Comparison of Quantitative Real-time PCR and Direct Immunofluorescence for the Detection of Pneumocystis Jirovecii

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2017 Jul 7

PMID 28683092

Citations 11

Authors

Bhavani Moodley

Stefano Tempia

John Andrew Frean

Affiliations

Soon will be listed here.

Abstract

Background: Pneumocystis pneumonia (PCP) is a serious risk for HIV-positive patients. Asymptomatic infection or colonisation with P. jirovecii has been shown to occur frequently. PCR assays frequently identify such cases, due to their high sensitivity. Quantitative real-time PCR (qPCR) gene copy number cut-off values have been suggested to differentiate colonisation and infection; these need to be standardised for routine use. We compared the results of qPCR with an immunofluorescence assay (IFA) to determine a specific cut-off value.

Methods: From March 2005 through June 2009, induced sputum specimens were collected from adult patients who were clinically suspected of having PCP, at the Chris Hani Baragwanath Hospital in Gauteng, South Africa. Laboratory diagnosis of PCP was done by a conventional direct IFA and a qPCR assay. A receiver operating characteristic (ROC) analysis was performed to determine a suitable copy number cut-off value.

Results: P. jirovecii was identified in 51% (156/305) and 67% (204/305) of specimens using IFA and qPCR, respectively. The cut-off value for the qPCR that best predicted the IFA results was 78 copies/5 μl (area under ROC curve 0.92). The sensitivity and specificity of qPCR using this cut-off was 94.6% and 89.1%, respectively, compared with the IFA.

Discussion: The results of the ROC curve analysis indicate an excellent predictive value of the qPCR using the proposed cut-off. However, the IFA test is an imperfect gold standard and so this cut-off should not be used in isolation; clinical data should also contribute to the interpretation of the qPCR result.

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References

Thomas Jr C, Limper A . Pneumocystis pneumonia. N Engl J Med. 2004; 350(24):2487-98. DOI: 10.1056/NEJMra032588. View

Carvalho M, Tondella M, McCaustland K, Weidlich L, McGee L, Mayer L . Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA. J Clin Microbiol. 2007; 45(8):2460-6. PMC: 1951257. DOI: 10.1128/JCM.02498-06. View

Fauchier T, Hasseine L, Gari-Toussaint M, Casanova V, Marty P, Pomares C . Detection of Pneumocystis jirovecii by Quantitative PCR To Differentiate Colonization and Pneumonia in Immunocompromised HIV-Positive and HIV-Negative Patients. J Clin Microbiol. 2016; 54(6):1487-1495. PMC: 4879311. DOI: 10.1128/JCM.03174-15. View

Fillaux J, Malvy S, Alvarez M, Fabre R, Cassaing S, Marchou B . Accuracy of a routine real-time PCR assay for the diagnosis of Pneumocystis jirovecii pneumonia. J Microbiol Methods. 2008; 75(2):258-61. DOI: 10.1016/j.mimet.2008.06.009. View

Robert-Gangneux F, Belaz S, Revest M, Tattevin P, Jouneau S, Decaux O . Diagnosis of Pneumocystis jirovecii pneumonia in immunocompromised patients by real-time PCR: a 4-year prospective study. J Clin Microbiol. 2014; 52(9):3370-6. PMC: 4313164. DOI: 10.1128/JCM.01480-14. View

Sing A, Trebesius K, Roggenkamp A, Russmann H, Tybus K, Pfaff F . Evaluation of diagnostic value and epidemiological implications of PCR for Pneumocystis carinii in different immunosuppressed and immunocompetent patient groups. J Clin Microbiol. 2000; 38(4):1461-7. PMC: 86465. DOI: 10.1128/JCM.38.4.1461-1467.2000. View

Khan M, Farrag N, Butcher P . Diagnosis of Pneumocystis carinii pneumonia: immunofluorescence staining, simple PCR or nPCR. J Infect. 1999; 39(1):77-80. DOI: 10.1016/s0163-4453(99)90106-8. View

Huang L, Crothers K, Morris A, Groner G, Fox M, Turner J . Pneumocystis colonization in HIV-infected patients. J Eukaryot Microbiol. 2004; 50 Suppl:616-7. DOI: 10.1111/j.1550-7408.2003.tb00651.x. View

Muhlethaler K, Bogli-Stuber K, Wasmer S, von Garnier C, Dumont P, Rauch A . Quantitative PCR to diagnose Pneumocystis pneumonia in immunocompromised non-HIV patients. Eur Respir J. 2011; 39(4):971-8. DOI: 10.1183/09031936.00095811. View

10.

Calderon E, Gutierrez-Rivero S, Durand-Joly I, Dei-Cas E . Pneumocystis infection in humans: diagnosis and treatment. Expert Rev Anti Infect Ther. 2010; 8(6):683-701. DOI: 10.1586/eri.10.42. View

11.

Huggett J, Taylor M, Kocjan G, Evans H, Morris-Jones S, Gant V . Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients. Thorax. 2007; 63(2):154-9. DOI: 10.1136/thx.2007.081687. View

12.

Alanio A, Desoubeaux G, Sarfati C, Hamane S, Bergeron A, Azoulay E . Real-time PCR assay-based strategy for differentiation between active Pneumocystis jirovecii pneumonia and colonization in immunocompromised patients. Clin Microbiol Infect. 2010; 17(10):1531-7. DOI: 10.1111/j.1469-0691.2010.03400.x. View

13.

Helweg-Larsen J . S-adenosylmethionine in plasma to test for Pneumocystis carinii pneumonia. Lancet. 2003; 361(9365):1237. DOI: 10.1016/S0140-6736(03)13027-9. View

14.

Fujisawa T, Suda T, Matsuda H, Inui N, Nakamura Y, Sato J . Real-time PCR is more specific than conventional PCR for induced sputum diagnosis of Pneumocystis pneumonia in immunocompromised patients without HIV infection. Respirology. 2009; 14(2):203-9. DOI: 10.1111/j.1440-1843.2008.01457.x. View

15.

Medrano F, Montes-Cano M, Conde M, de la Horra C, Respaldiza N, Gasch A . Pneumocystis jirovecii in general population. Emerg Infect Dis. 2005; 11(2):245-50. PMC: 3320436. DOI: 10.3201/eid1102.040487. View

16.

Larsen H, Masur H, Kovacs J, Gill V, Silcott V, Kogulan P . Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosing Pneumocystis carinii pneumonia. J Clin Microbiol. 2002; 40(2):490-4. PMC: 153364. DOI: 10.1128/JCM.40.2.490-494.2002. View

17.

Procop G, Haddad S, Quinn J, Wilson M, Henshaw N, Reller L . Detection of Pneumocystis jiroveci in respiratory specimens by four staining methods. J Clin Microbiol. 2004; 42(7):3333-5. PMC: 446244. DOI: 10.1128/JCM.42.7.3333-3335.2004. View

18.

Thomas Jr C, Limper A . Current insights into the biology and pathogenesis of Pneumocystis pneumonia. Nat Rev Microbiol. 2007; 5(4):298-308. DOI: 10.1038/nrmicro1621. View

19.

Takahashi T, Goto M, Endo T, Nakamura T, Yusa N, Sato N . Pneumocystis carinii carriage in immunocompromised patients with and without human immunodeficiency virus infection. J Med Microbiol. 2002; 51(7):611-692. DOI: 10.1099/0022-1317-51-7-611. View

20.

Dini L, du Plessis M, Frean J, Fernandez V . High prevalence of dihydropteroate synthase mutations in Pneumocystis jirovecii isolated from patients with Pneumocystis pneumonia in South Africa. J Clin Microbiol. 2010; 48(6):2016-21. PMC: 2884465. DOI: 10.1128/JCM.02004-09. View