COMPARISON OF 3 STAINING METHODS FOR DETECTING MICROSPORIDIA IN FLUIDS

Calcofluor white 2MR, modified trichrome blue, and indirect immunofluo rescent antibody (IFA) staining methods were evaluated and compared fo r detecting microsporidia in stool, Serial 10-fold dilutions of Enceph alitozoon (Septata) intestinalis were prepared in three formalinized s tool specimens or in Tris-buffered saline. Ten-microliter aliquots wer e smeared onto glass slides, fixed with methanol, stained, and read by at least three individuals. The results indicated that the calcofluor stain was the most sensitive method, required approximately 15 min to perform, but did generate some false-positive results due to similarl y staining small yeast cells. The modified trichrome blue stain was ne arly as sensitive as the calcofluor stain and allowed for easier disti nction between microsporidia and yeast cells. This stain, however, req uired approximately 60 min to perform. The IFA stain with polyclonal m urine antiserum against E. intestinalis was the least sensitive of the methods and required approximately 130 min to perform. The lower limi t of detection with the calcofluor and modified trichrome stains was a concentration of about 500 organisms in 10 mu l of stool to detect on e microsporidian after viewing 50 fields at a final magnification of x 1,000. Reliability was also addressed by use of 74 stool, urine, and intestinal fluid specimens, 50 of which were confirmed for the presenc e of microsporidia by transmission electron microscopy (TEM). All TEM- positive specimens were detected by calcofluor and modified trichrome blue staining. Ten specimens were not detected by the IFA stain. An ad ditional seven TEM-negative specimens were read positive for microspor idia with the calcofluor stain, and of these, five also were read posi tive with the modified trichrome blue stain, The resulting diagnostic paradigm was to screen specimens mens with the calcofluor stain and to confirm the results with the modified trichrome stain. IFA, which was less sensitive, may become useful for microsporidian species identifi cation as specific antibodies become available.