needed to clarify disease transmission in this region and to explore the role of the environment. We also suggest that in this area surveillance for leprosy cases should be intensified.

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.

Compreendendo a "alta em hanseníase": uma análise de conceito / Understanding "patient discharge in leprosy": a concept analysis

Resumo OBJETIVO Analisar o conceito de alta em hanseníase. MÉTODOS Estudo teórico pautado no referencial metodológico de análise de conceito. Realizou-se levantamento bibliográfico, de dezembro de 2015 a janeiro de 2016, nas bases SCOPUS, CINAHL, PUBMED, LILACS, SCIELO e BDENF, mediante uso dos descritores "Hanseníase" e "Alta do Paciente", obtendo-se 13 estudos. RESULTADOS Identificou-se alta por cura, alta medicamentosa, alta bacteriológica e pós-alta como possíveis usos do conceito. Os atributos definidos foram conclusão da poliquimioterapia, conclusão da poliquimioterapia para paucibacilares, conclusão da poliquimioterapia para multibacilares e cura da hanseníase. Como antecedentes, identificou-se infecção pelo M. leprae, acometimento de pele e de nervos periféricos, diagnóstico e tratamento e reações hansênicas. Saída do registro ativo de casos de hanseníase e continuidade de atenção em saúde foram os consequentes. Apresentou-se um caso modelo e um caso contrário. CONCLUSÕES A análise ampliou o conceito "alta em hanseníase", para além da clínica focada na poliquimioterapia.

Resumen OBJETIVO Analizar el concepto de alta lepra. MÉTODOS Análisis de concepto, propuesto por Walker y Avant. Literatura detenido en los meses de diciembre de 2015 y enero de 2016, las bases SCOPUS, CINAHL, PUBMED, LILACS, SCIELO y BDENF mediante el uso de descriptores "alta" del paciente asociados con el operador boleano AND, dando lugar a 13 estudios "lepra". RESULTADOS Se identificaron alta curación, drogas alta, alta bacteriológico y después del alta como posibles usos del concepto. Los atributos definidos eran finalización de la poliquimioterapia, la finalización de la poliquimioterapia para paucibacilar, la finalización de la poliquimioterapia para la curación de la lepra multibacilar y. Como antecedente, se identifico la infección por M. leprae, lapiel y La participación de los nervios periféricos, diagnóstico y tratamiento de las reacciones y la lepra. Activa La producción récord de casos de lepra, La continuidad del cuidado de la salud son consecuentes. CONCLUSIONES El término "alta lepra" se agranda, además de la terapia de múltiples fármacos.

Abstract OBJECTIVE To analyze the concept of patient discharge in leprosy. METHODS Theoretical study guided in the methodological framework of concept analysis. A bibliographical survey was held from December 2015 to January 2016 using the following bases: SCOPUS, CINAHL, PubMed, LILACS, SCIELO and BDENF, by use of the descriptors "Leprosy" and "Patient Discharge", resulting in 13 studies. RESULTS The following were identified as possible uses for the concept: discharge by cure, drug use discharge, bacteriological discharge and post-discharge. The attributes defined were completion of multidrug therapy, completion of multidrug therapy for paucibacillary leprosy, completion of multidrug therapy for multibacillary leprosy and cure from leprosy. The presence of an M. leprae infection, symptoms present in skin and peripheral nerves, diagnosis and treatment and leprosy reactions were identified as antecedents. Consequents were exclusion from the active leprosy record and continuity of health care. One case model and one opposing case were presented. CONCLUSIONS The analysis broadened the concept "discharge in leprosy", providing other meanings than the clinical definition of multidrug therapy.

Widespread nasal carriage of Mycobacterium lepraeamong a healthy population in a hyperendemic region of northeastern Brazil

A case-control study was conducted to determine the presence ofMycobacterium lepraeDNA in nasal secretions of leprosy cases and nonleprosy individuals in Fortaleza, Brazil. It included 185 cases identified by physicians at the Dona Libânia National Reference Centre for Sanitary Dermatology (CDERM). A control group (Co) (n = 136) was identified among individuals from CDERM not diagnosed as leprosy cases. To augment the spatial analysis of M. leprae specific repetitive element (RLEP) positive prevalence, an external group (EG) (n = 121), a convenience sample of healthy students, were included. Polymerase chain reaction for the RLEP sequence was conducted for all participants. Prevalence of RLEP positivity for cases and Co were 69.2% and 66.9%, respectively, significantly higher than for EG (28.1%), and reported elsewhere. Male sex, belonging to a lower socioeconomic status (D/E), history of a previous contact with a case and being older, were associated with being a leprosy case. Our geographical analysis demonstrated that the bacillus is widespread among the healthy population, with clusters of RLEP positive multibacillary cases concentrated in distinct areas of the city. Our results suggest that in endemic areas, as in Fortaleza, surveillance for both nonhousehold leprosy contacts and members of the general population living in cluster areas should be implemented.

Evaluación de diferentes muestras para la detección molecular de Mycobacterium leprae en Cuba / Evaluation of different samples for Mycobacterium leprae molecular detection in Cuba

Introducción: en la actualidad, la principal estrategia para controlar la infección por Mycobacterium leprae es la detección precoz y la multiterapia. El objetivo del presente estudio fue evaluar la utilidad del empleo de diferentes muestras clínicas en el diagnóstico molecular de la infección por M. leprae. Métodos: se evaluaron 22 pacientes con clínica sugestiva de lepra. Se tomaron muestras de linfa de cuatro puntos (ambos lóbulos auriculares y ambos codos) recogidas en una lámina porta objetos (lámina de baciloscopía) y en un hisopo, además muestra de hisopado nasal, muestra de sangre total y de tejido de la lesión. Las muestras se analizaron mediante baciloscopía, histología y PCR según correspondió en cada caso. Por otro lado, para determinar la sensibilidad y especificidad del método, se realizó un estudio de casos y controles en el que se emplearon 40 láminas de baciloscopías negativas de pacientes con lepra paucibacilar y 40 láminas negativas de personas sin lepra. Resultados: el 54,5 por ciento de los pacientes resultó positivo por baciloscopía. Solo en el 41 por ciento de los pacientes la histología tuvo resultados concluyentes de lepra. En el 100 por ciento de los pacientes se detectó la presencia de ADN de M. leprae a partir de la lámina de baciloscopía y el hisopado de linfa. En el 95,45 por ciento de los pacientes se pudo amplificar la secuencia diana a partir de la sangre total y solo en el 31,8 por ciento de los pacientes el hisopado nasal resultó positivo. El estudio de casos y controles mostró que la sensibilidad y especificidad de la PCR respecto al diagnóstico convencional fue de 100 por ciento. Conclusión: el diagnóstico de M. leprae mediante PCR, es de gran utilidad cuando las técnicas convencionales no son concluyentes. La lámina de baciloscopía y el hisopado de linfa constituyen las muestras clínicas más útiles para la confirmación molecular de la infección por M. leprae(AU)

Introduction: Currently, the early detection and treatment with multitherapy are the main strategy to control the infection by Mycobacterium leprae. The objective of the present study was to evaluate the usefulness of different clinical samples for molecular diagnosis of M. leprae infection. Methods: Twenty two patients with suggestive clinical leprosy were analyzed. Different clinical samples were taken by slit skin smear, histopathology and PCR. To determine the sensitivity and specificity of the PCR method, a case-control study was also performed using 40 slides from negative smears of patients with leprosy paucibacillary and 40 from individuals without leprosy. Results: From all patient studied fifty-four percent were positive by slit skin smear and 41 percent were conclusive of leprosy by histopathology. M. leprae DNA was detected in slit skin smears and lymph swabs in 100 percent of patients. In 95,45 percent of patients were detected M. leprae DNA in whole blood and in 31,8 percent of them in nasal swab. The sensitivity of PCR respect to conventional diagnostic was 100 percent, the specificity was 97.5 percent, and the positive predictive value was 97.56 and the negative predictive value was100 percent. Conclusion: The diagnosis of M. leprae by PCR is very useful when conventional techniques are inconclusive. The slit skin smears and lymph swabs are the most useful clinical samples for molecular confirmation of infection with M. leprae(AU)

Leprosy: a review of laboratory and therapeutic aspects - Part 2

Leprosy is a chronic infectious condition caused by Mycobacterium leprae(M. leprae). It is endemic in many regions of the world and a public health problem in Brazil. Additionally, it presents a wide spectrum of clinical manifestations, which are dependent on the interaction between M. leprae and host, and are related to the degree of immunity to the bacillus. The diagnosis of this disease is a clinical one. However, in some situations laboratory exams are necessary to confirm the diagnosis of leprosy or classify its clinical form. This article aims to update dermatologists on leprosy, through a review of complementary laboratory techniques that can be employed for the diagnosis of leprosy, including Mitsuda intradermal reaction, skin smear microscopy, histopathology, serology, immunohistochemistry, polymerase chain reaction, imaging tests, electromyography, and blood tests. It also aims to explain standard multidrug therapy regimens, the treatment of reactions and resistant cases, immunotherapy with bacillus Calmette-Guérin (BCG) vaccine and chemoprophylaxis.

Is drug-resistant Mycobacterium leprae a real cause for concern?: First approach to molecular monitoring of multibacillary Colombian patients with and without previous leprosy treatment / ¿Es la resistencia de Mycobacterium leprae a los medicamentos un verdadero motivo de preocupación?: Primera aproximación a la vigilancia molecular de pacientes colombianos multibacilares con tratamiento previo para lepra y sin él

Introduction: There is no information in Colombia on Mycobacterium leprae primary and secondary drug resistance in regards to the WHO-multidrug therapy regime. On the other hand, public health authorities around the world have issued various recommendations, one of which prompts for the immediate organization of resistance surveillance through simple molecular methods. Objective: To determine the prevalence of Mycobacterium leprae drug resistance to rifampicin, ofloxacin and dapsone in untreated and previously treated patients at the Centro Dermatológico Federico Lleras Acosta during the 1985-2004 period. Materials and methods: We conducted a retrospective study which included multibacillary patient biopsies through elective sampling: 381 of them from new patients and 560 from previously treated patients. Using a microtome, we obtained six slides from each skin biopsy preserved in paraffin, and we extracted M. leprae DNA. We amplified three molecular targets through PCR and obtained the patterns of drug resistance to dapsone, rifampicin and ofloxacin by reverse hybridization. Finally, we collected epidemiological, clinical and demographical data for analyses. Results: From 941 samples under study, 4.14% of them were resistant to one or more drugs, and 5.77 and 3.04% had resistant genotypes in new and previously treated patients, respectively. Total resistance for each drug was 0.43% for dapsone, 3.19% for rifampicin and 1.17% for ofloxacin. We found statistically significant differences for rifampicin and for the total population when comparing the results from untreated versus previously treated patients. Two thirds of the resistant samples were resistant to rifampicin alone or combined. Conclusions: The standard multidrug therapy schemes continue being effective for leprosy cases; however, it is necessary to guarantee adherence and regularity. Surveillance to drug resistance in new and previously treated leprosy cases should be established.

Introducción. Colombia no dispone de información sobre farmacorresistencia primaria y secundaria de Mycobacterium leprae al esquema de terapia múltiple de la Organización Mundial de la Salud (OMS) y las autoridades de salud pública del mundo han emitido varias recomendaciones, entre las cuales está organizar de inmediato la vigilancia a la resistencia empleando métodos moleculares simples. Objetivo. Determinar la prevalencia de la resistencia de M. leprae a rifampicina, ofloxacina y dapsona en pacientes del Centro Dermatológico Federico Lleras Acosta con tratamiento previo y sin él durante el período de 1985 a 2004. Materiales y métodos. Se realizó un estudio retrospectivo. Mediante muestreo electivo se incluyeron biopsias de pacientes multibacilares: 381 de pacientes nuevos y 560 de pacientes previamente tratados. Se obtuvieron con micrótomo seis cortes de cada biopsia de piel incluida en parafina, y se realizó la extracción de ADN de M. leprae. Se llevó a cabo la amplificación de tres blancos moleculares mediante PCR y se obtuvieron los patrones de resistencia a los medicamentos dapsona, rifampicina y ofloxacina por hibridación inversa. Se recolectaron datos epidemiológicos, clínicos y demográficos para llevar a cabo los análisis. Resultados. De las 941 muestras estudiadas, 4,14 % era resistente a uno o más fármacos, y se detectaron 5,77 y 3,04 % con genotipos resistentes en pacientes nuevos y previamente tratados, respectivamente. La resistencia total para cada fármaco fue de 0,43 % a dapsona, 3,19 % a rifampicina y 1,17 % a ofloxacina. Se encontró una diferencia estadísticamente significativa para rifampicina y para la población total al comparar los resultados de los pacientes no tratados con los de los pacientes tratados previamente. Dos tercios de las muestras resistentes lo fueron a rifampicina sola o combinada. Conclusiones. Los esquemas de terapia múltiple estándar siguen siendo efectivos para los casos de lepra; sin embargo, es necesario garantizar el cumplimiento y la regularidad y establecer la vigilancia de la farmacorresistencia en pacientes nuevos y previamente tratados.

Use of fine needle aspirate from peripheral nerves of pure-neural leprosy for cytology and PCR to confirm the diagnosis: A pilot study.

Background: The diagnosis of pure neural leprosy (PNL) remained subjective because of over-dependence of clinical expertise and a lack of simple yet reliable diagnostic tool. The criteria for diagnosis, proposed by Jardim et al., are not routinely done by clinicians in developing country as it involves invasive nerve biopsy and sophisticated anti-PGL-1 detection. We conducted a study using fi ne needle aspiration cytology (FNAC) coupled with Ziehl Neelsen staining (ZN staining) and Multiplex- Polymerase Chain Reaction (PCR) specifi c for M. leprae for an objective diagnosis of pure neural leprosy (PNL), which may be simpler and yet reliable. Aim: The aim of the study is to couple FNAC with ZN staining and multiplex PCR to diagnose pure neural leprosy patients rapidly, in simpler and yet reliable way. Methods: Thirteen patients of PNL as diagnosed by two independent consultants were included as case, and 5 patients other than PNL were taken as control in the study. Fine needle aspiration was done on the affected nerve, and aspirates were evaluated for cytology, ZN staining and multiplex- PCR. Results: Out of the 13 cases where fi ne needle aspiration was done, M. leprae could be elicited in the nerve tissue aspirates in 5 cases (38.4%) with the help of conventional acid-fast staining and 11 cases (84.6%) with the help of multiplex PCR. On cytological examination of the aspirates, only 3 (23%) cases showed specifi c epithelioid cells, whereas 8 (61.5%) cases showed non-specifi c infl ammation, and 2 (15.3%) cases had no infl ammatory cells. Conclusion: Our study demonstrates that in the fi eld of laboratory diagnosis of PNL cases, FNAC in combination with ZN staining for acid-fast bacilli (AFB) and Multiplex-PCR can provide a rapid and defi nitive diagnosis for the majority of PNL cases. FNAC is a less-invasive, outdoor-based and simpler technique than invasive nerve biopsy procedure. Thus, this study may enlighten the future path for easy and reliable diagnosis of PNL.

Detection of Mycobacterium leprae in saliva and the evaluation of oral sensitivity in patients with leprosy

The aim of this study was to investigate sensitivity disorders in the oral cavity related to the presence of Mycobacterium leprae in the saliva of treatment-naïve patients with leprosy in the state of Amazonas, Brazil. A cross-sectional study was conducted involving 45 subjects with leprosy. The subjects were interviewed to evaluate the sensitivity of the oral cavity. For the detection of M. leprae, saliva and slit-skin smear samples were collected. The samples were analysed using a bacteriological index (BI) protocol and the real-time quantitative polymerase chain reaction (qPCR). The results indicated that 15 of the 45 (33.3%) subjects with leprosy showed decreased oral sensitivity, which confirmed the importance of the oral cavity sensitivity evaluation. There was not a direct relationship between the presence of M. leprae in saliva and changes in oral sensitivity. Positive saliva qPCR results from six (31.6%) of 19 paucibacillary (PB) patients suggested the possibility of a new site for sample collection. Positive results using these diagnostic techniques (BI, slit-skin smear and saliva qPCR) increased to 55.5%, thus opening the possibility of combining these different techniques to increase the rate of positive diagnoses, especially in PB patients.

Mycobacterium leprae virulence-associated peptides are indicators of exposure to M: leprae in Brazil, Ethiopia and Nepal

Silent transmission of Mycobacterium leprae, as evidenced by stable leprosy incidence rates in various countries, remains a health challenge despite the implementation of multidrug therapy worldwide. Therefore, the development of tools for the early diagnosis of M. leprae infection should be emphasised in leprosy research. As part of the continuing effort to identify antigens that have diagnostic potential, unique M. leprae peptides derived from predicted virulence-associated proteins (group IV.A) were identified using advanced genome pattern programs and bioinformatics. Based on human leukocyte antigen (HLA)-binding motifs, we selected 21 peptides that were predicted to be promiscuous HLA-class I T-cell epitopes and eight peptides that were predicted to be HLA-class II restricted T-cell epitopes for field-testing in Brazil, Ethiopia and Nepal. High levels of interferon (IFN)-γ were induced when peripheral blood mononuclear cells (PBMCs) from tuberculoid/borderline tuberculoid leprosy patients located in Brazil and Ethiopia were stimulated with the ML2055 p35 peptide. PBMCs that were isolated from healthy endemic controls living in areas with high leprosy prevalence (EChigh) in Ethiopia also responded to the ML2055 p35 peptide. The Brazilian EChigh group recognised the ML1358 p20 and ML1358 p24 peptides. None of the peptides were recognised by PBMCs from healthy controls living in non-endemic region. In Nepal, mixtures of these peptides induced the production of IFN-γ by the PBMCs of leprosy patients and EChigh. Therefore, the M. leprae virulence-associated peptides identified in this study may be useful for identifying exposure to M. leprae in population with differing HLA polymorphisms.

Mycobacterium leprae in six-banded (Euphractus sexcinctus) and nine-banded armadillos (Dasypus novemcinctus) in Northeast Brazil

Human beings are the main reservoir of the causative agent of leprosy, Mycobacterium leprae. In the Americas, nine-banded armadillos (Dasypus novemcinctus) also act as a reservoir for the bacillus. In the state of Ceará (CE), which is located in Northeast Brazil and is an endemic area of leprosy, there are several species of armadillos, including D. novemcinctus and Euphractus sexcinctus (six-banded armadillo). Contact between humans and armadillos occur mainly through hunting, cleaning, preparing, cooking and eating. This study identified M. leprae DNA in the two main species of armadillos found in Northeast Brazil. A total of 29 wild armadillos (27 D. novemcinctus and 2 E. sexcinctus) were captured in different environments of CE countryside. Samples from the ear, nose, liver and spleen from each of these animals were tested by a nested M. leprae-specific repetitive element polymerase chain reaction assay. The samples that tested positive were confirmed by DNA sequencing. M. leprae was detected in 21% (6/29) of the animals, including five D. novemcinctus and one E. sexcinctus. This is the first Brazilian study to identify the presence of a biomarker of M. leprae in wild armadillos (D. novemcinctus and E. sexcinctus) in a leprosy hyperendemic area where there is continuous contact between humans and armadillos.

Concordance between expected and observed bacilloscopy results of clinical forms of leprosy: a 6-year retrospective study in Recife, State of Pernambuco, Brazil / Concordância entre os resultados observados e esperados da forma clinica da hanseníase de acordo com a baciloscopia: um estudo retrospectivo de seis anos em Recife, Estado de Pernambuco, Brasil

INTRODUCTION: Operational classification of leprosy based on the number of skin lesions was conceived to screen patients presenting severe forms of the disease to enable their reception of a more intense multidrug regimen without having to undergo lymph smear testing. We evaluated the concordance between operational classification and bacilloscopy to define multibacillary and paucibacillary leprosy. METHODS: We selected 1,213 records of individuals with leprosy, who were untreated (new cases) and admitted to a dermatology clinic in Recife, Brazil, from 2000 to 2005, and who underwent bacteriological examination at diagnosis for ratification of the operational classification. RESULTS: Compared to bacilloscopy, operational classification demonstrated 88.6% sensitivity, 76.9% specificity, a positive predictive value of 61.8%, and a negative predictive value of 94.1%, with 80% accuracy and a moderate kappa index. Among the bacilloscopy-negative cases, 23% had more than 5 skin lesions. Additionally, 11% of the bacilloscopy-positive cases had up to 5 lesions, which would have led to multibacillary cases being treated as paucibacillary leprosy if the operational classification had not been confirmed by bacilloscopy. CONCLUSIONS: Operational classification has limitations that are more obvious in borderline cases, suggesting that in these cases, lymph smear testing is advisable to enable the selection of true multibacillary cases for more intense treatment, thereby contributing to minimization of resistant strain selection and possible relapse.

INTRODUÇÃO: A classificação operacional da hanseníase baseada no número de lesões de pele foi concebida para selecionar pacientes que apresentam formas graves da doença para receber regime terapêutico mais intenso com múltiplas drogas sem o exame de baciloscopia da linfa. Nós avaliamos a concordância entre a classificação operacional e a baciloscopia para a definição de hanseníase multibacilar e paucibacilar. MÉTODOS: Nós selecionamos 1.213 registros de indivíduos com hanseníase não tratada (casos novos), atendidos em um Ambulatório de Dermatologia, em Recife, Brasil, no período de 2000 a 2005, que foram submetidos a exame bacteriológico ao diagnóstico para a ratificação da classificação operacional. RESULTADOS: Comparando com a baciloscopia, a classificação operacional baseada no número de lesões cutâneas mostrou sensibilidade de 88,6%, especificidade 76,9%, valor preditivo positivo de 61,8% e valor preditivo negativo de 94,1%, com uma precisão de 80% e um moderado índice kappa. Entre os casos com baciloscopia negativa, 23% tinham mais de cinco lesões de pele, recebendo um tratamento mais intensivo. Além disso, 11% dos casos baciloscopia positiva tinham até cinco lesões, o que induziriam casos multibacilares de serem tratados com hanseníase paucibacilar se a classificação operacional não tivesse sido confirmada pela baciloscopia. CONCLUSÕES: Concluímos que a classificação operacional tem limitações mais visíveis nos casos borderline, sugerindo que, nestes casos, o esfregaço seria aconselhável por permitir que os verdadeiros casos multibacilares fossem selecionados para um tratamento mais intenso, contribuindo para minimizar a seleção de cepas resistentes e uma possível recidiva.

IL-10 gene promoter polymorphisms and leprosy in a Colombian population sample / Polimorfismos en el gen promotor de IL-10 en una muestra de pacientes colombianos con lepra

Introduction. Polymorphisms in promoters of genes code for cytokines that affect transcription levels. Several have been associated with leprosy patients that have functional and clinical implications. Objective. Polymorphisms in the promoter of the IL10 gene of leprosy patients will be compared frequencies in normal population. Materials and methods. SNPs (single nucleotide polymorphism) -1082 A/G (rs1800896), -819C/T (rs1800871), and -592A/C (rs1800872) were identified in 100 leprosy patients and in a control group of 100 volunteers from a leprosy endemic region of Colombia. Results. The genotypes C/C and C/T in the SNP -819 were associated together with leprosy (OR=4.34, p<0.001).Similarly, the genotypes C/C and C/A in the -592 SNP showed an association (OR=4.3, p<0.001). The haplotypes -819C-519C and -1082A-819C-592C showed significant association (OR=4.34, p<0.001 and OR=6.25, p<0.001) respectively. These haplotypes in homozygosis conditions were also associated with leprosy: -819C-519C/-819C-519C (OR=4.34, p<0.001), -1082A -819C-592C/-1082A -819C-592C (OR=1.90, p=0.04). The SNP -1082 was not associated with leprosy in this population. Conclusions. The haplotypes associated with leprosy, -1082A-819C-592C/-1082A-819C-592C, have been reported as low producers of IL-10. Functionally, the low production of IL-10 may have immune response consequences and clinical implications. Additional haplotypes of IL-10 have been reported as markers for leprosy susceptibility or resistance in other ethnic populations. This suggests that differences in distribution of diverse IL-10 gene polymorphisms among ethnic groups may indicate important gene-disease associations.

Introducción. Se han reportado polimorfismos en los genes promotores que codifican para citocinas y que afectan los niveles de transcripción, con implicaciones clínicas y funcionales en pacientes con lepra. Objetivo. Detectar los polimorfismos en el gen promotor de la interleucina 10 (IL-10), de los polimorfismos de un solo nucleótido (Single Nucleotide Polymorphisms, SNP) -1082 A/G (rs1800896), -819C/T (rs1800871) y -592A/C (rs1800872), en 100 pacientes con lepra y un grupo control de 100 voluntarios, de una región endémica de Colombia. Resultados. Los haplotipos -819C-519C y -1082A-819C-592C mostraron asociación significativa con lepra: OR=4,34, p=1 x 10-3, y OR=6,25, p=5 x 10-4, respectivamente. Estos haplotipos en condiciones de homocigoto, están también asociados con lepra: -819C-519C/-819C-519C (OR=4,34 p=1 x 10-3), -1082A -819C-592C/-1082A -819C-592C (OR=1,90 y p=0,04). El SNP -1082 no se encontró asociado con lepra en esta población. Los genotipos C/C y C/T en el SNP -819, se encontraron asociados a lepra (OR=4,34, p=1 x 10-3); de igual manera, los genotipos C/C y C/A en el SNP -592 mostraron asociación (OR=4,34, p=1 x 10-3). Conclusiones. El haplotipo que encontramos asociado con lepra, -1082A-819C-592C/-1082A-819C-592C, se ha relacionado con baja producción de IL-10. Funcionalmente, esta baja producción de IL-10 puede tener consecuencias en la respuesta inmunitaria, además de implicaciones clínicas. Se han reportado diferentes haplotipos de IL-10 como marcadores de vulnerabilidad y resistencia de lepra en otras poblaciones, lo cual sugiere que las diferencias en la distribución de diversos polimorfismos del gen de IL-10 entre grupos étnicos, es un factor importante al determinar la asociación entre enfermedad y genes.