Development of single‐tube real‐time PCR assay for the rapid detection of Aspergillus and Fusarium—The two most common causative agents in fungal keratitis.

Background: To compare the performance of conventional, semi‐nested and real‐time panfungal ITS PCRs for diagnosing fungal keratitis (FK) and develop genus‐specific real‐time PCR for the most common aetiology of FK. Methods: This multicentric study includes 232 corneal samples from suspected FK patients from four centres across India between November 2019 through August 2021. A total of 87 corneal buttons were included for the comparison of conventional, semi‐nested and real‐time ITS PCRs, of which 68 were from confirmed FK patients. Of these 87 samples, 44 (microscopy and culture positive for Aspergillus sp. and/or Fusarium sp.) were used for the standardisation of genus‐specific real‐time primers/probes. Subsequently, the best method showing highest sensitivity and specificity was validated in 188 samples. Results: On Bayesian comparison, conventional ITS2 PCR showed best performance (sensitivity and specificity of 55.88% and 100%, respectively). Since, real‐time ITS2 PCR was also considerably efficient (sensitivity and specificity of 51.47% and 84.21%, respectively) in comparison with the conventional PCR but faster, cost‐effective, and less labor‐intensive, ITS‐2 real‐time PCR is a suitable method that can be applied along with culture and microscopy. During validation, real‐time PCR with genus‐specific primers showed 61.76% and 91.18% sensitivity with specificity of 98.05% and 79.22%, respectively, for Aspergillus sp. and Fusarium sp. Aspergillus probe, Fusarium probe and duplex PCR showed sensitivity of 52.94%, 50% and 54.41% with specificity of 92.86%, 82.47% and 75%, respectively. No cross‐reactivity of genus‐specific PCRs was observed during standardisation. Conclusions: ITS‐2 real‐time PCR can be applied as an adjunct with conventional methods for the diagnosis of FK. The genus‐specific duplex real‐time PCRs are rapid which reduces the turnaround time (TAT) avoiding the need for sequencing. [ABSTRACT FROM AUTHOR]