Abstract: SA-PO489

A Universal Real Time Quantitative Multiplex PCR Assay for the Non-Invasive Diagnosis of BK Polyomavirus Associated Nephropathy

Session Information

Category: Transplantation

  • 1702 Transplantation: Clinical and Translational

Authors

  • Dadhania, Darshana, Weill Cornell Medical College, New York, New York, United States
  • Snopkowski, Catherine, Weill Medical College of Cornell University, New York, New York, United States
  • Li, Carol Y., Weill Cornell Medical College, New York, New York, United States
  • Perry, Liana S., Weill Cornell Medical College, New York, New York, United States
  • Yang, Hua, Weill Cornell Medical College, New York, New York, United States
  • Lee, John Richard, Weill Cornell Medical College, New York, New York, United States
  • Muthukumar, Thangamani, Weill Cornell Medical College, New York, New York, United States
  • Suthanthiran, Manikkam, Weill Cornell Medical College, New York, New York, United States
Background

BK Polyomavirus associated nephropathy (BKVN) is an important cause of kidney allograft failure. Early detection & reduction of immunosuppression is the best strategy for mitigating BKVN associated graft dysfunction. Use of sensitivity DNA PCR and neighbor-joining method, a phylogenetic tree of BKV has been developed & population specific prevalence of BK has been emphasized. Existing BKV PCR assays fail to account for BKV subtypes, we designed & developed a multiplex quantitative PCR assay for detection of clinically significant subtypes.

Methods

We designed 3 sense & 3 antisense primers and 3 TaqMan probes, which in combination, amplified 7 BKV subtypes- BKV Dunlop, Ia, IC, III, 1v, V, and VI. Total RNA was isolated from 205 biopsy matched urine specimens reverse transcribed to cDNA and real-time quantitative multiplex PCR assays were established. All biopsies were stained for SV40: 36 were SV40 positive & classified as BKVN biopsies; remaining 169 were SV40 negative & classified as acute rejection (n=56); Normal (n=53); acute tubular injury (n=50); Other (n=10).Receiver-operating- characteristic curve analysis was used to calculate area under the curve (ROC-AUC), sensitivity and specificity for distinguishing BKVN biopsies from all other biopsy diagnoses.

Results

Analysis involving ROC curve demonstrated that BKVN diagnosis can be predicted with a sensitivity of 100% & a specificity of 95% with the use 4.54 x 108 copies of BKV mRNA per microgram of RNA as the cutpoint (ROC-AUC=0.98, 95% CI, 0.97 to 1.0, P<0.0001). (Figure 1)

Conclusion

We have designed and developed real time quantitative multiplex PCR assays for the detection of major subtypes of BKV and demonstrate its utility for the noninvasive diagnosis of BKVN. In view of population specific prevalence of BKV subtypes, the newly developed assay should have universal appeal.