The goal of the present work was to develop a novel reagent with potential for histoplasmosis diagnosis. For this purpose, the genetic sequence of the 100 kDa protein of Histoplasma capsulatum (Hcp100) was cloned and expressed as a secretory protein in Pichia pastoris. After optimizing the culture conditions and purifying by immobilized metal ion affinity chromatography, the highest yield of Hcp100 reached approximately 1.3 mg/l with > 90% purity in shake flasks using basal salt medium supplemented with casamino acids after 72 h of methanol induction. To investigate its potential for diagnosis, its detection in urine samples using specific polyclonal antibodies as reagent was evaluated by dot blot in 6 patients with progressive disseminated histoplasmosis (PDH), of whom all had AIDS. Antigen was detected in urine from all 6 (100%) PDH patients. Urine samples from a pool of 20 healthy individuals did not react with the anti-Hcp100 antibodies. The dot blot assay performed in this study provides preliminary data of a simple technology that can be performed in medical institutions with limited resources to facilitate the rapid diagnosis of histoplasmosis, particularly the disseminated forms. Hence, use of these assays may provide a rapid diagnostic tool of PDH in endemic areas for histoplasmosis where PDH-related mortality is high, hastening treatment and improving patient survival. Finally, this novel antigen and its specific antibodies may provide an alternative diagnostic reagent to the largely unknown and poorly characterized polysaccharide antigens (HPA, galactomannan, histoplasmin) frequently used in the diagnostic tests.
Few antigens are used as laboratory tools for the immunodiagnosis of histoplasmosis.
P. pastoris was an excellent system for recombinant Hcp100 expression.
Maximum expression levels of rHcp100 were achieved in BSM with 1% casamino acids.
Dot blot assays with anti-rHcp100 antisera can be successfully used for diagnosing PHD.
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This work was supported by the University of Buenos Aires (Grant UBACyT 20020130200222BA). DGM, ADN, and MLC are staff members of CONICET. MAT thanks the doctoral scholarship to CONICET.
This study was approved by the institutional review committee “Dr Vicente Federico Del Giúdice” at Hospital Nacional Alejandro Posadas, Buenos Aires, Argentina (Ref. 260 EMnP0S0/19). Informed consents were obtained from all participants. The procedures involving animals followed the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Comité Institucional para el Cuidado y Uso de Animales de Laboratorio (CICUAL-FFyB, Protocol number 1018-19).
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Toscanini, M.A., Maglio, D.G., Capece, P. et al. Histoplasma capsulatum 100-kilodalton antigen: recombinant production, characterization, and evaluation of its possible application in the diagnosis of histoplasmosis. Appl Microbiol Biotechnol 104, 5861–5872 (2020). https://doi.org/10.1007/s00253-020-10570-7
- Histoplasma capsulatum
- Recombinant Hcp100
- Pichia pastoris
- Antibodies anti-rHcp100
- Casamino acids