Serological Status of Vaccine and Hepatitis B Virus Exposure Among Children Under 5 and Aged 15–17 Years in Kampala, Uganda

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

 

The study is interesting but leaves a certain sense of incompleteness. I understand that conducting research in Uganda can be challenging, and I appreciate your effort in this study. Vaccination is a very effective preventive method, and HBV immunization was introduced several decades ago, with many publications confirming its effectiveness and the lack of need for additional doses. Starting with the study group, did you obtain any information regarding HIV infection? Congenital HIV affects vaccine efficacy, and Uganda, despite significant progress in the fight against HIV, still (according to WHO) remains a country where infection among women is prevalent. Did you include a question about HIV in the pre-study survey?

 

Additionally, I need more information about the conditions under which primary vaccinations are administered. Are they conducted in hospitals at birth or under the care of a family doctor? Furthermore, I am still determining the results you presented. The simultaneous presence of HBsAg and HBsAb may indicate low specificity of the tests. Similarly, regarding the conclusions, there is ongoing debate about the necessity of booster doses. More information about antibody clearance is also needed. Moreover, the Ugandan population is similar regarding immunological competence to countries where these vaccinations were introduced in the 1990s. The key to understanding the low seroprevalence is the specifics of vaccine administration.

 

Please elaborate on the limitations of the study you presented. A prospective study could provide a comprehensive view.

Author Response

Comment 1: Starting with the study group, did you obtain any information regarding HIV infection?

Response 1: Yes, we did test all participants for HIV using the Uganda National HIV RDT algorithm. However, HIV was an exclusion criteria. We have revised section 2.2 of the manuscript that deals with Inclusion and exclusion criteria for clarity.

Comment 2: Congenital HIV affects vaccine efficacy, and Uganda, despite significant progress in the fight against HIV, still (according to WHO) remains a country where infection among women is prevalent. Did you include a question about HIV in the pre-study survey?

Response 2: We thank the reviewer for pointing this out. Indeed it is true congenital HIV exposure does affect the babies immune maturation and responses to some infectious agents. We acknowledge this as a limitation (See Section "6. Limitations" of the revised manuscript) as we should have consented, enrolled and administered a socio-behavioural questionnaire tool to mothers as well, especially for the 0-5 year age-group.

Comment 3: Additionally, I need more information about the conditions under which primary vaccinations are administered. Are they conducted in hospitals at birth or under the care of a family doctor?

Response 3: The notion of family doctor in Uganda does not really exist, except for the affluent and emerging middle class. Instead, Immunization of children is done at facilities that deliver babies and at all levels of the public health facilities; daily at all facilities at hospital, health center IV and health center III, and only on scheduled immunization days at health center II (the lowest level of health facility). Private-not-for profit (mostly religious-based) and private for profit hospitals also are accredited by the Uganda National Expanded Program on Immunization (UNEPI) as part of their licensure as a hospital. Other private practitioners are accredited by UNEPI on a case-by-case basis to offer immunization based on establishment of facilities to support the service. We have added a sentence in line 62-64 of the revised manuscript to clarify this.

Comment 4: The simultaneous presence of HBsAg and HBsAb may indicate low specificity of the tests.

Response 4: We thank the reviewer for this comment. Indeed, we had similar worries of sub-optimal performance of HBV Combo RDT kits in our settings. Other than worries of stable availability of these test kits on the Ugandan market, it was the basis for concurrent use of 3 different Combo RDT kits in this study. Under section 3.2 (Lines 201-206) we show that all 3 different HBV Combo RDT kits were perfectly concordant in all the parameters measured, hence, we strongly believe that this simultaneous detection of HBsAg and anti-HBsAb was real and not a matter of specificity of the tests.

Comment 5: More information about antibody clearance is also needed. Moreover, the Ugandan population is similar regarding immunological competence to countries where these vaccinations were introduced in the 1990s. The key to understanding the low seroprevalence is the specifics of vaccine administration.

Response 5: Our group has consistently studied HBV and shows very worrying statistics in prevalence, incidence and history of exposure in Uganda (for example see Kitandwe et al., 2021 [doi: 10.1186/s12889-021-10428-1]. In this study, we show incidence of up to 10.5 cases/100 person years and 40% of participants aged 13-49 years having evidence of prior hepatitis B virus infection. Under these circumstances, the HBV vaccine should work as intended but UNEPI has not evaluated the effectiveness of the vaccine in its program since including it among the childhood immunizable diseases. Less than a handful of African countries (e.g Mauritius) have evaluated their HBV immunization program. In Muwanda et al., 2023 (doi: 10.1038/s41598-023-49674-1), we demonstrated the existence of very low prevalences of protective anti-HBs among African adolescents 15-17 years of age, an inaugural age-group following integration of HBV vaccines in EPIs.

While we agree with the reviewer that our population is similar in many ways regarding immunological competence to countries where these vaccinations were introduced in the 1990s, we also want to tell you that our group and others are starting to describe significant differences unique to Uganda (and a few other settings) that impede elicitation of adequate and quality immune responses not only to vaccines, but also other infectious diseases (See Muir et al., 2023 [doi: 10.1101/2023.02.24.23284435], Muyanja et al., 2014 doi: 10.1172/JCI77956, Elliot et al., 2010 doi: 10.1016/j.vaccine.2010.10.047, Kizito et al., 2013 doi: 10.1186/1471-2458-13-619). These differences call for a discussion on the doses (number, booster, antigen concentration) and we hope cumulative research including this manuscript can leads us there.

Comment 6: Please elaborate on the limitations of the study you presented. A prospective study could provide a comprehensive view.

Response 6: We have updated Section 6: Limitations We appreciate the proposal for a need for a prospective study. As you know cohort study designs are very expensive and appetite for their funding is very low currently. Triggered by your comment, we have started engaging a contemporary research group at the Uganda Virus Research Institute, Entebbe, who have a well documented and characterized Entebbe Mother and Baby Study (ISRCTN32849447) cohort. We have co-authored several papers with them and therefore believe use of this cohort's archived samples may be a cheaper alternative.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Your study is very important because in spite of having hepatitis B vaccines for many years a large percentage of patients and caregivers are not protected. You have recommended Anti-HBs titer determinations as well as some specific Hepatitis B Combo test kits. Are you utilizing these kits now?

Author Response

Comment 1: We thank the reviewer for appreciating the importance of our study.

Uganda and many low-income settings have very low access to anti-HBs titering testing, mostly due to prohibitive costs for the testing and establishment of the required laboratory infrastructure. Anti-HBs titering is already recommended and indeed done in assessing vaccine immunogenicity among people living with HIV, among HBV vaccine recipients for occupational health programs and if required by a clinician. In light of low proportions of children with detectable vaccine-specific anti-HBs titers seen in this and other studies, periodic anti-HBs titering even among EPI vaccination programs may be required to monitor effectiveness of the program. So, we are engaging policy makers to utilize these Hepatitis B Combo test kits as surrogates for titering in Uganda but we are conducting more research on the cut-offs and grey zones for determination of measurement uncertainty. So, we are not using Hepatitis B Combo test kits in this regard yet, but are being used in clinical diagnostics in our set up, and many medical laboratories in Uganda.

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, the authors examine the seroprevalence of HBV vaccination and infection among children under 5 and adolescents aged 15-17 years in Kawempe Division, Kampala, Uganda. They enrolled 501 participants from the younger group and 288 from the older group between May and August 2023. Using three HBV serologic rapid diagnostic tests, the authors assessed and compared the seroprevalence of five different HBV seromarkers in both age groups. Their findings indicate low levels of anti-HBs in both groups, particularly among adolescents aged 15-17 years, suggesting a need for a booster dose of the hepatitis B vaccine. Overall, the study is well-designed and the manuscript is well-written. However, some improvements in presentation are needed before publication. Here are a few suggestions for the authors to consider:

 

Specific points:

1.     Lines 91-92: It appears that "qualitative" should be referred to as "confirmatory" and "quantitative" as "screening." Please verify this terminology.

2.     Lines 171 and 185: Supplementary Table 2 should be placed after Supplementary Table 1 in the main text.

3.     Lines 219 and 241: Similarly, Figure 1C should follow Figure 1B in the sequence.

4.     Please enlarge the text in the figures for improved readability.

5.     Why was statistical analysis not performed to compare HBV infection status between children aged 15-17 and those aged 0-5 years?

6.     Lines 309-313: Did the authors attempt to amplify viral RNA from the HBV-positive specimens? If so, this should be mentioned in the Limitations section.

7.     Lines 370-374: These statements are not associated with the Conclusions of your study. Please consider removing them.

8.     Since "hepatitis B virus" has been abbreviated as "HBV" in line 55, please use "HBV" consistently throughout the main text.

 

Minor points:

1.     I think the Abstract section exceeds the 150 word limit. Consider omitting the sentence in lines 31-32.

2.     Line 40: Change “hepatitis B virus” to “HBV”

3.     Line 78: Define “UNEPI”

4.     Reference 19 is incomplete as it lacks the the journal name and page numbers

5.     Line 257: Change “hepatitis B virus” to “HBV”

6.     Line 281: Change “hepatitis B virus” to “HBV”

Comments on the Quality of English Language

Minor editing of English is recommended.

Author Response

Specific points:

Comment 1: Lines 91-92: It appears that "qualitative" should be referred to as "confirmatory" and "quantitative" as "screening." Please verify this terminology.

Response 1: We thank the reviewer for this observation. We accept that our use of the "screening" and "confirmatory" terminologies is confusing to the readership as diagnostic policies may vary according to country-specific hepatitis B virus diagnostic guidelines. In Uganda for instance, screening is by serological ELISA or RDT-based testing for presence of HBsAg in samples and the recommended confirmatory testing is by using a competitive ELISA serology method. In other countries, PCR-based demonstration of HBV genetic material in samples is acceptable as confirmatory. In light of country-specific variations in diagnostic guidelines, we have deleted the terminologies in the manuscript and adjusted the sentence for grammatical correctness (revised manuscript lines 93-94).

Comment 2: Lines 171 and 185: Supplementary Table 2 should be placed after Supplementary Table 1 in the main text.

Response 2: We thank the reviewer for seeing this discrepancy in our chronology. We have corrected this accordingly (see section 2.7 line 170-171) in the manuscript.

Comment 3: Lines 219 and 241: Similarly, Figure 1C should follow Figure 1B in the sequence.

Response 3: We agree with the reviewer on this chronological error. We have found that that maintenance of the chronology as recommended required merging sections 3.5 into section 3.4, and that is what we have done. In the tracked manuscript attached, this change runs from lines 220-251. The merger required a re-write of several sentences to maintain grammar, reflecting the extensive edits in the section indicated.

Comment 4: Please enlarge the text in the figures for improved readability.

Response 4: Thank you for this comment. We have made a significant improvement in the fonts on the Figure 1, and improved its clarity to 300 dpi quality. The old image has been replaced by the new improved image as figure 1 in the manuscript.

Comment 5: Why was statistical analysis not performed to compare HBV infection status between children aged 15-17 and those aged 0-5 years?

Response 5: We have compared the prevalence of HBV infection in the two groups and found no significant difference (Lines 229-230).

 

Comment 6: Lines 309-313: Did the authors attempt to amplify viral RNA from the HBV-positive specimens? If so, this should be mentioned in the Limitations section.

Response 6: No attempt to do viral RNA PCR was done. Instead, all the 12 serologically positive participants were referred to a public health center where hepatitis B virus infection services are provided free of charge.

Comment 7: Lines 370-374: These statements are not associated with the Conclusions of your study. Please consider removing them.

Response 7: We agree with the reviewer that the particular statements referenced in the conclusion were not supported with solid results. So, we have deleted them from the manuscript (Section 5, lines 375-378)

Comment 8: Since "hepatitis B virus" has been abbreviated as "HBV" in line 55, please use "HBV" consistently throughout the main text.

Response 8: We have harmonized the use of "HBV" in place of hepatitis B virus, wherever it is mentioned, except where it is in reference to "hepatitis" as a disease or in reference to the hepatitis B vaccine.

Minor points:

Comment 1: I think the Abstract section exceeds the 150 word limit. Consider omitting the sentence in lines 31-32.

Response 1: We thank  the reviewer for being this detailed. We have taken your advice and deleted the suggested sentence, and deleted some more words without changing the message being communicated. We have reduced the abstract to 244 words in the current version.

Comment 2: Line 40: Change “hepatitis B virus” to “HBV”

Response 2: This has been done, thank you.

Comment 3: Line 78: Define “UNEPI”

Response 3: UNEPI had already been defined in Line 37-38 of this revised manuscript attached

Comment 4: Reference 19 is incomplete as it lacks the the journal name and page numbers

Response 4: Thank you for this observation. We have corrected the reference as recommended in Pubmed

Comment 5: Line 257: Change “hepatitis B virus” to “HBV”

Response 5: This has been done

Comment 6: Line 281: Change “hepatitis B virus” to “HBV”

Response 6: This has also been done

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have properly addressed my comments in their revised manuscript. I don't have any further critical inquiries.