Quantitative Assessment of BK Virus-Associated Nephropathy from Renal Transplant Patient Biopsies by Real-Time PCR
Yuying Jiang, Kenneth Muldrew. University of Toledo Medical Center, Toledo, OH
Background: BK virus (BKV) is a member of human polyoma viruses. BK viral infection is ubiquitous in the general population and it maintains latency in renal tissues usually after a subclinical primary infection. Upon immunosuppression, BK viral infection can be reactivated and causes BKV nephropathy in renal transplanted patients. BKV nephropathy is a frequent reason for persistent graft dysfunction and frequent graft loss. As treatment for BKV nephropathy is different compared to that for renal graft rejection, it is important to have clinical assays to accurately detect active BKV infection in renal transplanted patients. Here we developed a clinical assay to evaluate BK viral load in the biopsy samples of renal transplanted patients by using real-time PCR.
Design: Sections of paraffin-embedded renal biopsy samples from renal transplant patients and autopsy kidney samples from non-transplant patients were de-paraffinized and DNA was extracted using Qiagen DNeasy Tissue Kit. Quantitative measurement of BK viral titer was made by real-time PCR assay using primers and a probe specific to the BK viral genome. Primers and probe specific to human Apolipoprotein B gene were used as internal control to normalize the sample size (number of renal cells per reaction). BK viral titers were correlated with previous immunostaining or in situ hybridization results as well as concurrent plasma BK viral load. Renal function was assessed by BUN and creatinine measurements.
Results: Significant high titer of BK viral DNA was detected in samples with previously confirmed BK viral nephropathy, whereas BK viral DNA was not detected in normal autopsy renal samples. Among BKV-positive patients, BKV was detected in renal transplant kidneys with a range of 2.82 to 4.59 log10 genome equivalents per cell, while concurrent plasma samples showed 5.3 to 7.5 log10 copies/per ml. Tissue and plasma BKV quantitation demonstrated a correlation coefficient of 0.985 for BKV-positive patients. The average serum creatinine level was 2.81 mg/dL for tissue BKV-positive patients.
Conclusions: Real-time PCR is an effective alternative approach to detect BKV infection from small kidney biopsy samples.
Monday, March 19, 2012 1:00 PM
Poster Session II # 291, Monday Afternoon