Background: Gaps remain in the detection of nucleic acid test (NAT) yield and occult hepatitis B virus (HBV) infection (OBI) by current HBV surface antigen (HBsAg) assays. The lack of detection may be due to HBsAg levels below current assay detection limits, mutations affecting HBsAg assays or HBsAg levels, or the masking of HBsAg by antibody to HBsAg (anti-HBs). In this study, we evaluate the incremental detection of NAT yield and OBI from five diverse geographic areas by an improved sensitivity HBsAg assay and characterize the samples relative to the viral load, anti-HBs status, and PreS1–S2–S mutations. Included is a comparison population with HBV DNA levels comparable to OBI, but with readily detectable HBsAg (High Surface–Low DNA, HSLD). Methods: A total of 347 samples collected from the USA, South Africa, Spain, Cameroon, Vietnam, and Cote D’Ivoire representing NAT yield (HBsAg(−), antibody to HBV core antigen (anti-HBc)(−), HBV DNA(+), N = 131), OBI (HBsAg(−), anti-HBc(+), HBV DNA(+), N = 188), and HSLD (HBsAg(+), anti-HBc(+), HBV DNA(+), N = 28) were tested with ARCHITECT HBsAg NEXT (HBsAgNx) (sensitivity 0.005 IU/mL). The sequencing of the PreS1–S2–S genes from a subset of 177 samples was performed to determine the genotype and assess amino acid variability, particularly in anti-HBs(+) samples. Results: HBsAgNx detected 44/131 (33.6%) NAT yield and 42/188 (22.3%) OBI samples. Mean HBV DNA levels for NAT yield and OBI samples were lower in HBsAgNx(−) (50.3 and 25.9 IU/mL) than in HBsAgNx(+) samples (384.1 and 139.5 IU/mL). Anti-HBs ≥ 10 mIU/mL was present in 28.6% HBsAgNx(+) and 45.2% HBsAgNx(−) OBI, and in 3.6% HSLD samples. The genotypes were A1, A2, B, C, D, E, F, and H. There was no significant difference between HBsAgNx(−) and HBsAgNx(+) in the proportion of samples harboring substitutions or in the mean number of substitutions per sample in PreS1, PreS2, or S for the NAT yield or OBI (p range: 0.1231 to >0.9999). A total of 21/27 (77.8%) of HBsAgNx(+) OBI carried S escape mutations, insertions, or stop codons. HSLD had more PreS1 and fewer S substitutions compared to both HBsAgNx(−) and HBsAgNx(+) OBI. Mutations/deletions associated with impaired HBsAg secretion were observed in the OBI group. Conclusions: HBsAgNx provides the improved detection of NAT yield and OBI samples. Samples that remain undetected by HBsAgNx have exceptionally low HBsAg levels below the assay detection limit, likely due to low viremia or the suppression of HBsAg expression by host and viral factors.

The cornerstone of functional cure for chronic hepatitis B (CHB) is hepatitis B surface antigen (HBsAg) loss from blood. HBsAg is encoded by covalently closed circular DNA (cccDNA) and HBV DNA integrated into the host genome (iDNA). Nucleos(t)ide analogs (NUCs), the mainstay of CHB treatment, rarely lead to HBsAg loss, which we hypothesized was due to continued iDNA transcription despite decreased cccDNA transcription. To test this, we applied a multiplex droplet digital PCR that identifies the dominant source of HBsAg mRNAs to 3,436 single cells from paired liver biopsies obtained from 10 people with CHB and HIV receiving NUCs. With increased NUC duration, cells producing HBsAg mRNAs shifted their transcription from chiefly cccDNA to chiefly iDNA. This shift was due to both a reduction in the number of cccDNA-containing cells and diminished cccDNA-derived transcription per cell; furthermore, it correlated with reduced detection of proteins deriving from cccDNA but not iDNA. Despite this shift in the primary source of HBsAg, rare cells remained with detectable cccDNA-derived transcription, suggesting a source for maintaining the replication cycle. Functional cure must address both iDNA and residual cccDNA transcription. Further research is required to understand the significance of HBsAg when chiefly derived from iDNA.

In vaccine clinical trials, both binding antibody (bAb) levels and neutralization antibody (nAb) titers have been shown to be correlates of SARS-CoV-2 vaccine efficacy. We report a strong correlation bAb and nAb responses against the SARS-CoV-2 Omicron (BA.1) variant in infection-naïve and previously infected (convalescent) individuals after one and two doses of BNT162b2 vaccination. The vaccine-induced bAb levels against Omicron were significantly lower compared to previous variants of concern in both infection-naive and convalescent individuals, with the convalescent individuals showing significantly higher bAb compared to the naïve individuals at all timepoints. The finding that bAb highly correlated with nAb provides evidence for utilizing binding antibody assays as a surrogate for neutralizing antibody assays. Our data also revealed that after full vaccination, a higher percentage of individuals had undetectable Omicron nAb (58.6% in naive individuals, 7.4% in convalescent individuals) compared to the percentage of individuals who had negative Omicron bAb (0% in naive individuals, 0% in convalescent individuals). The discordance between bAb and nAb activities and the high degree of immune escape by Omicron may explain the high frequency of Omicron infections after vaccination.

Background: Long-term studies of vaccine recipients are necessary to understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody durability and assess the impact of booster doses on antibody levels and protection from infection. The identification of vaccine breakthrough infections among fully vaccinated populations will be important in understanding vaccine efficacy and SARS-CoV-2 vaccine escape capacity. Methods: SARS-CoV-2 spike (S) receptor-binding domain and nucleocapsid (N) immunoglobulin (Ig) G levels were measured in a longitudinal study of 1000 Chicago healthcare workers who were infection naive or previously infected and then vaccinated. Changes in S and N IgG were followed up through 14 months, and vaccine breakthrough infections were identified by increasing levels of N IgG. Results: SARS-CoV-2 S IgG antibody levels among previously infected and previously noninfected individuals decreased steadily for 11 months after vaccination. Administration of a booster 8–11 months after vaccination increased S IgG levels >2-fold beyond those observed after 2 doses, resulting in S IgG levels that were indistinguishable between previously infected and uninfected individuals. Increases in N IgG identified vaccine breakthrough infections and showed >15% breakthrough infection rates during the Omicron wave starting in December 2021. Conclusions: These results demonstrate SARS-CoV-2 antibody changes after vaccination and breakthrough infections and identify high levels of vaccine breakthrough infections during the Omicron wave, based on N IgG increases.

Background: The loss of HBV HBsAg or functional cure is a desirable goal of hepatitis B management. The relative abundances of HBsAg isoforms may offer additional diagnostic and predicting values. To evaluate the clinical utility of HBsAg isoforms, we developed novel prototype assays on the ARCHITECT automated serology platform that specifically detects total-HBsAg (T-HBsAg), large (L-HBsAg), and middle (M-HBsAg) products of the S gene to determine the isoform composition of human specimens from acute and chronic HBV infection and during long-term nucleos(t)ide analog therapy. Results: In the early phase of acute HBV infection, L-HBsAg and M-HBsAg emerged within days and were in parallel to T-HBsAg during the entire course of infection. M-HBsAg levels were consistently higher than L-HBsAg levels. Patients with HBeAg(+) chronic hepatitis B had higher T-HBsAg, M-HBsAg, and L-HBsAg levels compared with HBeAg(−) patients. Correlations of M-HBsAg and L-HBsAg to T-HBsAg were similar in both. In contrast, there was no strong correlation between L-HBsAg or M-HBsAg with HBV DNA levels. During long-term nucleos(t)ide analog treatment, changes in HBsAg isoform abundance were proportional to T-HBsAg regardless of treatment responses for both HBeAg(+) and HBeAg(−) chronic hepatitis B. A larger sample size may be necessary to detect a significant difference. Conclusion: HBsAg isoform compositions parallel T-HBsAg levels in both acute and chronic hepatitis B infection. L-HBsAg and M-HBsAg individual biomarkers do not appear to provide an additional diagnostic benefit for staging chronic disease or monitoring response to treatment with current therapies.

The 2022 outbreak of the Clade IIb MPX virus and subsequent global spread lead to an urgent need for the development of high throughput, sensitive, and reproducible diagnostic tests. We developed 3 assays to detect MPX virus, two (MPXV + and MPXV) for m2000 RealTime and one (MPXV) for Alinity m platforms. Dual-targets in E9L and B6R (MPXV+) and J2L and B7R (MPXV) genes were selected to increase detection of emerging mutations. In silico prediction of MPXV + and MPXV target sequences suggest broad detection of Orthopox viruses with MPXV + and more specific MPX virus detection with MPXV assays, respectively. Each assay incorporates an internal control to confirm expected assay performance and the Alinity m MPXV assay also contains a cellular control which assesses adequacy of sample collection. m2000 RealTime MPXV + and MPXV assay sensitivity was determined to be 3.2 PFU/mL using a reference virus culture diluted into UTM. Alinity m MPXV lower limit of detection was 200 copies/mL using MPX plasmids in pooled UTM matrix. m2000 RealTime MPXV + and MPXV assays were validated with lesion swabs in UTM and 1:1 saliva:UTM mixtures without loss of sensitivity. Commercially available and remnant clinical lesion specimens in UTM were tested with RealTime MPXV+, RealTime MPXV and Alinity m MPXV assays and demonstrated high agreement to known MPX positive specimens. RealTime MPXV+, RealTime MPXV and Alinity MPXV are high throughput and sensitive assays used for the detection of MPX virus. These assays maybe useful during MPX outbreaks.