The study was conducted in the west Hararghe zone of the Oromia region from September to December 2020. West Hararghe is one of the 21 administrative zones in the Oromia regional state. Chiro town, which is 317 km to the east of Addis Ababa, serves as the zone's capital. Based on information from the 2007 Ethiopian CSA census, it is estimated that there are 2,667,000 people living in the west Hararghe zone. There are 17 woredas and 496 kebeles that make up the administrative division of the zone, of which 39 are urban and 457 are rural. In the zone, there are currently 4 public hospitals, 85 health centers, and 465 health posts. During the months of September to December 2020, the study was carried out at the public hospitals in Gelemso and Chiro (Source: 2019–2020 document profile from the West Hararghe Zonal Health Office).

A cross-sectional study design was conducted among pregnant mothers attending ANC clinics at public hospitals in west Hararghe. All confirmed pregnant mothers who were chosen through a systematic random sampling method and gave their written consent to take part in the study during the study period were included. Those who were mentally unconscious or who made a second ANC visit during the data collection period were excluded.

The sample size of the study was determined using a single population proportion formula by considering the prevalence of HBV infection in pregnant mothers at the Deder hospital in eastern Ethiopia (15), with a prevalence of 6.9%, a 95% confidence level (CI), and a margin of allowable error of 3%. To minimize the errors arising from the likelihood of noncompliance, 10% of the sample size was added. The final sample size was 302. Systematic random sampling was used until the required sample was reached.

A total of 1,358 pregnant mothers were expected to attend ANC clinics in two hospitals in the west Hararghe zone (460 pregnant mothers at the Chiro General Hospital and 898 pregnant mothers at the Gelemso General Hospital) for routine ANC from a previous registered annual number of clients served. A total of 302 study participants were selected proportionally from each hospital. A systematic random sampling technique was applied to select study participants. We took 3 months' average sampled population from the registration books, which were done in ANC clinic, where 460 (source population) pregnant mothers were divided for a sample size of 102 for the Chiro zonal hospital and 898 (source population) pregnant mothers were divided for a sample size of 200 for the Gelemso general hospital to get sample interval (kth value), which became 4 for both sites. Then, the first pregnant mother was randomly selected by the lottery method. Therefore, every 4th mother attending the clinics was enrolled in the study until the calculated sample size was achieved within 3 months of data collection. A total of 302 study participants were included in the study during the study period (Figure 1). Participants' identification number was used to avoid repetition.

A pretested and structured questionnaire adapted from the WHO checklist for the assessment of hepatitis B infection in antenatal patients (16) was used to collect information on sociodemographic information, circumcision, history of blood transfusion, risky sexual behavior, history of hospital admission and surgery, history of abortion, contact with HBV-infected individuals, and other factors. Data were collected by three trained midwives through a face-to-face interview. Data collectors introduced themselves and informed the participants of the purpose of the study. After the participants agreed to participate, they were asked to give written consent and were assigned a study identification number; then, the interview was held in a private room in the hospitals using a structured questionnaire.

At the time of arrival to the laboratory, all study participants' arms was cleaned with 70% denatured alcohol, and then approximately 3 ml of venous blood was collected using blood collection tubes following the standard operating procedure (SOP) by a trained laboratory technologist at the hospital laboratory. Blood collected by venipuncture was allowed to clot naturally. Serum was separated from whole blood cells and transferred to CryoTube and stored at −20°C. The serum sample was then transported to the Chiro Blood Bank Center Laboratory using a triple packing system with a vaccine carrier, where it was tested for HBsAg using an antibody sandwich enzyme-linked immunosorbent assay (ELISA) test kit (Human type, Germany) in accordance with the manufacturer's instructions. The test has a 99.9% specificity and 100% sensitivity. With monoclonal antibodies tailored to HBsAg, polystyrene microwell strips are used in the sandwich ELISA technique. A second antibody that was conjugated with horseradish peroxidase (HRP) and formed in the event that HBsAg was present in the sample, which is captured on the solid phase, was added to the microwells along with a patient sample. Since the color's intensity correlated with the antigen content of the sample, it was measured. Using a second AiDTM HBsAg ELISA, all reactive and some nonreactive results were confirmed.

The following operational definitions were used in this study:

Prevalence: Percentage of pregnant mothers that were affected with HBsAg during this study period at this study place.

Abortion: Spontaneous or deliberate ending of pregnancy before the fetus could be expected to survive.

Female circumcision: Comprises all procedures that involve partial or total removal of the external female genitalia traditionally.

Piercing: This is a type of body beautification where a sharp material punctures a hole in the body ears or nose parts where jewelry had been inserted.

Tattoo: Is any form of body art that is created when ink is inserted, using a needle or other sharp materials into the dermis layer of the skin.

Tonsillectomy: Traditional tonsillectomy: Complete or partial removal of tonsils traditionally.

Chronic HBV infection: Defined by the continued presence of HBsAg in the blood for longer than 6 months.

Knowledge: In this case, participants who scored 50% and above from all asked questions were labeled as have good knowledge and those who scored less than 50% were labeled as having poor knowledge on HBV infection and transmission (17).

The questionnaires were prepared in English and translated into Afaan Oromo language by an independent translator that could be understood by the study participants. Finally, the questionnaires were translated back to English to ensure their consistency with the previous initial version. Data collectors along with supervisors were trained on data collection procedures. The questionnaires were pretested on 15 pregnant mothers attending ANC clinics in the Chiro hospital to check their appropriateness and understandability by study participants before the actual data collection process to correct questionnaires. Supervisors and principal investigators checked the collected data for consistency and completeness. Codes were given to completed questionnaires. Oriented laboratory professionals collected blood specimens. Standardized procedures were followed during blood sample collection, storage, transportation, and analytical process. The ELISA test was performed by experienced laboratory personnel who were trained by Hema diagnosis laboratory with a Huma reader and Washer (Human, Germany). Quality control of serological tests, known positive and negative controls, was run in parallel with test samples. All laboratory procedures were carried out following SOP according to the manual instructions. The results were recorded with the patient's identification number, to avoid errors in the results of the test, and repeatedly checked before reporting.

Data were checked for completeness and consistency of the collected information, coded and entered into Epi-data version 3.1, and exported to Statistical Package for the Social Science (SPSS) version 22.0 software package for analysis. Descriptive statistical analysis was done using frequency tables, and proportions were used to summarize the data. Bivariate logistic regression analysis was carried out using cross-tabulation to see the association between HBsAg serostatus and independent variables while controlling for colinearity. Variables having a P-value less than 0.25 were included in the multiple logistic regression analysis at a 95% confidence interval. Then, multivariate logistic regression was done to control for possible confounders and identify the true effect of the selected predictor variables. The model adequacy was checked using the Hosmer–Lemeshow test to show the goodness of fitness. Finally, the strength of association between the outcome and predictor variables was assessed using an adjusted odd ratio (AOR) with a 95% confidence interval, and the significance of the association was declared at a P-value of less than 0.05.

Ethical clearance was obtained from the Institutional Health Research Ethics Review Committee (IHRERC) of the Haramaya University College of Health and Medical Science. The ethical letter was submitted to the West Hararghe Zonal Health office and official permission was sought from the West Hararghe Zonal Health Office and Gelemso and Chiro public hospitals’ administration; then, support letter was obtained from hospitals’ administration. Written informed voluntary consent was obtained after informing the purpose and importance of the study to each participant. To ensure the confidentiality of the participant's information, a code was used instead of the name of the participant. Participants were interviewed alone to maintain privacy. Individual test results were communicated with the attending physician for further care as national protocol and health education were given to clients on preventive measures to be taken as well as to protect others.

A total of 300 pregnant mothers were included in this study with an overall response rate of 99%. The mean age of the study participants was 28.6 (SD ± 6.88) years and it ranged from 18 to 47 years. More than half of the participants (65.3%) were urban residents, and 79.3% were pregnant between 6 and 9 months. Nearly all of the participants, 298 (99.3%), were married and out of which 28 (9.3%) were polygamous. A total of 86 (28.7%) and 148 (49.3%) participants were not able to read and write and were merchants, respectively (Table 1).

The majority of study participants (81.7%) had more than three children, had a history of body piercing (87%), and had previously undergone an abortion at a health facility [53 (74%)] and at home [19 (26%)], of which 5 (31%) at home and 11 (69%) in a health facility were HBsAg positive. More than half of the study participants had a history of female genital mutilation (FGM) (60.3%). Of the participants, 65% did not practice home delivery, and 56% had a history of health facility admission. A total of A total of 111 (37%), 50 (17%), 64 (21%), 68 (22.7%), and 125 (41.7%) had an experience of a tattoo, blood transfusion, surgical procedures, dental procedures, and tonsillectomy, respectively. About 49 (16.7%) study participants had multiple sexual partners (Table 2).

Out of the 300 study participants, 239 (79.7%) had poor knowledge. Of the participants, 35% and 38% had heard of a disease termed hepatitis B as a viral disease and affected liver function, respectively. Only 25% knew nausea, vomiting, and loss of appetite are common symptoms of hepatitis B. About 26%, 16%, and 33% of the study participants said there is no symptom of hepatitis B virus infection, HBV transmission can occur from mother to child during pregnancy, and HBV has a vaccine, respectively (Table 3).

The overall prevalence of HBV among pregnant mothers was 8% (24/300) (95% CI: 5.3–11.0). Among those HBV-seropositive pregnant mothers, 15 (62.5%) and 9 (37.5%) were from the Gelemso and Chiro Hospitals, respectively. The proportion of HBV was 9% among the 31–49 year age groups. Additionally, as one's level of education increases, the prevalence of HBV decreases.

In bivariate analysis, age, residence, educational status, history of home delivery, FGM, parity, history of abortion, tattooing, history of admission to the health facility, history of blood transfusion, history of surgical procedure, history of dental extraction, history of tonsillectomy, history of cesarean section, history of sexually transmitted diseases, history of multiple sexual partners, history of jaundice, history of contact with liver disease patient, and knowledge about HBV were candidate variables for multivariate analysis (Table 4).

In multivariable analysis, those pregnant mothers who had a tattoo, history of tonsillectomy, history of multiple sexual partners, and previous contact with liver disease patients were identified to have factors associated with HBV infection.

Pregnant mothers who had a tattoo had 4.3 times the odds of being HBsAg positive compared to those who did not have tattoos (adjusted odds ratio = 4.3; 95% CI: 1.1–17.0). Similarly, pregnant mothers who had a history of tonsillectomy had 5.7 times the odds of being HBsAg positive compared those who did not have a history of tonsillectomy (adjusted odds ratio = 5.7, 95% CI: 1.3–23.9). Mothers who had a history of contact with liver disease patients had 5.6 times the odds of being HBsAg positive compared to their counterparts (adjusted odds ratio = 5.6; 95% CI: 1.2–25.7). Pregnant mothers who had a history of multiple sexual partners had 10 times the odds of being HBsAg positive compared to those who did not have history of multiple sexual partners (adjusted odds ratio = 10.8; 95% CI: 2.5–45.9) (Table 5).