Design of a specific peptide against phenolic glycolipid-1 from Mycobacterium leprae and its implications in leprosy bacilli entry

BACKGROUND Mycobacterium leprae, the causative agent of Hansen's disease, causes neural damage through the specific interaction between the external phenolic glycolipid-1 (PGL-1) and laminin subunit alpha-2 (LAMA2) from Schwann cells. OBJECTIVE To design a LAMA2-based peptide that targets PGL-1 from M. leprae. METHODS We retrieved the protein sequence of human LAMA2 and designed a specific peptide using the Antimicrobial Peptide Database and physicochemical parameters for antimycobacterial peptide-lipid interactions. We used the AlphaFold2 server to predict its three-dimensional structure, AUTODOCK-VINA for docking, and GROMACS programs for molecular dynamics simulations. FINDINGS We analysed 52 candidate peptides from LAMA2, and subsequent screening resulted in a single 60-mer peptide. The mapped peptide comprises four β-sheets and a random coiled region. This peptide exhibits a 45% hydrophobic ratio, in which one-third covers the same surface. Molecular dynamics simulations show that our predicted peptide is stable in aqueous solution and remains stable upon interaction with PGL-1 binding. In addition, we found that PGL-1 has a preference for one of the two faces of the predicted peptide, which could act as the preferential binding site of PGL-1. MAIN CONCLUSIONS Our LAMA2-based peptide targeting PGL-1 might have the potential to specifically block this key molecule, suggesting that the preferential region of the peptide is involved in the initial contact during the attachment of leprosy bacilli to Schwann cells.

Estudo sorológico da co-infecção de leishmania infantum e mycobacterium leprae em área de alta endemicidade / Sorological sutdy of coinfection leishmania infantum e mmycobacterium leprae in area of endemicity high

Introdução: A região Nordeste é responsável por 55% dos casos de hanseníase e por quase 50% dos casos de Leishmaniose visceral no Brasil. O Ceará, em especial a capital Fortaleza, é responsável por um grande número de casos novos dessas doenças. Este fato é reforçado pela correlação na distribuição de casos dessas patologias por municípios do estado do Ceará,onde de acordo com os dados da Secretaria de Saúde do Estado (2013), observa-se forte correlação epidemiológica entre os casos de hanseníase e do Leishmaniose visceral nos 184 municípios principalmente em Fortaleza. Objetivos: Nosso objetivo foi analisar a produção de anticorpos IgM anti-PGL1 em pacientes com Calazar sem tratamento. Material e métodos: 28 pacientes com confirmação clínico-laboratorial para Leishmaniose visceral acompanhados no Hospital São José de Doenças Infecciosas. Resultados: Quanto ao gênero, 21 foram do sexo masculino e 7 do sexo feminino, com mediana de idade de 20,5 anos (var. 3 a 76 anos), dos quais 15 pacientes não necessitaram internamento e 13 foram internados por um período médio de 28 dias (var. 5 a 28 dias). A média e desvio-padrão do índice de IgM anti-PGL1 foi de 1,91 + 0,69, sendo 78,6% considerados soropositivos. Conclusão: Não foi observada qualquer diferença entre gênero,idade, necessidade ou não de internamento, ou tempo de tratamento. A alta frequência de IgM anti-PGL1 positiva pode ser secundária à ativiação policlonal que ocorre na Leishmaniose visceral, dificultando a possibilidade de detecção da infecção pelo M. leprae por avaliação sorológica em região de alta endemicidade para Leishmaniose visceral.

Introduction: The Northeast region accounts for 55% of leprosy cases and nearly 50% of cases of visceral leishmaniasis in Brazil. Ceará, in particular the Fortaleza capital is responsible for a large number of new cases of these diseases. This fact is reinforced by the correlation in the distribution of cases of these diseases in the state of Ceará counties where according to the data of the State Health Departament (2013), we observed strong epidemiological correlation between cases of leprosy and visceral leishmaniasis in 184 counties mostly in Fortaleza. Objectives: Our objective was to analyze the production of anti-PGL1 IgM antibodies in patients with visceral leishmaniasis untreated. Materials and Methods: 28 patients with clinical and laboratory confirmation for visceral leishmaniasis followed at SãoJosé Hospital for Infectious Diseases. Results: As togender, 21 were males and 7 females, with a median age of 20,5 years (var 3-76 years.), Of which 15 patients did not require hospitalization and 13 were hospitalized for an average 28 days (var. 5 to 28 days). The mean and standard deviation of the anti-IgM PGL1 index was 1.91 ± 0.69, and 78.6% considered seropositive. Conclusion: It was not observed any difference between gender, age, necessity or not hospitalization, or time treatment. The high frequency of positive IgM anti-PGL1,can be secondary to polyclonal activation occurring in kala-azar, hindering the possibility of detection of M. leprae infection by serologic evaluation in high endemicity area for visceral leishmaniasis.

Análise comparativa da reatividade anti-LID-1, NDO-LID, NDO-HSA e PGL-1 em hanseníase / Comparative analysis of the reactivity anti-LID-1, NDO-LID, NDO-HSA and PGL-1 in leprosy

A infecção subclínica pode ser avaliada por meio de teste sorológico, que determina imunoglobulinas circulantes. O objetivo do presente estudo foi analisar a reatividade de diferentes antígenos em casos novos de hanseníase, contatos domiciliares de casos e em população de área endêmica, com o intuito de identificar o melhor antígeno para o diagnóstico sorológico da hanseníase e detecção de indivíduos infectados pelo Mycobacterium leprae.Trata-se de um estudo transversal de natureza exploratória e analítica. A reatividade anti-LID-1, NDO-LID, NDO-HSA e PGL-1 foi avaliada por meio do enzyme-linked immunosorbentassay. Foram analisadas amostras de sangue total em papel de filtro Whatman de 2494indivíduos da população de sete municípios da microrregião de Almenara e de soro de 94casos novos de hanseníase e 104 contatos domiciliares de casos residentes no município de Uberlândia. O Banco de Dados foi criado no Software Epi Info versão 3.5.1 e análise realizada no software Statistical Package for the Social Sciences for Windows 18 e no GraphPad Prism versão 5. Para análise estatística foram utilizados os seguintes testes: Kolmogorov-Smirnov, Kruskal-Wallis one-way (H), Mann-Whitney (U) com correção de Bonferroni, kappa, Spearman (rho), teste Qui-quadrado de Pearson e regressão logística binária. Foi observado maior soropositividade no grupo de casos multibacilares (MB), em contatos domiciliares de casos MB e nos indivíduos residentes nos municípios de Almenara e Jequitinhonha. Obteve-se correlação positiva entre a sorologia e o índice baciloscópico,concordância substancial e significativa no grupo de casos novos de hanseníase e correlação positiva para todos os antígenos testados. Os testes anti-LID-1 e anti-NDO-LID apresentaram melhor performance para identificar os contatos domiciliares e ou indivíduos da população...

The subclinical infection can be evaluated by serologic test which determine circulating immunoglobulins. The aim of this study was to analyze the reactivity of different antigens inleprosy cases, household contacts of index cases and the population of the endemic area toidentify the best antigen for the diagnosis of leprosy and detection of individuals infected with Mycobacterium leprae. It is a cross-sectional study of exploratory and analytical nature. There activity anti-LID-1, NDO-LID, NDO-HAS e PGL-1 were evaluated using the enzyme linke dimmunosorbent assay. The whole blood in What man filter paper of 2494 individuals from the general population of seven municipalities in the micro-Almenara and serum of 94 patients with leprosy and 104 household contacts of patients residing in Uberlândia were analyzed. The database was created in Epi Info software version 3.5.1 and analysis in the software Statistical Package for Social Sciences for Windows 18 and GraphPad Prism version5. For statistical analysis the following tests were used: Kolmogorov-Smirnov, Kruskal Wallisone-way (H), Mann-Whitney (U) with Bonferroni correction, kappa, Spearman (rho), chisquaretest of Pearson and binary logistic regression. Identied higher seropositivity in the group of MB patients, household contacts of MB patients and in individuals living in the municipalities of Almenara and Jequitinhonha. Observed positive correlation between serology test and bacterial index, substantial agreement and significant in patients positive and positive correlation for all antigens. The LID-1 and NDO-LID antigens showed greater ability to identify household contacts or the general population infected with M. leprae, but the performance of the NDO-LID was better. The native PGL-1 had higher seropositivity than the NDO-HSA for all clinical forms of leprosy and household contacts. The seropositivity prevalence in the general population was higher than the detection rate of leprosy...

Clinico-histopathological correlation and detection of anti PGL-1 antibodies in leprosy patients.

Background: Leprosy (Hansen’s disease) is one of the major health problems of the world especially in developing countries. In India, it was first described in “Sushruta Samhita ” & treated by Chaulmoogra oil and caused by Mycobacterium Leprae. Early diagnosis of leprosy, an absolute necessity for control as well as effective therapy. For this, clinical diagnosis, skin smear examination is adequate coupled with histo-pathological examination of skin and nerve lesions with modified Fite Faraco stain for demonstration of acid fast bacilli. Moreover, bacillary index is required for adequate combined chemotherapy regimen. Detection of anti PGL-1 antibodies in serum gives an added advantage for detection and monitoring treatment. Materials & Methods : A total of 85 cases of leprosy who attended outpatient department of Skin & VD, Shri Sayaji General Hospital Baroda chosen for study during '07-08' period with 75 cases from leprosy hospital, Baroda which included 50 detected patients and 25 child contacts with 25 healthy voluntary blood donors from blood bank, SSGH selected.Clinical, past and family history taken with slit skin smears stained with Z-N stain,graded and histopathological evaluation done. Serological study done from serumof leprosy patients and healthy blood donors; tested by serodia kitsand interpretation made. Results : Most cases were in 2nd to 4th decade and males dominated. Clinically most cases were of indeterminate and tuberculoid type and histologically indeterminate and borderline tuberculoid. Clinico-histopathological correlation was found most in indeterminate followed by histoid type. Voluntary blood donors were seronegative. 21 out of 48 multibacillary cases and 6 out of 28 paucibacillary showed seropositivity for anti PGL-1 antibodies (p<0.001). Conclusion : . All suspected leprosy cases clinically should be subjected to slit skin smear examination with histopathological evaluation; bacillary study which helps in diagnosis and adequate treatment of patients. Detection of antibodies to PGL-1 in patients indicate pres-ence of leprosy bacilli and useful in preclinical diagnosis and determining progress of therapy.

Leprosy exposure, infection and disease: a 25-year surveillance study of leprosy patient contacts

Contact surveillance is a valuable strategy for controlling leprosy. A dynamic cohort study of leprosy contacts was initiated in 1987 at Oswaldo Cruz Foundation. The objective of this work was to review the data on the major risk factors leading up to the infectious stage of the disease, estimate incidence rates of leprosy in the cohort and characterise the risk factors for the disease among the contacts under surveillance. The incidence rate of leprosy among contacts of leprosy patients was estimated at 0.01694 cases per person-year in the first five years of follow-up. The following factors were associated with acquiring the disease: (i) not receiving the BCG vaccine, (ii) a negative Mitsuda reaction and (iii) contact with a patient with a multibacillary clinical form of leprosy. The contacts of index patients who had high bacilloscopic index scores > 1 were at especially high risk of infection. The following factors were associated with infection, which was defined as a seropositive reaction for anti-phenolic glicolipid-1 IgM: (i) young age (< 20 years), (ii) a low measured Mitsuda reaction (< 5 mm) and (iii) contact with an index patient who had a high bacilloscopic index. BCG vaccination and re-vaccination were shown to be protective among household contacts. The main conclusions of this study indicate an urgent need for additional leprosy control strategies in areas with a high incidence of the disease.

Correlation of zinc plasma and IgM anti-PGL-1 levels among close contact of leprosy.

Background: Previous study showed leprosy patients have low zinc status. Yet the status of zinc in close-contact, which indicated by IgM anti-PGL1 level, have not determined. The aim of the study was to determine the association of zinc plasma and IgM anti-PGL-1 levels among close contact of leprosy patients in Indonesia. Methods: This was an observational study. Subjects were 186 close-contact leprosy patients. Measurement of zinc plasma status used atomic absorption spectrophotometer while examination of IgM anti-PGL1 level used ELISA (Polyclonal rabbit anti human IgM/HRP/(Dako®). Results: The average level of IgM anti-PGL1 and zinc plasma were 804 unit/mL (± 439.4) and 16.6 μmol/L (± 3.5) consecutively. There was significant correlation between zinc plasma and IgM anti-PGL1 (r = - 0.230; p = 0.002). Conclusion: There is a significant correlation between zinc plasma and IgM anti-PGL1 in close contact of leprosy

Early detection of leprosy by examination of household contacts, determination of serum anti-PGL-1 antibodies and consanguinity

A cross-sectional clinical trial in which the serum anti-phenolic glycolipid (anti-PGL-1) antibodies were analysed in household contacts (HHC) of patients with leprosy as an adjunct early leprosy diagnostic marker was conducted. The families of 83 patients underwent clinical examination and serum anti-PGL1 measurement using enzyme-linked immunosorbent assay. Of 320 HHC, 98 were contacts of lepromatous leprosy (LL), 80 were contacts of borderline lepromatous (BL), 28 were contacts of borderline (BB) leprosy, 54 were contacts of borderline tuberculoid (BT), 40 were contacts of tuberculoid (TT) and 20 were contacts of indeterminate (I) leprosy. Consanguinity with the patients was determined for 232 (72.5 percent) HHC. Of those 232 contacts, 183 had linear consanguinity. Forty-nine HHC had collateral consanguinity. Fifty-eight contacts (18.1 percent) tested positive for anti-PGL1 antibodies. The number of seropositive contacts based on the clinical forms of the index case was 17 (29.3 percent) for LL, 15 (25.9 percent) for BL, one (1.7 percent) for BB, 14 (24.1 percent) for BT, three (5.2 percent) for TT and eight (13.7 percent) for I. At the one year follow-up, two (3.4 percent) of these seropositive contacts had developed BT leprosy. The results of the present study indicate that the serum anti-PGL-1 IgM antibody may be useful for evaluating antigen exposure and as a tool for an early leprosy diagnosis in HHC.

Use of anti-PGL-1 antibodies to monitor therapy regimes in leprosy patients

The suitability of IgM antibodies to PGL-1 for monitoring the response to multidrug therapy (MDT) was sequentially tested by ELISA in 105 leprosy patients, and bacterial indexes (BI) were also determined. Patients were divided into 3 groups: group 1, 34 multibacillary (MB) patients treated for 12 months with MDT-MB; group 2, 33 MB patients treated for 24 months with MDT-MB, and group 3, 38 paucibacillary (PB) patients treated for 6 months with MDT-PB. Untreated MB patients exhibited higher antibody levels (mean ± SEM): group 1 (6.95 ± 1.35) and group 2 (12.53 ± 2.02) than untreated PB patients (1.28 ± 0.35). There was a significant difference (P < 0.01) in anti-PGL-1 levels in group 1 patients: untreated (6.95 ± 1.35) and treated for 12 months (2.78 ± 0.69) and in group 2 patients: untreated (12.53 ± 2.02) and treated for 24 months (2.62 ± 0.79). There was no significant difference between untreated (1.28 ± 0.35) and treated (0.62 ± 0.12) PB patients. Antibody levels correlated with BI. The correlation coefficient (Pearson’s r) was 0.72 before and 0.23 (P < 0.05) after treatment in group 1 and 0.67 before and 0.96 (P < 0.05) after treatment in group 2. BI was significantly reduced (P < 0.01) after 12 and 24 months on MDT (group 1: 1.26-0.26; group 2: 1.66-0.36). Our data indicate that monitoring anti-PGL-1 levels during MDT may be a sensitive tool for evaluating treatment efficacy. These data also indicate that the control of leprosy infection can be obtained with 12 months of MDT in MB patients.

Survey to identify Mycobacterium leprae-infected household contacts of patients from prevalent regions of leprosy in Colombia

Leprosy in Colombia is in the post-elimination phase; nevertheless, there are regions of this country where the incidence is still around 3-4/100,000. Early detection of leprosy patients is a priority for achieving control and elimination of leprosy; however, the clinical exam is not very sensitive and thus, the majority of patients are diagnosed only when they demonstrate lesions, and damage to the nerves and skin has already occurred. The goal of the present study was to identify Mycobacterium leprae infection and immune responses in household contacts (HHC) of leprosy patients from three prevalent regions of Colombia. Clinical examination, the Mitsuda test, evaluation of IgM anti-PGL-I in the serum, the bacillar index (BI), and polymerase chain reaction (PCR) from nasal swabs (NS) were performed for 402 HHC of 104 leprosy patients during a cross-sectional survey. Positive titers for IgM anti-PGL1 were found for 54 HHC, and PCR-positive NS for 22. The Mitsuda reaction was negative for 38 HHC, although three were positive for IgM anti-PGL-1 titers. The data document that leprosy transmission among HHC is still occurring in a non-endemic country.

Uso do teste ML Flow em escolares diagnosticados com hanseníase no município de Paracatu, Minas Gerais / The use of ML Flow test in school children diagnosed with leprosy in the district of Paracatu, Minas Gerais

A detecção da hanseníase no município de Paracatu é elevada em menores de quinze anos, abrangendo cerca de 6,8/10.000 hab. em 2003 e é classificada como hiperendêmica. O estudo objetiva analisar a aplicação do teste sorológico do PGL-1 (ML Flow) em 56 de 68 pacientes escolares da rede pública, diagnosticados com hanseníase através da busca ativa de casos no município de Paracatu – MG (2004 a 2006), sendo 71%, paucibacilares. Cerca de 85,2% dos pacientes residiam na área urbana, 55,8% eram do sexo feminino e a doença predominava no grupo de 10 a 14 anos (IC95%:0,49-0,89%) e χ2=7,376, sendo que 15 (26,7%) com resultado do ML Flow positivo. Cinco pacientes tinham incapacidades do Grau 1, da forma clínica Dimorfa (40% ML Flow positivo). O percentual de casos de hanseníase entre os contatos intradomiciliares foi de 46,4%, sendo que 44,9% deles com resultado do teste do ML Flow positivo. O estudo sugere incorporar o teste ML Flow nos serviços de saúde, uma vez que o mesmo auxilia na classificação operacional da doença, controle de contatos intradomiciliares com resultado do teste positivo, visando à detecção precoce dos casos suspeitos de hanseníase.

The detection rate of leprosy in the district of Paracatu is high in the age group under 15 years-old, including about 6.8/10,000 inhabitants in 2003, and classified as hyperendemic. The study aimed to analyze the application of the PGL-1 (ML Flow) serological test in 56 of 68 school-age patients of the public school system, diagnosed with leprosy through active case finding in Paracatu, Minas Gerais State (2004 to 2006), with 71% classified as paucibacillary. Of these, 85.2% lived in urban areas, 55.8% were female and the disease was predominant in the 10 to 14 year-old age group (95%CI: 0.49-0.89; c2=7.373), with 15 (26.7%) presenting a positive ML Flow result. Five patients showed grade 1 disabilities in the borderline clinical form (40% ML Flow positive). The percentage of leprosy cases among household contacts was 46.4%, 44.9% presenting positive ML Flow test results. The study suggests incorporating the ML Flow test into the health services, since it assists in the operational classification of the disease and in the control of household contacts with positive test results, aimed at early detection of suspected leprosy cases.

The different levels of interferron gamma capacity production on several stages of leprosy.

Informations concerning imunological activity in subclinical leprosy is still lacking. Therefore, the objective of this study was to find out the prevalence of subclinical leprosy and interferon- (IFN-) production at several stages of leprosy. This observational study using a cross sectional approach was conducted in Semarang, Central Java, Indonesia. Twenty-six leprosy patients and 62 relatives who lived together with the leprosy patients were included in the study. The venous blood samples were taken from all (88) of the subjects. All of the venous blood samples were measured for IFN- levels by Elisa method. Sixty two blood samples from the relatives who lived together with the leprosy patients were measured for IgM anti PGL-1 to determine the subclinical stage of leprosy. Thus, there were three groups in this study: leprosy patients (LP), subclinical leprosy (SL) and sero-negative subjects (SN). Thirty three of the 62 relatives (53.2%) were in subclinical stage of leprosy, while 29 others were not (sero-negative). There was a significant difference (P= 0.001) in IFN- production capacity among the three groups (LP,SL and SN). Among the three groups, the LP group had the lowest, while SL group had the highest IFN- production capacity.