Breakthrough hepatitis B virus infection and its associated factors among vaccinated children in Northwest Ethiopia

Abstract

Despite the availability of an effective vaccine, the global prevalence of chronic hepatitis B (CHB) in children was estimated to range from 1.3 to 3.4%. In Ethiopia, the estimated seroprevalence of CHB in children under five years old was 2.21%. This study aimed to assess the prevalence of breakthrough hepatitis B virus (HBV) infection and its associated factors in northwest Ethiopia. A cross-sectional study was conducted in northwest Ethiopia from December 2023 to June 2024. Children aged 1–14 years who visited pediatric clinics for various medical reasons were enrolled. Hepatitis B surface antigen (HBsAg) was tested using an enzyme-linked immunosorbent assay (ELISA). Data were analyzed using SPSS version 23.0. Chi-square tests and logistic regression analyses were employed to identify predictors of breakthrough HBV infection, with statistical significance set at p < 0.05. Among 325 vaccinated children under 15 years of age, the prevalence of breakthrough HBV infection was 2.5% (95% CI: 0.9–4.3%). The age of the children and a history of contact with individuals with CHB were significantly associated with breakthrough infection. Children aged 5–9 years were 10.7 times (OR = 10.7; 95% CI: 1.1–99.6), and those aged 10–14 years were 20.7 times more likely to be infected with HBV (OR = 20.7; 95% CI: 2.0-215.8), compared to children aged 1–4 years. Additionally, children with a history of contact with CHB individuals were six times more likely to be infected with HBV (OR = 6.1; 95% CI: 1.4–27.6). This study found an intermediate prevalence of HBV infection among vaccinated children, which raises significant public health concerns. Age and contact with individuals with CHB were found to be significant factors associated with breakthrough HBV infection. These findings highlight the need for regular evaluation of the effectiveness of vaccination programs and the implementation of targeted preventive strategies to reduce transmission.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-02906-y.

Introduction

Hepatitis B virus (HBV) infection is a serious global health problem causing a substantial burden of both acute and chronic liver disease¹. Chronic infection typically develops when the immune response that usually clears the infection fails or is too weak. Thus, infections are almost always chronic in children younger than one year^2,3. Up to 25% of infants and older children with chronic hepatitis B (CHB) may develop cirrhosis and hepatocellular carcinoma (HCC)^4–6. Globally, more than 257 million people are chronically infected with HBV, and the virus causes about 887,000 deaths yearly, despite the availability of an effective vaccine³. In Africa, 61 million individuals live with CHB^7,8. In Ethiopia, the seroprevalence of CHB in children under five was 2.21% in 2015^9,10.

Hepatitis B is most commonly transmitted through mother-to-child transmission (MTCT) at birth (vertical transmission) and through horizontal transmission during early childhood¹¹. The risk of developing CHB among newly infected persons depends on their age at the time of infection. More than 90% of infected infants, 25–50% of infected children under five years of age, and 6–10% of acutely infected older children develop chronic infection⁶. Between the pre-vaccine era and 2015, the prevalence of CHB among children under five declined from 4.7 to 1.3%¹². However, the prevalence remained higher at 2.53% in the World Health Organization (WHO) African region¹³. The most effective strategy to reduce both MTCT and early childhood transmission of HBV is the timely administration of hepatitis B vaccination during infancy¹⁴. Globally, the current CHB prevalence among children is estimated to range from 1.3 to 3.4%¹⁵.

Moreover, breakthrough HBV infection was detected in vaccinated children¹⁶. There are increasing reports of breakthrough HBV infections in fully vaccinated children¹⁷. Breakthrough HBV infection was seen in 0.7% of healthy, fully vaccinated seven-year-old children in Taiwan¹⁸. Despite the availability of an effective vaccine, breakthrough HBV infection remains the leading public health concern, particularly among developing countries like Ethiopia^19,20. A decade after the introduction of hepatitis B vaccination as part of three-dose pentavalent vaccines in Ethiopia, the prevalence of HBV infection based on serum HBsAg ranged from 0.4 to 4.6%^21–25. While there is evidence of low to intermediate prevalence of HBV infection in both vaccinated and unvaccinated children in Ethiopia, there have been very few studies conducted in Ethiopia outlining which ages are associated with the highest prevalence of HBsAg positivity, considering the wide age range of 1–14 years. This study aimed to assess breakthrough HBV infection and its associated factors among vaccinated children in northwest Ethiopia. Specifically, the present study addressed the following research questions: (1) What is the prevalence of HBsAg positivity among vaccinated children aged 1–14 years? (2) Which age group (1–14 years) has the highest prevalence of HBV infection? (3) What factors contribute to breakthrough hepatitis B virus infections among children vaccinated during infancy in northwest Ethiopia?

Materials and methods

Study design, area, and period

A health facility-based cross-sectional study was conducted in Bahir Dar City, the capital of the Amhara National Regional State (ANRS), located in northwest Ethiopia (Figure S1)²⁶, from December 2023 to June 2024. The city is located 585 km from Addis Ababa, the capital of Ethiopia. In the public sector, healthcare services available in Bahir Dar include two specialized hospitals, one primary hospital, ten health centers, and thirty-nine health posts. For this study, three public health facilities in Bahir Dar (Felege Hiwot Comprehensive Specialized Hospital, Addis Alem Hospital, and Abay Health Center) participated in the research. The laboratory analyses were performed at the Amhara Public Health Institute (APHI), located in Bahir Dar, Ethiopia.

Study participants

The study population consisted of children aged 1 to 14 years who were attending selected health facilities during the study period. Children within this age range who visited pediatric clinics for various medical reasons were invited to participate in the study, provided their parents consented.

Eligibility criteria

To be eligible for this study, children should be 1 to 14 years old and willing to participate. Additionally, they should have completed the three doses of the hepatitis B vaccination as part of the pentavalent vaccine series (DPT-HepB-Hib) during infancy. Children who were unable to provide a blood sample due to severe illness or who were unwilling to participate in the study were excluded. Furthermore, children who did not receive the full dose of the vaccine were considered to have no vaccination history and were also excluded from the study. A breakthrough HBV infection occurs when a child is infected with HBV despite having received the three doses of hepatitis B vaccine.

Sampling procedures

A convenience sampling technique was used to select eligible children who were visiting healthcare facilities for various health conditions. Initially, children attending the pediatric clinics were assessed to determine if they had completed their hepatitis B vaccinations, using their vaccination cards or health registration records from each facility. Once eligible children were identified, data collectors provided mothers with a detailed explanation of the study before their child’s enrollment. If the mothers agreed to participate, an interview was conducted.

Data and sample collections

Trained nurses were responsible for completing pre-tested and structured questionnaires during children’s visits to selected health facilities. These questionnaires were administered in person and covered the following sections: sociodemographic information, family history of hepatitis B, vaccination history, and other related factors. The nurses conducted face-to-face interviews with the mothers to gather the necessary data. Additionally, trained sample collectors drew venous blood samples (ranging from two to five milliliters) from the children using serum separator tubes (SST).

The sera were separated by centrifugation after clotting within 8 h of collection at each health facility’s laboratory. The extracted sera aliquots were transported in a cold box at 2–4 °C to the APHI each day and stored at −80 °C until laboratory analysis. The temperature of the freezer at APHI was monitored daily using a temperature data logging device.

Laboratory analysis

All serum samples were serologically tested HBsAg using AiD™ enzyme-linked immunosorbent assay (ELISA) kits (Wanti Biological Pharmacy Enterprise Co., Ltd., Beijing, China). These kits have a reported sensitivity of 100% and a specificity of 99.92%. The lower limit of quantification (LLOQ) for this assay is 0.067 IU/mL, as specified by the manufacturer. Samples with HBsAg levels below the LLOQ were considered negative for HBsAg positivity. The testing was performed following the manufacturer’s instructions. Positive results were confirmed by analyzing duplicate HBsAg ELISA tests. An HBV infection was confirmed when a positive HBsAg ELISA test result was obtained in duplicate.

Data quality assurance

The questionnaire was initially developed in English, then translated into Amharic, and subsequently translated back into English to ensure consistency. A pretest was conducted prior to the actual data collection to validate the questionnaire and ensure the clarity and relevance of the questions. All data collectors and sample collection personnel received training from the principal investigator regarding the data collection procedures and sample drawing. The collected data were reviewed daily for consistency and accuracy. Standardized procedures were strictly followed during sample collection, storage, and laboratory analyses. The quality of test results was maintained by using known negative and positive samples as external quality controls. Each ELISA run included blank, negative, and positive controls to ensure the proper performance of reagents and the accuracy of test results.

Data analysis

Data from the questionnaires were entered, cleaned, categorized, and recoded using the Statistical Package for Social Sciences (SPSS) version 23. Variables of interest included the children’s sociodemographic, clinical, and biological factors potentially associated with HBV infection. Descriptive statistics were used to summarize frequency distributions and variability. Chi-square (χ2) or Fisher’s exact tests were used for the associations between categorical variables. Binary logistic regression analysis was also used to identify factors associated with HBsAg positivity. Odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated to estimate the strength of association between HBV infection and its potential determinants, and a P-value < 0.05 was considered statistically significant. All statistical analyses were performed with SPSS version 23.

Results

Sociodemographic characteristics

A total of 350 children were enrolled in this study. Of these, blood samples from 25 children were hemolyzed and subsequently rejected for serum analysis. As a result, 325 serum samples from children under 15 years of age were analyzed for HBsAg. The majority (69.8%) of the children were in the age group of 1–4 years. More than half (52.0%) of the children were male, and 58.8% were under school age. Two hundred fifty-eight (79.4%) of the children were urban dwellers. Among the 169 male participants, 128 (75.7%) were circumcised, with 89 (69.5%) of them having undergone traditional circumcision performed at home (Table 1).

Table 1.

Sociodemographic characteristics and breakthrough hepatitis B virus infection among vaccinated children living in Northwest Ethiopia, 2024 (n = 325).

Sociodemographic characteristics		Frequency	Percentage	Breakthrough HBV infection		P-value
				Yes (%)	No (%)
				8 (2.5; 95% CI: 0.9–4.3%)	317 (97.5)
Age group	1–4 years	227	69.8	1 (0.4)	226 (99.6)	< 0.001
	5–9 years	71	21.8	4 (5.6)	67 (94.4)
	10–14 years	27	8.4	3 (11.1)	24 (88.9)
Sex	Male	169	52.0	5 (3.0)	164 (97.0)	0.547
	Female	156	48.0	3 (1.9)	153 (98.1)
Education	Under school age	195	60	2 (1.0)	193 (99.0)	0.03
	Kindergarten	75	23	2 (2.7)	73 (97.3)
	Primary school	55	17	4 (7.3)	51 (92.7)
Residence	Urban	258	79.4	7 (2.7)	251 (97.3)	0.483
	Rural	67	20.6	1 (1.5)	66 (98.5)
Male circumcision	Yes	128	75.7	2 (1.6)	126 (98.4)	0.093
	No	41	24.3	3 (7.3)	38 (92.7)
Place of circumcision	Health institute	39	30.5	1 (2.6)	38 (97.4)	0.518
	Traditional	89	69.5	1 (1.1)	88 (98.9)

Breakthrough HBV infection in vaccinated children

Among the 325 vaccinated children included in the study, eight cases of breakthrough HBV infection were identified, yielding a prevalence of 2.5% (95% CI: 0.9–4.3%). Of the participants, three (11.1%) children, four (5.6%) children, and one (0.4%) child were tested positive for HBsAg in the age groups of 10–14 years, 5–9 years, and 1–4 years, respectively. Notably, an increased HBV breakthrough infection was observed when the age of children increased. Additionally, four (7.3%) children tested positive for HBsAg who were attending primary school, two (2.7%) children infected with HBV were attending kindergarten, and another two (1%) children positive for HBsAg were under school age. Furthermore, five (3%) of male children tested positive for HBsAg. By residence, seven (2.7%) children from urban dwellers were found to be HBsAg positive. Notably, concerning male circumcision status, three (7.3%) of uncircumcised male children were tested positive for HBsAg, while only one (2.6%) of those circumcised at a health institution tested positive for HBsAg (Table 1).

Determinants’ positivity among children with breakthrough HBV infection

Of the total HBsAg-positive children, four (50%) were 5–9 years old, and five (62.5%) were males. Five (62.5%) of the children positive for HBsAg had a history of contact with CHB family members, three (37.5%) had a history of hospital admission, and two (25%) had a history of circumcision. Two (25%) of the children positive for HBsAg had a history of jaundice. None of the children positive for HBsAg had a history of scarification or tattoos, a history of surgery, or a history of tooth extraction (Table 2).

Table 2.

Determinants’ positivity rates among children with breakthrough HBV infected children living in Northwest Ethiopia, 2024.

Determinants		Breakthrough HBV infection (%)	P-value
Age of children	1–4 years	1 (12.5%)	< 0.001
	5–9 years	4 (50%)
	10–14 years	3 (37.5%)
Sex of children	Male	5 (62.5%)	0.547
	Female	3 (37.5%)
History of hospital admission	Yes	3 (37.5%)	0.194
	No	5 (62.5%)
History of blood transfusion	Yes	0	1.000
	No	8 (100%)
History of tooth extraction	Yes	0	1.000
	No	8 (100%)
History of surgery	Yes	0	1.000
	No	8 (100%)
History of jaundice	Yes	1 (12.5%)	0.189
	No	7 (87.5%)
History of contact with CHB individuals	Yes	5(62.5%)	0.004
	No	3 (37.5%)
History of circumcision	Yes	2 (25%)	0.487
	N o	6 (75%)
History of tattooing	Yes	0	1.000
	No	8 (100%)

Determinants for HBV infection in vaccinated children

Binary logistic regression analyses were performed for each variable. In the multivariable logistic regression analysis, we found that the age of children and a history of contact with individuals with CHB were statistically significant factors associated with breakthrough HBV infection. Accordingly, children aged 5–9 years had 10.7 times higher odds of testing HBsAg-positive compared to those aged 1–4 years (OR = 10.7; 95% CI: 1.1–99.6), while those aged 10–14 years had 20.7 times higher odds (OR = 20.7; 95% CI: 2.0–215.8). Furthermore, children with a history of contact with individuals with CHB were six times more likely to test HBsAg-positive than those without such contact (OR = 6.1; 95% CI: 1.4–27.6). No statistically significant association was observed between HBsAg positivity and sex (P = 0.55) (Table 3).

Table 3.

Binary logistic regression analysis of the associated risk factors of breakthrough hepatitis B virus infection among vaccinated children living in Northwest Ethiopia, 2024.

Determinants		Breakthrough HBV infection		COR (95% CI)	P-value	AOR (95% CI)	P-value
		Yes (%)	No (%)
Age of children	1–4 years	1 (0.4)	226 (99.6)	1	0.017	1	0.039
	5–9 years	4 (5.6)	67 (94.4)	13.5 (1.5–122.8)		10.7 (1.1–99.6)
	10–14 years	3 (11.1)	24 (88.9)	28.2 (2.8–282.3)		20.7 (2.0–215.8)
Sex of children	Male	5 (3.0)	164 (97.0)	1.6 (0.4–6.6)	0.550	-	-
	Female	3 (1.9)	153 (98.1)	1		-
Education	Under school age	2 (1.0)	193 (99.0)	1	0.065	1	0.409
	Kindergarten	2 (2.7)	73 (97.3)	2.6 (0.4–19.1)		0.2 (0.01–3.6)
	Primary school	4 (7.3)	51 (92.7)	7.6 (1.3–42.5)		0.2 (0.01–3.7)
Residence	Urban	7 (2.7)	251 (97.3)	1.8 (0.2–15.2)	0.571	-	-
	Rural	1 (1.5)	66 (98.5)	1		-
History of living with CHB individuals	Yes	5 (9.1)	50 (90.9)	8.9 (2.1–38.4)	0.003	6.1 (1.4–27.6)	0.019
	No	3 (1.1)	267 (98.9)	1		1
History of jaundice	Yes	2 (5.7)	33 (94.3)	2.9 (0.6–14.8)	0.208	0.8 (0.1–5.4)	0.815
	No	6 (2.1)	284 (97.9)	1		1
History of hospital admission	Yes	3 (4.7)	61 (95.3)	2.5 (0.6–10.8)	0.215	1.9 (0.4–9.0)	0.418
	No	5 (1.9)	256 (98.1)	1		1

1: Reference; CI: Confidence Interval; COR: Crude Odds Ratio; AOR: Adjusted Odds Ratio.

Discussion

A breakthrough HBV infection occurs when an individual is infected with HBV, despite completing the HBV vaccine series. This infection has been reported in children born after the implementation of universal vaccination²⁷. The global prevalence of HBsAg in children under 5 years of age was approximately 1.3%, a significant decrease from about 4.7% before the widespread introduction of universal infant immunization^12,28. This study addressed the research questions of what is the prevalence of breakthrough HBV infection, which age group (1–14 years) has the highest prevalence of HBV infection, and what factors contribute to breakthrough HBV infections among children vaccinated during infancy in northwest Ethiopia?. The findings revealed a breakthrough HBV infection prevalence of 2.5%, with infection rates increasing with age. Additionally, two significant factors associated with breakthrough HBV infections were identified: the age of the children and a history of contact with family members who have CHB.

This study found that 2.5% of vaccinated children aged 1–14 years tested positive for HBsAg, indicating that the study area falls under the category of intermediate endemicity of HBV infection²⁹. This prevalence was consistent findings from other regions of Ethiopia, including with Gondar (4.2%)²⁴, Debre Markos (4.2%)³⁰, Jimma (2.1%)²³, Harar (1.1%)²², and Ethiopia (2.3%)³¹. It was also comparable with studies conducted in low- and high-income countries, such as Egypt (1.1%)³², Ghana (2.6%)³³, Burkina Faso (2.6%)³⁴, Senegal (1.1%)^35,36, Greenland (2.9%)³⁷, Vietnam (2.7%)³⁸, Thailand (4%)³⁹, China (0.9–2.1%)^40–43, and Taiwan (1–4%)^16,44.

The prevalence of breakthrough HBV infection observed in this study was higher than that of similar studies conducted in high-burden African countries, including Addis Ababa (0.4%)²¹, Egypt (0.4–0.6%)^45–47, and Nigeria (0.5%)⁴⁸. It also significantly higher than the seroprevalence of HBsAg reported among vaccinated children in non African countries, including Italy (0.6%)⁴⁹, Iran (0.6%)⁵⁰, Uzbekistan (0.8%)⁵¹, China (0.1%)⁵², and Singapore (0.3%)⁵³. Breakthrough HBV infection despite vaccination might be resulted from vaccine failure due to improper storage conditions, particularly the disruption of the cold chain, which can render the hepatitis B vaccine ineffective⁴³. However, the HBsAg prevalence in our study was lower than that reported in other Ethiopian regions, such as Addis Ababa (6.2%)⁵⁴ and Hawassa (4.4%)²⁵, as well as in countries like Gambia (4.5%)⁵⁵, and across West Africa (5%)⁵⁶. These discrepancies might be due to the differences in vaccination coverage, nutritional status, cultural practices, socioeconomic conditions, familial history of HBV, age groups of children, and healthcare system factors, such as the inclusion of a timely birth dose, clinical practices, quality of immunization programs, and variations in laboratory methods used for HBsAg detection.

In the current study, the prevalence of HBsAg was 11.1% in children aged 10–14 years, 5.6% in those aged 5–9 years, and 0.4% in 1–4-year-old children. This indicated that the prevalence of HBsAg was lower in the age group of 1–4 years vaccinated children compared to the period before vaccination, which was 3.1%⁵⁴. However, the prevalence of HBsAg was similar to the same study conducted before vaccination in the age group of 5–9-year-old children, which showed 5.0%⁵⁴. Conversely, the prevalence of HBsAg in this study was higher than the same study conducted before vaccination on 10–14-year-old children, which showed a 6.1% prevalence of HBsAg⁵⁴. Breakthrough HBV infection was increasing with the age of children, which was consistent with findings from similar studies conducted in Egypt, Sierra Leone, China, Singapore, and Nepal^{41,43,53,57–59}. This study, along with other similar studies, showed that HBV vaccination has a positive impact on reducing the MTCT of HBV infection. However, complete eradication of horizontal HBV infection has not yet been achieved despite the effectiveness of the universal HBV vaccination program⁴⁴.

A multivariable logistic regression analysis determined that age was a significant predictor of breakthrough HBV infection in vaccinated children in this study. This study revealed that children aged 5–9 years were 10.7 times more likely to be infected with HBV compared to those aged 1–4 years, while children aged 10–14 years were 20.7 times more vulnerable to the infection. This could be because HBV transmission in Africa primarily occurs horizontally during early childhood, particularly between the ages of two and ten⁶⁰. A previous study also reported that horizontal transmission in early childhood was higher than vertical transmission⁶¹. This result was consistent with a study conducted in China, which showed that older age was a determinant factor for HBsAg infection among children younger than 15⁴³.

Moreover, this study found that children who had contact with individuals with CHB were six times more likely to be infected with the HBV compared to those without such contact. This study was supported by a study conducted in northern Ethiopia⁶², which identified familial exposure to hepatitis B as a significant factor for HBV infection. Similarly, in Oman⁶³, interfamilial contact with HBV-infected individuals was identified as a common mode of transmission. In Nepal⁵⁹, children living with HBsAg-positive mothers were found to be more vulnerable than their counterparts. Furthermore, in India⁶⁴, the prevalence of HBsAg among household contacts of individuals with CHB was significantly higher than in the general population.

The current study indicated that male children had a higher prevalence of HBV infection (3.0%) compared to female children (1.9%), although this difference was not statistically significant. This finding was consistent with studies conducted in Debre Markos, Ethiopia (6.7% in males vs. 1.3% in females)³⁰, as well as in Senegal³⁶, Sierra Leone⁵⁸, Singapore⁵³, and Nepal⁵⁹. This might be because sex-based differences in immune responses play a role in HBV susceptibility⁶⁵. Additionally, male children may be more likely to engage in activities that increase their HBV exposure, such as playing in environments where hygiene is poor, engaging in rough play that could lead to cuts or injuries, or undergoing circumcision under non sterile conditions.

Our findings indicated that some well-known potential factors were not significantly associated with breakthrough HBV infection. This might be due to the fact that such potential factors are not commonly practiced in young children, or because health facilities in the study area properly sterilize and disinfect their equipment, thereby minimizing the risk of HBV transmission through medical or procedural exposure.

Limitations

The strength of this study lays in its inclusion of all age groups children after the introduction of the hepatitis B vaccine in the national expanded program on immunization (EPI). However, it is important to acknowledge some limitations. One notable limitation was the small number of breakthrough HBV infections detected in this study, which might impact the conclusions regarding the associated factors for the outcome variable. The study was cross-sectional which limited determining the causality of factors for HBV infection. Additionally, ELISA for anti-hepatitis B core antibody (anti-HBc) was not performed, which restricted our ability to determine whether the breakthrough infection was CHB. Furthermore, the lack of sequencing hinders our ability to determine mode of transmission and to identify vaccine escape mutants.

Significance

This study provides essential data to local and national health authorities to enhance strategies for the early detection and monitoring of breakthrough HBV infection. Additionally, it provides participants with important information that can lead to better health outcomes and earlier detection of infections, since early diagnosis and monitoring can prevent long-term complications. Furthermore, it raises community awareness of HBV transmission risks, encouraging more people to get tested, thus reducing overall transmission.

Conclusion and recommendations

The current study found an intermediate prevalence of HBV infection among vaccinated children in the study area, which raises significant public health concerns. The HBV breakthrough infection was increasing with the age of children suggesting horizontal transmission from family members. The main factors associated to HBV infection in vaccinated children were found to be the age of the children and contact with individuals who had CHB. Based on these findings, the study recommends that policymakers implement screening protocols for family members of children who test positive for HBsAg. Furthermore, these findings highlight the need for regular evaluation of the effectiveness of vaccination programs and the implementation of targeted preventive strategies to reduce transmission. Further study should be conducted with a large sample size, a prospective study, and including tests not performed but crucial for this type of study.

Electronic supplementary material

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Abbreviations

APHI

Amhara Public Health Institute

CHB

Chronic Hepatitis B

ELISA

Enzyme Linked Immunosorbent Assay

HBsAg

Hepatitis B surface Antigen

HBV

Hepatitis B Virus

MTCT

Mother to Child transmission

OR

Odds Ratio

WHO

World Health Organization

Author contributions

M.G.: conception and design of the work, data curation, analysis, interpretation, drafting the manuscript, revising the manuscript; A. H., and Y.W.: conception and design of the work, data curation, analysis, interpretation, revising the manuscript, and supervision. All authors reviewed the manuscript.

Funding

Not applicable.

Data availability

The data used for this study are available at the corresponding author, so interested readers can get the data from the corresponding author with a reasonable request.

Declarations

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Ethical considerations and consent to participate

The study protocol received approval from the scientific and ethical review committee of CDT-Africa and the Institutional Review Board of the College of Health Sciences, Addis Ababa University, Ethiopia. Informed consent was obtained from all participants and their legal guardians. The research poses no more than minimal risks to participants. The study complied with the Declaration of Helsinki.