Development and clinical application of a fully controlled quantitative PCR assay for cell-free cytomegalovirus in human plasma.
BACKGROUND
Cytomegalovirus (HCMV) disease continues to be a major problem in certain patient groups, including bone marrow transplant (BMT) recipients. The quantification of HCMV genome is clinically useful for the diagnosis of HCMV disease, for the virological surveillance of high-risk patients and for monitoring antiviral therapy.


OBJECTIVE
To develop a novel, robust, and fully controlled PCR (qPCR) for the quantification of HCMV DNA in plasma samples and to demonstrate its clinical usefulness in the BMT setting.


METHODS
The newly developed HCMV qPCR employs cell culture-derived murine CMV as an internal control for both extraction and amplification. Following amplification using common primers, detection of both internal control and patient HCMV amplicons is by specific probes and a chemiluminescence microtitre plate system. Its performance was evaluated using the routine non-quantitative nested HCMV PCR on whole blood (NQPCR) and correlated with clinical events such as disease and antiviral therapy.


RESULTS
A high level of concordance (85.1%) was found between the novel assay and the NQPCR, with the qPCR being slightly more sensitive. The samples giving discordant results generally had levels of HCMV DNA close to the limit of detectability or had been stored for prolonged periods.


CONCLUSIONS
The use of plasma as an analyte by the newly developed assay avoids the detection of cell-associated virus. On the other hand, testing a comparatively large volume of plasma ensures that sensitivity is not compromised by not detecting cell-associated HCMV. In a small preliminary evaluation in BMT recipients, changes in HCMV 'viral load' correlated with initiation and discontinuation of antiviral therapy and were biologically plausible.
Preiser
Wolfgang
W
Department of Virology, Royal Free and University College School of Medicine and University College London Hospitals NHS Trust (UCLH), Windeyer Building, 46 Cleveland Street, London W1P 6DB, UK. w.preiser@em.uni-frankfurt.de
Brink
Nicola S
NS
Ayliffe
Ursula
U
Peggs
Karl S
KS
Mackinnon
Stephen
S
Tedder
Richard S
RS
Garson
Jeremy A
JA
D001483:Base Sequence; D001793:Blood Preservation; D016026:Bone Marrow Transplantation; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000097:blood / Q000175:diagnosis*; D004279:DNA, Viral / Q000097:blood*; D005188:False Negative Reactions; D005500:Follow-Up Studies; D006801:Humans; D016867:Immunocompromised Host; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D018146:Muromegalovirus / Q000235:genetics / Q000302:isolation & purification; D010949:Plasma / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013997:Time Factors; D014184:Transplantation, Homologous; D019562:Viral Load; D014766:Viremia / Q000175:diagnosis* / Q000821:virology
D004279:DNA, Viral