Quantitative PCR in the Diagnosis and Monitoring of Human Cytomegalovirus Infection in Organ Transplant Patients

List of original publications 7 Abbreviations 8 Summary 9 Review of the literature 10 1. Human cytomegalovirus 10 1.1. Virus structure and genome organization 10 1.2. Virus replication 10 1.3. Epidemiology and transmission 11 1.4. Pathogenesis 12 2. Human cytomegalovirus infections in organ transplant patients 14 2.1. Clinical manifestations 14 2.2. Prevention and treatment of HCMV disease 15 2.2.1. Treatment of the disease 16 2.2.2. Prophylaxis 16 2.2.3. Pre-emptive therapy 17 2.2.4. Drug resistance 18 3. 1.2. Correlation of quantitative PCR assay and pp65 antigenemia test 36 1.3. Cobas Amplicor CMV Monitor test 37 2. Diagnostic validation and use of the Cobas Amplicor CMV Monitor assay (II, III) 37 2.1. Comparison of the quantitative assays and shell vial culture assay (III) 38 2.2. Diagnosis of symptomatic HCMV infection by the Cobas Amplicor CMV Monitor assay (II, III) 38 2.3. Monitoring of HCMV infection in individual patients (II, III) 39 3. HCMV real-time PCR assay (IV) 41 3.1. Technical characteristics of HCMV real-time assay 41 3.1.1. Detection limit, linearity and reproducibility of HCMV real-time assay 41 3.1.2. Automated extraction of nucleic acids 42 3.1.3. Turnaround time of HCMV real-time assay 42 3.2. Diagnostic validation and use of HCMV real-time PCR assay 43 3.2.1. Detection of HCMV DNA by real-time assay 43 3.2.2. Correlation between quantitative tests 45 3.2.3. Diagnosis and monitoring of HCMV infection in individual patients by HCMV real-time assay 46 Acknowledgements 47 References 48 7 List of original publications This thesis is based on the following original articles, which are referred to in the text by their Roman numerals.tenschlager I (2001). Comparison of plasma polymerase chain reaction and pp65-anti-genemia assay in the quantification of cytomegalovirus in liver and kidney transplant patients. (2002). Monitoring of viral load by quantitative plasma PCR during active cytomegalovirus infection of individual liver transplant patients. and Lautenschlager I. Quantitative PCR in the diagnosis of CMV infection and in the monitoring of viral load during the antiviral treatment in renal transplant patients. (submitted). (2004). Comparison of two quantitative CMV PCR tests, Cobas Amplicor CMV Monitor and TaqMan assay, and pp65-antigenemia assay in the determination of viral loads from peripheral blood of organ transplant patients. Journal of Clinical Virology (in press). 8 Abbreviations CMV cytomegalovirus CPE cytopathic effect cps/ml copies/ml dsDNA double stranded DNAE early gene product EIA enzyme-linked immunosorbent assay FRET fluorescence resonance energy transfer GCV ganciclovir HCMV human cytomegalovirus HCV hepatitis C virus HHV-6 human …