Molecular and Biotechnological Approaches in the Diagnosis of Leprosy

Leprosy is a worldwide health problem, which needs the development of new and innovative strategies to be controlled. Early diagnosis of leprosy is an important contribution to reducing the incidence of the disease; thus, the development of biotechnology platforms, which include the mapping of antigens with potential to be used in immunodiagnostic and molecular methods for the detection of Mycobacterium leprae, is an important tool to confirm the clinical diagnostic. Molecular biology and biotechnological methods have been used to assist in the diagnosis of this disease, each one with its advantages and drawbacks. Enzyme-linked immunosorbent assay (ELISA) is the used method for leprosy diagnosis, and it allows the detection of infection-related antigens. Alternatively, due to their versatility to perform the same functions as the protein and non-protein natural antigens, mimetic peptides are considered an important tool. On the other hand, lateral flow assay (LFA) and optical and electrochemical biosensors are rapid and portable methods, capable of performing diagnosis in the field without sample preparation. This chapter presents such techniques, their uses in the diagnosis and detection of M. leprae, as well as the potential for the development of new techniques and strategies that can help to control and understand mycobacteriosis

Subtractive phage display selection from canine visceral leishmaniasis identifies novel epitopes that mimic leishmania infantum antigens with potential serodiagnosis applications

Visceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy and Trypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected with Leishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n 31) compared to those from vaccinated dogs (n 21), experimentally infected dogs with cross-reactive parasites (n 23), and healthy controls (n 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no crossreactivity with T. cruzi- or Ehrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes of L. infantum antigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programsThis work was supported by grants from the Pró-Reitoria de Pesquisa of UFMG (supported 03/2013), the Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica (INCT Nano-Biofar), Rede Nanobiotec/Brasil-UFU (CAPES), PRONEX-FAPEMIG (APQ-01019- 09), FAPEMIG (APQ-00496-11 and APQ-00819-12), and CNPq (APQ- 472090/2011-9 and APQ-482976/2012-8). E.A.F.C. and L.R.G. are recipients of grants from CNPq. M.A.C.-F. is the recipient of a grant from FAPEMIG/CAPE

ASPECTOS EPIDEMIOLÓGICOS E PREVALÊNCIA DE ENTEROPARASITOSES EM CRIANÇAS DO BAIRRO JAMBEIRO, SÃO LUÍS, MA

A carência de condições básicas de higiene e saneamento, aliadas à falta de limpeza dos reservatórios de água e a não utilização de água fltrada ou fervida, intensifcam a ocorrência de problemas de saúde pública. Com o intuito de contribuir para a conscientização da população acerca dos problemas de saúde decorrentes da contaminação da água, realizou-se no Bairro do Jambeiro, localizado nas mediações da Universidade Federal do Maranhão, um projeto interdisciplinar que visou, pela divulgação dos dados da pesquisa científca, avaliar a água utilizada pelos moradores através de análise físico-química, microbiológica e parasitológica, de modo que os resultados permitissem a conscientização, através de atividades educacionais dos moradores. Os resultados endossam a precariedade de condições sanitárias encontradas nessa comunidade. Os poços, as torneiras e o córrego apresentam níveis de contaminação acima do recomendável pela Resolução CONAMA e ANVISA, sendo, portanto, impróprias para o consumo. Os exames parasitológicos revelaram alta prevalência (91%) de enteroparasitoses nas crianças. A partir desses resultados os integrantes do projeto e seus parceiros realizaram palestras destinadas à comunidade sobre os meios de tratamento da água, biologia dos parasitas e medidas profláticas das principais verminoses. Concomitante, ocorreu a entrega e esclarecimentos dos laudos da análise físico-química, microbiológica e parasitológica, onde houve a distribuição de remédios às crianças que estavam infectadas.Descritores:  Enteroparasitos; Epidemiologia; Prevalência.Abstract: The lack of basic conditions hygiene and sanitation, allied to grubbiness of water reservoirs and no use of fltered or boiled water, enhance the occurrence of health public problems. With the aim of contribute to awareness of the population about the health problems resulting from the water contamination, was held in the District of the Jambeiro, located near the Federal University of Maranhão, an interdisciplinary project the aimed, by disclosure of scientifc research, to evaluate the water used by population through physico-chemical, microbiological and parasitological analyses, so that the results allow awareness through educational activities of population. The results comproved the precarious sanitary conditions found in this community. The level contamination of the wells, taps and stream is higher than recommended by Resolution CONAMA and ANVISA, therefore, unft for consumption. The parasitological revealed a high prevalence (91%) of intestinal parasites in children. Based on these results the members of the project and its partners held talks for the community about the water treatment, biology of parasites and worms and of the main prophylactic measures. Concomitantly, there was the delivery and clarifcation of reports of physico-chemical, microbiological and parasitological, where there was the distribution of drugs to children who were infected.Descriptors: Enteroparasites; Epidemiology; Prevalence

Eficácia de intervenções imunomoduladoras para o tratamento da Esclerose Lateral Amiotrófica (ELA)

A esclerose lateral amiotrófica (ELA) é uma doença neurodegenerativa que afeta as células nervosas responsáveis pelo controle dos músculos voluntários, resultando em fraqueza muscular e atrofia. Nesse contexto, as intervenções imunomoduladoras têm como objetivo modular a resposta imune do organismo, ao reduzir a inflamação e possibilitar a neuroproteção, a partir do uso de anticorpos monoclonais, inibidores de citocinas e moduladores do sistema imunológico. No entanto, a eficácia de tais intervenções no tratamento da ELA ainda é incerta. Nesse sentido, o presente estudo tem como objetivo analisar a eficácia de intervenções imunomoduladoras para o tratamento da esclerose lateral amiotrófica. Para isso, foram selecionados cinco artigos que abordavam sobre a sua eficácia, por meio de uma estratégia de busca com recorte temporal entre 2017 e 2023, nas bases de dados PubMed (Medline), Cochrane Library e Embase. As intervenções imunomoduladoras, como o uso de inibidores de citocinas, têm demonstrado eficácia no tratamento da esclerose lateral amiotrófica (ELA). Além disso, há evidências de que a inflamação crônica pode estar envolvida em sua patogênese, o que sugere que a modulação do sistema imunológico pode ser uma abordagem terapêutica promissora. Em estudos clínicos recentes, a terapia com inibidores de citocinas mostrou-se capaz de reduzir a progressão da doença e melhorar a qualidade de vida dos pacientes com ELA. Ademais, há evidências que o uso de células-tronco pode melhorar o status funcional em pacientes com a doença. Entretanto, são necessários mais estudos, como ensaios clínicos randomizados e revisões sistemáticas com meta-análises, a fim de ratificar a eficácia das estratégias imunomoduladoras para a patologia

Desenvolvimento de peptídeos miméticos de antígenos do M. leprae e implicações no diagnóstico e prognóstico da hanseníase

Early diagnosis of leprosy is an important contribution to reducing the incidence of the disease. For its early detection, the development of new platforms that include the mapping of antigens with potential to be used in immunodiagnostic is of great interest. Among these antigens, the PGL-1 and epitopes derived from specific bacillus proteins have received great attention. Alternatively, due to their versatility to perform the same functions as the protein and non-protein natural antigens, mimetic peptides are considered an important tool. Thus, our goal was to produce mimetic peptides of Mycobacterium leprae antigens that are promising as serological markers, which will be explored in new diagnostic platforms. To produce peptide mimetics, phage display technology was used. In the first case, we used a monoclonal anti-PGL-1 (CS-38) aiming to obtain peptides that mimics the PGL-1. In the second case, the peptides were obtained having purified IgGs from patients with leprosy as target. The sequences of the selected peptides expressed on the phage surface were chemically synthesized. The synthetic peptides were validated by ELISA (case 1 and 2) and by an immunosensor based on Surface Plasmon Resonance (case 1). Aiming to confirm and identify the targets of the mimetic peptides, scFv antibodies were produced by reverse engineering. The PGL-1-M3 peptide that mimics the native PGL-1 had a sensitivity of 89.11% and specificity of 100.00% in the IgM detection, with positivity of 100% in lepromatous (LL). The IgG detection had positivity of 60% for tuberculoid (TT) and 39% for household contacts (HC). This peptide was used in assembling one biophotonics platform, which allowed the differentiation of all forms of leprosy (p <0.05). The anti-scFv-M3 PGL-1 recognized native PGL-1 and accurately detected the M. leprae in immunohistochemistry tests. The MPML11, MPML14 and MPML12 peptides that mimics M. leprae antigens detect IgG and IgA in patients and HC. In IgG detection, MPML11 peptide showed positivity in 52.2% of TT and 35% of HC, and is also a promising marker of type 2 reaction. MPML12 and MPML14 peptides showed a very similar behavior to the PGL-1, with positivity of 100% and 92.85% in LL, respectively. The three peptides detected IgA in the serum of patients, especially multibacillary (MBs); and IgA in saliva of MBs and HC which index case was multibacillary. Mimetic peptides obtained in this work were confirmed as true mimetics of M. leprae antigens and can be applied in the diagnosis of leprosy in different platforms.Conselho Nacional de Desenvolvimento Científico e TecnológicoDoutor em Genética e BioquímicaO diagnóstico precoce da hanseníase representa uma contribuição importante para diminuir a incidência da doença. Para isso é fundamental o desenvolvimento de novas plataformas, que incluam o mapeamento de antígenos com potencial para o imunodiagnóstico. Dentre estes destaca-se o PGL-1 e epítopos obtidos de proteínas específicas do bacilo. Alternativamente, peptídeos miméticos apresentam-se como uma importante ferramenta, pela versatilidade em desempenhar as mesmas funções que os antígenos naturais proteicos e não-proteicos. Dessa forma, nosso objetivo foi produzir peptídeos miméticos a antígenos do Mycobacterium leprae e que sejam promissores como marcadores sorológicos, os quais serão explorados em novas plataformas diagnósticas. Para produzir os peptídeos miméticos foi utilizada a tecnologia phage display. No primeiro processo utilizou-se um anticorpo monoclonal anti-PGL-1 (CS-38) para obter peptídeos miméticos ao PGL-1. No segundo processo, os peptídeos foram produzidos tendo como alvo IgGs purificadas de pacientes com hanseníase. As sequências dos peptídeos expressos nos fagos foram sintetizadas quimicamente. Os peptídeos sintéticos foram validados por ELISA (processo 1 e 2) e imunosensor baseado em Ressonância Plasmônica de Superfície (processo 1). Por meio de engenharia reversa, anticorpos scFv foram produzidos para confirmar e identificar alvos dos peptídeos miméticos. O peptídeo PGL-1-M3 mimético ao PGL-1 nativo apresentou sensibilidade de 89,11% e especificidade de 100,00% na detecção de IgM, com positividade de 100% em lepromatosos (LL), bem como títulos de IgG detectaram positividade de 60% para tuberculóides (TT) e 39% para contatos domiciliares (HC). Com este peptídeo foi montada uma plataforma biofotônica, que diferencia todas as formas clínicas de hanseníase (p<0,05). O scFv anti-PGL-1-M3 reconheceu PGL-1 nativo e detectou com precisão o M. leprae na imuno-histoquímica. Os peptídeos MPML11, MPML12 e MPML14 miméticos de antígenos do M.leprae detectam IgG e IgA em pacientes e HC. Na detecção de IgG, MPML11 apresentou positividade de 52,2% em TT e 35% em HC e também é um promissor marcador de reação tipo 2. MPML12 e MPML14 apresentaram um comportamento muito similar ao PGL-1, tendo 100% e 92,85% de positividade em LL, respectivamente. Os três peptídeos detectaram IgA nos soros de pacientes, especialmente multibacilares (MBs); bem como IgA na saliva de MBs e HC cujo caso índice era multibacilar. Os peptídeos miméticos obtidos nesse trabalho foram confirmados como miméticos verdadeiros de antígenos do M. leprae e podem ser aplicados no diagnóstico da hanseníase em diferentes plataformas

Seleção e caracterização de peptídeos recombinantes do Mycobacterium leprae ligantes à IgG por meio da tecnologia de phage display

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2012-07-30T21:26:19Z No. of bitstreams: 1 Mayara Ingrid Sousa Lima Seleção e caracterização de peptideos....pdf: 1915651 bytes, checksum: 4954d0969cc99ed5643d45ee27772173 (MD5)Made available in DSpace on 2012-07-30T21:26:19Z (GMT). No. of bitstreams: 1 Mayara Ingrid Sousa Lima Seleção e caracterização de peptideos....pdf: 1915651 bytes, checksum: 4954d0969cc99ed5643d45ee27772173 (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilA hanseníase é uma doença infecciosa crônica, causada pelo Mycobacterium leprae, que apresenta manifestações clínicas variadas. Essas variações refletem em diferenças que vão de uma forte resposta imune celular com controle do crescimento do bacilo, no pólo tuberculóide, a uma anergia em resposta celular, no pólo virchoviano. A caracterização do perfil antigênico do M. leprae frente a esse quadro de múltiplos aspectos clínicos representa uma ferramenta fundamental para o desenvolvimento de novas plataformas para um diagnóstico diferencial mais sensível e/ou desenvolvimento de unidades vacinais. Dessa forma, o objetivo desse trabalho foi selecionar e caracterizar peptídeos miméticos de antígenos do M. leprae reativos contra IgGs totais purificadas de pacientes com hanseníase. Para a seleção foi utilizada a tecnologia de phage display, usando bibliotecas randômicas de peptídeos expressos em fagos filamentosos. Foi realizada uma seleção com IgGs de pacientes Tuberculóides e outra com IgGs de pacientes Virchovianos. A validação dos peptídeos foi realizada utilizando o imunoensaio ELISA, o teste de redução de colônias e análise de bioinformática. Após a pré-validação e sequenciamento foram encontradas 17 mimotopos para o pólo Vichorviano e 12 no pólo Tuberculóide. Foram validados 4 peptídeos, sendo 2 do pólo Tuberculóide (T03, T04) e 2 do pólo Virchoviano (V06 e V13). Os peptídeos TALFPWL (T03) e YSTTLSY (T04) foram imunorreativos em soros de pacientes paucibacilares, bem como em pacientes Virchovianos, além de terem alinhado com proteínas de membrana do M. leprae com potencial antigênico. O peptídeo V06 apresentou especificidade de 100% e sensibilidade de 94,74%, o que se complementa com os dados do teste de redução da pIII, o qual obteve uma taxa de redução de 82% em soros Virchovianos. O peptídeo V13 também foi reativo e apresentou similaridades com chaperonas e proteínas de membrana. Este estudo aponta perspectivas para a identificação de novos antígenos, propiciando a descoberta de novos alvos biológicos com potencial diagnóstico e/ou terapêutico.Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which has varied clinical manifestations. These variations reflect differences that spans from a strong cellular mediated immunity and bacili growth control the tuberculoid pole to a poor T cell immunity at the lepromatous pole. The antigenic profile characterization in both clinical forms represents a fundamental tool for the development of new platforms for a differential diagnosis more sensitive and/or development of vaccine units. Thus, the objective was to select and characterize mimetics peptides antigens of M. leprae reactive against total IgG purified from leprosy patients. The phage display technology was used for selection using random peptides libraries expressed on filamentous phages. A selection was performed with IgGs from tuberculoid patients and other IgGs of lepromatous patients. Peptides validation was performed using the ELISA immunoassay, the plaque reduction test and bioinformatics analysis. After the pre-validation and sequencing were found 17 valid sequences for the lepromatous pole and 12 tuberculoid pole. Four peptides were validated, two of tuberculoid pole (T03, T04) and two lepromatous pole (V06 and V13). The peptides TALFPWL (T03) and YSTTLSY (T04) were imunoreatives in sera from paucibacillary patients and in lepromatous patients. They had alignment with membrane proteins of M. leprae antigenic potential. The V06 peptide showed 100% specificity and 94.74% sensitivity, which is supplemented with the plaque reduction test, who obtained a reduction rate of 82% in lepromatous sera. The V13 peptide was also reactive and showed similarities with chaperones and membrane proteins. This study presents insights for new antigens identification, leading to discovery of new biological targets with potential diagnostic or therapeuti

Anti-Lipoarabinomannan-Specific Salivary IgA as Prognostic Marker for Leprosy Reactions in Patients and Cellular Immunity in Contacts

Leprosy causes the most common peripheral neuropathy of infectious etiology, posing an important public health problem worldwide. Understanding the molecular and immunological mechanisms of nerve damage induced by M. leprae is mandatory to develop tools for early diagnosis and preventive measures. The phenolic glycolipid 1 (PGL-1) and lipoarabinomannan (LAM) antigens are major components of the bacterial surface and are implicated on leprosy immunopathogenesis and neural damage. Although the anti-PGL-1 serum IgM is highly used for operational classification of patients, the anti-LAM salivary IgA (sIgA) has not been investigated as diagnostic or prognostic marker in leprosy. Our aim was to assess the presence of anti-LAM sIgA in leprosy patients and their contacts in order to demonstrate whether such expression was associated with leprosy reactions. Distinct patterns of anti-LAM slgA were observed among groups, which were stratified into treatment-naïve patients (116), patients who completed multidrug therapy—MDT (39), household contacts (111), and endemic controls (11). Both anti-LAM sIgA and anti-PGL-I serum IgM presented similar prognostic odds toward leprosy reactions [(odds ratio) OR = 2.33 and 2.78, respectively]. Furthermore, the anti-LAM sIgA was highly correlated with multibacillary (MB) forms (OR = 4.15). Contrarily, among contacts the positive anti-LAM sIgA was highly correlated with those with positive Mitsuda test, suggesting that the presence of anti-LAM slgA may act as an indicator of cellular immunity conferred to contacts. Our data suggest that anti-LAM slgA may be used as a tool to monitor patients undergoing treatment to predict reactional episodes and may also be used in contacts to evaluate their cellular immunity without the need of Mitsuda tests

Systematic Review of Treatment Failure and Clinical Relapses in Leishmaniasis from a Multifactorial Perspective: Clinical Aspects, Factors Associated with the Parasite and Host

Leishmaniasis is a disease caused by protozoa of the genus Leishmania. Treatment options are limited, and there are frequent cases of treatment failure and clinical relapse. To understand these phenomena better, a systematic review was conducted, considering studies published between 1990 and 2021 in Portuguese, English, and Spanish. The review included 64 articles divided into three categories. Case reports (26 articles) focused on treatment failure and clinical relapse in cutaneous leishmaniasis patients (47.6%), primarily affecting males (74%) and children (67%), regardless of the clinical manifestation. Experimental studies on the parasite (19 articles), particularly with L. major (25%), indicated that alterations in DNA and genic expression (44.82%) played a significant role in treatment failure and clinical relapse. Population data on the human host (19 articles) identified immunological characteristics as the most associated factor (36%) with treatment failure and clinical relapse. Each clinical manifestation of the disease presented specificities in these phenomena, suggesting a multifactorial nature. Additionally, the parasites were found to adapt to the drugs used in treatment. In summary, the systematic review revealed that treatment failure and clinical relapse in leishmaniasis are complex processes influenced by various factors, including host immunology and parasite adaptation

Image_1_Anti-Lipoarabinomannan-Specific Salivary IgA as Prognostic Marker for Leprosy Reactions in Patients and Cellular Immunity in Contacts.tif

<p>Leprosy causes the most common peripheral neuropathy of infectious etiology, posing an important public health problem worldwide. Understanding the molecular and immunological mechanisms of nerve damage induced by M. leprae is mandatory to develop tools for early diagnosis and preventive measures. The phenolic glycolipid 1 (PGL-1) and lipoarabinomannan (LAM) antigens are major components of the bacterial surface and are implicated on leprosy immunopathogenesis and neural damage. Although the anti-PGL-1 serum IgM is highly used for operational classification of patients, the anti-LAM salivary IgA (sIgA) has not been investigated as diagnostic or prognostic marker in leprosy. Our aim was to assess the presence of anti-LAM sIgA in leprosy patients and their contacts in order to demonstrate whether such expression was associated with leprosy reactions. Distinct patterns of anti-LAM slgA were observed among groups, which were stratified into treatment-naïve patients (116), patients who completed multidrug therapy—MDT (39), household contacts (111), and endemic controls (11). Both anti-LAM sIgA and anti-PGL-I serum IgM presented similar prognostic odds toward leprosy reactions [(odds ratio) OR = 2.33 and 2.78, respectively]. Furthermore, the anti-LAM sIgA was highly correlated with multibacillary (MB) forms (OR = 4.15). Contrarily, among contacts the positive anti-LAM sIgA was highly correlated with those with positive Mitsuda test, suggesting that the presence of anti-LAM slgA may act as an indicator of cellular immunity conferred to contacts. Our data suggest that anti-LAM slgA may be used as a tool to monitor patients undergoing treatment to predict reactional episodes and may also be used in contacts to evaluate their cellular immunity without the need of Mitsuda tests.</p