NOVEL ELISA FOR THE DIAGNOSIS OF HISTOPLASMOSIS

AGUSTINA TOSCANINI; BRITO DEVOTO; IOVANNITTI CRISTINA; A. D. NUSBLAT; CUESTAS MARIA LUJAN

Bahia

Congreso; INFOCUS 2019; 2019

INFOCUS

Objectives: Histoplasmosisis a systemic and endemic mycosis caused by Histoplasma capsulatum. Thesignificance of histoplasmosis results from its worldwide distribution, itsability to mimic other serious disease entities and its propensity to causeserious disseminated infection in immunocompetent patients. Definitivediagnosis involves direct visualization of the fungus in tissue and/or by culture, which may take up to 4 weeks and lackssensitivity. The detection of antibodies offers a rapid alternative tomicrobiological means of diagnosis, and their detection by immunodiffusion (ID)and complement fixation (CF) is often used. Histoplasmin, the standard serodiagnosticreagent used is not ideal because of the presence of cross-reactive carbohydratecomponents which lead to a detrimental effect on the specificity andsensitivity of the CF and ID tests, respectively, particularly in the earlyacute stage of the disease. Moreover, current methods for its production aretime-consuming and problematic. An alternative approach to immunodiagnosis isto detect fungal antigens in urine or serum, which is particularly useful incases of disseminated disease. In this study, the application of a novelantigen, the 100kDa protein of H.capsulatum, Hcp100, for the development of an ELISA for the early diagnosisof histoplasmosis is described. Methods: Serum samples from 15histoplasmosis patients were studied. Five of them had the chronic form, onethe disseminated form and nine had AIDS and the disseminated form ofhistoplasmosis. The diagnosis for all individuals was confirmed either by cultureor histopathology. Only the AIDS patients studied were serology (ID) negative. Serumsamples from patients with paracoccidioidomycosis, coccidioidomycosis, cryptococcosis,tuberculosis and pneumocystosis were included. As negative controls, 100 serumsamples from healthy individuals were used. For the development of the novelELISA, the gene that encodes for Hcp100 was expressed in the Pichia pastorisX-33 strain as a soluble protein, purified by affinity chromatography and usedat a concentration of 0.5 µg/well for coating the ELISA plate. After blockingwith 5% (w/v) dried non-fat milk in PBS-Tween20 0.05%, serum samples were addedat a dilution of 1:1000 and incubated for 1h at room temperature. Then, goatanti-human IgG horseradish peroxidase conjugate at a dilution of 1:10000 wasused as a secondary antibody. The reaction was developed with OPD andterminated by addition of H2SO4 0.4N followed byabsorbance measurement at 492nm. Sensitivity, specificity, predictive valuesand the area under the curve (ROC) were estimated. Results: ROC analysisdetermined the optimal cutoff for Hcp100 antibody detection at which point thesensitivity was 80% and the specificity was 86%. Noteworthy, 6 out of 9 AIDSpatients tested positive by ELISA whilstnone were positive by ID. Positive and negative predictive values were 39% and 97%,respectively.Conclusion: Detection ofantibodies to Hcp100 has the potential to aid in the diagnosis ofhistoplasmosis, identifying cases that are falsely negative by ID. The ELISA developedherein can easily be adapted for use in-house in regional centers. Selectedpeptide epitopes will be useful in the development of more sensitive andspecific diagnostic tests.