Impacto da qPCR na avaliação diagnóstica de indivíduos com suspeição de hanseníase / Impact of qPCR on the diagnostic evaluation of individuals with suspected leprosy

Introdução: A hanseníase é uma infecção crônica causada pelo Mycobacterium leprae acometendo primariamente os nervos periféricos. Sabe-se que apesar da redução do número de casos novos, a transmissão continua ativa pelaincidência de casos em menores de 15 anos. Assim, torna-se relevante a busca de métodos diagnósticos mais sensíveis e específicos para a detecção de infecção subclínica. Objetivo: verificar através da qPCR, a presença do M. lepraeem raspados intradérmicos (RI) de indivíduos atendidos no CREDEN-PES/GV/MG. Método: Realizou-se coletas de amostras de raspado intradérmico (RI) de 411 participantes para determinação do índice baciloscópico (IB), extração e amplificação de DNA do M. leprae. Os participantes foram divididos em dois grupos: GRUPO 1, "Casos notificados", registrados no SINAN; GRUPO 2, "Indivíduos não notificados", que apresentavam quadro de suspeição diagnóstica para hanseníase. Do total de 411 amostras coletadas, 158 foram obtidas de casos notificados e 253 de indivíduos não notificados. Resultados: Verificou-se que do total de casos, 58.86% (masculino) e 41.14% (feminino). Entre os indivíduos não notificados, 53,36% (masculino) e 46,64% (feminino). Considerando a faixa etária, ambos os grupos apresentaram idade (40 a 69 anos). Quanto ao IB, 34,18% (casos) e 0% (não notificados) apresentaram IB positivo. Na qPCR, 82,28% dos casos e 47,43% dos indivíduos não notificados testaram positivo, com uma razão de chance de adoecimento de 5,14. Conclusão: É possível inferir, que os indivíduos com suspeição de hanseníase, com a qPCR positiva, podem ser portadores de infecção subclínica e potenciais transmissores do M. leprae para indivíduos mais susceptíveis.

Introduction: Leprosy is a chronic infection caused by Mycobacterium leprae, primarily affecting the peripheral nerves. It is known that despite the reduction in the number of new cases, transmission remains active due tothe incidence of cases in children under 15 years of age. Thus, the search for more sensitive and specific diagnostic methods for the detection of subclinical infection becomes relevant. Objective: to verify, through qPCR, the presence of M. leprae in intradermal scrapings (IS) of individuals treated at CREDEN-PES/GV/MG. Method: Intradermal scraping (IS) samples were collected from 411 participants to determine the bacilloscopic index (BI), extraction, and amplification of M. leprae DNA. Participants were divided into two groups: GROUP 1, "Notified cases," registered at SINAN; GROUP 2, "Individuals not notified," who had a suspected diagnosis of leprosy. Of the total of 411 samples collected, 158 were obtained from notified cases and 253 from not notified individuals. Results: It was found that of the total number of cases, 58.86% (male) and 41.14% (female). Among the not notified individuals, 53.36% (male) and 46.64% (female). Considering the age range, both groups were aged (40 to 69 years). As for the IS,34.18% (cases) and 0% (not notified) had a positive IS. In qPCR, 82.28% of cases and 47.43% of not notified individuals tested positive, with an illness odds ratio of 5.14. Conclusion:It is possible to infer those individualswith suspected leprosy, with positive qPCR, may be carriers of subclinical infection and potential transmitters of M. lepraeto more susceptible individuals.

Algorithm Design for a Cytokine Release Assay of Antigen-Specific In Vitro Stimuli of Circulating Leukocytes to Classify Leprosy Patients and Household Contacts.

BACKGROUND: Immunological biomarkers have often been used as a complementary approach to support clinical diagnosis in several infectious diseases. The lack of commercially available laboratory tests for conclusive early diagnosis of leprosy has motivated the search for novel methods for accurate diagnosis. In the present study, we describe an integrated analysis of a cytokine release assay using a machine learning approach to create a decision tree algorithm. This algorithm was used to classify leprosy clinical forms and monitor household contacts. METHODS: A model of Mycobacterium leprae antigen-specific in vitro assay with subsequent cytokine measurements by enzyme-linked immunosorbent assay was employed to measure the levels of tumor necrosis factor (TNF), interferon-γ, interleukin 4, and interleukin 10 (IL-10) in culture supernatants of peripheral blood mononuclear cells from patients with leprosy, healthy controls, and household contacts. Receiver operating characteristic curve analysis was carried out to define each cytokine's global accuracy and performance indices to identify clinical subgroups. RESULTS: Data demonstrated that TNF (control culture [CC]: AUC = 0.72; antigen-stimulated culture [Ml]: AUC = 0.80) and IL-10 (CC: AUC = 0.77; Ml: AUC = 0.71) were the most accurate biomarkers to classify subgroups of household contacts and patients with leprosy, respectively. Decision tree classifier algorithms for TNF analysis categorized subgroups of household contacts according to the operational classification with moderate accuracy (CC: 79% [48/61]; Ml: 84% [51/61]). Additionally, IL-10 analysis categorized leprosy patients' subgroups with moderate accuracy (CC: 73% [22/30] and Ml: 70% [21/30]). CONCLUSIONS: Together, our findings demonstrated that a cytokine release assay is a promising method to complement clinical diagnosis, ultimately contributing to effective control of the disease.

Associations between occupation, leprosy disability and other sociodemographic factors in an endemic area of Brazil.

BACKGROUND: In Brazil, new leprosy cases with grade-2 disability (G2D) have been increasing. Physical disability has been associated with experienced stigmatization, psychological distress, and social restriction. OBJECTIVES: To identify factors associated with leprosy disability in an endemic area of Brazil focusing on occupational and other sociodemographic factors. METHODS: Between July and December 2015, adult patients with multibacillary leprosy who attended a clinic in Belo Horizonte, Brazil were enrolled. Social, clinical, and demographic factors were collected from an administered questionnaire and medical charts. Occupations were categorized as manual vs non-manual. Descriptive statistics and multivariable logistic regression were performed to study associated factors with disability (Grade 1 disability (G1D) and G2D combined). FINDINGS: Seventy-three patients were enrolled with 48 (65.8%) presenting with either G1D or G2D at the time of enrollment. Twenty-nine (39.7%) had G2D. About half of the patients (n = 36, 49%) reported a manual labor occupation and reactions were common (n = 53, 73%). On univariate analyses, older age (p = 0.048) and low education (p = 0.007) were associated with disability. On multivariable analyses, only low education (primary or less) was associated with disability (OR = 6.34, 95% CI 1.37, 29.26). Greater distance from clinic, income, smoking, marital status, and occupation were not associated. MAIN CONCLUSIONS: Low education was associated with leprosy disability, consistent with prior studies, and therefore should be a focus for disability reduction programs. While the sample size of this study may have limited detection of associations between disability and social determinants tested, half of the patients reported a manual job, highlighting the need for more extensive studies on associations between occupation, disability, and related injuries.

Leprosy Screening Based on Artificial Intelligence: Development of a Cross-Platform App.

BACKGROUND: According to the World Health Organization, achieving targets for control of leprosy by 2030 will require disease elimination and interruption of transmission at the national or regional level. India and Brazil have reported the highest leprosy burden in the last few decades, revealing the need for strategies and tools to help health professionals correctly manage and control the disease. OBJECTIVE: The main objective of this study was to develop a cross-platform app for leprosy screening based on artificial intelligence (AI) with the goal of increasing accessibility of an accurate method of classifying leprosy treatment for health professionals, especially for communities further away from major diagnostic centers. Toward this end, we analyzed the quality of leprosy data in Brazil on the National Notifiable Diseases Information System (SINAN). METHODS: Leprosy data were extracted from the SINAN database, carefully cleaned, and used to build AI decision models based on the random forest algorithm to predict operational classification in paucibacillary or multibacillary leprosy. We used Python programming language to extract and clean the data, and R programming language to train and test the AI model via cross-validation. To allow broad access, we deployed the final random forest classification model in a web app via shinyApp using data available from the Brazilian Institute of Geography and Statistics and the Department of Informatics of the Unified Health System. RESULTS: We mapped the dispersion of leprosy incidence in Brazil from 2014 to 2018, and found a particularly high number of cases in central Brazil in 2014 that further increased in 2018 in the state of Mato Grosso. For some municipalities, up to 80% of cases showed some data discrepancy. Of a total of 21,047 discrepancies detected, the most common was "operational classification does not match the clinical form." After data processing, we identified a total of 77,628 cases with missing data. The sensitivity and specificity of the AI model applied for the operational classification of leprosy was 93.97% and 87.09%, respectively. CONCLUSIONS: The proposed app was able to recognize patterns in leprosy cases registered in the SINAN database and to classify new patients with paucibacillary or multibacillary leprosy, thereby reducing the probability of incorrect assignment by health centers. The collection and notification of data on leprosy in Brazil seem to lack specific validation to increase the quality of the data for implementations via AI. The AI models implemented in this work had satisfactory accuracy across Brazilian states and could be a complementary diagnosis tool, especially in remote areas with few specialist physicians.

Mycobacterium leprae-helminth co-infections and vitamin D deficiency as potential risk factors for leprosy: A case-control study in south-eastern Brazil.

BACKGROUND: Evidence suggests that biological mechanisms involved in helminth infections and vitamin deficiencies increase susceptibility to other infections. The aim of this study was to investigate the associations of helminth co-infection and select micronutrient deficiencies with leprosy using a case-control design. METHODS: From 2016 to 2018, individuals aged ≥3 years were recruited at clinics in and around Governador Valadares, Minas Gerais, Brazil in three groups: cases of leprosy, household contacts and community-matched (non-contact) controls. Helminths were diagnosed through stool Kato Katz examination and serum reactivity to anti-soluble adult worm antigen preparation IgG4. Serum ferritin, 25-OH vitamin D and retinol concentrations were measured. Multi-variate logistic regression was conducted to identify associations with active leprosy. RESULTS: Seventy-nine cases of leprosy, 96 household contacts and 81 non-contact controls were recruited; 48.1% of participants were male with a median age of 40 years. Helminths were found in 7.1% of participants on Kato Katz test, all but one of which were Schistosoma mansoni, and 32.3% of participants were positive for S. mansoni serology. On multi-variate analysis, cases were more likely to be infected with helminths (diagnosed by stool) than household contacts [adjusted odds ratio (aOR) 8.69, 95% confidence interval (CI) 1.50-50.51]. Vitamin D deficiency was common, and was more likely in cases compared with non-contact controls (aOR 4.66, 95% CI 1.42,-15.33). Iron deficiency was not associated with leprosy, and vitamin A deficiency was not detected. CONCLUSION: These associations suggest that the immune consequences of schistosomiasis and vitamin D deficiency may increase the risk of active leprosy. Comorbid conditions of poverty deserve further study as addressing co-infections and nutritional deficiencies could be incorporated into programmes to improve leprosy control.

The Burden of Helminth Coinfections and Micronutrient Deficiencies in Patients with and without Leprosy Reactions: A Pilot Study in Minas Gerais, Brazil.

Leprosy reactions are immune-mediated complications occurring in up to 50% of patients. The immune consequences of helminth infections and micronutrient deficiencies suggest a potential role in type 1 reactions (T1R) or type 2 reactions (T2R). We conducted a case-control study in Minas Gerais, Brazil, to evaluate whether comorbidities and other factors are associated with reactions in patients with multibacillary leprosy. Stool and serum were tested for helminth infections. Deficiencies of vitamin A, D, and iron were measured using serum retinol, 25-hydroxyvitamin D, and ferritin, respectively. Logistic regression models identified associations between reactions and helminth infections, micronutrient deficiencies, and other variables. Seventy-three patients were enrolled, 24 (33%) with T1R, 21 (29%) with T2R, 8 (15%) with mixed T1R/T2R, and 20 (27%) without reactions. Evidence of helminth infections were found in 11 participants (15%) and included IgG4 reactivity against Schistosoma mansoni, Strongyloides, and Ascaris antigens. Thirty-eight (52%) had vitamin D deficiency, eight (11%) had vitamin A insufficiency, 21 (29%) had anemia, and one (1.4%) had iron deficiency. Multivariable logistic regression showed no statistically significant associations between helminth coinfections and total reactions (adjusted odds ratios [aOR]: 1.36, 95% CI: 0.22, 8.33), T1R (aOR: 0.85, 95% CI: 0.17, 4.17), or T2R (aOR: 2.41, 95% CI: 0.29, 20.0). Vitamin D deficiency and vitamin A insufficiency were also not statistically associated with reactions. However, vitamin deficiencies and helminth infections were prevalent in these patients, suggesting a potential role for additional treatment interventions. Studying reactions prospectively may further clarify the role of comorbidities in the clinical presentation of leprosy.

A tale of two neglected tropical infections: using GIS to assess the spatial and temporal overlap of schistosomiasis and leprosy in a region of Minas Gerais, Brazil

BACKGROUND Despite public health efforts to reduce the global burden of leprosy, gaps remain in the knowledge surrounding transmission of infection. Helminth co-infections have been associated with a shift towards the lepromatous end of the disease spectrum, potentially increasing transmission in co-endemic areas. OBJECTIVES Using this biologically plausible association, we conducted a geographic information systems (GIS) study to investigate the spatial associations of schistosomiasis and leprosy in an endemic area of Minas Gerais (MG), Brazil. METHODS Data on new cases of Mycobacterium leprae and Schistosoma mansoni infections from 2007-2014 were retrieved from the Brazilian national notifiable diseases information system for seven municipalities in and surrounding Vespasiano, MG. A total of 139 cases of leprosy and 200 cases of schistosomiasis were mapped to a municipality level. For one municipality, cases were mapped to a neighborhood level and a stratified analysis was conducted to identify spatial associations. FINDINGS A relative risk of 6.80 [95% confidence interval (CI) 1.46 - 31.64] of leprosy was found in neighborhoods with schistosomiasis. Incidence rates of leprosy increased with corresponding incidence rates of schistosomiasis, and the temporal trends of both infections were similar. CONCLUSIONS The associations found in this project support the hypothesis that helminth infections may influence the transmission of leprosy in co-endemic areas.

A tale of two neglected tropical infections: using GIS to assess the spatial and temporal overlap of schistosomiasis and leprosy in a region of Minas Gerais, Brazil.

BACKGROUND: Despite public health efforts to reduce the global burden of leprosy, gaps remain in the knowledge surrounding transmission of infection. Helminth co-infections have been associated with a shift towards the lepromatous end of the disease spectrum, potentially increasing transmission in co-endemic areas. OBJECTIVES: Using this biologically plausible association, we conducted a geographic information systems (GIS) study to investigate the spatial associations of schistosomiasis and leprosy in an endemic area of Minas Gerais (MG), Brazil. METHODS: Data on new cases of Mycobacterium leprae and Schistosoma mansoni infections from 2007-2014 were retrieved from the Brazilian national notifiable diseases information system for seven municipalities in and surrounding Vespasiano, MG. A total of 139 cases of leprosy and 200 cases of schistosomiasis were mapped to a municipality level. For one municipality, cases were mapped to a neighborhood level and a stratified analysis was conducted to identify spatial associations. FINDINGS: A relative risk of 6.80 [95% confidence interval (CI) 1.46 - 31.64] of leprosy was found in neighborhoods with schistosomiasis. Incidence rates of leprosy increased with corresponding incidence rates of schistosomiasis, and the temporal trends of both infections were similar. CONCLUSIONS: The associations found in this project support the hypothesis that helminth infections may influence the transmission of leprosy in co-endemic areas.

A importância da busca ativa como estratégia de controle da hanseníase em territórios endêmicos / The importance of active surveillance as a strategy to control leprosy in endemic areas

De acordo com a literatura, contatos de casos de hanseníase apresentam maior risco de adoecimento e, nesse sentido, é primordial fortalecer e ampliar as ações de busca ativa de casos no âmbito da atenção primária de saúde, potencializando a ampliação do diagnóstico precoce, tratamento oportuno e demais medidas de vigilância, controle e reabilitação necessárias. Neste cenário, objetivou-se realizar a busca ativa de novos casos de hanseníase em um distrito rural do município de Mantena, MG, e caracterizar a situação socioeconômica e epidemiológica da doença na região. Foram realizadas visitas aos residentes do distrito e palestras informativas sobre a doença, sendo os moradores convidados a participar da pesquisa. Um total de 292 indivíduos foi examinado no período de julho de 2016 a fevereiro de 2017. Foram diagnosticados 27 casos novos, 22 dos quais eram multibacilares. Os graus de incapacidade 1 e 2 foram identificados em 74% da amostra. Sobre as variáveis socioeconômicas, a maioria dos casos possui baixa escolaridade e baixa renda familiar, além de todos os casos terem tido contato com a doença em algum momento. A busca ativa foi eficiente para a detecção de casos novos de hanseníase na população estudada e contribuiu para o controle da doença que é endêmica na região. Ademais, a busca ativa foi relevante, especialmente considerando a baixa instrução dos indivíduos e, portanto, menor acesso à informação.

Contacts of leprosy cases present a higher risk of illness and, therefore it is essential to strengthen and expand actions to actively search for cases in primary health care, thus intensifying early diagnosis, timely treatment and other surveillance measures, and improve control and rehabilitation. In this setting, we aimed to perform the active search of new cases of leprosy in the rural area of Mantena, MG, and to characterize the socioeconomic and epidemiological situation related to the disease in the region. For this purpose, visits were made to the residents of the region with informative talks about the disease, and subsequently inhabitants were invited to participate in the research. A total of 292 individuals were examined from July 2016 to February 2017. Twenty-seven new cases were diagnosed, 22 of which were multibacillary. Grade 1 and 2 physical disabilities were identified in 74% of the new cases. Regarding socioeconomic variables, the majority of the cases had little schooling and low family income. In addition, all cases had had contact with the illness, at some point. We conclude that the active search was efficient to detect new leprosy cases in the population, and it contributed to the control of the disease, which is endemic in the region. In addition, active search is an important methodology, especially when the population involved has little schooling and thus less access to information.