Sensitivity and specificity of potassium hydroxide and calcofluor white stain to differentiate between fungal and Pythium filaments in corneal scrapings from patients of Pythium keratitis

PURPOSE: To assess the sensitivity of potassium hydroxide and calcofluor white (KOH+CFW) mount in the diagnosis of Pythium keratitis and concordance among microbiologists. METHODS: Three microbiologists evaluated the microscopic images of KOH + CFW mounts of confirmed cases of Pythium and fungal keratitis seen between January 2019 and February 2021. The filaments were compared using specific differentiating features. The sensitivity and specificity of KOH + CFW in diagnosing Pythium infection were evaluated along with concordance among the microbiologists. RESULTS: Sixty consecutive cases with confirmed growth of fungus or Pythium insidiosum (n = 29) were evaluated. The sensitivity of KOH + CFW in the correct identification of Pythium filaments ranged from 79.3% to 96.5% among three microbiologists. There was good interobserver (k = 0.76–0.90) and intraobserver (k = 0.70–0.97) agreements among three microbiologists. The differentiating findings (P < 0.0001) suggestive of Pythium filaments were the absence of septae in 23 (79.3%) and collapsed walls in 22 (75.9%) cases. CONCLUSION: KOH + CFW has good sensitivity and specificity in the diagnosis of Pythium keratitis with good interobserver and intraobserver concordance.