Hepatitis B Virus Immunity Gap: A Six-Year Laboratory Data Review of Hepatitis B Serological Profiles in Gauteng Province, South Africa

Background In 1995, the hepatitis B vaccine in South Africa was incorporated into the childhood expanded programme of immunization. We report on immunity gaps of laboratory-based hepatitis B virus (HBV) among patients in public facilities in Gauteng Province from 1st January 2014 to 31st December 2019. Methodology. We analyzed HBV serological data extracted from the National Health Laboratory Services Central Data Warehouse (NHLS CDW). A descriptive analysis was performed for hepatitis B surface antigen (HBsAg), antibodies to HBV core (anti-HBc) total, anti-HBc IgM, and antibodies to HBV surface antigen (anti-HBs) according to annual distribution, age groups, and sex. Results The HBsAg positivity rate was 7.0% (75,596/1,095,561; p=0.001): 7.4% (96,532/944,077) in the 25 years and over age group and 4.0% (358/9,268 and 325/10,864) in the under 5 and 13–24 year age groups. The positivity rates of the other HBV serological markers were as follows: anti-HBc total was 37.0% (34,377/93,711; p  <  0.001), anti-HBc IgM was 2.4% (5,661/239,237; p=0.05), and anti-HBs was 37.0% (76,302/206,138; p ≤ 0.001). Naturally acquired HBV immunity was detected in 25.7% (11,188/43,536) of patients in the 25 years and over age group, and 9.7% and 8.2% (113/1,158 and 541/6,522) among those under 5 years and 13–24 year age group, respectively (p  <  0.001). Vaccine-induced immunity was 56.6% (656/1,158) in children under 5 years and 10.2% (4,425/43,536) among those 25 years and above (p  <  0.001). Fifty-six percent (29,404/52,581) of patients were HBV seronegative; predominantly among patients in the 13–24 year age group (60.6%; (3,952/6,522)) and 25 years and over (56.3% (24,524/43,536)) (p=<0.001). Conclusion The HBV infection seroprevalence remains high in South Africa, with Gauteng province having high intermediate endemicity. However, the HBV immunity gap has shifted from younger children to older children and adults.


Introduction
Hepatitis B virus (HBV) infection is a potentially lifethreatening, vaccine-preventable infection. It may acutely present as icteric hepatitis and less commonly as fulminant hepatitis or as chronic hepatitis that results in liver cirrhosis, liver failure, and hepatocellular carcinoma [1,2]. In 2019, an estimated 316 million people globally became chronically infected with HBV [3]. According to the World Health Organization (WHO) global hepatitis report, the African region was estimated to have 60 million chronic HBVinfected people, and tSe sub-Saharan region carried the highest disease burden. Furthermore, HBV infection was estimated to account for 88,000 deaths annually in Sub-Saharan Africa [1]. A systematic review of HBV prevalence by Schweitzer et al. estimated HBV prevalence in South Africa was high at 6.7%, and the number of people living with chronic HBV infection accounted for about 3.5 million [4].
Hepatitis B infection is acquired from HBV-infected bodily fuid (such as blood, saliva, or breastmilk) of an infected person [2]. Te modes of HBV transmission may follow various routes, which impact the geographical prevalence [2,5]. Te perinatal route (mother-to-child transmission) is the predominant mode of HBV spread globally [5]. In intermediate (2-8%) and high (>8%) HBVendemic countries, such as South Africa, the predominant mode of HBV transmission is the perinatal and horizontal route [2,5,6]. Furthermore, the majority of HBV infections occur in early childhood during the frst fve years of life [5][6][7].
Chronic hepatitis B infection has been defned as the persistence of the hepatitis B surface antigen (HBsAg) in the blood for six months or more [2]. Broad evidence available indicates that the risk of developing chronic hepatitis B infection is age-dependent [1][2][3][4]. Without efective HBV prophylaxis, about 90% of children who acquire HBV infection neonatally will become chronic carriers of HBV. About 20-60% amongst those are in the under 5 years of age group. HBV chronicity is estimated at 5-10% of older children (>5 years of age) and less than 5% of those infected after the age of 20 [1,6,7].
An efective hepatitis B vaccine has been available since the early 1980s. In addition, research has shown a persistent protective immune response post-vaccination for at least 20 years, with evidence of protective immunity in more than 90% of people after three doses [6,8]. In South Africa, the hepatitis B vaccine has been part of the childhood expanded programme of immunization (EPI) since 1995 [8,9]. At its inception, the hepatitis B vaccine was a monovalent antigen administered to infants at 6, 10, and 14 weeks of life [8][9][10]. However, in 2015, it was replaced by a more "baby-friendly" hexavalent vaccine administered at 6, 10, and 14 weeks, and the fourth dose at 18 months of life [8]. Nevertheless, HBV vaccination coverage has been variable. In 2019-2020, 83.5% of infants were reported to have received the third dose of the hepatitis B vaccine, while the full vaccine coverage declined to 76.8% by the fourth dose [11,12]. Meanwhile, the hepatitis B vaccine coverage among healthcare workers in Gauteng was reported to range from 15.4% to 90% and over [11].
Te positive impact of the hepatitis B vaccination programme has been the elimination of chronic hepatitis B, with a greater than 60% decline in HBV prevalence [8,10,11]. However, the prevalence of HBV remains high, depending on locality and population group [13]. Some studies estimated the prevalence of HBV monoinfection in South Africa at 1.0% in urban areas and approximately 10% in rural areas [14]. A population-based household survey study conducted in 2014-2015 reported an HBsAg prevalence of 4.0% in the 15-49 year age group in the KwaZulu-Natal province of South Africa [14], while the national HBV prevalence rate study showed the highest prevalence of HBV in Gauteng province [15]. Furthermore, a study by Prabdial-Sing et al. on children under 15 years of age in South Africa showed that 2.9% of the children had exposure to HBV [16].
Recently, the World Health Organisation (WHO) proposed strategies to eliminate HBV by 2030 [17]. Te proposed strategies aim to reduce the incidence of chronic hepatitis B by curbing the disease in children under fve years by 90.0% and HBV mortality by 65.0% [18]. Terefore, there is a need to identify existing gaps that negatively impact achieving HBV prevention, control, and elimination in South Africa.
Tus, this study aims to describe the epidemiological challenges driving the HBV prevalence (immunity gaps) determined from laboratory-based diagnosis of hepatitis B viral HBV infection among patients attending public health care facilities in Gauteng Province, South Africa, from January 1, 2014, to December 31, 2019.

Study Design and Site.
Tis was a retrospective descriptive cross-sectional study on qualitative hepatitis B serological diagnosis among patients who sought care at public health facilities in Gauteng Province from 1 January 2014 to December 31 December 2019. Tis study was conducted in the Department of Virology of the National Health Laboratory Service (NHLS) at Charlotte Maxeke Johannesburg Academic Hospital and the University of the Witwatersrand, Johannesburg, Gauteng Province, South Africa.

Study Population and Sample Selection.
Te study included the laboratory results of patients of all ages with HBV serological testing for the HBsAg, antibodies to HBV core (anti-HBc) total, anti-HBc IgM, and antibodies to HBV surface antigen (anti-HBs) performed at any of the NHLS Pathology laboratories in the Gauteng Province. Anonymized study data were obtained from the NHLS Central Data Warehouse (CDW). Te inclusion criteria in the study were patient results without missing or incomplete data for age and sex and a validated positive, negative, or equivocal result recorded. Equivocal results were interpreted as neither negative nor positive. Te HBV serological datasets without date of birth (age) and a missing observation for sex were excluded, including data generated from healthcare facilities outside the Gauteng province. Te patient records were retrieved per year from 2014 to 2019. Each patient's annual testing episodes were identifed using a unique patient folder number, which was then used to deduplicate the annual datasets.

Laboratory Testing and Interpretation.
Serological testing for HBsAg, anti-HBc total, anti-HBc IgM, and anti-HBs was conducted using the enzyme-linked immunosorbent assay on automated instruments: Architect (Abbott, Germany) or COBAS (Roche, USA) according to the

Age group (years) <5
manufacturer's instructions. Sample results that were equal or greater than sample cutof (S/CO), as specifed in the manufacturer's instruction or the laboratory standard operating procedure, were considered to be positive. Te S/CO ≥ 1 for HBsAg, anti-HBc total, and anti-HBc IgM was considered positive. In addition, the anti-HBs titre greater or equal to 10 milli-international units per mil (mUI/ ml) were assigned as positive and were associated with a measurable, signifcant correlate of protection [19].
Te descriptive interpretation of the test results was as follows: (i) anti-HBc IgM positive results in the presence of HBsAg and anti-HBc positive results are indicative of acute HBV infection; (ii) HBsAg positive, anti-HBc total positive, and anti-HBs negative results are indicative of HBV infection; (iii) HBsAg negative, anti-HBc total positive, and anti-HBs positive results are indicative of naturally acquired HBV immunity; (iv) HBsAg negative, anti-HBc total negative, and anti-HBs positive results were indicative of HBV vaccine-induced immunity; and (v) HBV seronegativity was indicated by HBsAg negative, anti-HBc total negative, and anti-HBs negative test results [19].

Statistical Analysis.
Variables included were the "test date," "year," "test site or location," "date of birth," "age," "sex," "HBV serology test-set markers," (HBsAg, anti-HBc total, anti-HBc IgM, and anti-HBs) and "test results." For descriptive analysis, we used Microsoft Excel 2016 and GraphPad Prism (version 7.0, California, USA). Te parametric data were presented as the mean and nonparametric data as the confdence interval and the interquartile range. Te p value was generated using the t-test (paired twosample for the means). Te estimated positivity rate for each test type was based on the number of positive cases among the total population.

Demographic Characteristics of the Study Population.
From 2014 to 2019, a total of 1,403,777 HBV serology datasets were registered on the NHLS CDW for patients who sought care at public health facilities in Gauteng. Of these, a total of 1,326,614 patients' HBV serology datasets with a registered date of birth (age) and sex were included in the study population. Te study population was divided into four age groups (Table 1).
Te total number of patients tested has fuctuated over the years. However, there was an increase from 2014 ( Table 1). Te majority (86.2% (944 077/1,095,561)) of the patients tested for HBsAg were of the age group 25 years and over. In contrast, among patients aged 0-24 years, the HBsAg testing rate was 12.0% (131,352/1,095,561) among the 13-24 age group and 1.0% (9,268/1,095,561 and 10,864/1,095,561) in the under 5 and 5-12 age groups, respectively.

Discussion
Tis study is a retrospective analysis of the HBV serological data to identify immunity gaps since the start of hepatitis B vaccination in South Africa. We reviewed the reactivity of HBV serology markers among patients who attended urban public health facilities in Gauteng province. Testing for HBV infection may have been necessary for several reasons. In children, HBV infection predominantly presents as subclinical. Terefore, HBV testing for these may have been for a medical workup for a medical condition or screening for perinatal maternal infection [15]. In contrast, adults are invariably symptomatic, with only ≤5.0% developing chronic disease [1,20]. Terefore, in a country with a high HIV prevalence, testing may have been part of HIV management since HIV-infected people have the highest rates of HBV infection [21]. Te study population consisted predominantly of females, and their ages varied, with the 25year-old and older age group comprising the majority. Te predominance of females in this study may be associated with a higher probability of symptomatic infection and health-seeking behaviour compared to males [15].   Overall, our data showed an HBsAg seroprevalence of 7.0% among our study population. Te detection of HBsAg in serum confrms the diagnosis of HBV infection [20][21][22]. A similar study on hepatitis B seroprevalence across South Africa reported a median HBV prevalence of 8.3% for the same province [15]. Although the annual total of positive cases of HBsAg showed a downward trend, the high seroprevalence observed in this study highlights that South Africa is still an intermediate-to-high HBV endemic country [20][21][22]. Countries with ≥8.0% HBV prevalence are regarded as having high endemicity, while those with a prevalence of less than 2.0% are of low endemicity [20,23]. In 2014 and 2015, the overall prevalence of HBV was lower in KwaZulu-Natal Province at 4.0% among 15-to 49-year-olds [14]. Our fnding showed an HBsAg seroprevalence almost double of what was expected for the Gauteng province since it is the country's business hub and people migrate from other provinces to Gauteng for work and better healthcare services.
Among the age groups eligible for hepatitis B vaccination (0-24 years), the HBV seroprevalence in the under-5 and 5-12-year age groups was 2.0% and 3.1%, respectively. Tese fndings were in agreement with a similar study by Moonsamy et al. [15], where the researchers reported a low to intermediate endemicity prevalence between 2.0% and 5.0% in children under 15 years of age. Te risk of developing chronic HBV infections is age-dependent. Perinatal and neonatal HBV infection results in up to 90% of chronic infection and decreases to 30.0%-50.0% in children between one and four years [6,20,23]. Early-life infection adds to the burden of chronic HBV infection despite the availability of efective vaccines. South Africa has not yet implemented the hepatitis B vaccine birth dose. Terefore, the cases of HBsAg in children under 5 years may stem from perinatal acquisition of HBV and horizontal transmission, which is the main route of HBV transmission in South Africa and the African region [8,20].
Tis study found a high HBV seroprevalence in males compared to females. A similar fnding for HBsAg and hepatitis B e-antigen (HBeAg), a marker for active HBV replication, was reported by Samsunder et al. in the HBV community seroprevalence survey conducted in KwaZulu-Natal in 2014-2015 [14]. Terefore, the higher seroprevalence of HBV among men reported here is not unexpected. Tis fnding was consistent with reported reviews that males are a risk factor for chronic HBV infection and rapid progress to HBV-associated hepatocellular carcinoma [13,24,25]. Te potential mechanisms for gender disparity in this study are not clear; nevertheless, a possible contributing factor may be an interplay between sex hormones and the immune response, as females have a higher proneness to symptomatic viral infection related to their intense infammatory responses [24].
Since antibodies to the hepatitis B core antigen persist for life, their presence provides evidence of natural exposure to circulating HBV. Terefore, these antibodies are found in patients with chronic HBV infection and individuals who recovered from the infection (naturally-acquired hepatitis B immunity) [19,26]. In this study, naturally-acquired hepatitis B immunity was observed in 23.0% of the patients ( Table 6). Although the highest seroprevalence of those with naturally-acquired HBV immunity was among those 25 years of age and older, the 10.0% of naturally-acquired HBV immunity among children under 5 years of age was also signifcant. Tis analysis may attest to an existing gap in preventing perinatal transmission through maternal HBsAg screening and treatment. Additionally, providing postexposure prophylaxis to newborns whose mothers are HBsAg positive [21,22], these gaps could be reduced by implementing HBV point-of-care diagnostic testing, the de facto proviso for HBsAg diagnosis during pregnancy, and vaccination strategies, i.e., the introduction of hepatitis B birth dose as outlined in the current Viral Hepatitis Guidelines for South Africa [21].
Te overall positivity rate of anti-HBc IgM was 2.4%. Te anti-HBc IgM detected in patients with positive HBsAg and anti-HBc Total indicates acute or reactivation of latent HBV infection [19,21,26]. During the study period, the annual   anti-HBc IgM positivity rate showed a downward trend and was less than 1.0% among those under 5 and in the 5-12 year age groups. Tis fnding may demonstrate the positive impact of the hepatitis B vaccination programme. Nonetheless, a higher anti-HBc IgM positivity rate was observed in the 13-24 age group (3.4%) and those 25 years and over (2.3%). Terefore, the high anti-HBc IgM levels in the 13-24 age group and those 25 years and over may have been overestimated since reactivation of latent HBV infection may cause low-level anti-HBc IgM positivity. However, our fndings may suggest engagement in high-risk behaviours such as unprotected sex, multiple sexual partners, or shared use of intravenous drug injections [15]. Since the two age groups mentioned above are also associated with high HIV prevalence (covariable) in South Africa, active management strategies to prevent secondary transmission of both HBV and HIV are needed [26][27][28].
Te overall positivity rate of anti-HBs was 37.0%. Te presence of anti-HBs in the absence of HBsAg and anti-HBc implies immunity against HBV infection through vaccination [8,21,29]. Tis study found that the overall seroprevalence of hepatitis B vaccine-induced immunity was 14.0%. Tis vaccine-induced immunity was higher at 57.0% and 51.5% in the under-5 and 5-12 year age groups, respectively, but lower than expected. Previous South African studies reported vaccine-induced immunity rates of 85.7% and 78.1% among HIV-negative and HIV-positive 5-24-month-old babies, respectively [10,30]. Our fndings were also lower than reported for a community-based surveillance South African study conducted in 2013 among children with febrile rash, which reported a vaccine-induced immunity of 81.8% for infants and 80.2% for children, respectively; however, their sample size was small [16]. Te lower hepatitis B vaccine-induced seroprotection observed in our study may imply missed vaccination, biological nonresponse, or waning anti-HBs concentration, which tends to fall of quickly after the prime immunization course [31]. In addition, a review study on undetectable anti-HBs antibodies in HIV-1-infected individuals reported a lower HBV vaccine response and a rapid loss of protection among these patients, including HIVexposed but uninfected children, compared to HIVunexposed children [32]. Terefore, in a country with a high burden of HIV infection and HIV-exposed children, such as South Africa, serological monitoring of anti-HBs antibodies should be part of the HBV preventative strategies.
In this study, 56.0% of patients were seronegative for all HBV markers (HBsAg, anti-HBc total, and anti-HBs). Tese results may indicate no exposure to HBV infection or a lack of hepatitis B vaccine-induced immunity, perhaps due to a lack of vaccination, vaccine nonresponse, or waning immunity [29,31,33]. Of these, the age group 25 years and over accounted for 56.0%. However, the subgroup analysis indicated a higher percentage, 83.4%. Te high seronegative rate among the 25year-old and older age group was expected, as these patients were not eligible for EPI, which began in 1995. However, the HBV seronegative rate among patients in the 13-24 age group was also signifcantly higher. Similar fndings were reported by Prabdial-Sing et al. [16] in children younger than 15 years. Of these, some seronegative patients may beneft from hepatitis B vaccination. However, booster doses are not recommended for those who have completed their vaccination doses, as this does not imply a loss of protection since an anamnestic response is induced following the booster dose [22,31].
Our study was possibly biased because the design used seroprevalence estimates obtained from patients who sought medical care in public health facilities but were not randomly selected. Terefore, our laboratory data may not be representative of the general population. However, since public health facilities cater to 80% of the national population, 80.0% of HBV serological testing is primarily conducted by national health laboratories. Additionally, our fndings were in agreement with other similar studies. Terefore, the fundamental strength of this study is its explanatory power in understanding the changing HBV epidemiology landscape in the South African context and the need to strengthen the prevention and control of HBV infection among diferent age groups.

Conclusion
Te introduction of the hepatitis B vaccine in the South African EPI schedule has positively impacted the under 5year HBV seroprevalence. Although there are gaps in the prevention of vertical and horizontal transmission, optimal implementation of the South African national guidelines for managing viral hepatitis, i.e., implementation of the HBV birth dose and optimum EPI coverage, may lead to the targeted goal of HBV prevalence <1.0% for children under 5 years. Terefore, cost-efective prevention and control strategies such as public awareness and education, HBV screening, and vaccination for high-risk age groups are recommended for immediate implementation. However, the HBV positivity rate of 7% in the Gauteng province still indicates high intermediate endemicity. From these data, we conclude that the HBV immunity gap has shifted from younger children to older children and adults.

Data Availability
Te Microsoft Excel spreadsheet data used to support the fndings of this study may be released upon application to the National Health Laboratory Services Central Data Warehouse, a division of NHLS Academic Afairs, and Research Management System, who can be contacted at CDWOfce@nhls.ac.za or collen.mukhithi@nhls.ac.za.

Ethical Approval
Ethical clearance was obtained from the University of Witwatersrand Human Research Ethics Committee (M201187). Permission to access NHLS CDW data was obtained from the NHLS Academic Afairs, Research Management System (AARMS) (reference number: SR3245157).

Disclosure
Te content hereof is the sole responsibility of the authors and does not necessarily represent the ofcial views of the SAMRC.