Optimization of the immunodiffusion assay using antigens obtained from native Paracoccidioides spp. isolates

CORREDOR L; ROJAS F; MUSSIN J; GARCÍA EFFRON G; GIUSIANO G

Foz do Iguazu

Congreso; 21st INFOCUS - Latin American Forum on Fungal Infections in Clinical Practice; 2023

Sociedade Brasileira de Infectologia

The aim was to obtain antigenic preparations of native isolates of Paracoccidioides spp. and to evaluate their performance in the double immunodiffusion assay against positive and negative sera tested with the reference antigen from patients with confirmed paracoccidioidomycosis (PCM).MethodsThe antigens were obtained from strains of Paracoccidioides spp. following protocols described by Fernandes (2023), with modifications. The isolates are deposited in the culture collection of the Mycology Department of the Instituto de Medicina Regional, Universidad Nacional del Nordeste, Argentina. Antigens were obtained after 7 days of culture at 35°C and concentrated by lyophilization for 24 hours. Double immunodiffusion (ID) was used to evaluate antigenic activity against 17 patient sera with microbiologically confirmed PCM, which showed no positive reaction with the reference antigen (RA) (Pb brasiliensis 339), and against 5 reactive sera. Similarly, the obtained antigens were evaluated against the reference polyclonal antiserum (RAb). The reaction between RAb and RA was used as positive control. To evaluate specificity, the antigens obtained were tested against sera from patients with histoplasmosis and aspergillosis.ResultsWe tested 8 antigenic preparations obtained from different native isolates. All were able to react with the 5 positive sera. Of the 17 false negative sera, 5 (30%) were positive with at least one of the antigens obtained. Reactivity of the antigens was variable against the different sera tested. Certain antigens were positive against a larger number of sera. No cross-reactivity was observed with sera from patients with histoplasmosis and aspergillosis against any of the antigens.ConclusionsNowadays, a new genotypic diversity of the genus Paracoccidioides is known. It is likely that serologic tests may yield false-negative results if the disease is caused by a different species or genotype than the RA used for the test. Currently, a new genotypic diversity of the genus Paracoccidioides is accepted. It is known that serological tests can give false-negative results when the disease is caused by a different species or genotype. This could be because RA used for serological testing, is obtained from a non-native strain of Paracoccidioides (Pb brasiliensis 339) that originated in Colombia. In Argentina, ID has a false-negative rate of 17% in patients with proven PCM. The results of this study showed that the use of native antigen increased the number of positive sera, improving the sensitivity of the immunodiffusion technique, which is sometimes the only diagnostic tool available. The specificity of the antigen preparations was validated, as no cross-reactivity with sera from patients with other mycoses was observed. The fact that not all sera reacted to all antigens indicates the need to use a pool to increase the sensitivity of the test.