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Use of different stains for microscopic evaluation for the diagnosis of Pythium keratitis
Sonali Sahoo1, Sanchita Mitra2, Ruchi Mittal3
1Cornea & Anterior Segment Service, L V Prasad Eye Institute, Bhubaneswar, Odisha, 751024, India.
Purpose:
To evaluate the efficacy of various staining techniques for detection of Pythium in keratitis cases.
Methods:
Data of nineteen consecutive culture-positive cases of Pythium keratitis were retrospectively analysed. Corneal scrapings and corneal buttons (in the cases which underwent therapeutic penetrating keratoplasty [TPK]) were sent for microbiological and histopathological examination. The direct smears were stained with Potassium hydroxide and calcofluor white (KOH + CFW), Gram and Iodine-Potassium Iodide-Sulphuric Acid (IKI-H2SO4) stains. The corneal buttons were stained with Gomori's Methanamine Silver (GMS), Periodic Acid-Schiff (PAS) and Iodine-Potassium Iodide-Sulphuric Acid (IKI-H2SO4) stains. The positivity of various stains in detecting Pythium was studied.
Results:
Gram and KOH + CFW staining from smear was done in 16 out of 19 (84.2%) cases. KOH + CFW and Gram stains were suggestive of Pythium in 10 (62.5%) and 7 (43.8%) cases, respectively. IKI-H2SO4 staining in scraping samples was positive for Pythium in all the 4 (100%) cases in which it was performed. Half corneal buttons were positive for Pythium with IKI-H2SO4 stain as well as GMS stain in all the 18 cases that underwent TPK (100%). PAS stain showed weak to faint pink staining of Pythium filaments in 7 out of 18 cases (38.9%).
Conclusion:
IKI-H2SO4 stain followed by KOH + CFW stain detects Pythium filaments most accurately in corneal scraping samples from keratitis patients, although the differences were not statistically significant. The positivity of the stains depends on astute observation by an experienced ocular microbiologist and pathologist.
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