A multiplex real-time PCR method for quantification of BK and JC polyomaviruses in renal transplant patients.
BACKGROUND
Infection of BK or JC human polyomavirus can lead to polyomavirus-associated nephropathy in renal transplant patients. Thus effective management of these patients requires early detection and quantification of these viruses in urine and blood.


METHODS
The aim of this study was to evaluate and validate a multiplex real-time PCR-based method for monitoring BK and/or JC viral loads in renal transplant patients. Analytic parameters such as limit of quantification, linear dynamic range, sensitivity and specificity, as well as reliability of the assays were determined. Seventy-six plasma or urine samples spiked with variable amounts of BK and JC viral DNA ranging from low (7.0 x 10(3) or 1.5 x 10(4)), to medium (1.0 x 10(6) to high (1.0 x 10(8) or 1.0109 copies/mL) levels of viruses were tested. In addition, 45 clinical urine or plasma samples with known copy numbers of BK or JC viruses, which were isolated from the renal transplant patients from 4 US medical centers, were also tested.


RESULTS
BK and/or JC viruses can be detected with distinguishable melting temperature of 64 degrees C or 68 degrees C, respectively. On the basis of the need for clinical monitoring of different types of specimens, the low limit of quantification for plasma or urine was set at 7.0 x 10(3) or 1.5 x 10(4) copies/mL, respectively with the linear dynamic range Z6 logs. The assay exhibits 100% specificity, 97.9% sensitivity with low intra-assay and interassay variability (coefficient of variation <4%).


CONCLUSIONS
This clinically validated method has the necessary utility to monitor BK and JC viremia and viruria in renal transplant patients.
Rubio
Luis
L
Molecular Diagnostic Laboratory, Department of Pathology, University of Maryland School of Medicine, University of Maryland Medical Center, Baltimore, MD 21201-1192, USA.
Pinczewski
Joel
J
Drachenberg
Cinthia B
CB
Vera-Sempere
Francisco J
FJ
Zhao
Richard Y
RY
D000046:Academic Medical Centers; D001739:BK Virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016867:Immunocompromised Host; D007577:JC Virus / Q000235:genetics / Q000302:isolation & purification*; D016030:Kidney Transplantation / Q000009:adverse effects; D010949:Plasma / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D027601:Polyomavirus Infections / Q000821:virology*; D012680:Sensitivity and Specificity; D014481:United States; D014556:Urine / Q000821:virology; D019562:Viral Load / Q000379:methods*