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
O diagnóstico de certeza de processos infecciosos como na paracoccidioidomicose (PCM) deriva da demonstração e do reconhecimento do agente etiológico em preparados histológicos, exame direto ou em cultivo. No entanto, em algumas situações, a pesquisa de anticorpos e antígenos específicos circulantes no soro de pacientes assume grande importância no diagnóstico indireto da infecção. Historicamente, na PCM, a sorologia, além de importante auxílio diagnóstico, tem a função de monitorar o curso da doença durante e após o tratamento através do acompanhamento dos títulos de anticorpos antifúngicos específicos. O Manual daVigilância da Paracoccidioidomicose do Estado de São Paulo preconiza, para o imunodiagnóstico de pacientes com suspeita clínica desta patologia, a utilização do teste de ELISA (Enzyme Linked Immunosorbent Assay) como triagem. Apesar destarecomendação, esta realidade não é observada na prática laboratorial, a técnica frequentemente utilizada é a imunodifusão dupla em gel de agarose (ID). O teste de ELISA tem sido utilizado para a detecção de anticorpos em quase todas as micosessistêmicas, senão em todas. Em relação ao imunodiagnóstico da PCM, a técnica de ELISA oferece ainda porcentagens de reatividade cruzada, porém, por ser um ensaio mais rápido, sua utilização como teste de triagem é de grande valia. Neste sentido, o objetivo deste trabalho foi otimizar e validar a metodologia de ELISAindireto para uso como método de triagem dos soros com suspeita clínica para PCM. A concentração proteica de antígeno para a sensibilização das placas foi de 10,0 µg/poço e as diluições...
The definitive diagnosis of infectious processes as in paracoccidioidomycosis (PCM) derives from the demonstration and recognition of the etiologic agent in histological preparations, direct examination or culture. However, in some situations, the search for circulating antigens and specific antibodies in the serum of patients is of great importance in the indirect diagnosis of infection. Historically, the PCM, serology, and an important diagnostic aid, has the function of monitoring the course of diseaseduring and after treatment through monitoring of titers of specific antibodies antifungal. The Manual of Surveillance Paracoccidioidomycosis from the State of São Paulo advocates for immunodiagnosis of patients with clinical suspicion of thispathology, the use of ELISA (Enzyme Linked Immunosorbent Assay) as screening. Despite this recommendation, this reality is not observed in laboratory practice, thecommonly used technique is the double immunodiffusion in agarose gel (ID). The ELISA method has been used to detect antibodies in nearly all systemic mycoses, if not all. Regarding the immunodiagnosis of PCM, the ELISA technique also provides percentages of cross-reactivity, however, because it is a faster test, its use as a screening test is of great value. Accordingly, the aim of this work was to optimize andvalidate the methodology of indirect ELISA for use as a screening of sera with clinical suspicion for PCM. The protein antigen concentration for sensitization of the plateswas 10.0 mg/well and the dilutions of serum and conjugate were 1:100 and 1:3,000, respectively. The antigen of choice was filtered culture isolate of P. brasiliensis B-339. 166 samples from the control group were used for the validation of the technique, they are 111 apparently healthy patients and 55 patients with PCM confirmed. To evaluate the performance of the technique, 205 samples of patientssuspected to PCM were used. The results of the control group demonstrated 67%...
Subject(s)
Validation Studies as Topic , Paracoccidioides , Paracoccidioidomycosis , Immunologic Tests , Enzyme-Linked Immunosorbent Assay , ImmunodiffusionABSTRACT
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.