Masked comparison of trypan blue stain and potassium hydroxide with calcofluor white stain in the microscopic examination of corneal scrapings for the diagnosis of microbial keratitis

Purpose: To evaluate the efficacy of trypan blue in direct microscopic examination of corneal scrapings in the diagnosis of non-viral microbial keratitis. Methods: In a prospective, interventional, masked study, 82 consecutive patients were investigated. Direct microscopic examination of the corneal scrapings involved three smears stained with potassium hydroxide with calcofluor white (KOH + CFW), Gram stain (not analyzed), and trypan blue stain and culture for bacteria, fungus, and Acanthamoeba. While KOH + CFW stained slides were examined under a fluorescence microscope, trypan blue-stained slides were examined by two microbiologists (masked to KOH + CFW and culture results) under normal light microscopy. Thirty samples were reexamined for interobserver and intraobserver variability. Results: Out of 82 samples, fungal/fungus-like elements were seen in 48 (58.5%) in KOH + CFW and 38 (46.3%) in trypan blue stain. One microsporidial case, detected in KOH + CFW was negative in trypan blue stain (culture not done). Fungal growth was positive in 23 out of 81 (28.3%) cases cultured, single bacterial species in 18 (22.2%), Pythium insidiosum in three, mixed bacteria and fungus/Pythium in 7 (8.6%), Acanthamoeba in none and 30 (37.0%) samples were sterile. With culture as gold standard, the respective sensitivity (84.9%, 75.7%) and specificity (90.9%, 68.2%) of KOH + CFW stain and trypan blue stain were comparable (p = 0.16, P = 0.06). The interobserver linear weighted kappa score between the two microbiologists was 1.00 while it was 0.86 for intraobserver agreement. Conclusion: Trypan blue stain, an easily available dye to ophthalmologists, is highly efficacious in the diagnosis of fungal keratitis.

Automated summary

Trypan blue stain is highly efficacious in the diagnosis of fungal keratitis, showing comparable sensitivity and specificity to traditional KOH + CFW stain in the diagnosis of non-viral microbial keratitis. The study also found good agreement between observers in interpreting trypan blue-stained samples.