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. 1997 Jul;35(7):1691–1695. doi: 10.1128/jcm.35.7.1691-1695.1997

Simplified sample processing combined with a sensitive one-tube nested PCR assay for detection of Pneumocystis carinii in respiratory specimens.

A Mathis 1, R Weber 1, H Kuster 1, R Speich 1
PMCID: PMC229823  PMID: 9196175

Abstract

Early diagnosis of Pneumocystis carinii pneumonia, a life-threatening complication in immunosuppressed patients, may lower morbidity and mortality. We have developed a one-tube nested PCR assay for the detection of P. carinii in respiratory specimens. Four primers were selected from the sequence of the small-subunit rRNA gene of P. carinii to amplify a 265-bp fragment, and their specificities for P. carinii were confirmed by both theoretical evaluations (by computer-assisted comparison with the sequences in GenBank) and empirical evaluations (with DNA from medically important fungi and diagnostic samples). The assay was optimized for routine diagnostic use. Processing of the clinical samples is rapid and simple (digestion with proteinase K directly in PCR buffer at room temperature in the presence of 10% Chelex 100 and no further purification steps). Bovine serum albumin (1 mg/ml) and glycerol (10%) in the amplification buffer reduced the number of samples inhibitory to the PCR, as assessed by control reactions containing a size-modified target. A total of 749 clinical specimens (312 bronchoalveolar lavage, 403 sputum or induced sputum, and 34 other specimens) from 507 patients (295 human immunodeficiency virus [HIV]-infected and 164 non-HIV-infected patients and 48 patients whose HIV status was unknown) were tested by PCR, and the results were compared with those of an indirect immunofluorescence assay (IFA). Concordant results were obtained for 732 samples (646 negative and 86 positive). There were 17 discrepant results: 12 were PCR positive and IFA negative, and 5 were PCR negative and IFA positive. After resolution of the discrepant results by review of the patients' clinical data, the sensitivity and specificity were 94.8 and 99.1%, respectively, for PCR and 93.8 and 100%, respectively, for IFA. In conclusion, the short procedure time and the technical ease of this PCR assay render it suitable for implementation in routine diagnostic laboratories.

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Selected References

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  1. Edman J. C., Kovacs J. A., Masur H., Santi D. V., Elwood H. J., Sogin M. L. Ribosomal RNA sequence shows Pneumocystis carinii to be a member of the fungi. Nature. 1988 Aug 11;334(6182):519–522. doi: 10.1038/334519a0. [DOI] [PubMed] [Google Scholar]
  2. Elvin K. Laboratory diagnosis and occurrence of Pneumocystis carinii. Scand J Infect Dis Suppl. 1994;94:1–34. [PubMed] [Google Scholar]
  3. Evans R., Joss A. W., Parratt D., Pennington T. H., Ho-Yen D. O. The role of a nested polymerase chain reaction in the diagnosis of Pneumocystis carinii pneumonia. Clin Mol Pathol. 1995 Dec;48(6):M347–M350. doi: 10.1136/mp.48.6.m347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Evans R., Joss A. W., Pennington T. H., Ho-Yen D. O. The use of a nested polymerase chain reaction for detecting Pneumocystis carinii from lung and blood in rat and human infection. J Med Microbiol. 1995 Mar;42(3):209–213. doi: 10.1099/00222615-42-3-209. [DOI] [PubMed] [Google Scholar]
  5. Kovacs J. A., Ng V. L., Masur H., Leoung G., Hadley W. K., Evans G., Lane H. C., Ognibene F. P., Shelhamer J., Parrillo J. E. Diagnosis of Pneumocystis carinii pneumonia: improved detection in sputum with use of monoclonal antibodies. N Engl J Med. 1988 Mar 10;318(10):589–593. doi: 10.1056/NEJM198803103181001. [DOI] [PubMed] [Google Scholar]
  6. Lu J. J., Bartlett M. S., Smith J. W., Lee C. H. Typing of Pneumocystis carinii strains with type-specific oligonucleotide probes derived from nucleotide sequences of internal transcribed spacers of rRNA genes. J Clin Microbiol. 1995 Nov;33(11):2973–2977. doi: 10.1128/jcm.33.11.2973-2977.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Lu J. J., Chen C. H., Bartlett M. S., Smith J. W., Lee C. H. Comparison of six different PCR methods for detection of Pneumocystis carinii. J Clin Microbiol. 1995 Oct;33(10):2785–2788. doi: 10.1128/jcm.33.10.2785-2788.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lundgren J. D., Barton S. E., Lazzarin A., Danner S., Goebel F. D., Pehrson P., Mulcahy F., Kosmidis J., Pedersen C., Phillips A. N. Factors associated with the development of Pneumocystis carinii pneumonia in 5,025 European patients with AIDS. AIDS in Europe Study Group. Clin Infect Dis. 1995 Jul;21(1):106–113. doi: 10.1093/clinids/21.1.106. [DOI] [PubMed] [Google Scholar]
  9. Mathis A., Deplazes P. PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs. J Clin Microbiol. 1995 May;33(5):1145–1149. doi: 10.1128/jcm.33.5.1145-1149.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Mehmann B., Brunner I., Braus G. H. Nucleotide sequence variation of chitin synthase genes among ectomycorrhizal fungi and its potential use in taxonomy. Appl Environ Microbiol. 1994 Sep;60(9):3105–3111. doi: 10.1128/aem.60.9.3105-3111.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Murray J. F. Pulmonary complications of HIV infection. Annu Rev Med. 1996;47:117–126. doi: 10.1146/annurev.med.47.1.117. [DOI] [PubMed] [Google Scholar]
  12. Ochert A. S., Boulter A. W., Birnbaum W., Johnson N. W., Teo C. G. Inhibitory effect of salivary fluids on PCR: potency and removal. PCR Methods Appl. 1994 Jun;3(6):365–368. doi: 10.1101/gr.3.6.365. [DOI] [PubMed] [Google Scholar]
  13. Ortiz-Rivera M., Liu Y., Felder R., Leibowitz M. J. Comparison of coding and spacer region sequences of chromosomal rRNA-coding genes of two sequevars of Pneumocystis carinii. J Eukaryot Microbiol. 1995 Jan-Feb;42(1):44–49. doi: 10.1111/j.1550-7408.1995.tb01538.x. [DOI] [PubMed] [Google Scholar]
  14. Pitkin A. D., Grant A. D., Foley N. M., Miller R. F. Changing patterns of respiratory disease in HIV positive patients in a referral centre in the United Kingdom between 1986-7 and 1990-1. Thorax. 1993 Mar;48(3):204–207. doi: 10.1136/thx.48.3.204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rossen L., Nørskov P., Holmstrøm K., Rasmussen O. F. Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int J Food Microbiol. 1992 Sep;17(1):37–45. doi: 10.1016/0168-1605(92)90017-w. [DOI] [PubMed] [Google Scholar]
  16. Skøt J., Lerche A. G., Kolmos H. J., Nielsen J. O., Mathiesen L. R., Lundgren J. D. Pneumocystis carinii in bronchoalveolar lavage and induced sputum: detection with a nested polymerase chain reaction. Scand J Infect Dis. 1995;27(4):363–367. doi: 10.3109/00365549509032732. [DOI] [PubMed] [Google Scholar]
  17. Speich R. Diagnosis of pulmonary problems in HIV-infected patients. Monaldi Arch Chest Dis. 1993;48(3):221–232. [PubMed] [Google Scholar]
  18. Speich R., Opravil M., Weber R., Hess T., Luethy R., Russi E. W. Prospective evaluation of a prognostic score for Pneumocystis carinii pneumonia in HIV-infected patients. Chest. 1992 Oct;102(4):1045–1048. doi: 10.1378/chest.102.4.1045. [DOI] [PubMed] [Google Scholar]
  19. Stringer J. R., Walzer P. D. Molecular biology and epidemiology of Pneumocystis carinii infection in AIDS. AIDS. 1996 Jun;10(6):561–571. doi: 10.1097/00002030-199606000-00001. [DOI] [PubMed] [Google Scholar]
  20. Teuscher A. U., Opravil M., Theiler R., Speich R., Kuster H., Siegenthaler W., Russi E. W., Lüthy R. Predictive value of bronchoalveolar lavage in excluding a diagnosis of Pneumocystis carinii pneumonia during prophylaxis with aerosolized pentamidine. Clin Infect Dis. 1993 Apr;16(4):519–522. doi: 10.1093/clind/16.4.519. [DOI] [PubMed] [Google Scholar]
  21. Tiley S. M., Marriott D. J., Harkness J. L. An evaluation of four methods for the detection of Pneumocystis carinii in clinical specimens. Pathology. 1994 Jul;26(3):325–328. doi: 10.1080/00313029400169761. [DOI] [PubMed] [Google Scholar]
  22. Wakefield A. E., Fritscher C. C., Malin A. S., Gwanzura L., Hughes W. T., Miller R. F. Genetic diversity in human-derived Pneumocystis carinii isolates from four geographical locations shown by analysis of mitochondrial rRNA gene sequences. J Clin Microbiol. 1994 Dec;32(12):2959–2961. doi: 10.1128/jcm.32.12.2959-2961.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wakefield A. E., Pixley F. J., Banerji S., Sinclair K., Miller R. F., Moxon E. R., Hopkin J. M. Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin. Mol Biochem Parasitol. 1990 Nov;43(1):69–76. doi: 10.1016/0166-6851(90)90131-5. [DOI] [PubMed] [Google Scholar]
  24. Wilson S. M., McNerney R., Nye P. M., Godfrey-Faussett P. D., Stoker N. G., Voller A. Progress toward a simplified polymerase chain reaction and its application to diagnosis of tuberculosis. J Clin Microbiol. 1993 Apr;31(4):776–782. doi: 10.1128/jcm.31.4.776-782.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wolfhagen M. J., Fluit A. C., Torensma R., Poppelier M. J., Verhoef J. Rapid detection of toxigenic Clostridium difficile in fecal samples by magnetic immuno PCR assay. J Clin Microbiol. 1994 Jul;32(7):1629–1633. doi: 10.1128/jcm.32.7.1629-1633.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. de Lamballerie X., Zandotti C., Vignoli C., Bollet C., de Micco P. A one-step microbial DNA extraction method using "Chelex 100" suitable for gene amplification. Res Microbiol. 1992 Oct;143(8):785–790. doi: 10.1016/0923-2508(92)90107-y. [DOI] [PubMed] [Google Scholar]

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