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Rapid detection of fungal keratitis with DNA-stabilizing FTA filter paper
Nardine Menassa1, Philipp P Bosshard, Claude Kaufmann
1Department of Ophthalmology, Cantonal Hospital Lucerne, Lucerne, Switzerland.
Abstract:
Purpose. Polymerase chain reaction (PCR) is increasingly important for the rapid detection of fungal keratitis. However, techniques of specimen collection and DNA extraction before PCR may interfere with test sensitivity. The purpose of this study was to investigate the use of DNA-stabilizing FTA filter paper (Indicating FTA filter paper; Whatman International, Ltd., Maidstone, UK) for specimen collection without DNA extraction in a single-step, nonnested PCR for fungal keratitis. Methods. Specimens were collected from ocular surfaces with FTA filter discs, which automatically lyse collected cells and stabilize nucleic acids. Filter discs were directly used in single-step PCR reactions to detect fungal DNA. Test sensitivity was evaluated with serial dilutions of Candida albicans, Fusarium oxysporum, and Aspergillus fumigatus cultures. Test specificity was analyzed by comparing 196 and 155 healthy individuals from Switzerland and Egypt, respectively, with 15 patients with a diagnosis of microbial keratitis. Results. PCR with filter discs detected 3 C. albicans, 25 F. oxysporum, and 125 A. fumigatus organisms. In healthy volunteers, fungal PCR was positive in 1.0% and 8.4% of eyes from Switzerland and Egypt, respectively. Fungal PCR remained negative in 10 cases of culture-proven bacterial keratitis, became positive in 4 cases of fungal keratitis, but missed 1 case of culture-proven A. fumigatus keratitis. Conclusions. FTA filter paper for specimen collection together with direct PCR is a promising method of detecting fungal keratitis. The analytical sensitivity is high without the need for a semi-nested or nested second PCR, the clinical specificity is 91.7% to 99.0%, and the method is rapid and inexpensive.
Insights
FTA filter paper simplifies fungal keratitis detection using polymerase chain reaction (PCR). This method bypasses DNA extraction, offering a rapid, sensitive, and cost-effective diagnostic approach for eye infections.
Area of Science:
- Ophthalmology
- Mycology
- Molecular Biology
Background:
- Polymerase chain reaction (PCR) is vital for rapid fungal keratitis diagnosis.
- Current methods involving specimen collection and DNA extraction can reduce PCR sensitivity.
Purpose of the Study:
- To evaluate DNA-stabilizing FTA filter paper for direct specimen collection and single-step PCR in fungal keratitis detection.
- To assess the sensitivity and specificity of this streamlined PCR method.
Main Methods:
- Ocular surface specimens were collected using FTA filter discs, which lyse cells and stabilize DNA.
- Filter discs were directly used in single-step, non-nested PCR assays.
- Sensitivity was tested with serial dilutions of fungal cultures; specificity was assessed in healthy individuals and patients with microbial keratitis.
Main Results:
- Direct PCR with FTA filter paper demonstrated high analytical sensitivity for detecting Candida albicans, Fusarium oxysporum, and Aspergillus fumigatus.
- Clinical specificity ranged from 91.7% to 99.0% in healthy volunteers and patients with bacterial keratitis.
- The method correctly identified fungal keratitis in 4 out of 5 cases, missing one case.
Conclusions:
- FTA filter paper combined with direct PCR is a promising, rapid, and inexpensive technique for diagnosing fungal keratitis.
- This approach eliminates the need for separate DNA extraction and nested PCR steps, enhancing efficiency.
