Histopathological evaluation of ocular microsporidiosis by different stains

Abstract

Background: There is limited data on comparing stains in the detection of microsporidia in corneal biopsies. Hence we wanted to evaluate various stains for their ability to detect microsporidia in corneal tissue sections.

Methods: Four cases diagnosed with microsporidiosis on Hematoxylin and Eosin and Periodic Acid Schiff's stained sections of the corneal button between January 2002 and December 2004, were included. Further sections were prospectively stained with calcofluor white, Gram, Giemsa, Masson's trichrome, acridine orange, Gomori's methenamine silver, Gram's chromotrope and modified acid fast stain. The stained sections were analyzed for the spore characteristics in terms of size, shape, color contrast, cell wall morphology, waist band in cytoplasm and ease of detection.

Results: All sections showed microsporidial spores as 3-5 microm, oval bodies. 1% acid fast, Gram's chromotrope and GMS stains provided a reliable diagnosis of microsporidia as diagnostic waist band could be identified and good contrast helped distinguish the spores from inflammatory debris.

Conclusion: Considering the ease of performance, cost effectiveness and rapidity of the technique, 1% acid fast stain and Gram's chromotrope stain are ideal for the detection of microsporidia.

Figure 1

Section of the corneal tissue shows epithelial ulceration, with inflammatory infiltrates in the anterior two-thirds of stroma (hematoxylin & eosin, × 1100) (b) Higher magnification shows polymorphonuclear cells (asterix) and faintly stained, ill-defined oval dot like structures (arrow) between the corneal lamellae (Hematoxylin and Eosin stain, × 400)

Figure 2

(a) Under higher magnification, the microsporidal spores are seen as pink oval structures (H & E stain, × 1000); (b) magenta pink oval structures in PAS stain (× 500), (c) deep blue oval structures with dark tip (arrow) in some spores Giemsa stain (× 500), (d) well defined brown oval spores with dark tip or band in Gomoris methenamine silver stain (× 500)

Figure 3

Microsporidal spores are seen as white fluorescent ring like structures in Calcofluor white stain (× 500), (b) dull oval orangish structures in Acridine orange stain (× 500), (c) oval well defined spores with a faint hollow around the spore in Gram stain (× 1000), (d) dark blue uniformely stained bodies in Masson's trichrome stain (× 500)

Figure 4

(a) Microsporidal spores are seen as well defined oval reddish bodies with a dark staining of the narrow end of the spore (black) or a waistband (white) closer to the tip of narrow end. Also seen are the unstained blue spores which possibly are immature or degenerating spores (1% acid fast stain, × 1000).(b) The spores are well delineated as purplish pink egg-shaped spores with a darker staining of the tip (white arrow). Even the degenerating spores show the darkly staining tip (black arrow) Gram's chromotrope stain (× 1000)