Diagnosis of progressive disseminated histoplasmosis using the novel recombinant antigen Hcp100.

TOSCANINIA MA; RODRIGUEZ LABOCCETTA C; VIDELA GARRIDO A; POSSE G; IOVANNITTI C; CAPECE P; FROLA C; GUELFAND L; VALDEZ RM; CHACON YA; NUSBLAT AD; CUESTAS ML

Congreso; 19TH INFOCUS.; 2021

Objectives: The diagnosis of progressive disseminated histoplasmosis (PDH), a life-threatening disease particularly for patients living with HIV, is still challenging in many countries where this disease is highly endemic. Definitive diagnosis is established by culture and/or visualization of the yeast cells by cytology or histopathology but both procedures have limited sensitivity and cultures are time-consuming. Antibodies (Ab) detection by immunodiffusion (ID) is often used but its sensitivity is low in the immunocompromised host. In addition, the standard serological reagent, the histoplasmin, is not ideal due to the presence of cross-reactive carbohydrates and for its problematic and time-consuming production. The only commercially available antigen (Ag) detection assay has high sensitivity in PDH cases; however, it is expensive and only performed in few laboratories. This study describes the potential use of the recombinant antigen, the 100kDa protein of Histoplasma capsulatum (Hcp100), for the development of novel Ab and Ag detection methods (ELISA and dotblot, respectively) for the diagnosis of PDH. Methods: Serum and urine samples from 29 patients with HIV/AIDS and PDH confirmed by culture or histopathology/cytology were studied for the detection of Ab and Ag respectively. To evaluate cross-reactivity, specimens from patients with other mycoses were studied. Specimens from 20 healthy individuals were used as negative controls. For Ab detection, ELISA plates were coated with 0.5 µg of recombinant Hcp100 per well. After blocking, serum samples were added at a dilution of 1:1000. Goat anti-human IgG horseradish peroxidase conjugate was used as the secondary antibody. The reaction was developed with OPD, terminated with H2SO4 followed by absorbance measurement at 492nm. The assay was compared with the immunodiffusion and an in-house ELISA using commercially histoplasmin (IMMY, USA) as reagent.For Ag detection, 1 µl of urine samples was spotted onto a nitrocellulose membrane. After blocking, mouse anti-Hcp100 IgG was used as the primary antibody and goat anti-mouse IgG horseradish peroxidase conjugate was used as the secondary antibody. Dots were visualized using ECL and densities were quantified by densitometry. The assay was compared with the Clarus Histoplasma GM ELISA (IMMY, USA).Sensitivity, specificity and percentage of cross-reactions were estimated for each assay. Results: Sensitivity and specificity of the Hcp100 Ab ELISA were higher than the histoplasmin Ab ELISA (Se: 82% vs 73% and Sp: 96% vs 95%). ID showed very low sensitivity (22.7%) and high specificity (100%). The Hcp100 dotblot showed higher sensitivity than the Clarus Histoplasma GM ELISA (95% v 86%), but lower specificity (86% vs 100%). Cross-reactivity was noted with other endemic mycoses in both assays.Conclusion: Recombinant Hcp100 appears to be a promising antigen to be used in antibody and antigen testing showing similar performance to commercial methods. The use of Hcp100 could be helpful in regional centers, particularly in those laboratories that are unable to acquire the only commercially available test for Histoplasma antigenuria. The selection of a specific region of the Hcp100 could improve the sensitivity and specificity of the diagnostic tests.