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Comparison of staining methods and a nested PCR assay to detect Histoplasma capsulatum in tissue sections
Ralf Bialek1, Friederike Ernst, Klaus Dietz
1Institute for Tropical Medicine, University Hospital Tübingen, Germany.
Abstract:
To optimize diagnosis of histoplasmosis in tissue sections, 30 spleen specimens from mice, experimentally infected with Histoplasma capsulatum, were examined by H&E, Grocott stain, anti-bacille Calmette-Guerin antibody immunostain, Fungiqual A fluorochrome stain (Drs Reinehr and Rembold, Kandern, Germany), and a nested polymerase chain reaction (PCR) assay. Results were compared with the tissue burden determined by quantitative culture. By applying logistic regression, the nested PCR assay was the most sensitive method, but not significantly more sensitive than the Grocott stain. The 50% quantile to achieve a positive result was determined to be 3 colony-forming units per milligram of spleen tissue for the PCR assay, 11 for the Grocott stain, 27 for the fluorochrome stain, 190 for immunostaining, and 533 for the H&E stain. The Grocott and fluorochrome stains did not differ significantly in detecting fungal elements. The PCR assay unambiguously identified H. capsulatum in tissue sections.
Insights
The nested polymerase chain reaction (PCR) assay offers the most sensitive method for diagnosing histoplasmosis in tissue samples. However, the Grocott stain also provides high sensitivity for detecting Histoplasma capsulatum.
Area of Science:
- Medical Mycology
- Diagnostic Pathology
- Molecular Diagnostics
Background:
- Histoplasmosis diagnosis in tissue sections can be challenging.
- Optimizing diagnostic methods is crucial for timely treatment.
- Histoplasma capsulatum is the causative agent of histoplasmosis.
Purpose of the Study:
- To compare the diagnostic sensitivity of various methods for Histoplasma capsulatum in mouse spleen tissue.
- To determine the limit of detection for each diagnostic assay.
Main Methods:
- Examination of 30 experimentally infected mouse spleen specimens.
- Utilized Hematoxylin and Eosin (H&E) stain, Grocott stain, anti-bacille Calmette-Guerin antibody immunostain, Fungiqual A fluorochrome stain, and nested polymerase chain reaction (PCR) assay.
- Compared results with quantitative culture for tissue burden determination.
Main Results:
- The nested PCR assay demonstrated the highest sensitivity, with a 50% positive result at 3 colony-forming units (CFU)/mg spleen tissue.
- Grocott stain showed comparable sensitivity to PCR, with a 50% positive result at 11 CFU/mg.
- Other methods like fluorochrome stain, immunostaining, and H&E stain had lower sensitivity and higher detection limits.
Conclusions:
- Nested PCR is the most sensitive diagnostic assay for Histoplasma capsulatum in tissue sections.
- Grocott stain remains a highly effective and sensitive method for histoplasmosis diagnosis.
- The study provides quantitative data on the detection limits of various diagnostic techniques.