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Early diagnosis of cytomegalovirus infection using monoclonal antibodies and DNA amplification
Human cytomegalovirus (HCMV) is ubiquitous and frequently infects normal individuals with few serious clinical consequences. However, patients with impaired cellular immunity frequently develop life-threatening opportunistic HCMV infections due to either primary infection or reactivation of latent virus. Pneumonia is the most common manifestation of HCMV visceral organ disease in solid organ and bonemarrow transplant recipients while retinitis is the most common manifestation in AlDS patients. Exposure of the fetus to HCMV in utero may lead to multi-organ system damage and permanent neurological sequelae. Early intervention with antiviral drugs depends on early sensitive and specific virological diagnosis. Detection of anti-CMV antibodies and virus isolation do not allow an early and presymptomatic diagnosis of HCMV infection.
The work described has focused on the development of methods to enable the rapid and simple detection of CMV immediate early (IE), early (E) and structural CMV proteins or the detection of amplified CMV DNA in urine, bronchoalveolar lavage (BAL) or blood samples obtained from immunocompetent or immunosuppressed patients. CMV mouse monoclonal antibodies were produced and three of them, (DDG9, CCH2 and AAC 10) were further characterized and used in the different assays. Urine samples from children attending day care centres and from kidney transplant recipients were used for the development of a new method for the detection of early CMV antigen in infected cell cultures. IF staining with the CCH2 monoclonal antibody at 24 hours post-inoculation using urine from kidney transplant recipients revealed a sensitivity of 70% compared to virus isolation. The sensitivity was increased to 85% by the use of biotin-streptavidin and a new immunoenzymatic staining (IENZ) procedure. However, viremia is a better marker for disease development and further studies concentrated on the detection of CMV in blood.
The CMV antigenemia assay, which can quantitatively detect CMV pp65 antigen in leukocytes during CMV infection, and a nested polymerase chain reaction (PCR) for amplification of CMV DNA from plasma were established and evaluated. We found plasma-PCR and the CMV antigenemia assay to be almost equally sensitive. However, CMV antigenemia gave the highest positive predictor value (ppv) (57%), for the development of symptomatic CMV infection in kidney transplant recipients, compared to 49% ppv for CMV DNA in plasma. In immunosuppressed patients with symptoms of pulmonary infection, amplification of CMV DNA by PCR from bronchoalveolar lavage (BAL) fluids was the most sensitive method for detection of CMV. The sensitivity was lower but the specificity was higher for the demonstration CMV E antigen and E antigen in infected cell cultures and for the direct detection of CMV antigens in BAL cells. For rapid diagnosis, a combination of these three methods are recommended. A negative PCR result had a 100% negative predictive value for the development of CMV pneumonia for the following two months.
Three CMV gene regions, (major immediate early (ME)), DNA polymerase, and glycoprotein B (gB)) were sequenced for 46 isolates obtained from four patient populations in order to assess the nucleotide stability and to evaluate the suitability of the three gene regions as targets for CMV DNA amplification. In diagnostic PCR assays, most laboratories use primers from the MIE gene region. However, we found the MIE to be the most variable gene region, indicating that this gene region is less suitable for diagnostic PCR primer selection compared to the more conserved DNA polymerase and gB gene regions. Our studies have contributed to the development of new, more optimal diagnostic methods for early diagnosis of CMV infection. The monoclonal antibodies have proved to be powerful tools for the detection of CMV in infected cells. With the new sequence data, new more optimal primer sequences can be selected and used in diagnostic PCR assays in order to detect the majority of CMV strains circulating in society.
History
Defence date
1997-02-07Department
- Department of Microbiology, Tumor and Cell Biology
Publication year
1997Thesis type
- Doctoral thesis
ISBN-10
91-628-2321-3Language
- eng