Relevant criteria for detecting microsporidia in stool specimens

Abstract

By using different staining techniques, 479 stool specimens from 212 diarrheic patients with AIDS were examined for microsporidian spores. Calcofluor fluorescence staining of 119 specimens revealed fluorescent ovoid structures of microsporidian size. Staining of these samples according to the method of Weber et al. (R. Weber, R. T. Bryan, R. L. Owen, C. M. Wilcox, L. Gorelkin, and G. S. Visvesvara, N. Engl. J. Med. 326:161-166, 1992) with trichrome produced six specimens with pinkish spores containing the characteristic microsporidian belt-like structure. The 6 specimens were processed for transmission electron microscopy, as were another 21 specimens which did not present the belt-like structure after trichrome staining but which looked highly suspicious after fluorescence staining. In these 21 samples, only fungal spores and, particularly, bacterial Clostridium spores were demonstrated, whereas in the 6 samples diagnosed positive after trichrome staining, the existence of microsporidia could be verified by electron microscopy. Based on our observations, we propose that the belt-like structure seen with the Weber stains in microsporidian spores corresponds to structures existing in priming-stage spores. The results suggest that routine microscopical fecal diagnosis for microsporidian infection should include a screening by fluorescence staining and, subsequently, a confirmatory viewing of fluorescence-positive samples after trichrome staining.

FIG. 1-4

Light micrograph of ovoid greenish-white spores (★) in a Calcofluor-stained smear of a human stool sample. Bar, 6 μm. Magnification, ×1,780. Fig. 2 Light micrograph of greenish-white spores outside (➩) and inside (➞) red-stained bacteria in a Calcofluor-stained smear of a human stool sample. Bar, 6 μm. Magnification, ×1,780. Fig. 3 Light micrograph of ovoid, greenish-white spores (★) and smaller yellow-orange spores (➞) in a Calcofluor-stained smear of a human stool sample. Bar, 6 μm. Magnification, ×1,780. Fig. 4 Smear of a human stool sample stained by Weber’s modified trichrome staining containing unstained, empty-looking spores (➩), as well as pink spores showing a belt-like structure with different internal localizations: equatorially (→) and diagonally (➞). Bar, 6 μm. Magnification, ×1,780.

FIG. 5

Electron microscopy cross section of a spore appearing greenish-white after Calcofluor staining as shown in Fig. 1. The spore wall consists of two distinct layers: an electron-dense layer (➩) and an electron-lucent one (➞). The spore cytoplasm contains randomly and densely packed ribosomes (asterisk). Bar, 0.5 μm. Magnification, ×60,000.

FIG. 6

Electron micrograph from a stool sample as shown by fluorescence microscopy in Fig. 2. The spores with a bilayered wall and electron-dense cytoplasm are situated in the interior of the bacteria (B). The characteristic binary fission of the bacteria can be seen (➞). Bar, 3 μm. Magnification, ×7,300.

FIG. 7

Electron micrograph of microsporidian spores from human stool samples as shown by light microscopy in Fig. 3 and 4. The characteristic polar tube can be seen in cross section (★). The bilayered spore wall consists of an outer electron-dense layer (exospore) (➩) and an inner electron-lucent layer (endospore) (➞). The spore cut in an axial plane shows two distinct electron-lucent compartments: an apical one (a) and caudal one (c) separated by a border of electron-dense cytoplasm containing polyribosomes (R) and part of the nuclear region (N). Bar, 0.5 μm. Magnification, ×60,000.

FIG. 8

Microsporidian spores from human stool samples as shown by light microscopy in Fig. 3 and 4. Two spores are empty looking (E). One spore shows a distinct compartmentation built up of two electron-lucent areas (a and c, with cross sections of the polar tube [★] shown in area c) separated by an electron-dense area. All three spores reveal the bilayered spore wall consisting of an outer electron-dense layer (exospore) (➩) and an inner electron-lucent layer (endospore) (➞). Bar, 1 μm. Magnification, ×30,000.