Cytomegalovirus (CMV) is a common viral infection. Upwards of 1 in 5 young children are infected, and in healthy individuals CMV becomes a lifelong asymptomatic latent infection. In patients receiving stem cell or solid organ transplants, latent CMV can reactivate, causing life-threatening infection. Patients who are at high risk of CMV reactivation are prophylactically treated with antiviral drugs; however, treatment can last for months, increasing the risk of CMV antiviral resistance, and exposing the patient to potentially toxic side-effects. Currently, basic antiviral resistance screening for two key genes involved in resistance is performed at a single national laboratory, delaying results, and limiting their clinical value. Here, we evaluated CMV diagnostics with capabilities for expanded antiviral resistance detection.
Three next generation sequencing assays; Vela Diagnostics ViroKey FLEX assay (amplicon, ion torrent), Agilent SureSelect XT Community Design assay (WGS, Illumina) and an in-house sequencing assay (amplicon, Illumina) were evaluated. We compared cost, workflow, and sequencing success directly from CMV-positive clinical samples.
The Vela assay cost ~$300 per sample, had a low sequencing success rate (30%, unrelated to viral load), required two days of semi-automated library preparation, and the automated bioinformatic pipeline failed to detect clinically relevant mutations. Agilent cost ~$220 per sample, resulted in high quality sequencing results down to 1000 IU/mL of CMV (for 85% of samples) and required two days library preparation for three day turnaround time. The in-house approach was cost-effective (~$120 per sample) and efficient (one day library preparation, two day turnaround); however, requires a higher viral load (~20,000 IU/mL) for 76% sequencing success. Both Agilent and In-house assays require manual interpretation of results.
Overall, the Agilent WGS assay is an excellent option for routine CMV resistance detection, however, the prohibitive cost of this assay may limit its utility within pathology settings. Refinement of the In-house amplicon sequence assay may provide a cost-effective and rapid alternative to Agilent WGS.