Atypical serologic profiles of hepatitis B virus infection across clinical cohorts of patients in Southwestern Nigeria

ABSTRACT Hepatitis B virus (HBV) infection follows a natural course of events predicted by a dynamic interaction between viral antigen and the host immune system, which forms the basis for HBV serological diagnosis. These interactions may deviate from the typical serologic patterns. This study investigates the types of atypical HBV serologic profiles (AHBSP) across clinical cohorts of patients with HBV infection in southwestern Nigeria. This is a cross-sectional, hospital-based, multi-centered study. Patients’ sera were analyzed for HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc IgM, and anti-HBc IgG by ELISA from 279 study participants attending selected gastroenterology clinics between August 2019 and December 2020. The prevalence of atypical HBV serologic profiles was 27% (n = 76). The mean age of patients was 35.7 ± 11.2 years. The gender distribution involved 183 females (65.6%) and 96 males (34.4%). Across clinical cohorts of patients with atypical serologic profiles, HBeAg Negative, anti-HBe positive with detectable HBV DNA had the highest prevalence of 21% followed by isolated anti-HBc antibody positive, HBsAg negative and detectable HBV DNA, 5%. The atypical serologic profiles, HBeAg positive, HBsAg negative with detectable HBV DNA and concurrent anti-HBs with HBsAg, had the lowest prevalence, 0.4%, respectively. This study identified the considerable presence of atypical HBV serologic profiles across clinical cohorts of HBV infection in southwestern Nigeria.


Introduction
Chronic hepatitis B virus (HBV) infection affects about 296 million individuals globally, [1] while it accounted for about 1.5 million new infections in 2019. [1] It is estimated that about 20 million Nigerians were infected with HBV [2] The clinical and serological outcomes of HBV infection involve interaction between HBV proteins and host immune components, this in turn is influenced by the selective pressure of HBV chemotherapeutic and prophylactic agents, host immune pressure, HBV variants, and serotypes. [3] A triple couplet of HBV antigen and host antibody system forms the basis of the serological evaluation of HBV infection. This includes hepatitis B surface antigen (HBsAg)-hepatitis B surface antibody (anti-HBs), hepatitis B e antigen (HBeAg)-hepatitis B e antibody (anti-HBe), hepatitis B core antigen (HBcAg) and anti-HBc antibodies (anti-HBc both IgM and IgG). [3] The hepatitis B virus surface antigen (HBsAg) plays an essential role in the serological diagnosis of HBV infection and predicts "infectiousness." [4] Chronic HBV infection is defined by the persistence of HBsAg in the serum for more than 6 months among other parameters, while in acute HBV infection, HBsAg is detectable in the serum within the first 6 months only. [4] Hepatitis B e antigen (HBeAg) may be detected in the acute phase of infection. The presence of HBeAg in the serum correlates with HBV replication in the liver [4] Detection of the anti-HBe and anti-HBs in the serum indicates HBV replication termination and infection resolution. [3,4] The anti-HBc IgM antibody is detectable in the acute phase of HBV infection and subsides within 6 months of infection following which anti-HBc IgG predominates. [3] The predictable established serological and clinical patterns of HBV infection involving interaction between hepatitis B virus proteins and human host immune response are known. [3] Deviations from this predictable pattern may occur due to viral factors such as infection with HBV mutant strains, serotypes, or host factors such as defects in cellular immune response and immunosuppression. [3][4][5] Atypical HBV serologic profiles (AHBSP) may therefore emerge. Some common atypical serologic types of HBV infection include the prolonged presence of HBsAg in the serum in the absence of all other serological markers of HBV infection, with detectable HBV DNA, [6][7][8][9] and the presence of anti-HBc IgM or IgG with all other serological markers negative, in the presence of detectable HBV DNA in the serum. [9][10][11][12][13] Other atypical patterns include HBeAg and anti-HBe positivity, with negative HBsAg serological findings, [11] coexistence of HBsAg and anti-HBs serologic patterns with identifiable HBV DNA in the serum. [14] The HBV serologic profiles that predict typical serological diagnosis and assessment of clinical patterns of HBV infection are already well established in Nigeria. There is, however, a paucity of reports in Nigeria and globally on the wholistic profiles of HBV atypical serologic findings across clinical cohorts of HBV-infected patients. This study aims to evaluate the pattern and prevalence of atypical HBV serologic types seen across clinical cohorts of HBV-infected patients in southwestern Nigeria.

Study design
We conducted a cross-sectional, hospital-based, multi-centered study between August 2019 and December 2020.

Study area and population
This research work was carried out in the southwestern region of Nigeria. We recruited study participants from the gastroenterology clinics of the University of Osun Teaching Hospital and State Specialist Hospital, Osogbo, Osun State, Ekiti State University Teaching Hospital, Ado-Ekiti, Federal Teaching Hospital, Ido-Ekiti, Ekiti State, Nigeria. All the adult participants (18 years and above) with hepatitis B virus surface antigen (HBsAg) or hepatitis B virus core antibody (anti-HBc IgM or IgG) positive serological outcomes who volunteered to participate and gave written informed consent were enrolled in the study, while we excluded those who do not fulfill the criteria for inclusion or declined consent.

Ethical permission
We obtained ethical approval for the research work from the Research and Ethics Committee of the University of Osun Teaching Hospital, Osogbo, Osun State, Ekiti State University Teaching Hospital (EKSUTH), Ado-Ekiti Nigeria, Federal Teaching Hospital Ido-Ekiti (FETHI), Ekiti State, Nigeria. The study design conformed to the 2013 declaration of Helsinki

Sample collection and analysis
We collected plasma samples analyzed in the study from patients attending gastroenterology clinics of selected tertiary health-care facilities in southwestern Nigeria between August 2019 and December 2020. We utilized selfadministered structured questionnaires to collect study participants' sociodemographic data. Five milliliters (5 mL) of whole blood was collected from each participant while ensuring sterile procedure was maintained; this was dispensed immediately into appropriately labeled sterile EDTA bottles and transported to the African Center of Excellence for Genomics of Infectious Diseases Research Laboratory, Redeemers University, Ede, Osun State, Nigeria, for laboratory analysis. Patients' plasma samples were separated and stored at −20°C until further analysis.
All plasma samples were subsequently subjected to hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs), hepatitis B e antigen (HBeAg), hepatitis B e antibody (anti-HBe), hepatitis B core IgG and IgM antibodies (anti-HBc IgG and anti-HBc IgM, respectively) by sandwich enzyme-linked immunosorbent assay (ELISA) technique (Melsin Diagnostic Kits, China). We carried out laboratory procedures in line with the manufacturer's instructions. We read each plasma sample optical density (OD) using the Emax endpoint ELISA microplate reader (Molecular Devices, California, USA) and interpreted the results in line with the manufacturer's instructions. The clinical specificity and sensitivity of the Melsin ELISA kits used were 99.88% and 100%, respectively. The Batch numbers of the kits used range from MID-00016 to MID-00028.
All study participants with positive HBsAg only based on cuft-of OD values were re-analyzed after 6 months of initial contact to identify those with persistent isolated HBsAg serological profiles.

DNA extraction and molecular detection of HBV DNA
One hundred and fifty patients' sera with either positive HBsAg or anti-HBc antibodies were randomly selected from 279 study participants recruited into the study. Total DNA was extracted from 200ul of HBsAg and anti-HBc positive plasma samples (n = 150) using the Qiagen DNEasy kit (Qiagen Germany) according to the manufacturer's instructions. Eluted DNA (50ul) was stored in −80°C until analyzed.
The molecular detection of HBV DNA was carried out through a nested PCR amplification of HBV S gene in a procedure targeting a 408 bp fragment, the partial 'S' gene region described earlier. [15,16] The first-round PCR primers used were HBV_S1F (5'-CTAGGACCCCTGCTCGTGTT-3') and HBV_S1R (5'-CGAACCACTGAACAAATGGCACT-3'), while the second-round primers were HBV_SNF (5'-GTTGACAAGAATCCTCACAATACC-3') and HBV_SNR (5'-GAGGCCCACTCCCATA-3'). The first and second-round PCR reaction conditions were similar except that the extracted DNA was used as the template for the first-round PCR, while the first-round PCR products were used as the template for the second-round PCR amplification. The PCR amplification was carried out using a 50 µl reaction containing 10 µl of Redload Taq (Solis Biodyne, USA), 2 µl of each of the primer stock (made in 24uM concentrations), 5 µl of DNA template, and RNase free water were added to make up the reaction volume to 50 µl. The PCR procedure was carried out using Eppendorf Thermal cycler (Eppendorf, United Kingdom) as follows: 94°C for 3 minutes, followed by 45 cycles (denaturation, 94°C for 30 seconds, annealing, 55°C for 60 seconds and elongation, 70°C for 40 second with a ramp of 40% from 55°C to 70°C). Reaction was further elongated at 72°C for 7 min and held at 4°C until the reaction was terminated. PCR products were resolved on 2% agarose gel stained with ethidium bromide and viewed using a UV transilluminator.

Statistical analysis
We checked self-administered structured questionnaires for accuracy and completeness and subsequently entered them into the spreadsheet. Patients' clinical-social variables were presented using frequencies and percentages. The Median and interquartile ranges (IQR) were calculated for non-normally distributed socio-demographic variables. We used student t-test or chisquared test when appropriate to compare and evaluate the association between socio-demographic variables and the different atypical serologic profiles. For all statistical analyses, a P-value of less than 0.05 was considered significant at 95% Confidence Interval (C.I). All statistical analyses were conducted in R statistical package (version 4.1.3) and GraphPad Prism (version 8.4.2, 2020).

Socio-demographic characteristics of study participants
A total of 300 participants were enrolled in the study; however, only 279 patients had appropriately filled out self-administered structured questionnaires and had their blood samples obtained for the study. The overall gender distribution of study participants was 183 females (65.6%) and 96 males (34.4%). The mean age of patients was 35.7 ± 11.2 years (age range: 18-78 years), while the 26-35 year age group had the highest frequency (n = 100) ( Table 1). Most (n = 213) of the participants were married (Table 1).
Among the enrollees with atypical HBV serologic profiles, the median age of participants was 34 years (interquartile range: 26-42 years) ( Table 2). Class five predominates in all the age groups ( Figure 1). The highest frequency of class 2 profile was found in the 21-30 year age group, followed by the 31-40 years and the 51-60 year age group, respectively ( Figure 1). Female gender preponderance was observed with prevalence of 56.6% (43 patients) and males 43.4% (33 patients) ( Table 2). A high proportion of enrollees were married (n = 61), while 13 were single. About two-thirds (n = 51) of this group of enrollees had never been transfused with blood ( Table 2).

Distribution of atypical HBV serologic categories among enrollees
Overall, the prevalence of atypical HBV serologic profiles among all the 279 study participants tested was 27% (n = 76). Class 5 AHBSP had the highest prevalence, 21% (59 patients) (Figure 2), followed by those with Class 2 AHBSP, 5.7% (15 patients) ( Figure 2). Class 3 and Class 4 AHBSP were observed in only one patient each, respectively ( Figure 2). None of the patients was classified under the Class 1 AHBSP (Figure 2).

Distribution of atypical HBV serologic profiles (AHBSP) across clinical cohorts of HBV infection
Of the study participants with AHBSP, class five predominates (n = 59) across the clinical cohort of patients with chronic asymptomatic HBV infection, followed by class two (n = 15) and class three (n = 1) patients (Table 3). Among the clinical cohort of patients with HBV induced hepatocellular carcinoma, the observed atypical HBV serologic profiles include class two (0.4%) and class five (0.4%), respectively (Table 3).
Of the 279 study participants enrolled 76 (27.2%) had atypical HBV serological profile, while 203 (72.8%) had the usual typical serological pattern. Chronic asymptomatic HBV infection clinical cohort predominates among patients with atypical serologic profiles, n = 6 ( Table 3). Among this clinical cohort, class 5 category of atypical HBV serologic profiles forms the bulk of patients with chronic HBV infection, n = 58 (20.4%) followed by the HBV induced hepatocellular carcinoma cohort, n = 1 (0.4%). Also, clinical cohort of patients with hepatocellular carcinoma was found predominantly in the class 2 atypical HBV serologic profile of patients (anti-HBc antibodies, negative HBsAg findings, and detectable HBV DNA occult hepatitis B infection) and class 5 atypical serologic profile (Table 4).

Discussion
In this study, 279 participants were enrolled, including patients with typical and atypical HBV serologic profiles (AHBSP). The overall gender disposition of participants showed a preponderance of the female gender (65.6%). This same pattern was observed across the AHBSP cohort with female gender preponderance (56.6%). This finding is at variance with some earlier studies conducted in Nigeria, where a higher prevalence of HBsAg was reported among sexually active males in comparison to females, [17] Fulani male nomads, [18] and prospective male blood donors in Nigeria. [19] The higher female gender seroprevalence of HBsAg and anti-HBc observed in this study  Generally, women tend to have better health-seeking behavior than men. [20] This might have skewed the study participants toward female gender predominance. In this study, however, on HBV clinical cohort sub-group specific analysis, male gender predominates among patients with symptomatic chronic HBV infection (HBV induced hepatocellular carcinoma and liver cirrhosis). Cases of deviations from the predictable typical serological patterns had been reported. In this study, the prevalence of identified atypical HBV serological findings among 279 respondents was 27.3% (76 patients). Among the patients with HBV atypical serological findings, category 5 (HBeAg negative, anti-HBe positive with detectable HBV DNA) had the highest prevalence of 21% followed by those with category 2 (isolated anti-HBc with negative HBsAg and detectable HBV DNA). No Category 1 (persistent isolated HBsAg) serological profile was recorded in the study despite a 6-month reevaluation of HBV serologic profiles of patients with isolated HBsAg positivity. Both categories 2 and 5 atypical serologic profiles were observed among participants within the 21-50 years age groups. T here is a paucity of data on the overall prevalence of AHBSP among a population of HBV infected patients against which comparable inference could be made with this study. Most reports on AHBSP are isolated, coincidental findings. There was a report on atypical HBV serologic findings in the United States of America on the persistence of anti-HBc IgM in an HIVpositive chronic hemodialysis patient, [21] in China, among treatment-naive chronic hepatitis b patients, concomitant HBsAg and anti-HBs atypical HBV serologic outcome was observed in 4.2% of study participants. [22] The enrolled  subjects were observed to be older, with a large proportion being HBeAg positive, higher levels of ALT, APRI Scores, FIB-4 scores, and liver stiffness values. They attributed concurrent HBsAg and anti-HBs with severe liver fibrosis and cirrhosis in treatment-naive patients with chronic hepatitis B infection. [22] A study on HBV serologic profiles conducted in Turkey reported the prevalence of atypical HBV serologic outcomes in a population of 592 HBV-positive patients as 5.2% (31 patients). Among these, concurrent positivity for both HBsAg and anti-HBs had the highest prevalence, 13 patients (2.1%), followed by the cohort with HBeAg positive, anti-HBe and HBV DNA negative results (1.5%). [16] The overall prevalence of AHBSP observed in the report from Turkey is remarkably lower than what we observed from our study in Nigeria (5.2% vs. 27.2%), and this may reflect the geographical variation in transmission dynamics of HBV.
This study was conducted in Nigeria, an HBV hyperendemic country in sub-Saharan Africa, where the main transmission dynamics of HBV involve perinatal and early childhood infection. [2,23] About 90% of individuals infected with HBV perinatally and during early childhood will develop chronic HBV infection. [24] Longstanding HBV infection has been implicated in the emergence of atypical serologic patterns of HBV. [25,26] The long infection period may enhance the evolution of HBV mutant strains under the host immune, drug, vaccine, and immunoglobulin selective pressure coupled with the lowreplicative fidelity of the HBV DNA polymerase. These mutations may be present with an altered antigenic conformation of the HBV antigens and unpredictable HBV antigen-antibody interaction that deviates from the usual typical patterns. To buttress this point, the highest prevalence of HBV atypical serologic patterns (categories 2 and 5) was observed among patients with chronic HBV infection, irrespective of whether they were symptomatic or not.
Geographical variation may occur in the pattern and prevalence of the HBV atypical serological profiles. For instance, the most common atypical pattern observed in Turkey was the concurrent HBsAg and anti-HBs serologic pattern (Category 4) (15) in contrast to findings from our study in Nigeria where the HBeAg negative, anti-HBe positive and detectable HBV DNA pattern predominates (category 5). An explanation for the observation above is that the nature of atypical serologic profiles circulating in an area may be dependent on the HBV mutants that predominate in the region; this invariably can be partly explained by the circulating genotype of HBV in that region. [27] Among all clinical cohorts of HBV infection, the acute hepatitis patient cohort understandably had the least prevalence (9%) of atypical serologic profiles (and all belong to category 5). This cohort of patients are most likely drug-naive, yet to be vaccinated, or had any immunoglobulin, with a relatively short period of time to allow for the evolution of HBV mutant strains, [25] it therefore suggests the possibility that this acute hepatitis clinical cohort was infected ab initio with HBeAg negative strains. This may give an idea of the circulating HBeAg negative variants of HBV in the Nigerian population. The prevalence of category 5 atypical serologic pattern in chronic asymptomatic HBV patients is more than twice what was observed in patients with chronic hepatitis with liver diseases (liver cirrhosis and hepatocellular carcinoma). This is remarkable, as the reverse would have been expected, as longer duration of time is involved in developing complications of hepatitis B virus infection, which would have allowed the evolution of more mutations across the genes of the hepatitis B virus. [25] However, this may suggest other patient clinical characteristics present among the chronic asymptomatic cohort, such as impaired cellular immunity, immunosuppression, age of participants, or viral characteristics such as different HBV serotypes. It is instructive to note that among all the three categories of atypical serologic profiles observed, category 5 of atypical serologic profiles (with HBeAg negative, anti-HBe positive, detectable HBV DNA) had the highest prevalence (21.1%). This further lends credence to the fact that a high proportion of HBeAg negative variants of HBV may be circulating among the Nigerian population.
Category 2 (anti-HBc antibody with/without anti-HBs, negative HBsAg, and detectable HBV DNA) serologic profile was observed to be highest among the chronic hepatitis B patients with chronic liver diseases (hepatocellular carcinoma). This pattern of atypical serologic profile is associated with persistent HBV infection commonly seen in occult HBV infection (OBI). Occult hepatitis B infection is characterized by episomal integration of replication competent covalently closed circular HBV DNA in the hepatocytes with or without HBV DNA in the blood and undetectable HBsAg when assessed using current assays. [28] Occult hepatitis B infection is implicated in the evolution of hepatocellular carcinoma (HCC) in individuals with chronic HBV infection, HBsAg seroclearance after HBV infection and cryptogenic liver diseases. [28,29] This study further validates this observation as the cases of OBI were highest among patients with hepatocellular carcinoma. The exact mechanism of hepatocarcinogenesis is unclear; however, the likelihood of HBV DNA integration in the hepatocytic cell cycle, increased synthesis of pro-oncogenic proteins, and persistent low-grade necroinflammation has been suggested. [28] Occult hepatitis B infection may also be associated with immunosuppressive states and conditions such as pregnancy, diabetes mellitus, malignancies, and HIV infection. In an HBV case-control molecular study conducted among 733 type 2 diabetes mellitus patients in Iran, occult hepatitis B virus infection was reported among type 2 diabetes mellitus patients with negative HBsAg and positive anti-HBc serological outcomes. None was recorded among the healthy controls. [30] The P120T and G145R HBV S gene mutations were associated with HBsAg diagnostic failure among those patients with occult hepatitis B infection in the study. In another study on prevalence, genotypic distributions, and mutational patterns of hepatitis B virus infection among pregnant women resident in the northern shores of Persian Gulf in Iran, occult hepatitis B infection was reported among the pregnant women. [31] This constitutes a higher risk of vertical transmission perinatally.
Concurrent HBsAg and anti-HBs constitute an atypical presentation of HBV and imply ongoing viral replication despite the presence of neutralizing antibodies against HBV surface antigen. Available evidence suggests the evolution of S gene escape mutants and [32][33][34] re-infection with a new variant. [3,14] Also, heterologous subtype-specific antibodies could be responsible for the atypical presentation above. The antibodies may target HBsAg subtypes different from the existing HBsAg. [3]

Conclusion
This study established the considerable presence of atypical HBV serologic profiles across clinical cohorts of HBV infection in southwestern Nigeria. The interpretation of HBV serological results and overall management of patients with HBV infection should be made with the understanding of the prevalence of these atypical profiles. Therefore, patients with HBV infection should have detailed molecular and serological evaluations carried out for optimum management.

Disclosure statement
No potential conflict of interest was reported by the author(s).