Comparison of molecular tests for detection and quantification of cell-associated cytomegalovirus DNA.
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 10(5) HFFs per 10(6) 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 log(10) 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 log(10) and 3.48 to at least 5.0 log(10) 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 log(10) target cells.
Caliendo
Angela M
AM
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA. acalien@emory.edu
Yen-Lieberman
Belinda
B
Baptista
Jovana
J
Andersen
Janet
J
Crumpacker
Clyde
C
Schuurman
Rob
R
Spector
Stephen A
SA
Bremer
James
J
Lurain
Nell S
NS
D003587:Cytomegalovirus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D004279:DNA, Viral / Q000032:analysis*; D005347:Fibroblasts / Q000821:virology; D005784:Gene Amplification; D006801:Humans; D008297:Male; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012867:Skin / Q000821:virology; D019562:Viral Load
D004279:DNA, Viral