Noninvasive diagnosis of BK virus nephritis by measurement of messenger RNA for BK VP1 virus in urine

We thank Dr. Hirsch for his thoughtful comments (1). He correctly states that decoy cells can arise not only from renal tubular cells but also from urothelial cells. We are in general agreement with his statement that “shedding of decoy cells is highly sensitive, but not specific for BK virus nephropathy.” Nickeleit et al. (2) claim, “The extraction of mRNA from urinary cells is technically challenging, highly susceptible to erroneous results and expensive.” We fail to see the relevance of these assertions to our investigation because we isolated total RNA and not mRNA from urinary cells. Isolation of RNA from urinary cells has not been a technical challenge in our hands and should not be for laboratories with appropriate skills for RNA isolation from clinical specimens. We have less faith than Nickeleit et al. about decoy cells being a specific marker. Indeed, we are surprised by their claim in light of their own report that decoy cells were present not only in the urine of 9 of 9 patients with BK nephritis but also in the urine of 16 of 16 patients without BK nephritis (data in Table 1 from reference 3). Moreover Nickeleit et al. have also reported “PV disease was associated with abundant decoy cell excretion in five of 28 (18%) recipients; twenty-three of twenty-eight (82%) were free of disease (no cytopathic changes in graft biopsies by light microscopy or IHC” [4]). Our investigation advances the idea that measurement of VP1 mRNA in urinary cells offers a noninvasive and accurate means of diagnosing BK virus nephritis. The important conceptual advance is that we used cDNA rather than DNA as the template in the real-time quantitative polymerase chain reaction (PCR) assay. The greater abundance of VP1 mRNA compared with VP1 genomic DNA allows the use of highly diluted cDNA in the PCR reaction. The dilution step may obviate positive tests caused by VP1 DNA in the urine samples. Jin et al. (5) found that 25.6% of urine samples from pregnant women and 31.8% of urine specimens from transplant recipients were positive for VP1 BKV DNA when 10 μl of resuspended urinary sediment was added to a 50-μl PCR mix. In our study, we used a 1:1000 dilution of cDNA, and the dilution step might have contributed to our prediction of BKV nephritis with a sensitivity of 93.8% and specificity of 87.5% using a BKV VP1 mRNA level of 6.54×105 copies per nanogram of total RNA. Clearly additional studies are needed and RNA-PCR (omitting the reverse transcription step) and DNase treatment of isolated RNA, as suggested by Hirsch (1), may further refine the assay we developed and explored in our article. Ruchuang Ding Manikkam Suthanthiran