Antibody levels and protection after Hepatitis B vaccine in adult vaccinated healthcare workers in northern Uganda.

Hepatitis B vaccine has contributed to the reduction in hepatitis B virus infections and chronic disease globally. Screening to establish extent of vaccine induced immune response and provision of booster dose are limited in most low-and-middle income countries (LMICs). Our study investigated the extent of protective immune response and breakthrough hepatitis B virus infections among adult vaccinated healthcare workers in selected health facilities in northern Uganda. A cross-sectional study was conducted among 300 randomly selected adult hepatitis B vaccinated healthcare workers in Lira and Gulu regional referral hospitals in northern Uganda. Blood samples were collected and qualitative analysis of Hepatitis B surface antigen (HBsAg), Hepatitis B surface antigen antibody (HBsAb), Hepatitis B envelop antigen (HBeAg), Hepatitis B envelop antibody (HBeAb) and Hepatitis B core antibody (HBcAb) conducted using ELISA method. Quantitative assessment of anti-hepatitis B antibody (anti-HBs) levels was done using COBAS immunoassay analyzer. Multiple logistic regression was done to establish factors associated with protective anti-HBs levels (≥ 10mIU/mL) among adult vaccinate healthcare workers at 95% level of significance. A high proportion, 81.3% (244/300) of the study participants completed all three hepatitis B vaccine dose schedules. Two (0.7%, 2/300) of the study participants had active hepatitis B virus infection. Of the 300 study participants, 2.3% (7/300) had positive HBsAg; 88.7% (266/300) had detectable HBsAb; 2.3% (7/300) had positive HBeAg; 4% (12/300) had positive HBeAb and 17.7% (53/300) had positive HBcAb. Majority, 83% (249/300) had a protective hepatitis B antibody levels (≥10mIU/mL). Hepatitis B vaccine provides protective immunity against hepatitis B virus infection regardless of whether one gets a booster dose or not. Protective immune response persisted for over ten years following hepatitis B vaccination among the healthcare workers.

Introduction

Elimination of hepatitis B virus (HBV) transmission is an achievable public health goal, particularly in the light of proven effectiveness and safety of hepatitis B vaccine [1]. Studies conducted in areas with high HBV endemicity have demonstrated declines in the prevalence of chronic HBV among children to < 2% after routine infant vaccination [1]. A substantial decline in HBV-related disease burden and prevalence of chronic HBV infection has been observed among children following introduction of universal infant hepatitis B vaccination [2]. However, the vaccine may not provide protection from exposure to hepatitis B virus later on in life due to waning of immune memory over time [3].

Persistence of hepatitis B antibodies (anti-HBs) and ability of the immune system to mount a response to exposure of HBV later in life is necessary for long term protection against hepatitis B virus infection [4]. Some studies have confirmed persistence of antibodies and immune memory following hepatitis B vaccination [5] while others confirm waning of antibody concentrations 13–15 years after primary vaccination among those vaccinated at birth [6]. The current vaccination of adult individuals against hepatitis B virus is premised on the fact that sufficient anti-HBs concentrations and immune memory is formed against HBV. However, unless routine post-vaccination serological testing is performed, it remains unclear what proportion of individuals who complete all 3 dose schedules of hepatitis B vaccine actually get protected, as was found in an earlier study where 22.9% of vaccinated children had undetected antibody levels [7].

Annually 5.9% of the healthcare workers are exposed to HBV corresponding to 66,000 preventable HBV infections globally [8]. Healthcare workers thus represent an important group in the population that need to be protected against HBV infection. While hepatitis B is a disease of public health concern in Uganda [9], healthcare workers (HCWs) in the country are at higher risk for transmission of hepatitis B virus (HBV) compared to the general population [10]. They have a higher risk of contracting the disease from exposure (eye, oral mucosa and skin) to potentially infectious patient’s blood and percutaneously from contaminated sharp objects such as needles [11].

Northern Uganda has disproportionately high prevalence of hepatitis B virus infection compared to other parts of the country [12]. As a preventive measure, all healthcare workers in Uganda are required to take hepatitis B vaccine as adults. A recent study by Ssekamatte et al., [13] reported a rather low hepatitis B vaccine dose completion of 57.8% only among healthcare workers in central Uganda. In spite of the low hepatitis B vaccine dose completion rate, no evaluation of hepatitis B vaccination has been done to assess immune response to the vaccine since the introduction of hepatitis B vaccination in Uganda in 2002. This could be due to the limited funding of the health sector, a common occurrence in most LMICs [14]. There is paucity of information on the extent of immune protection against hepatitis B virus infection among adult vaccinated individuals in Uganda despite the high risk of exposure. We therefore set out to determine the proportion of healthcare workers with protective levels of anti-HBs and also evaluate the prevalence of hepatitis B virus infection among those without protective anti-HBs post hepatitis B vaccination.

Methods

Ethical considerations

Ethical review and approval of the protocol was done by the Makerere University School of Biomedical Science Research and Ethic Review Committee (#SBS-REC 798). Additionally, administrative clearance was obtained from the hospitals prior to study initiation. A written informed consent was obtained from potential study participants prior to enrollment into the study.

Study design, setting and population

This was a cross sectional study done in Lira and Gulu regional referral hospitals in northern Uganda from October to December 2020. In Uganda, regional referral hospitals offer specialist clinical services such as psychiatry, Ear, Nose and Throat (ENT), ophthalmology, higher level surgical and medical services, and ancillary services (laboratory, medical imaging and pathology). They also provide general healthcare services including, preventive, promotive, curative, maternity, in-patient health services, surgery, blood transfusion, laboratory and medical imaging services. The regional referral hospitals are also involved in teaching and research. Hepatitis B screening and management services are also offered in regional referral hospitals in Uganda. Gulu and Lira regional referral hospital has 320, 420 healthcare workers respectively.

The study was conducted among adult vaccinated healthcare workers in Lira and Gulu regional referral hospitals in northern Uganda. We enrolled healthcare workers who had previously received primary Hepatitis B vaccine irrespective of when the vaccination was received.

Sample size determination

The sample size was calculated using Kish Leslie formula [15] applying a prevalence of non-immune protection of 22.9% [7], 95% level of significance and 10% non-response giving a sample size of 300 study participants.

Data collection

Interview data collection: The interview was conducted by two research assistants, a laboratory technologist and a nurse. The two research assistants were trained on the survey tool prior to conducting the interviews. Using simple random sampling, the healthcare workers in Lira and Gulu regional referral hospitals were approached for inclusion into the study. A sample frame of healthcare workers in each of the hospital was obtained from the hospital administrator. The name of each healthcare worker was then written on a separate piece of small paper which was folded and placed in a basket. With shaking of a basket at each point, one piece of paper was picked at a time without replacement until the required sample size was obtained. A healthcare worker whose name was picked was then approached for recruitment into the study. A written informed consent was obtained prior to enrollment into the study. All the healthcare workers who reported having taken a hepatitis B vaccine and consented for the study were recruited. Healthcare workers who were under hepatitis B treatment were excluded. Interview data was collected using interviewer administered questionnaires, which had been pretested on 10 healthcare workers at Mulago national referral hospital. The study tool collected data on, (i) socio-demographic characteristics, (ii) risk of exposure to HBV, (iii) HBV testing, (iv) HBV vaccine awareness, and (v) HBV vaccination (S1 Appendix).

Laboratory data collection: For each consenting healthcare worker, 4mls of venous blood was collected using ethylene diamine tetra acetic acid (EDTA) vacutainer tubes. The blood samples were immediately centrifuged (3000rpm) for five minutes and plasma separated from blood cells in to cryovials. The plasma was then screened using HBV Combo Rapid test (Vaxpert Inc Suite 355 Two South Biscaynne Blvd. Miami, FI, USA). This is a rapid test for qualitative detection of; Hepatitis B surface antigen (HBsAg) a protein on the surface of hepatitis B virus whose presence in serum is an indicator of acute or chronic hepatitis B virus infection; Hepatitis B surface antibody (HBsAb or Anti-HBs) a protein produced by the body’s immune system in response to the presence of Hepatitis B surface antigen; Hepatitis B envelop antigen (HBeAg) a viral protein made by the hepatitis B virus that is released from infected liver cells into the blood and is an indicator of active HBV replication; Hepatitis B envelop antibody (HBeAb) a protein produced by the body’s immune system in response to HBeAg a marker of resolution of illness; and Hepatitis B core antibody (HBcAb or Anti-HBc) a protein produced by the body’s immune system in response to hepatitis B virus and is an indicator of previous hepatitis B virus infection. e. The test was done following the manufacture’s guidelines. Briefly, the test cassette was removed from the sealed foil pouch and placed on a clean, leveled surface work-top in the laboratory. Holding the dropper vertically, three (3) full drops (approximately 75μl) of plasma was transferred to each sample well and the timer started. The results were read after 15 minutes. The appearance of a colored line in the control region (C) confirmed the viability of the test. For HBsAg, HBsAb and HBeAg tests, a positive result was confirmed with the presence of two distinct colored lines on the test cassette, one being the test region and the other the control region. While for HBeAb and HBcAb tests, a positive result was confirmed by presence of one colored line in the control region (C) and no colored line in the test region (T). Sample tests with no colored line in the control region (C) were repeated. In addition, known positive and negative control samples were run alongside the test samples for quality control. The plasma samples with detectable HBsAb were then transferred to Uganda Blood Bank Nakasero laboratory under 20°C, for analysis of HBsAb concentration. The COBAS Elecsys 2010 immunoassay analyzer was used in the analysis of HBsAb concentration. The analyzer was calibrated using control and sample plasma prior to the analysis. 50μl of plasma for each sample was processed in duplicate following manufacturer’s guidelines, the reaction mixture is then aspirated into the measuring cell. The HBsAb concentration was measured by comparing the electro-chem-luminescence signal obtained to that from the calibration. Hepatitis B antibody levels ≥ 10mIU/mL were considered protective [16].

Data management and analysis

At the end of each data collection day all the questionnaires were checked for completeness. Double data entry was done by two data entrants (OC and KR) into Epi-Data ver 3.1. Data was transferred to STATA ver 23 and cleaned prior to analysis. Categorical variables were analyzed using a modified Poisson regression with robust standard errors [17, 18]. Bivariate analysis was performed for each of the independent variables to determine whether they were independently associated with immune response to hepatitis B vaccine using prevalence ratios (PR) and p-values at 95% level of significance. All variables were entered and carried to the multivariate logistic regression using a backward elimination method. Confounding was assessed by comparing crude and adjusted PR, with a difference between crude and adjusted PR of greater than 10% considered as confounding.

Results

Socio-demographic characteristics of study participants

A total of 300 participants were sampled from Gulu and Lira regional referral hospital. Most participants were females (52%), aged between 26–35 years (43.3%), and were either nurses or midwives (45.5%). The main form of hepatitis B infection risk was IM/IV injection (86%). Most of the study participants (96.7%) received their last dose of hepatitis B vaccine shot between 2010 and 2020. Majority of the participants 244/300 (81.3%) completed all the three doses of hepatitis B vaccine. A high proportion (96.7%) of the study participants received Hepatitis B vaccine at 0, 1, 6 vaccine schedules (months). Majority (99%) did not receive a booster doze of hepatitis B vaccine (Table 1). Of the 300 study participants, 2.3% (7/300) had positive HBsAg; 88.7% (266/300) had positive HBsAb; 2.3% (7/300) had positive HBeAg; 4% (12/300) had positive HBeAb and 17.7% (53/300) had positive HBcAb.

Table 1

Descriptive characteristics of healthcare workers (n = 300) in Gulu and Lira regional referral hospitals, October–December 2020.

Characteristic	Description	Frequency, N = 300n (%)
Age (years)	18–25	89 (29.7)
	26–35	130 (43.3)
	36+	81 (27.0)
Sex	Male	144 (48.0)
	Female	156 (52.0)
Specialty	Doctor/Clinician/physician	17 (5.7)
	Nurse/Midwife	136 (45.5)
	Pharmacist	3 (1.0)
	Laboratory technician	70 (23.4)
	Others	73(24.5)
Risk exposure	Needle prick injury	58 (19.3)
	Blood splash	77 (25.7)
	Blood transfusion	10 (3.3)
	Injection (IM/IV)	258 (86.0)
	Conducted surgery	82 (27.3)
	Had a tattoo on the skin	3 (1.0)
	Had a surgical operation	33 (11.0)
	Others	2 (0.7)
Year hepatitis B vaccine intake	2000–2009	10 (3.33)
	2010–2020	290 (96.7)
Age at last hepatitis B vaccination	18–25	161 (53.7)
	26–35	102 (34.0)
	36+	37 (12.3)
Number of doses received	1	20 (6.7)
	2	36 (12.0)
	3	244 (81.3)
Completed hepatitis vaccine dose	Yes	244 (81.3)
	No	56 (18.7)
Time interval (months)	0, 1, 12	1 (0.4)
	0, 1, 6	236 (96.7)
	0, 2, 4	2 (0.8)
	0, 3, 6	4 (1.6)
	Do not recall	1 (0.4)
Received a booster dose	Yes	7 (2.3)
	No	293 (97.7)

Hepatitis B vaccine immune response among health workers

Of the 300 hepatitis B vaccinated healthcare workers screened, 270 (90%) had detectable anti-HBs, while 30 (10%) did not have detectable hepatitis B antibodies (HBsAb), two of whom were found to have active hepatitis B infection. Of the 270 who had detectable anti-HBs, antibody titre test was performed and the test was successful in 266 blood samples, where 93.6% (249/266, 95%CI: 89.9–96) had protective antibody titre (> 10mIU/mL) against hepatitis B virus infection.

Of the 266 participants with detectable anti-hepatitis B antibodies, 6.4% (17/266, 95%CI: 4–9.6) did not have protective (<10mIU/mL) antibody titre (non-responders), 15.4% (41/266, 95%CI: 11.5–20.3) had 10-99mIU/mL antibody titre (poor responders), 49.6% (132/266, 95%CI: 43.6–55.6) had 100-999mIU/mL antibody titre (moderate responder) and 28.6% (76/266, 95%CI: 23.4–34.3) had ≥1000mIU/mL antibody titre (strong responders). Overall, we found that the majority, 72% (216/300) of study participants had protective hepatitis B immunity (≥10mIU/mL) due to hepatitis B vaccine (HBsAg-ve, HBcAb-ve, HBsAb+ve). Furthermore, one in six vaccinated healthcare workers (51/300, 17%) had protective hepatitis B immunity (≥10mIU/mL) due to a natural infection (HBsAg-ve, HBcAb+ve, HBsAb+ve). A third of the study participants are susceptible to hepatitis B virus infection (HBsAg, and HBcAb, HBsAb all negative). Some of the participants (2/300, 0.7%) had active hepatitis B virus infection (HbsAg+ve, HbcAb+ve, HbsAb–ve).

Factors associated with hepatitis B virus immune response among healthcare workers in Gulu and Lira regional referral hospitals

In bivariate analyses, the factors that were significantly associated with protective anti-HBs levels include, year of last hepatitis B vaccine dose (p<0.001), time schedule for hepatitis B vaccine (0-1-6 months) (p<0.001), and booster hepatitis B vaccine dose (p<0.001) (Table 2).

Table 2

Factors associated with hepatitis B immune response among vaccinated healthcare workers (n = 266) in Gulu and Lira regional referral hospitals, October-December 2021.

Characteristic	Description	Immune response (≥10mIU/mL)n (%)	cPR	95% CI	aPR	95%CI	P-value
Age (years)	18–25	74 (27.8)	1		1
	26–35	110 (41.4)	1.00	0.93–1.08	0.99	0.92–1.07	0.854
	36+	65 (24.4)	0.99	0.91–1.08	0.93	0.79–1.09	0.357
Sex	Female	133 (50)	1		1
	Male	116 (43.6)	1.01	0.95–1.08	1.06	0.98–1.14	0.162
Specialty	Allied Health	25 (9.4)	1		1
	Doctors	14 (5.3)	0.91	0.74–1.11	0.86	0.66–1.13	0.277
	Nurse/Midwife	116 (43.6)	1.01	0.85–1.08	1.04	0.94–1.15	0.463
	Pharmacist	2 (0.8)	0.69	0.31–1.55	0.51	0.13–2.03	0.341
	Lab. Technician	56 (21.1)	0.94	0.84–1.05	0.96	0.85–1.08	0.500
	Others	35 (13.2)	0.96	0.85–1.08	0.99	0.88–1.13	0.992
Year vaccine intake	2000–2009	9 (3.4)	1		1
	2010–2020	240 (90.2)	0.93	0.90–0.96	0.94	0.9–0.97	<0.001
Age at vaccination	15–25	133 (50)	1		1
	26–35	85 (31.9)	0.97	0.90–1.04	1.02	0.92–1.14	0.693
	36+	31 (11.7)	1.03	0.95–1.11	1.10	0.93–1.31	0.264
Vaccine schedule (months)	0, 1,12	1 (0.4)	1		1
	0, 1, 6	202 (75.9)	0.94	0.91–0.97	1.07	1.0–1.11	<0.001
	0, 2, 4	2 (0.8)	1	1			1.000
	0, 3, 6	1 (0.4)	1	1			1.000
	Do not recall	1 (0.4)	1	1			1.000
Received booster	No	246 (92.5)	1
	Yes	3 (1.1)	1.07	1.04–1.10	1.07	1.0–1.11	<0.001

Majority of the participants, 50% (133/266) who were 15–25 years at the time of vaccination had sufficient immune response (≥10mIU/mL).

After adjusting for other covariates in multivariable analysis, there was no predictor of participants having protective anti-HBs titre (>10mIU/mL). There was no significant difference in the odds of having protective anti-HBs titre (>10mIU/mL) in participants who received the vaccine between 2000–2009 and those who received the vaccine between 2010–2020 (aPR = 0.94, p<0.001). The odds of having protective anti-HBs titre (>10mIU/mL) did not significantly differ by vaccine schedule (aPR = 1.07, p<0.001) and receiving of booster dose (aPR = 1.07, p<0.001) (Table 2).

Discussion

In this study we found a fairly high hepatitis B vaccine three-dose completion rate (81.3%) among healthcare workers in northern Uganda. This was an improvement when compared to a previous report of 57.8% among healthcare workers in central Uganda and another report from other LMICs with completion rates of 40–90% (16–19) [13]. The high Hepatitis B vaccine completion rate found in this study could be due to the higher prevalence of Hepatitis B virus infection in northern Uganda compared to the rest of the country [12]. Whereas there is an increase in public awareness through promotion of hepatitis B vaccine and the requirement by the ministry of health for all healthcare workers to receive hepatitis B vaccine prior to engagement in clinical work including all health professional students [9], our findings indicate that the vaccination rate still falls short of the 100% completion among healthcare workers as recommended by the WHO [19]. The likelihood of spread of hepatitis B virus infection between healthcare workers and their patients become more apparent especially given the unknown hepatitis B vaccine coverage in the general population in Uganda. Of concern was the increased risk of transmission where we found that the majority of the healthcare workers got intramuscular/intravenous injections, or conducted surgical procedures and accidentally had blood splashed on their bodies during those procedures, further emphasizing the need for completion of hepatitis B vaccination doses.

On qualitative analysis, we found a high proportion of healthcare workers with detectable anti-HBs indicative of either recovery from hepatitis B infection or immunity secondary to hepatitis B vaccination. After quantification of the anti-HBs, we found over 90% of the healthcare workers with detectable anti-HBs had protective antibody concentration (≥ 10mIU/mL). These findings are similar to reports from previous studies [20] that reported presence of protective immune response among individuals vaccinated with hepatitis B vaccine. The high proportion of healthcare workers with detectable anti-HBs is a confirmation of Hepatitis B vaccination among the study participants. In addition, protective anti-HBs titers found in majority (72%) of the study participants is an indicator of effective immune response to the hepatitis B vaccine.

The findings that healthcare workers who received hepatitis B vaccine 20 years back as adults still had protective anti-HBs levels is an indicator of long-term protection against hepatitis B virus infection even though the majority never received a booster vaccine dose. While the Uganda Ministry of Health does not conduct periodic screening of healthcare workers to assess for immune protection, our findings in this study are in line with evidence from previous studies [20, 21] that reported more 30 years of immune protection from hepatitis B vaccine among individuals vaccinated as children and young adults. Our findings show that regular screening to assess extent of hepatitis B immune protection among individuals who have completed all the three hepatitis B vaccine dose schedules may not be necessary due to the limited resources in most LMICs. However, for the few individuals who had weak or no protective immune response (<10mIU/mL) against HBV following complete vaccination, provision of a challenge dose of the vaccine is recommended as this has previously been shown to prompt development of immunity[22].

While only three of the healthcare workers had received a booster dose of the hepatitis B vaccine, we found no significant difference in the presence of protective immune response between individuals who received a booster dose and those who did not. A similar finding was reported in a previous study by Bruce et al. [20] which found development of protective immune response (≥10mIU/mL) among fully vaccinated individuals who did not receive a booster hepatitis B vaccine dose. In our study we included individuals who reported to have received hepatitis B vaccines in the past 20 years. Although there is currently no screening to establish immune response to hepatitis B vaccine in Uganda, it is likely that despite the booster dose of the vaccine, vaccinated individuals already had sufficiently high levels of anti-HBs in the body. This could be due to the persistence of anti-hepatitis B antibodies in vaccinated individuals [22]. Our findings indicate that completion of all three hepatitis B vaccine doses is sufficient for the development of protective anti-HBs titers.

A low proportion (2.3%) of healthcare workers in this study had detectable HBsAg an indicator of acute or chronic hepatitis B virus infection, while 1 in every 6 healthcare workers had a positive HBcAb indicative of previous or ongoing hepatitis B virus infection, possibly due to non-response to the hepatitis B vaccine as all study participants had been vaccinated. This is supported by the findings that a low proportion of hepatitis B virus infection occurred among healthcare workers who had undetectable anti-HBs on qualitative screening. The lack of protective anti-HBs titre following vaccination found in our study is similar to findings from a previous study [7].

While our study had some limitations where the HBV Combo Rapid test was used for qualitative screening of HBsAg, HBsAb, HBeAg, HBeAb and HBcAb, we were able to transfer samples with positive HBsAb to the national blood bank laboratory (Nakasero blood bank) for quantitative measurement of the HBsAb levels in the plasma samples. Additionally, we also referred participants with positive HBsAg for further tests to confirm Hepatitis B viral infection. It was also not possible to ascertain from the study participants what kind of hepatitis B vaccine that they had received, whether plasma derived or recombinant vaccine. However, results from previous studies have shown that antibody response and the effectiveness of the plasma-derived vaccine are similar to that of recombinant hepatitis B vaccine [19, 23].

Conclusion and recommendation

Most vaccinated healthcare workers in northern Uganda developed protective anti-HBs levels and the protective immune response persisted for over ten years following vaccination. Some vaccinated healthcare had weak/no immunity and developed breakthrough hepatitis B virus infection which would require booster hepatitis B vaccination. There may be need for post-vaccination testing to assess immunological priming from hepatitis B vaccine among healthcare workers.

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Reviewer #1: The paper is well written and is very important in the field of Hep B prevention and control.

I would combine tables 3 and 4 together so one can compare both crude and adjusted effects easitly.

I was also keen to see the explanation of what the recent vaccnations 2010-2020 are least likely to have protective anti-HB titres. One would think that recent vaccination might be associated with protection as opposed those vaccinated a long time ago.

Could it be the type of vaccine used is diffrent, or what was picked up as high titres for older vaccination would be a boost from natural infection.

Reviewer #2: Firstly, I would like to congratulate the authors for addressing such an important public health topic with a relevant methodology. Generally, the paper is well written and structured. However, further efforts are required to make the study more attractive to the readers. I have provided and raised numerous comments and questions on the manuscript that has to be addressed by the authors. Authors can get my comments and questions from my attachment.

Reviewer #3: General comments: I would like to thank the authors for this wonderful article. It addresses a public health concern that is to some extent ignored in many low resource settings. The article also builds on previous work undertaken in Uganda. The paper is well-written. Nonetheless, there are a few issues that the authors need to address to make the paper more scientifically informative.

1) Methods: Provide sufficient detail on the study setting. Where are the study facilities located? How are the staffing levels, cadres and services provided? Do the healthcare facilities provide Hepatitis B screening and management services?

2) Methods: Why was Mulago national referral hospital chosen for the pre-test? Is Mulago hospital and the two study healthcare facilities comparable?

3) A section on quality assurance and control for both serological testing and interviews with health workers is missing. Were the research assistants trained?

4) Discussion: Generally, the authors need to strengthen the discussion. Whereas they have attempted to provide implications for the findings, there is no explanation for most of the findings. For example, what explains the high proportion of healthcare workers with detectable anti-HBs?

5) Discussion: The authors point out that there was an improvement in the hepatitis B vaccination rate compared to a previous study by Ssekamatte et al. Both studies are recent and were conducted in Uganda. What explains the improvement? Are there any contextual differences between healthcare facilities in Wakiso and Lira and Gulu hospitals? What explains the high vaccine completion rates in Gulu and Lira referral hospitals? Remember that regional referral hospitals also have treatment centres for Hepatitis B.

6) Discussion: The authors mentioned that ‘’ While only three of the healthcare workers had received a booster dose of the hepatitis B vaccine, we found no significant difference in the presence of protective immune response between individuals who received a booster dose and those who did not’’. However, the authors do not provide an explanation to the findings. Why is it so?

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Reviewer #2: Yes: Tefera Alemu

Reviewer #3: No

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Submitted filename: Comment to the authors.docx

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Submitted filename: Comments to authors_HBV in northern Uganda.docx

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28 Sep 2021

RESPONSE TO REVIEWERS COMMENTS ON MANUSCRIPT PONE-D-21-17608

Editor’s comment: Thank you for stating in the text of your manuscript "A written informed consent was obtained prior to enrollment into the study. ". Please also add this information to your ethics statement in the online submission form.

Response: We are grateful for the comment, the statement "A written informed consent was obtained prior to enrollment into the study” has been added to the ethics statement in the online submission form.

Ethical considerations

Ethical review and approval of the protocol was done by the Makerere University School of Biomedical Science Research and Ethic Review Committee (#SBS-REC 798). Additionally, administrative clearance was obtained from the hospitals prior to study initiation. A written informed consent was obtained from potential study participants prior to enrollment into the study.

Editor’s comment: Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed the survey or questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. If the questionnaire is published, please provide a citation to the (1) questionnaire and/or (2) original publication associated with the questionnaire.

Response: Thanks for the comment, the questionnaire used for data collection in the study has been added as supplementary material in the revised manuscript (Additional file_1).

Editor’s comment: We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

Response: The details of the grant has been provided. However, I am not the primary recipient of the grant which provided financial support for this study.

Editor’s comment: Please amend the manuscript submission data (via Edit Submission) to include author David Katete.

Response: Thanks for the observation, David Katete has been added as an author in the manuscript submission system.

Comments Reviewer #1: The paper is well written and is very important in the field of Hep B prevention and control.

I would combine tables 3 and 4 together so one can compare both crude and adjusted effects easitly.

Response: Thanks for the comment, tables 3 and 4 have been combined to enable easy comparison of crude and adjusted odds ratios in the revised manuscript

Comments Reviewer #1: I was also keen to see the explanation of what the recent vaccnations 2010-2020 are least likely to have protective anti-HB titres. One would think that recent vaccination might be associated with protection as opposed those vaccinated a long time ago.

Could it be the type of vaccine used is diffrent, or what was picked up as high titres for older vaccination would be a boost from natural infection.

Response: We agree with the concern raised by the reviewer, the adjusted odds ratio of 0.94 is close to 1.0 and therefore the interpretation of this result was incorrect. We have adjusted the interpretation in the revised manuscript, this is now interpreted as no significant difference in the odds of immune protection between individuals who received the hepatitis vaccine between 2000-2009 and 2010-2020.

Comments Reviewer #2: Firstly, I would like to congratulate the authors for addressing such an important public health topic with a relevant methodology. Generally, the paper is well written and structured. However, further efforts are required to make the study more attractive to the readers. I have provided and raised numerous comments and questions on the manuscript that has to be addressed by the authors. Authors can get my comments and questions from my attachment.

Response: Thanks for the compliment, the comments have been addressed in the revised manuscript

Comments Reviewer #2: In my opinion, it is better if you modify your title to make it easily understandable, attention grasping and in line with your study objectives.

Response: Thanks for the comment, the title of the manuscript has been adjusted in the revised manuscript. The current title is “Antibody levels and protection after Hepatitis B vaccine in adult vaccinated healthcare workers in northern Uganda”. This has been incorporated in the revised manuscript.

Comments Reviewer #2: Your sample size should be mentioned in the abstract section in between line number 28 and 29.

Response: Thanks for the comment, the sample size of 300 healthcare workers has been mentioned in the abstract of the revised manuscript.

Comments Reviewer #2: You put too many information under abstract section. Results that answer your objectives are enough.

Response: Thanks, some of the information has been removed from the abstract of the revised manuscript.

Comments Reviewer #2, Introduction section: You stated that “However, the vaccine may not provide protection from exposure to hepatitis B virus later on in life due to waning of immune memory over time (3).” This idea needs a strong scientific evidence and it should be cited with additional and credible source to convince the readers.

Response: Additional references have been provided in the revised manuscript

1. Cocchio S, Baldo V, Volpin A, Fonzo M, Floreani A, Furlan P, et al. Persistence of Anti-Hbs after up to 30 Years in Health Care Workers Vaccinated against Hepatitis B Virus. Vaccines. 2021; 9:323.

2. Bruce MG, Bruden D, Hurlburt D, Zanis C, Thompson G, Rea L, et al. Antibody Levels and Protection After Hepatitis B Vaccine: Results of a 30-Year Follow-up Study and Response to a Booster Dose. The Journal of Infectious Diseases. 2016; 214:16-22.

Comments Reviewer #2, Methods and Materials: The tittle in line number 105 should be “Method and Materials”, instead of method.

Response: Thanks, the title “Methods and Materials” has been incorporated in the revised manuscript

Comments Reviewer #2, Methods and Materials: Your sampling technique is not stated

Response: Simple random sampling was used and is stated under the section on data collection. A sample frame of healthcare workers in the hospital was obtained from the hospital administrator. The name of each healthcare worker was then written on a separate piece of small paper which was folded and placed in a basket. With shaking of a basket at each point, one piece of paper was picked at a time without replacement until the required sample size was obtained.

Comments Reviewer #2, Methods and Materials: No exclusion or inclusion criteria?

Response: Thanks for the comment, we included all healthcare workers who reported having taken a hepatitis B vaccine. Healthcare workers who were under hepatitis B treatment were not included in the study.

Comments Reviewer #2, Methods and Materials: The following terms should be defined under method section. Otherwise, it will be difficult to understand the results.

Hepatitis B surface antigen (HBsAg), Hepatitis B surface antibody (HBsAb), Hepatitis B envelop antigen (HBeAg), Hepatitis B envelop antibody (HBeAb), and Hepatitis B core antibody (HBcAb).”

Response:

Hepatitis B surface antigen (HBsAg)- a protein on the surface of hepatitis B virus; it can be detected in high levels in serum during acute or chronic hepatitis B virus infection. The presence of HBsAg indicates that the person is infectious

Hepatitis B surface antibody (HBsAb or Anti-HBs)- a protein produced by the body’s immune system in response to the presence of the surface protein of hepatitis B virus (HBsAg)

Hepatitis B envelop antigen (HBeAg)- This is a viral protein made by the hepatitis B virus that is released from infected liver cells into the blood and is an indicator of active HBV replication.

Hepatitis B envelop antibody (HBeAb)- a protein produced by the body’s immune system in response to HBeAg. The development of HBeAb during acute Hepatitis B infection is a marker of resolution of illness

Hepatitis B core antibody (HBcAb or Anti-HBc)- a protein produced by the body’s immune system in response to hepatitis B virus. This antibody does not provide any protection or immunity against the hepatitis B virus. A positive or "reactive" test indicates that a person may have been infected with the hepatitis B virus at some point in time.

Comments Reviewer #2, Methods and Materials: Line number 148 “Hepatitis B antibody levels ≥ 10mIU/mL were considered protective.” The recommendation for this cut off value should be cited.

Response: A reference for the protective Hepatitis B antibody levels has been provided in the revised manuscript.

Jack AD, Hall AJ, Maine N, Mendy M, Whittle HC (1999): What Level of Hepatitis B Antibody Is Protective? The Journal of Infectious Diseases; 179:489–92

Poovorawan Y, Chongsrisawat V, Theamboonlers A, Leroux-Roels G, Kuriyakose S, Leyssen M, et al. Evidence of protection against clinical and chronic hepatitis B infection 20 years after infant vaccination in a high endemicity region. J Viral Hepat. 2011; 18:369-75.

Comments Reviewer #2, Results:

�  Which one is your research objective? The first or the second sentence? Is immune development status due to vaccination and natural infection the same?

“Where 93.6% (249/266, 95%CI: 89.9-96) had protective immune response (> 10mIU/mL) against hepatitis B virus.”

“Overall, we found that the majority, 72% (216/300) of the study participants had protective hepatitis B immunity 192 (≥10mIU/mL) due to hepatitis B vaccine.”

Response: The extent of development of protective immune response from natural infection is not the same as that from vaccination. In our study, 93.6% (249/266) of the study participants developed protective immune response (> 10mIU/mL) from both natural infection and vaccination. Of the individuals with protective immune response (> 10mIU/mL), 72% (216/300) was due to hepatitis B vaccine. Therefore, the second statement “Overall, we found that the majority, 72% (216/300) of the study participants had protective hepatitis B immunity 192 (≥10mIU/mL) due to hepatitis B vaccine” is the main objective of our study. This has been clarified in the revised manuscript.

Comments Reviewer #2, Results: In line number 192and 193 you said that “due to hepatitis B vaccine” and “due to a natural infection”. These should be defined under definition of terms of the methodology part and readers can understood easily it meaning.

Response: ‘due to natural infection’- The Hepatitis B antibodies produced by the body as a result of hepatitis B virus infection.

‘due to hepatitis B vaccine’- The Hepatitis B antibodies produced by the body as a result of Hepatitis B vaccine.

This has been incorporated in the revised manuscript

Comments Reviewer #2, Results: You have duplication of ideas in between line number 184 up to 187.

Response: This has been adjusted in the revised manuscript

Comments Reviewer #2, Results: All the points in table 2 are described under the paragraphs above. So, what is the importance of this table? I think it is duplications of efforts and the interpretations in table 2 are ideas to be mentioned under definition of terms/operational definition. You can simply remove the last column (frequency) and put it under method section as operational definition or you can modify it.

Response: Thanks for the comment, we agree the information in table 2 has been incorporated and provided in a descriptive form in the revised manuscript.

Comments Reviewer #2, Results: In line number 206 and 207, you stated “Factors associated with hepatitis B virus immune response among 207 healthcare workers in Gulu and Lira Regional Referral hospitals”. It is too long and seems table tittle.

Results: Thanks for the comment, this is a section header and has been adjusted in the revised manuscript

Comments Reviewer #2, Results: There is no need to prepare different tables for bivariate and multivariate regressions models. Please merge table 3 and 4.

Response: Thanks for the comment, table 3 and 4 have been merged into one in the revised manuscript

Comments Reviewer #2, Results: In multivariate analysis, your two-by-two table did not fulfill the Chi-square assumption for regression. There are empty cell and your CI also includes 1, but your p value is less than 5%. How can it be possible? I don’t think so that you have a significant predictor.

Response: In our analysis, we used modified poisson regression which does not set to full fil the chi-square assumption as required in chi-square test. Using poisson regression helps take care of the small and empty cells. In addition to addressing convergence when all the observations are small or empty cells, poisson regression is also useful when the prevalence of the outcome of interest is high.

1. HUANG, F. L. 2019. Alternatives to logistic regression models in experimental studies. The Journal of Experimental Education, 1-16.

2. ZOU, G. 2004. A modified poisson regression approach to prospective studies with binary data. Am J Epidemiol, 159, 702-6.

Comments Reviewer #2, Results: Your statement under table 4 is difficult to understand, for instance “were 7% times” and the likes. The paragraph from line number 119 up to 229 are also not easily understandable.

Response: This has been adjusted in the revised manuscript

Comments Reviewer #2, Results: Be consistent throughout. Example, for your dependent variable, use either “protective anti-HBs levels” or “hepatitis B immune response” throughout.

Response: Thanks for the comment, we have adhered to using ‘protective anti-HBs levels’ throughout the revised manuscript

Comments Reviewer #2, Recommendation: In your recommendation, you did not talk about the factors of protective anti-HBs levels. Is that not your study objective from the outset or not?

Response: This has been adjusted in the revised manuscript

Comments Reviewer #2: Grammar and punctuation errors are her and there in the paper and sometimes your idea is difficult to understand and not coherent. Please get assistance from someone who is fluent in English language and/or manuscript editing software’s.

Results: Thanks for the comment, this has been adjusted in the revised manuscript

Comments Reviewer #3: General comments: I would like to thank the authors for this wonderful article. It addresses a public health concern that is to some extent ignored in many low resource settings. The article also builds on previous work undertaken in Uganda. The paper is well-written. Nonetheless, there are a few issues that the authors need to address to make the paper more scientifically informative.

Response: Thanks for the compliment

Comments Reviewer #3, Methods: Provide sufficient detail on the study setting. Where are the study facilities located? How are the staffing levels, cadres and services provided? Do the healthcare facilities provide Hepatitis B screening and management services?

Response: The study was conducted in Lira and Gulu regional referral hospitals. In Uganda, regional referral hospitals offer specialist clinical services such as psychiatry, Ear, Nose and Throat (ENT), ophthalmology, higher level surgical and medical services, and ancillary services (laboratory, medical imaging and pathology). They also provide general healthcare services including, preventive, promotive, curative, maternity, in-patient health services, surgery, blood transfusion, laboratory and medical imaging services. The regional referral hospitals are also involved in teaching and research. Hepatitis B screening and management services are also offered in regional referral hospitals in Uganda.

Government of Uganda. Health sector strategic and investment plan- Promoting people’s health to enhance socio-economic development 2010/11-2014/15. Kampala: Ministry of Health; 2010.

Comments Reviewer #3, Methods: Why was Mulago national referral hospital chosen for the pre-test? Is Mulago hospital and the two study healthcare facilities comparable?

Response: Mulago hospital is a national referral hospital and is not comparable to the regional referral hospitals in terms of the extent and level of services offered. However, both Mulago national referral hospital and the two study healthcare facilities provide Hepatitis B screening, treatment and vaccination services.

Comments Reviewer #3, Methods: A section on quality assurance and control for both serological testing and interviews with health workers is missing. Were the research assistants trained?

Response: The interview was conducted by two research assistants, a laboratory technologist and a nurse. The two research assistants were trained on the survey tool prior to conducting the interviews.

The description of quality control measures for both the interviews and laboratory experiments have been incorporated in the revised manuscript.

Comments Reviewer #3, Discussion: Generally, the authors need to strengthen the discussion. Whereas they have attempted to provide implications for the findings, there is no explanation for most of the findings. For example, what explains the high proportion of healthcare workers with detectable anti-HBs?

Response: The high proportion of healthcare workers with detectable anti-HBs is a confirmation of Hepatitis B vaccination among the study participants. In addition, protective anti-HBs titers were found in majority (72%) of the study participants an indicator of effective immune response to the hepatitis B vaccine.

This has been incorporated in the discussion section of the revised manuscript.

Comments Reviewer #3, Discussion: The authors point out that there was an improvement in the hepatitis B vaccination rate compared to a previous study by Ssekamatte et al. Both studies are recent and were conducted in Uganda. What explains the improvement? Are there any contextual differences between healthcare facilities in Wakiso and Lira and Gulu hospitals? What explains the high vaccine completion rates in Gulu and Lira referral hospitals? Remember that regional referral hospitals also have treatment centres for Hepatitis B.

Response: Thanks, we agree with the comment. Contextual factors may explain the higher vaccine completion rate in Lira and Gulu compared to Wakiso. For example, northern Uganda has the highest prevalence of hepatitis B virus infection in the country and this is likely to have driven more healthcare workers in the region to take up the hepatitis B vaccine compared to the rest of the country.

This has been incorporated in the revised manuscript

Comments Reviewer #3, Discussion: The authors mentioned that ‘’ While only three of the healthcare workers had received a booster dose of the hepatitis B vaccine, we found no significant difference in the presence of protective immune response between individuals who received a booster dose and those who did not’’. However, the authors do not provide an explanation to the findings. Why is it so?

Response: A recent study by Cocchio et al., 2021 showed that Hepatitis B antibodies persist in vaccinated healthcare workers for up to 30 years. In our study we included individuals who reported to had received hepatitis B vaccines in the past 20 years (2000 – 2020) it is thus likely that despite the booster dose of the vaccine, vaccinated individuals still had sufficiently high levels of anti-HBs in the body.

This has been incorporated in the revised manuscript.

Comments Reviewer #3: Details of the ethics approval are lacking. Kindly provide information on the ethics clearance for the current study.

Response: Ethical review and approval of the protocol was done by the Makerere University School of Biomedical Science Research and Ethic Review Committee (#SBS-REC 798). Additionally, administrative clearance was obtained from the hospitals prior to study initiation. A written informed consent was obtained from potential study participants prior to enrollment into the study.

This has been incorporated in the adjusted manuscript.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

17 Dec 2021

Antibody levels and protection after Hepatitis B vaccine in adult vaccinated healthcare workers in northern Uganda

PONE-D-21-17608R1

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Reviewer #1: Yes: Michelo Simuyandi

23 Dec 2021

PONE-D-21-17608R1

Antibody levels and protection after Hepatitis B vaccine in adult vaccinated healthcare workers in northern Uganda

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