Background
The disease burden associated with Hepatitis B (HBV) necessitates accurate diagnosis and it is important for effective disease management and prevention. The standard approach to HBV diagnosis is hepatitis B surface antigen (HBsAg) screening. However, the rising detection of HBV DNA in HBsAg negative specimens indicates a challenge. The underdiagnosis of HBV due to reliance on HBsAg screening poses a significant public health risk, as individuals with undetected HBV infection unknowingly transmit the virus. Moreover, these individuals remain at risk of liver disease progression without appropriate intervention. Addressing this diagnostic gap is critical for improving patient outcomes, enhancing the accuracy of HBV surveillance, and reducing the overall disease burden associated with HBV. The aim of this study was to investigate HBV DNA and mutations in HBsAg negative specimens at Dr George Mukhari National Health Laboratory Services (NHLS/DGM).
Methods
The study included 444 stored residual serum samples that tested negative for HBsAg during routine diagnosis, from March 2021 to May 2022 at National Health Laboratory Services, Dr George Mukhari (NHLS/DGM). Majority (33%) of the samples were from the 20-29 age group. Two hundred and fourteen (68%) were females. HIV status was known in 158 of 320 samples, of which 92% (146/158) were HIV positive and 8% (12/158) were HIV negative. HBV DNA was detected using real-time polymerase chain reaction (rt-PCR) with a 45 IU/ml (252 copies/ml) detection limit. Subsequently, conventional PCR was performed to amplify and sequence the surface region.
Results
Sixty-nine of 444 (16 %) samples tested positive for HBV DNA. Of these, 28 (41%) had viral loads (VL) below 200 IU/ml with a median VL of 80 IU/ml [61 IU/ml -129 IU/ml], while the remaining 41 (59%) had VL above 200 IU/ml with a median VL of 688 IU/ml [383 IU/ml -7101 IU/ml]. Sequences mutational analysis identified L209V as the most prevalent mutation in 61.3% of sequences, along with D144A causing epitope conformational changes. Immune escape mutations, influencing HBsAg antigenicity such as D144A, Y100C, P142L, N131S and P120T were identified. Genotyping identified six sequences as genotype A1, 20 genotype A2, and four genotype G.
Conclusions
These findings suggest revising HBV diagnostic criteria, recommending that diagnosis should not solely rely on HBsAg screening. Alternatively diagnostic assays, as cost effective as HBsAg screening but with more sensitivity are needed.