In situ detection of DNA and mRNA of human cytomegalovirus to distinguish different forms of viral infection in leukocytes
Languages of publication
In situ PCR and in situ reverse transcription PCR (RT-PCR) were applied to discriminate between latent and productive infection of human cytomegalovirus (HCMV) in leukocytes. We investigated 28 samples, in which viral pp65 antigen was detected only in the cytoplasm of leukocytes. Additionally we assayed 12 specimens lacking pp65 antigen. Using nested PCR (nPCR), viral DNA was detected in 27 samples. In six samples the results of nPCR were unreadable due to the presence of polymerase inhibitors. By application of in situ PCR, we were able to confirm the presence of viral DNA in the nucleus and/or cytoplasm. Productive infection was recognized in 20 samples in which transcripts for late viral genes were detected. Among the 20 samples negative by in situ RT-PCR, we recognized phagocytosis of viral particles in eight and the latent form of HCMV infection in five.
- Alford CA, Britt WJ (1996) Cytomegalovirus. In Virology (Fields BN, Knipe DM, eds) pp 2494-2512, Lippincott-Raven Publishers, Philadelphia.
- Amorim ML, Cabeda JM, Seca R, Mendes AC, Castro AP, Amorim JM (2001) Cytomegalovirus infection of liver transplant recipients; comparison of antigenemia and molecular biology assays. Liver Transplant 11: 1004-1007.
- Boeckh M, Boivin G (1998) Quantitation of CMV; methodological aspects and clinical applications. Clin Microbiol Rev 11: 533-554.
- Deiman B, van Aarle P, Sillekens P (2002) Characteristics and applications of nucleic acid sequence-based amplification (NASBA). Mol Biotechnol 20: 163-179.
- Gerna GM, Revello G, Percivalle E, Morini F (1992) Comparison of different immunostaining techniques and monoclonal antibodies to the lower matrix phosphoprotein (pp65) for optimal quantification of human cytomegalovirus antigenemia. J Clin Microbiol 30: 1232-1237.
- Gerna G, Baldanti F, Pesea M, Alessandrino E, Pagani A, Locatelli F, Middeldorp J, Revello MG (2000) Human cytomegalovirus immediate early mRNA detection by nucleic acid sequence-based amplification as a new parameter for preemptive therapy in bone marrow transplant recipients. J Clin Microbiol 38: 1845-1853.
- Gozlan J, Caburet F, Trancrede C, Petit JC (1992) A reverse polymerase chain reaction method for detection of human cytomegalovirus late transcripts in cells infected in vitro. J Virol Methods 40: 1-10.
- Hengel H, Brune B, Koszinowski UH (1998) Immune evasion by cytomegalovirus-survival strategies of a highly adapted opportunist. Trends Microbiol 6: 190-197.
- Ikewaki J, Ohtsuka E, Kawano R, Ogata M, Kikuchi H, Nasu M (2005) Real-time PCR assays based on distinct genomic regions for cytomegalovirus reactivation following hematopoietic stem cell transplantation. Bone Marrow Transplant 35: 403-410.
- Kas-Deelen AM, The TH, Bloom N, van der Strate BW, De Maar EF, Smit J, van Son WJ, Harmsen MC (2001) Uptake of pp65-positive polymorphonuclear cells mediated by phagocytosis and cell fusion? Intervirology 44: 8-13.
- Komminoth P, Long AA (1995) In situ polymerase chain reaction-methodology, application and non-specific pathways. In PCR Applications Manual, pp 99-106, Boehringer Mannheim GmbH.
- Kosz-Vnenchak M, Grabiec A, Zawilinska B (2001) Application of PCR in situ method for detection of HCMV DNA. Acta Microbiol Pol 50: 169-173.
- Lewis F (1996) An Approach to in situ PCR. PE Applied Biosystems, A Division of The Perkin-Elmer Corporation.
- Mitchell SM, Fox JD, Tedder RS, Gazzard BG, Lightman S (1994) Vitrous fluid sampling and viral genome detection for the diagnosis of viral retinitis in patients with AIDS. J Med Virol 43: 336-340.
- Nitsche A, Oswald O, Schetelig J, Radonic A, Thulke S, Siegert W (2003) Quantitative real-time PCR compared with pp65 antigen detection for cytomegalovirus (CMV) in 1122 blood specimens from 77 patients after allogenic stem cell transplantation: which test better predicts CMV disease development? Clin Chem 49: 1683-1685.
- Nuovo GJ (1996) PCR in situ Hybridization. Protocolos and Applications, 3th edn, Lippincott, Raven Press New York.
- Perez JL, Mariscal D, Tubau F, Niubo J, Martin R (1994) Evaluation of the CMV-vue antigenemia assay for rapid detection of cytomegalovirus in peripheral blood leukocytes. Diagn Microbiol Infect Dis 19: 15-18.
- Piatkowska-Jakubas B, Zawilinska B, Kopec J, Rojek-Zakrzewska D, Koprynia M, Polus A, Skotnicki A (2003) Antigenemia pp65 assay and qualitative CMV DNA PCR assay for monitoring cytomegalovirus infection in allogeneic stem cell transplant recipients-own experience. Acta Haematol Polon 34: 339-346.
- Preiser W, Brauninger S, Schwerdtfeger R, Ayliffe U, Garson JA, Brink NS, Franck S, Doerr HW, Rabenau HF (2001) Evaluation of diagnostic methods for the detection of cytomegalovirus in recipients after allogenic stem cell transplants. J Clin Virol 20: 59-70.
- Razonable RR, Paya CV, Smith TF (2002) Role of the laboratory in diagnosis and management of cytomegalovirus infection in hematopoietic stem cell and solid-organ transplant recipients. J Antimicrob Chemoter 49: 243-253.
- Schafer P, Tenschert W, Gutensohn K, Laufs R (1997) Minimal effect of delayed sample processing on results of quantitative PCR for cytomegalovirus DNA in leukocytes compared to results of an antigenemia assay. J Clin Microbiol 35: 741-744.
- Streblow DN, Nelson JA (2003) Models of HCMV latency and reactivation. Trends Microbiol 11: 293-295.
- Taylor-Wiedeman JA, Sissons JPG, Borysiewicz IK, Sinclair JH (1991) Monocytes are a major site of persistence of human cytomegalovirus in peripheral blood mononuclear cells. J Gen Virol 72: 2059-2064.
- The TH, van der Berg AP, Harmsen MC, van der Bij W, van Son WJ (1995) The cytomegalovirus antigenemia assay: a plea for standarization. Scand J Infect Dis 99: 25-29.
- Yakushiji K, Gondo H, Kamezaki K, Shigematsu K, Hayashi S, Kuroiwa M, Taniguchi S, Ohno Y, Takase K, Numata A, Aoki K, Kato K, Nagafuji K, Shimoda K, Okamura T, Kinukawa N, Kasuga N, Sata M, Harada M (2002) Monitoring of cytomegalovirus after allogenic stem cell transplantation: comparison of an antigenemia assay and quantitative real-time polymerase chain reaction. Bone Marrow Transplant 29: 599-606.
- Zaia JA, Gallez-Hawkins G, Li X, Yao ZQ, Lomeli N, Molinder K, La Rosa C, Diamond DJ (2001) Infrequent occurrence of natural mutations in the pp65495-503 epitope sequence presented by HLA A*0201 allele among human cytomegalovirus isolates. J Virol 75: 2472-2474.
Publication order reference