Trypan blue staining for fungal keratitis diagnosis: a comparative study in rural India

Fungal keratitis is a significant public health concern in rural areas, where access to advanced healthcare facilities is limited. This study aims to evaluate and compare the sensitivity and specificity of trypan blue (0.06%) with 10% potassium hydroxide (KOH) and Gram stain in the diagnosis of fungal keratitis in a resource-poor setting.

Single-center, unmasked, comparative, cross-sectional study at a rural secondary center in south India from April to September 2023. All patients with clinically diagnosed microbial keratitis who presented to the clinic were enrolled, and the corneal scrapings from the infiltrate were collected on three slides and stained with Gram stain, 10% KOH, and 0.06% Trypan blue in a random sequence, and assessed for the presence of fungal filaments. Cases showing fungal filaments on any one of the 3 slides, were enrolled in the study, while the rest were excluded.

A total 86 /108 patients with fungal keratitis were enrolled. The rate of detection of fungal filaments was 96.5% with Gram stain, 83.7% with 10% KOH, and 85.9% with Trypan Blue. Sensitivity was found to be 88%, specificity 66.67%, positive likelihood ratio 2.64 (95% C.I 0.53,13) and negative likelihood ratio was 0.18 (95% C.I. 0.07,0.49) for Trypan blue. The concordance between Trypan Blue and KOH stain was 77.3% (Cohen's kappa = 0.17), and between Trypan Blue and Gram stain was 87.21% (Cohen's kappa = 0.22).

The Trypan blue staining technique seems to provide a reliable, easy to learn alternative for the detection of fungal filaments in the diagnosis of fungal keratitis, particularly in resource-poor settings.