Skip to main content
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1994 Jan;32(1):5–10. doi: 10.1128/jcm.32.1.5-10.1994

Application of PCR to multiple specimen types for diagnosis of cytomegalovirus infection: comparison with cell culture and shell vial assay.

M J Miller 1, S Bovey 1, K Pado 1, D A Bruckner 1, E A Wagar 1
PMCID: PMC262960  PMID: 8126204

Abstract

Human cytomegalovirus (CMV) is a herpesvirus that is responsible for significant morbidity and mortality in congenitally infected infants and immunocompromised patients. Antiviral therapies are available, thus making timely diagnosis of significant importance to at-risk patients. A PCR system was devised. The newly devised system, unlike previously described systems, can be applied to a wide variety of specimen types in a clinical microbiology laboratory setting. Specimens from all sites routinely accepted for CMV culture were shown to be acceptable for CMV PCR. Sensitivity and specificity were established in comparison with those of both monolayer culture and shell vial assay (SVA). The sensitivity and specificity of PCR for detection of CMV in specimens exclusive of urine and blood were 97.5 (77 of 79 specimens) and 87.2% (41 of 47 specimens), respectively. The sensitivity and specificity of PCR for urine and blood specimens were 100 (10 of 10) and 95.7% (45 of 47) and 66.7 (4 of 6) and 78.8% (41 of 52), respectively. Discrepancies of positive PCR results with negative culture or SVA results occurred for specimens flanked chronologically by other culture- or SVA-positive specimens and were likely culture failures, increasing the specificity (100%) of PCR. Discrepancies of negative PCR results with positive culture or SVA results occurred in specimens with few cells or infectious foci by SVA or culture and may represent sampling variability associated with low virus titers.

Full text

PDF
5

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bauer H. M., Ting Y., Greer C. E., Chambers J. C., Tashiro C. J., Chimera J., Reingold A., Manos M. M. Genital human papillomavirus infection in female university students as determined by a PCR-based method. JAMA. 1991 Jan 23;265(4):472–477. [PubMed] [Google Scholar]
  2. Beutler E., Gelbart T., Kuhl W. Interference of heparin with the polymerase chain reaction. Biotechniques. 1990 Aug;9(2):166–166. [PubMed] [Google Scholar]
  3. Brytting M., Sundqvist V. A., Stålhandske P., Linde A., Wahren B. Cytomegalovirus DNA detection of an immediate early protein gene with nested primer oligonucleotides. J Virol Methods. 1991 May;32(2-3):127–138. doi: 10.1016/0166-0934(91)90043-y. [DOI] [PubMed] [Google Scholar]
  4. Brytting M., Xu W., Wahren B., Sundqvist V. A. Cytomegalovirus DNA detection in sera from patients with active cytomegalovirus infections. J Clin Microbiol. 1992 Aug;30(8):1937–1941. doi: 10.1128/jcm.30.8.1937-1941.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buffone G. J., Hine E., Demmler G. J. Detection of mRNA from the immediate early gene of human cytomegalovirus in infected cells by in vitro amplification. Mol Cell Probes. 1990 Apr;4(2):143–151. doi: 10.1016/0890-8508(90)90015-r. [DOI] [PubMed] [Google Scholar]
  6. Chou S. Effect of interstrain variation on diagnostic DNA amplification of the cytomegalovirus major immediate-early gene region. J Clin Microbiol. 1992 Sep;30(9):2307–2310. doi: 10.1128/jcm.30.9.2307-2310.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Demmler G. J., Buffone G. J., Schimbor C. M., May R. A. Detection of cytomegalovirus in urine from newborns by using polymerase chain reaction DNA amplification. J Infect Dis. 1988 Dec;158(6):1177–1184. doi: 10.1093/infdis/158.6.1177. [DOI] [PubMed] [Google Scholar]
  8. Einsele H., Ehninger G., Steidle M., Vallbracht A., Müller M., Schmidt H., Saal J. G., Waller H. D., Müller C. A. Polymerase chain reaction to evaluate antiviral therapy for cytomegalovirus disease. Lancet. 1991 Nov 9;338(8776):1170–1172. doi: 10.1016/0140-6736(91)92032-w. [DOI] [PubMed] [Google Scholar]
  9. Einsele H., Steidle M., Vallbracht A., Saal J. G., Ehninger G., Müller C. A. Early occurrence of human cytomegalovirus infection after bone marrow transplantation as demonstrated by the polymerase chain reaction technique. Blood. 1991 Mar 1;77(5):1104–1110. [PubMed] [Google Scholar]
  10. Emanuel D., Peppard J., Stover D., Gold J., Armstrong D., Hammerling U. Rapid immunodiagnosis of cytomegalovirus pneumonia by bronchoalveolar lavage using human and murine monoclonal antibodies. Ann Intern Med. 1986 Apr;104(4):476–481. doi: 10.7326/0003-4819-104-4-476. [DOI] [PubMed] [Google Scholar]
  11. Gerna G., Zipeto D., Parea M., Revello M. G., Silini E., Percivalle E., Zavattoni M., Grossi P., Milanesi G. Monitoring of human cytomegalovirus infections and ganciclovir treatment in heart transplant recipients by determination of viremia, antigenemia, and DNAemia. J Infect Dis. 1991 Sep;164(3):488–498. doi: 10.1093/infdis/164.3.488. [DOI] [PubMed] [Google Scholar]
  12. Gleaves C. A., Meyers J. D. Rapid detection of cytomegalovirus in bronchoalveolar lavage specimens from marrow transplant patients: evaluation of a direct fluorescein-conjugated monoclonal antibody reagent. J Virol Methods. 1989 Dec;26(3):345–349. doi: 10.1016/0166-0934(89)90117-1. [DOI] [PubMed] [Google Scholar]
  13. Gleaves C. A., Smith T. F., Shuster E. A., Pearson G. R. Comparison of standard tube and shell vial cell culture techniques for the detection of cytomegalovirus in clinical specimens. J Clin Microbiol. 1985 Feb;21(2):217–221. doi: 10.1128/jcm.21.2.217-221.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gleaves C. A., Smith T. F., Shuster E. A., Pearson G. R. Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by using low-speed centrifugation and monoclonal antibody to an early antigen. J Clin Microbiol. 1984 Jun;19(6):917–919. doi: 10.1128/jcm.19.6.917-919.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ho M. Epidemiology of cytomegalovirus infections. Rev Infect Dis. 1990 Sep-Oct;12 (Suppl 7):S701–S710. doi: 10.1093/clinids/12.supplement_7.s701. [DOI] [PubMed] [Google Scholar]
  16. Holland G. N., Sidikaro Y., Kreiger A. E., Hardy D., Sakamoto M. J., Frenkel L. M., Winston D. J., Gottlieb M. S., Bryson Y. J., Champlin R. E. Treatment of cytomegalovirus retinopathy with ganciclovir. Ophthalmology. 1987 Jul;94(7):815–823. doi: 10.1016/s0161-6420(87)33534-1. [DOI] [PubMed] [Google Scholar]
  17. Jiwa N. M., Van Gemert G. W., Raap A. K., Van de Rijke F. M., Mulder A., Lens P. F., Salimans M. M., Zwaan F. E., Van Dorp W., Van der Ploeg M. Rapid detection of human cytomegalovirus DNA in peripheral blood leukocytes of viremic transplant recipients by the polymerase chain reaction. Transplantation. 1989 Jul;48(1):72–76. doi: 10.1097/00007890-198907000-00017. [DOI] [PubMed] [Google Scholar]
  18. Keay S., Bissett J., Merigan T. C. Ganciclovir treatment of cytomegalovirus infections in iatrogenically immunocompromised patients. J Infect Dis. 1987 Dec;156(6):1016–1021. doi: 10.1093/infdis/156.6.1016. [DOI] [PubMed] [Google Scholar]
  19. Khan G., Kangro H. O., Coates P. J., Heath R. B. Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J Clin Pathol. 1991 May;44(5):360–365. doi: 10.1136/jcp.44.5.360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kolk A. H., Schuitema A. R., Kuijper S., van Leeuwen J., Hermans P. W., van Embden J. D., Hartskeerl R. A. Detection of Mycobacterium tuberculosis in clinical samples by using polymerase chain reaction and a nonradioactive detection system. J Clin Microbiol. 1992 Oct;30(10):2567–2575. doi: 10.1128/jcm.30.10.2567-2575.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leland D. S., Hansing R. L., French M. L. Clinical experience with cytomegalovirus isolation using conventional cell cultures and early antigen detection in centrifugation-enhanced shell vial cultures. J Clin Microbiol. 1989 Jun;27(6):1159–1162. doi: 10.1128/jcm.27.6.1159-1162.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Martin W. J., 2nd, Smith T. F. Rapid detection of cytomegalovirus in bronchoalveolar lavage specimens by a monoclonal antibody method. J Clin Microbiol. 1986 Jun;23(6):1006–1008. doi: 10.1128/jcm.23.6.1006-1008.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Myerson D., Hackman R. C., Meyers J. D. Diagnosis of cytomegaloviral pneumonia by in situ hybridization. J Infect Dis. 1984 Aug;150(2):272–277. doi: 10.1093/infdis/150.2.272. [DOI] [PubMed] [Google Scholar]
  24. Paya C. V., Wold A. D., Ilstrup D. M., Smith T. F. Evaluation of number of shell vial cell cultures per clinical specimen for rapid diagnosis of cytomegalovirus infection. J Clin Microbiol. 1988 Feb;26(2):198–200. doi: 10.1128/jcm.26.2.198-200.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Paya C. V., Wold A. D., Smith T. F. Detection of cytomegalovirus infections in specimens other than urine by the shell vial assay and conventional tube cell cultures. J Clin Microbiol. 1987 May;25(5):755–757. doi: 10.1128/jcm.25.5.755-757.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ruano G., Pagliaro E. M., Schwartz T. R., Lamy K., Messina D., Gaensslen R. E., Lee H. C. Heat-soaked PCR: an efficient method for DNA amplification with applications to forensic analysis. Biotechniques. 1992 Aug;13(2):266–274. [PubMed] [Google Scholar]
  27. Rubin R. H. Impact of cytomegalovirus infection on organ transplant recipients. Rev Infect Dis. 1990 Sep-Oct;12 (Suppl 7):S754–S766. doi: 10.1093/clinids/12.supplement_7.s754. [DOI] [PubMed] [Google Scholar]
  28. Schooley R. T. Cytomegalovirus in the setting of infection with human immunodeficiency virus. Rev Infect Dis. 1990 Sep-Oct;12 (Suppl 7):S811–S819. doi: 10.1093/clinids/12.supplement_7.s811. [DOI] [PubMed] [Google Scholar]
  29. Shibata D., Martin W. J., Appleman M. D., Causey D. M., Leedom J. M., Arnheim N. Detection of cytomegalovirus DNA in peripheral blood of patients infected with human immunodeficiency virus. J Infect Dis. 1988 Dec;158(6):1185–1192. doi: 10.1093/infdis/158.6.1185. [DOI] [PubMed] [Google Scholar]
  30. Umana J. P., Mutimer D. J., Shaw J. C., McLeish P. J., Buchan A., Martin B., Neuberger J. M., Elias E., McMaster P. Cytomegalovirus surveillance following liver transplantation: does it allow presymptomatic diagnosis of cytomegalovirus disease? Transplant Proc. 1992 Dec;24(6):2643–2645. [PubMed] [Google Scholar]
  31. Volpi A., Whitley R. J., Ceballos R., Stagno S., Pereira L. Rapid diagnosis of pneumonia due to cytomegalovirus with specific monoclonal antibodies. J Infect Dis. 1983 Jun;147(6):1119–1120. doi: 10.1093/infdis/147.6.1119. [DOI] [PubMed] [Google Scholar]
  32. Walmsley S. L., Chew E., Read S. E., Vellend H., Salit I., Rachlis A., Fanning M. M. Treatment of cytomegalovirus retinitis with trisodium phosphonoformate hexahydrate (Foscarnet). J Infect Dis. 1988 Mar;157(3):569–572. doi: 10.1093/infdis/157.3.569. [DOI] [PubMed] [Google Scholar]
  33. Zipeto D., Revello M. G., Silini E., Parea M., Percivalle E., Zavattoni M., Milanesi G., Gerna G. Development and clinical significance of a diagnostic assay based on the polymerase chain reaction for detection of human cytomegalovirus DNA in blood samples from immunocompromised patients. J Clin Microbiol. 1992 Feb;30(2):527–530. doi: 10.1128/jcm.30.2.527-530.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zurlo J. J., O'Neill D., Polis M. A., Manischewitz J., Yarchoan R., Baseler M., Lane H. C., Masur H. Lack of clinical utility of cytomegalovirus blood and urine cultures in patients with HIV infection. Ann Intern Med. 1993 Jan 1;118(1):12–17. doi: 10.7326/0003-4819-118-1-199301010-00003. [DOI] [PubMed] [Google Scholar]
  35. Zweygberg Wirgart B., Landqvist M., Hökeberg I., Eriksson B. M., Olding-Stenkvist E., Grillner L. Early detection of cytomegalovirus in cell culture by a new monoclonal antibody, CCH2. J Virol Methods. 1990 Feb;27(2):211–219. doi: 10.1016/0166-0934(90)90137-5. [DOI] [PubMed] [Google Scholar]
  36. van der Bij W., Torensma R., van Son W. J., Anema J., Schirm J., Tegzess A. M., The T. H. Rapid immunodiagnosis of active cytomegalovirus infection by monoclonal antibody staining of blood leucocytes. J Med Virol. 1988 Jun;25(2):179–188. doi: 10.1002/jmv.1890250208. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES