MicroRNA biomarkers in leprosy: insights from the Northern Brazilian Amazon population and their implications in disease immune-physiopathology

Leprosy, or Hansen’s Disease, is a chronic infectious disease caused by Mycobacterium leprae that affects millions of people worldwide. Despite persistent efforts to combat it leprosy remains a significant public health concern particularly in developing countries. The underlying pathophysiology of the disease is not yet fully understood hindering the development of effective treatment strategies. However, recent studies have shed light on the potential role of microRNAs (miRNAs), small non-coding RNA molecules that can regulate gene expression, as promising biomarkers in various disease, including leprosy. This study aimed to validate a set of nine circulating miRNAs to propose new biomarkers for early diagnosis of the disease. Hsa-miR-16-5p, hsa-miR-106b-5p, hsa-miR-1291, hsa-miR-144-5p, and hsa-miR-20a-5p showed significant differential expression between non-leprosy group (non-LP) and leprosy group (LP), accurately discriminating between them (AUC > 0.75). In addition, our study revealed gender-based differences in miRNA expression in LP. Notably, hsa-miR-1291 showed higher expression in male LP, suggesting its potential as a male-specific biomarker. Similarly, hsa-miR-16-5p and hsa-miR-20a-5p displayed elevated expression in female LP, indicating their potential as female-specific biomarkers. Additionally, several studied miRNAs are involved in the dysregulation of apoptosis, autophagy, mitophagy, cell cycle, and immune system in leprosy. In conclusion, the validation of miRNA expression highlights several miRNAs as potential biomarkers for early diagnosis and provides new insights into the pathogenesis of the disease

Silent peripheral neuropathy determined by high-resolution ultrasound among contacts of patients with Hansen's disease

IntroductionHansen's disease (HD) primarily infects peripheral nerves, with patients without HD being free of peripheral nerve damage. Household contacts (HHCs) of patients with HD are at a 5–10 times higher risk of HD than the general population. Neural thickening is one of the three cardinal signs that define a case of HD according to WHO guidelines, exclusively considering palpation examination that is subjective and may not detect the condition in the earliest cases even when performed by well-trained professionals. High-resolution ultrasound (HRUS) can evaluate most peripheral nerves, a validated technique with good reproducibility allowing detailed and accurate examination.ObjectiveThis study aimed to use the peripheral nerve HRUS test according to the HD protocol as a diagnostic method for neuropathy comparing HHCs with healthy volunteers (HVs) and patients with HD.MethodsIn municipalities from 14 different areas of Brazil we selected at random 83 HHC of MB-patients to be submitted to peripheral nerve ultrasound and compared to 49 HVs and 176 HD-patients.ResultsHousehold contacts assessed by HRUS showed higher median and mean absolute peripheral nerve cross-sectional area (CSA) values and greater asymmetries (ΔCSA) compared to HVs at the same points. Median and mean absolute peripheral nerve CSA values were higher in patients with HD compared to HCCs at almost all points, while ΔCSA values were equal at all points. Mean ± SD focality (ΔTpT) values for HHCs and patients with HD, respectively, were 2.7 ± 2.2/2.6 ± 2.2 for the median nerve, 2.9 ± 2.7/3.3 ± 2.9 for the common fibular nerve (p > 0.05), and 1.3 ± 1.3/2.2 ± 3.9 for the ulnar nerve (p < 0.0001).DiscussionConsidering HRUS findings for HHCs, asymmetric multiple mononeuropathy signs (thickening or asymmetry) in at least 20% of the nerves evaluated could already indicates evidence of HD neuropathy. Thus, if more nerve points are assessed in HHCs (14 instead of 10), the contacts become more like patients with HD according to nerve thickening determined by HRUS, which should be a cutting-edge tool for an early diagnosis of leprosy cases

Epidemiologia espacial e sorológica da hanseníase no estado do Pará

Leprosy remains a severe public health problem in the State of Pará, Brazil. Over 80,000 cases were detected during the last 20 years in Pará, and currently, the annual case detection rate (50/100,000 inhabitants) is three-fold higher than the Brazilian average. The main objective of this study was to develop a method combining anti-PGL-I serology and spatial epidemiology as a tool for reducing the leprosy disease burden in Pará. An initial cross-sectional survey was conducted in eight municipalities of Pará at the residences of people reported to be affected by leprosy during the last five to six years. A group of researchers with experience treating leprosy patients, including dermatologists, nurses, physical therapists and lab technicians, performed a dermatoneurologic clinical examination and collected blood samples to test for anti-PGL-I IgM in 1,945 household contacts (HHC) of the 531 reported cases. Additionally, 1,592 school children (SC), aged 6-20 years, from 37 randomly selected elementary and secondary public schools underwent the same clinical and serologic evaluation. The residential addresses of reported leprosy cases and the residences of the examined SC were georeferenced to determine the spatial distribution pattern of leprosy. Two years later, based on the previous serological data, we returned to two cities to re-examine the same subjects. To evaluate the significance of geographic information in detecting new cases, we also selected two new public schools located in high-risk areas for leprosy. High-risk areas were determined by the spatial analysis of the distribution of cases in one municipality. During the initial survey, 156 (8%) HHC and 63 (4%) SC were diagnosed as new leprosy cases; 806 (41.4%) HHC and 777 (48.8%) SC tested positive for anti-PGL-I. Spatial analysis of one selected municipality demonstrated heterogeneity in the distribution of leprosy cases, with spatial clusters of high and low detection rates in specific regions of the city (p<0.01). Additionally, 94.7% of the initially examined SC lived within less than 200 meters of a leprosy case registered during the six years prior to this study. During follow-up, the incidence of leprosy was significantly higher among seropositive individuals (22.3%) when compared to seronegative individuals (9.4%) (OR = 2.7; 95%CI = 1.29 – 5.87; p = 0.01); leprosy rates were also significantly higher among dwellers of residences with at least one seropositive subject (17.4%), compared with dwellers of residences with no seropositive subjects (7.4%) (OR = 2.6; 95%CI = 1.18 – 5.91; p = 0.02). Selecting schools located in areas of the city at high-risk of leprosy increased the efficiency of detecting new cases among SC (8.2%) when compared to randomly selected schools (4%) (p = 0.04). The data indicate a high rate of undiagnosed leprosy cases and of subclinical infection with M. leprae in the State of Pará. Anti-PGL-I serology and spatial epidemiology are effective tools to increase the early detection of new cases, and these methods should be used by the municipalities of Pará to help reach leprosy control targets.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorDECIT - Departamento de Ciência e Tecnologia da Secretaria de Ciência, Tecnologia e Insumos Estratégicos do Ministério da Saúde do BrasilCNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESPA - Fundação Amazônia de Amparo a Estudos e PesquisasMALTALEP - Ordem de MaltaMais de 80.000 casos de hanseníase foram diagnosticados nos últimos 20 anos no Pará e, ainda hoje, com um coeficiente de detecção anual de 50/100.000 habitantes (três vezes superior à média nacional) a doença permanece como um grave problema de saúde pública neste Estado. O objetivo geral deste estudo foi desenvolver um método integrando a epidemiologia espacial e sorológica como ferramenta de combate à hanseníase no Pará. Inicialmente, foram realizadas visitas domiciliares a famílias de pessoas afetadas pela hanseníase, diagnosticadas nos últimos cinco a seis anos, em oito municípios de diferentes regiões do Estado. A equipe de pesquisadores com experiência no manejo da hanseníase, composta por médicos dermatologistas, enfermeiros, fisioterapeutas e técnicos de laboratório, realizou exame clínico dermatoneurológico em 1.945 contatos intradomiciliares de 531 casos notificados e coletou amostra de sangue para pesquisa sorológica de anticorpos IgM anti-PGL-I. Além disso, 1.592 estudantes de 37 escolas públicas do ensino fundamental e médio, com idade entre 6 e 20 anos, também foram selecionados aleatoriamente para serem submetidos à mesma avaliação. As residências dos casos notificados, bem como a dos estudantes incluídos no estudo foram georreferenciadas para a análise da distribuição espacial da hanseníase. Dois anos mais tarde, com base na informação sorológica prévia, a equipe de pesquisadores retornou a dois municípios para reavaliar os indivíduos incluídos no estudo. Adicionalmente, duas novas escolas públicas localizadas em áreas de alto risco de hanseníase, determinadas pela análise da distribuição espacial da doença em um dos municípios, foram selecionadas para avaliar-se a importância da informação geográfica na detecção de casos novos. Na avaliação inicial, 156 (8%) contatos e 63 (4%) estudantes foram diagnosticados como casos novos de hanseníase; 806 (41,4%) contatos e 777 (48,8%) estudantes foram soropositivos para anti-PGL-I. A análise da distribuição espacial dos casos registrados da doença em um dos municípios selecionados indicou que a hanseníase apresenta um padrão heterogêneo, com clusters de alta e baixa taxa de detecção anual em áreas específicas da cidade (p < 0,01), e que 94,7% dos estudantes examinados residiam a menos de 200 metros de um caso registrado durante os seis anos anteriores ao estudo. No seguimento, a incidência de hanseníase foi significativamente maior entre os indivíduos soropositivos (22,3%) quando comparados aos soronegativos (9.4%) (OR = 2,7; IC95% = 1,29 – 5,87; p = 0,01); também foi significativamente mais alta entre moradores de residências com pelo menos um sujeito soropositivo (17,4%), comparada aos de residências sem nenhum morador soropositivo (7,4%) (OR = 2,6; IC95% = 1,18 – 5,91; p = 0,02). A seleção de escolas localizadas em áreas de maior risco dentro do município aumentou significativamente a eficiência na detecção de casos novos entre escolares (8,2%), quando comparada aos resultados obtidos em escolas selecionadas aleatoriamente (4%) (p = 0,04). Os dados mostram alta taxa de prevalência oculta de hanseníase e de infecção subclínica pelo M. leprae no Pará. A epidemiologia espacial e sorológica são ferramentas eficazes para aumentar a detecção precoce de casos novos e deveriam ser utilizadas pelos municípios do Pará para que o Estado possa finalmente alcançar as metas de controle da hanseníase

Efeito da laserterapia de baixa intensidade sobre a cicatrização de úlceras hansênicas: ensaio clínico randomizado

Neuropathic ulcer is one of the most stigmatical sequels of leprosy. Its presence is very disabling to the patient, and can cause deformity and/or amputation of the affected limb. Different methods of treatments have been used in the ulcers management, but the results are frequently dissatisfactory, resulting in many people living with chronic wounds for a long time. Low Level Laser Therapy (LLLT) is used in many services to accelerate wound healing, but its results are controversial, and several published works are inconclusive. The main goal of this study was to evaluate the effect of LLLT on wound healing of leprosy patients in a randomized clinical trial. This study was conducted at the dressing service of Dr Marcello Candia Reference Unit in Sanitary Dermatology of the State of Para. First, in order to analyze the clinical and epidemiological characteristics of 51 patients (97 ulcers) we conducted anamnesis, identification of the regions affected by the lesions (including digital photographic register), measurement of ulcer depth and surface area, using the software ImageTool 3.0. The most frequent comorbidity was systemic high blood pressure with 13 cases. To evaluate laser therapy, 25 subjects were randomly allocated into two groups of intervention. The control group (CG), with 12 patients (14 ulcers) received the standard treatment with daily simple dressings, use of topic 1% silver sulfadiazine cream, and orientations about self-care and prevention of disabilities. The experimental group (EG), with 13 subjects (17 ulcers), received standard treatment plus LLLT 3 times per week during 12 weeks. The LLLT consisted of 4 J/point in the wound edges, using a punctual contact technique and 2 J/cm² in the wound bed, using a scanning technique. The LLLT equipment was an AlGaInP diode laser (660 nm) with a power density of 1 W/cm². The analyzed variables were: ulcer surface area in square centimeters (cm²), depth in millimeters (mm), and PUSH score. The mean ulcer surface in the CG was 5,3 cm² (± 9,2) before and 4,4 cm² (± 8,5) after treatment, the depth was 6,3 mm (± 5,4) before and 5,4 (± 5,7) after, and the PUSH score was 9,7 (± 3,4) before and 8,4 (± 5,3) after treatment. In the EG, mean ulcer surface was 4,2 cm² (± 5,9) before and 3,8 cm² (± 5,7) after treatment, the depth was 6,2 mm (± 5,1) before and 4,1 mm (± 3,9) after, and the PUSH score was 9,6 (± 3,3) before and 7,9 (± 5,3) after treatment. The statistical analysis did not show any significant difference (p > 0.05) in none of the variables analyzed before and after treatment. Considering the parameters used in this study, in comparison with usual leprosy neuropathic ulcer treatment, LLLT did not demonstrate any additional benefits to wound healing. The study suggests that 1) special attention should be given to the control of blood pressure on these patients, and 2) diminish or avoid weight bearing on the affected area should continue as key recommendation for ulcer healing.SCTIE - Secretaria de Ciência, Tecnologia e Insumos Estratégicos do Ministério da Saúde do BrasilCNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoFINEP - Financiadora de Estudos e ProjetosÚlcera neuropática é uma das sequei as mais estigmatizantes da hanseníase. Sua presença é bastante incapacitante para o indivíduo, podendo levar a deformação e/ou amputação do membro afetado. Diversas técnicas de tratamento têm sido utilizadas no manejo destas úlceras, no entanto, os resultados nem sempre são satisfatórios, ocasionando a existência de pessoas que convivem há vários anos com ferimentos crônicos. A laserterapia de baixa intensidade (LBI) tem sido utilizada em diversos países no tratamento de úlceras de difícil cicatrização, entretanto seus resultados são inconclusivos e muitos dos trabalhos publicados apresentam falhas metodológicas. O objetivo deste estudo foi avaliar o efeito da LBI sobre o processo de cicatrização de úlceras em pacientes hansenianos, em um teste clínico controlado e randomizado. O estudo foi realizado no ambulatório de curativos da Unidade de Referência Estadual em Dermatologia Sanitária do Pará - Marcello Candia, a partir de um levantamento clínico-epidemiológico para caracterização da população estudada, por meio de anamnese, identificação da localização das úlceras, registro fotográfico digital, avaliação da área das lesões utilizando o programa ImageTool 3.0, e medida da profundidade das úlceras de 51 pacientes, totalizando 97 lesões. A comorbidade mais frequente nesta população foi a hipertensão arterial sistêmica (13 casos). Em seguida, 25 sujeitos foram distribuídos aleatoriamente em dois grupos de estudo. O grupo controle (GC), formado por 12 pacientes (14 Úlceras) permaneceu recebendo o tratamento de rotina, composto por curativos simples diários, uso de sulfadiazina de prata 1 %, e orientações para autocuidados e prevenção de incapacidades. O grupo experimental (GE), formado por 13 sujeitos (17 úlceras), permaneceu recebendo o mesmo tratamento de rotina do GC mais a aplicação de LBI três vezes por semana, durante um período de 12 semanas. A LBI foi aplicada com um equipamento de laser diodo A1GaInP (660 nm), na dose de 2 J/cm² com a técnica de varredura sem contato no leito da lesão, e 4 J/ponto nas bordas da lesão com aplicações pontuais com contato. A densidade de potência foi de 1 W/cm². As variáveis estudadas foram: área da lesão em cm², profundidade em milímetros e escore PUSH. No GC a média da área das úlceras foi de 5,3 (±9.2) antes e 4,4 (± 8,5) depois do tratamento, a profundidade foi de 6,3 (± 5,4) antes e 5,4 (± 5.7) depois, o escore PUSH foi 9,7 (± 3,4) antes e 8,4 (± 5.3) depois. No GE a média da área das Úlceras foi de 4,2 (± 5,9) antes e 3,8 (± 5,7) depois do tratamento, a profundidade foi de 6.2 (± 5.1) antes e 4,1 (± 3,9) depois, o escore PUSH foi 9,6 (± 3,3) antes e 7,9 (± 5,3) depois. A análise estatística não evidenciou diferença significativa (p > 0,05) em nenhuma das variáveis estudadas antes e depois do tratamento. A LBI, dentro dos parâmetros utilizados neste estudo, não demonstrou benefícios adicionais para a cicatrização de úlceras em hansenianos. A diminuição do peso exercido sobre a área da lesão e o controle da pressão arterial são medidas importantes no manejo destas úlceras

Spatial analysis spotlighting early childhood leprosy transmission in a hyperendemic municipality of the Brazilian Amazon region.

BACKGROUND: More than 200,000 new cases of leprosy were reported by 105 countries in 2011. The disease is a public health problem in Brazil, particularly within high-burden pockets in the Amazon region where leprosy is hyperendemic among children. METHODOLOGY: We applied geographic information systems and spatial analysis to determine the spatio-temporal pattern of leprosy cases in a hyperendemic municipality of the Brazilian Amazon region (Castanhal). Moreover, we performed active surveillance to collect clinical, epidemiological and serological data of the household contacts of people affected by leprosy and school children in the general population. The occurrence of subclinical infection and overt disease among the evaluated individuals was correlated with the spatio-temporal pattern of leprosy. PRINCIPAL FINDINGS: The pattern of leprosy cases showed significant spatio-temporal heterogeneity (p<0.01). Considering 499 mapped cases, we found spatial clusters of high and low detection rates and spatial autocorrelation of individual cases at fine spatio-temporal scales. The relative risk of contracting leprosy in one specific cluster with a high detection rate is almost four times the risk in the areas of low detection rate (RR = 3.86; 95% CI = 2.26-6.59; p<0.0001). Eight new cases were detected among 302 evaluated household contacts: two living in areas of clusters of high detection rate and six in hyperendemic census tracts. Of 188 examined students, 134 (71.3%) lived in hyperendemic areas, 120 (63.8%) were dwelling less than 100 meters of at least one reported leprosy case, 125 (66.5%) showed immunological evidence (positive anti-PGL-I IgM titer) of subclinical infection, and 9 (4.8%) were diagnosed with leprosy (8 within 200 meters of a case living in the same area). CONCLUSIONS/SIGNIFICANCE: Spatial analysis provided a better understanding of the high rate of early childhood leprosy transmission in this region. These findings can be applied to guide leprosy control programs to target intervention to high risk areas

miRNome Expression Analysis Reveals New Players on Leprosy Immune Physiopathology

Leprosy remains as a public health problem and its physiopathology is still not fully understood. MicroRNAs (miRNA) are small RNA non-coding that can interfere with mRNA to regulate gene expression. A few studies using DNA chip microarrays have explored the expression of miRNA in leprosy patients using a predetermined set of genes as targets, providing interesting findings regarding the regulation of immune genes. However, using a predetermined set of genes restricted the possibility of finding new miRNAs that might be involved in different mechanisms of disease. Thus, we examined the miRNome of tuberculoid (TT) and lepromatous (LL) patients using both blood and lesional biopsies from classical leprosy patients (LP) who visited the Dr. Marcello Candia Reference Unit in Sanitary Dermatology in the State of Pará and compared them with healthy subjects. Using a set of tools to correlate significantly differentially expressed miRNAs with their gene targets, we identified possible interactions and networks of miRNAs that might be involved in leprosy immunophysiopathology. Using this approach, we showed that the leprosy miRNA profile in blood is distinct from that in lesional skin as well as that four main groups of genes are the targets of leprosy miRNA: (1) recognition and phagocytosis, with activation of immune effector cells, where the immunosuppressant profile of LL and immunoresponsive profile of TT are clearly affected by miRNA expression; (2) apoptosis, with supportive data for an antiapoptotic leprosy profile based on BCL2, MCL1, and CASP8 expression; (3) Schwann cells (SCs), demyelination and epithelial–mesenchymal transition (EMT), supporting a role for different developmental or differentiation gene families, such as Sox, Zeb, and Hox; and (4) loss of sensation and neuropathic pain, revealing that RHOA, ROCK1, SIGMAR1, and aquaporin-1 (AQP1) may be involved in the loss of sensation or leprosy pain, indicating possible new therapeutic targets. Additionally, AQP1 may also be involved in skin dryness and loss of elasticity, which are well known signs of leprosy but with unrecognized physiopathology. In sum, miRNA expression reveals new aspects of leprosy immunophysiopathology, especially on the regulation of the immune system, apoptosis, SC demyelination, EMT, and neuropathic pain

Clusters of leprosy in Castanhal.

<p>(A) The spatial distribution of individual leprosy cases overlying the respective Kernel density estimation layer, representing areas with a high and low density of cases per km². (B) LISA test (local Moran's I) characterizing areas with a statistically significant (p<0.05) positive spatial association according to the raw detection rate. The areas marked as high-high indicate a high rate in an area surrounded by high values of the weighted average rate of the neighboring areas, and low-low represents areas with a lower rate surrounded by lower values. (C) The most likely cluster of leprosy detected by the Kulldorff's spatial scan statistics (<i>p</i><0.01).</p