OF CLINICAL MICROBIOLOGY, Feb. 1990, p. 393-394 0095-1137/90/020393-02$02.00/0 Copyright © 1990, American Society for Microbiology
JOURNAL
Vol. 28, No. 2
Rapid Detection of Pneumocystis carinii in Bronchoalveolar Lavage Samples by Using Cellufluor Staining VICKIE S. BASELSKIl.2* MARY K. ROBISON,2 LINDA W. PIFER,3 AND DIANE R. WOODS3 Departments of Pathologyl and Pediatrics, University of Tennessee, Memphis, Memphis, Tennessee 38163, and Regional Medical Center, Memphis, Tennessee 381032* Received 20 July 1989/Accepted 18 October 1989
Cellufluor (Calcofluor white) has been found to be a useful, rapid chemofluorescent stain for detection of Pneumocystis carinii cysts in bronchoalveolar lavage samples. When compared with toluidine blue O and Giemsa stains on 45 specimens (22 positive and 23 negative), the sensitivity and specificity of the Cellufluor stain were 95 and 100%, respectively.
Bronchoalveolar lavage (BAL) has proven to be very useful in establishing the etiology of pneumonia in immunosuppressed patients (4, 20). BAL specimens are particularly useful for the detection of Pneumocystis carinii, the most common pulmonary pathogen in this group of patients. In the Microbiology Laboratory of the Regional Medical Center, a BAL profile has been established in which a variety of rapid stains are performed on cytocentrifuged preparations of BAL samples to detect common etiologic agents. For detection of fungi, a Cellufluor (Calcofluor white) stain is routinely performed (1, 9, 18). Cellufluor is a chemofluorescent optical brightening agent (19) which nonspecifically binds to P-linked polysaccharide polymers (17) found in fungi and other organisms. When viewed with a properly equipped fluorescence microscope, fungal elements fluoresce an intense apple green. During routine examination of Cellufluor stains in BAL profiles, fluorescing structures which were morphologically compatible with Pneumocystis cysts were also noted. To verify this finding, a prospective study was undertaken to compare Cellufluor with two conventional stains (toluidine blue O [TBO] and Giemsa) for detection of P. carinii. The methods used to process BAL specimens for Cellufluor stains were similar to protocols previously published (7, 12, 16). Briefly, after immediate transport to the laboratory, the specimens (generally 30 to 80 ml) were centrifuged at 1,100 x g for 30, min, the supernatant was decanted, and the sediment was suspended in 10 ml of Hanks balanced salt solution. After being vortexed, the cell suspension was used for preparation of cytocentrifuged smears for staining (Cytospin 2; Shandon, Inc., Pittsburgh, Pa.). For each smear, 0.25 ml was centrifuged at 600 rpm for 15 min by using Shandon disposable filter units. After slide removal, the smears were air dried and fixed with absolute methanol for 1 to 2 min to dryness. The methanol step has been found to be essential to fix cells onto the slides, and it also results in improved fluorescence by Cellufluor. For Cellufluor staining, a commercially available kit was used (Fungi-Fluor; Polysciences, Inc., Warrington, Pa.). Smears were stained with a few drops of Fungi-Fluor solution A (Cellufluor) for 1 to 2 min, rinsed gently with tap water, and wet mounted for observation. Alternatively, slides may be air dried and viewed directly. Although the Fungi-Fluor kit contains a counterstain, solution B, for tissue contrast, the reagent was not found to be necessary in *
this study. Slides were observed with a Leitz Laborlux D fluorescence microscope (Wild Leitz USA, Inc.; Rockleigh, N.J.) equipped with filter module H3 (excitation, 420 to 490 nm; barrier, 515 nm). It should be noted that this varies from the Cellufluor optimal excitation wavelength (250 to 400 nm) but is adequate for use with low-background samples (10). Slides were initially scanned at x500 oil magnification, and x 1,000 oil magnification was used for morphotype verification. Conventional stains were performed in the research laboratory of one of us (L.W.P.). For these stains, an untreated portion of the BAL samples was used to prepare smears from routinely centrifuged sediments. A TBO stain was performed as previously described (5), along with a Giemsa stain containing Triton X-100 for all TBO-negative samples. Figure 1 shows the characteristic morphologies observed with Cellufluor for Pneumocystis cysts and Histoplasma capsulatum yeasts. Pneumocystis cysts were 5 to 7 ,um in diameter, generally round, fairly uniform in size, and nonbudding. A highly characteristic staining pattern with peripheral staining of the cyst wall and intense staining of an internal "double-parenthesis-like" structure was noted. This pattern most closely resembles the staining pattern seen with methenamine silver stains (2, 13) where these structures, believed to play a role in excystment (8, 11), are also evident. Occasionally, forms with a more homogeneous staining pattern were observed, but never in the absence of the more typical predominant form, and occasionally, free double-parenthesis forms were seen, presumably because of cyst degeneration. In contrast, the iHistoplasma yeasts were smaller (3 to 5 ,um in diameter), ovoid, and frequently budding and showed only intense peripheral staining. Other, larger yeasts (Candida sp.) could also be easily differentiated. Occasional background staining of some cell types, macrophage inclusions, some bacterial morphotypes, and various tissue elements (e.g., collagen, elastin, and keratin) (9, 18, 19) also posed no interpretive difficulties. A prospective comparison of Cellufluor with TBO and Giemsa stains for detection of P. carinii was performed on 45 clinical BAL specimens. Cellufluor was positive in 21 (95%) of 22 TBO- or Giemsa-positive samples and negative in 23 (100%) of 23 TBO- and Giemsa-negative samples. The single discrepancy was in a sample with very low cyst numbers. Review of additional slides by Cellufluor failed to resolve the discrepancy, which is presumed to represent sampling error. By using both rat lung control slides and known positive BAL samples, several different Calcofluor white preparations were tested for the ability to stain P. carinii. In addition
Corresponding author. 393
394
J. CLIN. MICROBIOL.
NOTES
clinical microbiologists should also be alert to the possibility of detection of P. carinii in respiratory samples with this stain. We acknowledge Brian J. Harrington for providing samples of Calcofluor white powders for testing.
FIG. 1. Typical morphology of P. carinii cysts (top left) and H. capsulatum yeasts (lower right) observed by Cellufluor staining of a cytocentrifuged preparation of a BAL specimen visualized with a Leitz Laborlux D microscope with an H cube filter (x 1,000 oil magnification).
to the Fungi-Fluor kit, prepared stains from Difco Laboratories (Detroit, Mich.) and Remel (Lenexa, Kans.) and 0.1% aqueous solutions of Calcofluor white powders from Polysciences, Inc. (Cellufluor M2R purified and Cellufluor purified) and Sigma Chemical Co. (Fluorescent Brightener 28; St. Louis, Mo.) were tested. Although all solutions stained Candida yeast control slides, only Fungi-Fluor showed characteristic Pneumocystis cyst staining. The reasons for this difference are unclear, since the Fungi-Fluor kit uses an aqueous solution of the Polysciences Cellufluor product, but differences in Cellufluor staining characteristics for fungi have also been observed (1, 9, 14, 18). In summary, the Fungi-Fluor Cellufluor stain was found to be an accurate, rapid method for detection of P. carinii cysts in BAL specimens. This is the first report of use of this stain for detection of P. carinii. However, given the biochemical similarities of the cyst wall to fungal cell walls, properties responsible for staining of both P. carinii and fungi with other stains such as TBO and methenamine silver (2, 8), this is not a surprising finding. Studies are in progress to determine whether the stain may also be reliably used for other respiratory specimens, such as sputum (3). Compared with other commonly used stains, such as
TBO, Giemsa, and methenamine silver, Cellufluor offers a number of advantages, including rapidity, ease of stain performance, and low cost ($0.08 per test) (2). The highly characteristic morphology revealed by Cellufluor makes smear interpretation relatively easy, particularly for differentiation of Pneumocystis cysts from nonbudding yeast cells. Although a comparison with monoclonal fluorescentantibody staining (6, 21) was not performed, these stains seem not to be required for BAL samples and would also be more costly and more difficult to perform and interpret (15). Lastly, use of a prepared kit overcomes problems of reagent stability which have been described with in-house preparation of Cellufluor aqueous solutions (14). Since many laboratories now use Cellufluor for rapid detection of fungi,
LITERATURE CITED 1. Al-Doory, Y., R. J. Yankey, and M. L. Elgart. 1985. A rapid microscopic method for fungi in clinical specimens. Lab. Manage. 23:63-68. 2. Bedrossian, C. W. M., M. R. Mason, and P. K. Gupta. 1989. Rapid cytologic diagnosis of Pneumocystis: a comparison of effective techniques. Semin. Diagn. Pathol. 6:245-261. 3. Blumenfeld, W., and J. M. Griffiss. 1988. Pneumocystis carinii in sputum. Comparable efficacy of screening stains and determination of cyst density. Arch. Pathol. Lab. Med. 112:816-820. 4. Broaddus, C., M. D. Dake, M. S. Stulbarg, W. Blumenfeld, W. K. Hadley, J. A. Golden, and P. C. Hopewell. 1985. Bronchoalveolar lavage and transbronchial biopsy for the diagnosis of pulmonary infections in the acquired immunodeficiency syndrome. Ann. Intern. Med. 102:747-752. 5. Chalvardjian, A. M., and L. A. Grawe. 1963. A new procedure for the identification of Pneumocystis carinii cysts in tissue sections and smears. J. Clin. Pathol. 16:383-384. 6. Gill, V. J., G. Evans, F. Stock, J. E. Parrillo, H. Masur, and J. A. Kovacs. 1987. Detection of Pneumocystis carinii by fluorescent-antibody stain using a combination of three monoclonal antibodies. J. Clin. Microbiol. 25:1837-1840. 7. Gill, V. J., N. A. Nelson, F. Stock, and G. Evans. 1988. Optimal use of the cytocentrifuge for recovery and diagnosis of Pneumocystis carinii in bronchoalveolar lavage and sputum specimens. J. Clin. Microbiol. 26:1641-1644. 8. Gutierrez, Y. 1989. The biology of Pneumocystis carinii. Semin. Diagn. Pathol. 6:203-211. 9. Hageage, G. J., and B. J. Harrington. 1984. Use of calcofluor white in clinical mycology. Lab. Med. 15:109-112. 10. Hughes, J., and M. E. McCully. 1975. The use of an optical brightener in the study of plant structure. Stain Technol. 50: 319-329. 11. Hughes, W. T., R. A. Price, H.-K. Kim, T. P. Coburn, D. Grigsby, and S. Feldman. 1973. Pneumocystis carinii pneumonitis in children with malignancies. J. Pediatr. 82:404-415. 12. Kahn, F. W., and J. M. Jones. 1988. Analysis of bronchoalveolar lavage specimens from immunocompromised patients with a protocol applicable in the microbiology laboratory. J. Clin.
Microbiol. 26:1150-1155.
13. Kim, H.-K., and W. T. Hughes. 1973. Comparison of methods for identification of Pneumocystis carinii in pulmonary aspirates. Am. J. Clin. Pathol. 60:462-466. 14. Koch, H. H., and M. Pimsler. 1987. Evaluation of Uvitex 2B: a nonspecific fluorescent stain for detecting and identifying fungi and algae in tissue. Lab. Med. 18:603-606. 15. Linder, J., and S. J. Radio. 1989. Immunohistochemistry of Pneumocystis carinii. Semin. Diagn. Pathol. 6:238-244. 16. Linder, J., and S. Rennard. 1988. Bronchoalveolar lavage, p. 17-44. American Society of Clinical Pathologists, Chicago. 17. Maeda, H., and N. Ishida. 1967. Specificity of binding of hexopyranosyl polysaccharides with fluorescent brightener. J. Biochem. 62:276-278. 18. Monheit, J. E., D. F. Cowan, and D. G. Moore. 1984. Rapid detection of fungi in tissues using calcofluor white and fluorescence microscopy. Arch. Pathol. Lab. Med. 108:616-618. 19. Paton, A. M., and S. M. Jones. 1971. Techniques involving optical brightening agents, p. 135-144. In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. SA. Academic Press, Inc., New York. 20. Stover, D. E., M. B. Zaman, S. I. Hajdu, M. Lange, J. Gold, and D. Armstrong. 1984. Bronchoalveolar lavage in the diagnosis of diffuse pulmonary infiltrates in the immunosuppressed host. Ann. Intern. Med. 101:1-7. 21. Walzer, P. D. 1988. Diagnosis of Pneumocystis carinii pneumonia. J. Infect. Dis. 157:629-632.
JOURNAL
Vol. 28, No. 2
Rapid Detection of Pneumocystis carinii in Bronchoalveolar Lavage Samples by Using Cellufluor Staining VICKIE S. BASELSKIl.2* MARY K. ROBISON,2 LINDA W. PIFER,3 AND DIANE R. WOODS3 Departments of Pathologyl and Pediatrics, University of Tennessee, Memphis, Memphis, Tennessee 38163, and Regional Medical Center, Memphis, Tennessee 381032* Received 20 July 1989/Accepted 18 October 1989
Cellufluor (Calcofluor white) has been found to be a useful, rapid chemofluorescent stain for detection of Pneumocystis carinii cysts in bronchoalveolar lavage samples. When compared with toluidine blue O and Giemsa stains on 45 specimens (22 positive and 23 negative), the sensitivity and specificity of the Cellufluor stain were 95 and 100%, respectively.
Bronchoalveolar lavage (BAL) has proven to be very useful in establishing the etiology of pneumonia in immunosuppressed patients (4, 20). BAL specimens are particularly useful for the detection of Pneumocystis carinii, the most common pulmonary pathogen in this group of patients. In the Microbiology Laboratory of the Regional Medical Center, a BAL profile has been established in which a variety of rapid stains are performed on cytocentrifuged preparations of BAL samples to detect common etiologic agents. For detection of fungi, a Cellufluor (Calcofluor white) stain is routinely performed (1, 9, 18). Cellufluor is a chemofluorescent optical brightening agent (19) which nonspecifically binds to P-linked polysaccharide polymers (17) found in fungi and other organisms. When viewed with a properly equipped fluorescence microscope, fungal elements fluoresce an intense apple green. During routine examination of Cellufluor stains in BAL profiles, fluorescing structures which were morphologically compatible with Pneumocystis cysts were also noted. To verify this finding, a prospective study was undertaken to compare Cellufluor with two conventional stains (toluidine blue O [TBO] and Giemsa) for detection of P. carinii. The methods used to process BAL specimens for Cellufluor stains were similar to protocols previously published (7, 12, 16). Briefly, after immediate transport to the laboratory, the specimens (generally 30 to 80 ml) were centrifuged at 1,100 x g for 30, min, the supernatant was decanted, and the sediment was suspended in 10 ml of Hanks balanced salt solution. After being vortexed, the cell suspension was used for preparation of cytocentrifuged smears for staining (Cytospin 2; Shandon, Inc., Pittsburgh, Pa.). For each smear, 0.25 ml was centrifuged at 600 rpm for 15 min by using Shandon disposable filter units. After slide removal, the smears were air dried and fixed with absolute methanol for 1 to 2 min to dryness. The methanol step has been found to be essential to fix cells onto the slides, and it also results in improved fluorescence by Cellufluor. For Cellufluor staining, a commercially available kit was used (Fungi-Fluor; Polysciences, Inc., Warrington, Pa.). Smears were stained with a few drops of Fungi-Fluor solution A (Cellufluor) for 1 to 2 min, rinsed gently with tap water, and wet mounted for observation. Alternatively, slides may be air dried and viewed directly. Although the Fungi-Fluor kit contains a counterstain, solution B, for tissue contrast, the reagent was not found to be necessary in *
this study. Slides were observed with a Leitz Laborlux D fluorescence microscope (Wild Leitz USA, Inc.; Rockleigh, N.J.) equipped with filter module H3 (excitation, 420 to 490 nm; barrier, 515 nm). It should be noted that this varies from the Cellufluor optimal excitation wavelength (250 to 400 nm) but is adequate for use with low-background samples (10). Slides were initially scanned at x500 oil magnification, and x 1,000 oil magnification was used for morphotype verification. Conventional stains were performed in the research laboratory of one of us (L.W.P.). For these stains, an untreated portion of the BAL samples was used to prepare smears from routinely centrifuged sediments. A TBO stain was performed as previously described (5), along with a Giemsa stain containing Triton X-100 for all TBO-negative samples. Figure 1 shows the characteristic morphologies observed with Cellufluor for Pneumocystis cysts and Histoplasma capsulatum yeasts. Pneumocystis cysts were 5 to 7 ,um in diameter, generally round, fairly uniform in size, and nonbudding. A highly characteristic staining pattern with peripheral staining of the cyst wall and intense staining of an internal "double-parenthesis-like" structure was noted. This pattern most closely resembles the staining pattern seen with methenamine silver stains (2, 13) where these structures, believed to play a role in excystment (8, 11), are also evident. Occasionally, forms with a more homogeneous staining pattern were observed, but never in the absence of the more typical predominant form, and occasionally, free double-parenthesis forms were seen, presumably because of cyst degeneration. In contrast, the iHistoplasma yeasts were smaller (3 to 5 ,um in diameter), ovoid, and frequently budding and showed only intense peripheral staining. Other, larger yeasts (Candida sp.) could also be easily differentiated. Occasional background staining of some cell types, macrophage inclusions, some bacterial morphotypes, and various tissue elements (e.g., collagen, elastin, and keratin) (9, 18, 19) also posed no interpretive difficulties. A prospective comparison of Cellufluor with TBO and Giemsa stains for detection of P. carinii was performed on 45 clinical BAL specimens. Cellufluor was positive in 21 (95%) of 22 TBO- or Giemsa-positive samples and negative in 23 (100%) of 23 TBO- and Giemsa-negative samples. The single discrepancy was in a sample with very low cyst numbers. Review of additional slides by Cellufluor failed to resolve the discrepancy, which is presumed to represent sampling error. By using both rat lung control slides and known positive BAL samples, several different Calcofluor white preparations were tested for the ability to stain P. carinii. In addition
Corresponding author. 393
394
J. CLIN. MICROBIOL.
NOTES
clinical microbiologists should also be alert to the possibility of detection of P. carinii in respiratory samples with this stain. We acknowledge Brian J. Harrington for providing samples of Calcofluor white powders for testing.
FIG. 1. Typical morphology of P. carinii cysts (top left) and H. capsulatum yeasts (lower right) observed by Cellufluor staining of a cytocentrifuged preparation of a BAL specimen visualized with a Leitz Laborlux D microscope with an H cube filter (x 1,000 oil magnification).
to the Fungi-Fluor kit, prepared stains from Difco Laboratories (Detroit, Mich.) and Remel (Lenexa, Kans.) and 0.1% aqueous solutions of Calcofluor white powders from Polysciences, Inc. (Cellufluor M2R purified and Cellufluor purified) and Sigma Chemical Co. (Fluorescent Brightener 28; St. Louis, Mo.) were tested. Although all solutions stained Candida yeast control slides, only Fungi-Fluor showed characteristic Pneumocystis cyst staining. The reasons for this difference are unclear, since the Fungi-Fluor kit uses an aqueous solution of the Polysciences Cellufluor product, but differences in Cellufluor staining characteristics for fungi have also been observed (1, 9, 14, 18). In summary, the Fungi-Fluor Cellufluor stain was found to be an accurate, rapid method for detection of P. carinii cysts in BAL specimens. This is the first report of use of this stain for detection of P. carinii. However, given the biochemical similarities of the cyst wall to fungal cell walls, properties responsible for staining of both P. carinii and fungi with other stains such as TBO and methenamine silver (2, 8), this is not a surprising finding. Studies are in progress to determine whether the stain may also be reliably used for other respiratory specimens, such as sputum (3). Compared with other commonly used stains, such as
TBO, Giemsa, and methenamine silver, Cellufluor offers a number of advantages, including rapidity, ease of stain performance, and low cost ($0.08 per test) (2). The highly characteristic morphology revealed by Cellufluor makes smear interpretation relatively easy, particularly for differentiation of Pneumocystis cysts from nonbudding yeast cells. Although a comparison with monoclonal fluorescentantibody staining (6, 21) was not performed, these stains seem not to be required for BAL samples and would also be more costly and more difficult to perform and interpret (15). Lastly, use of a prepared kit overcomes problems of reagent stability which have been described with in-house preparation of Cellufluor aqueous solutions (14). Since many laboratories now use Cellufluor for rapid detection of fungi,
LITERATURE CITED 1. Al-Doory, Y., R. J. Yankey, and M. L. Elgart. 1985. A rapid microscopic method for fungi in clinical specimens. Lab. Manage. 23:63-68. 2. Bedrossian, C. W. M., M. R. Mason, and P. K. Gupta. 1989. Rapid cytologic diagnosis of Pneumocystis: a comparison of effective techniques. Semin. Diagn. Pathol. 6:245-261. 3. Blumenfeld, W., and J. M. Griffiss. 1988. Pneumocystis carinii in sputum. Comparable efficacy of screening stains and determination of cyst density. Arch. Pathol. Lab. Med. 112:816-820. 4. Broaddus, C., M. D. Dake, M. S. Stulbarg, W. Blumenfeld, W. K. Hadley, J. A. Golden, and P. C. Hopewell. 1985. Bronchoalveolar lavage and transbronchial biopsy for the diagnosis of pulmonary infections in the acquired immunodeficiency syndrome. Ann. Intern. Med. 102:747-752. 5. Chalvardjian, A. M., and L. A. Grawe. 1963. A new procedure for the identification of Pneumocystis carinii cysts in tissue sections and smears. J. Clin. Pathol. 16:383-384. 6. Gill, V. J., G. Evans, F. Stock, J. E. Parrillo, H. Masur, and J. A. Kovacs. 1987. Detection of Pneumocystis carinii by fluorescent-antibody stain using a combination of three monoclonal antibodies. J. Clin. Microbiol. 25:1837-1840. 7. Gill, V. J., N. A. Nelson, F. Stock, and G. Evans. 1988. Optimal use of the cytocentrifuge for recovery and diagnosis of Pneumocystis carinii in bronchoalveolar lavage and sputum specimens. J. Clin. Microbiol. 26:1641-1644. 8. Gutierrez, Y. 1989. The biology of Pneumocystis carinii. Semin. Diagn. Pathol. 6:203-211. 9. Hageage, G. J., and B. J. Harrington. 1984. Use of calcofluor white in clinical mycology. Lab. Med. 15:109-112. 10. Hughes, J., and M. E. McCully. 1975. The use of an optical brightener in the study of plant structure. Stain Technol. 50: 319-329. 11. Hughes, W. T., R. A. Price, H.-K. Kim, T. P. Coburn, D. Grigsby, and S. Feldman. 1973. Pneumocystis carinii pneumonitis in children with malignancies. J. Pediatr. 82:404-415. 12. Kahn, F. W., and J. M. Jones. 1988. Analysis of bronchoalveolar lavage specimens from immunocompromised patients with a protocol applicable in the microbiology laboratory. J. Clin.
Microbiol. 26:1150-1155.
13. Kim, H.-K., and W. T. Hughes. 1973. Comparison of methods for identification of Pneumocystis carinii in pulmonary aspirates. Am. J. Clin. Pathol. 60:462-466. 14. Koch, H. H., and M. Pimsler. 1987. Evaluation of Uvitex 2B: a nonspecific fluorescent stain for detecting and identifying fungi and algae in tissue. Lab. Med. 18:603-606. 15. Linder, J., and S. J. Radio. 1989. Immunohistochemistry of Pneumocystis carinii. Semin. Diagn. Pathol. 6:238-244. 16. Linder, J., and S. Rennard. 1988. Bronchoalveolar lavage, p. 17-44. American Society of Clinical Pathologists, Chicago. 17. Maeda, H., and N. Ishida. 1967. Specificity of binding of hexopyranosyl polysaccharides with fluorescent brightener. J. Biochem. 62:276-278. 18. Monheit, J. E., D. F. Cowan, and D. G. Moore. 1984. Rapid detection of fungi in tissues using calcofluor white and fluorescence microscopy. Arch. Pathol. Lab. Med. 108:616-618. 19. Paton, A. M., and S. M. Jones. 1971. Techniques involving optical brightening agents, p. 135-144. In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. SA. Academic Press, Inc., New York. 20. Stover, D. E., M. B. Zaman, S. I. Hajdu, M. Lange, J. Gold, and D. Armstrong. 1984. Bronchoalveolar lavage in the diagnosis of diffuse pulmonary infiltrates in the immunosuppressed host. Ann. Intern. Med. 101:1-7. 21. Walzer, P. D. 1988. Diagnosis of Pneumocystis carinii pneumonia. J. Infect. Dis. 157:629-632.
