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Duplex droplet digital PCR (ddPCR) improves the diagnosis of Aspergillus and Fusarium keratitis
Yamini Tawde1, Sourav Das1, Shreya Singh2
1Department of Medical Microbiology, Post Graduate Institute of Medical Education & Research (PGIMER), Chandigarh, India.
Abstract:
Fungal keratitis (FK) is a serious corneal infection leading to blindness. The present study aims to develop a duplex ddPCR for the detection of Aspergillus sp. and Fusarium sp. causing FK and also to enhance the diagnostic accuracy by evaluating the sensitivity of ddPCR in comparison to qPCR. This Prospective, multicentric study was conducted from November 2019 to August 2021, including three healthcare facilities across India. All patients suspected with FK were included in the study. A part of the collected corneal samples was used for routine microbiological workup and another part of the sample was used for the standardization and validation of ddPCR. A total of 42 corneal buttons were used for standardisation. Aspergillus primer concentration of 750nM showed highest sensitivity of 81.48 % with 93.33 % specificity at ct-value 14.05 while 900 nM primer concentration was best for Fusarium with 80 % sensitivity and 96.3 % specificity at ct-value 83.65. Probe concentrations for both Aspergillus and Fusarium were 250 mM. Validation cohort included 74 corneal samples, Aspergillus ddPCR demonstrated 84.21 % sensitivity with 94.64 % specificity while Fusarium ddPCR had 75 % sensitivity with a specificity of 96.3 %. The comparative analysis demonstrated an improvement in diagnostic performance by ddPCR for both Aspergillus and Fusarium compared to recently reported genus-specific real-time PCR. The ddPCR noticeably enhanced the diagnostic performance for both Aspergillus and Fusarium FK in comparison to the genus-specific real-time PCR. Thus, ddPCR can serve as a rapid technique holding a challenge over conventional and qPCR for FK diagnosis.
Insights
A new duplex digital droplet PCR (ddPCR) method accurately detects Aspergillus and Fusarium fungal keratitis (FK). This advanced technique offers improved sensitivity and specificity over traditional qPCR, aiding faster FK diagnosis and preventing blindness.
Area of Science:
- Ophthalmology
- Molecular Biology
- Mycology
Background:
- Fungal keratitis (FK) is a severe corneal infection that can lead to irreversible blindness.
- Accurate and rapid diagnosis of FK is crucial for effective treatment and visual outcome.
- Current diagnostic methods, including culture and qPCR, have limitations in sensitivity and speed.
Purpose of the Study:
- To develop and validate a duplex digital droplet PCR (ddPCR) assay for simultaneous detection of Aspergillus and Fusarium species in corneal samples.
- To compare the diagnostic performance (sensitivity and specificity) of the developed ddPCR assay against conventional methods and quantitative PCR (qPCR).
- To assess the potential of ddPCR as a rapid and accurate diagnostic tool for fungal keratitis.
Main Methods:
- A prospective, multicentric study involving patients suspected of FK across three Indian healthcare facilities.
- Standardization and validation of a duplex ddPCR assay using corneal button samples (n=42 for standardization, n=74 for validation).
- Optimization of primer and probe concentrations for Aspergillus and Fusarium detection via ddPCR, followed by comparative analysis with genus-specific real-time PCR.
Main Results:
- Optimized ddPCR assay demonstrated high sensitivity and specificity for both Aspergillus (84.21% sensitivity, 94.64% specificity) and Fusarium (75% sensitivity, 96.3% specificity) in the validation cohort.
- The ddPCR assay showed improved diagnostic performance compared to genus-specific real-time PCR for both fungal species.
- Specific primer and probe concentrations were determined for optimal detection of Aspergillus and Fusarium.
Conclusions:
- The developed duplex ddPCR assay is a sensitive and specific tool for the rapid detection of Aspergillus and Fusarium in fungal keratitis.
- ddPCR offers enhanced diagnostic accuracy and speed compared to conventional microbiological workup and qPCR.
- This technique holds significant promise for improving the clinical management and outcomes of fungal keratitis patients.
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