Specific detection of human BK polyomavirus in urine samples of immunocompromised patients

A semiquantitative PCR assay for the detection of BK virus in urine was developed using primers for BK virus that specifically amplified BK but not JC virus. DNA was extracted from urine through treatment with proteinase K followed by DNA precipitation with sodium acetate. Semiquantitation was achieved by amplifying serial dilutions (1:1, 1:10, 1:100, and 1:1,000) of the urine specimens. Each assay included both positive (stock BK virus and previously positive patient urine) and negative (no template) controls. A urine sample was interpreted as positive if any of the serial dilutions showed amplification of the DNA fragment of the expected size. For some patient-derived samples, amplification of the expected-size fragment was achieved with a dilute template whereas no amplification was achieved with a concentrated template. This was attributed to interfering substances in the urine. PCR results were compared with urine cytology and shown to be more sensitive. Validation studies were performed at the University of Nebraska Medical Center, utilizing a separate qualitative PCR assay that detects both BK and JC virus and distinguishes between them by restriction enzyme digestion patterns. Of 46 urine samples analyzed using both methods, 22 were positive by both assays, 18 were negative by both assays, 5 were positive only by the Nebraska method, and 1 was positive only by our method. In comparison with the Nebraska PCR, our PCR assay had a sensitivity of 81% and specificity of 95%. For twenty-one (43%) of 49 immunocompromised patients, tests were positive when specimens were submitted because of clinical suspicion of BK virus infection.