Ultrasensitive PCR system for HBV DNA detection: Risk stratification for occult hepatitis B virus infection in English blood donors

Abstract

Occult hepatitis B (HBV) infection (OBI), characterized by low viral loads, accounts for much of the risk of HBV transfusion‐transmitted infection. With anticore antibodies (anti‐HBc) screening introduced in England, the imperative to identify OBI donors has increased. We aimed to develop an ultra‐sensitive PCR system and investigate risk factors for HBV DNA presence in blood donations. Seven extraction methods and three PCR assays were compared. The optimal system was sought to determine HBV DNA presence in anti‐HBc‐positive donations. Predictors of DNA positivity were subsequently investigated. Extraction from 5 mL of plasma increased sample representation and resulted in HBV DNA detection in low viral load samples (~0.5 IU/mL). Screening of 487 763 donations in 2022 identified two OBI donors and 2042 anti‐HBc‐positive donors, 412 of the latter with anti‐HBs < 100 mIU/mL. Testing of 134 anti‐HBc‐positive donations utilizing the 5 mL extraction method identified two further HBV DNA‐positive donations. Higher anti‐HBc titer and anti‐HBs negativity were significant predictors of DNA detectability in anti‐HBc‐positive donations. An ultrasensitive PCR assay identified potentially infectious donations increasing HBV DNA detection in anti‐HBc‐positive donors from 0.5% to 1.9%. Anti‐HBc titers may further complement the risk stratification for DNA positivity in anti‐HBc screening and minimize unnecessary donor deferral.

Biomarkers of transfusion transmitted occult hepatitis B virus infection: Where are we and what next?

“…We recently showed that a higher anti-HBc titre was predictive of DNA presence in the plasma of anti-HBc positive donors, 44 corroborating other findings that higher anti-HBc levels were independently associated with detecting cccDNA. 52 Increased anti-HBc levels suggest the production of more HBV core antigens and may correlate with increased production of infectious virions, translating to higher TTI risk.…”

“…43 We recently developed an ultrasensitive HBV DNA Polymerase chain reaction (PCR) system with a 50% LOD of 0.12 IU/mL, where assay sensitivity was improved by increasing sample representation and extraction volume to 5 mL of plasma. 44 Indeed, testing DNA extracts from at least 1 mL of sample was also previously recommended. 3 Increasing the sample volume in automated testing in diagnostic services would significantly improve the sensitivity of HBV DNA detection, enabling further DNA-positive OBI donors to be identified.…”

“…Accumulating evidence suggests that the infectivity of blood transfusions from OBI donors is significantly reduced and potentially neutralised when anti-HBs antibodies are present compared to donors with isolated anti-HBc status. 6,8,53,[57][58][59][60] Indeed, studies found HBV DNA detection highest in anti-HBc positive and anti-HBs negative blood donors, 37,44 where anti-HBs in the bloodstream may neutralise infectious HBV particles. 54 TTI studies corroborate this, where a European study found all recipients of anti-HBs-negative fresh frozen plasma (FFP) were infected, whilst none of the three FFP recipients of anti-HBs positive FFP had markers of infection.…”

“…72 However, our recent findings suggested that his approach had relatively low sensitivity and specificity for predicting detectable HBV DNA. 44…”

“…Such sensitive detection is challenging, considering current NAT assays have 95% LODs ranging from 4 to 12 IU/mL 43 . We recently developed an ultrasensitive HBV DNA Polymerase chain reaction (PCR) system with a 50% LOD of 0.12 IU/mL, where assay sensitivity was improved by increasing sample representation and extraction volume to 5 mL of plasma 44 . Indeed, testing DNA extracts from at least 1 mL of sample was also previously recommended 3 .…”