Comparison of Droplet Digital PCR and Metagenomic Next-Generation Sequencing Methods for the Detection of Human Herpesvirus 6B Infection Using Cell-Free DNA from Patients Receiving CAR-T and Hematopoietic Stem Cell Transplantation

Abstract

Purpose The aim of this study was to examine and compare the differences between droplet digital PCR (ddPCR) and metagenomic next-generation sequencing (mNGS) in the detection of human herpesvirus 6B (HHV-6B). Long-term monitoring of HHV-6B viral load in patients receiving chimeric antigen receptor-modified T-cell (CAR-T) therapy and hematopoietic stem cell transplantation (HSCT) can be used to identify immune effector cell-associated neurotoxicity syndrome (ICANS) and guide drug therapy. Methods Twenty-seven patients with suspected HHV-6B infection who had both mNGS and ddPCR test results were analyzed retrospectively, including 19 patients who received CAR T-cell therapy and 8 who received HSCT. The HHV-6B probe and primers were designed, and the performance of the ddPCR assay was evaluated. Subsequently, ddPCR was performed utilizing blood and urine. Data on clinical information and mNGS investigations were collected. Results The ddPCR test results correlated significantly with the mNGS test results ( P < 0.001, R 2 = 0.672). Of the 27 time-paired samples, ddPCR showed positive HHV-6B detection in 20 samples, while mNGS alone showed positive HHV-6B detection in 12 samples. ddPCR detected additional HHV-6B infections in 8 samples that would have been missed if only mNGS were used. In addition, the first HHV-6B infection event was detected at a median of 14 days after CAR T-cell infusion (range, 8 to 19 days). Longitudinal monitoring of HHV-6B by ddPCR was performed to assess the effectiveness of antiviral therapy. The data showed that with antiviral treatment HHV-6B viral load gradually decreased. Conclusion Our results indicated that ddPCR improved the HHV-6B positive detection ratio and was an effective adjunct to mNGS methods. Furthermore, the longitudinal detection and quantification of HHV-6B viral load in patients undergoing CAR T-cell therapy and HSCT may serve as a guide for drug treatment.

Improving the safety of CAR‐T‐cell therapy: The risk and prevention of viral infection for patients with relapsed or refractory B‐cell lymphoma undergoing CAR‐T‐cell therapy

“…83 Although many HHV-6 infections are asymptomatic and represent initial infections, reactivations, or external source reinfections, the virus can affect the nervous system and trigger severe illnesses, particularly in individuals with compromised immune systems. 84 Lareau et al found that HHV-6 reactivated ex vivo in a CAR-independent manner, correlating with T-cell activation and proliferation. 85 The actual HHV-6 infection rate is likely underreported.…”

“…Human herpesvirus 6 (HHV‐6), a commonly found β‐herpesvirus, can infect various cell types and establish lifelong latent infections in humans, similar to other herpesviruses 83 . Although many HHV‐6 infections are asymptomatic and represent initial infections, reactivations, or external source reinfections, the virus can affect the nervous system and trigger severe illnesses, particularly in individuals with compromised immune systems 84 . Lareau et al found that HHV‐6 reactivated ex vivo in a CAR‐independent manner, correlating with T‐cell activation and proliferation 85 .…”