Vol. 29, No. 3
JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1991, p. 645-647
0095-1137/91/030645-03$02.00/0 Copyright C) 1991, American Society for Microbiology
Comparison of Monoclonal Antibody and Calcofluor White Stains for the Detection of Pneumocystis carinii from Respiratory Specimens NICOLAS STRATTON, JANET HRYNIEWICKI, SANDRA L. AARNAES, GRACE TAN, LUIS M. DE LA MAZA, AND ELLENA M. PETERSON* Medical Microbiology Division, Department of Pathology, University of California, Irvine, Medical Center, 101 City Drive, Orange, California 92668 Received 23 August 1990/Accepted 13 November 1990
Three monoclonal antibody staining kits, from Genetic Systems, Disease Detection International, and Meridian Diagnostics, were compared with calcofluor white for the direct detection of Pneumocystis carinii in respiratory specimens. Of the 150 specimens tested, 23 were found positive for P. carinii by any of the four stains; 13 were bronchoalveolar lavage, 7 were induced sputum and 3 were expectorated sputum specimens. All stains detected the positive bronchoalveolar lavage specimens, the Genetic Systems stain detected six induced sputum specimens, and the other stains each detected four induced sputum specimens. The monoclonal antibody stains detected all three expectorated specimens, while the calcofluor stain detected only one. Overall, the sensitivities of the stains were 96% for Genetic Systems, 87% for both Disease Detection International and Meridian Diagnostics, and 78% for calcofluor.
slides were air dried, and two were fixed in acetone, one was fixed in methanol, and one was heat fixed. Two indirect fluorescent-MAb stains, Merifluor (Meridian Diagnostics Inc., Cincinnati, Ohio) and Fluoroslide (Disease Detection International, Irvine, Calif.), and a direct fluorescent-MAb stain from Genetic Systems (Seattle, Wash.) were compared. These stains detect cysts, trophozoites, and extracellular matrix material. The Fluoroslide stain has three MAbs, the Genetic Systems stain has two MAbs, and the Merifluor stain has one MAb. The acetone-fixed slides were used with the Genetic Systems and Fluoroslide stains, while the heat-fixed slide was used with the Merifluor stain. Manufacturer's instructions were followed for the use and interpretation of each MAb stain. The methanol-fixed slide was used with the calcofluor stain. One drop of calcofluor (Fungi-Fluor stain solution A; Polysciences, Inc., Warrington, Pa.) was placed on the cell preparation for 1 min, and the slide was examined immediately. The characteristic morphology of P. carinii, as described previously (1), was used in the interpretation of the calcofluor-stained slides. All slides were viewed with an epifluorescence microscope (Olympus, Tokyo, Japan). An excitation filter of 455 to 490 nm was used for the fluorescein-labeled stains, as were a 510-nm dichroic mirror and a 520-nm long-band-pass barrier filter. The calcofluor-stained slides were viewed with a
With the advent of AIDS, the number of specimens submitted for the laboratory detection of the opportunistic pathogen Pneumocystis carinii has greatly increased. Paralleling this increase has been the development of rapid laboratory detection methods and effective treatment protocols. Among the laboratory advances has been the development of monoclonal antibodies (MAbs) for the direct detection of this pathogen in patient material (3). Fluorescencetagged MAb stains have been shown to be equally as sensitive as or, in some instances, more sensitive than traditional stains, such as methenamine silver nitrate and toluidine blue 0, for the detection of P. carinii (2-6). There has been a recent report on the use of calcoflour white for the detection of P. carinii in bronchoalveolar lavage specimens (BALs) (1). As compared with toluidine blue 0 and Giemsa stains, it was 95% sensitive and 100% specific for the detection of P. carinii. The chief advantage of this inexpensive stain is that in addition to being extremely rapid, it can also serve to identify fungal structures in patient material. In this report, we compared this stain to three commercially available MAb staining kits for the detection of P. carinii in expectorated and induced sputum specimens as well as BALs. Upon receipt in the laboratory, specimens were diluted 1:2 with Sputolysin (Behring Diagnostics Inc., Somerville, N.J.), mixed by vortexing, and allowed to incubate for 10 min at 37°C. Specimens were centrifuged at 1,000 x g for 5 min, and the pellet was resuspended in phosphate-buffered saline (0.01 M, pH 7.4) to a no. 4 McFarland standard. In preliminary studies, this adjustment yielded a concentration of approximately 3 x 105 to 5 x 105 leukocytes per ml and was found to be optimal for yielding an even monolayer upon centrifugation in a Cytospin 2 (Shandon, Inc., Pittsburgh, Pa.). Subsequently, a 0.2-ml specimen was aliquoted into each of four cytofunnels equipped with white filters and centrifuged in a Cytospin 2 at 1,200 rpm for 10 min. The
TABLE 1. Overall detection of samples containing P. carinii by four stains Specimen type
Total BAL Induced sputum
Corresponding author. 645
Total no. of No. (%) of specimens found positive by: MenCalcospecimens Genetic Fluorofluor fluor slide systems positive
23 13 7 3
22 13 6 3
(96) 20 (87) 20 (87) 18 (78) (100) 13 (100) 13 (100) 13 (100) (86) 4 (57) 4 (57) 4 (57) (100) 3 (100) 3 (100) 1 (33)
J. CLIN. MICROBIOL.
TABLE 2. Predictive values of four stains for the detection of P. carinii % Predictive value
Genetic Systems Fluoroslide Merifluor Calcofluor
95.6 87.0 87.0 78.3
100 100 100 100
100 100 100 100
99.2 97.7 97.7 96.3
405-nm excitation filter, a 455-nm dichroic mirror, and a 455to 515-nm barrier filter. The 150 specimens examined included 53 BALs, 47 induced sputum specimens, 48 expectorated sputum specimens, 1 lung tissue specimen, and 1 respiratory secretion. Of these specimens, 15% (23 of 150) were found positive for P. carinii by at least one of the stains used. These 23 specimens were from 21 patients, and 57% were BALs, 30% were
FIG. 1. Use of calcofluor and MAb stains to detect the cysts of P. carinii in a BAL. (A) Typical calcofluor stain of a small clump of cysts; note the double parenthesislike structure inside the cysts. (B) BAL stained with the Genetic Systems stain; the distinctive honeycomb appearance of P. carinii is evident. (C) The addition of calcofluor to the mounting medium in the same field as that in panel B revealed the typical cysts of P. carinii when the filters were changed to those needed to visualize the calcofluor stain. (D) Fluorescent clump of material detected in a BAL by Merifluor; the material lacked the typical honeycomb appearance seen in panel B. (E) Same clump as that in panel D after calcofluor was added and the filters were changed; in this case, the typical cysts seen with calcofluor confirmed that the clump seen with Merifluor was positive for P. carinii. Magnification, x 1,520.
VOL. 29, 1991
induced sputum specimens, and 13% were expectorated sputum specimens. All four stains detected all of the BALs positive for P. carinii (Table 1). For the induced sputum samples, the Genetic Systems stain detected six, while all of the other stains detected four. It is interesting to note that one of the induced specimens was detected only by the calcofluor stain. With this one specimen, a rare cyst was revealed by calcofluor and, because of its distinctive morphology, could readily be identified as P. carinii. However, technologists viewing the smear stained with the Genetic Systems stain, while seeing rare fluorescent extracellular matrix material, were not able to definitively assign a positive reading to the smear. With the other two stains, no fluorescent material indicative of P. carinii was seen. This specimen was from a patient who was positive for human immunodeficiency virus type 1 antibodies, had chest X-ray findings consistent with P. carinii pneumonia, and responded to trimethoprim-sulfamethoxazole (Bactrim; Hoffmann-La Roche) therapy. The MAb stains detected all positive expectorated samples, while the calcofluor stain failed to detect two. Because of the presence of more fungal structures as well as the higher background, expectorated samples were difficult to examine with the calcoflour stain. While P. carinii might have been present, it was most likely obscured by the high background of the samples. The overall sensitivities and specificities of the four stains are shown in Table 2. For data analysis, any specimen that was found positive by at least one stain and was also from a patient who had either a previous laboratory-proven episode of P. carinii pneumonia or chest X-ray findings consistent with P. carinii pneumonia and who responded to appropriate therapy was considered a true-positive. Only three specimens were found positive by only one or two of the stains. Of these specimens, one, as discussed above, was only calcofluor positive, the second was found positive only by the Genetic Systems stain, and the third was found positive by the Genetic Systems stain and calcofluor. All three of these were induced sputum specimens. Since fluorescein and calcofluor use different sets of excitation and barrier filters, we were interested in determining whether the two stains could be used to view the same smear. Therefore, with selected smears found positive by any of the three MAb stains, we added 1 drop of calcofluor stain under the coverslip. When material resembling a cystic structure was revealed by the MAb stain, the filters were switched and the same material was visualized with the violet filters used with the calcofluor stain (Fig. 1). The
distinctive cyst morphology with the parenthesislike structure revealed by the calcofluor stain confirmed the material as P. carinii. This confirmation by calcofluor may be useful in selected cases in which there is doubt about the material being stained by the MAb stain, especially in smears with a high background. In summary, the Genetic Systems stain was found to be the most sensitive and was the easiest to read because of the low background staining. The other two MAb stains were comparable in performance. While all four stains performed well with BALs, the two indirect MAb stains and the calcofluor stain were less reliable when used with induced sputum specimens. This result could have been due to the higher background experienced with these stains. While the calcofluor stain could serve as a rapid, inexpensive stain for BALs, it was less useful in the primary screening of sputum samples, possibly because of the background and staining of fungal structures present in these samples. Therefore, calcofluor could serve as a primary screening stain for BALs and could be used as a confirmatory stain for sputum samples because of the distinctive nature of the cyst morphology.
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