For this study, we used HCW serum samples collected in 2020 for the investigation of COVID-19 serology. The sample collection was detailed previously [23]. In brief, HCWs were recruited from central, provincial, and district hospitals in Vientiane (capital), Oudomxay, Luangprabang, Savannakhet, and Champasak provinces. Participants were randomly selected in each department and included clinicians, nurses, and laboratory technicians. We also included other nonclinician workers (administrative HCWs) in order to see if there was a trend in HBV exposure according to patient contact. After individual informed consent was obtained, a structured questionnaire was used to collect information on demographics and 5 mL of whole blood was collected. Serum was separated by centrifugation and stored at 4 °C for a maximum of 7 days and at −80 °C for long term.

Ethics approval was obtained from the Lao National Ethics Committee for Health Research (reference #052/2020). Informed consent was obtained from all participants involved in the study, and the consent forms from the study stated that any remaining serum might be used for testing serology against other infectious diseases. All participant data are deidentified, with personal identifiers removed and replaced by unique codes. Serum samples are stored securely, and access is restricted to authorized personnel only. Data are protected through encrypted storage and secure handling procedures. There was no compensation for the participants in the study according to the Lao National Ethic Committee regulations.

Anti-HBc and anti-HBs antibodies were measured by commercial enzyme-linked immunosorbent assays (ELISAs) according to the manufacturers’ instructions (Diasorin and Biorad). All anti-HBc–positive and anti-HBs–negative samples were tested for HBsAg (Biorad) [8]. All HBsAg-positive samples were tested for anti-HDV antibodies (indicating exposure to HDV) by ELISA (DIA.PRO). Individuals who were HBsAg positive were defined as having “acute or chronic infection,” individuals who were anti-HBc positive were defined as having been “previously exposed,” and individuals who were anti-HBc negative but anti-HBs positive were defined as having “serological evidence of vaccination.” Individuals who were anti-HBs negative had no serological evidence of protection and were defined as “susceptible to infection,” although it is possible that a small proportion were protected by the presence of memory B cells in the absence of detectable antibodies.

Descriptive statistics, bivariate analysis, and multivariable analysis were conducted using Stata (version 14; StataCorp). Descriptive statistics were used to summarize categorical and continuous variables. Bivariate analysis assessed the associations between the dependent variable (HBV serostatus) and independent variables (sociodemographics and work status). Results of the bivariate analyses are presented as crude odds ratios (cORs). Multivariable analysis, using logistic regression, was applied to variables with a P value ≤.20 from the bivariate analysis. Thus, in the final multivariate analysis model, the presence of anti-HBc antibody was compared with 4 factors: sex, age group, ethnicity, and province. No factors were found to be significantly linked to HBsAg positivity following bivariate analysis (all P>.20; Table 1). The serological profile of vaccination had only 2 variables in the multivariable analysis model: sex and age groups (Table 2). Both cORs and adjusted odds ratios (aORs) were reported from these analyses.

The total number of participants was 666. The median age was 34 (range 20-65) years, 77.8% (n=518) were female, and 96.5% (n=643) were of Lao-Tai ethnicity. Most participants were living in Vientiane capital (n=410, 61.6%) and 53.6% (n=357) had ≤10 years work experience. The majority of the participants had direct contact with patients (n=620, 93.1%; Table 3).

Overall, 40.1% (267/666) HCWs were positive for anti-HBc antibodies (exposure), with no significant difference between male and female individuals (P=.10). HBV exposure in those aged ≤30 years was 32.7% (81/248), and the likelihood of the exposure was approximately 3 times higher in those aged ≥50 years (66/118, 55.9%; aOR 2.91, 95% CI 1.83‐4.61; P<.001). HCWs from Oudomxay province had higher HBV exposure than those living in Vientiane (21/31, 67.7% vs 160/410, 39%; aOR 3.69, 95% CI 1.68‐8.12; P=.001; Table 1 and Multimedia Appendices 1 and 2).

Overall, 36 (5.4% when extrapolated to the total study population) individuals were HBsAg positive (currently infected). The HBsAg prevalence was higher among men than women, although not significantly different (12/148, 8.1% and 24/518, 4.6%, respectively; cOR 0.48, 95% CI 0.26‐1.12; P=.10). HBsAg prevalence increased with age from 3.6% (9/248) in those aged ≤30 years to 6.8% (8/118) in those aged ≥50, although this also did not reach statistical significance (P=.10; Table 1 and Multimedia Appendix 1).

The overall prevalence of serological evidence for vaccination (anti-HBs positive and anti-HBc negative) was 28.7% (191/666). After multivariable analysis, individuals aged 31‐49 years had significantly higher odds (104/300, 34.7%; aOR 1.70, 95% CI 1.16‐2.49; P=.006) to have this profile than those aged ≤30 years (58/248, 23.4%; Table 2 and Multimedia Appendix 1).

A total of 284 (42.6%) participants were anti-HBs negative and susceptible to infection (Multimedia Appendix 3). All 36 HBsAg–positive samples tested negative for anti-HDV antibodies.

This study reported a moderate prevalence of 5.4% (36/666) HBsAg positivity among HCWs. This is similar to previous studies of Lao HCWs, with prevalence ranging from 3% to 12% [9,11]. However, it is lower than the prevalence in the adult general population (8%‐10%) [8,13], perhaps due to the fact that two-thirds of the participants were female individuals, who have lower exposure and HBsAg prevalence as compared to male individuals [11,14,24,25]. Indeed, in this study, male HCWs had higher prevalence of HBsAg positivity than female HCWs, although this was not statistically significant. This difference could be due to differential immune response, hormones, or risks practices [26,27]. Also, we found that HBsAg positivity increased with age. This is similar to what we reported previously in HCWs and the general population [9,24]. Our data show that a substantial proportion of Lao HCWs are chronically infected with HBV, and therefore, appropriate precautions are needed to reduce the risk of onward transmission to patients. All HCWs should be tested for HBsAg, and HCWs who are HBsAg positive should get access to counseling and treatment. Furthermore, the high proportion of HCWs with no serological evidence of protection indicates that the health care facilities need to implement routine HBV vaccination, with 3 doses and boosters as necessary for all HCWs. After receiving the hepatitis B vaccine, anti-HBs immunoglobulin G levels can remain detectable for several years, usually lasting a minimum of 5 to 10 years, and in some cases, even longer [5].

None of the individuals who were HBsAg positive had anti-HDV antibodies in this study, suggesting that HDV prevalence might be low in Lao PDR. However, since HDV prevalence varies with geographical regions and study cohorts (eg, in neighboring Vietnam, the prevalence ranged from 0% to 43% among individuals who are HBsAg positive [28-31], and in Thailand, from 0% to 65% [32-34]), further studies are needed to determine whether there are any subpopulations with HDV exposure in Lao PDR.

Anti-HBc seroprevalence, indicating exposure, was 40.1% (267/666). This high level is similar to other studies in Lao PDR [8,10,13]. As expected, HBV exposure increased with age. This was in accordance with previous published studies in the Lao general population [8,12,13,25,35] and HCWs [9,11] and may reflect exposure in the workplace or elsewhere. There was no correlation of exposure with the length of time in the job or type of job, as reported in a previous study of Lao HCWs [9]. However, the distinction between staff with and without contact with patients was not always clear, as some administration staff may have previously worked in a clinical setting. HBV exposure was higher in those living in the northern provinces. In particular, HCWs from Oudomxay province had significantly higher exposure than HCWs from other provinces. The data agreed with studies of HBV in Lao blood donors, where significantly higher exposure was found in the northern provinces [14,35]. The reason for this geographical variation is not clear. The rate of HBsAg positivity was not significantly higher in the northern provinces in this study. This may be due to the lower proportion of participants from the northern provinces in this study and the overall lower numbers of individuals who were HBsAg positive; due to HBV infection happening during adulthood, when chronic infection is less likely to occur; or a combination of both reasons.

This study found a low percentage of HCWs who had the serological evidence of vaccination (191/666, 28.7%). This profile was more common in those 31‐49 years of age, suggesting that this age group has been vaccinated against HBV as an adult or perhaps during an ad hoc HBV vaccination campaign in their hospitals. Although the rate of participants with serological evidence of vaccination in this study is higher than other studies conducted among HCWs in Lao PDR (13.6% [11] and 21% [9]) and Indonesia (11.6%) [36], it remains inadequate and reveals a large number of HCWs without protective anti-HBs antibodies (31.2%; Multimedia Appendix 3). Importantly, many of the participants with anti-HBc and anti-HBs antibodies may also have been vaccinated, but there is no way to distinguish these from the individuals exposed to HBV without vaccination based on the laboratory results.

This study has several limitations. First, we did not comprehensively measure the risk factors and practices of the participants. Understanding the intensity of exposure and the nature of contact with patients could provide valuable insights into the factors contributing to HBV exposure among HCWs. Second, the reason for higher HBV exposure in the northern populations has not been addressed—an issue that could facilitate effective management strategies. Lastly, the sample size and locations were limited. A more comprehensive, nationwide survey is needed at all health facilities in order to establish HBV testing, treatment, and vaccination practices as well as serological profiles.

According to the findings of this study, HBsAg prevalence is intermediate with no evidence of HDV coinfection in HCWs in Lao PDR. Importantly, a high proportion of HCWs remains susceptible to infection. Therefore, there is a need to implement mandatory pre-employment hepatitis B testing, in accordance with the Lao National Strategic Plan on Viral Hepatitis 2024‐2030. This would reduce the risk of HBV infection for both HCWs and patients. The obstacles to implementation of routine, comprehensive nationwide testing, vaccination, and treatment for HCWs include low awareness and lack of financial and human resources. These issues require an investment in HCW training, financial investment, and political will. Furthermore, the high proportion of HCWs with no serological evidence of protection indicates that the health care facilities need to implement routine HBV vaccination, with 3 doses and boosters as necessary for all HCWs.