Full Access
Multiplex polymerase chain reaction for the evaluation of cytomegalovirus DNA load in organ transplant recipients
Cristina Nazzari,
Aurelia Gaeta,
Marina Lazzarini,
Tiziana Delli Castelli,
Carlo Mancini,
Corresponding Author
Cristina Nazzari
I Chair of Clinical Microbiology, School of Medicine, La Sapienza University, Rome, Italy
I Chair of Clinical Microbiology, School of Medicine, La Sapienza University, P. le A. Moro 5, 00185 Rome, Italy===Search for more papers by this authorAurelia Gaeta
I Chair of Clinical Microbiology, School of Medicine, La Sapienza University, Rome, Italy
Search for more papers by this authorMarina Lazzarini
I Chair of Clinical Microbiology, School of Medicine, La Sapienza University, Rome, Italy
Search for more papers by this authorTiziana Delli Castelli
I Chair of Clinical Microbiology, School of Medicine, La Sapienza University, Rome, Italy
Search for more papers by this authorCarlo Mancini
I Chair of Clinical Microbiology, School of Medicine, La Sapienza University, Rome, Italy
Search for more papers by this authorCristina Nazzari,
Aurelia Gaeta,
Marina Lazzarini,
Tiziana Delli Castelli,
Carlo Mancini,
Corresponding Author
Cristina Nazzari
I Chair of Clinical Microbiology, School of Medicine, La Sapienza University, Rome, Italy
I Chair of Clinical Microbiology, School of Medicine, La Sapienza University, P. le A. Moro 5, 00185 Rome, Italy===Search for more papers by this authorAurelia Gaeta
I Chair of Clinical Microbiology, School of Medicine, La Sapienza University, Rome, Italy
Search for more papers by this authorMarina Lazzarini
I Chair of Clinical Microbiology, School of Medicine, La Sapienza University, Rome, Italy
Search for more papers by this authorTiziana Delli Castelli
I Chair of Clinical Microbiology, School of Medicine, La Sapienza University, Rome, Italy
Search for more papers by this authorCarlo Mancini
I Chair of Clinical Microbiology, School of Medicine, La Sapienza University, Rome, Italy
Search for more papers by this authorFirst published: 28 April 2000
Citations: 6
Abstract
Because of the considerable impact of human cytomegalovirus (HCMV) infection, sensitive, specific, and standardized methods are required for rapid and accurate evaluation of viral load in monitoring transplant recipients. The aim of the present study was to evaluate the usefulness of a multiplex polymerase chain reaction (PCR) for the coamplification of HCMV-DNA and β-globin genomic sequence in polymorphonuclear leukocytes (PMNL). Analysis and quantification of PCR products were carried out by a DNA enzyme immunoassay (DEIA), which is based on the hybridization of amplified DNA with a single-stranded DNA probe, which coats microtitre wells. Colorimetric detection of the DNA-antibody complex was carried out and optical density (O.D.) was recorded at 450/630 nm. To quantify HCMV/DNA load, a standard curve to which samples O.D. refer was obtained by amplifying serial dilutions of recombinant pGEM-3Z plasmid DNA containing a genomic fragment of glycoprotein B. 340 PMNL specimens from 102 solid organ recipients were tested for the detection of pp65 antigen and HCMV-DNA. The results showed a good correlation between viral load and clinical symptoms of HCMV infection; high specificity and predictive values for HCMV disease were found by PCR, using a cut-off limit of 103 genomic copies per 2 × 105 PMNL. These findings indicate that the system described is an efficient and reproducible diagnostic method easy to apply for routine diagnosis and therapeutic monitoring of transplanted patients. J. Med. Virol. 61:251–258, 2000. © 2000 Wiley-Liss, Inc.
REFERENCES
- Allen RD, Pellett PE, Stewart JA, Koopmans M. 1995. Nonradioactive PCR-enzyme-linked immunosorbent assay method for detection of human cytomegalovirus DNA. J Clin Microbiol 33: 725–728.
- Bitsch A, Kirchner H, Dennin R. 1993a. The long persistence of CMV DNA in the blood of renal transplant patients after recovery from CMV infection. Transplantation 56: 108.
- Bitsch A, Kirchner H, Dupke R, Bein G. 1993b. Cytomegalovirus transcripts in peripheral blood leukocytes of actively infected transplant patients detected by reverse transcription polymerase chain reaction. J Infect Dis 167: 740–743.
- Boeckh M, Boivin G. 1998. Quantitation of cytomegalovirus: methodologic aspects and clinical applications. Clin Microbiol Rev 11: 533–554.
- Boeckh M, Woogerd PM, Stevens Ayers T, Ray CG, Bowden RA. 1994. Factors influencing detection of quantitative cytomegalovirus antigenemia. J Clin Microbiol 32: 832–834.
- Boivin G, Olson CA, Quirk MR, Kringstad B, Hertz MI, Jordan MC. 1996. Quantitation of cytomegalovirus DNA and characterization of viral gene expression in bronchoalveolar cells of infected patients with and without pneumonitis. J Infect Dis 173: 1304–1312.
- Boivin G, Handfield J, Murray G, Toma E, Lalonde R, Lazar JG, Bergeron MG. 1997. Quantitation of cytomegalovirus (CMV) DNA in leukocytes of human immunodeficiency virus-infected subjects with and without CMV disease by using PCR and the SHARP signal detection system. J Clin Microbiol 35: 525–526
- Boivin G, Handfield J, Toma E, Murray G, Lalonde R, Bergeron MG. 1998. Comparative evaluation of the cytomegalovirus DNA load in polymorphonuclear leukocytes and plasma of human immunodeficiency virus-infected subjects. J Infect Dis 177: 355–360.
- Boland G J, De Weger RA, Yilanus MGJ, Ververs C, Bosboom-Kalsbeek K, De Gast GC. 1992. Detection of cytomegalovirus (CMV) in granulocytes by polymerase chain reaction compared with the CMV antigen test. J Clin Microbiol 30: 1763.
- Caballero OL, Menezes CLP, Costa MCSL, Fernandes SC, Anacleto TM, de Oliveira RM, Viotti EA, Tavora ERF, Vilaca SS, Sabbaga E, de Paula FJ, Tavora PF, Villa LL, Simpson AJ. 1997. Highly sensitive single-step PCR protocol for diagnosis and monitoring of cytomegalovirus infection in renal transplant recipients. J Clin Microbiol 35: 3192–3197.
- Cagle PT, Buffone G, Holland VA, Samo T, Demmler GJ, Noon GP, Lawrence EC. 1992. Semiquantitative measurement of cytomegalovirus DNA in lung and heart-lung transplant patients by in vitro DNA amplification. Chest 101: 93–96.
- Chan A, Zhao J, Krajden M. 1994. Polymerase chain reaction kinetics when using a positive internal control target to quantitatively detect cytomegalovirus target sequences. J Virol Methods 48: 223–236
- Chow S. 1992. Effect of interstrain variation on diagnostic DNA amplification of the cytomegalovirus major immediate-early gene region. J Clin Microbiol 30: 2307–2310.
- Delgado R, Lumbreras C, Alba C, Pedraza MA, Otero JR, Gomez R, Moreno E, Norirga AR, Paya CV. 1992. Low prediction value of polymerase chain reaction for diagnosis of cytomegalovirus disease in liver transplant recipients. J Clin Microbiol 30: 1876–1878.
- Drouet E, Boibieux A, Michelson S, Ecochard R, Biron F, Peyramond D, Colimon R, Denoyel G. 1993. Polymerase chain reaction detection of cytomegalovirus DNA in peripheral blood leukocytes as a predictor of cytomegalovirus disease in HIV-infected patients. AIDS 7: 665–668.
- Drouet E, Colimon R, Michelson S, Fourcade N, Niveleau A, Ducere C, Boibieux A, Chevallier M, Denoyel G. 1995. Monitoring level of human cytomegalovirus DNA in blood after liver transplantation. J Clin Microbiol 33: 389–394.
- Fox JC, Kidd IM, Griffiths PD, Sweny P, Emery VC. 1995. Longitudinal analysis of cytomegalovirus load in renal transplant recipients using a quantitative polymerase chain reaction: correlation with disease. J Gen Virol 76: 309–319.
- Gaeta A, Nazzari C, Angeletti S, Lazzarini M, Mazzei E, Mancini C. 1997. Monitoring for cytomegalovirus infection in organ transplant recipients: analysis of pp65 Antigen, DNA and Late mRNA in peripheral blood leukocytes. J Med Virol 53: 189–195.
10.1002/(SICI)1096-9071(199711)53:3<189::AID-JMV2>3.0.CO;2-4 CAS PubMed Web of Science® Google Scholar
- Gerna G, Revello MG, Percivalle E, Morini F. 1992. Comparison of different immunostaining techniques and monoclonal antibodies to lower matrix phosphoprotein (pp65) for optimal quantitation of human cytomegalovirus antigenemia. J Clin Microbiol 30: 1232–1237.
- Ghisetti V, Barbui A, Donegani E, Bobbio M, Caimmi P, Pansini S, Zattera G, Pucci A, di Summa A, Marchiaro G. 1996. Comparison of polymerase chain reaction and pp65 antigen test for early detection of human cytomegalovirus in blood leukocytes of cardiac transplant recipients. Clin Microbiol Infect 1: 195–202.
- Gozlan J, Salord JM, Chouaid C, Duvivier C, Picard O, Meyohas PC, Petit JC. 1993. Human cytomegalovirus (HCMV) late-mRNA detection in peripheral blood of AIDS patients: diagnostic value for HCMV disease compared with those of viral culture and HCMV DNA detection. J Clin Microbiol 31: 1943–1945.
- Grefte A, van der Giessen M, van der Gun BTF, van Son W, The TH. 1991. The predominant viral antigen present in peripheral blood leukocytes during an active cytomegalovirus (CMV) infection is the lower matrix protein pp65. In: MP Landini, editor. Progress in cytomegalovirus research. International Congress Series. Amsterdam: Excepta Medica. p 233–236.
- Griner PF, Mayewski RJ, Mushlin AI, Greenland P. 1981. Selection and interpretation of diagnostic test and procedures: principles and applications. Ann Intern Med 94: 553.
- Grundy JE, Ayles HM, McKeating JA, Butcher RG, Griffiths PD, Poulter LW. 1988. Enhancement of class I HLA antigen expression by cytomegalovirus: role in amplification of virus infection. J Med Virol 25: 483.
- Haberhausen G, Pinsil J, Kuhn CC, Markert-Hahn C. 1998. Comparative study of different standardization concepts in quantitative competitive reverse transcription-PCR assays. J Clin Microbiol 36: 628–633.
- Ho M. 1993. Cytomegalovirus infection after solid organ transplantation. In: Y Beker, G Darai, E-S Huang, editors. Molecular aspect of human cytomegalovirus disease. Berlin: Springer-Verlag. p 163–170.
- Kanj SS, Sharara AI, Clavien PA, Hamilton JD. 1996. Cytomegalovirus infection following liver transplantation: review of the literature. Clin Infect Dis 22: 537–549.
- Kuhn JE, Wendland T, Schafer P, Mohring K, Wieland U, Elgas M, Eggers HJ. 1994. Monitoring of renal allograft recipients by quantitation of human cytomegalovirus genomes in peripheral blood leukocytes. J Med Virol 44: 398–405.
- Kwok S. 1990. Procedures to minimize PCR-product carry-over. In: MA Innis, DH Gelfand, JJ Sninsky, TJ White, editors. PCR protocols. A guide to methods and applications. San Diego, CA: Academic Press. p 142–145.
10.1016/B978-0-12-372180-8.50021-4 Google Scholar
- Landry ML, Ferguson D, Cohen S, Huber K, Wetherill P. 1995. Effect of delayed specimen processing on cytomegalovirus antigenemia test results. J Clin Microbiol 33: 257–259.
- Mantero G, Zonaro A, Albertini A, Bertolo P, Primi D. 1991. DNA enzyme immunoassay: general method for detecting products of polymerase chain reaction. Clin Chem 37: 422–429.
- Mendez J C, Espy MJ, Smith TF, Wilson JA, Paya CV. 1998. Evaluation of PCR primers for early diagnosis of cytomegalovirus infection following liver transplantation. J Clin Microbiol 36: 526–530.
- Meyer-Konig U, Serra A, von Laer D, Kirste G, Wolff C, Haller O, Neumann-Haefelin D, Hufert FT. 1995. Human cytomegalovirus immediate early and late transcripts in peripheral blood leukocytes: diagnostic value in renal transplant recipients. J Infect Dis 171: 705–709.
- Mutimer D, Matyi-Toth A, Shaw J, Elias E, O'Donnell K, Stalhandske P. 1997. Patterns of viremia in liver transplant recipients with symptomatic cytomegalovirus infection. Transplantation 63: 68–73.
- Pasternack MS, Medearis DN, Rubin NH. 1990. Cell-mediated immunity in experimental cytomegalovirus infection: a perspective. Rev Infect Dis 12: 720-726.
- Payan C, Veal N, Sarol L, Villarme M, Ngohou C, Riberi P, Francois S, Ifrah N, Loison J, Chennebault JM, Pichard E, Kouyoumdjian S, Lunel F. 1997. Human cytomegalovirus DNA kinetics using a novel HCMV DNA quantitative assay in white blood cells of immunocompromised patients under Ganciclovir therapy. J Virol Methods 65: 131–138.
- Randawa PS, Manez R, Frye B, Erlich GD. 1994. Circulating immediate-early mRNA in patients with cytomegalovirus infection after solid organ transplantation. J Infect Dis 170: 1264–1267.
- Resnick RM, Cornelissen MTE, Wright DK, Eichinger GH, Fox HS, Schegget J, Manos M. 1990. Detection and typing of human papillomavirus in archival cervical cancer specimens by DNA amplification with consensus primers. J Natl Cancer Inst 82: 1477–1484.
- Roberts TC, Buller RS, Gaudreault-Keener M, Sternhell KE, Garlock K, Singer GG, Brennan DC, Storch GA. 1997. Effects of storage temperature and time on qualitative and quantitative detection of cytomegalovirus in blood specimens by shell vial culture and PCR. J Clin Microbiol 35: 2224–2228.
- Schafer P, Laufs R. 1996. Experience with quantitative PCR for the management of HCMV disease. Intervirology 39: 204–212.
- The TH, van der Ploeg M, van der Berg AP, Viegler AM, van der Giessen M, van Son WL. 1992. Direct detection of cytomegalovirus in peripheral blood leukocytes-a review of the antigenemia assay and the polymerase chain reaction. Transplantation 54: 193–198.
- van der Berg AP, van der Bij W, van Son WJ, van der Giessen AJ, Schirm J, Tegzess AM, The TH. 1989. Cytomegalovirus antigenemia as a useful marker of symptomatic cytomegalovirus infection after renal transplantation—a report of 130 consecutive cases. Transplantation 48: 991–995.
- von Willebrand E, Petterson E, Ahonen J, Hayvry P. 1986. CMV infection, class II antigen expression, and human kidney allograft rejection. Transplantation 42: 364.
- Weber B, Nestler U, Ernst W, Rabenau H, Braner J, Birkenbach A, Scheurmann EH, Schoeppe W, Doerr HW. 1994. Low correlation of human cytomegalovirus DNA amplification by polymerase chain reaction with cytomegalovirus disease in organ transplant recipients. J Med Virol 43: 187–193.
- Zaia J A, Gallez-Hawkins GM, Tegtmeyer BR, ter Veer A, Li X, Niland JC, Forman SJ. 1997. Late cytomegalovirus disease in marrow transplantation is predicted by virus load in plasma. J Infect Dis 176: 782–785.
- Zipeto D, Revello MG, Silini E, Parea M, Percivalle E, Zavattoni M, Milanesi G, Gerna G. 1992. Development and clinical significance of a diagnostic assay based on the polymerase chain reaction for detection of human cytomegalovirus DNA in blood samples from immuno-compromised patients. J Clin Microbiol 30: 527–530.
- Zipeto D, Baldanti F, Zella D, Furione M, Cavicchini A, Milanesi G, Gerna G. 1993. Quantification of human cytomegalovirus DNA in peripheral blood polymorphonuclear leukocytes of immunocompromised patients by the polymerase chain reaction. J Virol Methods 44: 45–55.
- Zipeto D, Morris S, Hong C, Dowling A, Wolitz R, Merigan TC, Rasmussen L. 1995. Human cytomegalovirus (CMV) DNA in plasma reflects quantity of CMV DNA present in leukocytes. J Clin Microbiol 33: 2607–2611.
Citing Literature
