Abstract
Introduction: Immunocompromised patients are prone to reactivations and (re-)infections of multiple DNA viruses. Viral load monitoring by single-target quantitative PCRs (qPCR) is the current cornerstone for virus quantification. In this study, a metagenomic next-generation sequencing (mNGS) approach was used for the identification and load monitoring of transplantation-related DNA viruses. Methods: Longitudinal plasma samples from six patients that were qPCR-positive for cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK polyomavirus (BKV), adenovirus (ADV), parvovirus B19 (B19V), and torque teno-virus (TTV) were sequenced using the quantitative metagenomic Galileo Viral Panel Solution (Arc Bio, LLC, Cambridge, MA, USA) reagents and bioinformatics pipeline combination. Qualitative and quantitative performance was analysed with a focus on viral load ranges relevant for clinical decision making. Results: All pathogens identified by qPCR were also identified by mNGS. BKV, CMV, and HHV6B were additionally detected by mNGS, and could be confirmed by qPCR or auxiliary bioinformatic analysis. Viral loads determined by mNGS correlated with the qPCR results, with inter-method differences in viral load per virus ranging from 0.19 log10 IU/mL for EBV to 0.90 log10 copies/mL for ADV. TTV, analysed by mNGS in a semi-quantitative way, demonstrated a mean difference of 3.0 log10 copies/mL. Trends over time in viral load determined by mNGS and qPCR were comparable, and clinical thresholds for initiation of treatment were equally identified by mNGS. Conclusions: The Galileo Viral Panel for quantitative mNGS performed comparably to qPCR concerning detection and viral load determination, within clinically relevant ranges of patient management algorithms.
Highlights
Immunocompromised patients are prone to reactivations andinfections of multiple DNA viruses
We investigated the additional detection of DNA viruses identified by the broad metagenomic next-generation sequencing (mNGS) approach, for which no targeted quantitative PCRs (qPCR) had initially been ordered
After demultiplexing of the sequence reads using bcl2fastq (Illumina, San Diego, CA, USA), FASTQ files were uploaded to the Galileo Analytics web application [13,15] which automatically processes data for quality assessment and pathogen detection using a custom database of DNA viruses involved in transplant-associated infections: ADV, CMV, Epstein-Barr virus (EBV), HHV-6A, HHV-6B, HSV-1, HSV-2, JCV, VZV, B19V, and torque teno-virus (TTV)
Summary
Opportunistic viral infections frequently occur after solid organ or hematopoietic cell transplantation, with associated morbidity and mortality of up to 40% [1]. The monitoring of opportunistic viral infections in transplant patients is most frequently performed by multiple single-plex quantitative PCRs. Metagenomic next-generation sequencing (mNGS) is increasingly being applied for the identification of pathogens in undiagnosed cases suspected of infection [2,3,4]. The Galileo Viral Panel (Arc Bio, LLC, Cambridge, MA, USA) has been designed as a quantitative mNGS approach for ten transplant-related DNA viruses [13,14]. This all-inclusive approach encompasses the library preparation kit, controls, calibration reagents, and cloud-based user-friendly software for bioinformatic analysis. We investigated the additional detection of DNA viruses identified by the broad mNGS approach, for which no targeted qPCR had initially been ordered
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