Use of the Quantitative Karius® Plasma Next Generation Sequencing Cell-Free Pathogen DNA Test to Detect and Monitor Cytomegalovirus Infection in Allogeneic Stem-Cell Transplant Recipients

Abstract

Background Allogeneic stem-cell transplant (allo-SCT) recipients are at risk of developing severe CMV infection. The pre-emptive approach to CMV prevention, weekly monitoring with CMV PCR with treatment for positive viral load (VL), is standard of care. The quantitative Karius plasma Next Generation Sequencing test (KT) can identify DNA from >1,000 human pathogens. We report on the clinical validation and utility of KT for detecting and monitoring CMV viremia in allo-SCT patients. Methods Plasma samples negative or positive by CMV PCR (COBAS® AmpliPrep/COBAS® TaqMan® CMV Test [CAP/CTM] or cobas 6800/8800 [cobas® CMV]) were tested with KT for analytical comparison. For KT, after cell-free DNA (cfDNA) was sequenced, human-derived reads were excluded bioinformatically, and remaining DNA sequences were aligned to a curated microbial pathogen database. KT results were reported as molecules per microliter (MPM). Linear regression was used to correlate log10 transformed CMV VL results within the overlapping quantifiable range of comparison assays. Using the regression equation to convert KT results from MPM to IU/mL, percent agreement was calculated and assay result agreement was analyzed with Bland-Altman plots. Allo-SCT recipients with CMV viremia were selected from the prospective observational DISCOVER trial (NCT02804464), and their plasma specimens were tested with KT. Mean differences in MPM between ≥7 days before start of antiviral treatment (D-7) and start of treatment (D0), and between D0 and ≥14 days after start of treatment (≥D14) were examined with Wilcoxon rank-sum tests. Results Among 125 residual samples tested by all 3 assays, 1 sample failed KT sequencing and was excluded from the analysis. Percent agreement between KT and cobas CMV (n = 124) was 94% using the cobas cut-off for negative samples (VL Conclusions High correlation was observed between KT and two FDA-approved CMV VL assays. KT identified significant increases in CMV VL at start of treatment and significant decreases at ≥14 post-treatment in allo-SCT recipients, showing potential clinical utility of KT as a monitoring assay, with the added value of detecting >1,000 pathogens in the same blood draw.