Optimization and validation methodology of indirect ELISA for the immunodiagnosis of paracoccidioidomycosis caused by Paracoccidioides brasiliensis

Abstract

 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 disease  during 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 this pathology, the use of ELISA (Enzyme Linked Immunosorbent  Assay) as screening. Despite this recommendation, this reality is not observed in laboratory practice, the  commonly 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 and validate 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 plates was 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 patients suspected to PCM  were used. The results of the control group demonstrated 67% sensivity and 96% and specificity of  100% and 95% for ID and indirect ELISA, respectively. The comparison of the two techniques, and  ID gold standard resulted in co-positivity 100% and co-negativity of 70%. The agreement between the  techniques was rated as good, according to kappa index. The analysis of serum samples from patients  with other lung diseases (aspergillosis, histoplasmosis and tuberculosis) showed a 50% cross-reactivity.  The results show that the methodology of indirect ELISA, due to its high sensitivity, can be used as  serological screening test; there by giving more speed in the release of reports of patients with absence  of reactivity to P. brasiliensis.