Factors interfering with the production of Histoplasma capsulatum antigens / Fatores que interferem na produção de antígenos de Histoplasma capsulatum

Histoplasma capsulatum causes systemic mycosis that depends on host susceptibility, fungal virulence, and factors associated with the infectious process. We evaluated the possible interference of the phenotype of 12 samples of H. capsulatum isolated from HIV-positive and negative patients in obtaining antigens, aiming at the serological diagnosis through the gender-specific recognition of the H and M fractions. The antigens were evaluated by double immunodiffusion against H. capsulatum anti-antigen polyclonal antibody and serum samples from patients with histoplasmosis.The phenotypic evaluation revealed differences in the identification of the fungal agent and in the expression of H and M antigens, considered serological markers of the disease, associated with pigmentation and the production of conidia. It was found that antigenic preparations obtained from H. capsulatum isolated from HIV-positive patients may have satisfactory antigenic capacity. The patient's immune status does not seem to interfere with the expression of antigenic proteins secreted by H. capsulatum. However, we suggest that prolonged use of antiretrovirals drugs or steroids can cause important phenotypic alterations. We showed that some fungal samples from patients with a long history of immunosuppressive drugs produced atypical cellular elements and low reactivity against the H and M fractions. (AU)

Histoplasma capsulatum causa micose sistêmica endêmica que depende da suscetibilidade do hospedeiro, da virulência fúngica e de fatores associados ao processo infeccioso. Avaliamos a possível interferência do fenótipo de 12 amostras de H. capsulatumisolados de pacientes HIV positivos e negativos na obtenção de antígenos, visando o diagnóstico sorológico por meio do reconhecimento gênero-específico das frações H e M. Os antígenos foram avaliados por imunodifusão dupla, frente a anticorpo policlonal anti-antígeno de H. capsulatum e frente a amostras de soro de pacientes com histoplasmose. A avaliação fenotípica revelou diferenças, não só na identificação do agente fúngico, mas também na expressão dos antígenos H e M, considerados marcadores sorológicos da doença, associados à pigmentação e produção de conídios. Verificou-se que preparações antigênicas obtidas de H. capsulatum isoladas de pacientes HIV positivos podem ter capacidade antigênica satisfatória. O estado imunológico do paciente parece não interferir na expressão de proteínas antigênicas secretadas por H. capsulatum. No entanto, sugerimos que o uso prolongado de antirretrovirais e/ou esteróides pode causar alterações fenotípicas importantes. Verificou-se que algumas amostras fúngicas isoladas de pacientes com longo histórico de uso de imunossupressores produziram elementos celulares atípicos e baixa reatividade sorológica contra as frações H e M de H. capsulatum. (AU)

Multiparametric assay of screening for the diagnosis of mycoses of interest in Public Health: standardization of methodology / Ensaio multiparamétrico de triagem para diagnóstico de micoses de interesse em Saúde Pública: padronização da metodologia

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).

Perfil sorológico dos pacientes com suspeita clínica de paracoccidiodomicose avaliados pelo Instituto Adolfo Lutz no ano de 2016 / Serological profile of patients with clinical suspicion of paracoccidiodomycosis evaluated by Instituto Adolfo Lutz in 2016

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)

Perfil sorológico dos pacientes com suspeita clínica de paracoccidiodomicose avaliados pelo Instituto Adolfo Lutz no ano de 2016 / Serological profile of patients with clinical suspicion of paracoccidiodomycosis evaluated by Instituto Adolfo Lutz in 2016

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.

Standardization and validation of Dot-ELISA assay for Paracoccidioides brasiliensis antibody detection

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.

Evaluation of the “shelf life” of nitrocellulose membranes immobilized with Paracoccidioides brasiliensis antigen for performing the immunodiagnosis of paracoccidioidomycosis / Avaliação do “tempo de prateleira” de membranas de nitrocelulose imobilizadas com antígeno de Paracoccidioides brasiliensis para o imunodiagnóstico da paracoccidioidomicose

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.

Standardization and validation of Dot-ELISA assay for Paracoccidioides brasiliensis antibody detection

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)

Immunological assays employed for the elucidation of an histoplasmosis outbreak in São Paulo, SP

Several reports showed outbreaks of histoplasmosis acquired while bat-inhabited caves were visited by tourists, miners or researchers. We evaluated the performance of double immunodifusion (DI) and immunoblotting (IB) assays, employed for the histoplasmosis outbreak elucidation occurred in Vale do Paraíba, São Paulo. The existence of epidemiologic link, four patients with clinical signs suggestive of histoplasmosis and mycological confirmation has made that all 35 individuals involved to the cave visit were subjected to serological evaluation. By DI, we observed reactivity against H. capsulatum antigen in a single serum examined nearly 20 days after exposure to fungal propagules. On the other hand, IB showed reactivity against H and M fractions in 50% of samples evaluated. The analysis of the second sample batch, collected two months after the exposure showed that 96.7% were reactive by DI with antibodies titers ranging from 1 to 16 and 100% of reactivity against H and M fractions, by IB, suggesting an acute infection. The analysis of the overall agreement between the methods showed to be reasonable (κ = 0.37). This study confirms the importance and efficacy of more sensitive methodologies, such as IB assay, to early elucidation of disease, especially in cases of patients without mycological information.

Importance of the association of molecular and immunological diagnosis in immunocompetent patient with Histoplasma capsulatum and Cryptoccocus neoformans infection: a case report

This case reports an immunocompetent 29-year-old woman with suspected pneumonia, suggestive of fungal infection. Immunoblotting analysis reactivity againstHistoplasma capsulatum and Paracoccidioides brasiliensis were observed. Nested-PCR in blood employing species-specific primers was positive for H. capsulatum andCryptococcus neoformans. The evaluation of paucisymptomatic patients with positive results for H. capsulatum and C. neoformans could be relevant for the prevention as well as the possible evaluation of the reactivated quiescent foci. In conclusion, the associated methodology may have contributed to the monitoring endogenous reactivation of these diseases.(AU)

Importance of the association of molecular and immunological diagnosis in immunocompetent patient with Histoplasma capsulatum and Cryptoccocus neoformans infection: a case report

This case reports an immunocompetent 29-year-old woman with suspected pneumonia, suggestive of fungal infection. Immunoblotting analysis reactivity against Histoplasma capsulatum and Paracoccidioides brasiliensis were observed. Nested-PCR in blood employing species-specific primers was positive for H. capsulatum and Cryptococcus neoformans. The evaluation of paucisymptomatic patients with positive results for H. capsulatum and C. neoformans could be relevant for the prevention as well as the possible evaluation of the reactivated quiescent foci. In conclusion, the associated methodology may have contributed to the monitoring endogenous reactivation of these diseases.

Cryptic species of Paracoccidioides brasiliensis: impact on paracoccidioidomycosis immunodiagnosis

We aimed to evaluate whether the occurrence of cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii, has implications in the immunodiagnosis of paracoccidioidomycosis (PCM). Small quantities of the antigen gp43 were found in culture filtrates of P. lutzii strains and this molecule appeared to be more variable within P. lutzii because the synonymous-nonsynonymous mutation rate was lower, indicating an evolutionary process different from that of the remaining genotypes. The production of gp43 also varied between isolates belonging to the same species, indicating that speciation events are important, but not sufficient to fully explain the diversity in the production of this antigen. The culture filtrate antigen AgEpm83, which was obtained from a PS3 isolate, showed large quantities of gp43 and reactivity by immunodiffusion assays, similar to the standard antigen (AgB-339) from an S1 isolate. Furthermore, AgEpm83 was capable of serologically differentiating five serum samples from patients from the Botucatu and Jundiaí regions. These patients had confirmed PCM but, were non-reactive to the standard antigen, thus demonstrating an alternative for serological diagnosis in regions in which S1 and PS2 occur. We also emphasise that it is not advisable to use a single antigen preparation to diagnose PCM, a disease that is caused by highly diverse pathogens.

Histoplasmose: um risco ocupacional entre pesquisadores que realizam trabalho de campo? / Is the histoplasmosis an occupational hazard between the investigators who perform field work?

A histoplasmose é micose sistêmica causada por Histoplasma capsulatum. A infecção humana ocorre pela inalação de microconídios encontrados em solo contendo excretas de aves e/ou morcegos. Neste estudo foi enfatizado o potencial das atividades profissionais praticadas em cavernas em possibilitar risco de infecção por Histoplasma. Utilizando-se questionário semi estruturado, foram obtidas informações sobre o trabalho de campo executado por um grupo de 15 biólogos. A presença de anticorpos séricos anti-H.capsulatum foi avaliada em amostras de sangue coletadas desses profissionais. Na entrevista, 87 por cento dos indivíduos relataram frequentes visitas a cavernas, 53 acampamento no interior das grutas; 33 por cento captura de morcegos e 53 por cento de coleta de amostras de solo. A maioria das cavernas situa-se nas regiões sudeste e centro-oeste do Brasil. A detecção de anticorpos por imunodifusão dupla demonstrou que 20 por cento dos soros reagiram com o antígeno de H. capsulatum. Por immunoblotting, houve 94,1 por cento de reconhecimento específico da fração de 94 kDa, sugerindo-se que os indivíduos entraram em contato com H. capsulatum. Este estudo sugere fortemente que a prática da espeleologia propicia a exposição dos indivíduos a espécies fúngicas que habitam saprofiticamente o meio ambiente, especialmente H. capsulatum, configurando-se como potencial fator de risco para infecções.

Punçäo aspirativa de ganglio linfático: revisäo de 283 casos puncionados no Hospital das Clínicas da F. M. de Botucatu / Aspiration puncture of lymph node: review of 283 cases punctured in the Hospital das Clínicas of Botucatu F. M

Com o objetivo de avaliar a eficácia do método de punçäo aspirativa por agulha fina (PAAF) em nódulos linfáticos, bem como avaliar a distribuiçäo das diferentes lesöes diagnosticadas, estudamos 283 casos aspirados no HC da FMB. Tivemos 187 casos negativos para células neoplásicas (66,07//) 10 casos suspeitos (3,53//) e 86 casos positivos para neoplasia (30,38//). Os casos de material insuficiente näo foram considerados nesta análise. Das lesöes benignas, a maior parte correspondia a linfadenites. Estas foram caracterizadas em três grandes grupos: 32 casos de linfadenite aguda (11,31//), 83 casos de linfadenite reacional inespecífica (29,32//) e 46 casos de processo inflamatório crônico granulomatoso (16,25//), 1 caso de histoplasmose e 1 caso sugestivo de doença de Kikuchi. As neoplasias foram subdivididas em primárias e metastáticas, sendo que das primárias 13 eram linfomas de Hodgkin (4,59// e 14 linfomas näo Hodgkin (4,94//). Mais recentemente temos introduzido técnicas imunocitoquímicas para melhor caracterizaçäo dos linfomas e como auxiliar na detecçäo de sítios primitivos em punçöes de metástases