Near-Full-Length Hepatitis B Virus Genomes Isolated from Cerebrospinal Fluid of People Living with HIV and Cryptococcal Meningitis in Botswana

Background/Objectives: Increasing evidence suggests that the hepatitis B virus (HBV) can be detected in extrahepatic compartments. Data on the molecular characteristics of HBV in cerebrospinal fluid (CSF) remain limited, particularly among people living with HIV (PLWH) and cryptococcal meningitis (CM). This study aimed to characterize HBV genomes from plasma and CSF and compare paired sequences for genetic similarity and potential compartment-specific differences. Methods: Archived plasma and CSF samples from PLWH with CM from Botswana enrolled in AMBITION-CM trial (2018–2021) were sequenced using Oxford Nanopore Technology to generate near-full-length HBV genomes. Genotyping and mutational analysis were performed and paired plasma–CSF sequences were compared. Results: Four sequences were generated, including one plasma–CSF pair and two CSF-only sequences. The sequences clustered with genotypes A1 and D3. The single paired plasma–CSF sequences shared 98.8% nucleotide similarity, and clustered together with strong phylogenetic support (SH-aLRT/ultrafast bootstrap = 99.6/100). Variant analysis identified 492 and 545 high-quality variants (Q ≥ 30) in plasma and CSF, respectively. In the single paired sequences, hepatocellular carcinoma (HCC)-associated mutation T1858C was detected in plasma but not in the CSF. Mutations were also identified in CSF-derived sequences, including A1762T/G1764A, G1896A, G1899A, associated with increased HCC risk, rtS184L, rtF194L, rtP236T, linked to antiviral resistance and sP127L, sQ129L, sM133L, sS136P, associated with immune escape. Conclusions: Near-full-length HBV genomes were successfully recovered and genetically characterized from the CSF of PLWH with CM. The single plasma–CSF paired sample, showed high genetic similarity and had a compartment-specific mutation. Clinically relevant mutations were observed in the CSF warranting further investigation of HBV in the CSF to confirm these observations in PLWH with CM. These preliminary findings from a limited number of participants provide a basis for larger-scale studies of near-full-length HBV genome characterization in CSF.