Received on September 7, 2003
Accepted on February 12, 2004

Molecular Diagnostics and Genetics

Quantitative Real-Time PCR with Automated Sample Preparation for Diagnosis and Monitoring of Cytomegalovirus Infection in Bone Marrow Transplant Patients

¹ Department of Pathology, Oregon Health & Science University, Portland, OR 97201, and Current address: Laboratory of Population Genetics, National Cancer Institute, Bethesda, MD 20892
² Department of Pathology, Oregon Health & Science University, Portland, OR 97201

^* To whom correspondence should be addressed. E-mail: pressr{at}ohsu.edu.

Background: In bone marrow and stem cell transplant patients, the widespread use of preemptive cytomegalovirus (CMV) antiviral therapy necessitates faster, more precise, and more sensitive quantitative laboratory methods for serial viral load monitoring.

Methods: We quantified CMV viral load assay by real-time PCR after plasma DNA was prepared by an automated robotic workstation. Fluorescent hybridization probes were directed at the glycoprotein B (gB) gene (or EcoRI D region) of CMV. The -globin gene was amplified in parallel to control for the efficiency of the extraction and PCR steps. Samples were collected from patients for whom final diagnoses were divided based on other tests and clinical history.

Results: The assay was linear (R = 0.999) from 125 to 5 x 10⁹ copies/mL with a PCR efficiency of 1.975 (gB) or 2.02 (EcoRI D). The viral loads determined by PCRs directed at these two different viral targets were no different (n = 53; R = 0.928). The interassay CV was 3.5%, and the intraassay CV was 1-4%. Compared with a commercially available quantitative competitive PCR assay (Roche Monitor; R = 0.59), the mean results were 3.1 times higher (mean ratio; P = 0.002). The diagnostic sensitivity and specificity were 96% and 100%, respectively (n = 147), compared with 74% and 98% for a qualitative competitive PCR assay (Roche Amplicor). On a subset of samples, sensitivity of viral culture was 50% (n = 44). Of 1115 clinical referral samples from 252 patients, 10% of the samples and 18% of the patients had low-level CMV viremia (median, 500 copies/mL). In this predominantly (85%) bone marrow transplant testing cohort, serial CMV viral load results were the predominant clinical trigger for the initiation, monitoring, and cessation of preemptive antiviral therapy.

Conclusions: The combination of automated DNA preparation and semiautomated real-time fluorescent PCR detection allows for a sensitive, precise, and accurate high-throughput assay of CMV viral load that can be used as the laboratory trigger for preemptive antiviral therapy.