Modified technique to recover microsporidian spores in sodium acetate-acetic acid-formalin-fixed fecal samples by light microscopy and correlation with transmission electron microscopy

Abstract

Microsporidia are an emerging cause of significant disease, particularly in the immunocompromised host. Until recently, the diagnosis of enteric infections has required invasive sampling, the use of expensive technology, and considerable technological expertise. The purpose of the present study was to examine three modifications to the processing of fecal specimens for light microscopy (LM) examination for microsporidian spores: the use of pretreatment with potassium hydroxide, modified centrifugation conditions, and a modified staining technique. A sodium acetate-acetic acid-formalin-fixed fecal sample containing numerous microsporidian spores confirmed to be positive by transmission electron microscopy (TEM) was used in all studies performed. A simulation of a heavy to lightly infected individual was used. The results of LM were correlated with those of TEM. Duplicate smears were stained with Weber's modified trichrome and Giemsa (GS) stains. The stained slides were randomized and examined blindly by LM at x 625 and x 1,250 magnifications. A portion of the dilutions after centrifugation were fixed for TEM. The Weber modified trichrome stain performance rating was higher than the Giemsa stain rating because of ease of interpretation, and material stained with Weber modified trichrome stain required less examination time at a lower magnification. The number of positive smears and the quantity of spores detected were significantly higher following pretreatment of the sample with KOH. TEM was positive only when numerous spores were present, but the quality of the photomicrographs was superior after pretreatment with KOH. Pretreatment of sodium acetate-acetic acid-formalin-fixed fecal samples with 10% KOH and then a 5-min centrifugation time and staining with Weber modified trichrome stain provide for the excellent recovery of microsporidia in the routine diagnostic parasitology laboratory.