ABSTRACT
The standardization and validation of a multiplex assay requires the combination of important parameters such as sensitivity and specificity, acceptable levels of performance, robustness, and reproducibility. We standardized a multiparametric Dot-blot aimed at the serological screening of paracoccidioidomycosis, histoplasmosis, and aspergillosis. A total of 148 serum were evaluated: 10 from healthy subjects, 36 from patients with paracoccidioidomycosis, 62 from patients with histoplasmosis, and 40 from patients with aspergillosis. It was found that the multiparametric Dot-blot showed a high percentage of cross-reactivity. However, when evaluated individually, in the serological screening of histoplasmosis, a good performance was observed when compared to the double immunodiffusion assay, considered the gold standard test, with 100% co-positivity and 83.3% co-negativity. The performance of serological screening for aspergillosis was not satisfactory when compared to double immunodiffusion, showing 71.4% co-positivity and 100% co-negativity. The evaluation of the stability of nitrocellulose membranes showed that membranes sensitized with H. capsulatum antigen remained stable for 90 days and those sensitized with A. fumigatus antigen for 30 days. We conclude that the use of crude antigens was not suitable for the standardization of the multiparametric Dot-blot assay, due to the high cross-reactivity, and that further tests should be performed with purified proteins (AU).
A padronização e validação de um ensaio multiplex requer a combinação de parâmetros importantes, como sensibilidade e especificidade, níveis aceitáveis de desempenho, robustez e reprodutibilidade. Este trabalho padronizou um Dot-blot multiparamétrico visando a triagem sorológica da paracoccidioidomicose, histoplasmose e aspergilose. Foram avaliadas 148 amostras de soro: 10 de indivíduos saudáveis, 36 de pacientes com paracoccidioidomicose, 62 de pacientes com histoplasmose e 40 de pacientes com aspergilose. Verificou-se que o Dot-blot multiparamétrico apresentou elevado percentual de reatividade cruzada. Entretanto, quando avaliado individualmente, na triagem sorológica da histoplasmose observou-se bom desempenho quando comparado ao ensaio de imunodifusão dupla, considerado o teste padrão ouro, com 100% de co-positividade e 83,3% de co-negatividade. O desempenho da triagem sorológica da aspergilose não foi satisfatório quando comparado a imunodifusão dupla, apresentando 71,4% de co-positividade e 100% de co-negatividade. A avaliação da estabilidade das membranas de nitrocelulose mostrou que membranas sensibilizadas com antígeno de H. capsulatum permaneceram estáveis por 90 dias e as sensibilizadas com antígeno de A. fumigatus, por 30 dias. Concluímos que o uso de antígenos brutos não foi adequado para a padronização do ensaio de Dot-blot multiparamétrico, devido ao alto índice de reatividade cruzada, e que novos testes devem ser realizados com proteínas purificadas (AU).
Subject(s)
Paracoccidioidomycosis , Aspergillosis , Reference Standards , Immunologic Tests , Public Health , Methodology as a Subject , Histoplasmosis , Mycoses/diagnosisABSTRACT
Oitenta porcento dos casos de paracoccidioidomicose (PMC) ocorrem no Brasil. As regiões brasileiras com maior número de casos são: sul, sudeste e centro-oeste, sendo emergente no norte e nordeste. A imunodifusão dupla em gel de agarose assume grande importância no diagnóstico, por permitir o monitoramento da doença e por oferecer subsídios para levantamentos soroepidemiológicos. O objetivo deste trabalho foi de avaliar e caracterizar os pacientes atendidos no Laboratório de Imunodiagnóstico das Micoses do Instituto Adolfo Lutz de São Paulo, em 2016. Trata-se de um estudo retrospectivo realizado utilizando-se dados secundários e avaliando-se as seguintes informações: idade, sexo, procedência do pedido médico, resultado e histórico sorológico dos pacientes. Dos 1.408 pacientes, 12,8% apresentaram reatividade sorológica para Paracoccidioides brasiliensis. Destes, 42,5% não possuiam histórico sorológico, sendo considerados como casos novos da doença. A classificação dos pacientes reagentes por gênero demonstrou que 83,4% eram do sexo masculino, com razão de masculinidade de 5:1. A faixa etária variou de um (1) a 92 anos, e aproximadamente 40% dos pacientes eram da faixa etária de 41 a 60 anos. Este estudo demonstra e reforça a importância da implementação dos estudos soroepidemiológicos como ferramenta auxiliar para nortear as ações de vigilância e políticas em saúde na PCM. (AU)
Eighty percent of paracoccidioidomycosis (PMC) cases occur in Brazil. The highest numbers occur in south, southeast and center-west region, being emergent in the north and northeast areas. The double immunodiffusion in agarose gel is valuable for its diagnosis, as it allows the monitoring of the disease and offers subsidies for the seroepidemiological surveys. This study evaluated and characterized the patients attended in 2016 at the Mycoses Immunodiagnosis Laboratory of Adolfo Lutz Institute of São Paulo. This retrospective study, based on the secondary data, evaluated the information: age, sex, medical request origin, result and serological history of the patients. Of 1,408 patients, 12.8% presented positive serological reactivity for Paracoccidioides brasiliensis. Of them, 42.5% had no serological history and they were considered as new cases. The classification of reactive patients by gender showed that 83.4% were males, being the masculinity ratio of 5:1. The age range was one (1) to 92 years old, and ±40% of the patients were of age ranging from 41-60 years old. This study reinforces the importance of the implementation of the seroepidemiological studies as to guide the surveillance actions and the public health politics in PCM. (AU)
Subject(s)
Paracoccidioides , Paracoccidioidomycosis , Patients , Immunologic TestsABSTRACT
Oitenta porcento dos casos de paracoccidioidomicose (PMC) ocorrem no Brasil. As regiões brasileiras com maior número de casos são: sul, sudeste e centro-oeste, sendo emergente no norte e nordeste. A imunodifusão dupla em gel de agarose assume grande importância no diagnóstico, por permitir o monitoramento da doença e por oferecer subsídios para levantamentos soroepidemiológicos. O objetivo deste trabalho foi de avaliar e caracterizar os pacientes atendidos no Laboratório de Imunodiagnóstico das Micoses do Instituto Adolfo Lutz de São Paulo, em 2016. Trata-se de um estudo retrospectivo realizado utilizando-se dados secundários e avaliando-se as seguintes informações: idade, sexo, procedência do pedido médico, resultado e histórico sorológico dos pacientes. Dos 1.408 pacientes, 12,8% apresentaram reatividade sorológica para Paracoccidioides brasiliensis. Destes, 42,5% não possuiam histórico sorológico, sendo considerados como casos novos da doença. A classificação dos pacientes reagentes por gênero demonstrou que 83,4% eram do sexo masculino, com razão de masculinidade de 5:1. A faixa etária variou de um (1) a 92 anos, e aproximadamente 40% dos pacientes eram da faixa etária de 41 a 60 anos. Este estudo demonstra e reforça a importância da implementação dos estudos soroepidemiológicos como ferramenta auxiliar para nortear as ações de vigilância e políticas em saúde na PCM.
Eighty percent of paracoccidioidomycosis (PMC) cases occur in Brazil. The highest numbers occur in south, southeast and center-west region, being emergent in the north and northeast areas. The double immunodiffusion in agarose gel is valuable for its diagnosis, as it allows the monitoring of the disease and offers subsidies for the seroepidemiological surveys. This study evaluated and characterized the patients attended in 2016 at the Mycoses Immunodiagnosis Laboratory of Adolfo Lutz Institute of São Paulo. This retrospective study, based on the secondary data, evaluated the information: age, sex, medical request origin, result and serological history of the patients. Of 1,408 patients, 12.8% presented positive serological reactivity for Paracoccidioides brasiliensis. Of them, 42.5% had no serological history and they were considered as new cases. The classification of reactive patients by gender showed that 83.4% were males, being the masculinity ratio of 5:1. The age range was one (1) to 92 years old, and ±40% of the patients were of age ranging from 41-60 years old. This study reinforces the importance of the implementation of the seroepidemiological studies as to guide the surveillance actions and the public health politics in PCM.
Subject(s)
Humans , Paracoccidioides/isolation & purification , Paracoccidioidomycosis/diagnosis , Paracoccidioidomycosis/blood , Serotyping , Brazil , Immunodiffusion , Immunologic TestsABSTRACT
Abstract Background Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). Methods In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. Results The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. Conclusion Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.
ABSTRACT
Considerando-se que o immunoblotting para o imunodiagnóstico da paracoccidioidomicose (PCM) é uma metodologia in house e laboriosa envolvendo duas etapas iniciais, SDS-PAGE e Western blot, neste estudo foi avaliado o tempo de prateleira das membranas de nitrocelulose sensibilizadas com antígeno de P. brasiliensis, armazenadas a -20 oC durante 7, 15, 30, 45, 60 e 90 dias. Vinte e oito amostras de soro foram analisadas em dois grupos de membranas de nitrocelulose (membranas previamente bloqueadas com PBS-leite 5 % e as não-bloqueadas). Não houve diferença no padrão de reatividade quando os soros foram avaliados frente a ambos os grupos, especialmente para membranas armazenadas por 7, 15, 30, 45 e 60 dias. A boa estabilidade do antígeno utilizado para sensibilizar as membranas fez com que estas pudessem ser armazenadas a -20 °C até 60 dias. Estas características contribuem para efetuar o diagnóstico rápido da PCM, bem como as perspectivas dessas membranas sensibilizadas serem encaminhadas para os laboratórios, que não possuam infraestrutura necessária para executar as etapas que antecedem a realização de immunoblotting, como a produção de antígeno, as técnicas de SDS PAGE e Western blot. Este procedimento contribui substancialmente para melhorar o diagnóstico sorológico da PCM, pois poderá fornecer resultados reprodutíveis nas unidades componentes da Rede de Laboratórios.
The immunoblotting reaction for performing the paracoccidioidomycosis (PCM) immunodiagnosis is an in-house methodology; and being a laborious task involving two previous steps, SDS-PAGE and Western blot, we evaluated the shelf life of nitrocellulose membranes containing the immobilized P. brasiliensis antigens, stored at -20 oC for 7, 15, 30, 45, 60 and 90 days. Twenty-eight serum samples were analyzed on two nitrocellulose membranes groups: (a) membranes previously blocked with PBS-5 % non-fat dry milk and (b) the priory non-blocked membranes. No difference was detected in the reactivity pattern in serum samples evaluated in the both membrane groups, especially for those stored for 7, 15, 30, 45 and 60 days. It might be emphasized that a good stability of P. brasiliensis antigens, immobilized on the nitrocellulose membranes, enable them to be stored up to 60 daysat -20 oC. This finding contributes to the rapid diagnosis of PCM, and for sending them to other laboratories without adequate infrastructure for carrying out the steps that precede the immunodetection as the antigen production, SDS-PAGE and Western blot techniques. This scheme contributes substantially to improve the quality of PCM serodiagnosis, as it provides reproducible results in the units of the Laboratory Network.
Subject(s)
Immunoblotting , Paracoccidioides , Paracoccidioidomycosis , Serologic TestsABSTRACT
Background Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). Methods In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. Results The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. Conclusion Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life - membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories - and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.(AU)
Subject(s)
Paracoccidioides , Paracoccidioidomycosis , Enzyme-Linked Immunosorbent Assay , Predictive Value of Tests , Reference StandardsABSTRACT
A paracoccidioidomicose (PCM), micose profunda de natureza crônicagranulomatosa, apresenta maior incidência no Brasil. O diagnóstico de certezaderiva da visualização do agente etiológico, o fungo Paracoccidioides spp emamostras biológicas. No entanto, em algumas situações o acesso ao local dalesão impede a coleta do material biológico. Assim, as técnicas imunosorológicaspermitem inferir diagnóstico com certo grau de certeza, otimizandoo tempo utilizado para se obter resultados. A técnica sorológica amplamenteutilizada para o imunodiagnóstico da PCM é a imunodifusão dupla em gel deagarose (ID), com especificidade e sensibilidade variando de 65 a 100%; sendode fácil execução e não necessitando de automação. A técnica de Dot-Blot (DB) tem sido utilizada com sucesso no diagnóstico de inúmeras doençasparasitárias e infecciosas, como a toxoplasmose e a leishmaniose visceral. Nodiagnóstico da PCM, a metodologia mostrou-se promissora noacompanhamento de pacientes durante o tratamento anti-fúngico e eminquéritos soroepidemiológicos. Diante do exposto, o objetivo deste trabalho foipadronizar o ensaio de Dot-Blot visando o diagnóstico rápido da PCM,propondo que o mesmo seja uma ferramenta de triagem dos soros comsuspeita clínica para a doença. A padronização da técnica de Dot-Blot apresentou melhores resultados quando se utilizou antígeno obtido de filtradode cultura de P. brasiliensis do isolado B-339 para a sensibilização demembranas de nitrocelulose. As diluições de soro e conjugado foram de 1:40 e1:2000, respectivamente, incubando ambos em solução PBS-L 3%. Para apadronização do ensaio de Dot-Blot, 143 amostras de soro foram utilizadas echamadas de grupo controle. Destas, 23 amostras de soro foram de pacientesaparentemente sadios, 77 amostras de soro de pacientes com PCM confirmadae 43 amostras de soros com outras doenças (tuberculose, aspergilose ehistoplasmose)...
The paracoccidioidomycosis (PCM) is the greatest mycosis of chronicgranulomatous nature in Brazil. The definitive diagnosis derives from the view ofthe etiologic agent, Paracoccidioides spp in biological samples. However, insome situations, access to the site of injury prevents the collection of biologicalmaterial. Thus, immuno- serological techniques allow inferring diagnosis with adegree of certainty and optimizing time taken to get results. The serologicaltechnique widely used for immunodiagnosis of PCM is double immunodiffusionin agarose gel (DI), with a sensitivity and specificity ranging from 65 to 100%,being easy to perform and not requiring automation. The Dot-blot (DB)technique has been used successfully in the diagnosis of many infectious andparasitic diseases such as visceral leishmaniasis and toxoplasmosis. On thediagnosis of PCM, this methodology showed promise results in monitoringpatients during treatment with anti-fungal and seroepidemiological surveys.Given the above, the objective of this study was to standardize the Dot-blotassay targeting the rapid diagnosis of PCM, suggesting that it is a screeningtool for sera with clinical suspicion for the disease. The standardization of Dot-Blot showed better results when using antigen obtained from culture filtrate of P.brasiliensis isolated from B-339 to sensitize nitrocellulose membranes. Theserum and conjugated dilutions were...