Occult hepatitis B virus infection among hepatitis B surface antigen negative vaccinated healthcare workers in East Gojjam zone hospitals

Abstract

Occult hepatitis B infection (OBI) is defined as the presence of hepatitis B virus (HBV) DNA in the serum of individuals who test negative for hepatitis B surface antigen (HBsAg). OBI poses a significant public health challenge, especially in hyperendemic regions like Ethiopia, where it can persist even among vaccinated populations. The phenomenon is of particular concern among healthcare workers, who are at increased risk of HBV infection. This study aimed to determine OBI and its associated factors among fully vaccinated, hepatitis B surface antigen (HBsAg)-negative healthcare workers in hospitals of East Gojjam Zone, Northwest Ethiopia. An institution-based cross-sectional study was conducted from March 25 to November 30, 2024 among 399 fully vaccinated healthcare workers in eleven hospitals in East Gojjam Zone. Socio-demographic and clinical data were collected using a self-administered questionnaire. Five up to seven milliliters of venous blood were collected from each study participant and 100 µL serum samples was used to screen HBsAg via enzyme-linked immunosorbent assay (ELISA), and HBsAg-negative samples underwent HBV DNA detection and quantification using the Abbott real-time polymerase chain reaction (PCR). Data were analyzed using SPSS version 25 and crude prevalence ratio (CPR) and adjusted prevalence ratio (APR) were calculated at 95% confidence intervals. Statistical significance was set at p < 0.05. Of the 399 fully vaccinated, HBsAg-negative healthcare workers, 39 (9.8%; 95% CI: 7.0–13.0%) were found to have detectable HBV DNA, confirming OBI. Among these, 31/39 (79.5%) had low-level viremia (< 200 IU/mL), while 8/39 (20.5%) had higher viral loads (> 200 IU/mL). Alcohol use (APR: 2.5, 95% CI: 2.1 to 7.2, p < 0.021) and multiple sexual contacts (APR: 3.7, 95% CI: 2.8 to 6.4, p < 0.003) were independent risk factors for OBI. Despite full vaccination, a significant number of healthcare workers in this study were found to have OBI. This highlights the limitations of relying only on HBsAg screening for HBV detection.

Introduction

Occult hepatitis B infection (OBI) refers to the presence of replication-competent hepatitis B virus (HBV) DNA in the liver or serum (typically at low levels, < 200 IU/mL) in individuals testing negative for hepatitis B surface antigen (HBsAg) and this low-level HBV persistence, driven by intrahepatic cccDNA¹. OBI represents a significant global public health challenge due to its high prevalence. OBI in individuals who test negative for HBsAg following immunization remains a significant clinical and public health concern, despite the success of universal vaccination strategies². The burden of OBI among healthcare providers worldwide shows notable prevalence, reflecting their increased occupational risk to HBV infection compared to the general population. The prevalence of OBI among healthcare workers varies by region, ranging approximately from 3.3% to 11%³. The prevalence of OBI tends to be higher in countries with low socio-demographic index (SDI), reaching as high as approximately 30.94% in low SDI countries versus much lower in high SDI countries⁴.

In addition, the prevalence and impact of occult OBI infection among HBsAg-negative populations in sub-Saharan Africa, particularly Ethiopia, are both significant and concerning⁵. In Ethiopia, studies report that OBI prevalence ranges from approximately 0.4% to 20%⁶. The observed variation in OBI prevalence among different studies and sites reflects differences in diagnostic approaches, population risk factors, and endemicity levels. The data underscore that OBI poses a serious public health challenge in the region^7,8. In other words, OBI is a significant public health issue because it involves hidden viral infections that standard tests may miss, posing risks of transmission and disease progression. Vaccinated individuals who test negative for surface antigen are important to study since they may still carry and unknowingly spread the virus, highlighting the need for improved detection and monitoring strategies⁹.

Furthermore, the detection of OBI has undergone significant advancements over the years, evolving from reliance on conventional HBsAg assays to more sensitive molecular techniques¹⁰. Initially, OBI was challenging to identify due to the absence or very low levels of detectable HBsAg despite the presence of replication-competent HBV DNA¹¹. However, the advent and increasing use of nucleic acid testing (NAT) and polymerase chain reaction (PCR) technologies have revolutionized OBI detection by enabling the identification and quantification of low-level viral DNA (HBV DNA < 200 IU/mL) in the blood and liver tissue, even in individuals who test negative for HBsAg by standard assays¹². Low-level viremia, defined as a viral load below 200 IU/mL, is generally considered not infectious, meaning it poses minimal risk for transmitting the virus to others¹³. However, in the context of OBI, especially in HBsAg-negative vaccinated individuals, this low-level viral presence remains clinically important¹⁴.

Despite the absence of detectable surface antigen, the virus can persist at low levels, which may contribute to cryptic or hidden viral transmission and complicate diagnosis and management. Therefore, even with low-level viremia, careful monitoring and awareness are essential to address potential risks and ensure effective control of HBV infection HBV infection¹⁵. The molecular assays have improved sensitivity and specificity, uncovering a substantial burden of hidden infections that were previously missed, especially among vaccinated populations and in high-risk groups such as healthcare workers¹⁶.

Despite these advances, challenges remain due to varying assay sensitivities, lack of standardized testing protocols, and limited access to these sophisticated diagnostic tools in resource-limited settings¹⁷. Consequently, integrating sensitive HBV DNA-based screening methodologies into routine diagnostic algorithms is crucial to accurately detect OBI, guide clinical management, and prevent cryptic transmission, thereby addressing a significant gap left by traditional serological testing methods¹⁸.

While several studies have investigated the prevalence of OBI among different at-risk populations in Ethiopia, including pregnant women, HIV-positive individuals, and blood donors, there is a significant lack of data specifically addressing OBI among fully vaccinated, HBsAg-negative healthcare workers^19,20. Most existing researches have focused on unvaccinated or general populations^20–22. Furthermore, the effectiveness of current HBV screening protocols, which primarily rely on HBsAg detection, remains largely unexamined in the context of vaccinated healthcare workers in the study setting. Moreover, the impact of behavioral and sociodemographic factors on OBI risk in this group also remains poorly understood²³.

Moreover, healthcare workers are at increased risk of both acquiring and transmitting HBV due to occupational exposures, making accurate detection and prevention of HBV transmission in this group a public health priority²⁴. In other words, healthcare workers are at increased risk because their job involves close and frequent contact with patients, including those who may carry infectious diseases. This occupational exposure increases the likelihood of being infected by blood borne viruses²⁵. Moreover, healthcare workers often involve handling sharp instruments and bodily fluids, which can lead to accidental injuries and infections²⁶.

Vaccine inefficacy refers to the situation when a vaccine fails to provide adequate protection against infection. This can occur for several reasons, including primary non-response, where an individual’s immune system does not produce a sufficient response after vaccination²⁷; the emergence of escape mutants, which are virus variants that evade the immune response triggered by the vaccine; or waning immunity, where the protective effects of the vaccine decrease over time, leaving individuals more susceptible to infection. Understanding these factors is important for addressing gaps in vaccine effectiveness and improving long-term protection²⁸.

The persistence of OBI despite full vaccination raises concerns about the adequacy of current vaccination and screening strategies, especially in regions with diverse HBV genotypes and high endemicity²⁹. Understanding the prevalence and determinants of OBI among vaccinated, HBsAg-negative healthcare workers is essential for informing policy, improving screening protocols (such as integrating nucleic acid testing), and ultimately reducing the risk of cryptic HBV transmission in healthcare settings³⁰. Hence, this study aimed to determine OBI among fully vaccinated, HBsAg-negative healthcare workers in eleven hospitals of East Gojjam Zone, Northwest Ethiopia.

Materials and methods

Study design, setting, and period

An institution-based cross-sectional study was applied among healthcare workers who had completed the full hepatitis B vaccination series in eleven hospitals of East Gojjam Zone, Northwest Ethiopia, from March 25 to November 30, 2024.

Population and eligibility criteria

All fully vaccinated healthcare workers (those who have received three doses of the hepatitis B vaccine series) employed in hospitals throughout the East Gojjam Zone comprised the source population. Those who met the eligibility criteria and were present at their respective hospitals during the data collection period formed the study population. Moreover, individuals who provided written informed consent were included in the study. However, individuals with a known history of chronic HBV infection, and those who tested positive for HBsAg were excluded from the study. In addition, janitors or cleaners were excluded from this study due to specific study design choices that prioritized clinical staff with direct patient contact and mandatory HBV vaccination.

Sample size determination and sampling method

The required sample size for this study was calculated using a 50% proportion via the single population proportion formula: .

Where n = sample size.

p = proportion.

q = 1-p.

Z = confidence interval.

p = 0.5 q = 0.5 with confidence interval of 95% Z = 1.96.

n= (1.96)²*0.5*0.5 = 384.

(0.05)².

The final sample size was calculated as follows:

Sample size (n) = Original estimate (384) + 10% non-response allowance (38) → Total n = 422.

The total sample size was proportionally allocated to each participating hospital and profession based on the total number of fully vaccinated healthcare workers in each. Study participants were then recruited using simple random sampling. However, laboratory analysis of all 422 samples via enzyme-linked immunosorbent assay (ELISA) revealed that 23 participants tested positive for HBsAg and were subsequently excluded from the study. The remaining 399 participants, who tested negative for HBsAg, underwent HBV DNA testing. Consequently, the final sample size for this study was 399.

Data collection and laboratory procedures

Self-administrated questionnaire was used to collect socio-demographic and clinical data and. In addition, 5–7 milliliters of venous blood were collected from each study participant by venipuncture. All samples from the various hospitals in East Gojjam Zone were transported to the molecular laboratory at Debre Markos Comprehensive Specialized Hospital (DMCSH) in a cold box with ice packs to ensure proper storage. Serum was separated from each blood sample by centrifugation and then stored at −80 °C until laboratory analysis was performed.

A qualitative sandwich ELISA [Monolisa™ HBs Ag ULTRA, Bio-Rad, France] was used to screen for HBsAg in 100 µL serum sample at Debre Markos Blood Bank Laboratory, with spectrophotometric analysis performed at 450/620–700 nm wavelengths³¹. Then, samples that tested negative for HBsAg by ELISA were subsequently subjected to HBV DNA detection and viral load quantification using 200 µL of serum. This was performed with the ABBOTT real-time automated DNA extraction and amplification system (ABBOTT real-time PCR, Abbott Molecular Inc.). The Abbott assay had an upper limit of quantification of 10⁹ IU/mL, while its lower limit of quantification matched the limit of detection (15 IU/mL for a 200 µL of serum sample). Specimens with results reported as “not detected” were considered negative, and those with values reported as “< 15 IU/mL” were assumed to be below the detection threshold. Samples exhibiting HBV DNA levels exceeding the upper limit of 10⁹ IU/mL were diluted and retested at suitable concentrations.

The assay targets a highly conserved region within the surface gene of the HBV genome, ensuring specificity across HBV genotypes. Primers which was specific to HBV polymerase was designed to minimize mismatches within this region. Then, the concentration of HBV DNA in each sample and control was determined using either a stored calibration curve or a curve generated by calibrators within the current calibration or sample run. The Abbott m2000rt system automatically reports the results on its workstation. Assay results were expressed in IU/mL, with an average conversion factor of 3.41 copies per IU (1 IU = 3.41 copies).

Laboratory quality control

A pretest was conducted prior to data collection to optimize the experimental setup, utilizing samples obtained from 5% of study participants. Standard operating procedures (SOPs) were strictly adhered to during sample collection, transportation, and processing. Additionally, each ELISA and HBV DNA kit was validated using known positive and negative control samples. In addition, an internal control DNA was introduced during the sample preparation process and processed alongside the calibrators, controls, and samples. The presence of amplified HBV DNA and the internal control was detected during the extension and annealing steps. The amplification cycle at which a standardized fluorescent signal was detected by the Abbott m2000rt system was inversely proportional to the logarithm of the HBV DNA concentration in the original sample. Each sample was then quantified using an external calibration curve. To ensure consistency, 10% of the samples were re-checked.

Data analysis

The data were entered into EpiData software version 3.1 and exported to SPSS version 25 for analysis. Results were presented in both text and tables. The crude prevalence ratio (CPR) was calculated using bivariate analysis to examine the relationship between each independent variable and the outcome using the chi-square test, along with the computation of risk estimates and 95% confidence intervals. Variables with a p-value less than 0.25 were considered for inclusion in the multivariable model. In the multivariable analysis, generalized linear models with a Poisson distribution, log link function, and robust standard errors were used, with results reported as adjusted prevalence ratio (APR) and 95% confidence intervals. A p-value below 0.05 was considered statistically significant.

Ethical approval

The study received ethical approval from the Institutional Research Ethics Review Committee (IRERC) of the College of Health Sciences, Debre Markos University, in accordance with the Declaration of Helsinki (protocol number: R/C/S/D/317/01/16). In addition, written informed consent was obtained from all study participants prior to their enrollment.

Results

Socio-demographic characteristics

Among the 399 participants, the majority (77.7%) were over 30 years old. Males made up 54.6% of the group. Moreover, most respondents were married (50.6%). A large proportion (79.7%) reported having a family size of 1–5 members. Additionally, most participants resided in urban areas (77.7%), and a significant majority (79.4%) held a degree or higher educational qualification (Table 1).

Table 1.

Socio-demographic characteristics of the study participants.

Socio-demographic variables	Age category	Frequency	Percent
Age	≤ 30	89	22.3
	> 30	310	77.7
Sex	Male	218	54.6
	Female	181	45.4
Marital status	Single	179	44.9
	Married	202	50.6
	Divorced	18	4.5
Family size	1–5	318	79.7
	≥ 6	81	20.3
Residence	Rural	89	22.3
	Urban	310	77.7
Profession	Nurse	81	20.3
	Midwife	55	13.8
	Doctor	67	16.8
	Laboratory	71	17.8
	Pharmacy	48	12.0
	Radiology	35	8.8
	Anesthesia	26	6.5
	Environmental health	16	4.0
Educational status	Diploma	82	20.6
	Degree and above	317	79.4
Total		399	100.0

Prevalence of OBI and distribution of HBV DNA with socio-demographic characteristics

The overall prevalence of OBI among HBsAg negative vaccinated healthcare workers in this study was 39/399 (9.8%) (95% CI: 7.0–13.0%). Among the study participants, HBV DNA were detected in 32/310 (10.3%) individuals aged over 30 years. Males accounted for 51.3% of HBV DNA detections. Furthermore, the majorities of HBV DNA detections were among married individuals (64.1%). Most cases with HBV DNA detection had a family size of 1–5 members (74.4%) and resided in urban areas (89.7%). In terms of HBV DNA concentration, 77.4% of those with < 200 MU/mL were degree holders or above, and 54.8% were female, while higher concentrations (≥ 200 MU/mL) were more common among males (75.0%) and those with a diploma (50.0%) (Table 2).

Table 2.

Prevalence and viral load distribution of HBV DNA with Socio-Demographic Characteristics.

Variables	Category	HBV DNA			HBV DNA concentration (MU/mL)
		Detected (%)	Not detected (%)	Total	<200	≥200	Total
Age	≤ 30	7(17.9)	82(22.8)	89	5(16.1)	2(25.0)	89
	> 30	32(82.1)	278(77.2)	310	26(83.9)	6(75.0)	310
Sex	Male	20(51.3)	198(55.0)	218	14(45.2)	6(75.0)	218
	Female	19(48.7)	162(45.0)	181	17(54.8)	2(25.0)	181
Marital status	Single	12(30.8)	167(46.4)	179	9(29.0)	3(37.5)	179
	Married	25(64.1)	177(49.2)	202	20(64.5)	5(62.5)	202
	Divorced	2(5.1)	16(4.4)	18	2(6.5)	0(0.0)	18
Family Size	1–5	29(74.4)	289(80.3)	318	24(77.4)	5(62.5)	318
	≥ 6	10(25.6)	71(19.7)	81	7(22.6)	3(37.5)	81
Residence	Rural	4(10.3)	85(23.6)	89	3(9.7)	1(12.5)	89
	Urban	35(89.7)	275(76.4)	310	28(90.3)	7(87.5)	310
Profession	Nurse	11(28.2)	70(19.4)	81	8(25.8)	3(37.5)	81
	Midwife	7(17.9)	48(13.3)	55	4(12.9)	3(37.5)	55
	Doctor	3(7.7)	64(17.8)	67	2(6.5)	1(12.5)	67
	Laboratory	4(10.3)	67(18.6)	71	4(12.9)	0(0.0)	71
	Pharmacy	6(15.4)	42(11.7)	48	6(19.4)	0(0.0)	48
	Radiology	4(10.3)	31(8.6)	35	3(9.7)	1(12.)	35
	Anesthesia	3(7.7)	23(6.4)	26	3(9.7)	0(0.0)	26
	Environmental health	1(2.6)	15(4.2)	16	1(3.2)	0(0.0)	16
Educational status	Diploma	11(28.2)	71(19.7)	82	7(22.6)	4(50.0)	82
	Degree and above	28(71.8)	289(80.3)	317	24(77.4)	4(50.0)	317

HBV DNA detection with clinical and behavioral variables

HBV DNA was detected in 7.7% of smokers. Participants with a family history of hepatitis had an HBV DNA detection rate of 20.5%. Participants with a history of alcohol use had a 41.0% rate of HBV DNA detection. Additionally, individuals with multiple sexual contacts and those with a history of hospital admission had higher HBV DNA detection rates of 33.3% and 38.5%, respectively (Table 3).

Table 3.

Prevalence and viral load distribution of HBV DNA with participants ‘clinical and behavioral variables.

Clinical/behavioral variables	Category	HBV DNA			HBV DNA concentration (MU/mL)
		Detected (%)	Not detected (%)	Total	<200	≥200	Total
Smoking cigarette	Yes	3(7.7)	15(4.2)	18	3(9.7)	0(0.0)	18
	No	36(92.3)	345(95.8)	381	28(90.3)	8(100.0)	381
Family history of hepatitis	Yes	8(20.5)	30(8.3)	38	5(16.1)	3(37.5)	38
	No	31(79.5)	330(91.7)	361	26(83.9)	5(62.5)	361
Khat chewing	Yes	7(17.9)	44(12.2)	51	6(19.4)	1(12.5)	51
	No	32(82.1)	316(87.8)	348	25(80.6)	7(87.5)	348
Alcohol use	Yes	16(41.0)	62(17.2)	78	13(41.9)	3(37.5)	78
	No	23(59.0)	298(82.8)	321	18(58.1)	5(62.5)	321
Injectable medications	Yes	11(28.2)	67(18.6)	78	10(32.3)	1(12.5)	78
	No	28(71.8)	293(81.4)	321	21(67.7)	7(87.5)	321
Multiple sexual contact	Yes	13(33.3)	48(13.3)	61	11(35.5)	2(25.0)	61
	No	26(66.7)	312(86.7)	338	20(64.5)	6(75.0)	338
Hospital admission	Yes	15(38.5)	73(20.3)	88	13(41.9)	2(25.0)	88
	No	24(61.5)	287(79.7)	311	18(58.1)	6(75.0)	311
Blood transfusion	Yes	7(17.9)	14(3.9)	21	7(25.6)	0(0.0)	21
	No	32(82.1)	346(96.1)	378	24(77.4)	8(100.0)	378
Sharp materials sharing	Yes	8(20.5)	32(8.9)	40	7(22.6)	1(12.5)	40
	No	31(79.5)	328(91.1)	359	24(77.4)	7(87.5)	359
Body piercing	Yes	12(30.8)	61(16.9)	73	12(38.7)	0(0.0)	73
	No	27(69.2)	299(83.1)	326	19(61.3)	8(100.0)	326
Body tattooing	Yes	10(25.6)	46(12.8)	56	8(25.8)	2(25.0)	56
	No	29(74.4)	314(87.2)	343	23(74.2)	6(75.0)	343
Dialysis	Yes	1(2.6	5(1.4	6	1(3.2)	0(0.0)	6
	No	38(97.4	355(98.6	393	30(96.8)	8(100.0)	393
Surgery	Yes	6(15.4	40(11.1	46	4(12.9)	2(25.0)	46
	No	33(84.6	320(88.9	353	27(87.1)	6(75.0)	353
Dental extraction	Yes	16(41.0	118(32.8	134	13(41.9)	3(37.5)	134
	No	23(59.0	242(67.2	265	18(58.1)	5(62.5)	265

Risk factors associated with HBV DNA detection

Several factors show significant crude associations with HBV DNA detection including family history of hepatitis, alcohol use, multiple sexual contacts, hospital admission, blood transfusion, sharing sharp materials, body piercing, and tattooing. However, after adjusting for other variables, only alcohol use (APR: 2.5, 95% CI: 2.1 to 7.2, p = 0.021) and multiple sexual contacts (APR: 3.7, 95% CI: 2.8 to 6.4, p = 0.003) remain significant independent risk factors for HBV DNA detection (Table 4).

Table 4.

Crude and adjusted prevalence ratio analysis of risk factors for HBV DNA detection.

Variables	Category	HBV DNA
		Detected (%)	Not detected (%)	CPR(95%CI)	P-V	APR(95%CI)	P-V
Family history of hepatitis	Yes	8(20.5)	30(8.3)	4.2(3.4, 7.2)	0.014*	0.6(−0.6, 1.8)	0.306
	No	31(79.5)	330(91.7)	Ref
Alcohol use	Yes	16(41.0)	62(17.2)	2.2(2.0, 6.5)	0.001*	2.5(2.1, 7.2)	0.021**
	No	23(59.0)	298(82.8)	Ref
Injectable medications	Yes	11(28.2)	67(18.6)	2.1 (1.9, 6.3)	0.151*	−0.34(−0.9, 0.8)	0.936
	No	28(71.8)	293(81.4)	Ref
Multiple sexual contact	Yes	13(33.3)	48(13.3)	3.0(4.8, 7.4)	0.001*	3.7(2.8, 6.4)	0.003**
	No	26(66.7)	312(86.7)	Ref
Hospital admission	Yes	15(38.5)	73(20.3)	4.1(2.3, 8.2)	0.009*	0.4(−0.4, 1.2)	0.356
	No	24(61.5)	287(79.7)	Ref
Blood transfusion	Yes	7(17.9)	14(3.9)	4.3(3.0, 8.1)	0.001*	0.4(−0.6, 1.4)	0.461
	No	32(82.1)	346(96.1)	Ref
Sharp materials sharing	Yes	8(20.5)	32(8.9)	3.7(2.1, 5.4)	0.022*	−0.6(−1.8, 0.7)	0.364
	No	31(79.5)	328(91.1)	Ref
Body piercing	Yes	12(30.8)	61(16.9)	4.5(2.2, 7.1)	0.034*	0.5(−0.3, 1.2)	0.229
	No	27(69.2)	299(83.1)	Ref
Body tattooing	Yes	10(25.6)	46(12.8)	2.6(1.8, 4.4)	0.028*	0.2(−0.8, 1.1)	0.739
	No	29(74.4)	314(87.2)	Ref

NB: *: Candidate variable for multivariate analysis at P < 0.25, CPR: crude prevalence ratio, **: significant variable by the multivariate analysis at P < 0.05, APR: adjusted prevalence ratio, CI: confidence interval, P-V: p-value, Ref: reference.

Discussion

The detection of OBI in vaccinated, HBsAg-negative healthcare workers carries significant public health implications, especially concerning blood safety, healthcare worker screening, and vaccination booster policies³². Importantly, the study advocates for the integration of NAT into routine HBV diagnostic algorithms, particularly for healthcare workers, to improve the detection of OBI and minimize the risk of cryptic viral transmission³. Additionally, the detection of OBI in vaccinated individuals suggests that vaccine-induced immunity may wane over time or be insufficient in some cases, underscoring the potential need for booster vaccinations to maintain protective immunity³.

This study investigated the prevalence and determinants of OBI among fully vaccinated, HBsAg-negative healthcare workers in East Gojjam Zone Hospitals, Northwest Ethiopia. Despite universal vaccination and negative HBsAg status, 39/399 (9.8%) (95% CI: 7.0–13.0%) of the study population were found to have detectable HBV DNA. Notably, the majority of these cases, 31/39 (79.5%) had low-level viremia (< 200 IU/mL). Low-level viremia often reflects occult or suppressed hepatitis B virus infection, where viral replication is minimal or controlled by the host immune response or antiviral therapy³³. Clinically, this low-level viremia may be associated with a lower risk of liver disease progression and reduced infectivity, although it still poses a risk for viral reactivation, especially in immunocompromised individuals³⁴.

In contrast, high-level viremia, defined as HBV DNA levels exceeding 200 IU/mL, indicates active viral replication, which is typically associated with increased liver inflammation, higher risk of liver damage, and greater potential for disease progression to cirrhosis or hepatocellular carcinoma³⁵. High-level viremia also signifies greater infectivity and the need for closer clinical monitoring and often antiviral treatment to prevent complications. Hence, distinguishing between low- and high-level viremia is crucial for guiding clinical decisions regarding prognosis, monitoring frequency, and therapeutic interventions in hepatitis B management³⁶.

The observed OBI prevalence of 9.8% is lower than that reported in a study conducted in Gondar, Ethiopia, which found a prevalence ranging from 19.1% to 20.3%³⁷. Conversely, the current prevalence is higher than a study from Addis Ababa, Ethiopia, which reported an OBI rate of 0.41%³⁸, and China (6.0%)³⁹. Additionally, this finding aligns with previous study from the Gambia (9.4%)⁵. The differences in OBI prevalence observed between studies can be attributed to several factors. For instance, geographic and regional variability plays a crucial role, as HBV endemicity differs across various parts of Ethiopia and globally, influenced by aspects such as vaccination coverage, public health policies, and local transmission dynamics⁴⁰. Moreover, diagnostic methodologies significantly contribute to the variation; for example, differences in the sensitivity and specificity of HBV DNA assays, including the types of PCR used and their detection limits, impact OBI detection rates⁴¹.

Consequently, studies that utilize liver tissue samples or highly sensitive molecular techniques often report higher prevalence compared to those relying on serum samples or less sensitive testing methods^42,43. Additionally, host and viral factors, such as the genetic diversity of HBV, viral mutations including escape mutants, and individual immune responses, further explain discrepancies in OBI rates, particularly in populations where viral mutations are more common^37,44. Furthermore, variations in sample sizes, study periods, and inclusion criteria also affect the reported prevalence, highlighting the importance of considering multiple interconnected factors when interpreting OBI epidemiological data^45,46.

OBI may persist in vaccinated individuals due to several factors. One key reason is vaccine non-response, where some individuals fail to develop adequate protective immunity despite vaccination, leaving them susceptible to latent or low-level HBV infection^47,48. Another mechanisms for OBI infection among vaccinated healthcare workers include waning immunity post-vaccination, where anti-HBs levels decline over time, particularly after 15–20 years, leaving individuals susceptible despite initial protection, a process accelerated by prolonged occupational exposure in HCWs with extended work durations and low anti-HBs titers⁴⁹. Pre-vaccination infection can also persist as occult due to viral integration into the host genome or low-level replication that evades HBsAg detection¹⁹.

Furthermore, genotype mismatch between the vaccine strain and circulating HBV genotypes can reduce vaccine effectiveness, as vaccines are primarily based on genotype A2, while in many regions, other genotypes predominate, potentially allowing breakthrough infections^50,51. In addition, immune evasion mechanisms also contribute to OBI persistence; HBV can undergo mutations, including in the surface antigen genes (preS/S genes), which enable the virus to evade immune detection by altering epitopes targeted by antibodies and T cells⁵². These escape mutants undermine both natural and vaccine-induced immunity, facilitating ongoing viral replication at levels below detection by standard HBsAg assays⁵³. Together, these factors explain why OBI can remain prevalent even among vaccinated populations, emphasizing the importance of ongoing surveillance, improved vaccine formulations, and sensitive molecular diagnostics to manage and control HBV infection effectively¹⁹.

In addition, the study found that OBI was more common among older participants, > 30 years (82.1%), males (51.3%), married individuals (64.1%), and those residing in urban areas (89.7%). As people age, their immune system undergoes immunosenescence, which reduces the ability to control and clear HBV effectively and allowing viral DNA to persist despite negative HBsAg⁵⁴. Furthermore, older individuals may have waning vaccine-induced immunity, making them more susceptible to viral persistence³⁰.

Furthermore, certain HBV genotypes exhibit genetic variations that enable the virus to persist at low levels and evade immune detection by producing mutated surface antigens and viral proteins that interfere with host antiviral responses⁵⁵. These mutations can alter key viral epitopes, preventing recognition by immune cells, and suppress interferon signaling pathways, thereby allowing HBV to escape immune clearance⁵⁶. This immune evasion leads to low-level viral replication without detectable surface antigen, causing occult HBV infection⁵⁶. Additionally, viral proteins like HBx actively inhibit immune sensors and signaling molecules, further promoting viral persistence despite immune pressure. Thus, genetic variability of HBV combined with immunosenescence in older people increases their susceptibility to occult HBV infection⁵⁷.

Moreover, estrogen in females provides protective effects by enhancing immune responses and defending hepatic cells, while males lack this hormonal advantage, making them more prone to chronic infection and disease progression^58,59. In other words, males generally have a weaker immune response to HBV, partly because androgens suppress immune functions while estrogens in females enhance antiviral immunity⁵⁸. This sexual dimorphism affects viral clearance, allowing HBV, especially certain genotypes prone to immune evasion, to persist at low levels without detectable surface antigen, characteristic of occult infection⁶⁰. Additionally, genetic factors such as androgen response elements in the HBV genome may promote viral replication more in males. These differences contribute to higher rates of occult HBV infection and related complications in males compared to females⁶¹.

Furthermore, no specific HBV genotypes have been identified that uniquely suppress immunity more in males than females due to androgen secretion⁶²; instead, literature highlights general sexual dimorphism in HBV infection, where males exhibit higher chronicity and complications driven by androgen-enhanced replication across genotypes without genotype-specific attribution⁶³. Two androgen response elements (AREs) exist in the HBV genome, primarily located in the enhancer I (EnhI) region upstream of the core promoter⁶⁴, where androgen receptor binding upregulates HBV mRNA transcription, protein production, and replication, with additional minor AREs potentially contributing and these primary sites mediating effects independently of HBx in some models⁶⁵. Androgen receptor and HBx enhance HBV transcription via EnhI AREs, promoting low-level replication that persists as covalently closed circular DNA (cccDNA)⁶⁶, a hallmark of OBI, contributing to male-biased OBI persistence and reactivation risks under immunosuppression, though not genotype-specific; given the absence of genotyping data in our study on OBI among vaccinated healthcare workers in East Gojjam Zone Hospitals, direct links cannot be made⁶⁷.

Married individuals are more likely to develop OBI because living closely with an infected spouse facilitates the transmission of HBV genotypes capable of immune evasion and viral persistence⁶⁸. The causal relationship between area of residence (urban vs. rural) and OBI prevalence remains incompletely established due to confounding factors and limited evidence from cross-sectional studies like ours in East Gojjam Zone hospitals, where urban healthcare workers showed higher OBI rates potentially linked to greater occupational and environmental exposures (e.g., needle stick injuries, high patient volumes) and population density facilitating cryptic transmission, yet these associations are associative rather than proven causal without longitudinal data controlling for variables such as, socioeconomic status, healthcare access disparities and genetic/epidemiological variations across regions⁶⁹. Reverse causation or unmeasured confounders, like urban migration of high-risk individuals or differential screening sensitivity, could inflate apparent urban prevalence. Thus, while urban settings plausibly amplify OBI risks among vaccinated HBsAg-negative HCWs through intensified exposure, definitive causality requires prospective cohort studies with genotyping and multivariate adjustments beyond our descriptive analysis³².

In this study, multivariate analysis of generalized linear models confirmed that alcohol use (APR: 2.5, 95% CI: 2.1 to 7.2, p = 0.021) and multiple sexual contacts (APR: 3.7, 95% CI: 2.8 to 6.4, p = 0.003) were independently associated with OBI. Alcohol is known to impair immune function and liver health, potentially facilitating viral persistence⁷⁰. In other words, this association may be attributed to the immunosuppressive effects of alcohol, which can impair both innate and adaptive immune responses, potentially facilitating the persistence or reactivation of low-level HBV replication even in vaccinated individuals⁷¹.

In addition, alcohol consumption increases oxidative stress and induces liver cell damage, which in turn promotes viral transcription and replication, especially through upregulation of liver-specific transcription factors that activate HBV genes⁷². Moreover, alcohol weakens immune surveillance by impairing dendritic cell and B cell functions, reducing the ability to clear infected cells⁷³. Furthermore, chronic alcohol intake impairs the immune response to HBV vaccination by suppressing B-cell function, inhibiting cytokine responsiveness (e.g., IL-2 and IL-4), and reducing anti-HBs production, potentially accelerating waning titers through enhanced viral replication and cytotoxicity⁷⁴.

The genetic variability of HBV further enables some genotypes to evade immune detection, and alcohol-induced immune suppression amplifies this effect, supporting viral persistence and occult infection⁷⁵. In addition, studies indicate alcohol exacerbates HBV pathogenesis by increasing cccDNA levels via ethanol metabolism, oxidative stress, and endoplasmic reticulum disruption⁷⁶. Alcohol hepatitis could theoretically contribute to OBI persistence by fostering liver inflammation that sustains low-level cccDNA transcription, though confirmatory data from vaccinated cohorts like healthcare workers is limited, warranting targeted investigation⁷².

Sexual contacts, meanwhile, increase HBV exposure to potentially heterologous strains that challenge vaccine efficacy, particularly if immunity has waned, as frequent partners elevate breakthrough infection odds in low anti-HBs vaccinates without directly accelerating titer decline but amplifying re-exposure⁷⁷. Furthermore, multiple sexual contacts significantly increase the risk of exposure to OBI as certain HBV genotypes, such as genotype A (including subtypes A1 and A2), are more common in populations with multiple sexual contacts⁷⁸. In addition, sexual transmission facilitates exposure to diverse HBV genotypes and variants, increasing the likelihood of infection with mutated or drug-resistant strains⁷⁹. In other words, Multiple sexual contacts heighten exposure to diverse HBV genotypes like A (subtypes A1/A2), prevalent in such populations and prone to chronicity over genotype C’s aggressive liver pathology⁸⁰, while sexual transmission introduces genotypic variants and mutations (e.g., precore/core promoter) that evade detection and sustain intrahepatic cccDNA, distinguishing OBI from overt chronic HBV³⁰.

Studies have shown that individuals with multiple sexual partners have a significantly higher risk of OBI, as sexual contact facilitates the transfer of HBV with distinct genotypic and mutational profiles that can persist without overt symptoms^37,81. Specifically, genotype variability, such as the predominance of HBV genotype D in occult infections, along with mutations that confer resistance to antiviral drugs or increase reactivation risk, underlines the complexity of occult infection and its transmission through sexual contact⁸². Therefore, sexual behavior involving multiple partners increases exposure to genetically diverse HBV, facilitating occult infection via viral mutations that allow immune escape and low-level replication undetectable by routine screening.

While the current study design effectively estimated the prevalence of OBI and identified correlations with potential risk factors such as alcohol use and multiple sexual contacts, it inherently limited the ability to draw causal inferences about these risk factors⁸³. Specifically, the cross-sectional nature meant that exposure to risk factors and detection of OBI was assessed simultaneously, making it impossible to confirm whether these factors preceded the development of OBI or resulted from it⁸⁴. For example, alcohol use, identified as an independent risk factor, might have varied over time, and it remains unclear if it directly contributed to OBI or was associated with other unmeasured variables influencing infection risk⁸⁵.

Moreover, without longitudinal follow-up, the study could not determine whether OBI status changed over time in relation to exposure variables, nor could it capture the dynamic interplay between vaccination status, immune response, and infection⁸⁶. Highlighting the need for prospective cohort studies or case-control designs to better establish causal relationships would help guide future research in this important area of cryptic HBV transmission among vaccinated healthcare workers⁸⁷.

Limitation of the study

While the study provides valuable insights, several limitations must be acknowledged. Anti-HBs and anti-HBc were not determined due resource and time constraints. The lack of HBV genotyping and detection of vaccine-escape mutants precludes assessment of vaccine efficacy against diverse viral strains. In other words, presence of vaccine-escape mutants (non-A2 genotypes), mutations in the “a” determinant of HBsAg was not detected resource constraints. Moreover, the hepatitis B virus infection status of the study participants prior to vaccination was not known due to a lack of information. Additionally, the cross-sectional design limits causal inference regarding risk factors.

Conclusion and recommendation

Despite being fully vaccinated, a notable proportion of healthcare workers in the study setting harbor OBI. In other words, this study provides compelling evidence that OBI remains a significant public health concern among fully vaccinated, HBsAg-negative healthcare workers. Moreover, the majority of the case exhibited low-level viremia, underscoring the risk of potential transmission. Importantly, a history of alcohol use and multiple sexual contacts were identified as independent risk factors for OBI among individuals who have completed the hepatitis B vaccination series, suggesting that behavioral factors contribute to the persistence of occult infection even in vaccinated populations.

These results emphasize the need to integrate NAT into routine HBV screening protocols, particularly for healthcare workers, to enhance detection and reduce the risk of cryptic transmission. In other words, routine screening for HBV DNA in high-risk vaccinated groups may improve early detection and help prevent further spread. This finding also underscores the need for ongoing surveillance, improved vaccine formulations, and consideration of booster doses or alternative vaccination strategies in high-risk populations. In addition, it is better to consider post-vaccination serological testing to confirm protective immunity among healthcare workers.

Author contributions

AA: Involved in conceptualization, methodology, software, formal analysis, investigation, resources, data curation, writing-original draft, editing, visualization, and validation. DA: Involved in conceptualization, methodology, software, and investigation. TM: Involved in conceptualization, data acquisition, analysis, and interpretation. MJ: Involved in conceptualization, methodology, software, and writing-original draft.

Funding

Debre Markos University was funded only for material and personal cost.

Data availability

Data will be made available by the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

The Institutional Research Ethics Review Committee (IRERC) of the College of Health Sciences at Debre Markos University approved the study, which was conducted in accordance with the Declaration of Helsinki. The protocol number for this approval is [R/C/S/D/317/01/16].

Informed consent

Written informed consent was obtained from all study participants.