Comparison of three staining methods for detecting microsporidia in fluids

Abstract

Calcofluor white 2MR, modified trichrome blue, and indirect immunofluorescent antibody (IFA) staining methods were evaluated and compared for detecting microsporidia in stool. Serial 10-fold dilutions of Encephalitozoon (Septata) intestinalis were prepared in three formalinized stool specimens or in Tris-buffered saline. Ten-microliter aliquots were 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 similarly staining small yeast cells. The modified trichrome blue stain was nearly as sensitive as the calcofluor stain and allowed for easier distinction between microsporidia and yeast cells. This stain, however, required approximately 60 min to perform. The IFA stain with polyclonal murine antiserum against E. intestinalis was the least sensitive of the methods and required approximately 130 min to perform. The lower limit of detection with the calcofluor and modified trichrome stains was a concentration of about 500 organisms in 10 l of stool to detect one microsporidian after viewing 50 fields at a final magnification of ؋1,000. Reliability was also addressed by use of 74 stool, urine, and intestinal fluid specimens, 50 of which were confirmed for the presence 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 additional seven TEMnegative specimens were read positive for microsporidia with the calcofluor stain, and of these, five also were read positive with the modified trichrome blue stain. The resulting diagnostic paradigm was to screen specimens 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 identification as specific antibodies become available.

Waterborne protozoan pathogens

“…The organisms can be detected in tissue sections by using hematoxylin-eosin, Gram, periodic-acid Schiff, Gomori methenamine silver, Warthin-Starry, acid-fast, and Giemsa stains. Calcofluor white, Uvitex-2B, polarized light, and the most commonly used method, modified trichrome with Chromotrope 2R, are used for organism detection in stool specimens (28,32,54,57,68,127,128,132,136,190,198). Slides must be prepared with a very thin layer of specimen, stained, and examined under at least ϫ1,000 magnification.…”

“…Currently, no specific antibodies are available for use in its detection; however, there is a reported cross-reactivity between Encephalitozoon antibodies and E. bieneusi antigens. Through this cross-reaction, polyclonal antibodies have been somewhat successfully used to demonstrate spores of E. bieneusi in stool and intestinal biopsy tissue (8,28,57,194,196,205). Because no in vitro cultivation methods for E. bieneusi are available, it appears that the future of diagnosis for this organism may be limited to the use of diagnostic rRNA probes and PCR (55,62,65,187,203,204).…”

Symptomatic respiratory Encephalitozoon cuniculi infection in renal transplant recipients

“…The standard Calcofluor M2R staining method was used to detect microsporidial spores (Didier et al, 1995).…”

Diagnosis of disseminated microsporidian Encephalitozoon hellem infection by PCR-Southern analysis and successful treatment with albendazole and fumagillin

“…Histochemistry. Smears from the urine sediment and conjunctival swabs were prepared on glass slides, fixed with methanol for 10 min, and stained with calcofluor, modified trichrome blue, and indirect immunofluorescent antibody (IFA) as previously described (16).…”