Evaluation of a Next-Generation Sequencing Metagenomics Assay to Detect and Quantify DNA Viruses in Plasma from Transplant Recipients

Abstract

Viral infections are major causes of morbidity and mortality in solid-organ and hematopoietic stem cell transplant recipients. This study evaluated the performance of the Galileo Pathogen Solution metagenomics Next-Generation sequencing assay to detect and quantify 11 DNA viruses (cytomegalovirus, Epstein-Barr virus, BK virus, human adenovirus, JC virus, herpes simplex virus 1 and 2, varicella zoster virus, human herpesvirus 6A and 6B, and parvovirus B19) and to qualitatively detect torque teno virus. DNA extracted from 47 plasma samples of viremic transplant recipients were subjected to DNA library preparation with pathogen enrichment/human background depletion, sequencing, and automated data analysis. The viral loads were determined with the Galileo assay using a standard curve generated from a calibration panel. All of the samples tested had a 100% agreement with the real-time quantitative PCR (qPCR) assays in detecting the primary virus targets and the majority of the quantified samples had a viral load difference within 0.46 log₁₀ IU/mL or copies/mL. The mean difference for cytomegalovirus between the Galileo and qPCR assays was 0.21 log₁₀ IU/mL (SD, ±0.43 log₁₀ IU/mL). The mean difference for BK virus between the Galileo and qPCR assays was 0.17 log₁₀ cp/mL (SD, ±0.67 log₁₀ cp/mL). Additionally, 75 co-infections were detected in 31 samples by the Galileo assay. The study findings show that the Galileo assay can simultaneously detect and quantify multiple viruses in transplant recipients with results that are comparable with standard-of-care qPCR assays.

Figure 1

Galileo Pathogen Solution (GPS) workflow. The DNA extracted is converted into next-generation sequencing libraries using the Galileo kit (Arc Bio, LLC). After sequencing, the Galileo Analytics informatics pipeline produces quality control (QC) and pathogen identification (ID) reports from demultiplexed fastq files.

Figure 2

Log₁₀-transformed quantitative viral load agreement of cytomegalovirus (CMV) determined by both the Galileo Pathogen Solution (GPS) and real-time quantitative PCR (qPCR) assays. The linear regression line was calculated as follows: y = 0.89x + 0.63 and R² = 0.83. The Bland–Altman plot showed a mean difference of 0.21 log₁₀ IU/mL. A: CMV linear regression. B: CMV Bland–Altman plot. Diff, difference.

Figure 3

Log₁₀-transformed quantitative viral load agreement of BK virus (BKV) determined by both the Galileo Pathogen Solution (GPS) and real-time quantitative PCR (qPCR) assays. The linear regression line was calculated as follows: y = 0.77x + 0.81 and R² = 0.78. The Bland–Altman plot showed a mean difference of 0.17 log₁₀ copies/mL. A: BKV linear regression. B: BKV Bland–Altman plot. Diff, difference.

Figure 4

Log₁₀-transformed quantitative viral load agreement for all samples determined by both the Galileo Pathogen Solution (GPS) and real-time quantitative PCR (qPCR) assays. The linear regression line was calculated as follows: y = 0.58x + 1.60 and R² = 0.46. The Bland–Altman plot showed a mean difference of 0.91 log₁₀ copies/mL or IU/mL. A: Linear regression plot for all samples. B: Bland–Altman plot for all samples. B19, parvovirus B19; BKV, BK virus; CMV, cytomegalovirus; EBV, Epstein–Barr virus; HHV, human herpesvirus; HSV, human herpesvirus; JCV, JC virus; VZV, varicella zoster virus; Diff, difference.

Figure 5

Log₁₀-transformed viral signal score by Galileo Pathogen Solution (GPS) and viral load measurements determined by real-time quantitative PCR (qPCR) assays. The linear regression line was calculated as follows: y = 0.87x + 0.55 and R² = 0.82. A: Torque teno virus (TTV) linear regression plot. B: TTV Bland–Altman plot. Diff, difference.