continue dehydration in 100% alcohol for 10 min; (step 9) clear in ... One hundred slides known to be positive for microsporidia .... USD, Incorporated, Stone.
Vol. 32, No. 4
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1994, P. 1974-1075 0095-1 137/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Modified Technique for Efficient Detection of Microsporidia EVELYNE KOKOSKIN,* THERESA W. GYORKOS, ANN CAMUS, LYNE CEDILOTIE, TERRY PURTILL, AND BRIAN WARD McGill University Centre for Tropical Diseases, Montreal General Hospital, Montreal, Canada H3G 1A4 Received 18 November 1993/Returned for modification 6 January 1994/Accepted 18 January 1994
Changes in temperature (from room temperature to 50°C) and staining time (from 90 to 10 min) were evaluated as a means of improving the detection of microsporidia from stool specimens. A blinded and independent comparison of 50 known positive matched-specimen pairs by three technologists resulted in consistently easier microscopic detection. The background is clearer, and spores stain more intensely. Staining time is reduced by 80 min.
The prevalence of microsporidia can exceed 20% in AIDS patients with chronic diarrhea (3). In our center, we have found a prevalence of 12% in these patients (6). Routine screening of stool specimens for microsporidia by the modified trichrome staining technique (7) can consume an important portion of laboratory resources because of the requirement for prolonged staining time and the difficulty in identifying such a small parasite (approximately 1 [Lm in size), especially in lightly infected patients. This has highlighted the need for an improved laboratory technique for the rapid diagnosis of microsporidial infections. The standard staining technique used to detect microsporidia in stool specimens is the modified trichrome stain of Weber et al. (7). This technique uses formalin-preserved stool specimens and takes approximately 2 h to complete. The steps in the modified trichrome stain technique of Weber et al. (7) follow: (step 1) apply a small amount of preserved unconcentrated liquid stool specimen to a glass slide (stool specimen should be barely visible on the slide); (step 2) dry thoroughly; (step 3) fix in methanol for 5 min; (step 4) stain with modified trichrome stain at room temperature for 90 min; (step 5) rinse with acid alcohol for 10 s; (step 6) rinse briefly with 95% alcohol; (step 7) dehydrate in 95% alcohol for 5 min; (step 8) continue dehydration in 100% alcohol for 10 min; (step 9) clear in Hemo-De (Fisher Scientific Ltd.) for 10 min; and (step 10) apply mounting fluid, cover with coverslip, dry, and examine. (For step 1, it has been our experience that the detection of the specimen and organisms on the slide can be facilitated by applying a few thicker bands of stool at the end of the barely visible smear.) In an effort to improve upon this technique, Ryan et al. (4) have examined changes to the composition of the stain, and we have examined the effects of temperature on staining time and quality of the stain. A preliminary assessment was undertaken to determine the optimal test staining time and temperature. Staining times of 5, 10, 15, and 20 min and temperatures of 37, 45, 50, 55, 60, and 65°C were evaluated. The best combination (determined on the basis of minimum time in arriving at the best stain quality) was found to be 10 min of staining time (a modification of step 4 in the standard Weber technique) at a temperature of 50°C. These conditions produced a reddish staining organism with clearly defined edges, a belt-like stripe (7), and vacuoles on a
green background. At lower temperatures, the organisms were a paler pink and the background was pinkish. At higher temperatures, the organisms were intensely stained but were distorted. No other changes to the Weber technique were made. This modified technique was then compared with the routine Weber technique. One hundred slides known to be positive for microsporidia from sodium acetate-acetic acid-formalin (SAF) (8) preserved specimens (50 slides processed by our modified technique and 50 slides by the routine Weber technique) were randomly assigned and independently examined by three technologists. Each slide was evaluated for the following characteristics and graded as follows: staining intensity (grade 1, pale, difficult to detect; grade 2, pinkish; grade 3, reddish, intense, easy to detect), morphology (grade 1, no distortion; grade 2, few distorted; grade 3, some distortion; grade 4, majority distorted), background contrast (grade 1, colorless; grade 2, green; grade 3, green, with pink area; grade 4, pinkish), and overall grading (grade 1, poor, organisms hard to detect; grade 2, satisfactory for experienced reader but organisms not obvious; grade 3, good with only minor problems; grade 4, excellent, easy to detect and identify). The technologists were blinded to staining technique. Each technologist had a minimum of 12 months of experience in the routine reading of microsporidial stains. Data were entered into a computer by using Epi Info (2) software and converted to Quattro Pro (1) for analysis. In addition to descriptive statistics, comparisons were made by using the Wilcoxon matched-pair signed-rank test (5). The following improvements were noted. The modified technique facilitated slide processing; the heated stain was less viscous, making the decolorization process faster and more consistent. The modified technique was ranked superior to the original technique by all three technologists on two of the three attributes studied (i.e., staining intensity and background contrast), with the single exception of borderline statistical significance by one technologist on the background contrast variable (Table 1). The color of the organisms tended to be more vibrant and distinct against a sharper and clearer background. Morphological characteristics were less distorted by the modified technique, but the difference was not statistically significant. In addition, the measure of overall assessment classified the modified technique as significantly better than the original. The modifications described permit faster processing, easier reading, and a reduction in the time-to-diagnosis from 120 to 40 min, for a saving of 80 min. The resulting increase in efficiency maximizes laboratory output and enhances clinical management of patients because of a faster turnaround time in
* Corresponding author. Mailing address: McGill University Centre for Tropical Diseases, Montreal General Hospital, 1650 Cedar Ave., Montreal, Quebec, Canada H3G lA4. Phone: (514) 937-6011, ext. 3870. Fax: (514) 937-0803.
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TABLE 1. Comparison of modified technique and Weber technique for the staining of intestinal microsporidia Staining intensity
Technologist
Grade'
no.
1 2 3
Grade
pI,
Weber
Modified
1.69 2.30 1.99
2.22 2.70 2.59
