Are We There Yet? Impact of the First International Standard for Cytomegalovirus DNA on the Harmonization of Results Reported on Plasma Samples.
BACKGROUND
Interassay harmonization of cytomegalovirus (CMV) DNA measurement is important for infection management. Uncertainty exists regarding the result harmonization achievable in patient plasma samples using quantitative polymerase chain reaction (qPCR) assays with calibrators now traceable to the First World Health Organization International Standard (IS) for CMV DNA.


METHODS
Serial dilutions of the IS and a blinded panel of 40 genotyped CMV DNA-positive pooled plasma samples and 10 negative plasma samples were tested by 6 laboratories using 10 qPCR assays calibrated to the IS. Each clinical sample was constructed using plasma from a single unique transplant recipient.


RESULTS
The variance for individual CMV DNA-positive samples was greater for clinical samples (median, 1.50 [range, 1.22-2.82] log10 IU/mL) than for IS dilutions (median, 0.94 [range, 0.69-1.35] log10 IU/mL) (P < .001); 58.9% of all clinical sample results and 93.6% of IS dilution results fell within ±0.5 log10 IU/mL of the mean viral load of each sample. Result variability was not impacted by either genotype or quantitative levels of CMV DNA. Testing procedure differences can significantly influence results, even when analyte-specific reagents are identical. For clinical samples, all assays demonstrated result bias (P < .008). Assays with amplicon sizes ≤86 bp had significantly higher results compared to assays with larger amplicon sizes (≥105 bp) (P < .001).


CONCLUSIONS
The variability in CMV DNA results reported on individual samples has been reduced by the IS, but ongoing clinically relevant variability persists, preventing meaningful interassay result comparison.
Department of Medicine, University of Alberta, Edmonton, Canada.
Jutta K
JK
Preiksaitis
Department of Pathology, St Jude Children's Research Hospital, Memphis, Tennessee.
Randall T
RT
Hayden
Department of Medicine, University of Alberta, Edmonton, Canada.
Yupin
Y
Tong
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Canada.
Xiaoli L
XL
Pang
National Institute for Biological Standards and Control, Hertfordshire, United Kingdom.
Jacqueline F
JF
Fryer
National Institute for Biological Standards and Control, Hertfordshire, United Kingdom.
Alan B
AB
Heath
Department of Laboratory Medicine, University of Washington Medical Center, Seattle.
Linda
L
Cook
Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.
Astrid K
AK
Petrich
Focus Diagnostics Inc., Cypress, California.
Brian
B
Yu
Department of Medicine, Alpert Medical School at Brown University, Providence, Rhode Island.
Angela M
AM
Caliendo
D003587:Cytomegalovirus / Q000235:genetics* / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000097:blood / Q000175:diagnosis / Q000821:virology*; D004279:DNA, Viral / Q000097:blood*; D060005:Genotyping Techniques; D006801:Humans; D038622:Internationality; D058889:Molecular Typing; D012015:Reference Standards; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000592:standards
D004279:DNA, Viral
cytomegalovirus; international standard; result harmonization; viral load