Droplet Digital PCR Development for Adenovirus Load Monitoring in Children after Hematopoietic Stem Cell Transplantation

Human adenovirus (AdV) reactivation after hematopoietic stem cell transplantation (HSCT) is associated with life-threatening clinical manifestations. Although real-tme quantitative PCR (qPCR) has been widely used to measure AdV loads, it has not been standardized for AdV. Droplet digital PCR (ddPCR) is a novel pathogen detection technology that enables the absolute quantification of viral loads. ddPCR would enable a more accurate AdV DNA detection compared with qPCR. In this study, ddPCR was developed for AdV DNA and its performance characteristics compared with those of qPCR. AdV DNAemia incidence during the first 12 weeks after allogenic HSCT was then retrospectively examined by qPCR and ddPCR in 97 HSCT procedures using the preserved 545 DNA samples. ddPCR exhibited better reproducibility and sensitivity, as well as equivalent quantifiability, compared with qPCR. AdV DNA among HSCT patients was detected in 11 (2.0%) and 49 (9.0%) of 545 samples by qPCR and ddPCR, respectively. AdV DNA levels >1000 copies/mL were observed in five cases by qPCR and/or ddPCR. However, two patients developed fulminant hepatitis and died; other patients remained asymptomatic with subsequently undetectable AdV DNA. In conclusion, ddPCR was more sensitive and reproducible in detecting AdV DNA among pediatric HSCT recipients than qPCR. ddPCR offers the potential to provide a more accurate DNAemia detection, determine cutoff values for treatment initiation, and enable antiviral efficacy assessment.