An Optimized In-House Protocol for Cryptococcus neoformans DNA Extraction from Whole Blood: Comparison of Lysis Buffer and Ox-Bile Methods

Author: mycolabadmin
6/4/2025
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Summary

Researchers developed a simple, low-cost method to extract DNA from Cryptococcus neoformans fungal cells found in blood samples. The new method uses a homemade salt buffer solution instead of expensive commercial kits, making it accessible to laboratories in developing countries. Testing showed this method works better than an alternative ox-bile method, detecting very small numbers of fungal cells. This breakthrough could help doctors more quickly diagnose a serious fungal infection called cryptococcal meningitis in resource-limited settings.

Background

Cryptococcus neoformans is an encapsulated yeast that causes opportunistic infections in immunosuppressed individuals, particularly those with HIV/AIDS. Traditional diagnostic methods like culture and India ink staining of cerebrospinal fluid are invasive and have low sensitivity. Molecular techniques like PCR provide high sensitivity but rely on expensive commercial DNA extraction kits that limit affordability in low-resource settings.

Objective

This study aimed to develop a cost-effective, in-house method for extracting Cryptococcus neoformans DNA from whole blood. The objective was to compare an in-house lysis buffer method with an ox-bile method for red blood cell lysis and evaluate their effectiveness in DNA extraction quality, quantity, and limit of detection.

Results

The lysis buffer method yielded superior DNA quality (A260/A280 of 1.87-1.90) and higher quantity (up to 853.47 ng/µL) compared to ox-bile (A260/A280 of 1.46-1.85, up to 266.73 ng/µL). The lysis buffer method detected DNA from as low as 62 CFU/mL, while ox-bile detected an average of 284 CFU/mL. PCR primers were selective to C. neoformans genome with no amplification from other fungal or bacterial species tested.

Conclusion

The in-house lysis buffer method provides a cost-effective, high-yield alternative for C. neoformans DNA extraction from whole blood that outperforms ox-bile in both DNA quality and sensitivity. This method is suitable for molecular diagnostics in resource-limited settings and can complement existing cryptococcal antigen screening tests. Further validation with clinical samples is recommended.

Published in:Journal of Fungi,
Study Type:Comparative Laboratory Study,
Source: PMID: 40558942, DOI: 10.3390/jof11060430