Optimal mode for delivery of seasonal malaria chemoprevention in Ouelessebougou, Mali: A cluster randomized trial

Background Seasonal malaria chemoprevention (SMC), the administration of complete therapeutic courses of antimalarials to children aged 3–59 months during the malaria transmission season, is a new strategy recommended by the World Health Organization (WHO) for malaria control in Sahelian countries such as Mali with seasonal transmission. The strategy is a highly cost-effective approach to reduce malaria burden in these areas. Despite the substantial benefits of SMC on malaria infection and disease, the optimal approach to deliver SMC remains to be determined. While fixed-point delivery (FPD) and non-directly observed treatment (NDOT) by community health workers are logistically attractive, these need to be evaluated and compared to other modes of delivery for maximal coverage. Methods To determine the optimal mode fixed-point (FPD) vs door-to-door delivery (DDD); directly observed treatment (DOT) vs. non- directly observed treatment (NDOT)), 31 villages in four health sub-districts were randomized to receive three rounds of SMC with Sulfadoxine-pyrimethamine plus Amodiaquine (SP+AQ) at monthly intervals using one of the following methods: FPD+DOT; FPD+NDOT; DDD+DOT; DDD+NDOT. The primary endpoint was SMC coverage assessed by cross-sectional survey of 2,035 children at the end of intervention period. Results Coverage defined as the proportion of children who received all three days of SMC treatment during the three monthly rounds based information collected by interview (primary endpoint) was significantly higher in children who received SMC using DDD 74% (95% CI 69% - 80%) compared to FPD 60% (95% CI 50% - 70%); p = 0.009. It was similar in children who received SMC using DOT or NDOT 65%, (95% CI 55% - 76%) versus 68% (95% CI 57% - 79%); p = 0.72. Conclusions In summary, door-to-door delivery of SMC provides better coverage than FPD. Directly observed therapy, which requires more time and resources, did not improve coverage with SMC. Trial registration ClinicalTrials.gov NCT02646410

and will then be implemented in villages of two additional sub-districts. Villages in these two sub-districts will be randomized in two groups. Children in the first group will received three rounds of SMC and those in the second group will receive four rounds of SMC to determine the optimal frequency of SMC based on the incidence rate of clinical malaria as measured by passive surveillance. Children in the four sub-districts selected in Year 1 will continue to receive three rounds of SMC in Year 2 using the optimal mode of delivery. In Year 3, children in the randomly selected sub-districts will received SMC by the optimal delivery system determined in Years 1 -2. Immune responses will be measured and compared between the children receiving SMC to a cohort of children not receiving SMC, to assess the impact of SMC on key antimalarial antibody responses over the three year period using cross-sectional surveys at the beginning and the end of the transmission season. Prevalence of malaria infection, anemia and molecular markers of P. falciparum resistance to SP and AQ will be measured during these cross-sectional surveys as secondary endpoints in randomly selected sample of the children.

Expected Outcomes (How this study will inform implementation/scale-up):
The anticipated outcome of the study will be a qualified optimal approach to deliver SMC in Mali, and an expanded understanding of its impact on health outcomes and antimalarial immunity.

Background
More than 200 million people live in areas where malaria transmission is highly seasonal.
Malaria remains a major cause of morbidity and mortality and is estimated to cause between 610 000-971000 deaths globally per year. Sub-Saharan Africa is disproportionately affected, suffering 91% of global malaria deaths with 88% occurring in children under 5 y of age [WHO 2012]. In absence of a vaccine, simple and effective control strategies are urgently needed to reduce the malaria burden in sub-Saharan Africa. Vector control, using insecticide-treated bednets (ITNs), insecticide-treated curtains, or indoor residual spraying (IRS), can reduce mortality and morbidity from malaria substantially [Lengeler 2004], but in high transmission settings, these interventions provide only partial protection and additional control measures are needed.
We recently showed that Seasonal Malaria Chemoprevention (SMC), previously known as The strategy has been shown to be highly effective, safe and cost-effective for preventing malaria in children under 5 years of age in areas with highly seasonal malaria transmission such as the Sahel region in Africa [WHO GMP 2012].
Although several potential approaches can be used to implement the strategy, there is insufficient evidence to recommend a standard strategy to deliver SMC [WHO implmentation guidelines].
Two studies have compared the delivey of SMC through community health workers and health facilities in Ghana  The pivotal efficacy studies in Mali, Senegal and Burkina Faso that have lead to the evaluation and the recommendation of the strategy have assesed the efficacy of two or three treatment rounds of the SMC, and there is no data to support additional benefit provided by a 4th treatment round of SMC in the WHO recommendation. In this study we will evaluate the efficacy of 4 th

Rationale for This Study
round of the SMC treatment against clinical malaria episodes in Mali.
Our previous studies demonstarted that SMC reduces malaria infection and disease by more than 80% in Malian children. In addition to the protective effect of the intermittenet treatment other factors may have contributed to the success of these trials such as the method of treatment delivery. WHO policy to implement SMC is based on trials conducted in Mali and other sites, however, critical factors such as the optimal method of delivery that can be implemented on a country level and will achieve a similar success rate as observed in the clinical trials remained to be determined.
This study is design to answer the primary objective, that is to identify the most effective and efficient methods to deliver SMC in Mali that is urgently needed to support SMC implementation. An optimal delivery of the strategy will make a substantial contribution toward immunity to malaria when given for more than one transmission season. Under conditions of long term intervention, we hypothesize that immunity to liver-stage malaria antigens (reflecting exposure to infected mosquito bites) will be similar among children receiving SMC to that of children who will not receive SMC, but immunity to blood stage antigens will be markedly decreased (reflecting protection from blood stage infection). In this proposal, children will receive intervention in a stepped wedge fashion and be followed for a period up to 3 years. We are currently studying the acquisition of immunity to malaria among children recruited at birth as part of an immuno-epidemiological (IMEP) cohort study in Ouelessebougou, where we plan to enroll up to 2000 children who will be followed up for 5 years. We will leverage this cohort of children as a control group for the proposed intervention study. This will provide insights into the acquisition of malaria immunity and may indicate whether the cessation of longterm SMC will increase risk of subsequent malaria and warrant additional control measures.
dose will have an additional benefit over 3 rounds of treatment.
Another specific objective of this study is to determine whether SMC will increase the spread of SP and AQ resistant parasites. In our previous study that evaluated the impact of SMC during one season on resistance to SP, there was no difference in the frequency of SP resistance markers between parasites collected in treatment and non-treatment villages. It is unknown if longer implementation of SMC will accelerate the accumulation of resistant parasites, nor the status of development of resistance to AQ. Our recent evaluation of IPTp in Tanzania, an area with widespread resistance to SP, IPTp was associated with increased placental parasite density and inflammation. The current study will allow comparing the frequencies of SP and AQ resistance markers at the beginning of the study and during each subsequent year. The success of SMC in West Africa is due to limited spread of drug resistant parasites, however, changes in parasite population need to be monitored to ensure the effectiveness of this program.

Potential Use of Findings
SMC is a new intervention approved by WHO as policy in the March 2012. The main and urgent objectives of the project are to identify the most effective method to deliver SMC, and to obtain more information on the longterm impact of SMC on malaria immunity. Mali is one of the countries supported by the US President's Malaria Initiative (PMI) implemented through USAID. The aim of PMI is to reduce malaria-related deaths by 50 percent in selected countries by achieving 85 percent coverage of proven preventive and curative interventions, in support of the National Malaria Control Program. Finding the optimal delivery method of the SMC will allow optimal deployment of the strategy through the country for higher impact on the mortality and mortality from malaria in Mali and contribute in achievement toward the millimun development goal and the PMI objectives. The project will also assess the effect of SMC on malaria immunity when given over three consective years, so that better of understdaning of the malaria immunity will be gained and appropriate measures could be taken to accompagn the implementation of the strategy if needed The project will be carried out through local health system. Staff of the health center in selected villages will be trained to carry out the SMC interventions with support of the project team composed of MRTC, LMIV and the National Malaria Control Program. The project will collaborate with the President Malaria Initiative (PMI) of the USAID mission in Bamako. The MRTC is currently assuring the training, monitoring and evaluation of US President Malaria Initiative (PMI) program to Mali funded through USAID and the collaboration will continue through this project.

Overall objective
The overall objective is to identify the most effective method to deliver SMC, and to obtain more information on the long term impact of SMC on malaria immunity.

Overall design
The study will use a cluster randomized design over three years. Eight sub-districts will be randomly selected among the 13 sub-districts of Ouelessebougou but excluding the sub district of the Ouelessebougou where the IMEP cohort study is ongoing. These eight sub-districts will be randomized so that the intervention begins in four districts during the first year (Year 1), in two sub-districts in the second year (Year 2) and in two remaining sub-districts in third year (Year 3).
The target population is children aged 3-59 months of age in the district of the Ouelessebougou in Koulikoro region, in Mali.
In Year 1, the implementation of SMC will occur in four randomly selected sub-districts in Ouelessebougou. Villages in these sub-districts will be randomized at 1:1 ratio to receive either a door-to door delivery (DDD) or fixed point distribution (FPD) of SMC. They will also be randomized to receive SMC as directly observed therapy (DOT) or non-directly observed therapy (NDOT). A census of the target population (children aged 3-59 months) will be carried out in all the selected villages prior to the intervention. During this census, blood samples will be collected in a randomly selected subset of study population to gather baseline data on cellular and humoral responses to malaria antigens, parasites prevalence and density on blood smears and frequency of the molecular markers of the resistance to SP and AQ at the start of the transmission season. The census list will be used to deliver the intervention and will be regularly updated. SMC will consist of the administration of full treatment course of sulfadoxinepyrimethamine plus amodiaquine (SP+AQ) at monthly interval during the malaria transmission starting in August 2014. Children will be followed passively for the determination of the incidence of the malaria disease (uncomplicated and severe malaria). A cross-sectional survey will be carried after the third round in November/December to determine the coverage of SMC, the prevalence of the malaria parasitemia and anemia as well as humoral and cellular immune responses in a randomly selected number of children. Data collected in year 1 will be analyzed to determine the optimal delivery method (DDD+ DOT or DDD+NDOT or FPD+ DOT or FPD + NDOT) based primarily on the coverage of SMC and secondarily on the incidence of clinical malaria. The difference in SMC coverage is expected to be reflected in the incidence of clinical malaria, and the latter will be used to support the data on coverage of SMC. Should there be no statistically significant difference between the arms in coverage and incidence of the disease, the easiest method of the delivery will be recommended for Year 2.
In Year 2, SMC will be implemented using the optimal delivery system (determined in Year 1) in two sub-districts assigned during Year 2 after a census of children under 5 years of age.
Villages in these two newly selected sub-districts will be randomized to receive either three or four SMC rounds during the transmission season starting in August. Baseline data on malaria parasitemia, markers of resistance to SP and AQ and immune responses to malaria antigens will be collected at baseline prior to the intervention in these two sub districts through a crosssectional survey. In order to assess the effect of the fourth dose of SP and AQ, children will be followed passively to measure incidence rate of clinical malaria, and a cross-sectional survey will be carried out at the end of the malaria transmission season in November/December in a randomly selected sample of children to determine the prevalence of malaria infection, anemia, and humoral and cellular immune responses. Children that participated in Year 1 will continue to receive three rounds of SMC in Year 2 using the best delivery method identified in Year 1. The choice of the optimal number of doses will be based on the incidence of clinical malaria between the two arms of children in the two newly selected districts. An absolute difference of 7.5 episodes per 100 children per year will be considered meaningful.
In Year 3, all study children in the eight selected sub-districts will receive SMC by optimal method and frequency determined in Year 1 and Year 2 and will be followed passively for clinical malaria episodes through the end of the season. Venous blood samples will be collected at the beginning (July/August) and the end of the transmission season (November/ December) in a subset of randomly selected children to assess humoral and cellular responses to malaria antigens as well as the prevalence of malaria parasitemia among children who received SMC during one, two and three malaria transmission seasons. These outcomes will also be compared to those among children in the IMEP cohort who never received SMC, in a separate analysis taking into account the differences and similarities between the cohorts.

Detailed Description of Study Population
The study population will be children aged 3-59 months living in the districts of Ouelessebougou. The study will include up to eight sub-districts in the districts of Ouelessebougou estimated and up to 10,800 children less than five years of age.

Community consent
The objectives of the study and the method to conduct the study will be discussed with the Ministry of Health and district health officials prior to its commencement. Community approval will be sought through meetings with leaders of the study communities at the sub-district level as well as the village level. During these meetings the study procedures (including information on SMC delivery) will be explained to the leaders. There will be no special advertisement for a particular method. Parents will be informed to make the children in the target age group available at home in villages randomized to door to door delivery or to bring them to a specified fixed point in the villages randomized to fixed point delivery. Leaders will have the opportunity to ask questions and provide input on the best ways to encourage community participation. They will be asked together with the community health workers to pass the information to members of the communities.

Census and randomization
Once community consent to the trial is obtained, a household census will be conducted and all households within the study areas with children under 5 years of age will be identified. At the time of census, a preliminary screening of potentially eligible children will be undertaken.
Eligible children will be identified and written informed consent will be obtained from their caretakers for their inclusion in the trial. Children entered into the trial will be assigned a unique ID number and their demographic data (date of birth and/or age, and gender) will be collected.
The census will be updated at each SMC round and potentially eligible participants (such as children who did not reach the age of three months during the previous round) will be identified and screened for eligibility and enter into the study. This updated census will collect information on migration and death of children enrolled into the study for precise estimation of person-time of follow-up.
The unit of randomization will be village and all eligible children in a village will be allocated to the same study arm (DDD+ DOT or DDD+NDOT or FPD+ DOT or FPD + NDOT) to avoid confusion in Year 1. Villages in the selected sub-districts will be randomized at 1:1 ratio to receive either a door -to-door delivery (DDD) or fixed point distribution (FPD) of SMC. They will also be randomized to receive SMC as directly observed therapy (DOT) or a non-directly observed therapy (NDOT). In Year 2, villages in the two newly randomly selected districts will be randomized into two groups to receive either three or four rounds of SMC.

Inclusion and exclusion criteria
Children of either sex aged 3-59 months of age at the start of each period of drug administration will be eligible for inclusion in the trial, provided that parental consent is obtained. Children under the age of three months on the date of the start of drug administration will be excluded that year, as SMC is not recommended for children in this age group whose risk of acquiring malaria is low. Children who reach the age of 59 months or more at the end of the first or second years of treatment will not receive SMC in subsequent years. Exclusion criteria will be as limited as possible to make the results of the trial broadly applicable and will include i) a severe, chronic illness, ii) a known allergy to one of the study drugs, iii) known HIV positive subjects using Cotrimoxazole. Children with acute clinical malaria at the time of SMC drug administration will be treated by ACT and will not be given SMC drugs during that round as per WHO recommendation. They will receive SMC drugs during subsequent cycles.

Interventions
SMC will consist of the administration of full treatment courses of sulfadoxine-pyrimethamine plus amodiaquine (SP+AQ) at monthly intervals during the peak of the malaria transmission season (August -through October or November depending on the year of the study and study arm). During each round, children aged 3-11 months will receive 75 mg of AQ given once daily for 3 days plus a single dose of 250/12.5 mg of SP, while children aged 12-59 months will received 150 mg AQ base given once daily for 3 days and a single dose of 500/25 mg of SP. The single dose of SP will be given only on the first day, at the same time as the first dose of AQ.
Children will be observed for 30 minutes after drug administration. If vomiting occurs during this period, drugs will be re-administered. If vomiting recurs this will be noted but the drugs will not be given on a third occasion. Such children will not be excluded from the trial and they will be eligible to receive drugs at the subsequent monthly treatment.
In villages assigned to door -to-door delivery (DDD), community health workers (CHW) will visit each compound to administer SMC drugs. In the villages assigned to fixed point distribution (FPD), children will receive SMC at a central fixed point in the village. The administration of the three days treatments at each round will be done by the CHW for children assigned to directly observed therapy (DOT) while for children assigned to non-directly observed therapy (NDOT), the treatment of first day will be given by the CHW and treatments for second and third days will be given by the caregiver at home for each round. SMC will be implemented using

Follow-up and measurement of outcomes
Surveillance for clinical attacks of malaria will start at the time of the first administration of study drugs in August and continue until the end of the third malaria transmission (November/December 2016). Clinical episodes of malaria will be detected using a passive surveillance system. Data will be collected by health care workers including the community health workers. Clinical malaria episodes occurring in the study health facilities or in the study communities will be recorded throughout the study period in all the study villages. Children with an illness suggestive of malaria will be tested for malaria with RDT and/or with blood smear by the heath center staff and/or community health workers and treated according to the recommendations of the National Malaria Control Program. Cases of clinical malaria will be recorded on standardized case report forms. Refresher training of staff will be conducted each year to ensure standardization in the recording of symptoms and signs that are used to make a diagnosis and categorisation of clinical malaria episodes.
Each year, at the onset and the end of malaria transmission season, a cross-sectional survey will be undertaken in a random sample of children to assess the prevalence of malaria infection, anemia and immunological parameters. Coverage of SMC will be assessed at the cross-sectional survey at the end of the transmission seasons through interview of the caregivers and based on information in the child SMC administration card. Children will be examined, and a blood sample will be obtained for measurement of including hemoglobin concentration, preparation of a blood smear for microscopy, and analysis of cellular and humoral immune responses to known (CSP, MSP1, AMA1) and novel liver and blood stage P. falciparum antigens.

Definitions
The following definitions will be used during the course of the study. Anaemia. Anaemia will be defined as Hb concentration <11 g/dl, moderate anaemia as Hb concentration < 8 g/dL and severe anaemia as Hb concentration < 5 g/dl. (pfmdr1) will be monitored.

Laboratory Procedures
Detection of malaria. RDT will be used for the initial diagnosis of malaria and to guide treatment according the guidelines of the NMCP. Parasite prevalence during cross-sectional surveys will be determined using blood smears. Blood films collected at the same time will be read subsequently by two microscopists. Slides which are judged to be discordant for either positivity or parasite density will be read by a third reader. The final result will be the median of the two or three readings.
Measurement of haemoglobin concentration. Haemoglobin concentration will be measured colorimetrically using hemoglobin analyzer (Hemocue AB, Angelholm, Sweden). Immunologic assays will include humoral antimalarial immune response to blood stage and liver stage antigens, cellular antimalarial immune responses to liver stage antigens, levels of cytokines and other host responses (e.g., erythropoietin) measured in sera/plasma or in immune cells / immune cell subsets. Our primary immune endpoints for this study will be humoral immune responses to the well-characterized pre-erythrocytic antigen CSP, and blood stage antigens MSP1 and AMA1. All three antigens are candidate antigens for malaria vaccines, and the Laboratory for Malaria Immunology and Vaccinology developed a highly standardized assays to support preclinical and clinical development of vaccines based on these antigens. Responses to CSP reflect host exposure to the preerythrocytic parasite stages (ie, sporozoites and intrahepatocytic forms), while responses to AMA1 and MSP1 reflect exposure to the blood stage forms of the parasite.
In general, the immune mechanisms that mediate naturally acquired protection from malaria are not conclusively established. For this reason, we will survey a number of humoral and cellular immune responses as secondary or exploratory aspects of the study. For example, preerythrocytic forms of the parasite, when attenuated, can induce sterile immunity in humans, and available evidence suggests that different cellular and humoral immune responses may contribute to protection. For secondary immunological endpoints of this study, we will survey humoral and cellular immune responses to CSP and novel preerythrocytic antigens that may have a role in protective immunity. LMIV has identified several P. falciparum antigens whose orthologues confer protective preerythrocytic immunity in mouse malaria models, including PFL1995c, LISP1, and SAP1.
For blood stage immunity, it is known that naturally acquired IgG antibodies can clear parasite and resolve symptoms when given as passive therapy, but the targets of these antibodies are unknown. In addition to our primary endpoints of antibody levels against AMA1 nd MSP1, we will also measure functional activity of blood stage antibodies as secondary endpoints. These include antibodies that will block parasitized red cells from binding to endothelial and other receptors, which many scientists believe should reduce parasite sequestration and therefore malaria-related disease.
Based on the results of our primary and secondary immunologic studies, and the clinical findings of the trial, we may undertake more exploratory assays. For example, if the laboratory or clinical evidence suggests that SMC modulates protective antimalarial immunity, LMIV has genome based tools, such as transcriptomic or proteomic approaches, to broadly survey host responses (plasma, serum, cellular) that may be involved in malaria infection and disease.

Study discontinuation
Study may be discontinued if there is: 1. Evidence of high level of resistance of P. falciparurum to SP and AQ determined as prevalence of dhps 540 mutation > 50% or in vivo resistance of P. falciparum resistance to SP+AQ > 10% 2. Drastic reduction of the incidence clinical malaria to less than 0.1 episode per child per year without SMC intervention.

Subjects withdrawal criteria and handling
Withdrawal criteria will include: -Withdrawal of consent, HIV positive subjects under cotrimoxazole, -Allergy or intolerance of SMC drugs and -Travel outside the study villages.
For these subjects the administration of SMC drugs will be discontinued, but subjects will continue to receive routine health care services provided through the heath centers in the areas.

Quality Assurance/ Quality Control of Data and Collection
An independent GCP monitor will be appointed by the sponsor (PEER-Health or NIAID/NH) to ensure that the study is conducted according to the study protocol, GCP, ethical and regulatory requirements standards are met. The monitor will conduct an initiation visit, a close out visit and at least one additional visit each year. S/he will examine a random selection of clinical and laboratory records during each visit.
SMC implementation tools will be developed in collaboration with the district health authorities.
Standard operating procedures will be developed for each of the key activities including (consent, census, SMC administration, clinical exam, diagnosis and treatment of the malaria, sample collections and processing and lab techniques. Activities will be carried out by competent and trained staff. Initial training on the study protocol, GCP and GLP will be provided to the health staff in the intervention villages, as well additional staff of the project. Refresher training will be provided at least one year. Data will be collected in standardized forms. Quality control will be performed on sample of the RDTs in a routine manner and feedback and refresher training will be provided according to the procedures of the district health center. SMC drugs will be sourced through reliable channels to ensure quality. SMC drugs, ACT for treatment of uncomplicated malaria, injectable quinine and artesunate for treatment of severe malaria, and RDT for malaria diagnosis will be provided by the USAID PMI as part of their support for SMC implementation and malaria control in Mali. Quality control of the drug will be performed by independent laboratory through USAID-PMI and CDC. Diagnosis and treatment for malaria in under -5 children is free in Mali.

Patient Safety Considerations
Risks to participants in the trial are small. The trial will use only licensed products and does not involve any new methods. Over 800,000 courses of SP and AQ have been given for SMC without any report of a drug associated severe adverse event (SAE) including when enhanced surveillance was used [

Definitions and Procedures for recording and reporting Adverse Events (AE) and Serious Adverse Events (SAE)
An adverse event (AE) is defined as any clinical symptom or sign that occurs in study children after administration of the study drugs that may or may not have a causal relationship with the study drugs. A serious adverse event (SAE) is any clinical condition that fulfils at least one of the following criteria: results in death, is life-threatening (the child was at risk of death at the time of the adverse event), results in disability/incapacity. AE and SAE will be classified by severity, causality and outcome as described below Severity. The severity of a clinical adverse event is to be scored according to the following scale: • Mild: Awareness of sign or symptom, but easily tolerated.
• Moderate: Discomfort enough to cause interference with usual activity.
• Severe:Incapacitating with inability to perform usual activity.
• Life-threatening: Patients at risk of death at the time of the event.
• Death Assessment of Causality and Outcome. The relationship between the study drugs and the occurrence of each AE/SAE will be determined based on their clinical judgment. Alternative causes, such as the natural history of the underlying diseases, concomitant therapy, other risk factors, and the temporal relationship of the event to the study drug will be considered and investigated. The relationship of an adverse event to study drug is to be assessed according to the following definitions: • Definitely unrelated: events that had occurred prior to administration of the study drugs or events that are obviously unrelated to the study (e.g. accidental injury).
• Unlikely: There is no reasonable temporal association between the study drug and the suspected event and the event could have been produced by the child's clinical state or other concomitant medications.
• Possible: The suspected adverse event may or may not have a reasonable temporal association with the administration of study drug but the nature of the event is such that an association with the study drug cannot be ruled out. The event could be related to the child's clinical state or by concomitant medications.
• Probable: The suspected adverse event follows a reasonable temporal sequence after administration of study drugs, abates upon discontinuation of the drug, and cannot be reasonably explained by the known clinical state of the child.
• Definitely related: events that have no uncertainty in their association to the administration of study drugs.
Outcome. The outcome of each AE will be assessed according to the following classification: • Completely recovered : The child has fully recovered with no observable residual effects • Not yet completely recovered : the child's condition has improved, but still has some residual effects The AE remains the same as at onset • Unknown : The outcome of the AE is not known because of lost to follow-up

Disaster Recovery Plans
Study data will be regularly entered in to DataFax database that is supported and maintained by the NIAID. As with ongoing IMEP study data will maintained by the DataFax support team at NIAID/NIH with rigorous the back-up systems. The system has the advantage of generating automatically pdf copies of the forms that can be printed to replace the records in case of disaster.

A. Sample Size and Power Calculation(s)
Cross-sectional surveys. Sample size for cross-sectional surveys will be based on the SMC coverage. Assuming 60% average coverage of SMC by the combination of nonoptimal methods (pools of two groups), 300 children/arm will be needed to detect 10% increase in average coverage for two pooled groups with at least 85% power correcting for multiple tests and using

Passive survaillance.
Assuming a clinical malaria attack rate of 0.3 per child per year in "3 rounds" arm, to detect an absolute difference in attack rate of the 0.075 episode/chil/ year (corresponding to 25% reduction) in the "4 rounds" arm, with a one sided p-value of 0.025 and power of 80%, using a quasi poisson model accounting for 20 equally sized clusters per arm with intraclass correlation of 0.01 and 5% missing data we will need 1386 per arm, or 2,772 subjects total.
Immunologocal parameters. We will base our sample size calculations for immunological endpoints on the humoral immune response to AMA1. We select this response because AMA1 is highly immunogenic from among malaria antigens. Also, the intervention in this trial is directed at reducing the blood stage burden of parasites during the malaria season, and the response to (before vaccination) were 40 (25-75) antibody units. Based on these data, 467 subjects per group will provide 80% power, to detect a 2 fold or greater reduction in peak AMA1 antibody levels among the children receiving SMC, accounting for cluster sampling in 10 equally sized clusters with 0.01 intraclass correlation as well as 10% missing data given that samples may be inadequate or spoil during routine collection and transport.

B. Planned Interim Analyses (if applicable)
At the end of the first transmission season, data will be analyzed to determine the optimal mode of delivery by comparing primarily the coverage of SMC and secondarily the incidence rate of the clinical malaria between the four intervention arms. In addition, descriptive analysis of the baseline frequencies of the immunological parameters, and molecular markers of the resistance of P. falciparum to SP and AQ, will also be performed at the end of Year 1.
Data collected in Year 2 will be used to determine the optimal frequency of the SMC rounds by comparing primarily the incidence of clinical malaria and secondarily the parasite prevalence at the end of season between the 2 arms. Descriptive analysis of immunological parameters and markers of drug resistance will also be performed.
Details of the statistical methods are provided below in the final analysis plan.

C. Final Analysis Plan
Describe statistical methods that will be used to analyze data.
Data will be entered and verified using DataFax. The cleaned database will be exported to Stata (Houston Texas USA) or SAS for analysis. An intention-to-treat analysis will be used except for the immunological endpoints for which the exact number of the SMC treatments received will be considered.
SMC coverage will be determined using the information on the SMC card and from interview of parent or care giver and will be determined as the proportion of children who received all the treatments at all the SMC rounds during the transmission season. The proportion of children who received at least the first treatment dose of the SMC will be also estimated as secondary endpoints for SMC coverage. SMC coverage will be compared between arms and years of interventions with 95% confidences intervals adjusted for cluster design (using cluster option in Stata for example). The DDD+NDOT arm will be considered as control group.
The census of the children will be updated at each round and at the end of the transmission season to allow a good estimation of person-time of follow-up. Cases of the clinical malaria cases will be recorded and the incidence rate of the clinical malaria defined as the number of clinical malaria episodes divided by total child days at risk. Children will not be considered at risk 21 days after a clinical malaria episode. Incidence rates of clinical malaria will be compared between treatment arms using generalized estimating equation to estimate the incidence rate ratio, with adjustment for age, previous SMC regimens and the lack of independence among repeated episodes in the same child.
Proportions of other secondary endpoints such as parasite prevalence, prevalence of anemia and frequency of molecular markers of resistance to SP and AQ and adverse events associated with study drugs will be estimated and compared between groups using generalized linear models with 95% confidence intervals, adjusted for cluster design and potential confounding variables such age and use of ITN.
Differences at baseline will be compared using Chi-squared or Fisher's exact tests as appropriate for categorical variables and by analysis of the variances for quantitative variables that are normally distributed. Nonparametric tests will be used for non-normally distributed data.

A. Ethical Standard
The study will be conducted in compliance with the protocol, current Good Clinical Practice (GCP) guidelines, and all applicable regulatory requirements.

B. Human Subjects Considerations i. Potential Risks
Risks to participants in the trial are small. The trial will use only licensed and recommended drugs and does not involve any new methods. The risks to the participants are limited to those associated with the use of SP + AQ and blood sampling. Risks occasionally associated with finger and heel pricks and venipuncture include pain and bruising at the site of the prick, and rarely infection. Use of SP +AQ can be associated with side effects of the drugs including allergy. Subjects with known allergy to the study drugs will be excluded. Over 800,000 courses of SP and AQ have been given for SMC without any report of a drug associated severe adverse benefits conveyed by the intervention. Samples collected on filter paper will be tested for markers of the resistance to SP and AQ at baseline and two years after to monitor the impact of the sensitivity of the malaria parasites to the study drugs as per the WHO recommendations.
Conduct of the trial will not impose any additional costs on the local health services. The project will contribute to the additional costs associated related to the study and the strengthening the health system in the study areas.

ii. Potential Benefits
The benefits of participation to the study include the protection against clinical malaria conferred by SMC and improved supervision of the health care provided by the CHW and the health staff.
Finding the optimal delivery method of the SMC will allow optimal deployment of the strategy through the country for higher impact on malaria morbidity and mortality in Mali and other countries. The study will also provide insights into the acquisition of malaria immunity and may indicate whether the cessation of longterm SMC will increase risk of subsequent malaria and warrant additional control measures.

C. Institutional Review Board (IRB)
The protocol and consent forms and any subsequent amendment will be submitted for approval to Ethical Committee of the Faculty of Medicine, Pharmacy and Dentistry of the University of Bamako (US, DHHS/OHRP Federal Wide Assurance #: FWA00001769). Individual consent will be obtained before inclusion in the study.

Community Consent
The previous studies conducted by the MRTC in Ouelessebougou have permitted extensive contacts with the village population that has led to the development of mutual trust and the establishment of an ongoing informed consent process. The community has become familiar with the informed consent process, including written, signed consent forms. The community informed consent process goes through the following steps: 1.
Explanation and clarification to village leaders, including the village chief and elders, of the purposes, procedures, and risks to the study subjects.

2.
Allow time for village leaders to communicate with community members and relay any additional questions or concerns.

3.
Thorough protocol explanation to the family heads.

Individual Consent
The informed consent document will be used to explain the risks and benefits of study participation to the parent or guardian in simple terms before a subject is enrolled in the study. The informed consent document contains a statement that the consent is freely given, that the subject is aware of the risks and benefits of entering the study, and that the subject is free to withdraw from the study at any time. Written consent will be given by the parent/guardian, after receiving detailed information on the study. Written informed consent must be obtained for each subject prior to enrolment in the study. The informed consent form will be signed and personally dated by the child's parent or guardian. The person who conducts the informed consent discussion should also sign and date the inform consent form. A copy of original signed informed consent form will be retained in the participant's chart and another will be provided to him. A participant who is unable to read or write will place an imprint of his finger in the place of a signature; in addition, an independent witness will sign the consent form to attest that the information in the consent form was orally conveyed to the participant. A copy of the inform consent document in English is provided in Annex 4.

i. Minors or subjects with cognitive impairment
Minor subjects (under 5 years of age) with or without cognitive impairment will be enrolled into this study after inform consent granted by the parent or guardian.

E. Subject Confidentiality
The investigator will ensure that the subject's anonymity is maintained. Participants will not be identified in any publicly released reports of this study. All records will be kept confidential to the extent provided by laws and regulations. The study monitors and other authorized representatives of the regulatory authorities may inspect all documents and records required to be maintained by the Investigator. All laboratory specimens, evaluation forms, reports, and other records that leave the site will be identified only by a coded number in order to maintain subject confidentiality.

F. Study Discontinuation
Implementation of SMC has already started the district of Koutiala, in Mali. The plan is to expand it to additional districts each year. It is expected that by the end of this project in 2017, SMC implementation will be expended to the whole country including the district of Ouelessebougou. PMI is one of key partners in Mali that is now supporting SMC implementation in Mali. Should the extension of the SMC implementation delayed, the investigators will work with the National Malaria Control Program and PMI to insure that implementation of the SMC using the optimal approach in Ouelessebougou continue.

Waste Management and Monitoring Plan
MRTC has been conducting clinical research with NIAID/NIH for more than 20 years. As results of this collaboration the MRTC is equipped with CAP certified clinical laboratory. The staff is well aware and trained on the waste management and monitoring. Waste management and monitoring will performed according the SOP provided in Annex 2. The primary waste associated with the study includes biohazard waste (solid and liquid). Clinical and laboratory staff are responsible for safe disposable of biohazard waste according to the SOP. This responsibility includes segregating non-hazardous waste (like paper) from hazardous waste to minimize environment impact. Laboratory and clinics are supplied with protective clothing such as lab coat and disposable gloves and material to collect biohazard waste. IMEP program facility located at Ouelessebougou is equipped with an incinerator for the treatment of solid waste.
Waste produced by outlined clinics will be treated locally if incinerator is available or will be brought to the central lab facility at Ouelessebougou. Sites equipped with incinerator will serve as the final disposal sites. A plan to vaccinate team members with hepatitis B vaccine will be implemented as part of the overall safety management prior to the study initiation. As with all clinical, laboratory and data procedure, SOP adherence is monitored on a regular basis by the program PIs.

Vertebrate Animal Welfare
Not applicable.

Capacity Development
The project will build capacity in the local health system and at MRTC. Staff of the health center in selected villages and at the district levels will be trained to carry out the SMC interventions with support of the project team composed of MRTC, LMIV and the National Malaria Control Program. Implementations, monitoring and evaluations tools will be developed and could be used in other districts. Cases of malaria will be properly diagnosed, treated and reported. Integrated diseases surveillance system will be strengthened. Junior scientists at MRTC will work on this project as part of the continued training and capacity building at MRTC. The project will contribute to acquire new clinical lab and IT equipment and to maintain existing equipment that will be used for the project at MRTC. The data collected will be used as part of Master, MD, PharmD and PhD thesis at the University of Bamako and elsewhere. .

Data Dissemination Plan and Knowledge Transfer
The study findings will be presented and discussed during the regular meetings of the project

Detailed Timeline
Timelines of the key study activities are summarized in Table 1. 28

Detailed Budget
Provide a detailed budget for the study and a detailed justification/narrative of the budget. Identify all sources of funding.
Les Un autre objectif spécifique est de déterminer le nombre de rounds de traitement. Efficace dans les essais qui ont servi de base pour l'élaboration de la politique de CPS par l'OMS, les enfants ont reçu trois rounds de traitement par an, la politique de l'OMS suggère jusqu'à quatre rounds de traitement par an. En tenant compte de la logistique associée à chaque round de traitement, il est essentiel de déterminer si un 4ème round (passage) aura un avantage supplémentaire par rapport à trois rounds (passages) de traitement. Dans notre précédente étude qui a évalué l'impact de la CPS au cours d'une saison sur la résistance à la SP, il n'y avait pas de différence dans la fréquence des marqueurs de résistance de SP entre les parasites recueillis dans les groupes qui ont reçu le traitement et les groupes qui non pas reçu le traitement. On ne sait pas si la mise en oeuvre de la CPS à long terme ne va pas accélérer l'accumulation de parasites résistants, ou le développement de la résistance à SP et AQ. L'évaluation du TPI pendant la grossesse en Tanzanie, une zone avec une résistance généralisée à la SP, a montré que le TPI pendant la grossesse était associé à une augmentation de la densité parasitaire placentaire et l'inflammation. L'étude actuelle va permettre la comparaison des fréquences des marqueurs de résistance à SP et à AQ au début de l'étude et à chaque année de suivi. Le succès de la CPS en Afrique de l'Ouest est dû à une propagation limitée de parasites résistants aux médicaments, cependant, les changements dans la population de parasites doivent être surveillés pour s'assurer de l'efficacité de cette stratégie.

Objectifs spécifiques
 Déterminer le mode optimal et la fréquence optimale d'administration de la CPS  Comparer les mesures quantitatives de l'immunité chez les enfants qui ont reçu la CPS et les enfants qui n'ont pas reçu la CPS.
 Evaluer l'impact de la CPS à long terme sur la fréquence des marqueurs moléculaires de la résistance de P. falciparum à la SP et AQ.

Les critères de jugement principaux sont les suivants:
 La couverture de la CPS pour la détermination du mode optimal de distribution lors de la première année. La couverture de la CPS sera définie comme la proportion d'enfants ayant reçu les trois doses de traitements à chacun des trois passages de la CPS.

Recensement et randomisation
Une fois la permission communautaire est obtenue, un recensement des enfants
Les enfants atteints de paludisme clinique aigu au moment de l'administration du médicament de la CPS seront traités par CTA et ne recevront pas les médicaments de la CPS pendant ce round conformément à la recommandation de l'OMS. Ils recevront des médicaments de la CPS au cours des rounds ultérieurs.

Analyses intermé diaires prévues:
A la fin de la première saison de transmission, les données seront analysées pour déterminer le mode optimal de distribution en comparant principalement la couverture de la CPS et secondairement le taux d'incidence des accès palustres entre les quatre groupes d'intervention. En outre, une analyse descriptive des fréquences de base des paramètres immunologiques et les marqueurs moléculaires de la résistance de P.

Avantages potentiels
Les avantages de la participation à l'étude comprennent la protection contre le paludisme clinique conférée par la CPS et l'amélioration de la supervision des soins de santé fournis par l'ASC et le personnel de santé.
Trouver la méthode de distribution optimale de la CPS permettra un déploiement optimal de la stratégie à travers le pays pour un impact plus important sur la morbidité et la mortalité palustre au Mali et dans d'autres pays. L'étude devra également fournir des informations sur l'acquisition de l'immunité contre le paludisme et peut indiquer si l'arrêt à long terme de la CPS n'augmentera pas le risque de paludisme ultérieur et justifier des mesures de contrôle supplémentaires.

Fin de l'Étude
La mise en oeuvre de la CPS a déjà commencé dans le district de Koutiala au Mali ainsi que dans quatre autres districts. Le plan est de l'étendre à d'autres districts chaque année. Il est prévu que d'ici la fin de ce projet en 2017, la mise en oeuvre de la CPS sera étendue sur l'ensemble du pays, y compris le district de Ouéléssébougou. PMI est un des principaux partenaires du Mali qui soutiennent la mise en oeuvre de la CPS au Mali. Si l'extension de la mise en oeuvre de la CPS est retardée, les investigateurs travailleront avec le PNLP et le PMI pour s'assurer que la mise en oeuvre de la CPS utilisant l'approche optimale continue à Ouéléssébougou à la fin de cette étude.