Microscopic diagnosis of Pneumocystis jirovecii pneumonia in bronchoalveolar lavage and oropharyngeal wash samples of immunocompromised patients with pneumonia
Keywords:
Pneumonia, Pneumocystis/diagnosis, fluorescent antibody technique, direct, tolonium chloride, oropharynx, bronchoalveolar lavage, immunocompromised host
Abstract
Introduction. The diagnosis of Pneumocystis jirovecii pneumonia is based on observation of the microorganism using several staining techniques in respiratory samples, especially bronchoalveolar lavage and induced sputum. Recently, the fungus also has been detected in oropharyngeal wash samples, but only using molecular tests.Objective. The diagnostic yield of two microscopic stains, toluidine blue O and direct fluorescent antibody, was compared in bronchoalveolar lavage and oropharyngeal wash samples for the detection of P. jirovecii in immunocompromised patients with pneumonia.
Materials and methods. Cross-sectional evaluation diagnostic tests were used in 166 immunossupressed patients with suspected P. jirovecii. By protocol, bronchoscopic bronchoalveolar lavage and oropharyngeal wash samples were prepared by cytocentrifugation, and slides were stained with toluidine blue and fluorescent antibody. The proportion of positive results from each stain and concordance between them were determined.
Results. Twenty-four cases (14.5%) of P. jirovecii were detected in bronchoalveolar lavage samples. Of them, 21 were positive by both toluidine blue and fluorescent antibody stains, whereas 3 cases were detected by fluorescent antibody alone. None of the 166 oropharyngeal wash samples were positive by either of these techniques. No significant differences were found between proportions from positive results (p=0.63). Concordance (kappa coefficient) between both stains was 0.92 (95% CI: 0.84-1.00).
Conclusions. Both techniques were useful to diagnose P. jirovecii in bronchoalveolar lavage samples. However, toluidine blue stain did not detect 12% of fluorescent antibody positive cases. Oropharyngeal wash samples do not provide sufficient material for the microscopic identification of this fungus.
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References
1. Morris A, Lundgren JD, Masur H, Walzer PD, Hanson DL, Frederick T, et al. Current epidemiology of Pneumocystis pneumonia. Emerg Infect Dis. 2004;10:1713-20.
2. Casanova K, Sáez A, Navas T, Vera R, Panizo M, Chiriboga D. Epidemiología de la neumocistosis. Medicina Interna (Caracas). 2006;22:207-26.
3. Benfield TL, Helweg-Larsen J, Bang D, Junge J, Lundgren JD. Prognostic markers of short-term mortality in AIDS-associated Pneumocystis carinii pneumonia. Chest. 2001;119:844-51.
4. Thomas CF Jr, Limper AH. Pneumocystis pneumonia. N Engl J Med. 2004;350:2487-98.
5. Vélez L, Correa LT, Maya MA, Mejía P, Ortega J, Bedoya V, et al. Diagnostic accuracy of bronchoalveolar lavage samples in immunosuppressed patients with suspected pneumonia: Analysis of a protocol. Respir Med. 2007;101:2160-7.
6. Walzer PD, Evans HE, Copas AJ, Edwards SG, Grant AD, Miller RF. Early predictors of mortality from Pneumocystis jirovecii pneumonia in HIV-infected patients: 1985-2006. Clin Infect Dis. 2008;46:625-33.
7. Pulvirenti J, Herrera P, Venkataraman P, Ahmed N. Pneumocystis carinii pneumonia in HIV-infected patients in the HAART era. AIDS Patient Care STDS. 2003;17:261-5.
8. Huang L. Clinical presentation and diagnosis of Pneumocystis pneumonia in HIV-infected patients. En: Walzer PD, Cushion MT, editors. Pneumocystis pneumonia. Third edition. New York: Marcel Dekke; 2005. p. 373.
9. Meyer KC. Bronchoalveolar lavage as a diagnostic tool. Semin Respir Crit Care Med. 2007;28:546-60.
10. Walser PD, Smulian AG. Pneumocystis Species. En: Mandell GL, Bennett JE, Dolin R, editores. Principles and practice of infectious diseases. Seventh edition. Philadelphia, PA: Churchill Livingstone Elsevier; 2010. p. 3377-90.
11. Helweg-Larsen J, Jensen JS, Benfield T, Svendsen UG, Lundgren JD, Lundgren B. Diagnostic use of PCR for detection of Pneumocystis carinii in oral wash samples. J Clin Microbiol. 1998;36:2068-72.
12. Larsen HH, Huang L, Kovacs JA, Crothers K, Silcott VA, Morris A, et al. A prospective, blinded study of quantitative touch-down polymerase chain reaction using oral-wash samples for diagnosis of Pneumocystis pneumonia in HIV-infected patients. J Infect Dis. 2004;189:1679-83.
13. Wakefield AE, Miller RF, Guiver LA, Hopkin JM. Oropharyngeal samples for detection of Pneumocystis carinii by DNA amplification. Q J Med. 1993;86:401-6.
14. Matos O, Costa MC, Lundgren B, Caldeira L, Aguiar P, Antunes F. Effect of oral washes on the diagnosis of Pneumocystis carinii pneumonia with a low parasite burden and on detection of organisms in subclinical infections. Eur J Clin Microbiol Infect Dis. 2001;20:573-5.
15. Atzori C, Agostoni F, Angeli E, Mainini A, Orlando G, Cargnel A. Combined use of blood and oropharyngeal samples for noninvasive diagnosis of Pneumocystis carinii pneumonia using the polymerase chain reaction. Eur J Clin Microbiol Infect Dis. 1998;17:241-6.
16. De Oliveira A, Unnasch TR, Crothers K, Eiser S, Zucchi P, Moir J, et al. Performance of a molecular viability assay for the diagnosis of Pneumocystis pneumonia in HIV-infected patients. Diagn Microbiol Infect Dis. 2007;57:169-76.
17. Nevez G, Totet A, Pautard JC, Raccurt C. Pneumocystis carinii detection using nested-PCR in nasopharyngeal aspirates of immunocompetent infants with bronchiolitis. J Eukaryot Microbiol. 2001;Suppl:122S-3.
18. Ricaurte O, Caldas ML, López F, Gutiérrez L, Nicholls S, Castañeda E. Pneumocystis carinii. Bogotá: Instituto Nacional de Salud; 1992.
19. Correa LT, Durango GE, López E, Hurtado HE, Vélez LA. Evaluación de cuatro técnicas de coloración para el diagnóstico de neumonía por Pneumocystis jirovecii (carinii) en pacientes inmunocomprometidos. Infectio. 2004;8:99.
20. Muñoz CO, Tobón AM, Zuluaga A, Restrepo A, Cano LE, González A. Diagnóstico por PCR anidada de Pneumocystis jirovecii en muestras respiratorias de pacientes con neumonía, infectados con el VIH o sin infectar. Infectio. 2006;10:135-6.
21. Kahn FW, Jones JM. Analysis of bronchoalveolar lavage specimens from immunocompromised patients with a protocol applicable in the microbiology laboratory. J Clin Microbiol. 1988;26:1150-5.
22. Gosey LL, Howard RM, Witebsky FG, Ognibene FP, Wu TC, Gill VJ, et al. Advantages of a modified toluidine blue O stain and bronchoalveolar lavage for the diagnosis of Pneumocystis carinii pneumonia. J Clin Microbiol. 1985;22:803-7.
23. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics. 1977;33:159-74.
24. Cregan P, Yamamoto A, Lum A, van Der Heide T, MacDonald M, Pulliam L. Comparison of four methods for rapid detection of Pneumocystis carinii in respiratory specimens. J Clin Microbiol. 1990;28:2432-6.
25. Bedrossian CW, Mason MR, Gupta PK. Rapid cytologic diagnosis of Pneumocystis: A comparison of effective techniques. Semin Diagn Pathol. 1989;6:245-61.
26. Gal AA, Koss MN, Strigle S, Angritt P. Pneumocystis carinii infection in the acquired immune deficiency syndrome. Semin Diagn Pathol. 1989;6:287-99.
27. Grocott RG. A stain for fungi in tissue sections and smears using Gomori's methenamine-silver nitrate technic. Am J Clin Pathol. 1955;25:975-9.
28. Kim HK, Hughes WT. Comparison of methods for identification of Pneumocystis carinii in pulmonary aspirates. Am J Clin Pathol. 1973;60:462-6.
29. Bava AJ, Moreno D, Bellegarde E. Comparison of 3 tests for the diagnosis of pulmonary pneumocystosis in AIDS patients. Rev Argent Microbiol. 2005;37:150-2.
30. Kovacs JA, Ng VL, Masur H, Leoung G, Hadley WK, Evans G, et al. Diagnosis of Pneumocystis carinii pneumonia: Improved detection in sputum with use of monoclonal antibodies. N Engl J Med. 1988;318:589-93.
31. Caliendo AM, Hewitt PL, Allega JM, Keen A, Ruoff KL, Ferraro MJ. Performance of a PCR assay for detection of Pneumocystis carinii from respiratory specimens. J Clin Microbiol. 1998;36:979-82.
32. Lautenschlager I, Lyytikainen O, Jokipii L, Jokipii A, Maiche A, Ruutu T, et al. Immunodetection of Pneumocystis carinii in bronchoalveolar lavage specimens compared with methenamine silver stain. J Clin Microbiol. 1996;34:728-30.
33. Banoo S, Bell D, Bossuyt P, Herring A, Mabey D, Poole F, et al. Evaluation of diagnostic tests for infectious diseases: General principles. Nat Rev Microbiol. 2006;4 (Suppl.): S20-32.
34. Procop GW, Haddad S, Quinn J, Wilson ML, Henshaw NG, Reller LB, et al. Detection of Pneumocystis jiroveci in respiratory specimens by four staining methods. J Clin Microbiol. 2004;42:3333-5.
35. Aderaye G, Woldeamanuel Y, Asrat D, Lebbad M, Beser J, Worku A, et al. Evaluation of toluidine blue O staining for the diagnosis of Pneumocystis jiroveci in expectorated sputum sample and bronchoalveolar lavage from HIV-infected patients in a tertiary care referral center in Ethiopia. Infection. 2008;36:237-43.
36. Ng VL, Yajko DM, McPhaul LW, Gartner I, Byford B, Goodman CD, et al. Evaluation of an indirect fluorescent-antibody stain for detection of Pneumocystis carinii in respiratory specimens. J Clin Microbiol. 1990;28:975-9.
37. Manterola C, Pineda V. El valor de "p" y la "significación estadística". Aspectos generales y su valor en la práctica clínica. Rev Chil Cir. 2008;60:86-9.
38. Cushion MT, Beck JM. Summary of Pneumocystis research presented at the 7th International Workshop on Opportunistic Protists. J Eukaryot Microbiol. 2001;48(Suppl.):101S-5S.
39. Helweg-Larsen J. Pneumocystis jiroveci. Applied molecular microbiology, epidemiology and diagnosis. Dan Med Bull. 2004;51:251-73.
40. Witebsky FG, Andrews JW, Gill VJ, MacLowry JD. Modified toluidine blue O stain for Pneumocystis carinii: Further evaluation of some technical factors. J Clin Microbiol. 1988;26:774-5.
41. Pinlaor S, Mootsikapun P, Pinlaor P, Phunmanee A, Pipitgool V, Sithithaworn P, et al. PCR diagnosis of Pneumocystis carinii on sputum and bronchoalveolar lavage samples in immuno-compromised patients. Parasitol Res. 2004;94:213-8.
42. Stratton N, Hryniewicki J, Aarnaes SL, Tan G, de la Maza LM, Peterson EM. Comparison of monoclonal antibody and calcofluor white stains for the detection of Pneumocystis carinii from respiratory specimens. J Clin Microbiol. 1991;29:645-7.
43. Aliouat-Denis CM, Martinez A, Aliouat el M, Pottier M, Gantois N, Dei-Cas E. The Pneumocystis life cycle. Mem Inst Oswaldo Cruz. 2009;104:419-26.
44. Gill VJ, Evans G, Stock F, Parrillo JE, Masur H, Kovacs JA. Detection of Pneumocystis carinii by fluorescent-antibody stain using a combination of three monoclonal antibodies. J Clin Microbiol. 1987;25:1837-40.
45. Gracia JD, Miravitlles M, Mayordomo C, Ferrer A, Álvarez A, Bravo C, et al. Empiric treatments impair the diagnostic yield of BAL in HIV-positive patients. Chest. 1997;111:1180-6.
2. Casanova K, Sáez A, Navas T, Vera R, Panizo M, Chiriboga D. Epidemiología de la neumocistosis. Medicina Interna (Caracas). 2006;22:207-26.
3. Benfield TL, Helweg-Larsen J, Bang D, Junge J, Lundgren JD. Prognostic markers of short-term mortality in AIDS-associated Pneumocystis carinii pneumonia. Chest. 2001;119:844-51.
4. Thomas CF Jr, Limper AH. Pneumocystis pneumonia. N Engl J Med. 2004;350:2487-98.
5. Vélez L, Correa LT, Maya MA, Mejía P, Ortega J, Bedoya V, et al. Diagnostic accuracy of bronchoalveolar lavage samples in immunosuppressed patients with suspected pneumonia: Analysis of a protocol. Respir Med. 2007;101:2160-7.
6. Walzer PD, Evans HE, Copas AJ, Edwards SG, Grant AD, Miller RF. Early predictors of mortality from Pneumocystis jirovecii pneumonia in HIV-infected patients: 1985-2006. Clin Infect Dis. 2008;46:625-33.
7. Pulvirenti J, Herrera P, Venkataraman P, Ahmed N. Pneumocystis carinii pneumonia in HIV-infected patients in the HAART era. AIDS Patient Care STDS. 2003;17:261-5.
8. Huang L. Clinical presentation and diagnosis of Pneumocystis pneumonia in HIV-infected patients. En: Walzer PD, Cushion MT, editors. Pneumocystis pneumonia. Third edition. New York: Marcel Dekke; 2005. p. 373.
9. Meyer KC. Bronchoalveolar lavage as a diagnostic tool. Semin Respir Crit Care Med. 2007;28:546-60.
10. Walser PD, Smulian AG. Pneumocystis Species. En: Mandell GL, Bennett JE, Dolin R, editores. Principles and practice of infectious diseases. Seventh edition. Philadelphia, PA: Churchill Livingstone Elsevier; 2010. p. 3377-90.
11. Helweg-Larsen J, Jensen JS, Benfield T, Svendsen UG, Lundgren JD, Lundgren B. Diagnostic use of PCR for detection of Pneumocystis carinii in oral wash samples. J Clin Microbiol. 1998;36:2068-72.
12. Larsen HH, Huang L, Kovacs JA, Crothers K, Silcott VA, Morris A, et al. A prospective, blinded study of quantitative touch-down polymerase chain reaction using oral-wash samples for diagnosis of Pneumocystis pneumonia in HIV-infected patients. J Infect Dis. 2004;189:1679-83.
13. Wakefield AE, Miller RF, Guiver LA, Hopkin JM. Oropharyngeal samples for detection of Pneumocystis carinii by DNA amplification. Q J Med. 1993;86:401-6.
14. Matos O, Costa MC, Lundgren B, Caldeira L, Aguiar P, Antunes F. Effect of oral washes on the diagnosis of Pneumocystis carinii pneumonia with a low parasite burden and on detection of organisms in subclinical infections. Eur J Clin Microbiol Infect Dis. 2001;20:573-5.
15. Atzori C, Agostoni F, Angeli E, Mainini A, Orlando G, Cargnel A. Combined use of blood and oropharyngeal samples for noninvasive diagnosis of Pneumocystis carinii pneumonia using the polymerase chain reaction. Eur J Clin Microbiol Infect Dis. 1998;17:241-6.
16. De Oliveira A, Unnasch TR, Crothers K, Eiser S, Zucchi P, Moir J, et al. Performance of a molecular viability assay for the diagnosis of Pneumocystis pneumonia in HIV-infected patients. Diagn Microbiol Infect Dis. 2007;57:169-76.
17. Nevez G, Totet A, Pautard JC, Raccurt C. Pneumocystis carinii detection using nested-PCR in nasopharyngeal aspirates of immunocompetent infants with bronchiolitis. J Eukaryot Microbiol. 2001;Suppl:122S-3.
18. Ricaurte O, Caldas ML, López F, Gutiérrez L, Nicholls S, Castañeda E. Pneumocystis carinii. Bogotá: Instituto Nacional de Salud; 1992.
19. Correa LT, Durango GE, López E, Hurtado HE, Vélez LA. Evaluación de cuatro técnicas de coloración para el diagnóstico de neumonía por Pneumocystis jirovecii (carinii) en pacientes inmunocomprometidos. Infectio. 2004;8:99.
20. Muñoz CO, Tobón AM, Zuluaga A, Restrepo A, Cano LE, González A. Diagnóstico por PCR anidada de Pneumocystis jirovecii en muestras respiratorias de pacientes con neumonía, infectados con el VIH o sin infectar. Infectio. 2006;10:135-6.
21. Kahn FW, Jones JM. Analysis of bronchoalveolar lavage specimens from immunocompromised patients with a protocol applicable in the microbiology laboratory. J Clin Microbiol. 1988;26:1150-5.
22. Gosey LL, Howard RM, Witebsky FG, Ognibene FP, Wu TC, Gill VJ, et al. Advantages of a modified toluidine blue O stain and bronchoalveolar lavage for the diagnosis of Pneumocystis carinii pneumonia. J Clin Microbiol. 1985;22:803-7.
23. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics. 1977;33:159-74.
24. Cregan P, Yamamoto A, Lum A, van Der Heide T, MacDonald M, Pulliam L. Comparison of four methods for rapid detection of Pneumocystis carinii in respiratory specimens. J Clin Microbiol. 1990;28:2432-6.
25. Bedrossian CW, Mason MR, Gupta PK. Rapid cytologic diagnosis of Pneumocystis: A comparison of effective techniques. Semin Diagn Pathol. 1989;6:245-61.
26. Gal AA, Koss MN, Strigle S, Angritt P. Pneumocystis carinii infection in the acquired immune deficiency syndrome. Semin Diagn Pathol. 1989;6:287-99.
27. Grocott RG. A stain for fungi in tissue sections and smears using Gomori's methenamine-silver nitrate technic. Am J Clin Pathol. 1955;25:975-9.
28. Kim HK, Hughes WT. Comparison of methods for identification of Pneumocystis carinii in pulmonary aspirates. Am J Clin Pathol. 1973;60:462-6.
29. Bava AJ, Moreno D, Bellegarde E. Comparison of 3 tests for the diagnosis of pulmonary pneumocystosis in AIDS patients. Rev Argent Microbiol. 2005;37:150-2.
30. Kovacs JA, Ng VL, Masur H, Leoung G, Hadley WK, Evans G, et al. Diagnosis of Pneumocystis carinii pneumonia: Improved detection in sputum with use of monoclonal antibodies. N Engl J Med. 1988;318:589-93.
31. Caliendo AM, Hewitt PL, Allega JM, Keen A, Ruoff KL, Ferraro MJ. Performance of a PCR assay for detection of Pneumocystis carinii from respiratory specimens. J Clin Microbiol. 1998;36:979-82.
32. Lautenschlager I, Lyytikainen O, Jokipii L, Jokipii A, Maiche A, Ruutu T, et al. Immunodetection of Pneumocystis carinii in bronchoalveolar lavage specimens compared with methenamine silver stain. J Clin Microbiol. 1996;34:728-30.
33. Banoo S, Bell D, Bossuyt P, Herring A, Mabey D, Poole F, et al. Evaluation of diagnostic tests for infectious diseases: General principles. Nat Rev Microbiol. 2006;4 (Suppl.): S20-32.
34. Procop GW, Haddad S, Quinn J, Wilson ML, Henshaw NG, Reller LB, et al. Detection of Pneumocystis jiroveci in respiratory specimens by four staining methods. J Clin Microbiol. 2004;42:3333-5.
35. Aderaye G, Woldeamanuel Y, Asrat D, Lebbad M, Beser J, Worku A, et al. Evaluation of toluidine blue O staining for the diagnosis of Pneumocystis jiroveci in expectorated sputum sample and bronchoalveolar lavage from HIV-infected patients in a tertiary care referral center in Ethiopia. Infection. 2008;36:237-43.
36. Ng VL, Yajko DM, McPhaul LW, Gartner I, Byford B, Goodman CD, et al. Evaluation of an indirect fluorescent-antibody stain for detection of Pneumocystis carinii in respiratory specimens. J Clin Microbiol. 1990;28:975-9.
37. Manterola C, Pineda V. El valor de "p" y la "significación estadística". Aspectos generales y su valor en la práctica clínica. Rev Chil Cir. 2008;60:86-9.
38. Cushion MT, Beck JM. Summary of Pneumocystis research presented at the 7th International Workshop on Opportunistic Protists. J Eukaryot Microbiol. 2001;48(Suppl.):101S-5S.
39. Helweg-Larsen J. Pneumocystis jiroveci. Applied molecular microbiology, epidemiology and diagnosis. Dan Med Bull. 2004;51:251-73.
40. Witebsky FG, Andrews JW, Gill VJ, MacLowry JD. Modified toluidine blue O stain for Pneumocystis carinii: Further evaluation of some technical factors. J Clin Microbiol. 1988;26:774-5.
41. Pinlaor S, Mootsikapun P, Pinlaor P, Phunmanee A, Pipitgool V, Sithithaworn P, et al. PCR diagnosis of Pneumocystis carinii on sputum and bronchoalveolar lavage samples in immuno-compromised patients. Parasitol Res. 2004;94:213-8.
42. Stratton N, Hryniewicki J, Aarnaes SL, Tan G, de la Maza LM, Peterson EM. Comparison of monoclonal antibody and calcofluor white stains for the detection of Pneumocystis carinii from respiratory specimens. J Clin Microbiol. 1991;29:645-7.
43. Aliouat-Denis CM, Martinez A, Aliouat el M, Pottier M, Gantois N, Dei-Cas E. The Pneumocystis life cycle. Mem Inst Oswaldo Cruz. 2009;104:419-26.
44. Gill VJ, Evans G, Stock F, Parrillo JE, Masur H, Kovacs JA. Detection of Pneumocystis carinii by fluorescent-antibody stain using a combination of three monoclonal antibodies. J Clin Microbiol. 1987;25:1837-40.
45. Gracia JD, Miravitlles M, Mayordomo C, Ferrer A, Álvarez A, Bravo C, et al. Empiric treatments impair the diagnostic yield of BAL in HIV-positive patients. Chest. 1997;111:1180-6.
How to Cite
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Rodiño J, Rincón N, Aguilar YA, Rueda ZV, Herrera M, Vélez LA. Microscopic diagnosis of Pneumocystis jirovecii pneumonia in bronchoalveolar lavage and oropharyngeal wash samples of immunocompromised patients with pneumonia. biomedica [Internet]. 2011 Mar. 7 [cited 2024 May 18];31(2):222-31. Available from: https://revistabiomedica.org/index.php/biomedica/article/view/307
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