The cientificWorldJOURNAL Review Article Immunodiagnostic Methods: What Is Their Role in Areas of Low Endemicity?

Worldwide Schistosomiasis mansoni continues to be a serious public health problem. Over the past decades, control programmes have made remarkable progress in reducing S. mansoni infections to a relatively low level in Brazil and African countries. Endemic regions are currently circumscribed in certain core areas where reinfection and repeated chemotherapy are frequent and, consequently, are related to residents with low parasite load. At present, diagnosis is predominately a key step for final disease control although low endemicity area residents are hardly detected by most of the available assays. In this paper, we review the current status and efforts made aiming at the improvement of diagnostic tools for S. mansoni in low endemicity infections. The establishment of diagnostic assays-simple, affordable, sensitive, and specific for field diagnosis of S. mansoni-is essential and should be given high priority

Diagnosis of \u3ci\u3eSchistosoma mansoni\u3c/i\u3e Infections: What are the Choices in Brazilian Low-Endemic Areas?

The population of Brazil is currently characterised by many individuals harbouring low-intensity Schistosoma mansoni infections. The Kato-Katz technique is the diagnostic method recommended by the World Health Organization (WHO) to assess these infections, but this method is not sensitive enough in the context of low egg excretion. In this regard, potential alternatives are being employed to overcome the limits of the Kato-Katz technique. In the present review, we evaluated the performance of parasitological and immunological approaches adopted in Brazilian areas. Currently, the diagnostic choices involve a combination of strategies, including the utilisation of antibody methods to screen individuals and then subsequent confirmation of positive cases by intensive parasitological investigations

Avaliação da resposta imune a vacinas de antígeno particulado de Leishmania sp e sua associação com vacina de DNA pCI-neo-p36(lack) em camundongos balb/c desafiados com Leishmania chagasi.

A leishmaniose visceral é uma enfermidade crônica que pode atingir 98% de mortalidade em humanos não tratados. Pela sua gravidade e alta prevalência, a vacinação é um meio importante de proteção. Alguns tipos de vacinas vêm sendo testados, dentre eles, as vacinas de antígeno particulado e a de DNA. Estas vacinas demonstraram a capacidade de reduzir a carga parasitária no fígado e no baço em modelo murino e induzir a produção de IFN- , com indução de uma resposta celular prolongada, em alguns casos. No nosso estudo, camundongos BALB/c foram vacinados com três doses de uma vacina subcutânea contendo 100 μg de antígeno particulado de L. chagasi, L. braziliensis ou L. amazonensis e 50 μg de saponina como adjuvante, associada ou não a uma vacina intramuscular, em duas doses, com 100 μg de pCI-neop36( LACK). Esta vacina de DNA continha um gene que codifica a proteína LACK (homóloga de Leishmania de receptores de proteína kinase C ativada), uma proteína de 36 kDa, conservada nas várias espécies e formas do ciclo de vida da Leishmania. Nos dois protocolos de vacinação, os camundongos foram desafiados 4 ou 12 semanas após a administração da vacina com 1 x 107 formas promastigotas de L. chagasi e sacrificados 5 semanas após o desafio para análise da capacidade protetora das vacinas, no fígado e no baço, e de indução da produção de IFN- e IL-4 pelos esplenócitos. Os dados mostraram que a vacina pCI-neo-p36(LACK) associada à vacina de antígeno particulado de L. chagasi induziu maior grau de proteção que a vacina de antígeno particulado administrada isoladamente, especialmente no fígado quando o desafio foi feito 4 semanas após a vacinação. Além disso, a vacina de antígeno de L. amazonensis, quando associada ou não a vacina pCI-neo-p36(LACK), induziu uma maior redução da carga parasitária do que a de L. braziliensis, no entanto nenhuma destas duas vacinas induziu proteção maior que a vacina de antígeno de L. chagasi. As vacinas induziram a produção significativa de IFN-g mas não de IL-4 que, em alguns casos, foi suprimida. Apesar da capacidade da associação das vacinas em aumentar a proteção contra o parasito, problemas relacionados com o uso de vacinas de DNA precisam ser melhor compreendidos.American visceral leishmaniasis is a chronic disease that can reach 98% of human mortality when not treated. Due to its severity and high incidence, vaccination has become an important approach to protection. Several vaccines have been tested, amongst then, freeze-thawed antigen and DNA vaccines. These two vaccines have demonstrated the capacity to reduce the parasite burden in liver and spleen in murine model and to induce the production of IFN- , with the induction of a long-term cellular immunity in some cases. In our study, BALB/c mice were vaccinated with a three doses subcutaneous vaccine containing 100 μg of L. chagasi, L. braziliensis or L. amazonensis freeze-thawed antigen with 50 μg of saponin as an adjuvant, associated or not with an intramuscular one, in two doses, with 100 μg of pCI-neo-p36(LACK). This DNA vaccine was constituted of LACK (Leishmania homologue of receptors for activated C kinase) expression gene, a 36 kDa protein highly conserved in all species and life cycle stages of Leishmania. In the two vaccines protocols, mice were challenged intravenously with 1 x 107 L. chagasi promastigotes 4 or 12 weeks after booster and sacrified 5 weeks after challenge for the analysis of the vaccines protective capacity in liver and spleen and the measure of the production of IFN-g and IL-4 by splenocytes. Data showed that the pCI-neo-p36(LACK) associated to L. chagasi freeze-thawed antigen induced a higher protection than the freeze-thawed antigen alone, specially in liver when the challenge was performed 4 weeks after booster. In addiction, L. amazonensis vaccine induced a greater reduction in parasite burden than L. braziliensis. The vaccines induced a significant production of IFN-g but not of IL-4, that was suppressed is some cases. Despite the showed capability of the vaccines association in increase the protection against the parasite, problems with the use of DNA vaccines must be reviewed

Desenvolvimento e padronização de novas metodologias aplicadas ao diagnóstico e monitoração de cura da esquistossomose mansoni na fase inicial (aguda) e crônica

Submitted by Nuzia Santos ([email protected]) on 2013-01-07T16:58:40Z No. of bitstreams: 1 4. Tese Final RQ3Banca1.pdf: 2857998 bytes, checksum: 113972bf6018f1468776705c406f2b79 (MD5)Made available in DSpace on 2013-01-07T16:58:40Z (GMT). No. of bitstreams: 1 4. Tese Final RQ3Banca1.pdf: 2857998 bytes, checksum: 113972bf6018f1468776705c406f2b79 (MD5)Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil.Neste projeto, foram testados diferentes antígenos candidatos a padronização de novos métodos diagnósticos a serem usados nas diferentes fases da infecção esquistossomótica. Inicialmente, foram analisados os antígenos brutos do parasito visto que seu baixo custo e simplicidade de obtenção merecem novas abordagens. Assim, antígenos de vermes adultos, ovos e tegumento de esquistossômulos foram analisados com soro de pacientes submetidos a rigoroso diagnóstico parasitológico para determinação de infecção e/ou diagnóstico clínico e imunológico. Ao serem aplicados no método indireto de ELISA, estes antígenos apresentaram alta sensibilidade e especificidade em fases distintas da infecção murina. Através desses resultados foi possível confirmar que o uso de antígenos obtidos de diferentes formas evolutivas do parasito serve como ferramenta potencial para análise da evolução cronológica da infecção. Quando aplicados em amostras humanas, o uso de antígenos de vermes adultos mostrou-se promissor para o diagnóstico de pacientes residentes de áreas endêmicas que apresentavam baixa carga parasitária, com índices de sensibilidade e especificidade de 95%. Tendo sido, nestas condições, superior aos antígenos de ovos. O estudo de pacientes em fase aguda da infecção permitiu a validação da técnica indireta de ELISA com antígenos de tegumento de esquistossômulos. Esta técnica apresentou uma significativa sensibilidade na identificação de grande parte dos pacientes recentemente infectados. A outra abordagem adotada por este tarbalho envolveu o uso do Antígeno Catódico Circulante (CCA), antígeno este secretado/excretado por vermes jovens e adultos, que foram direcionados para o desenvolvimento de novas e promissoras metodologias diagnósticas. Para este propósito, o CCA foi utilizado em diferentes formas antigênicas: como glicoproteína purificada a partir de vermes, como proteína recombinante e como peptídeos imunogênicos de 20 aminoácidos. Estes antígenos foram usados na padronização do Método de Separação Imunomagnética, denominado IMS. O uso de CCA recombinante no método de IMS indireto levou aos índices mais significativos de sensibilidade e especificidade, sem que qualquer resultado falso-negativo fosse detectado. Por outro lado, o uso da glicoproteína CCA purificada demonstrou ser superior no diagnóstico para monitorização de cura. A partir destes resultados, mais promissores que a ELISA convencional, partimos para a padronização final desta técnica para a detecção direta do CCA nas mesmas amostras, de forma a permitir somente a identificação de infecções ativas. Para isto, anticorpos monoclonais específicos para a glicoproteína CCA foram produzidos e conjugados a marcadores. A escolha do clone foi baseada na reduzida especificidade deste pela porção responsável pelas reações cruzadas do CCA, a porção glicídica Lewis x. O novo método IMS para detecção direta demonstrou alta sensibilidade de 94% e especificidade de 100%, apresentando correlação direta com a carga parasitária destes pacientes determinada pela contagem de ovos nas fezes. Os excelentes resultados na detecção de antígeno circulante obtidos no presente trabalho, que contrapõem os obtidos em outros trabalhos publicados, se devem a nova metodologia empregada que utiliza a concentração destes antígenos ao invés da diluição de amostras. Por fim, idealizamos um último método, denominado FluoIMS, destinado a identificação qualitativa da presença de CCA através da microscopia de fluorescência. Este método, de detecção direta e de execução bastante simples, apresentou significativos índices de sensibilidade quando três lâminas individuais para cada amostra foram analisadas. Nossos resultados trazem grandes expectativas para a melhoria do diagnóstico dos muitos pacientes infectados por baixas cargas do Schistosoma mansoni, em diferentes fases da infecção, e apontam novas perspectivas para aplicação destes métodos no controle de cura pós-tratamento.In this project, various candidate antigens for standardization of new diagnostic methods were tested for use at different phases of schistosome infection. Initially, the crude antigens of the parasite were analyzed, since their low cost and ease of obtaining deserve new approaches. Thus, antigens of adult worms, egg antigens, and tegument antigens of schistosomula were analyzed by means of sera of patients submitted to a rigorous parasitological diagnosis for determination of infection. When applied to the indirect method of ELISA, these antigens presented high sensitivity and specificity levels at different phases of murine infection. Based on these results, it was possible to confirm that the use of antigens obtained at different evolutive phases of the parasite acts as a potential tool for analysis of the chronological evolution of infection. When applied to human samples, the use of adult worm antigens was promising for diagnosis of patients living in endemic areas, and presenting low worm burden, with sensitivity and specificity levels of 95%. Under these conditions, they were considered superior than the egg antigens. The study related to tourists presenting acute phase of infection allowed the validation of the indirect technique of ELISA, with tegument antigens of schistosomula. This technique showed a significant sensitivity for identification of a large part of recently infected patients. Another approach used in this study involved the use of Circulating Cathodic Antigen (CCA), which was secreted/excreted by juvenile and adult worms, that were directed to development of new and promissing diagnostic methodologies. For this purpose, the CCA was used at different forms of antigens: as purified glycoprotein obtained from worms, as recombinant protein and as immunogenic peptides of 20 aminoacids. These antigens were used for standardization of the Immunomagnetic Separation Method, named IMS. The use of recombinant CCA in the indirect method of IMS showed the most significant levels of sensitivity and specificity, and no false-negative results could be detected. On the other hand, the use of purified glycoproteinCCA demonstrated to be superior for diagnosis of cure control. Based on these results, which were more promissing than the conventional ELISA, we started the final standardization of this technique for the direct detection of CCA in the same samples, in order to allow only the identification of active infections. For this purpose, specific monoclonal antibodies for glycoprotein CCA were produced and conjugated to merchandises. The choice of the clone was based on the lack of its specificity by the portion responsible for the cross-reactions of CCA, the glicidic portion Lewis x, and in order that a low level of cross-reactivity could be detected by this method, in future analyses. The new method IMS demonstrated high levels of sensitivity (94%) and specificity (100%), reaching superior levels than the ones showed by the current immunological methods, as well as presenting a direct correlation with those patients´worm burdens, which were obtained by fecal egg counts. The excellent results obtained in the present study regarding the detection of circulating antigen, that did not corroborate the results obtained in other published papers, are due to the new methodology used, that utilizes the concentration of these antigens and not the dilution of samples. Finally, we planned another method, named FluoIMS, for the qualitative identification of the presence of CCA by means of fluorescence microscopy. This method, offering direct detection and ease of execution, showed significant levels of sensitivity, when three individual glass-plates for each sample were analyzed. Our results offer great expectancy for the improvement of diagnosis of infected patients with low Schistosoma mansoni burdens, at different phases of infection, and indicate new perspectives for application of these methods in the post-treatment cure contro

Vaccine Self-assembling Immune Matrix is a new delivery platform that enhances immune responses to rHBsAg in mice

Submitted by Nuzia Santos ([email protected]) on 2016-01-28T16:44:54Z No. of bitstreams: 1 Vaccine Self-Assembling Immune Matrix Is a New Delivery Platform That Enhances Immune Responses to Recombinant HBsAg in Mice.pdf: 5355417 bytes, checksum: 1fa209aaf586972d0ef3ef9827f73fe2 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2016-01-28T16:49:40Z (GMT) No. of bitstreams: 1 Vaccine Self-Assembling Immune Matrix Is a New Delivery Platform That Enhances Immune Responses to Recombinant HBsAg in Mice.pdf: 5355417 bytes, checksum: 1fa209aaf586972d0ef3ef9827f73fe2 (MD5)Made available in DSpace on 2016-01-28T16:49:40Z (GMT). No. of bitstreams: 1 Vaccine Self-Assembling Immune Matrix Is a New Delivery Platform That Enhances Immune Responses to Recombinant HBsAg in Mice.pdf: 5355417 bytes, checksum: 1fa209aaf586972d0ef3ef9827f73fe2 (MD5) Previous issue date: 2015University of Georgia. College of Veterinary Medicine and The Center for Tropical and Emerging Global Diseases. Department of Infectious Diseases. Athens, Georgia, USA/Fundação Oswaldo Cruz. Centro de Pesquisa Rene Rachou. Laboratorio de Esquistossomose. Belo Horizonte, MG, BrasilUniversity of Georgia. College of Veterinary Medicine and The Center for Tropical and Emerging Global Diseases. Department of Infectious Diseases. Athens, Georgia, USAUniversity of Georgia. College of Veterinary Medicine and The Center for Tropical and Emerging Global Diseases. Department of Infectious Diseases. Athens, Georgia, USAUniversity of Georgia. College of Veterinary Medicine and The Center for Tropical and Emerging Global Diseases. Department of Infectious Diseases. Athens, Georgia, USAVaccination remains the most effective public health tool to prevent infectious diseases. Many vaccines are marginally effective and need enhancement for immunocompromised, elderly, and very young populations. To enhance immunogenicity, we exploited the biphasic property of the (RADA)4 synthetic oligopeptide to create VacSIM (vaccine self-assembling immune matrix), a new delivery method. VacSIM solution can easily be mixed with antigens, organisms, and adjuvants for injection. Postinjection, the peptides self-assemble into hydrated nanofiber gel matrices, forming a depot with antigens and adjuvants in the aqueous phase. We believe the depot provides slow release of immunogens, leading to increased activation of antigen-presenting cells that then drive enhanced immunogenicity. Using recombinant hepatitis B virus surface antigen (rHBsAg) as a model immunogen, we compared VacSIM delivery to delivery in alum or complete Freund's adjuvant (CFA). Delivery of the rHBsAg antigen to mice via VacSIM without adjuvant elicited higher specific IgG responses than when rHBsAg was delivered in alum or CFA. Evaluating IgG subtypes showed a mixed Th1/Th2 type response following immunization with VacSIM, which was driven further toward Th1 with addition of CpG as the adjuvant. Increased specific IgG endpoint titers were observed in both C57BL/6 and BALB/c mice, representative of Th1 and Th2 environments, respectively. Restimulation of splenocytes suggests that VacSIM does not cause an immediate proinflammatory response in the host. Overall, these results suggest that VacSIM, as a new delivery method, has the potential to enhance immunogenicity and efficacy of numerous vaccines

Antígenos de tegumento de esquistossômulos são candidatos em potencial para o diagnóstico de fase aguda da esquistossomose mansoni

Submitted by Nuzia Santos ([email protected]) on 2018-11-20T15:10:37Z No. of bitstreams: 1 The schistosomula tegument antigen.pdf: 100464 bytes, checksum: fccef39afb63f172735e675c258d5118 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2018-11-20T15:18:12Z (GMT) No. of bitstreams: 1 The schistosomula tegument antigen.pdf: 100464 bytes, checksum: fccef39afb63f172735e675c258d5118 (MD5)Made available in DSpace on 2018-11-20T15:18:12Z (GMT). No. of bitstreams: 1 The schistosomula tegument antigen.pdf: 100464 bytes, checksum: fccef39afb63f172735e675c258d5118 (MD5) Previous issue date: 2013Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Esquistossomose. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Esquistossomose. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Esquistossomose. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Esquistossomose. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Pesquisas Clínicas. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Esquistossomose. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Esquistossomose. Belo Horizonte, MG, BrazilA detecção da infecção pelo helminto Schistosoma mansoni quando realizada nas fases iniciais, especialmente antes da oviposição nos tecidos do hospedeiro, pode impedir de forma eficiente o desenvolvimento de graves lesões patológicas. Baseado nisto, foi desenvolvido um ensaio imunoenzimático indireto para detecção de anticorpos IgG específicos contra antígenos de esquistossômulos (ELISA-SmTeg). Este ensaio foi aplicado em amostras sorológicas de camundongos não infectados, da mesma forma que de camundongos recentemente infectados, após sete e 15 dias de infecção. Os resultados foram comparados com o número de vermes adultos obtidos por perfusão do sistema hepático murino 50 dias pós-infecção. A sensibilidade e a especificidade do novo método, denominado ELISA-SmTeg, foram de 100% (p = 0,0032, 0,0048, respectivamente, durante sete e 15 dias de infecção) com um valor de corte de 0,15 (p = 0,0002). Nossos resultados mostraram que um ensaio de baixo custo, que utiliza antígenos de fácil obtenção, é capaz de discriminar a esquistossomose mansoni em modelo experimental de forma precoce, incluindo sete dias pós-infecção. If Schistosoma mansoni infection could be detected in its early stages, especially before the egg deposition in the host tissues, the development of severe pathologic lesions could be efficiently prevented. We therefore developed an indirect enzyme-linked immunosorbent assay based on the detection of specific IgG against schistosomula antigens (ELISA-SmTeg). The assay was applied in sera samples from non-infected and infected mice collected seven and 15 days post-infection. The results were compared to the number of adult worms obtained by perfusion of the murine hepatic system 50 days post-infection. The sensitivity and specificity of the ELISA-SmTeg were 100% (p = 0.0032 and 0.0048 respectively for seven and 15 days of infection) with a cutoff value of 0.15 (p = 0.0002). Our findings show a novel low-cost serological assay using antigens which are easy to obtain, which was able to detect all the infected mice as early as seven days post-infection

Antischistosomal activity of a calcium channel antagonist on schistosomula and adult Schistosoma mansoni worms

Current schistosomiasis control strategies are largely based on chemotherapeutic agents and a limited number of drugs are available today. Praziquantel (PZQ) is the only drug currently used in schistosomiasis control programs. Unfortunately, this drug shows poor efficacy in patients during the earliest infection phases. The effects of PZQ appear to operate on the voltage-operated Ca2+channels, which are located on the external Schistosoma mansoni membrane. Because some Ca2+channels have dihydropyridine drug class (a class that includes nifedipine) sensitivity, an in vitro analysis using a calcium channel antagonist (clinically used for cardiovascular hypertension) was performed to determine the antischistosomal effects of nifedipine on schistosomula and adult worm cultures. Nifedipine demonstrated antischistosomal activity against schistosomula and significantly reduced viability at all of the concentrations used alone or in combination with PZQ. In contrast, PZQ did not show significant efficacy when used alone. Adult worms were also affected by nifedipine after a 24 h incubation and exhibited impaired motility, several lesions on the tegument and intense contractility. These data support the idea of Ca2+channels subunits as drug targets and favour alternative therapeutic schemes when drug resistance has been reported. In this paper, strong arguments encouraging drug research are presented, with a focus on exploring schistosomal Ca2+channels

Immunodiagnostic Methods: What Is Their Role in Areas of Low Endemicity?

Worldwide Schistosomiasis mansoni continues to be a serious public health problem. Over the past decades, control programmes have made remarkable progress in reducing S. mansoni infections to a relatively low level in Brazil and African countries. Endemic regions are currently circumscribed in certain core areas where reinfection and repeated chemotherapy are frequent and, consequently, are related to residents with low parasite load. At present, diagnosis is predominately a key step for final disease control although low endemicity area residents are hardly detected by most of the available assays. In this paper, we review the current status and efforts made aiming at the improvement of diagnostic tools for S. mansoni in low endemicity infections. The establishment of diagnostic assays—simple, affordable, sensitive, and specific for field diagnosis of S. mansoni—is essential and should be given high priority