Development of a digital droplet PCR assay to measure HBV DNA in patients receiving long-term TDF treatment.

The COBAS TaqMan assay has a lower limit of quantification (LLOQ) of 169 HBV copies/mL and a lower limit of detection (LLOD) of 58 copies/mL. HBV DNA below the TaqMan LLOQ is classified as target not detected (TND) (<58 copies/mL) or target detected (TD) (between 58 and 169 copies/mL). Here we have developed a more sensitive digital droplet PCR (ddPCR) assay to evaluate the impact of long-term tenofovir disoproxil fumarate (TDF) treatment in patients that did or did not achieve HBsAg seroconversion. A ddPCR assay was developed to detect HBV DNA to 8 copies/mL. HBV DNA levels in plasma from patients with or without HBsAg seroconversion were assessed by ddPCR. For patients who did not achieve HBsAg seroconversion, the majority of TD samples (33/58, 57%) were HBV DNA positive by ddPCR while (10/37, 27%) of TND samples were positive. In contrast, for patients who achieved HBsAg seroconversion, HBV DNA was rarely detected by ddPCR after HBsAg seroconversion (1/28, 3.6%). ddPCR is a sensitive method to evaluate low-level viral replication in plasma samples. Frequent detection of HBV DNA by ddPCR among patients who did not achieve HBsAg seroconversion suggests new agents may be needed to suppress low levels of replicating HBV.