Skip to main content

Advertisement

SpringerLink
Log in

A new method for the detection of Pneumocystis jirovecii using flow cytometry

  • Article
  • Published:
European Journal of Clinical Microbiology & Infectious Diseases Aims and scope Submit manuscript

Abstract

Pneumocystis jirovecii is an opportunistic pathogen responsible for severe pneumonia in immunocompromised patients. Its diagnosis has been based upon direct microscopy either by classic staining (Gomori Grocott) or by epifluorescence microscopy (immunofluorescence staining, IFS), both of which are time-consuming and low on sensitivity. Our aim was to develop a flow cytometric (FC) protocol for the detection of P. jirovecii on respiratory samples. In our study, 420 respiratory samples were analysed in parallel by IFS and FC, and compared from clinical diagnosis to its resolution upon specific anti-Pneumocystis therapy. The optimum specific antibody concentration for FC analysis was determined to be 10 µg/ml, without any cross-reactions to bacteria or fungi. All positive cases detected by IFS were positive by FC; however, FC classified eight samples to be positive which were classified as negative by routine technique. These samples were obtained from patients with respiratory symptoms who responded favourably to Pneumocystis-specific therapy and were subsequently considered to be true-positives. Using clinical diagnosis as a reference method, FC showed 100% sensitivity and specificity, whereas IFS showed 90.9% sensitivity and 100% specificity. According to our results, a new diagnostic approach is now available to detect P. jirovecii in respiratory samples.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Murray PR, Rosenthal KS, Pfaller MA (2005) Fungal classification, structure, and replication. In: Murray PR, Rosenthal KS, Pfaller MA (eds) Medical microbiology, 5th edn. Mosby, Philadelphia, Pennsylvania, pp 67–73

    Google Scholar 

  2. Stringer JR, Beard CB, Miller RF, Wakefield AE (2002) A new name (Pneumocystis jiroveci) for Pneumocystis from humans. Emerg Infect Dis 8:891–896

    PubMed  Google Scholar 

  3. Kelley CF, Checkley W, Mannino DM, Franco-Paredes C, Del Rio C, Holguin F (2009) Trends in hospitalizations for AIDS-associated Pneumocystis jirovecii pneumonia in the United States (1986 to 2005). Chest 136:190–197

    Article  PubMed  Google Scholar 

  4. Su YS, Lu JJ, Perng CL, Chang FY (2008) Pneumocystis jirovecii pneumonia in patients with and without human immunodeficiency virus infection. J Microbiol Immunol Infect 41:478–482

    PubMed  Google Scholar 

  5. Wazir JF, Ansari NA (2004) Pneumocystis carinii infection. Update and review. Arch Pathol Lab Med 128:1023–1027

    PubMed  Google Scholar 

  6. Krajicek BJ, Thomas CF Jr, Limper AH (2009) Pneumocystis pneumonia: current concepts in pathogenesis, diagnosis, and treatment. Clin Chest Med 30:265–278

    Article  PubMed  Google Scholar 

  7. Morris A, Sciurba FC, Norris KA (2008) Pneumocystis: a novel pathogen in chronic obstructive pulmonary disease? COPD 5:43–51

    Article  PubMed  Google Scholar 

  8. Maschmeyer G, Beinert T, Buchheidt D, Cornely OA, Einsele H, Heinz W, Heussel CP, Kahl C, Kiehl M, Lorenz J, Hof H, Mattiuzzi G (2009) Diagnosis and antimicrobial therapy of lung infiltrates in febrile neutropenic patients: guidelines of the infectious diseases working party of the German Society of Haematology and Oncology. Eur J Cancer 45:2462–2472

    Article  CAS  PubMed  Google Scholar 

  9. Morris A, Netravali M, Kling HM, Shipley T, Ross T, Sciurba FC, Norris KA (2008) Relationship of pneumocystis antibody response to severity of chronic obstructive pulmonary disease. Clin Infect Dis 47:e64–e68

    Article  PubMed  Google Scholar 

  10. Nevez G, Chabé M, Rabodonirina M, Virmaux M, Dei-Cas E, Hauser PM, Totet A (2008) Nosocomial Pneumocystis jirovecii infections. Parasite 15:359–365

    CAS  PubMed  Google Scholar 

  11. Procop GW, Haddad S, Quinn J, Wilson ML, Henshaw NG, Reller LB, Artymyshyn RL, Katanik MT, Weinstein MP (2004) Detection of Pneumocystis jiroveci in respiratory specimens by four staining methods. J Clin Microbiol 42:3333–3335

    Article  CAS  PubMed  Google Scholar 

  12. Durand-Joly I, Chabé M, Soula F, Delhaes L, Camus D, Dei-Cas E (2005) Molecular diagnosis of Pneumocystis pneumonia. FEMS Immunol Med Microbiol 45:405–410

    Article  CAS  PubMed  Google Scholar 

  13. Jiancheng W, Minjun H, Yi-jun A, Lan S, Zengzhu G, Jianrong S, Xixiong K (2009) Screening Pneumocystis carinii pneumonia in non-HIV-infected immunocompromised patients using polymerase chain reaction. Diagn Microbiol Infect Dis 64:396–401

    Article  PubMed  Google Scholar 

  14. Alvarez-Martínez MJ, Miró JM, Valls ME, Moreno A, Rivas PV, Solé M, Benito N, Domingo P, Muñoz C, Rivera E, Zar HJ, Wissmann G, Diehl AR, Prolla JC, de Anta MT, Gatell JM, Wilson PE, Meshnick SR (2006) Sensitivity and specificity of nested and real-time PCR for the detection of Pneumocystis jiroveci in clinical specimens. Diagn Microbiol Infect Dis 56:153–160

    Article  PubMed  Google Scholar 

  15. Robberts FJL, Liebowitz LD, Chalkley LJ (2007) Polymerase chain reaction detection of Pneumocystis jiroveci: evaluation of 9 assays. Diagn Microbiol Infect Dis 58:385–392

    Article  CAS  PubMed  Google Scholar 

  16. Barbosa JM, Costa-de-Oliveira S, Rodrigues AG, Hanscheid T, Shapiro H, Pina-Vaz C (2008) A flow cytometric protocol for detection of Cryptosporidium spp. Cytometry A 73:44–47

    PubMed  Google Scholar 

  17. Barbosa J, Costa-de-Oliveira S, Rodrigues AG, Pina-Vaz C (2008) Optimization of a flow cytometry protocol for detection and viability assessment of Giardia lamblia. Travel Med Infect Dis 6:234–239

    Article  PubMed  Google Scholar 

  18. Barbosa J, Rodrigues AG, Pina-Vaz C (2009) Cytometric approach for detection of Encephalitozoon intestinalis, an emergent agent. Clin Vaccine Immunol 16:1021–1024

    Article  CAS  PubMed  Google Scholar 

  19. Libertin CR, Woloschak GE, Wilson WR, Smith TF (1984) Analysis of Pneumocystis carinii cysts with a fluorescence-activated cell sorter. J Clin Microbiol 20:877–880

    CAS  PubMed  Google Scholar 

  20. Lapinsky SE, Glencross D, Car NG, Kallenbach JM, Zwi S (1991) Quantification and assessment of viability of Pneumocystis carinii organisms by flow cytometry. J Clin Microbiol 29:911–915

    CAS  PubMed  Google Scholar 

  21. D’Avignon LC, Schofield CM, Hospenthal DR (2008) Pneumocystis pneumonia. Semin Respir Crit Care Med 29:132–140

    Article  PubMed  Google Scholar 

  22. Morris A, Wei K, Afshar K, Huang L (2008) Epidemiology and clinical significance of Pneumocystis colonization. J Infect Dis 197:10–17

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

J. Barbosa was supported by a PhD grant (SFRH/BD/44346/2008) from Fundação para a Ciência e a Tecnologia (FCT).

Author information

Authors and Affiliations

  1. Department of Microbiology, Porto Faculty of Medicine, University of Porto, Al. Prof. Hernâni Monteiro, 4200-319, Porto, Portugal

    J. Barbosa, C. Bragada, S. Costa-de-Oliveira, E. Ricardo, A. G. Rodrigues & C. Pina-Vaz

  2. Escola Superior de Saúde Jean Piaget, Vila Nova de Gaia, Portugal

    J. Barbosa

  3. Burn Unit, Department of Plastic and Reconstructive Surgery, Hospital S. João, Porto, Portugal

    A. G. Rodrigues

  4. Department of Microbiology, Hospital S. João, Porto, Portugal

    C. Pina-Vaz

  5. Cardiovascular Research and Development Unit, Faculty of Medicine, University of Porto, Porto, Portugal

    S. Costa-de-Oliveira, E. Ricardo, A. G. Rodrigues & C. Pina-Vaz

Authors
  1. J. Barbosa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C. Bragada
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S. Costa-de-Oliveira
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. E. Ricardo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A. G. Rodrigues
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C. Pina-Vaz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. Barbosa.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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 29, 1147–1152 (2010). https://doi.org/10.1007/s10096-010-0980-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10096-010-0980-5

Keywords

Search

Navigation