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ABSTRACT

Background: The hepatitis B virus (HBV) remains a major public health concern. The interpretation of HBV serological markers is essential for determining infection status, assessing disease activity, and informing screening strategies.

Aim: This study aimed to estimate the prevalence of HBV infection among individuals undergoing screening at a reference laboratory in Tripoli, Libya, and to explore its association with selected demographic factors, particularly gender and nationality.

Methods: A cross-sectional study was conducted at the Reference Medical Laboratory in Tripoli, Libya, between October and November 2021. A total of 184 serum samples were analyzed using a third-generation enzyme-linked immunosorbent assay. Demographic data, including sex and nationality, were obtained from clinical records. Associations between categorical variables were assessed using Fisher’s exact test, and prevalence odds ratios were calculated for exploratory subgroup comparison.

Results: Serological reactivity was identified in 83 of 184 samples (45.1%). The study sample was predominantly male (92.9%, 171/184). Among the reactive cases, hepatitis B surface antigen (HBsAg) was detected in 33.7% (28/83), antibodies against HBsAg in 51.8% (43/83), IgM antibody to hepatitis B core antigen in 4.8% (4/83), and hepatitis B e antigen in 9.7% (8/83). Libyan nationals accounted for 85.5% (71/83) of the reactive cases, whereas foreign nationals accounted for 14.5% (12/83). Although a higher number of HBsAg-positive cases was observed among males, this association did not reach statistical significance.

Conclusion: This study provides a facility-based characterization of HBV serological patterns among individuals in Tripoli, Libya, indicating the presence of both current infection markers and evidence of acquired immunity. Although higher frequencies of infection markers were observed in certain subgroups, these differences were not statistically significant and should be interpreted with caution. The findings support the importance of strengthening HBV screening and ensuring appropriate clinical follow-up.

Keywords: Hepatitis B virus, HBV serology, ELISA, Epidemiology, Gender, Libya.

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Introduction

Hepatitis B virus (HBV) infection remains a major global public health challenge and a leading cause of cirrhosis, hepatocellular carcinoma (HCC), and liver-related mortality (Chen et al., 2006; Ott et al., 2012; World Health Organization, 2024).According to the World Health Organization, approximately 254 million people were living with chronic HBV infection worldwide in 2022, and HBV resulted in an estimated 1.1 million deaths, mostly from cirrhosis and HCC (World Health Organization, 2024). Although effective vaccines have substantially reduced HBV transmission in many settings, the infection continues to persist in several regions, particularly where vaccination coverage, screening, and linkage to care remain suboptimal.

HBV is primarily transmitted through exposure to infected blood or body fluids, particularly when skin or mucous membrane integrity is compromised. In settings with a high prevalence of chronic infection, mother-to-child transmission at birth remains a major route of HBV infection. Although largely preventable, healthcare-associated transmission continues to occur in settings where infection control practices are inconsistently implemented (Song et al., 2014). Although universal childhood vaccination has reduced the incidence of HBV in many regions, transmission persists where vaccine coverage is incomplete, population mobility is high, or access to screening, vaccination, and healthcare services remains limited (Song et al., 2014; World Health Organization, 2024).

HBV epidemiology in the Middle East and North Africa (MENA) shows marked variation across countries, many of which have historically been categorized as areas of intermediate or high endemicity (Ott et al., 2012). Hepatitis D virus (HDV) represents an additional concern among HBV-infected individuals in North Africa. A systematic review and meta-analysis estimated that approximately 5% of hepatitis B surface antigen (HBsAg)-positive individuals in the general population had HDV infection, whereas prevalence exceeding 20% was reported among patients with chronic liver disease. These findings indicate that HDV remains present in the region and may contribute to severe liver outcomes in HBV-endemic populations (Daw et al., 2018; Cornberg et al., 2020). Earlier population-based studies in Libya, conducted before the introduction of universal infant vaccination, reported the prevalence of HBsAg ranging from approximately 0.8% to 2.7% in the general population. Higher prevalence was consistently observed among adults and certain high-risk groups (Daw and El-Bouzedi, 2014).

The epidemiological pattern of HBV in Libya reflects a contrast between the impact of universal infant vaccination and infection persistence in older, previously unvaccinated cohorts. Following the introduction of universal infant vaccination in the early 1990s, the prevalence of HBV among pediatric cohorts declined substantially; however, older birth cohorts may continue to include individuals with chronic infection acquired before widespread vaccine coverage (World Health Organization, 2024). In addition, years of instability, population movement, and variable access to health care services may have affected HBV transmission patterns and surveillance activity continuity. Available surveillance data may also lack detailed serological and demographic information. The true burden of HBV infection and the distribution of HBV serological markers in Libyan populations remain incompletely defined (Daw and El-Bouzedi, 2014; World Health Organization, 2024).

HBV is an enveloped, partially double-stranded DNA virus belonging to the Hepadnaviridae family. The infectious virion, known as the Dane particle, consists of an outer lipid envelope containing HBsAg and an inner nucleocapsid composed mainly of hepatitis B core antigen, which encloses the viral genome and polymerase (Bruss, 2007). HBsAg is the principal HBV infection marker and represents the major antigenic target of vaccine-induced protective immunity. Antibodies against HBsAg (anti-HBs) generally indicate immunity following vaccination or recovery from previous infection. IgM antibody to hepatitis B core antigen (anti-HBc IgM) is commonly associated with recent or acute infection, whereas hepatitis B e antigen (HBeAg), a secreted product related to the core gene, is associated with active viral replication and increased infectivity. Therefore, the combined interpretation of these markers is essential for distinguishing current infection, previous exposure, immune status, and phases of higher viral replication (Terrault et al., 2018; CDC, 2023).

The effective diagnosis and management of HBV infection depend on the combined interpretation of serological markers. Persistence of HBsAg for more than 6 months is generally used to define chronic infection, whereas anti-HBc IgM is commonly associated with recent or acute infection. HBeAg is associated with active viral replication and increased infectivity, whereas anti-HBs reflects immunity acquired either after infection recovery or through vaccination. Facility-based sero-epidemiological studies can help describe these patterns, identify immunity gaps, and detect groups requiring clinical follow-up or targeted public health interventions (Yuen et al., 2016; EASL, 2017; Shambesh et al., 2018; Seto et al., 2018; Terrault et al., 2018; Cornberg et al., 2020).

In this context, updated facility-based epidemiological data from Tripoli—Libya’s largest urban center and a nexus for internal and cross-border movement—are needed to improve the understanding of HBV serological patterns among tested individuals. Therefore, this study was designed to estimate the prevalence of HBV infection and characterize the serological profile of HBV markers among individuals undergoing testing at a major reference laboratory in Tripoli, Libya. The study further explored associations with selected demographic variables, particularly sex and nationality, to provide facility-based evidence that may inform surveillance, screening strategies, and targeted public health interventions.

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Materials and Methods

Study design and setting

A cross-sectional study was conducted at the Reference Medical Laboratory in Tripoli, Libya, a major diagnostic referral center receiving specimens from both public and private health care facilities in Tripoli and surrounding areas. The laboratory processes samples from both Libyan and non-Libyan patients referred for routine screening or clinical evaluation. Data collection was conducted between October and November 2021.

Study sample

The study included 184 individuals who underwent serological testing for HBV during the study period. Samples were obtained from individuals referred to the laboratory for routine screening, such as pre-employment or general health examinations, or for clinical evaluation of liver function.

The inclusion criteria comprised serum samples submitted for HBV testing with available demographic information, including age, gender, and nationality. All samples were included regardless of clinical presentation. The exclusion criteria included hemolyzed, lipemic, or insufficient-volume samples, as these conditions could affect the reliability of the analytical results. Samples that did not meet these criteria were excluded before analysis.

All eligible samples received during the defined study period and meeting the prespecified analytical and data-completeness criteria were included consecutively to reduce selection subjectivity at the investigator level. The investigators performed no additional participant selection after sample receipt. Nevertheless, because the study was laboratory-based and relied on individuals referred for screening or clinical evaluation, selection bias related to health care access, referral patterns, and testing practices cannot be excluded. Exclusions were applied during routine laboratory quality control before dataset assembly; thus, the exact number of excluded samples could not be retrospectively verified.

Sample collection and processing

Approximately 5 ml of venous blood was collected from each participant via aseptic venipuncture into plain tubes without anticoagulant. The blood samples were allowed to clot at room temperature and then centrifuged at 3,000 rpm for 10 minutes to obtain serum. The serum was aliquoted into sterile tubes and stored at −20°C until further analysis. Sample collection, serum separation, aliquotting, storage, and handling were performed according to standard laboratory biosafety procedures and the manufacturer’s recommendations for enzyme-linked immunosorbent assay kits. The selection and interpretation of HBV serological markers were guided by the Centers for Disease Control and Prevention recommendations for hepatitis B serological testing and clinical interpretation (CDC, 2023).

Serological testing

Serological testing was performed using a third-generation enzyme-linked immunosorbent assay kit (DIALAB GmbH, Vienna, Austria). The markers assessed included HBsAg, anti-HBs, anti-HBc IgM, and HBeAg. These markers were selected to identify current HBV infection, immunity following vaccination or previous exposure, recent infection, and active viral replication. The optical density was measured using an automated microplate reader, and the results were interpreted according to the manufacturer’s instructions.

Data management and statistical analysis

Data were analyzed using the Statistical Package for the Social Sciences version 22.0. Categorical variables are summarized as frequencies and percentages. Associations between categorical variables were assessed using Fisher’s exact test because of small subgroup sizes and the presence of zero cell counts in several comparisons. Because this was a cross-sectional study, association estimates were interpreted as prevalence odds ratios (PORs), rather than odds ratios reflecting incident disease risk. Given the relatively high prevalence of HBV serological reactivity in this study, PORs may overestimate the magnitude of association compared with the prevalence ratios (Zocchetti et al., 1997; Pearce, 2004). Therefore, PORs were retained for exploratory subgroup comparison only and were interpreted cautiously, particularly where confidence intervals were wide or statistical significance was not reached. Where applicable, Haldane’s continuity correction was applied to address zero-cell-count-related instability. All statistical tests were two-tailed, and a p-value < 0.05 was considered statistically significant. Active HBV infection was defined as positive for HBsAg. PORs were not used for causal inference.

Ethical approval

Ethical approval for this study was obtained from the local ethics committee in Tripoli, Libya. This study was conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants prior to sample collection. All samples and associated clinical records were anonymized using unique identification codes, and no personal identifiers were accessible to the investigators during data analysis.

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Results

Demographic characteristics

This study included 184 serum specimens from individuals who underwent HBV serological testing. Most participants were males (171/184, 92.9%), whereas females accounted for 7.1% (13/184). The majority of participants were Libyan nationals, with a smaller proportion of non-Libyan residents.

Overall serological reactivity to HBV markers

Of the 184 specimens, 83 were reactive for at least one HBV serological marker (45.1%; 95% CI: 37.9–52.3). The remaining 101 samples (54.9%) were nonreactive for all tested markers (Table 1).

Distribution of the serological markers of HBV

Table 2 presents the distribution of specific HBV serological markers among the 83 reactive samples. Anti-HBs was detected in 43 (51.8%) cases, HBsAg in 28 (33.7%), HBeAg in 8 (9.7%), and anti-HBc IgM in 4 (4.8%).

Demographic distribution of positive cases

Of the 83 serologically reactive cases, 71 (85.5%) were Libyan nationals, and 12 (14.5%) were foreign nationals. HBsAg and anti-HBc IgM were observed only among Libyan participants, whereas anti-HBs was detected in both groups. HBeAg was identified among male participants from both Libyan and foreign groups (Table 3).

Table 1. Overall distribution of HBV serological reactivity among the individuals tested.

Table 2. Distribution of HBV serological markers among reactive cases (n=83).

Association between gender and positivity for HBsAg

The absolute number of HBsAg-positive cases was higher among males; however, the proportion of HBsAg positivity was higher among females than males. (POR for females relative to males=2.722; 95% CI: 0.777–9.543; p=0.117). This estimate should be interpreted cautiously because of the cross-sectional design, small number of female participants, and wide confidence interval (Table 4).

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Discussion

Epidemiological interpretation and viral persistence of the disease

The findings indicate ongoing HBV circulation among the tested individuals, with an overall serological reactivity rate of 45.1%, indicating both current and previous exposure. HBsAg positivity, which indicates current HBV infection, was observed in 15.2% of participants, while anti-HBs antibodies were detected in 23.4%, consistent with immunity acquired through previous infection or vaccination. Approximately 6.5% of the study sample exhibited isolated anti-HBc positivity, which may indicate previous HBV exposure, waning anti-HBs levels, or possible occult infection in some contexts.

The observed serological patterns reflect HBV circulation among individuals attending the laboratory; however, these findings should not be interpreted as representative of population-level endemicity. The results are best understood as indicative of serological patterns among tested individuals rather than as a direct measure of community prevalence, given the facility-based sampling framework. Several regional studies from Libya have documented anti-HBc reactivity among blood donors and heterogeneity in HBV prevalence across different regions of the country, as reported in a large national population-based survey (Habas et al., 2010; Daw and El-Bouzedi, 2014; Shambesh et al., 2018).

Table 3. Distribution of HBV serological markers stratified by sex and nationality among reactive cases (n=83; exploratory analysis).

Table 4. Association between sex and HBsAg positivity in the study sample.

These findings indicate ongoing HBV circulation among individuals in Tripoli with detectable markers of population immunity. This is reflected by the proportion of participants with detectable anti-HBs antibodies (23.4% of the study sample), a finding that may be related to the long-term implementation of the HB vaccination program in Libya since the early 1990s (Daw and El-Bouzedi, 2014). In contrast, the persistence of HBsAg positivity in 15.2% of the study samples indicates that current HBV infection remains present among individuals undergoing testing. Therefore, ongoing surveillance and focused preventive efforts are warranted, especially among populations that may have missed routine childhood vaccination (World Health Organization, 2024).

Isolated anti-HBc positivity was identified in 6.5% of the study samples, which warrants careful diagnostic consideration. This serological pattern is commonly associated with resolved HBV infection or waning anti-HBs levels; however, it has also been associated with occult hepatitis B infection (OBI), in which HBV DNA may persist despite the absence of detectable HBsAg (Raimondo et al., 2019). OBI has also been reported in regional studies among Libyan blood donors, suggesting that reliance on HBsAg screening alone may underestimate previous or ongoing HBV exposure in some settings (Habas et al., 2010; Shambesh et al., 2018). Furthermore, in the MENA region, particularly in North Africa, HBV epidemiology may be complicated by the co-infection of HDV. Although HDV was not assessed in the present study, the observed HBsAg positivity warrants attention because HDV has been reported among individuals with chronic HBV infection in the region and is associated with a more rapid progression of liver disease (Daw et al., 2018).

Taken together, the persistence of HBV infection markers among tested individuals indicates that HBV infection remains present in this population despite the long-standing implementation of vaccination programs in Libya. This finding highlights that HBV prevention and control efforts require continued strengthening. This pattern may be related to several factors, including older birth cohorts who were not covered by universal infant vaccination, possible ongoing horizontal transmission, and interruptions in healthcare and immunization services during periods of sociopolitical instability. These findings support the need to complement vaccination programs with strengthened screening, infection control measures, linkage to clinical care, and broader surveillance activities (World Health Organization, 2024).

Gender disparities

The marked predominance of males (92.9%) among the tested individuals likely reflects the characteristics of local screening practices rather than the underlying demographic structure of Tripoli. This imbalance may be related to institutional screening requirements, which are more frequently applied to men, such as health clearance procedures for military service or employment. Although a higher number of HBsAg-positive cases was observed among males, the association between sex and infection was not statistically significant (p=0.117). This result should be interpreted cautiously, as the small number of female participants may have reduced statistical power and constrained the ability to detect potential gender-related differences in prevalence.

Previous studies have reported sex-related differences in the outcomes of HBV infection, which may reflect a combination of biological, immunological, hormonal, and socio-behavioral factors, including differences in occupational exposure and healthcare engagement. However, this study was not designed or powered to establish true sex-based differences in the burden of HBV. The marked underrepresentation of females in this cohort highlights limitations in current testing patterns and supports the interpretation that the observed gender distribution may reflect unequal access to screening or variations in healthcare utilization rather than a true gender-specific disease burden (Schweitzer et al., 2015; Ruggieri et al., 2018).

Although Schweitzer et al. (2015) provided an important global reference point, epidemiological patterns do not necessarily translate directly into local settings. The observed HBV distribution in Tripoli may reflect context-specific factors related to health care access, service utilization, institutional screening requirements, and socio-cultural influences that shape testing behavior and case detection. These contextual dynamics indicate that the variations identified in this cohort should be interpreted primarily as facility-based patterns rather than as definitive evidence of underlying gender-specific epidemiological differences.

Role of foreign nationals

Foreign nationals accounted for 14.5% of all serologically reactive cases. HBeAg, a marker associated with active viral replication, was detected among foreign male participants despite the subgroup’s relatively small size. This observation should be interpreted cautiously because of the limited number of foreign participants and the absence of statistically significant subgroup differences.

The serological patterns observed among foreign participants may reflect multiple factors, including HBV endemicity, migration history, access to vaccination, and healthcare use. Some migrant populations may originate from regions with higher HBV endemicity, including parts of sub-Saharan Africa and Southeast Asia, which could influence HBV serological profiles in host countries (Schweitzer et al., 2015). However, the present study did not collect detailed country-of-origin, vaccination, or migration-history data; therefore, no definitive conclusions regarding the drivers of HBV marker distribution among foreign nationals in this cohort can be drawn.

These findings highlight the importance of inclusive HBV screening strategies that ensure equitable access to confirmatory testing, clinical evaluation, vaccination when appropriate, and antiviral treatment for all tested populations, including migrant groups, rather than being interpreted as evidence of a specific transmission risk attributable to nationality. Such integrated approaches are consistent with WHO targets for viral hepatitis elimination and may strengthen national HBV control efforts (World Health Organization, 2024).

Clinical significance of serological markers

The detection of IgM antibodies against hepatitis B core antigen (anti-HBc IgM) in 4.8% of reactive cases may indicate recent acute HBV infection or an acute flare in the context of chronic infection. Distinguishing between these clinical phases is important because it can influence clinical follow-up, prognostic assessment, and public health decision-making (Shambesh et al., 2018; CDC, 2023).

Accurate HBV infection staging cannot be established using isolated serological markers alone. Interpretation requires the integrated evaluation of HBsAg, anti-HBs, anti-HBc IgM, and HBeAg, together with clinical history and, where available, molecular testing. Concurrent assessment of these markers can help differentiate between current infection, previous exposure, immune status, recent infection, and active viral replication markers. In this context, combined serological interpretation supports appropriate clinical follow-up and is consistent with internationally recognized standards for HBV diagnosis and management (EASL, 2017; Terrault et al., 2018; CDC, 2023).

Nationality and gender-specific HBV serological marker patterns

The distribution of HBV serological markers according to nationality and sex showed variation across subgroups; however, none of these differences reached statistical significance. Therefore, these findings should be interpreted cautiously and regarded as descriptive rather than causal, particularly given the cross-sectional design, small subgroup sizes, and presence of zero-cell counts in several comparisons.

The absence of HBsAg among foreign nationals in this cohort should be interpreted with caution, as the limited sample size and potential selection bias may have contributed to underrepresentation. In addition, HBsAg negativity does not fully exclude the possibility of OBI, particularly in individuals with previous exposure or resolved infection. Although molecular testing was not performed in the present study, previous reports have documented OBI among HBsAg-negative individuals, especially when sensitive nucleic acid testing methods are applied (Samal et al., 2012; Raimondo et al., 2019). These considerations highlight the limitations of relying solely on serological markers in certain contexts and underscore the importance of interpreting negative findings with caution.

The detection of HBsAg and HBeAg among Libyan males in this cohort indicates the presence of current infection markers and possible viral replication within this subgroup. However, these observations should be interpreted cautiously because the subgroup comparisons were not statistically significant and were affected by limited sample size and imprecise estimates. The observed pattern may reflect underlying transmission dynamics, but confirmation in larger and more representative cohorts is required. These findings are broadly consistent with the current WHO guidance, emphasizing continued case identification, confirmatory evaluation, and linkage to care (World Health Organization, 2024).

Although based on a limited sample, the detection of HBeAg-positive cases among foreign nationals highlights the potential importance of including migrant populations within equitable HBV screening and follow-up strategies. However, this observation should not be interpreted as evidence of nationality-based transmission risk, given the small subgroup size and the absence of statistically significant differences. In this context, integrating screening with appropriate clinical follow-up, confirmatory testing, vaccination when indicated, and access to antiviral therapy remains a key component of HBV control efforts, as reflected in current international guidance (EASL, 2017; World Health Organization, 2024).

Study limitations

Although the findings provide useful insights, they should be interpreted within the context of several methodological limitations. The relatively small sample size—particularly among foreign nationals (n=12) and female participants (n=11), Libyan females, with no foreign female representation)—likely reduced statistical power and constrained subgroup analyses by gender and nationality. The study was not specifically powered for gender or nationality subgroup comparisons. Accordingly, the wide confidence intervals (CIs) observed in several analyses likely reflect limited precision rather than evidence of no underlying difference.

Furthermore, the cross-sectional design limits the ability to assess temporal variation in HBV serological patterns or to infer disease progression. The association measures reported in this study should be interpreted as PORs rather than risk ratios or causal effect estimates because the data were cross-sectional. Given the relatively common outcome prevalence, these estimates may overestimate the magnitude of association compared with prevalence ratios and were therefore retained only for the exploratory subgroup comparison.

No formal sensitivity analysis by referral category could be undertaken because the retrospective dataset did not consistently retain a standardized classification that distinguished pre-employment screening from clinically indicated testing. Another limitation is the reliance on serological markers without molecular confirmation. The absence of HBV DNA quantification, viral genotyping, and detailed vaccination histories restricts the accurate classification of infection status and limits the assessment of viral activity and transmission dynamics. Consequently, occult HBV infection could not be definitively evaluated within the study framework.

Furthermore, the laboratory-based sampling framework may have introduced selection bias related to health care access, referral practices, and symptom-driven testing patterns, which may limit the generalizability of the findings. Accordingly, the results reflect the serological patterns among the individuals tested rather than the population-level prevalence. The presence of zero-cell counts in several subgroup analyses necessitated the use of Fisher’s exact test and contributed to wide CIs, indicating limited precision in some estimates. These methodological constraints support the cautious interpretation of the findings and underscore the need for larger, multicenter studies incorporating molecular data to improve the understanding of HBV epidemiology.

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Conclusion

This study provides a facility-based characterization of HBV serological patterns among individuals in Tripoli, Libya, revealing evidence of current infection markers, previous exposure, and acquired immunity. Although higher frequencies of infection markers were observed among males and certain subgroups, these differences did not reach statistical significance and should be interpreted with caution.

The findings underscore the importance of strengthening HBV screening strategies and ensuring linkage to appropriate clinical evaluation and follow-up care. In this context, the inclusion of extended serological markers, such as HBeAg and anti-HBc IgM, may support more accurate clinical interpretation and help identify individuals requiring further assessment.

Future research should focus on larger, multicenter studies with more balanced demographic representation and longitudinal designs to better capture infection dynamics over time. The integration of molecular diagnostics, including HBV DNA quantification, viral load assessment, and genotyping, together with comprehensive epidemiological data, would further enhance the understanding of HBV transmission and support national control efforts in alignment with WHO elimination targets (World Health Organization, 2024).

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Acknowledgments

The authors would like to acknowledge the staff of the Reference Medical Laboratory (RML), Tripoli, Libya, for their technical support and assistance with sample collection and laboratory analysis. The authors are also grateful to all participants for their willingness to participate in this study.

Conflict of interest

The authors declare no competing financial interests or personal relationships that could have influenced the work reported in this paper.

Funding

The authors declare that no specific financial support or grants were received from any funding agency in the public, commercial, or not-for-profit sectors.

Authors’ contributions

Saad A.A. contributed to the conception and design of the study, wrote the manuscript, and supervised data analysis. Marium M. Hussein contributed to manuscript writing, data analysis, and manuscript submission coordination. Muhannad Marzouk Hussein was responsible for data collection, data analysis, and manuscript writing. Nagat Alkmishi, Khadija Ali, and Firuz Elaswad performed the laboratory work, including sample processing and serological testing. AI-based tools were solely used for language improvement. The authors take full responsibility for the content and integrity of the manuscript. All authors critically reviewed the manuscript for important intellectual content and approved the final version for submission.

Data availability

The data supporting the findings of this study are available upon reasonable request from the corresponding author.

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