Comparison of two quantitative CMV PCR tests, Cobas Amplicor CMV Monitor and TaqMan assay, and pp65-antigenemia assay in the determination of viral loads from peripheral blood of organ transplant patients.
BACKGROUND
Quantitative PCR assays have become the most common methods in the determination of viral load during cytomegalovirus (CMV) infection of transplant patients. However, usually these tests are still quite time-consuming and labor-intensive which diminishes their utility of these tests in routine diagnostic laboratories.


OBJECTIVE
The objective of this study was to develop a quantitative CMV PCR test which is time-saving and easy to perform for the detection and monitoring of CMV infection of transplant patients.


METHODS
The quantitative real time CMV PCR assay using TaqMan chemistry and an automated sample preparation system, MagNA Pure LC, was developed. The designed quantitative CMV test was compared to commercial quantitative PCR test, Cobas Amplicor Monitor, in the determination of CMV DNA loads in plasma samples of liver and kidney transplant patients. The results were also correlated with the CMV pp65-antigenemia test. The clinical material of 270 blood specimens of transplant patients were tested using these two PCR methods and pp65-antigenemia test in parallel. Plasma samples were used for PCR assays and leucocytes for the antigenemia test.


RESULTS
The TaqMan assay described was easy to perform, it was rapid (3-4 h) and hands-on time needed for performing the test was short. The detection limit of the assay was 250 copies/ml (cps/ml) plasma and the linear range up to 25,000,000 cps/ml. TaqMan assay was the most sensitive test detecting 92% of the CMV positive findings. Cobas Monitor detected 80% and pp65 test 88% of the positive findings. The correlations between TaqMan and antigenemia assays, and between Cobas Amplicor and antigenemia were statistically significant and high, R = 0.84 (P < 0.0001) and R = 0.80 (P < 0.0001), respectively. Also correlation between two PCR tests was statistically significant (R = 0.64, P < 0.0001). Of the 27 patient studied, 19 demonstrated CMV antigenemia and DNAemia in their blood during the post transplant monitoring. Thirteen of these patients developed a symptomatic CMV infection and were treated with ganciclovir. The peak viral loads of symptomatic patients were statistically higher by all three methods than those of asymptomatic patients.


CONCLUSIONS
The developed real time TaqMan assay was rapid and easily performed and could be the best alternative for the diagnosis of CMV infection and monitoring of liver and kidney transplant patients.
Piiparinen
H
H
Department of Virology, Helsinki University Central Hospital and University of Helsinki, Haartmaninkatu 3, FIN-00290 Helsinki, Finland.
Höckerstedt
K
K
Grönhagen-Riska
C
C
Lautenschlager
I
I
D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000175:diagnosis / Q000821:virology; D004279:DNA, Viral / Q000097:blood; D006801:Humans; D016030:Kidney Transplantation / Q000009:adverse effects*; D016031:Liver Transplantation / Q000009:adverse effects*; D010750:Phosphoproteins / Q000097:blood; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D019914:Taq Polymerase; D019562:Viral Load; D014763:Viral Matrix Proteins / Q000097:blood
D004279:DNA, Viral; D010750:Phosphoproteins; D011933:Reagent Kits, Diagnostic; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; D019914:Taq Polymerase