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. 2022 Sep 21;60(9):e0055522.
doi: 10.1128/jcm.00555-22. Epub 2022 Aug 23.

Matrix Matters: Assessment of Commutability among BK Virus Assays and Standards

R T Hayden  1 , Y Su  2 , J Boonyaratanakornkit  3 , L Cook  4 , Z Gu  1 , K R Jerome  4   5 , B A Pinsky  6   7 , S S Sam  8 , S K Tan  6   7 , H Zhu  1 , L Tang  2 , A M Caliendo  8
Affiliations

Matrix Matters: Assessment of Commutability among BK Virus Assays and Standards

R T Hayden et al. J Clin Microbiol. .

Abstract

Quantitative testing of BK virus (BKPyV) nucleic acid has become the standard of care in transplant patients. While the relationship between interassay harmonization and commutability has been well characterized for other transplant-related viruses, it has been less well studied for BKPyV, particularly regarding differences in commutability between matrices. Here, interassay agreement was evaluated among six real-time nucleic acid amplification tests (NAATs) and one digital PCR (dPCR) BKPyV assay. Differences in the commutability of three quantitative standards was examined across all assays using a variety of statistical approaches. Panels, including 40 samples each of plasma and urine samples previously positive for BKPyV, together with one previously negative plasma sample and four previously negative urine samples, were tested using all assays, with each real-time NAAT utilizing its usual quantitative calibrators. Serial dilutions of WHO, National Institute for Standards and Technology (NIST), and commercially produced (Exact/Bio-Rad) reference materials were also run by each assay as unknowns. The agreement of the clinical sample values was assessed as a group and in a pairwise manner. The commutability was estimated using both relativistic and quantitative means. The quantitative agreement across assays in the urine samples was within a single log10 unit across all assays, while the results from the plasma samples varied by 2 to 3 log10 IU/mL. The commutability showed a similar disparity between the matrices. Recalibration using international standards diminished the resulting discrepancies in some but not all cases. Differences in the sample matrix can affect the commutability and interassay agreement of quantitative BKPyV assays. Differences in commutability between matrices may largely be due to factors other than those such as amplicon size, previously described as important in the case of cytomegalovirus. Continued efforts to standardize viral load measurements must address multiple sources of variability and account for differences in assay systems, quantitative standards, and sample matrices.

Keywords: BK virus; commutability; digital PCR; quantitative PCR; real-time PCR; standardization; viral load.

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Conflict of interest statement

The authors declare a conflict of interest. R. T. Hayden has served on advisory boards for Roche Diagnostics, Quidel Corporation, Inflammatix, and T2 BioSciences; A. M. Caliendo has served on advisory boards for Cepheid, First Light, Visby, ID Connect, Quidel and Chromacode; B.A. Pinsky has received research funds from altona Diagnostics; S.K. Tan is currently an employee of and holds stock in Vir Biotechnology, Inc. J. Boonyaratanakornkit was an employee of Bio-Rad Laboratories/Exact Diagnostics. None of the other authors have potential conflicts of interest to disclose.

Figures

FIG 1
FIG 1
BK virus values from 7 assays performed on plasma (PT) samples: Luminex (blue line), Qiagen (brown line), Abbott (black line), DiaSorin (purple line), LDT (orange line), ELITech (green line), and dPCR (red line). The real-time NAATs and digital PCR (dPCR) assay are reported in number of log10 international units per milliliter and log10 copies per milliliter, respectively.
FIG 2
FIG 2
BK virus values from 7 assays performed on urine (PT) samples: Luminex (blue line), Qiagen (brown line), Abbott (black line), DiaSorin (purple line), LDT (orange line), ELITech (green line), and dPCR (red line). The real-time NAATs and digital PCR (dPCR) assay are reported in number of log10 international units per milliliter and log10 copies per milliliter, respectively.
FIG 3
FIG 3
Variability in clinical samples across 6 real-time NAATs in plasma (A) and urine (B) matrices, illustrated using an ellipse to represent the 95% prediction area. Commutability of the WHO standard material for BK virus was examined across the 6 NAATs using correspondence analysis. Clinical sample projections, WHO standard dilution projections, and method projections are shown on the first significant factorial plane. The axes indicate how much of the total information in the clinical samples is accounted for by the first two dimensions, with most of that attributed to the first dimension. The proportion of the variation represented by each dimension is reported in parentheses.
FIG 4
FIG 4
Quantitative BK virus values (left) yielded by the real-time NAATs and dPCR assay (red solid line) compared to the recalibrated values (right) based on the WHO standard material against the dPCR values in plasma (A) and urine (B) matrices. Values are shown for Luminex (blue line), Qiagen (brown line), LDT (orange line), and ELITech (green line) assays with available CT values.
FIG 5
FIG 5
Distribution of BKPyV fragments across the viral genome (top) (red and green lines represent sequencing reads in forward and reverse directions, respectively) and frequency distribution of a sequenced BKPyV fragment, arranged by increasing base pair size (bottom), in plasma (A) and urine (B). NA, not applicable.
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