Use of different stains for microscopic evaluation of corneal scrapings for diagnosis of microsporidial keratitis

Abstract

Retrospective evaluation of potassium hydroxide plus calcofluor white (KOH+CFW), Gram, Giemsa, and modified Ziehl-Neelsen (1% H(2)SO(4), cold) stains for the detection of microsporidia in corneal scrapings from 30 patients showed KOH+CFW and acid-fast stains to be most efficient (29/30 [96.7%] and 28/30 [93.3%], respectively) in the diagnosis of microsporidial keratitis.

FIG. 1.

Microsporidal spores as observed under various stains on corneal scrapings. (a) KOH+CFW stain (magnification, ×1,000). Organisms were seen as bright turquoise to white oval bodies, often clustered in groups, against a relatively dark background. The spores displayed variable fluorescence intensities. Depending on the orientation of the microsporidia, the anterior end appeared concave. (b) Gram stain (magnification, ×1,000). Spores appeared ovoid and refractile and bright purple, resembling gram-positive organisms. The spores were scattered or highly clustered within the cytoplasm of occasional epithelial cells. Microsporidial spores show a dark staining belt girding them either diagonally or equatorially. (c) Giemsa stain (magnification, ×1,000). This stain is not taken up by the cell wall, and only the cytoplasm gets stained. The spores appear smaller than those in the other stains. There was also poor differentiation from other bacteria and debris. The darkly stained belt could be identified in 18/30 cases, aiding preliminary diagnosis. (d) Modified Ziehl-Neelsen stain (magnification, ×1,000). Except for two, all cases of microsporidial spores were acid fast (1% H₂SO₄). The acid-fast spores appeared bright red on a blue background, and a posterior vacuole and central diagonal strip within the spores were often visible. Bacteria and other cell debris appeared blue, owing to methylene blue counterstain.