Gates Open Res Gates Open Research 2572-4754 F1000 Research Limited London, UK 10.12688/gatesopenres.14287.2 Study Protocol Articles A hybrid effectiveness-implementation study protocol to assess the effectiveness and chemoprevention efficacy of implementing seasonal malaria chemoprevention in five districts in Karamoja region, Uganda

[version 2; peer review: 2 approved, 1 approved with reservations]

 Kajubi Richard Investigation Supervision Writing – Review & Editing https://orcid.org/0000-0002-9179-9182 1 Ainsworth Jennifer Data Curation Supervision Writing – Original Draft Preparation 2 Baker Kevin Conceptualization Funding Acquisition Investigation Methodology Resources Supervision Visualization Writing – Review & Editing https://orcid.org/0000-0002-2040-3662 a 2 3 Richardson Sol Conceptualization Data Curation Methodology Validation Writing – Review & Editing 2 Bonnington Craig Conceptualization Investigation Methodology Software Validation Writing – Review & Editing 2 Rassi Christian Conceptualization Funding Acquisition Project Administration Resources Supervision Writing – Original Draft Preparation Writing – Review & Editing 2 Achan Jane Investigation Methodology Writing – Review & Editing https://orcid.org/0000-0001-8111-025X 2 Magumba Godfrey Funding Acquisition Project Administration Resources Supervision Writing – Review & Editing 1 Rubahika Denis Supervision Writing – Review & Editing 4 Nabakooza Jane Methodology Supervision Writing – Review & Editing 4 Tibenderana James Conceptualization Funding Acquisition Investigation Methodology Project Administration Resources Supervision Visualization Writing – Review & Editing 2 Nuwa Anthony Conceptualization Funding Acquisition Investigation Methodology Project Administration Supervision Writing – Original Draft Preparation Writing – Review & Editing 1 Opigo Jimmy Resources Supervision Writing – Review & Editing 4 Technical, Malaria Consortium, Kampala, Uganda Technical, Malaria Consortium, London, UK Department of Global Public Health, Karolinska Institute, Stockholm, Sweden National Malaria Control Division, Ministry of Health of Uganda, Kampala, Uganda k.baker@malariaconsortium.org

No competing interests were disclosed.

 18 12 2023 2023 7 14 8 6 2026 Copyright: © 2023 Kajubi R et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background

The World Health Organization (WHO) recommends seasonal malaria chemoprevention (SMC) with sulfadoxine-pyrimethamine and amodiaquine (SPAQ) for children aged 3 to 59 months, living in areas where malaria transmission is highly seasonal. However, due to widespread prevalence of resistance markers, SMC has not been implemented at scale in East and Southern Africa. An initial study in Uganda showed that SMC with SPAQ was feasible, acceptable, and protective against malaria in eligible children in Karamoja region. Nonetheless, exploration of alternative regimens is warranted since parasite resistance threats persist.

 Objective

The study aims to test the effectiveness of SMC with Dihydroartemisinin-piperaquine (DP) or SPAQ (DP-SMC & SPAQ-SMC), chemoprevention efficacy as well as the safety and tolerability of DP compared to that of SPAQ among 3-59 months old children in Karamoja region, an area of Uganda where malaria transmission is highly seasonal.

 Methods

A Type II hybrid effectiveness-implementation study design consisting of four components: 1) a cluster randomized controlled trial (cRCT) using passive surveillance to establish confirmed malaria cases in children using both SPAQ and DP; 2a) a prospective cohort study to determine the chemoprevention efficacy of SPAQ and DP (if SPAQ or DP clears sub-patent infection and provides 28 days of protection from new infection) and whether drug concentrations and/or resistance influence the ability to clear and prevent infection; 2b) a sub study examining pharmacokinetics of DP in children between 3 to <6 months; 3) a resistance markers study in children 3–59 months in the research districts plus the standard intervention districts to measure changes in resistance marker prevalence over time and finally; 4) a process evaluation.

 Discussion

This study evaluates the effects of SPAQ-SMC versus DP-SMC on clinical malaria in vulnerable children in the context of high parasite SP resistance, whilst informing on the best implementation strategies.

 Conclusion

This study will inform malaria policy in high-burden countries, specifically on utility of SMC outside the sahel, and contribute to progress in malaria control.

 Seasonal Malaria Chemoprevention SMC SPAQ DP chemoprevention efficacy malaria Karamoja Uganda East Africa Southern Africa cRCT resistance markers GiveWell This work was supported by the Gates Foundation [INV-039889]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Amendments from Version 1

Revisions were undertaken in response to the reviewers' suggestions for refining the manuscript. Primarily, amendments were made to enhance coherence in the discussion and conclusion section within the abstract, aligning them more accurately with the outcomes detailed in the body of the manuscript. An additional line was incorporated into the introduction section, specifically addressing the historical application of SMC solely within the Sahel region. Furthermore, the references were updated to include the WHO's implementation manual released in 2023, denoted as WHO-UCN-GMP-2023.01-eng, ensuring the manuscript reflects the most current and relevant sources. The errors and typos identified in the process evaluation section were corrected for accuracy and clarity. Additionally, efforts were made to enhance reader comprehension by including comprehensive explanations for all abbreviations used at the outset. To maintain consistency and coherence throughout, the references to therapeutic combinations employed were standardized to ensure uniformity and clarity across the manuscript. These revisions collectively aimed to strengthen the manuscript's clarity, accuracy, and overall coherence in presenting the manuscript, aligning with the reviewers' valuable recommendations for improvement.

 Introduction

Seasonal malaria chemoprevention (SMC) is a very potent community-based intervention to avert malaria infections due to Plasmodium falciparum in areas such as the Sahel region, where malaria morbidity and mortality are high and malaria transmission is seasonal 1– 3 . It entails the intermittent administration of antimalarials to at-risk populations during the period of greatest risk: usually coinciding with the rainy season. Since 2012, SMC has been recommended for children aged 3-59 months using sulfadoxine-pyrimethamine (SP) and amodiaquine by the World Health Organization (WHO) 4 . Owing to concerns over widespread parasite resistance to SP and AQ across east and southern Africa, the Sahel region of west and central Africa has historically been prioritised for the scale-up of SMC. In 2020, SMC was implemented in 13 Sahelian countries targeting over 33 million children 5 . Consolidated guidelines for malaria published by WHO in 2023 no longer provide specific parameters for the deployment of SMC in terms of seasonality, drug regimen or therapeutic efficacy of the antimalarials used, emphasising the importance of context and local evidence 6 .

There is therefore a need to explore if SMC can be a viable malaria prevention strategy in new geographies, including those where resistance to SP and AQ is high, especially since the definition is not clear regarding the relation between resistance and the effectiveness of SMC. Studies have shown that across east and southern Africa P falciparum responses to SP are seriously compromised 7– 9 , and SP is no longer recommended for treatment of malaria episodes. Several mutant alleles that confer parasite resistance to SP are commonly found in the region, including in P falciparum dihydrofolate reductase (pfdhfr) gene at codons 51, 59 and 108, and in P falciparum dihydropteroate synthase (pfdhps) gene at codons 437 and 540 10 . The combination of three mutations in pfdhfr (51I, 59R, and 108N) and two in pfdhps (437G and 540E) leads to an intermediate level of SP resistance 11 . Addition of either pfdhfr164L or pfdhps 581G leads to higher-level SP resistance 11 . These additional mutations have been rare in Africa, but some reports have noted moderate prevalence of the pfdhfr 164L mutation in parasites from south-western Uganda 12– 15 , and of the pfdhps581G mutation in parasites from Uganda and Tanzania 8, 9, 16 . For another malaria chemoprevention strategy, intermittent preventive treatment in pregnancy (IPTp), which also uses SP, it has been suggested that parasite resistance may undermine the effectiveness of the intervention 17 . Markers associated with amodiaquine resistance have also been found, including in the P falciparum multidrug resistance 1 (Pfmdr1) gene and the chloroquine resistance transporter (PfCRT). Even if the effectiveness of SMC in the context of high parasite existence can be demonstrated, it will also be important to identify alternative SMC drug regimens that could replace SPAQ in the future.

The Uganda National Malaria Reduction Strategic Plan 2021-2025 recommends the implementation of SMC in areas with highly seasonal malaria to accelerate progress towards malaria elimination 18 . In response to this recommendation, the National Malaria Control Division (NMCD) and Malaria Consortium are conducting SMC implementation research in Karamoja subregion. In 2021, SMC using SPAQ was implemented in two districts with a combined target population of 85,000 children 3–59 months. Five monthly SMC cycles were implemented between May and September. Village Health Teams, a recognised cadre of community health workers, served as community distributors, with supervision provided by health facility-based health workers. The intervention was found to be feasible and acceptable, with high coverage achieved through the standard door-to-door delivery mechanism. There were also indications that the intervention was effective in preventing malaria cases among targeted children. However, there remains a need to generate robust evidence of effectiveness using appropriate study designs. There is also a need to further explore if SMC using SPAQ can provide a sustained, prolonged protective effect against malaria in the context of parasite resistance.

In 2022, SMC will be implemented in eight districts of Karamoja, targeting 270,000 children 3–59 months. As in the previous year, five monthly SMC cycles will be implemented using a door-to-door delivery model. The majority of children will receive SMC with SPAQ, while around 15,000 children will receive SMC with dihydroartemisinin-piperaquine (DP). As there is currently no age-based dosing regimen for SMC, DP will be given according to a weight-based dosing regimen. In Uganda, DP is used as second-line treatment for uncomplicated malaria and remains highly effective and safe for the treatment of uncomplicated malaria 19, 20 . It has also been used successfully in mass drug administration and to reduce malaria incidence in emergency settings. DP could be an alternative SMC drug regimen primarily due to its long half-life. However, there is insufficient evidence of its tolerability in very young children as safety studies have typically involved only children above 6 months.

 Protocol Aims and objectives

This study phase aims to test the feasibility, effectiveness, and chemoprevention efficacy of SMC with SPAQ and DP in Karamoja region in Uganda, where malaria transmission is highly seasonal, and inform malaria policy in Uganda. Specific objectives include: 1) to determine the effectiveness of SMC with SPAQ and DP in terms of its reduction in incidence of malaria infection among children aged 3–59 month for SPAQ and children 6–59 months for DP; 2a) to conduct a prospective chemoprevention efficacy cohort study to determine if SPAQ and DP clear existing parasitemia and provide 28 days of protection from infection and whether drug concentrations and/or SPAQ or DP drug resistance influence the protection duration of these two SMC regimen; 2b) to assess the safety of DP in children 3–6 months through pharmacometric analysis; 3) to conduct a resistance markers study in children 3–59 months in the two research districts plus the three standard intervention districts to measure changes in resistance marker prevalence over time; and 4) to assess the impact of the SMC implementation model, determining process, costing and acceptability outcomes for the intervention.

 Methods Study design

This type 2 hybrid effectiveness-implementation study uses a convergent mixed-methods approach, as previously described in other similar studies conducted by our group 21, 22 . SMC with SPAQ and DP will be implemented in five monthly cycles between June 2022 and October 2022 in five districts of Karamoja. Over 85,795 children aged 3–59 months, in Kotido and Moroto for phase 1 of the study, will continue to receive SMC in phase 2, and in addition, 60,000 children in three districts (Nabilatuk, Amudat, and Nakapiripirit) will receive SMC for the first time. The research will involve the following components: 1) Conducting a cluster randomized controlled trial (cRCT) using passive surveillance to establish confirmed malaria cases among participating children; 2) conducting a prospective chemoprevention efficacy cohort study to determine whether SPAQ and/or DP provides 28 days of protection from infection, and whether drug concentrations and/or resistance influence the duration of protection; 3) conducting a resistance markers study in children aged 3–59 months in the three research districts plus the two standard intervention districts to describe changes in resistance marker prevalence over time; and 4) conduct a process evaluation of the SMC implementation looking at cost and process outcomes 6 .

 Study setting

The study will be conducted in five districts of Karamoja region as shown in Figure 1 below. The cRCT will be conducted in Amudat. The chemoprevention component will be conducted in Nakapiripirit because SMC has not been previously implemented in this location therefore drug resistance selection pressure from a previous year of SMC implementation will be avoided or minimized and malaria rates are expected to be higher allowing for a greater detection of breakthrough infections. The resistance markers component will be conducted in all five districts inclusive of Nabilatuk, Kotido and Moroto, as well as the process evaluation for assessment of the SMC implementation model.

Area of implementation of Year 2 SMC in Uganda. Population

Eligible participants for receiving SMC will be male or female children with no fever, aged 3–59 months for any of the SMC cycles, staying in the districts of Kotido, Moroto and Nabilatuk districts (where delivery is continuing from Phase 1 and where coverage surveys will be conducted in Phase 2), and randomly-selected areas in Amudat and Nakapiripirit.

Additionally, other key stakeholders who will be sampled include health workers participating in implementing SMC, health officials at different levels of the health system, community leaders, and caregivers of children below ten years of age, (see Table 1 for estimated population sizes).

Sample sizes for each objective.
Objective Study population Estimated sample size
1 cRCT: Sample of eligible children in Amudat district of age 3–59 months, resident in the study area for at least a month, planned for DP and SPAQ 4,270
2a Chemoprevention efficacy cohort study – children under 3–59 months for SPAQ and 6-59 months for DP 1,250
2b Safety of DP in 3–6 month old children through pharmacometrics to low dose DP. 30
3 Resistance Marker study – children under 5 1,500
4 Assessment of the SMC implementation model, deciding the process, costing and acceptability outcomes of the intervention 144
Total number of participants: 7,194
Recruitment and data collection

cRCT component. In the control arm, one eligible child will be recruited at random in each household of which, researchers from selected communities will have randomly selected. Informed consent will be obtained from caregivers, then children will be enrolled and their eligibility confirmed using a short baseline questionnaire to collect individual data on each child.

For the intervention arms, VHTs will be followed by a researcher as they administer SMC. Households in selected communities will be randomly sampled from those visited by the VHTs. Before SMC is initiated, one eligible child will be recruited, and a short baseline questionnaire will be administered to collect individual data and to confirm their eligibility. Informed consent will be obtained from caregivers and then a questionnaire used for collecting individual child data, to which all household questions from the end-of-round survey will be added (e.g. on household wealth, language, migration status, education and level of literacy of caregivers and heads-of-household, etc.), and to determine their eligibility. Blood samples will be taken for haemoglobin concentration measurement using a Hemocue machine.

The primary endpoint will be recorded with passive surveillance conducted by study clinicians in intervention and control areas throughout the five-month study period.

Children recruited into the study presenting at clinics or to VHTs will be identified using information on their SMC record cards, and data on clinic visits including suspected malaria cases and results of RDTs will be matched to baseline questionnaire data to build a database for analysis. Date of clinic attendance and confirmation of malaria cases using rapid diagnostic tests will be recorded to allow for calculation of follow-up time and time to malaria events for fitting of Cox proportional hazards regression model. This will be facilitated through matching of codes on children's SMC cards with records of the same codes in clinic logbooks.

Chemoprevention efficacy cohort study. Children will be screened at the community level by research assistants with the help of VHTs and referred to a designated study clinic for further assessment and non-randomized recruitment. At the study clinic, study clinicians will confirm eligibility criteria through conversation with the caretaker and obtain informed consent. Enrolled children will receive SMC via directly observed therapy (DOT) over the first three days and will have malaria thick smears and dry blood spots (DBS) taken at day 0, 2, 7, 14, 21 and 28 throughout and after administering a single cycle of SPAQ or DP. In addition, the study clinician will assess if the child received other treatment or experienced any problem during all the scheduled sample collection days (Day 0, 7, 14, 21, 28) as well as bed net usage. Caregivers and guardians will also be reminded at each visit to seek care at a health facility or from a community health worker if the child becomes febrile. Children who become febrile and are confirmed on HPR2-pLDH RDT between day 0 and day 28 will have an additional DBS taken and then treated using standard guidelines. All DBS samples will be processed for all of the SMC drugs on days 7 and for sulfadoxine, amodiaquine and piperaquine for day 28. Individual surveys on day 28 after the final DBS is taken will be conducted to determine if the child received other treatment or experienced illness over the past month of study implementation. On day 0 and day 28, they will also be processed to ascertain low density parasitaemia estimates through quantitative polymerase chain reaction (qPCR). Figure 2 below depicts the planned sample collection time points and subsequent sample processing sequence). We aim to recruit participants from Nakapiripit, one of the two research districts.

Planned sample collection time points and subsequent sample processing sequence.

Resistance markers study. We will monitor changes of SP, AQ, resistance in the three intervention districts, receiving SMC using only SPAQ (Nabilatuk, Kotido and Moroto) plus the two research districts where the SMC will be implemented using both SPAQ and DP (Amudat and Nakapiripirit), while the DP resistance will be monitored only in the later. A baseline cross-sectional survey will be conducted based at the health facilities before SMC project implementation as well as an endline survey following one complete round of SMC distribution to measure the prevalence of molecular markers associated with resistance to SP/DP in symptomatic children under five years of age with a positive RDT attending selected health facilities in the intervention and control areas.

The key markers of resistance to be monitored are: dihydrofolate reductase (dhfr): codons 108, 51, 59 and 164; dihydropteorate synthetase (dhps): codons 431, 437, 540, 581 and 613; Pf chloroquine resistance transporter gene (pfcrt): codons 72-76; Pf multidrug resistance gene 1 (pfmdr1): codons 86, 184 and 1246, Plasmepsin 2 (plm2) and K13 mutations of significance

Process evaluation component. Once interview participants are identified for potential recruitment, they will be approached by a research assistant who will share more information about the study and invite their participation. If willing, a meeting will then be set up at a time and location that is convenient for the individual, where further information will be shared about the study including a written information sheet provided to all participants.

In-depth interviews and focus group discussions based on topic guides will be utilized to explore insights on SMC including acceptability and thoughts regarding the experience of implementation.

 Data analysis

cRCT component. We will accomplish data analysis using Stata and excel. The proportion command, with 95% confidence intervals calculated using a logit transform will be used to compute coverage. Population size weights will be applied with the svy: command as appropriate for the estimates of coverage indicators whereby a self-weighting sample was not possible to achieve. The pertinent indicators will be calculated using proportions per district and an average across both districts. The confidence interval (CI) of 95% will be used to provide a range of values around the estimate within which selected result will be expected to fall.

Data from HMIS will be retrospectively analysed to identify epidemiological trends in malaria in the study districts by age group. Summary statistics will be computed and data will be graphically presented by month over the study period.

Chemoprevention efficacy cohort study. Relevant mutations distributions and proportions will be analysed comparing parasitological efficacy between groups of mutations. Analysis of the blood drug concentrations will be done as a cohort by mean, median and standard deviation, and statistical tests of their associations with treatment outcomes, precisely drug levels on day seven. Outliers with low levels of drug concentration based on the outcome measures described will be focused upon. Positivity on day 28 will be associated with the antimalarial drug resistance genotype. Chemoprevention failure rates will be reported on as the cumulative failure rate using Kaplan-Meier analysis of the proportion. Time to event analysis will be incorporated to assess duration of SMC protection.

Resistance markers study. The prevalence of Pfdhps, Pfdhfr Pfcrt and Pfmdr1 and K13 alleles will be measured in parasites obtained from smear positive children. Additionally, the copy number of Pfplm2 will also be assessed.

Process evaluation component. The data will be analysed thematically. Both key informant interviews and FGDs will be audio-recorded and thereafter transcribed verbatim i.e. in the real words (or equivalent translations) used by the informants. These will be organised according to findings relating to SMC acceptability, implementation, and views and experiences with SMC more broadly. Initially, open, descriptive coding will be used in order to ensure interpretations are grounded in participants’ accounts as much as possible, and that findings are generated inductively, as well as exploring pre-determined areas of investigation (for example, relating to acceptability of SMC). The transcribed data will be entered into Nvivo 12 for analysis and coded based on both pre-determined themes as well as those that emerged from the data. Codes and themes will continuously be refined as the analysis progresses. The coded text under each code / theme will provide the basis for determining the patterns and meanings in the data, comparing across data from different respondent groups, and for making arguments and conclusions. Relevant quotations will also be identified and included in the analysis report.

 Data management

For the cRCT component (1), using computer aided personal interview (CAPI), which allows for in-field data entry and server synchronisation, data collected will be verified for quality assurance purposes by the quality assurance officer in field and uploaded daily to the SurveyCTO platform. The uploaded files will undergo additional consistency checks, cleaned and saved into Stata. Extensive data cleaning will be done at the end of the fieldwork.

For the chemoprevention efficacy and safety studies, all clinical data will be recorded onto standardized case record forms (CRFs) by IDRC study clinicians. Data will then be entered directly from CRFs into a computerized database or transferred from the CRFs onto standardized data extraction forms and then into a computerized database. All computerized data will be double entered to verify accuracy of entry.

For the resistance markers study, data will initially be managed at health facilities-level by field workers with support from the IDRC Data Management Team (DMT). They will send the pVHTr questionnaires to the IDRC once sample collection has been completed. Data will then be entered by a data entry clerk using specific software for clinical data management. Rigorous consistency checks will be created to reduce errors during data entry.

For the process evaluation, data will be stored without any participant-identifying information (with use of pseudonyms and removal of job title or other identifying information), in a password-protected format. Raw data collected and analysed will not be shared with anyone other than the researchers of this study.

 Eligibility criteria

Inclusion and exclusion criteria for each objective are detailed in Table 2 below.

Eligibility Criteria.
Inclusion criterion (1) (2) (3) (4)
Children between 3–59 months x x x
Being resident in the study area x x x
Afebrile with no other malaria associated symptoms in the past 48 hours or at time of recruitment X X
Consent to participate in the study obtained X X X
Willingness and ability of the child’s guardians to comply with the study protocol for the duration of the study including attending a designated health centre if their child has malaria symptoms during the data collection period x X
Can comply with 3 days DOT of standard SPAQ or DP regimen (day 0–2 x
Clinical signs and symptoms suggestive of malaria infection: fever (axillary temperature ≥37.5ºC) or history of fever in the preceding 24 hours. X
Positive RDT x
A person aged 18 years or older with primary or caring responsibility for the feeding and daily care of at least one child aged 3–59 months, who has been resident in Nabilatuk, Kotido, Moroto, Amudat or Nakapiripirit prior to the beginning of the SMC pilot x
Community members, community leaders, religious leaders X
Village Health Teams (VHTs) involved in the distribution of SMC in Nabitatuk, Kotido, Moroto, Amudat and Nakapiripirit X
Community Health Workers, Community Distributors X
District level health officials (District Health Officers, Malaria Focal Persons) X
Provincial level health officials X
Implementing partners X
National level policy makers X
Gender experts and/or gender advocates x
Exclusion criterion (1) (2) (3) (4)
Malaria symptoms (tympanic fever ≥ 37.5°C or history of fever in past 48 hours) X X
Known allergy to medicine provided X X
Receiving a sulfa-based medication for treatment or prophylaxis, including co-trimoxazole (trimethoprim– sulfamethoxazole) X X
Individuals receiving azithromycin due to the antimalarial activity of azithromycin X x
Severe malnutrition according to WHO guidelines X X
Recruited in any other SMC study element X X
Children with HIV or ARV use X X
Children with a history of severe adverse reaction to SP, AQ or DP or have used SP within the previous month before recruitment x X
Treatment of uncomplicated malaria with DP in the past 28 days X
Chronic illness of any kind X
Recruited in cRCT or any other studies X
Treatment with an ACT in previous 2 weeks X
Any signs or symptoms of severe malaria X
Negative RDT x
Any individual who refuses consent to participate x x x X
Individuals aged under 18 years x
Ethical considerations

Ethical approval for this study was obtained from the Mbale Regional Referral Hospital-REC on 28 April 2022 (Ref: MRRH-2022-168) and by the Uganda National Council for Science and Technology (UNCST) on 5 May 2022 (Ref: HS2212ES). Only participants that meet the inclusion criteria and whose caregivers provide written informed consent will be included in this study. The trial has been registered on clinicaltrials.gov (Ref: NCT05323721).

 Study status

Research activities commenced in June 2022 after approval of Ethics Committees. Data collection has been completed and cleaning and preliminary analysis is on-going. Final analysis is expected to be completed by April 2023.

 Dissemination

Malaria Consortium will develop a research uptake plan to help the study team identify relevant stakeholders and consider appropriate messages and activities, along with respective budgets and timelines, to ensure these stakeholders are kept engaged and informed of progress throughout the research cycle. Results will be disseminated through agreed peer-review publications and international conferences. There will also be collaboration with the Ministry of Health National Malaria Control Division to disseminate findings nationally.

 Conclusions

This study will contribute strong evidence on the effectiveness of SMC in the context of high parasite resistance in areas outside the Sahel which had previously not been prioritized for scale-up, using appropriate study designs. The findings will be crucial for future SMC research and deployment by understanding if drug dosing or drug choice may need to be altered to provide continued efficacious SMC in the local context. Ultimately, this will influence policy change in malaria control.

 Data availability

No data are associated with this article.

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Reference Source World Health Organization: World Malaria Report 2021. World Health Organization, Geneva,2021. Reference Source World Health Organization: WHO Guidelines for Malaria. World Health Organization, Geneva,2023. Reference Source Desai M Gutman J Taylor SM : Impact of sulfadoxine-pyrimethamine resistance on effectiveness of intermittent preventive therapy for Malaria in pregnancy at clearing infections and preventing low birth weight. Clin Infect Dis. 2016;62(3):323–333. 26486699 10.1093/cid/civ881 4762476 Sendagire H Kyabayinze D Swedberg G : Plasmodium falciparum: higher incidence of molecular resistance markers for sulphadoxine than for pyrimethamine in Kasangati, Uganda. Trop Med Int Health. 2005;10(6):537–5. 15941416 10.1111/j.1365-3156.2005.01414.x Mbonye AK Birungi J Yanow SK : Prevalence of Plasmodium falciparum resistance markers to sulfadoxine-pyrimethamine among pregnant women receiving intermittent preventive treatment for malaria in Uganda. 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PLoS One. 2018;13(3): e0193296. 29505578 10.1371/journal.pone.0193296 5837084 Antwi GD Bates LA King R : Facilitators and barriers to uptake of an extended seasonal malaria chemoprevention programme in Ghana: a qualitative study of caregivers and community health workers. PLoS One. 2016;11(11): e0166951. 27898699 10.1371/journal.pone.0166951 5127521 Kateera F Nsobya SL Tukwasibwe S : Molecular surveillance of Plasmodium falciparum drug resistance markers reveals partial recovery of chloroquine susceptibility but sustained sulfadoxine-pyrimethamine resistance at two sites of different malaria transmission intensities in Rwanda. Acta Trop. 2016;164:329–336. 27647575 10.1016/j.actatropica.2016.09.008 Chico RM Chandramohan D : Intermittent preventive treatment of malaria in pregnancy: at the crossroads of public health policy. Trop Med Int Health. 2011;16(7):774–785. 21477099 10.1111/j.1365-3156.2011.02765.x Uganda National Malaria Control Division (NMCD): National Malaria Control and Elimination Strategic Plan 2021–2025. 2020. Ministry of Health Uganda: Uganda Clinical Guidelines 2016: National Guidelines for Management of Common Conditions.Ministry of Health Uganda, Kampala,2016. Reference Source Uganda National Malaria Control Division (NMCD): National Malaria Control and Elimination Policy. 2018. Wharton-Smith A Baker K Roca-Feltrer A : Assessment of the Feasibility, Acceptability, and Impact of Implementing Seasonal Malaria Chemoprevention in Nampula Province, Mozambique: Protocol for a Hybrid Effectiveness-Implementation Study. JMIR Res Protoc. 2021;10(9): e27855. 34524109 10.2196/27855 8482168 Baker K Aide P Bonnington CA : Feasibility, Acceptability, and Protective Efficacy of Seasonal Malaria Chemoprevention Implementation in Nampula Province, Mozambique: Protocol for a Hybrid Effectiveness-Implementation Study. JMIR Res Protoc. 2022;11(9): e36403. 36149743 10.2196/36403 9547334 10.21956/gatesopenres.16472.r35688 Reviewer response for version 2 Guntur, Ph.D Robertus Dole 1 Referee https://orcid.org/0000-0002-1597-3644 Department of Mathematics Faculty of Science and Engineering, Nusa Cendana University, Kupang, East Nusa Tenggara, Indonesia

Competing interests: No competing interests were disclosed.

 26 2 2024 Copyright: © 2024 Guntur, Ph.D RD 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

Question 1

If you could provide the flow chart for recruiting your participants, it would be great to improve the article. The flow chart is a kind of picture so it could not add the number of words to this article. Then, the authors said, “we make the flow chart available in a future publication after data collection”.  Meanwhile on the study status section of this protocol paper, the author said that “Data collection has been completed “. Would you please to clarify this?

Question 2

The rationale in determining sample size is very important in a study. The data in Table 1 is like a magic number if the authors could not provide the reasons in choosing those numbers.

Question 3

The questionnaire is an important aspect in assessing this article as the authors collected individual data from the children. The consistency between your statement in the article and the questionnaire is important to be evaluated!!! The authors could include the questionnaires as the supplementary material for this submission.

Question 4

Plan analysis is one of the essential components on a protocol paper. The authors could provide general plan on data analysis to achieve your aims as each aim of this study has a different statistical method.

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

No

Are the datasets clearly presented in a useable and accessible format?

Not applicable

Reviewer Expertise:

Epidemiology and Biostatistics, Malaria

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

 10.21956/gatesopenres.16472.r35687 Reviewer response for version 2 Nogueira Fatima 1 Referee https://orcid.org/0000-0003-0313-0778 Universidade Nova de Lisboa, Lisbon, Lisbon, Portugal

Competing interests: No competing interests were disclosed.

 31 1 2024 Copyright: © 2024 Nogueira F 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

Changes were made.

Is the study design appropriate for the research question?

Partly

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

No

Are the datasets clearly presented in a useable and accessible format?

Not applicable

Reviewer Expertise:

malaria drug resistance

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.21956/gatesopenres.16472.r35689 Reviewer response for version 2 Poku-Awuku Abena 1 Referee Medicines for Malaria Venture, Geneva, Geneva, Switzerland

Competing interests: No competing interests were disclosed.

 31 1 2024 Copyright: © 2024 Poku-Awuku A 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

Keywords: I'm not sure if it makes sense to state both Seasonal Malaria Chemoprevention and SMC as two separate keywords.

Conclusion in abstract: Sahel should start with a capital S

Introduction:  Plasmodium falciparum and all other mentions of P falciparum or scientific names must be italicized.

Line 7 of introduction: West and Central Africa should start with capital letters as they are the names of the regions; western and southern Africa, not as they are not names

Recruitment and data collection (resistance markers)- later in line 3 of this section should be "latter"

Process evaluation component: Please state out what FGD is before using the acronym

Overall, the script is fine but requires proof reading.

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

Yes

Are the datasets clearly presented in a useable and accessible format?

No

Reviewer Expertise:

Malaria, advocacy

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.21956/gatesopenres.15598.r33878 Reviewer response for version 1 Guntur, Ph.D Robertus Dole 1 Referee https://orcid.org/0000-0002-1597-3644 Department of Mathematics Faculty of Science and Engineering, Nusa Cendana University, Kupang, East Nusa Tenggara, Indonesia

Competing interests: No competing interests were disclosed.

 17 7 2023 Copyright: © 2023 Guntur, Ph.D RD 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The method section lacks details. The author should provide as many details as possible in the methods section.

Please provide a flow chart of a recruitment strategy for your participants.

Please provide sample size calculation to justify your sample size in Table 1.

The authors said that the questionnaire is used for collecting individual child data. Where is your questionnaire? Please attach as supporting documents.

In the analysis section, the procedure on how the authors analyse the data of 7,194 participants is not clear. How do you analyse your data to achieve your aim of study? Please breakdown your analysis plan following your objective 1 to 4. How do you analyse the socio-demographic data of your participants? Furthermore, in the part of resistance markers study, what is your statistical analysis to achieve the objective number 3?

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

No

Are the datasets clearly presented in a useable and accessible format?

Not applicable

Reviewer Expertise:

Epidemiology and Biostatistics, Malaria

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Kajubi Richard Malaria Consortium, Uganda

Competing interests: I have no competing interests to declare

 25 7 2023

Dear Robertus Dole Guntur,

Thank you for your interest and valuable feedback to our article. Please see below responses to your comments.

The method section lacks details. The author should provide as many details as possible in the methods section.

Please provide a flow chart of a recruitment strategy for your participants.

Thank you for your comment. We appreciate your interest in having a flow chart of our recruitment strategy. However, due to space limitations in th is protocol paper, we were unable to include visual content like flow charts. We understand the value of a recruitment flow chart for readers to better understand the study's participant selection process. However, we provid ed a detailed description of the recruitment strategy in the "Methods" section to enhance transparency and clarity. Your suggestion is valuable, and we make the flow chart available in a future publication after data collection.

Please provide sample size calculation to justify your sample size in Table 1.

Thank you for your valuable feedback. We agree that providing a sample size calculation is essential to justify the chosen sample size in Table 1. However, the sample size was determined based on prior research in the field, feasibility constraints, and the statistical requirements for detecting meaningful effects in our study. Due to word count limitations in the article, we were unable to include the sample size calculation in the main text.  To address this concern, we will include a detailed sample size calculation in the supplementary materials or as an online appendix to ensure transparency and support the robustness of our study. This will allow readers to better understand the rationale behind the selected sample size and the statistical power of our analysis.

The authors said that the questionnaire is used for collecting individual child data. Where is your questionnaire? Please attach as supporting documents                                         Thank you for your comment and interest in our study. We apologize for any confusion. While we understand the importance of providing the questionnaire for transparency and reproducibility, due to the word limit constraints, we were unable to include the complete questionnaire in the article.  However, we want to assure you that the questionnaire used in the study is available for reference and upon request. To ensure accessibility and facilitate reproducibility, we will make the questionnaire available as supplementary material or on request by contacting the corresponding author.

In the analysis section, the procedure on how the authors analyse the data of 7,194 participants is not clear. How do you analyse your data to achieve your aim of study? Please breakdown your analysis plan following your objective 1 to 4. How do you analyse the socio-demographic data of your participants? Furthermore, in the part of resistance markers study, what is your statistical analysis to achieve the objective number 3?      Thank you for your valuable feedback and for seeking clarification on our data analysis procedures. We understand the importance of providing a clear and detailed analysis plan to support the study's objectives. However, due to the word limit constraints, we were unable to include that information in th is article . We will ensure that these details are available in all subsequent publications with regard to each of the objectives.

10.21956/gatesopenres.15598.r33622 Reviewer response for version 1 Nogueira Fatima 1 Referee https://orcid.org/0000-0003-0313-0778 Universidade Nova de Lisboa, Lisbon, Lisbon, Portugal

Competing interests: No competing interests were disclosed.

 28 6 2023 Copyright: © 2023 Nogueira F 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

Include the meaning of abbreviations in full at the beginning of the document, such as SPAQ.

Homogenize the reference to the therapeutic combinations used.

In the “Methods Study design” refer to and comply with the WHO guidelines (WHO-UCN-GMP-2023.01-eng).

Consider including gametocyte count/carriage.

Is the study design appropriate for the research question?

Partly

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

No

Are the datasets clearly presented in a useable and accessible format?

Not applicable

Reviewer Expertise:

malaria drug resistance

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Kajubi Richard Malaria Consortium, Uganda

Competing interests: I have competing interests to declare

 25 7 2023

Dear Fatima Nogueira,

Thank you for your valuable insights and feedback. Please see the responses to your concerns below.

Include the meaning of abbreviations in full at the beginning of the document, such as SPAQ.

Thank you for your suggestion. We recognize the importance of providing clarity to readers by including the full meaning of abbreviations at the beginning of the document. Please note that We used the wording "sulfadoxine-pyrimethamine and amodiaquine" at the beginning of both the abstract and the main body of the article.

Homogenize the reference to the therapeutic combinations used.

Thank you for your suggestion to homogenize the reference to the therapeutic combinations used. We understand the importance of maintaining consistency and clarity in scientific writing. We will review all references to therapeutic combinations throughout the article and ensure that they are presented in a uniform manner. This may involve standardizing the naming conventions, abbreviations, or any other relevant details related to the therapeutic combinations.

In the “Methods Study design” refer to and comply with the WHO guidelines (WHO-UCN-GMP-2023.01-eng).

Thank you for your suggestion to refer to and comply with the WHO guidelines (WHO-UCN-GMP-2023.01-eng) in the "Methods Study design" section. We recognize the significance of adhering to established guidelines to ensure the credibility and validity of our research.We will review and add the specific reference to the WHO guidelines (WHO-UCN-GMP-2023.01-eng) and describe how our study design aligns with the recommended protocols and methodologies outlined in the guidelines.

Consider including gametocyte count/carriage.

Thank you for your suggestion to include gametocyte count/carriage in the study. We recognize the importance of assessing gametocyte count/carriage as a significant parameter in malaria research, especially in understanding transmission dynamics. Unfortunately, in this particular study, we did not include gametocyte count/carriage as one of the study variables hence we did not collect that data.

10.21956/gatesopenres.15598.r33624 Reviewer response for version 1 Poku-Awuku Abena 1 Referee Medicines for Malaria Venture, Geneva, Geneva, Switzerland

Competing interests: No competing interests were disclosed.

 15 6 2023 Copyright: © 2023 Poku-Awuku A 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The discussion and conclusion put in the abstract are too vague and do not reflect the outcomes found in the body of the article.

Introduction:

Lines 1-3: It would be good to state upfront that SMC is widely implemented in the Sahel and has been used only there. Lines 1-3 make it seem as if SMC can be implemented in all malaria-endemic areas.

Lines 8-9: WHO's implementation manual published in 2023 provides parameters for seasonality. Please check and refer to accordingly.

Lines 13-15: I would remove the part of the sentence that says "the definition is not clear regarding relation between resistance and effectiveness of SMC" because it contradicts what is said 2 sentences later. 

Recruitment and data collection: Line 6: is it only one child or one child per household?

Line 10: what were the end of round demographic survey results used for?

Process evaluation component: Line 5 typo: possible60; line 9: "progresses" not "progressed".

This paper would be stronger and more complete if the final data results would be available. If this is not ready, I'm not clear if the objective of the study has been achieved or if results to substantiate the objectives can be proven.

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

Yes

Are the datasets clearly presented in a useable and accessible format?

No

Reviewer Expertise:

Malaria, advocacy

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Kajubi Richard Malaria Consortium, Uganda

Competing interests: No competing interests were disclosed.

 25 7 2023

Dear Abena Poku-Awuku,

Thank you for your valuable feedback. Please see the responses to your concerns below.

The discussion and conclusion put in the abstract are too vague and do not reflect the outcomes found in the body of the article.

Thank you for your valuable feedback. We appreciate your concern regarding the clarity of the discussion and conclusion in the abstract. While we understand your point, the purpose of the abstract is to provide a concise overview of the study and its key findings. However, this being a protocol article, we had not yet collected complete data at the time of submission hence we didn’t have any key findings to discuss and make a more compelling conclusion. We will ensure to do that in the subsequent manuscript after all data collection is complete.

Introduction:

Lines 1-3: It would be good to state upfront that SMC is widely implemented in the Sahel and has been used only there. Lines 1-3 make it seem as if SMC can be implemented in all malaria-endemic areas.

Thank you for your insightful comment. We appreciate your suggestion to clarify the geographic scope of SMC implementation in the article. However, we did clarify that SMC applies to all malaria-endemic areas where transmission is seasonal, and the same paragraph clearly stipulates that SMC was implemented in 13 Sahelian countries and had been used only there until we conceived these studies. We feel that can suffice.

Lines 8-9: WHO's implementation manual published in 2023 provides parameters for seasonality. Please check and refer to accordingly.

Thank you for bringing this to our attention. We appreciate your diligence in pointing out the availability of the WHO's implementation manual published in 2023, which provides parameters for seasonality in the context of our study. However, at the time of submitting our protocol, some of the study justifications had been based on Consolidated guidelines for malaria published by WHO in 2022, hence we felt that it was more appropriate to cite the 2022 guidelines.

Lines 13-15: I would remove the part of the sentence that says "the definition is not clear regarding relation between resistance and effectiveness of SMC" because it contradicts what is said 2 sentences later.

Thank you for your careful observation. We appreciate your suggestion to remove the part of the sentence in lines 13-15 that implies uncertainty about the relation between resistance and the effectiveness of SMC. Upon further analysis, however, we feel that there is no contradiction because chemoprevention is different from treatment. Indeed, SP is no longer recommended for the treatment of malaria episodes, but it’s still recommended for chemoprevention in pregnancy despite high resistance. That is what we mean by the “relation between resistance and effectiveness of SMC.”

Recruitment and data collection: Line 6: is it only one child or one child per household?

Thank you for seeking clarification on this point. We meant one eligible child from each household as stated.

Line 10: what were the end of round demographic survey results used for?

Thank you for your question regarding the use of the end of round demographic survey results mentioned in line 10. By incorporating the end of round demographic survey, we aimed to provide information on coverage and quality, suitability of design, the extent of attainment of targets, and assess planning and general implementation of processes as required in the SMC protocol

Process evaluation component: Line 5 typo: possible60; line 9: "progresses" not "progressed".

Thank you for catching those typos in the process evaluation component. We appreciate your attention to detail. We will make the necessary corrections in line 5, changing "possible" to "possible, ," and in line 9, replacing "progressed" with "progresses."

This paper would be stronger and more complete if the final data results would be available. If this is not ready, I'm not clear if the objective of the study has been achieved or if results to substantiate the objectives can be proven.

Thank you for your feedback. We understand your concern about the completeness and strength of the paper without access to the final data results. Please note that this is a protocol paper that was submitted at the time when data collection was not complete.

While the article currently lacks the final data results, we want to assure you that the study's objectives have been pursued rigorously and with meticulous methodology. The data collection processes have since been completed, and we aim to present comprehensive and conclusive results in future publications.

We acknowledge the importance of substantiating the study's objectives with concrete evidence, and we will work diligently to finalize the data analysis and interpretation. Once the results are available, we will consider publishing a follow-up paper to share the outcomes and their implications.

Thank you for your understanding and valuable input.