Real-Time Automated PCR for Early Diagnosis and Monitoring of Cytomegalovirus Infection after Bone Marrow Transplantation

Abstract

The purpose of this study was to assess the usefulness of real-time automated PCR as a quantitative, highly reproducible, and sensitive method to detect cytomegalovirus (CMV) DNA in blood specimens. Intra- and interassay precision rates were 0.89% (small number of copies [L]), 1.43% (middle number of copies [M]), and 1.12% (high number of copies [H]), and 4.46% (L), 1.51% (M), and 2.28% (H), respectively. The linearity of this assay was obtained between 10 and 107 copies/well, with a minimum detection limit of 20 copies/well. Specimens from 55 of 70 healthy subjects were found to be positive for CMV antibody, but CMV DNA was not detected in any of them. In the qualitative assessment of each specimen, the results of the CMV antigenemia assay and those of the real-time PCR assay agreed in 80% (plasma specimens), 79% (all nucleated cells), and 86% (blood) of the cases examined. For eight patients diagnosed as having CMV infection or disease, no sample was positive in the antigenemia assay earlier than in the real-time PCR assay. Furthermore, the results of this assay could be obtained within 8 h. We concluded that the real-time PCR assay is useful for rapid diagnosis of CMV infection and monitoring of clinical courses.

Monitoring low cytomegalovirus viremia in transplanted patients by a real-time PCR on plasma

“…In order to evaluate the presence of PCR inhibitors, a commercial DNA control was chosen. This internal control or equivalent, comparable from one laboratory to another and for long duration, was not developed in numerous previous studies in this field [Machida et al, 2000;Tanaka et al, 2000;Kearns et al, 2001;Leruez-Ville et al, 2003;Mengelle et al, 2003a, as examples]. Because of mostly low CMV viremia in the patients followed-up in our laboratory, each extract was tested either for CMV DNA or for the internal DNA control (b-globin gene), which was introduced in a separate tube for each sample to avoid any competition between the two PCR systems.…”

“…Various reports led to an open discussion of CMVrelated risk monitoring [Machida et al, 2000;Griscelli et al, 2001;Guiver et al, 2001;Tanaka et al, 2002;Yakushiji et al, 2002;Geddes et al, 2003;Gourlain et al, 2003;Matthes-Martin et al, 2003;Yun et al, 2003]. Breakpoints of CMV viremia correlated with a risk of CMV disease were not definitely established to monitor various groups of transplanted patients.…”

Real-time PCR in the microbiology laboratory

“…Conventional quantitative PCR has already proven that the application of nucleic acid amplification to the monitoring of viral load provides a useful marker of disease progression and the efficacy of antiviral compounds [97,204,[206][207][208][209][210]. Because disease severity and viral load are linked, the use of real-time PCR quantitation has proven beneficial when studying the role of viral reactivation or persistence in the progression of disease [40,102,107,108,119,158,165,171,183,184,187,211,[211][212][213][214][215][216]. Alterations to a microbe's tropism or its replication, and the effects that these changes have on a host cell, can also be followed using real-time PCR [217][218][219].…”

Real-time Fluorescent PCR Techniques to Study Microbial–Host Interactions

“…Quantitative real-time PCR has similarly examined the interaction between virus and host and monitored changes in viral load resulting from antiviral therapy ultimately impacting on the treatment regimen selected (Nitsche et al, 1999;Clementi, 2000;Limaye et al, 2000). Because disease severity and viral load are linked, microbial quantitation by real-time PCR has proven beneficial when studying the impact of viral reactivation, persistence or isolated gene expression on the progression of disease (Ohyashiki et al, 2000;Chen et al, 2003;Kearns et al, 2001c;Furuta et al, 2001;Limaye et al, 2001;Tanaka et al, 2000a,b;Lallemand et al, Real-time Fluorescent PCR Techniques to Study 2000;Laue et al, 1999;Kimura et al, 1999;Chang et al, 1999;Lo et al, 1999;Hawrami and Breur, 1999;Hoshino et al, 2000;Nitsche et al, 2000;Machida et al, 2000;Limaye et al, 2001;Najioullah et al, 2001;Gault et al, 2001). Altered microbial tropism or replication and the effect of these changes on the host cell can also be followed using real-time PCR (Kennedy et al, 1998a(Kennedy et al, , 1999a.…”