Objective: Comparing the nested polymerase chain reaction (PCR) technique with the standard diagnostic culture method for Pythium insidiosum.

Material and Method: Eighty-three patients with suspected fungal keratitis were enrolled in this observational, crosssectional study from the Faculty of Medicine Siriraj Hospital between February 2011 and February 2014. Patient symptoms, associated diseases, duration of ulcers, precipitating causes, best-corrected visual acuity, intraocular pressure, and other clinical findings were recorded. Corneal scrapings were taken for Gram staining, bacterial and fungal cultures, staining with potassium hydroxide preparation, and DNA extraction for nested PCR. The sensitivity, specificity, accuracy, and agreement of the nested PCR analysis and culture diagnosis of P. insidiosum were compared.

Results: Five patients had a positive result for nested PCR amplification of P. insidiosum, while only one of these was also positive for culture growth of Pythium. Nested PCR sensitivity was 50% (95% confidence interval [95% CI] 1.3-98.7), specificity 94.7% (95% CI 86.9-98.5), and accuracy 93.5% (95% CI 85.7-97.2) with a fair agreement (kappa 0.258, p = 0.011).

Conclusion: Therefore, nested PCR may be an appropriate test for P. insidiosum in diagnosing Pythium keratitis with high accuracy, despite small amounts of corneal specimen.

Keywords: Accuracy, Nested PCR, Pythium insidiosum keratitis, Sensitivity, Specificity