Cytomegalovirus (CMV) disease is a relatively frequent and often serious complication in immunocompromised CMV-infected patients. In the last few years it has become apparent that in order to differentiate between subclinical viral shedding and large scale viral replication occurring during the prodrome before the onset of active disease it is necessary to utilize sequential monitoring with a quantitative assay. Several studies have shown that CMV quantitative polymerase chain reaction (PCR) assays are more sensitive than buffy coat CMV antigen detection assays. This extra sensitivity can in some cases give an additional week of warning before the onset of CMV disease in a patient. Instituting antiviral therapy at an earlier time point in the prodromal stage may decrease the chance that the patient will go on to develop active CMV disease.We have completed development of a competitive quantitative PCR assay for the detection of CMV in buffy coat cells. A standard amount of mimic of the DNA target sequence is included in the reaction mixture of each PCR tube to detect and account for variations in tube-to-tube PCR efficiency in the calculations of viral copy number made from the measured signal strength. The assay is capable of detecting as few as three to five viral genome equivalents in an amplification reaction. Preliminary comparisons of the quantitative PCR protocol with p65 antigenemia determinations in a series of patient samples demonstrates that the PCR assay has greater sensitivity and permits an earlier detection of the CMV prodrome before the onset of CMV disease. The coefficient of variance (CV) of our assay is about 40 percent, in line with other published descriptions of assays of this type.To develop an assay with improved performance, and, therefore, better potential predictive value for disease onset or progression in patients, we have worked on a Oreal-timeO PCR version of our assay. Assays of this design often have CVs of 10 percent or less. Development of one version of a real-time PCR assay using our existing validated primers and probe sequences is complete.
| Cortez, Karoll J; Fischer, Steven H; Fahle, Gary A et al. (2003) Clinical trial of quantitative real-time polymerase chain reaction for detection of cytomegalovirus in peripheral blood of allogeneic hematopoietic stem-cell transplant recipients. J Infect Dis 188:967-72 |
