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Published on: July 9, 2014
Application of Real-Time PCR Assays for the Diagnosis of Histoplasmosis in Human FFPE Tissues Using Three Molecular
Luisa F López1, Ángela M Tobón2, Diego H Cáceres1,3
1Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas (CIB), Medellín 050034, Colombia.
Abstract:
Histoplasmosis is a fungal infection caused by the thermally dimorphic fungus Histoplasma capsulatum. This infection causes significant morbidity and mortality in people living with HIV/AIDS, especially in countries with limited resources. Currently used diagnostic tests rely on culture and serology but with some limitations. No molecular assays are commercially available and the results from different reports have been variable. We aimed to evaluate quantitative real-time PCR (qPCR) targeting three protein-coding genes of Histoplasma capsulatum (100-kDa, H and M antigens) for detection of this fungus in formalin-fixed paraffin-embedded (FFPE) samples from patients with proven histoplasmosis. The sensitivity of 100-kDa, H and M qPCR assays were 93.9%, 91% and 57%, respectively. The specificity of 100-kDa qPCR was 93% when compared against samples from patients with other mycoses and other infections, and 100% when samples from patients with non-infectious diseases were used as controls. Our findings demonstrate that real-time PCR assays targeting 100-kDa and H antigen showed the most reliable results and can be successfully used for diagnosing this mycosis when testing FFPE samples.
Insights
Quantitative real-time PCR (qPCR) offers a promising molecular diagnostic tool for histoplasmosis, a serious fungal infection. Assays targeting 100-kDa and H antigens in FFPE samples show high accuracy for detecting Histoplasma capsulatum.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Histoplasmosis, caused by Histoplasma capsulatum, is a significant opportunistic infection in HIV/AIDS patients, particularly in resource-limited settings.
- Current diagnostic methods like culture and serology have limitations, and no commercial molecular assays are available.
- Variable results from existing molecular detection methods necessitate reliable diagnostic tools.
Purpose of the Study:
- To evaluate the efficacy of quantitative real-time PCR (qPCR) for detecting Histoplasma capsulatum in formalin-fixed paraffin-embedded (FFPE) samples.
- To assess qPCR assays targeting three specific protein-coding genes (100-kDa, H, and M antigens) of Histoplasma capsulatum.
- To determine the sensitivity and specificity of these molecular targets for diagnosing histoplasmosis.
Main Methods:
- Utilized quantitative real-time PCR (qPCR) to analyze FFPE tissue samples from patients diagnosed with histoplasmosis.
- Targeted three protein-coding genes of Histoplasma capsulatum: 100-kDa antigen, H antigen, and M antigen.
- Evaluated assay performance by comparing results against clinical diagnoses and control groups including other mycoses, infections, and non-infectious diseases.
Main Results:
- The qPCR assays targeting the 100-kDa and H antigens demonstrated high sensitivity (93.9% and 91%, respectively).
- The M antigen assay showed lower sensitivity (57%).
- The 100-kDa qPCR assay exhibited high specificity (93% against other infections, 100% against non-infectious diseases).
Conclusions:
- Real-time PCR assays targeting the 100-kDa and H antigens are reliable for diagnosing histoplasmosis.
- These molecular assays can be effectively applied to FFPE samples for improved detection of Histoplasma capsulatum.
- qPCR represents a valuable advancement in the molecular diagnostics of histoplasmosis, especially in resource-limited areas.
