Diagnosis of histoplasmosis with a novel ELISA based on detection of antigen M in urine samples

ABUD, J.E.; CABEZA, MATÍAS; LOAIZA-OLIVA M; GARCÍA-EFFRON, G; RODRÍGUEZ HORACIO ADOLFO

Santiago del Estero

Congreso; 2nd International Meeting on Endemic Mycoses of the Americas (IMEMA) and the 1st International Symposium on Implantation Mycoses (ISIM); 2023

IMEMA

The dimorphic fungus Histoplasma capsulatum (H. capsulatum), the etiological agent of the disease known as histoplasmosis, is distributed worldwide. It is highly endemic in Latin America and represents the most frequent endemic mycosis in Argentina. The methods currently employed for the diagnosis of histoplasmosis are very limited in terms of specificity and sensitivity. Additionally, a few antigen detection kits are available in our country and they are all based on the detection of membrane polysaccharides, which exhibited cross-reactivity with other related fungi.The presence of the glycoprotein M has been demonstrated in both mycelial and yeast forms, and it has also been postulated that it is released from dead cells during infection. Immunoassays based on the detection of the protein M of H. capsulatum hold potential for the rapid and accurate diagnosis of histoplasmosis. Thus, our primary objective is to develop a specific ELISA for diagnostic of histoplasmosis targeting the glycoprotein M of H. capsulatum instead of polysaccharides.Based on the immunogenic characteristics of the glycoprotein M, recombinant antigens of selected immunogenic domains were generated in Escherichia coli. Subsequently, a panel of monoclonal antibodies (MAbs) was obtained and their specificity was evaluated against the recombinant proteins used as antigens, histoplasmin, and antigens of Paracoccidioides brasiliensis (PCM) by ELISA and Western blotting. Finally, a prototype anti-M sandwich ELISA (EIA anti-M) was established, and functional parameters including specificity, detection limit (0.4 ng/ml), linear range (0.15 µg/ml – 0.01 µg/ml), and calibration sensitivity were determined (Figure 1). ELISA performance was established with 55with 55 urine samples. The majority of these 55 samples were obtainedcame from transplant patients (n=46), whereas a fewer amount were was obtained from HIV (n=6) or leukemia patients with Aspergillosis (n=3). Out of all these samples, only 2 were considered confirmed as positive by the antigen detection kit Clarus Histoplasma GM EIA/IMMY. The ELISA developed in this study demonstrated a correlation of XXX% for positive samples and XXX% for negative samples. Therefore, our results supported the high potential of this new ELISA to be adapted to a point-of-care format such as lateral flow immunochromatography.