Elif Gözde DURGUT, Semra KUŞTİMUR, Merve AYDIN, Ayşe KALKANCI, Funda DOĞURMAN AL

Comparison of real-time PCR, indirect immunofluorescence antibody assay, and different staining techniques for the diagnosis of Pneumocystis jirovecii

Aim: Pneumocystis pneumonia (PCP), caused by Pneumocystis jirovecii (P. jirovecii), is an opportunistic infection with a severeprogression, often observed in immunocompromised patients. The diagnosis is difficult due to the non-specific clinical andradiological findings. Therefore, rapid and accurate diagnosis of the agent is important in terms of timely implementation of thetreatment. In this study, it was aimed to detect P. jirovecii by Giemsa staining, Modified Toluidine Blue O staining (MTolB), indirectimmunofluorescent antibody (IIFA) assay, and real-time polymerase chain reaction (PCR) in clinical samples obtained from patientssuspected of having PCP.Material and Methods: Respiratory tract samples (23 oral wash, 19 bronchoalveolar lavage fluid, and eight induced sputum samples)of 50 patients referred to the Microbiology Laboratory of Gazi University Health Application and Research Hospital with the suspicionof PCP were analyzed. The presence of P. jirovecii in the respiratory tract samples was investigated by Giemsa staining, MTolBstaining, IIFA (Pneumocell, Cellabs Pty Ltd, Australia), and real-time PCR (the primers targeting the DHFR gene).Results: Of the 50 samples included in the study, four (8%) with MTolB, five (10%) with Giemsa, seven (14%) with IIFA, and seven(14%) with real-time PCR were positive. When real-time PCR was accepted as the gold standard, the sensitivity and specificity valueswere found to be 85.7% and 97.7%, respectively for IIFA, 71.4% and 100%, respectively for Giemsa and 57% and 100%, respectively forMTolB. There was almost perfect agreement between the results of real-time PCR and IIFA (κ=0.92). In the comparison between PCRand cytochemical staining methods, Giemsa had almost perfect agreement with PCR (κ=0.92) and had a higher coefficient comparedto MTolB (κ=0.88).Conclusion: It is considered that it would be more beneficial to use IIFA and real-time PCR tests together in the diagnosis of P.jirovecii.

PDF

___

1. Kaur R, Wadhwa A, Bhalla P, et al. Pneumocystis pneumonia in HIV patients: a diagnostic challenge till date. Med Mycol 2015;53:587-92.
2. Barbosa J, Bragada C, Costa-de-Oliveira S, et al. A new method for the detection of Pneumocystis jirovecii using flow cytometry. Eur J Clin Microbiol Infect Dis 2010;29:1147-52.
3. Tasaka S. Pneumocystis pneumonia in Human Immunodeficiency Virus-infected adults and adolescents: Current concepts and future directions. Clin Med Insights Circ Respir Pulm Med 2015;9:19-28.
4. Ma L, Cissé OH, Kovacs JA. A molecular window into the biology and epidemiology of Pneumocystis spp. Clin Microbiol Rev 2018;31:e00009-18.
5. Edman JC, Kovacs JA, Masur H, Santi DV, Elwood HJ, Sogin ML. Ribosomal RNA sequence shows Pneumocystis carinii to be a member of the fungi. Nature 1988;334:519-22.
6. Stringer JR, Beard CB, Miller RF, Wakefield AE. A new name (Pneumocystis jiroveci) for Pneumocystis from humans. Emerg Infect Dis 2002;8:891-6.
7. Uemura N, Makimura K, Onozaki M, Otsuka Y, Shibuya Y, Yazaki H, et al. Development of a loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia. J Med Microbiol 2008;57:50-7.
8. Morris A, Norris KA. Colonization by Pneumocystis jirovecii and its role in disease. Clin Microbiol Rev 2012;25:297-317.
9. Tasaka S, Tokuda H. Pneumocystis jirovecii pneumonia in non-HIV-infected patients in the era of novel immunosuppressive therapies. J Infect Chemother 2012;18:793-806.
10. Robert-Gangneux F, Belaz S, Revest M, et al. Diagnosis Pneumocystis jirovecii pneumonia in immunocompromised patients by real-time PCR: a 4-year prospective study. J Clin Microbiol 2014;52:3370-6.
11. Tasaka S, Hasegawa N, Kobayashi S, et al. Serum indicators for the diagnosis of pneumocystis pneumonia. Chest 2007;131:1173-80.
12. Samuel CM, Whitelaw A, Corcoran C, et al. Improved detection of Pneumocystis jirovecii in upper and lower respiratory tract specimens from children with suspected pneumocystis pneumonia using real-time PCR: a prospective study. BMC Infect Dis 2011;11:329.
13. Oren I, Hardak I, Finkelstein R, et al. Polymerase chain reaction-based detection of Pneumocystis jirovecii in bronchoalveolar lavage fluid for the diagnosis of pneumocystis pneumonia. Am J Med Sci 2011;342:182-5.
14. Roux A, Canet E, Valade S, et al. Pneumocystis jirovecii pneumonia in patients with or without AIDS, France. Emerg Infect Dis 2014;20:1490-7.
15. Linssen CF, Jacobs JA, Beckers P, et al. Interlaboratory comparison of three different real-time PCR assays for the detection of Pneumocystis jiroveci in bronchoalveolar lavage fluid samples. J Med Microbiol 2006;55:1229-35.
16. Sokulska M, Kicia M, Wesołowska M, et al. Pneumocystis jirovecii—from a commensal to pathogen: clinical and diagnostic review. Parasitol Res 2015;114:3577-85.
17. Durand-Joly I, Chabé M, Soula F, et al. Molecular diagnosis of Pneumocystis pneumonia. FEMS Immunol Med Microbiol 2005;45:405-10.
18. Robberts FJ, Liebowitz LD, Chalkley LJ. Polymerase chain reaction detection of Pneumocystis jiroveci: evaluation of 9 assays. Diagn Microbiol Infect Dis 2007;58:385-92.
19. Rohner P, Jacomo V, Studer R, et al. Detection of Pneumocystis jirovecii by two staining methods and two quantitative PCR assays. Infection 2009;37:261- 5.
20. Gosey LL, Howard RM, Witebsky FG, 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.
21. Walker J, Conner G, Ho J, et al. Giemsa staining for cysts and trophozoites of Pneumocystis carinii. J Clin Pathol 1989;42:432-4.
22. IBM Corp. Released 2010. IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY: IBM Corp.
23. Altman DG. Practical statistics for medical research. 1 st editon. Boca Raton: Chapman and Hall/CRC Press; 1990.
24. Yanık K, Karadağ A, Usta E, et al. Evaluation of respiratory samples results of the direct fluorescent antibody test sending with suspicion of Pneumocystis jirovecii pneumonia to the parasitology laboratory of Ondokuz Mayıs University Medical School between the years 2003-2011. Türk Mikrobiyol Cem Derg 2012;42:132-6.
25. Flori P, Bellete B, Durand F, et al. Comparison between real-time PCR, conventional PCR and different staining techniques for diagnosing Pneumocystis jiroveci pneumonia from bronchoalveolar lavage specimens. J Med Microbiol 2004;53:603-7.
26. Tekinşen FF, Koç AN. Investigation of Pneumocystis jirovecii in clinical specimens by different methods. Mikrobiyol Bul 2013;47:658-67.
27. Tomás AL, Matos O. Pneumocystis jirovecii pneumonia: Current advances in laboratory diagnosis. OBM Genetics 2018;2:49.
28. White PL, Backx M, Barnes RA. Diagnosis and management of Pneumocystis jirovecii infections. Expert Rev Anti Infect Ther 2017;15:435-47.
29. Reid AB, Chen SC, Worth LJ. Pneumocystis jirovecii pneumonia in non-HIV infected patients: new risks and diagnostic tools. Curr Opin Infect Dis 2011;24:534-44.
30. Tuncer S, Ergüven S, Kocagöz S, et al. Comparison of cytochemical staining, immunofluorescence and PCR for diagnosis of Pneumocystis carinii on sputum samples. Scand J Infect Dis 1998;30:125-8.
31. Fillaux J, Malvy S, Alvarez M, et al. Accuracy of a routine real-time PCR assay for the diagnosis of Pneumocystis jirovecii pneumonia. J Microbiol Methods 2008;75:258-61.
32. Pennington K, Wilson J, Limper AH, et al. Positive Pneumocystis jirovecii sputum PCR results with negative bronchoscopic PCR results in suspected Pneumocystis pneumonia. Canadian Respiratory Journal. Published Online: April 3, 2018.
33. Kern M, Böhm S, Deml L, et al. Inhibition of Legionella pneumophila PCR in respiratory samples: a quantitative approach. J Microbiol Methods 2009;79:189-93.
34. Döskaya M, Caner A, Degirmenci A, et al. Degree and frequency of inhibition in a routine real-time PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey. J Med Microbiol 2011;60: 937-44.