Detection of the hepatitis B surface antigen in patients with occult hepatitis B using an assay with enhanced sensitivity

Patients with occult hepatitis B infection (OBI) have undetectable hepatitis B surface antigen (HBsAg) by conventional assays but detectable hepatitis B virus (HBV) DNA in blood/liver. We evaluated the key performance characteristics of a sensitive HBsAg assay (ARCHITECT HBsAg Next Qualitative Assay, referred as NEXT) with respect to HBsAg detection. Assay precision, sample carryover and seroconversion sensitivity of NEXT were evaluated. HBsAg was measured by NEXT in 1,138 individuals, including 1,038 patients who attended liver clinics in a tertiary hospital (200 HBV DNA-positive blood donors whose HBsAg was undetectable by conventional assays, and 38 patients receiving immunosuppressive therapy, 800 chronic hepatitis B patients with HBsAg seroclearance) and 100 HBsAg-negative subjects recruited from a community project. The within-run and within-laboratory coefficients of variation were <6% for the positive sample pools. In 9 seroconversion panels tested, NEXT allowed an earlier HBsAg detection than conventional assays. NEXT detected HBsAg in 10/200 (5%) HBsAg-negative blood donors, 1/20 (5%) and 0/18 HBsAg-negative patients with and without HBV reactivation respectively, and 59/800 (7.3%) patients with HBsAg seroclearance. HBsAg was detectable by NEXT in 27.8%, 8.2%, 6.9%, 3.8% and 1.9% samples at <3, 3–5, >5–8, >8–11, and >11 years after HBsAg seroclearance, respectively. Seven out of 100 HBsAg-negative community identified subjects was tested positive by NEXT. Comparing with conventional HBsAg assays, NEXT demonstrated a higher sensitivity and conferred an increment of 5–7% detection rate in patients with OBI, thereby helping in identifying HBV carriers and prevention of OBI-associated HBV transmission and reactivation.

Detection of occult hepatitis B virus in patients undergoing chemotherapy in Iran

Aim: Occult hepatitis B infection (OBI) is life threatening and has a high mortality rate despite applying antiviral treatments in cancer patients. This study aimed to investigate the prevalence of OBI in patients undergoing chemotherapy in Iran. Materials & methods: A total of 342 patients undergoing chemotherapy were enrolled. OBI detection in anti-HBc positive individuals was conducted using nested-PCR. Results: Among 342 subjects, 103 (30.1%) were positive for anti-HBc. Fifteen (14.6%) cases of 103 anti-HBc positive samples were also positive for HBsAg. Overall, HBV DNA was positive in three (3.4%) of 88 anti-HBc subjects. Conclusion: Our results indicated that OBI might occur in almost one in 25 anti-HBc-positive patients undergoing chemotherapy.

E2 Site Mutations in S Protein Strongly Affect Hepatitis B Surface Antigen Detection in the Occult Hepatitis B Virus

The mechanism of occult hepatitis B infection (OBI) has not yet been fully clarified. Our previous research found that novel OBI-related mutation within S protein, E2G, could cause the hepatitis B surface antigen (HBsAg) secretion impairment, which resulted in intracellular accumulation in OBI of genotype B. Here, to further explore the role of E2 site mutations in the occurrence of OBI, we analyzed these site mutations among 119 OBI strains identified from blood donors. Meanwhile, 109 wild-type HBV strains (HBsAg positive/HBV DNA positive) were used as control group. Furthermore, to verify the E2 site mutations, two conservative 1.3-fold full-gene expression vectors of HBV genotype B and C (pHBV1.3B and pHBV1.3C) were constructed. Then, the E2 mutant plasmids on the basis of pHBV1.3B or pHBV1.3C were constructed and transfected into HepG2 cells, respectively. The extracellular and intracellular HBsAg were analyzed by electrochemical luminescence and cellular immunohistochemistry. The structural characteristics of S proteins with or without E2 mutations were analyzed using relevant bioinformatics software. E2 mutations (E2G/A/V/D) existed in 21.8% (26/119) of OBIs, while no E2 mutations were found in the control group. E2G/A/V/D mutations could strongly affect extracellular and intracellular level of HBsAg (p &lt; 0.05). Notably, unlike E2G in genotype B that could cause HBsAg intracellular accumulation and secretion decrease (p &lt; 0.05), E2G in genotype C could lead to a very significant HBsAg decrease both extracellularly (0.46% vs. pHBV1.3C) and intracellularly (11.2% vs. pHBV1.3C) (p &lt; 0.05). Meanwhile, for E2G/A mutations, the relative intracellular HBsAg (110.7–338.3% vs. extracellular) and its fluorescence intensity (1.5–2.4-fold vs. with genotype-matched pHBV1.3B/C) were significantly higher (p &lt; 0.05). Furthermore, N-terminal signal peptides, with a typical cleavage site for peptidase at positions 27 and 28, were exclusively detected in S proteins with secretion-defective mutants (E2G/A). Our findings suggest that: (1) E2G/A/V/D mutations were confirmed to significantly influence the detection of HBsAg, (2) the underlying mechanism of OBI caused by E2G mutation is quite different between genotype B and genotype C, and (3) E2G/A could produce a N-terminal truncated S protein, which might attribute to the HBsAg secretion impairment in the OBIs.

HBV - infection in children with perinatal infection. Clinical report of familial hepatitis B

Hepatitis B, despite of being a controlled infection today, is one of the most common form of hepatitis in the world. According to the experts' evaluation there are about 3 million patients with chronic hepatitis B in our country. The global strategy of the World Health Organization includes the elimination of viral hepatitis by 2030.The program used in the North-West Federal District to eliminate acute hepatitis B has reduced the incidence rate due to the widespread vaccination coverage of children and the annual increase in adult immunization coverage.However, the relevance of HBV-infection in children still remains high which is associated with a high infection by hepatitis B virus in women of childbearing age and the possibility of the transmission of the infection from mother to her child. In case of perinatal infection the formation of chronic hepatitis B in children reaches up to 90%. The natural course of chronic hepatitis B is characterized by a change in pathogenetically determined phases, and HBsAg-negative infection, which is a latent (occult) form of hepatitis, was added to them in 2009. Occult hepatitis B is an epidemiological danger, for the child as well, if the mother suffers from this form of chronic hepatitis B. Monitoring of pregnant women is often limited to identifying only HBsAg, which is not enough to detect occult hepatitis B. Lately diagnosed occult HBV-infection can become a source of the infection for the baby, especially in cases when due to some reasons there are disorders in hepatitis B vaccination schedule after birth. The article presents an interesting clinical case of family hepatitis B.

Occult Hepatitis B Virus Infection among β-Thalassemia Major Patients in Ahvaz City, Iran

Occult Hepatitis B Infection (OBI) is a critical risk factor for triggering post-transfusion hepatitis (PTH), cirrhosis, hepatocellular carcinoma, and hepatitis B virus (HBV) reactivation, which β-thalassemia major (BTM) patients are at risk of it due to multiple blood transfusions. This study was aimed at determining the prevalence of OBI among BTM patients from Khuzestan Province, Iran. In this cross-sectional study, 90 thalassemia patients, who have received blood 36 to 552 times, participated referred to the Shafa hospital of Ahvaz city from January 2018 to April 2019. ELISA for determining serological markers (HBsAg, anti-HBc, anti-HBs, and anti-HCV) and real-time PCR for detecting HBV-DNA were performed; Nested PCR was conducted for DNA sequencing and determining the genotype of OBI case. Phylogenetic and statistical analyses were done by R package. Of 90 subjects enrolled in this study; 95.5% (86/90) were HBsAg negative, and the frequency of OBI among them was 1.16% (1/86). The anti-HBs, anti-HBc, and anti-HCV were detected in 80.00%, 7.78%, and 12.2% of patients, respectively. HBV-DNA was assessed at four HBsAg-positive subjects as well, and all of them were negative. The phylogenetic analysis showed that the detected HBV DNA in the OBI case belongs to the genotype D. This research, for the first time, demonstrated that OBI is present among β-thalassemia patients in Iran. Also, further studies are necessary to determine the actual prevalence of OBI among BTM patients in Iran to decisions concerning OBI screening, especially in transfusion centers.