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Journal of Clinical Microbiology, August 2003, p. 3509-3513, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3509-3513.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Comparison of Molecular Tests for Detection and Quantification of Cell-Associated Cytomegalovirus DNA

Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia,¹ Department of Clinical Pathology, Cleveland Clinic Foundation, Cleveland, Ohio,² Department of Immunology/Microbiology, Statistics and Data Analysis Center, Harvard School of Public Health,³ Division of Infectious Diseases, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts,⁴ Department of Virology, Eykman Winkler Institute for Clinical Microbiology, Utrecht University Hospital, Utrecht, The Netherlands,⁵ Department of Pediatric Infectious Diseases, University of California at San Diego, San Diego, California,⁶ Department of Immunology/Microbiology, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois⁷

Received 28 February 2003/ Returned for modification 25 April 2003/ Accepted 28 May 2003

A cell-based standard was developed to compare the COBAS Amplicor CMV Monitor test, the Hybrid Capture System CMV DNA test, and the NucliSens CMV test. The standard was prepared by infecting human foreskin fibroblasts (HFFs) with cytomegalovirus (CMV) strain AD169 at low multiplicity of infection (0.03) and harvesting the cells at 6 h postinfection. Buffy coat cells were added to produce concentrations of from 0 to 10⁵ HFFs per 10⁶ total cells. Five laboratories performed the Amplicor PCR test and two laboratories performed the NucliSens and Hybrid Capture tests. The Amplicor PCR test was 1.5 to 2.0 log₁₀ more sensitive than the Hybrid Capture test. The specificities of the Amplicor PCR and Hybrid Capture tests were 100 and 93.8%, respectively. The linear range of the Amplicor PCR and Hybrid Capture tests were 2 to 4.48 log₁₀ and 3.48 to at least 5.0 log₁₀ CMV target cells, respectively. The standard deviations of the Amplicor PCR and Hybrid Capture tests varied throughout their linear range, and for both tests the variability was greater for lower concentrations of input CMV DNA. These data allow the direct comparison of viral load values between the Amplicor and Hybrid Capture tests. The analytical sensitivity of the NucliSens test could not be determined by using the 6-h standard, because the low multiplicity of infection and short culture time did not allow for adequate transcription of pp67 late mRNA measured in the test. Extending the incubation time of the standard to 24 h increased the analytical sensitivity of the NucliSens test to 3.0 log₁₀ target cells.

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