In a new study Tanmay N Gokhale, Joveeta Joseph and colleagues explore if a blue dye routinely used in cataract surgeries could also be used to diagnose fungal keratitis in low resource settings.
About a third of all eye infections in rural areas are caused by fungi, leading to a condition called fungal keratitis. It is a sight-threatening condition that affects the cornea, the clear, dome-shaped outer layer of the eye. Fungal keratitis is difficult to differentiate from the more common cause of eye infections: bacteria. These infections are common in agricultural workers who are susceptible to injuries from leaves, twigs, or other vegetative matter. Rural hospitals are not equipped to identify the active organism, and so doctors end up prescribing antibiotics as the first line of treatment. Antibiotics are powerful tools against bacteria, but have no effect on fungi, prolonging and exacerbating the infection. Therefore, an early and accurate diagnosis is important to develop a treatment plan to preserve vision. In low resource settings like rural hospitals, clinicians turn to staining techniques to identify pathogens.
Staining involves dyeing the otherwise transparent structures of biological specimens to make them visible under the microscope. Stains like Gram stain and potassium hydroxide (KOH) are some commonly used stains each with their own strength and limitations. Gram staining has been the ‘gold standard’ in low-resource settings, but it requires reagents that are expensive and need frequent replacement. KOH is simpler to perform, but it produces low contrast, making fungal filaments difficult to recognize without expertise. Trypan blue--the dye often used to visualize the lens capsule during cataract surgery--can also stain fungal filaments blue which appear as distinct branching structures under the microscope. But it is less commonly used as a diagnostic stain, despite it being an easy and comparable alternative: it is a single step process, has a better contrast than KOH and is readily available everywhere cataract surgeries are performed. Its functionality as a diagnostic tool in rural clinical settings, however, remains to be established.
In a new study published in International Ophthalmology, researchers from LVPEI compare the diagnostic accuracy of gram stain, 10% KOH, and 0.06% Trypan blue for detecting fungal keratitis. Among 108 patients with microbial keratitis, 86 (53 men) confirmed fungal keratitis cases were included in the analysis. Of the 86 cases, gram stain detected 83 (96.5%) fungal keratitis cases, while KOH and Trypan blue detected 71 (83.7%) and 73 (85.9%) cases, respectively. Trypan blue also showed higher specificity (66.7%) than KOH (33.3%), meaning it was better at ruling out non-fungal cases, reducing the chance of misdiagnosis.
There was no significant statistical difference between KOH and Trypan blue when it came to accurately identifying the samples with fungal infection (sensitivity). The findings suggest that both KOH and Trypan blue can be used as alternatives to gram staining. But Trypan blue’s better contrast, single step use, and ready availability in eye care settings due to its routine use in cataract surgery make it a useful diagnostic tool where resources are scarce.
‘Fungal eye infections are highly time sensitive. Any delay in diagnosis can cost vision, making early, on-site detection crucial in resource limited settings,' notes Dr. Tanmay Gokhale, ophthalmologist at LVPEI and first author of the study.
Citation
Gokhale TN, Joseph J, Rathi VM, Sharma S, Bagga B. Trypan blue staining for fungal keratitis diagnosis: a comparative study in rural India. Int Ophthalmol. 2025 Oct 3;45(1):403. doi: 10.1007/s10792-025-03749-2. PMID: 41042281.
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