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 detec-tion 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 so that variations in tube-to-tube PCR efficiency are detected and accounted for in 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 quanti-tative 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 pre-dictive value for disease onset or progression in patients, we have developed a second version of a real-time PCR assay. Assays of this design often have CVs of 10 percent or less. This improved real-time PCR assay incorporates several new technical advances that have just become available. The assay utilizes a new instrument, the LightCycler, which uses airflow to heat and cool capillary tubes for very rapid cycling times. The instrument also measures fluorescence intensity (positive signal) from each capillary tube as the assay is running. We have also designated a hybridization probe for use with the quantitative CMV assay that uti-lizes the recently described molecular beacon configuration. Preliminary studies with this system have been successful. Validation studies with a series of blood samples are planned.
| 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 |
