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

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FONTE

American Society for Microbiology

RESUMO

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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