Duplex droplet digital PCR (ddPCR) improves the diagnosis of Aspergillus and Fusarium keratitis.

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.