c.i.-fluorescent-brightening-agent-28 and AIDS-Related-Opportunistic-Infections

Topics: AIDS-Related Opportunistic Infections; Animals; Antibodies, Monoclonal; Antibodies, Protozoan; Azo Compounds; Benzenesulfonates; Encephalitozoon; Encephalitozoonosis; Eosine Yellowish-(YS); Fluorescent Antibody Technique, Indirect; Humans; Lung; Methyl Green; Predictive Value of Tests; Sensitivity and Specificity; Staining and Labeling; Urine

Calcofluor white (CFW), a chemofluorescent agent, has proven effective in the recognition of Pneumocystis carinii cysts in respiratory fluids and secretions. However, its usefulness in the recognition of P. carinii cysts in tissue preparations has not been established. We studied 68 formalin-fixed, paraffin-embedded, transbronchial tissue biopsy specimens from individuals seropositive for the human immunodeficiency virus and stained them with the CFW stain and the conventional Gomori methenamine silver (GMS) stain to determine the concordance rate of the two stains. CFW-positive specimens showed light peripheral staining and a unique double parenthesis-like structure near the center of the cysts. Thirty-six (52.9%) of the 68 specimens were CFW and GMS positive, whereas 27 (39.7%) of the specimens were negative by both techniques, yielding a concordance rate of 92.6%. Five (7.4%) of the 68 specimens showed disparate results, and, of these, four (5.9%) were CFW positive and GMS negative, whereas one (1.5%) was CFW negative and GMS positive. We conclude that the CFW stain is suitable and useful for the demonstration of P. carinii cysts in tissue preparations.

Topics: AIDS-Related Opportunistic Infections; Benzenesulfonates; Biopsy; Fluorescent Dyes; Humans; Lung; Paraffin Embedding; Pneumocystis; Pneumonia, Pneumocystis; Silver Staining; Tissue Fixation

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 microliters of stool to detect one microsporidian after viewing 50 fields at a final magnification of x1,000. 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 TEM-negative 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.

Topics: AIDS-Related Opportunistic Infections; Animals; Benzenesulfonates; Body Fluids; Diagnostic Errors; Encephalitozoon; Evaluation Studies as Topic; Feces; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Humans; Microscopy, Electron; Microsporida; Microsporidiosis; Reproducibility of Results; Sensitivity and Specificity; Staining and Labeling