Diagnosis of Latent Histoplasmosis Using Interferon-γ Release Assays
Methods: Healthy volunteers from endemic areas with and without known latent histoplasmosis were recruited to provide blood as potential cases and controls. Peripheral blood mononuclear cells (PBMCs) were harvested, separated, counted and used in assays to measure IFNγ release after exposure to multiple Histoplasma antigen preparations, using ELISA and ELISpot, with phytohemagglutinin (PHA) as a positive control. Antigen-specific spot-forming units or concentration of IFNγ (pg/mL) were calculated for cells tested. One volunteer with known latent histoplasmosis had a very robust response and was used for optimization and reproducibility studies.
Results: Screening of volunteers identified PBMCs from 2 cases exhibiting robust responses to yeast cell lysate (YCL) from H. capsulatum strain G217B, with less response to YCL from a different strain and C-antigen. Assay optimization produced reproducible measurements of approximately 100 spot-forming units/105 cells with ELISpot and measureable quantities (25-50 pg/mL) of IFNγ released by ELISA. Positive responses were reproducible with repeated assay and differential blood draws. Controls with no known exposure or latent disease demonstrated no response to Histoplasma YCL.
Conclusion: This study provides proof of concept that latent histoplasmosis is detectable by measurement of INFγ expressed in and/or released from reactive T-cells after exposure to H. capsulatum antigens. Optimization of such assays could better enable development of IGRAs for diagnosis of latent infection, enabling therapeutic strategies to prevent reactivation of histoplasmosis in individuals with impending immune suppression.
R. Larue Jr.,
J. Nosanchuk, None
K. A. Marr, Astellas: Consultant and Scientific Advisor, Consulting fee
Merck: Consultant, Consulting fee
Pfizer: Consultant, Consulting fee
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