Detection of Human Cytomegalovirus (HCMV) — DNA from Granulocytes and Peripheral Blood Mononuclear Cells (PBMNC) of Immunosuppressed Patients by Nested PCR: Lack of Correlation to Isolation of Infectious Virus
A rapid and very sensitive nested PCR method with primer oligonucleotides from the immediate early gene region of HCMV laboratory strain AD 169 was used to detect HCMVDNA from PBMC, granulocytes, urine, CSF, bronchoalveolar lavage and autopsy samples from bone marrow or kidney transplant recipients with clinically suspected primary or recurrent CMV-infection. Results of nested DNA-PCR were compared to isolation of infectious virus by conventional cell culture and culture-immunoperoxidase staining. Prior to amplification DNA was isolated from all specimens. Native urine was inhibitory to the action of Taq polymerase. Therefore urine samples were first concentrated with PEG 6000/ NaCl. In total 60 different specimens from 20 patients were analyzed. In 43 samples HCMV-DNA could be detected. Detection rates of HCMV-DNA from PBMC (14/21) and granulocytes (11/17) were nearly identical (67% and 65% respectively). Infectious virus however could be isolated in only one case. The initial detection rate of virus isolation from leukocytes prior to ganciclovir treatment was 19% (4/21). As shown for three patients ganciclovir treatment had no influence on HCMV-DNA detection from granulocytes, PBMC and urine (within the indicated time) but detection of initially infectious virus failed after ganciclovir administration. These results clearly demonstrate that detection of HCMV-DNA from granulocytes and PBMC (containing up to 20% monocytes) from immunosuppressed patients by DNA-PCR does not correlate to viremia, which has been established as predictive indicator for HCMV disease in renal and bone marrow transplantation. In this context the persistence of HCMVDNA in monocytes and granulocytes of asymptomatic seropositive subjects has to be discussed.
KeywordsHepatitis Chloroform Glycol Sedimentation Electrophoresis
Unable to display preview. Download preview PDF.
- Gema G, Zipeto D, Parea M, Revello MG, Silini E, Percivalle M, Zavattoni M and 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, 164: 488–98CrossRefGoogle Scholar
- Gema G, Zipeto D, Percivalle E, Parea M, Revello MG, Maccario R, Peri G and Milanesi G. Human cytomegalovirus infection of the major leukocyte subpopulations and evidence for initial viral replication in polymorphonuclear leukocytes from viremic patients. J. Infect. Dis. 1992, 166: 1236–44CrossRefGoogle Scholar
- Hamprecht K, Sorg G, Steinmaßl M, and Gerth H-J and Jahn G. submittedGoogle Scholar
- Jiwa NM, Van Gernert GW, Raap AK, Van de Rijke FM, Mulder A, Lens PF, Salimans MMM, Zwaan FE, Van Dorp W and 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, 48: 72–76PubMedCrossRefGoogle Scholar
- Ratnamohan VM, Mathys JM, McKenzie A and Cunnigham AL. HCMV-DNA is detected more frequently than infectious virus in blood leucocytes of immunocompromised patients: a direct comparison of culture-immunofluorescence and PCR for detection of HCMV in clinical specimens. J. Med. Virol. 1992, 38: 252–59PubMedCrossRefGoogle Scholar