Calibration of BK Virus Nucleic Acid Amplification Testing to the 1st WHO International Standard for BK Virus

Abstract

Significant interassay variability in the quantification of BK virus (BKV) DNA precludes establishing broadly applicable thresholds for the management of BKV infection in transplantation. The 1st WHO International Standard for BKV (primary standard) was introduced in 2016 as a common calibrator for improving the harmonization of BKV nucleic acid amplification testing (NAAT) and enabling comparisons of biological measurements worldwide. Here, we evaluated the Altona RealStar BKV assay (Altona) and calibrated the results to the international unit (IU) using the Exact Diagnostics BKV verification panel, a secondary standard traceable to the primary standard. The primary and secondary standards on Altona had nearly identical linear regression equations (primary standard, Y = 1.05X - 0.28, R² = 0.99; secondary standard, Y = 1.04X - 0.26, R² = 0.99) and conversion factors (primary standard, 1.11 IU/copy; secondary standard, 1.09 IU/copy). A comparison of Altona with a laboratory-developed BKV NAAT assay in IU/ml versus copies/ml using Passing-Bablok regression revealed similar regression lines, no proportional bias, and improvement in the systematic bias (95% confidence interval of intercepts: copies/ml, -0.52 to -1.01; IU/ml, 0.07 to -0.36). Additionally, Bland-Altman analyses revealed a clinically significant reduction of bias when results were reported in IU/ml (IU/ml, -0.10 log₁₀; copies/ml, -0.70 log₁₀). These results indicate that the use of a common calibrator improved the agreement between the two assays. As clinical laboratories worldwide use calibrators traceable to the primary standard to harmonize BKV NAAT results, we anticipate improved interassay comparisons with a potential for establishing broadly applicable quantitative BKV DNA load cutoffs for clinical practice.

Keywords: molecular methods; polyomavirus.

FIG 1

Comparison of the Exact Diagnostics BK virus verification panel and the 1st World Health Organization International Standard for BK virus on the Altona RealStar BK virus assay. The line of identity is shown as a dashed line.

FIG 2

Quantitative comparisons of clinical specimens tested with the Altona RealStar BK virus assay (Altona) and our prior laboratory developed assay (VP1 BKV). (A) Passing-Bablok regression analysis of 93 plasma specimens quantifiable by both the Altona and VP1 BKV and compared in log₁₀ copies/ml. The regression line (solid line) and 95% confidence intervals (dashed lines) are displayed. (B) Passing-Bablok regression analysis of the same 93 plasma specimens compared in log₁₀ IU/ml. The copy-to-IU conversion factor for VP1 BKV was 0.28. The regression line (solid line) and 95% confidence intervals (dashed lines) are displayed. (C) Bland-Altman plot comparing Altona with VP1 BKV in copies/ml. The bias (solid line) was −0.70 log₁₀ copies/ml. The 95% limits of agreement (gray dashed lines) and zero line (dotted black line) are also shown. (D) Bland-Altman plot comparing Altona with VP1 BKV in IU/ml. The bias (solid line) was −0.10 log₁₀ IU/ml. The 95% limits of agreement (gray dashed lines) and zero line (dotted black line) are also shown.