Isheeta Shah1*, Natasha Browne-Marke2, Sarah Holt2, Cinzia Pultrone3, Davide Rigamonti3, Alessandro Di Cosimo3,
Maddalena Gilardi3 and Gavin Wall4
1 QIAGEN Sciences Inc., 19300 Germantown Rd, Germantown, MD 20874, United States;
2 QIAGEN Manchester Ltd, Citylabs 2.0. Hathersage Road, Manchester, M13 0BH, UK
3 Sentinel Diagnostics, Via Robert Koch, 2, 20152 Milano MI, Italy;
4 QIAGEN GmbH, QIAGEN Straße 1, 40724 Hilden, Germany
* Presenting author
BK virus (BKV) infection is linked to two major complications: BKV associated nephropathy (BKVAN) in kidney transplant patients and BKV associated hemorrhagic cystitis in hematopoietic stem cell transplant patients. In the absence of BKV specific therapy and limited treatment options for advanced BKVAN, active screening of BKV replication in urine and blood combined with pre-emptive modulation of immunosuppression therapy are essential measures to prevent BKVAN. PCR-based viral load quantitation in plasma and urine is the standard clinical tool for monitoring BKV reactivation and response to treatment. Studies reporting quantitative BKV PCR results demonstrate a positive correlation between higher viral loads and an increased probability of developing BKVAN. The NeuMoDx® BKV Quant Assay enables rapid quantitative and specific detection of BKV in urine and plasma matrices.
The NeuMoDx BKV Quant Assay combines automated DNA extraction, amplification and detection by real-time PCR. Performance of the NeuMoDx BKV Quant Assay was characterized in both urine and plasma using a specimen input volume of 550 μl. The performance of the NeuMoDx BKV Quant Assay was demonstrated across key analytical metrics including analytical sensitivity (LoD), linearity, precision, turnaround time and specificity. Evaluation of analytical sensitivity was performed using the 1st WHO International Standard for BKV, and the limits of quantitation (LLoQ/ULoQ) were determined using the TAE ≤1.0 criterion. The study was performed over
3 days across multiple systems with multiple lots of NeuMoDx reagents. Each system processed 42 replicates at each dilution level (positive samples) and 8 replicates for negative samples per day.
LoD
Analytical sensitivity was characterized by testing a dilution series of the EDX BKV Verification Panel, calibrated against the 1st WHO International Standard in BKV, to determine the LoD on the NeuMoDx Systems. For plasma/serum and urine, LoD was defined as the closest target level, above the concentration determined by Probit -style analysis with 95% CI. The study was performed over 3 days across multiple systems with multiple lots of NeuMoDx reagents.
The LoD of the NeuMoDx BKV Quant Assay in plasma/serum (550 μl workflow) was 20 IU/ml (1.3 Log10 IU/ml) with 95% CI: 11.03. In urine, the LoD was 20.0 IU/ml (1.3 Log10 IU/ml) with 95% CI: 13.09.
LLoQ and ULoQ
Lower limit of quantitation (LLoQ) and upper limit of quantitation (ULoQ) are defined as the upper target levels at which >95% detection is achieved AND the total analytical error* (TAE) ≤1.0. To determine LLoQ and ULoQ, the TAE was calculated for each of the BKV target levels >95% detection.
*TAE: Bias + 2*SD (Westgard Statistic)
Based on this data set and the determined LoD, the LLoQ and ULoQ were determined to be 20 IU/ml (1.3 Log10 IU./ml) and 7.58 x 107 IU/ml (here approximated to 8 Log10 IU/ml), respectively for plasma/serum 550 μl and urine.
Linearity
Linearity was established in plasma/serum and urine by preparing a dilution series using BKV synthetic plasmid with established traceability to the 1st WHO International Standard for BK virus.
11 serial dilutions, prepared in BKV-negative Base Matrix 53 or pooled BKV-negative human urine, were created to span a concentration range of 8–1.58 Log10 IU/ml for plasma/serum 550 μl and urine.
Linearity of three BKV genotypes (BK Virus Dunlop, BK Virus Gardner, BK Virus AB269822_FIN-2) was characterized by testing four different concentrations of each genotype of BKV. Linearity across three BKV genotypes is presented in the table.
Cross-reactivity
Analytical specificity was demonstrated by screening 22 organisms commonly found in plasma/serum or urine specimens, as well as species phylogenetically similar to BKV for cross-reactivity. No cross-reactivity was observed with any of the organisms tested, confirming 100% analytical specificity of the NeuMoDx BKV Quant Assay.
Interfering substances
The NeuMoDx BKV Quant Assay was evaluated in the presence of typical exogenous and endogenous interfering substances encountered in BKV clinical plasma/serum or urine specimens.
Each substance was spiked with 3 Log10 IU/ml BKV and samples were analyzed for interference.
None of the exogenous and endogenous substances affected the specificity of the NeuMoDx BKV Quant Assay.
Within-lab precision
Determined using a 5-member panel of BKV prepared with BKV plasmid to assess performance on one NeuMoDx 96 Molecular System across three separate runs. Maximum overall bias was 0.27 Log10 IU/ml. Equivalent performance was demonstrated across lots as quantitation of all panel members was within tolerance specification.
Lot-to-lot reproducibility
Precision was determined by testing two replicates of a 5-member panel of BKV specimens prepared with BKV plasmid twice daily for 20 days. Excellent precision was demonstrated across days and runs.
Instrument-to-instrument reproducibility
Determined using three different systems (two NeuMoDx 288 Molecular System and one NeuMoDx 96 Molecular System). A 5-member panel of BKV prepared with BKV plasmid was used to assess performance. Testing was performed in parallel
on the systems for 5 days. The variation within-day and between systems was characterized, and the overall standard deviation was determined to be ≤0.30 Log10 IU/ml. Equivalent performance was demonstrated across systems as SD in quantitation of all panel members was within tolerance specification.
The NeuMoDx BKV Quant Assay provides a rapid, easy-to-use and automated method
for detection and quantification of BKV DNA in plasma and urine specimens.