Development and utilization of a quantitative polymerase chain reaction assay to evaluate human polyomavirus JC DNA load.
OBJECTIVE
Quantitative polymerase chain reaction (PCR) analysis to evaluate virus load in comparison with the patient's base-line virus levels would be an optimal diagnostic approach to monitoring human polyomavirus infections and to investigate their possible involvement in the onset of nephropathy in this patient group. Studies on the correlation between viral burden and renal disease have pointed to the incidence of JC virus (JCV) related progressive multifocal leukoencephalopathy (PML) occurring in renal and haematopoietic stem cell transplant recipients.


METHODS
We developed a reliable internally-controlled quantitative PCR assay to measure JCV-DNA in fluid samples of urine, serum and cerebrospinal fluid (CSF) by densitometric analysis of the amplification products. The assay was also used to evaluate the JCV load in CFS samples from patients with suspected demyelinating syndrome and in urine and serum samples from healthy subjects and renal transplant recipients.


RESULTS
All CSF samples from the 51 patients with suspected demyelinating syndrome tested JCV-DNA negative: none of them had a diagnosed PML. Analysis of the prevalence of JCV-viruria and JCV-viraemia confirmed our previous data. JCV-viruria was detected in 17% of renal transplant recipients and 26.6% of healthy controls; JCV-viraemia was found in 3.4% of transplant patients and 0% in controls. Noteworthy was a lower prevalence of JCV-viraemia in the 116 (3.4%) renal transplant patients than the prevalence previously reported for the 51 (11.8%) patients with suspected demyelinating syndrome. The mean viral load of viruria was much higher in the healthy controls than in the transplant recipients [104020 DNA copies/mL (DS+/-62284) vs 4136 DNA copies/mL (DS+/-77371)].


CONCLUSIONS
The quantitative PCR assay developed in our lab offers in 2 h time a reliable true quantification of viral DNA by densitometric analysis of the amplification product. To check for the possible presence of potential Taq polymerase inhibitors an internal control (the homemade pJCV-C plasmid) is used. The relation between polyomavirus infections and their possible involvement in post-transplant pathologies need further investigation. It would be useful to monitor the JCV-DNA load in urine and serum from more renal transplant recipients, including patients with nephropathy or active graft rejection over a longer period of time.
Cavallo
R
R
Virology Unit, Department of Public Health and Microbiology, University of Turin, Turin, Italy.
Bergallo
M
M
Costa
C
C
Piana
F
F
Burdino
E
E
Merlino
C
C
Negro Ponzi
A
A
D001483:Base Sequence; D016022:Case-Control Studies; D004279:DNA, Viral / Q000032:analysis / Q000134:cerebrospinal fluid / Q000235:genetics*; D006801:Humans; D007577:JC Virus / Q000235:genetics* / Q000302:isolation & purification; D016030:Kidney Transplantation; D007968:Leukoencephalopathy, Progressive Multifocal / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards
D004279:DNA, Viral