Candidate epistatic mutation clusters in occult hepatitis B virus genotype D: a critical narrative review of the evidence gap and a proposed testable framework

Abstract

Hepatitis B virus (HBV) genotype D is the dominant genotype across the Middle East. It forms the background against which diagnostic assays, blood-screening algorithms, and vaccination programmes operate in the region. Occult hepatitis B infection (OBI), the presence of replication-competent HBV DNA in individuals who test negative for hepatitis B surface antigen (HBsAg), remains a persistent challenge for diagnosis, for transfusion and transplant safety, and for the management of immunosuppressed patients. Individual surface-protein escape mutations are well characterised, yet they account only incompletely for the full spectrum of OBI. It is not yet established whether combinations of co-occurring surface mutations interact non-additively, that is epistatically, to influence HBsAg detectability, secretion, or replication in genotype D, whether such combinations are enriched in Middle Eastern populations, or whether they are shaped by HIV or HCV coinfection. The functional and structural data required to answer these questions are largely absent. We critically appraise the available evidence, separate established findings from extrapolation and hypothesis using an explicit grading scheme, and propose a testable bioinformatic and experimental framework to determine whether candidate epistatic clusters contribute to OBI in genotype D. We propose and apply a four-level evidence hierarchy distinguishing co-occurrence, phylogenetically corrected covariation, experimental single-versus-combination comparison, and mechanistic validation. Against this hierarchy, published data on HBsAg mutation clusters in genotype D reach Level 1–2 at most; the decisive Level 3 experiment, site-directed reconstruction of candidate clusters versus their constituent single mutants on a defined genotype D backbone, has not been performed. Genotype-D-specific and Middle-East-specific datasets adequate for cluster-level analysis are limited, direct evidence linking coinfection to selection of mutation clusters is absent, and structural inference remains constrained because recent cryo-EM structures of HBsAg subviral particles do not resolve the ‘a’ determinant loops at the resolution required to interpret mutation-cluster-level conformational perturbations. Epistasis is a biologically plausible, under-tested mechanism that may help explain why single mutations incompletely account for OBI. It should not be presented as an established driver. We define the sequencing, functional, structural, and clinical studies required to test the hypothesis directly, and we identify the methodological safeguards, particularly phylogenetic correction, that any such effort must adopt.