Sickle cell anemia (SCA), one of most prevalent monogenic diseases worldwide, is caused by a glutamic acid to valine substitution on the beta globin protein of hemoglobin, which leads to hemolytic anemia. Hydroxyurea, the only disease-modifying therapy approved by the Food and Drug Administration for SCA, has proven to be a viable therapeutic option for SCA patients in resource-rich settings, given clinical improvements experienced while taking the medication and its once-daily oral dosing. Significant studies have demonstrated its safety and clinical efficacy among children and adults in developed countries. In Sub-Saharan Africa, however, the risk of malaria, hematologic toxicities, and safety of hydroxyurea in children with SCA living in malaria-endemic areas are unknown.Study objectives include determining the incidence of malaria in SCA patients taking hydroxyurea versus placebo; establishing the frequency of hematologic toxicities and adverse events (AEs) in children with SCA treated with hydroxyurea versus placebo; and defining the relationships between hydroxyurea treatment and fetal hemoglobin, soluble intracellular adhesion molecule-1, and nitric oxide levels, and between levels of these factors and risk of subsequent malaria.Novel use Of Hydroxyurea in an African Region with Malaria  is a prospective, randomized, placebo-controlled, double-blinded phase III trial to compare risk of malaria with oral hydroxyurea versus placebo. Children will be recruited from the Mulago Hospital Sickle Cell Clinic in Kampala, Uganda.Two hundred Ugandan children aged between 1.00 and 3.99 years with confirmed SCA will be randomized into treatment groups by order of entry in the study, based on a predetermined blinded randomization list. The primary outcome of the trial is malaria incidence in the 2 study groups, defined as episodes of clinical malaria occurring over the 1-year randomized study treatment period.NOHARM will be the first prospective randomized, placebo-controlled clinical trial investigating the use of hydroxyurea for children with SCA in a malaria-endemic region within Africa. The results of this trial have the potential to significantly advance understanding of how to safely and effectively use hydroxyurea in children with SCA in malaria-endemic areas.Clinicaltrials.gov NCT01976416; https://clinicaltrials.gov/ct2/show/NCT01976416 (Archived by WebCite at http://www.webcitation.org/6hmoilZnp) Sickle cell anemia (SCA) is a genetic blood disorder where sickling of red blood cells, due to a genetic alteration in hemoglobin, leads to chronic hemolysis, organ damage, vaso-occlusive events, and other potentially life-threatening complications . It is aDespite being the most prevalent genetic disease in the African region, disease prevention and management for SCA are inadequate . With thHydroxyurea, an inducer of fetal hemoglobin (HbF), saw its first clinical application for SCA in 1984. The drug has been shown to dramatically reduce vascular crises, hospitalizations, and the need for transfusions among SCA patients. It is inexpensive, can be given with once-daily dosing, and has been shown to be safe and effective from the age of 9 months to adulthood [At the time of this paper’s publication, however, hydroxyurea is not the standard of care in Sub-Saharan Africa for SCA, as the safety of the drug in a malaria-endemic region is not known. Studies have shown that children with SCA have higher mortality rates from malaria than children with either sickle cell trait  or children with normal hemoglobin. Children with HbAS are strongly protected from severe malaria , and thiMany of the factors relating to vaso-occlusive crises and other complications in SCA are important in the pathogenesis of severe malaria. Factors associated with severe malaria that may be affected by hydroxyurea in children with SCA include intracellular adhesion molecule-1 (ICAM-1) ,14, vascPlasmodium falciparum growth in vitro [decreased risk of malaria in children taking hydroxyurea. However, the effect of hydroxyurea on markers of endothelial activation is less clear. Early studies found that hydroxyurea raised concentrations of ICAM-1 in an in vitro model of SCA [Plasmodium species other than P. falciparum, which may interact differently with ICAM-1 than P. falciparum. In vitro studies suggest that hydroxyurea decreases VWF incorporation into the endothelial cell extracellular matrix [Hydroxyurea is thought to prevent vaso-occlusive crises and pain episodes in children with SCA in part through generation of NO ,27,28. Tin vitro  and is tin vitro , and hydl of SCA , raisingl of SCA , and anol of SCA . A subsel of SCA , but ther matrix , but humr matrix . Early sr matrix , but oner matrix , and anor matrix . A recenr matrix , but it The hematologic toxicities of hydroxyurea, particularly neutropenia, are additional important safety concerns for children with SCA in malaria-endemic areas of Sub-Saharan Africa. The recently completed BABY-HUG study reported hematologic toxicities with hydroxyurea treatment that included severe neutropenia (5%), severe anemia (1%), and thrombocytopenia (11%), although similar toxicities were also noted in the placebo-treated arm . MalariaIn summary, some changes associated with hydroxyurea treatment (increased NO and HbF and improved splenic function) would be expected to protect against malaria, but the data on hydroxyurea-related endothelial changes thought to be important in severe malaria pathogenesis  are less clear, with some studies suggesting that these factors might be increased with hydroxyurea and others suggesting no difference or even a decrease. The frequency, severity, and consequences of the hematologic toxicities of hydroxyurea in children with SCA in malaria-endemic areas are also unknown. Thus, a prospective clinical trial to determine definitively whether hydroxyurea poses special risks to children with SCA in malarial regions is warranted.The objectives of the study are:1. To determine the incidence of malaria in children with SCA treated with hydroxyurea versus placebo: The working hypothesis of this aim is that incidence of malaria is not greater in children with SCA treated with hydroxyurea in comparison to those treated with placebo. We will test this hypothesis by comparing malaria incidence over a 1-year period in children with SCA in the hydroxyurea versus placebo treatment groups.2. To establish the frequency of hematologic toxicities and AEs in children with SCA treated with hydroxyurea versus placebo: The working hypothesis of this aim is that children with SCA treated with hydroxyurea will have more medication-related hematologic toxicities, such as neutropenia, but no increase in SCA-related AEs  compared with children treated with placebo. We will test this hypothesis by comparing hematologic toxicities and AEs in children with SCA in the hydroxyurea versus placebo treatment groups.3. To define the relationship between hydroxyurea treatment and HbF, sICAM-1, and NO levels and between levels of these factors and risk of subsequent malaria. The working hypotheses of this aim are that: (1) hydroxyurea will increase HbF and plasma NO levels and decrease plasma sICAM-1 levels and (2) HbF and plasma NO levels will inversely correlate, and plasma sICAM-1 levels will positively correlate, with subsequent malaria incidence. We will test this hypothesis by testing the associations between (1) hydroxyurea and change in levels of HbF, sICAM-1, and NO at 2-, 4-, and 12-month follow-up and (2) change in levels of HbF, sICAM-1, and NO at 2 and 4 months and risk of subsequent malaria.We are conducting a prospective, randomized, placebo-controlled, double-blinded phase III trial to compare oral hydroxyurea with placebo among Ugandan children with SCA. The trial opened to accrual in September 2014, and we estimate that the 2-year follow-up will be completed in December 2017.Study participants will be recruited from the Mulago Hospital Sickle Cell Clinic (MHSCC) in Kampala, Uganda. The MHSCC is the first and largest specialized clinic for the treatment of SCA in Uganda. The clinic was established by Professor Christopher Ndugwa in 1968, and since its inception, over 11,000 SCA patients have been registered at the MHSCC. Currently, approximately 3,500 patients are actively treated at this clinic, about 20% of whom are aged younger than 4 years. Kampala is an area of mesoendemic malaria transmission, but the outpatient and inpatient malaria burden is still high. Malaria is the primary diagnosis in more than 2,500 children admitted annually to Mulago Hospital and more than 20,000 children seen as outpatients at Mulago Hospital.Children were recruited for the Novel use Of Hydroxyurea in an African Region with Malaria (NOHARM) study through the MHSCC Database of active patients, using the following criteria:1. Pediatric patients with documented SCA .2. Age range of 1.00-3.99 years, inclusive, at the time of enrollment.3. Weight at least 5.0 kg at the time of enrollment.4. Willingness to comply with all study-related treatments, evaluations, and follow-up.Children who meet the aforementioned criteria, but who are acutely ill at the time of recruitment will be asked to return to clinic at a later date to establish baseline laboratory values and ensure that they do not meet any of the following exclusion criteria:1. Active use of hydroxyurea on a regular basis.2. Known chronic medical condition .3. Severe malnutrition determined by impaired growth parameters as defined by WHO (weight for length and height < −3 z-scores below the median WHO growth standards).9/L, ARC <80 × 109/L with Hb <7.0 gm/dL, platelets <80 × 109/L, and absolute neutrophil count (ANC) <1.0 × 109/L.4. Preexisting severe hematologic toxicity: Hb <4.0 gm/dL, Hb <6.0 gm/dL with absolute reticulocyte count (ARC) <100 × 105. Alanine aminotransferase or creatinine >2× the upper limit of normal for age.6. Blood transfusion within 30 days before enrollment.Medical evaluation and treatment for standard clinical issues will be performed as per the MHSCC guidelines. Control and treatment groups will receive the same standard care and will complete evaluations in the same time frame. Standard preventive measures provided in the MHSCC for children with SCA include the following: (1) folic acid (1 mg of PO) daily; (2) penicillin prophylaxis (250 mg PO) daily for all children aged younger than 5 years; (3) sulfadoxine-pyrimethamine prophylaxis, once monthly; and (4) anthelmintic treatment every 6 months. All children in the study will also receive an insecticide-treated bednet (ITN) because of the documented benefits of ITNs in population-based studies of malaria .Eligibility screening and Baseline Evaluations will occur together at month 0. Study treatment initiation will also occur at month 0 or within 1 to 3 days of being found eligible for randomization. Study treatment will commence at 20 ± 2.5 mg/kg of PO daily , with appropriate adjustments as needed for hematologic toxicities as outlined in the following section. The appropriate dose will be achieved using a combination of 100 mg and 1000 mg (4 × 250 mg scored) tablets.Using the participant’s weight at month 0, the daily dose will be calculated using available tablet sizes and a goal of 20 ± 2.5 mg/kg/day using dosing tables based on the participant’s weight. At each interval visit, laboratory studies will be used to assess treatment toxicity, typically anemia or neutropenia, but possibly also reticulocytopenia or thrombocytopenia. The daily dose will be held or lowered based on protocol treatment toxicity guidelines. Medication adherence will be assessed at each visit, and attempts will be made to collect treatment compliance data.Ethical Considerations section.Due to the concerns about potentially increasing the risk of severe malaria with the use of hydroxyurea, a placebo-controlled randomized clinical trial is the only way the issue of hydroxyurea safety in this patient population can be resolved. The risk of death from malaria makes it imperative that the effects of hydroxyurea on malaria be studied in Ugandan children with SCA who are at risk of malaria. The study design was discussed and refined with local academic leaders before submission and approval by the Makerere School of Medicine Research Ethics Committee and the other institutional review boards (IRBs) listed in the following Plasmodium species on microscopy. All parents or guardians are asked to bring their children to the MHSCC for any illness, and we anticipate compliance to be high because free evaluation and care by study personnel are available at the clinic. Children will be evaluated for malaria by a study clinician, and microscopy will be done by study personnel trained in malaria microscopy. All blood smears will be read independently by 2 readers, and any reading with a discrepancy between the 2 readers will have a third reading done to establish final diagnosis.The primary outcome of the NOHARM trial is malaria incidence, defined as episodes of clinical malaria occurring over the 1-year randomized study treatment period. Clinical malaria is defined as a history of fever or presence of measured fever (axillary temperature ≥37.5°C) in a child with a blood smear positive for P. falciparum parasitemia >1000 parasites/µL; (2) measured axillary temperature ≥37.5°C; (3) P. falciparum parasitemia >1000 parasites/µL and measured axillary temperature ≥37.5°C; and (4) malaria requiring hospital admission. The following covariates will be assessed: (1) local council area or village (location of household where child lives), as a surrogate for malaria exposure. Malaria exposure in Kampala is heterogeneous. Randomization should effectively place children from different areas of exposure risk evenly in the 2 treatment groups, but we will assess this; (2) socioeconomic status (SES). We have developed a simple tool for evaluation of SES of the families of children in our malaria studies. SES is often related to malaria exposure; (3) age and gender.To assess incidence of malaria by additional criteria that may add specificity (at the cost of some sensitivity), we will add the following definitions as secondary outcomes. These definitions will include all children with the primary definition but with the added criteria of the following: (1) Dose Toxicity section.The primary outcomes include: (1) composite outcome: one or more of the SCA-related AEs summarized in The secondary outcomes include: (1) individual AEs, including painful events and (2) toxicities for hemoglobin, reticulocytes, neutrophils, or platelets. The following covariates will be assessed: age; gender; and weight and height for age, weight for height (nutrition).The primary outcome will be change in percent HbF and plasma concentration of sICAM-1 and NO.The secondary outcome will be change in concentration of plasma VCAM, VWF, and TNF. HbF levels and initial hemoglobin S testing will be performed by capillary electrophoresis (Sebia MINICAP) in the Uganda Cancer Institute laboratory. sICAM-1 and soluble VCAM-1 will be assessed by ELISA (R&D Systems), VWF activity by ELISA (Corgenix), and TNF-α by cytometric bead assay (EMD-Millipore). All these tests will be done in the University of Minnesota or University of California—San Francisco or Makerere University laboratory in Kampala, Uganda. NO levels will be measured by fluorometric assay of total plasma nitrate and nitrite (EMD-Millipore). NO levels will be tested at the Indiana University because a fluorometric assay reader is not available on the Makerere campus.Subjects will have clinic visits and laboratory assessments at specific time points as detailed in We aimed to randomize 200 children with SCA into study arms (100 in each treatment arm). Twelve months of age was chosen as the minimum age based on published experience from the phase III infant hydroxyurea (BABY HUG) clinical trial . An uppeFor Specific Aim 1, the detectable difference in malaria incidence depends on incidence in the control group. Few relevant data are available for children with SCA in Uganda, so, we present a range of detectable differences corresponding to a range of 0.3 to 1.3 malaria incidents per child in a year in the control group . With 100 children per group, these control-group rates imply 90% power to detect between 59% and 34% difference between groups, respectively in malaria incidence For Specific Aim 2, the outcome of greatest interest for this aim is the occurrence of 1 or more AEs. We estimate that approximately 50% of control-group children will have 1 or more of the AEs summarized in For Specific Aim 3, to test the association of study treatment with each marker, we have 80% power to detect a difference between groups of 0.40 standard deviations in the marker (this is conservative and is based on having a single follow-up marker measurement per child instead of the 3 we will have). To analyze the association between markers and malaria episodes, the detectable difference in rate of malaria episodes depends on the rate of episodes per child. For a low rate of 0.3 episodes per child in 10 months, we have 80% power to detect a rate ratio of 2.1 for a 1-standard-deviation change in the marker; for a high rate of 1.2 episodes per child, the detectable rate ratio is 1.5.Families of recruited subjects will be informed of study procedures and the expected follow-up schedule. Subjects will be enrolled by the study nurse or medical officer based on meeting inclusion criteria, not meeting exclusion criteria, and being able to maintain the follow-up schedule. Subjects will be enrolled after written informed consent has been obtained from the parent or guardian, according to the guidelines of the main IRB and the local ethics committees , 3 and 4Block randomization will be used and will occur via the OnCore Electronic Data Capture system. OnCore is a software system widely used in many clinical research centers across the United States and internationally. The data coordinating center (DCC) will have uploaded randomization “block files” into the system so that participants can be randomized into the study.Study participants will be randomized by the DCC staff into treatment groups by order of entry in the study, based on the predetermined blinded randomization list. The child’s study identification number will be recorded, and treatment group may only be determined by comparing the child’s study identification number to the blinded list, which only the DCC staff will have access to until the study is completed or stopping rules are reached and unblinding is required. The study pharmacist will have identical appearing hydroxyurea or placebo tablets and will provide the appropriate medication to the child. Dosage adjustment will be done according to the aforementioned guidelines so that if lower counts occur on placebo, placebo dose will also be lowered. Neither study participants nor their care providers, investigators, study personnel treating the participants, study coordinators, and outcomes assessors will know which arm a child is in. Laboratory values for scheduled visits are entered in the database and provided to the clinicians taking care of the study participant only if the laboratory value is a critical value.Before site activation, the study investigators will ensure that adequate clinical, laboratory, pharmacy, data management facilities, and operations are in place. In addition, adequate training in all aspects of the trial, including electronic data entry, will be conducted. Research staff will undergo retraining as needed to ensure sound practices are maintained.OnCore is Web based and password protected. The DCC has been using it for clinical trials management since 2009. Security privileges will be assigned in OnCore based on team roles and duties, within the context of the protocol. OnCore allows for validation of electronic case report forms, which allows data to be locked once they are verified.Data from this study will be reviewed in real time by the study team members. Source documents will be reviewed, and data entries will be confirmed. Inconsistencies will be examined with the data manager, and corrective actions will be taken as needed. In regard to subject confidentiality, coded numbers will be used for identification of participant records and laboratory specimen. Clinical information will not be released by the NOHARM Medical Coordinating Center (MCC) or DCC without written permission from the subject, parent, or guardian, except as necessary for monitoring. Clinical information can be shared by the site investigators and staff for patient care reasons, for example, local consultations. The final trial dataset will be kept by the DCC, and no one outside the DCC, including the Principal Investigator, will have access to the randomization information or to the full trial dataset until the end of the randomized trial, unless findings in interim analysis lead the data safety and monitoring board (DSMB) to request study un-blinding. The DCC and MCC have a written agreement clarifying these roles.Efforts for participant retention in the trial will be encouraged through the regular contact via visits for refill of hydroxyurea or placebo and provision of ongoing clinical care for the duration of the study. Outcome data will be recorded for those who withdraw from the study up to the time of withdrawal.Specific Aim 1, we will compare the hydroxyurea versus placebo groups according to total malaria incidence (inpatient and outpatient) over the 12-month treatment period, using an intention-to-treat analysis. The comparison will use negative binomial regression with adjustment for age (a known predictor of outcome), gender (if associated with outcome), local council area or village, and SES. This choice is based on analysis of preliminary data indicating significant overdispersion compared with Poisson-distributed counts. Secondary outcomes will be tested using similar methods. We will record whether a child was admitted for malaria, and the complication that led to admission, but the final number of episodes of malaria recorded as the primary outcome will be all episodes, whether treated as outpatient or requiring admission. Episodes requiring admission will be assessed in secondary analysis .For Specific Aim 2 is to establish the frequency of hematologic toxicities and AEs in children with SCA treated with hydroxyurea versus placebo. The working hypothesis is that children with SCA treated with hydroxyurea will have more medication-related hematologic toxicities, such as neutropenia, but no increase in SCA-related AEs  than children treated with placebo. We will test this hypothesis by comparing hematologic toxicities and AEs in children with SCA in the hydroxyurea versus placebo treatment groups. The frequency and incidence of each of the SCA-related hematologic toxicities listed in “Dose Toxicities” will be compared between the hydroxyurea and placebo groups using the chi-square test and negative binomial regression, respectively. Fractions of children having 1 or more AEs will be compared between hydroxyurea and placebo groups using chi-square tests, and incidence of all AEs per child will be compared between groups using negative binomial regression . Secondary analysis will assess incidence and frequency of individual AEs.Specific Aim 3 is to define the relationship between hydroxyurea treatment and HbF, sICAM-1, and NO levels and between levels of these factors and risk of subsequent malaria. The working hypotheses of this aim are that (1) hydroxyurea will increase HbF and plasma NO levels and decrease plasma sICAM-1 levels and (2) HbF and plasma NO levels will inversely correlate, and plasma sICAM-1 levels will positively correlate, with subsequent malaria incidence. We will test this hypothesis by testing the association between (1) hydroxyurea and change in levels of HbF, sICAM-1, and NO at 2-, 4-, and 12-month follow-up and (2) change in levels of HbF, sICAM-1, and NO at 2 and 4 months and risk of subsequent malaria.The hypothesized causal pathway is hydroxyurea → marker changes → increased malaria risk. To assess the first step in the causal pathway, the hydroxyurea and control groups will be compared according to change in and absolute level of HbF, sICAM-1, and NO levels. Markers will be analyzed separately; for each marker, the analysis will use a mixed linear model with child as the random effect and with fixed effects being group (hydroxyurea versus control) and visit , possibly after transforming the marker, so, its distribution is more symmetric. Analysis for the second step in this pathway, association between change in or absolute level of HbF, sICAM-1, and NO levels and malaria risk, will use negative binomial regression as in Specific Aim 1. Each marker will be analyzed separately, and then, all 3 will be analyzed simultaneously. The predictors will be change in child's 2-month and 4-month marker measurements, and the outcome will be the child's counts of malaria episodes in the 10-month period between 2 and 12 months of follow-up, with adjustment for age, gender, local council area or village, and SES. For these analyses, hydroxyurea and placebo groups will be analyzed separately. Analysis of secondary predictors will be done analogously.All analyses will be done as randomized analyses. Missing data will be recorded as missing and will not be used in analysis.The primary study end point will be evaluated after 12 months of study treatment (hydroxyurea or placebo). After these 12 months, children will enter a follow-up phase, during which they can receive an additional 12 months of open-label hydroxyurea treatment (through the full period of study follow-up) if they or their parents wish to do so, after consultation with local physicians at the MHSCC, and a clear explanation of the potential benefits and problems of treatment with hydroxyurea. We are working with the Ministry of Health to see if hydroxyurea can be subsidized, if it is safe and effective, so that children can continue to receive it after the trial is completed. Parents or guardians who are concerned that their child suffered harm from study participation are encouraged to talk to study personnel, who will review the issue with the ethics review committee and determine whether compensation is required.Hydroxyurea is the only disease-modifying therapy that is approved by the Food and Drug Administration (FDA) for SCA ,36. Appr9/L), study treatment will be held and weekly blood counts performed. If the hematologic toxicity resolves within 2 weeks, the daily dose will resume at 20 ± 2.5 mg/kg/day. If the toxicity persists for 2 weeks or occurs twice within a 3-month period, the study treatment dose will be reduced by 5 mg/kg/day to 15 ± 2.5 mg/kg/day.Hematologic toxicities and occasional dose adjustments are expected with hydroxyurea therapy. Laboratory toxicities from hydroxyurea will manifest primarily as reversible and transient myelosuppression, especially of granulocytes . Careful9/L. For anemia, the thresholds will be any of the following: if the Hb concentration falls to <4.0 g/dL; or Hb <6.0 g/dL with the ARC <100 × 109/L; or Hb <7.0 g/dL with the ARC <80 × 109/L, study treatment should be withheld until weekly counts document recovery. If the toxicity resolves within 2 weeks, the daily dose will resume at 20 ± 2.5 mg/kg/day. If the toxicity persists for 2 weeks or occurs twice within a 3-month period, the study treatment dose will be reduced by 5 mg/kg/day to 15 ± 2.5 mg/kg/day. Study treatment will also be held during acute hepatic or renal toxicity .A similar algorithm will be used for platelet toxicity, defined as <80 × 10An independent DSMB will supervise the trial. The DSMB will consist of a group of experienced investigators and a lay advocate. Among these individuals, there will be expertise in international pediatric hematology clinical research and specifically malaria and the use of hydroxyurea in SCA, as well as representation in biostatistics, ethics, clinical research, and patient advocacy.In our trial, we expect AEs to occur, but we expect that children with SCA treated with hydroxyurea will have more medication-related hematologic toxicities, but no increase in SCA-related AEs. After obtaining written consent from study participants, all AEs and serious adverse events (SAE) will be collected and reported to the MCC and the DCC using the correct data collection forms. The Common Terminology Criteria for Adverse Events version 4.0, available since 2009, will be used for AE reporting. All AEs are categorized by organ system and graded by severity. SAE reporting will occur in a timely manner to the DSMB and to the ethical boards that approved this study. All SAEs will be followed until resolution or stabilization.Periodic review of data will occur with the external DSMB, and the NOHARM study will be discontinued if at any time the DSMB or study team feels that it is in the best interests of the study subjects. Because NOHARM is a phase III trial in a potentially vulnerable patient population, it is important that an independent group have access to the feasibility, safety, and efficacy data. Review by the DSMB will be critical to ensure that the subjects are protected from harm, while also ensuring that the study integrity is not compromised.Interim analyses will occur when 50, 100, and 150 participants have completed 1 year of follow-up . Clear stopping rules, presentation of data to the DSMB, and regular meetings of the DSMB will ensure that the NOHARM trial does indeed protect its study participants from harm, by determining whether differences in severe malaria or death rates between the 2 treatment arms are large enough to demonstrate superiority of one treatment arm over the other. Stopping rules will be created to evaluate: (1) excess risk of severe malaria  and (2) the excess risk of death (the primary unexpected AE) in one treatment arm versus the other. The DSMB will review the protocol, including stopping rules, and decide on a schedule of meetings.P value at early interim analyses . Other spending functions  require somewhat smaller differences between groups at early interim analyses compared with O'Brien–Fleming, at the price of requiring somewhat larger differences in later interim analyses. These other spending functions can also provide slightly greater statistical power. P values required to stop at the 3 planned interim analyses  and the final analysis , for some candidate spending functions. These are also the risk of a type I  error at each analysis, if in fact, the groups do not differ in the chance of an AE  See P values required to stop at each interim analysis.Each of the 2 harm outcomes listed previously will be monitored using a stopping rule based on the Lan–DeMets alpha-spending function approach. For each outcome, the total alpha (type I error risk) will be 0.025, so that the total chance of a false-positive finding (combining the 2 outcomes) is the usual 0.05. The stopping rule will be 2 sided. Different choices of the alpha-spending function make different allocations of the type I error risk between earlier and later interim analyses. The best-known spending function, the O'Brien–Fleming boundaries, is conservative in the sense of requiring a very small To maximize the power for early detection of excess risk without unduly sacrificing power at the final analysis , we prefer the H-S-D spending function with γ=−3.The NOHARM trial enrolled its first participants in September 2014, and enrollment was completed in November 2015. A total of 213 consented participants were enrolled, of which 208 were randomized into the blinded study treatment phase , the Mulago Hospital Research and Ethics Committee , the University of Minnesota Institutional Review Board , Indiana University Institutional Review Board , Cincinnati Children’s Hospital Medical Center , the Uganda National Council on Science and Technology , and the Uganda National Drug Authority. In the event of the need for protocol amendments, such changes will be discussed with all investigators on the trial, and once agreed on, a protocol amendment form or a change in protocol request will be made to the appropriate IRB or ethics committees.Informed consent is obtained after discussion between at least 1 parent or guardian of a prospective study participant and trained study team personnel. Every consent discussion will include: purpose of the study, study procedures , and potential benefits or risks of hydroxyurea. An additional consent for sharing and storing blood samples is also obtained. All discussions and informed consent processes will be completed in either English or Luganda, at an education-appropriate level.When the open-label phase is completed and the study analysis done, we will provide a forum for all interested study participants to review the study findings. We will review them in clear, nontechnical terms in the local language and leave time for questions and ongoing discussion after the session. The final study findings will be published in a peer-reviewed publication and presented at 1 or more national or international meetings, including meetings in Uganda. We will use standard International Committee of Medical Journal Editors criteria for authorship. Through this publication, the public will have access to the study protocol. Participant level data will be maintained in our database until fully analyzed. If permitted by the study IRBs, we will eventually make participant-level data available to researchers on request in a deidentified dataset.Progress to Date subsection of the Methods section, as of March 28, 2016, 90 participants have completed the blinded treatment phase of the study, and all study participants are expected to have completed the blinded treatment phase of the study by November 2016. We anticipate analysis of results for the blinded phase to be completed by early to mid-2017. The open-label phase of the study will be completed in November 2017, and we are working with the Ministry of Health and other partners to see how children in the study can continue hydroxyurea if it proves to be safe and effective . A culturally sensitive, language-appropriate informed consent has been developed and aligns with the format recommended by the SOMREC. Aside from the ethical challenges, ensuring adequate laboratory infrastructure has been essential, especially with the frequency of blood draws in our study. Qualified laboratory staff performed blood draws, and topical anesthetics were used to minimize discomfort related to these procedures.For this trial, we received local ethics approval from the SOMREC, at Makerere University College of Health Sciences before trial commencement (in addition to approval from multiple other institutions listed in the One limitation of this trial pertains to our ability to assess the primary end point, namely, the effects of 12 months of hydroxyurea versus placebo treatment on malaria incidence. We will provide ITNs and standard malaria prophylaxis to all NOHARM study children, which should lower the incidence of malarial infections, and thus could potentially impact our primary end point. Prevalence data obtained for sample size calculations were collected from populations in which children did receive ITNs; however, these populations may live in areas with higher malaria incidence because some children in those neighborhoods developed severe malaria. If malaria incidence in the study population is low, we may have power to detect only very large differences in malaria incidence between the treatment and placebo groups. In this circumstance, valuable data will still be gained about safety, incidence of neutropenia and infections other than malaria, and effectiveness against vascular crises and other complications of SCA. We will be able to provide estimates of the differences in malaria incidence, but these estimates will have wide confidence intervals, so, further study in areas of higher malaria transmission may be required to determine more precisely the risk of malaria and severe malaria with hydroxyurea treatment in SCA.Other potential limitations pertain to randomization and blinding. Ideally, persons treating study participants would be separate from the trial, so that changes in laboratory values will not lead study personnel to guess the study arm in which the child is enrolled. In this study, to minimize the potential confusion of having multiple physicians and nurses involved in evaluation and care of the child, we instead instituted a system where laboratory results for all scheduled visits came to the data manager, and the clinicians taking care of the child saw laboratory values only if they were critical values. Thus, most laboratory values were not seen by clinical study personnel and made it unlikely that they would or could accurately guess the study arm for most study children.In summary, the NOHARM trial is a randomized controlled clinical trial of hydroxyurea for children with SCA in a malaria-endemic region. If the study demonstrates a clear benefit of hydroxyurea with no increased malaria risk or AEs in children with SCA, these critical data could lead to transformation of treatment practices for SCA across Africa.The trial is currently ongoing. Children are currently in either the randomized treatment phase or open-label treatment phase of follow-up.


Plasmodium falciparum significantly decreased the incidence of the disease in many endemic countries including Senegal. Surprisingly, in Kedougou  P. falciparum malaria remains highly prevalent and the relative contribution of other Plasmodium species to the global malaria burden is very poorly documented, partly due to the low sensitivity of routine diagnostic tools. Molecular methods offer better estimate of circulating Plasmodium species in a given area. A molecular survey was carried out to document circulating malaria parasites in Kedougou region.Control efforts towards malaria due to Plasmodium species were characterized by a nested PCR-based approach targeting the 18S small sub-unit ribosomal RNA genes of Plasmodium spp.A total of 263 long-term stored sera obtained from patients presenting with acute febrile illness in Kedougou between July 2009 and July 2013 were used for malaria parasite determination. Sera were withdrawn from a collection established as part of a surveillance programme of arboviruses infections in the region. Plasmodium genomic DNA was amplifiable by nested PCR from 62.35% (164/263) of samples. P. falciparum accounted for the majority of infections either as single in 85.97% (141/164) of Plasmodium-positive samples or mixed with Plasmodium ovale  or Plasmodium vivax . All 19 (11.58%) P. ovale-infected patients were mixed with P. falciparum, while no Plasmodium malariae was detected in this survey. Four patients (2.43%) were found to be infected by P. vivax, two of whom were mixed with P. falciparum. P. vivax infections originated from Bandafassi and Ninefesha villages and concerned patients aged 4, 9, 10, and 15 years old, respectively. DNA sequences alignment and phylogenetic analysis demonstrated that sequences from Kedougou corresponded to P. vivax, therefore confirming the presence of P. vivax infections in Senegal.Of the 263 sera screened in this study, P. falciparum in Kedougou and provide the first molecular evidence of P. vivax infections in Senegal. These findings pave the ways for further investigations of P. vivax infections in Senegal and its contribution to the global burden of malaria disease before targeted strategies can be deployed.The results confirm the high prevalence of  Plasmodium  known to infect humans [P. falciparum remains the deadliest in Africa [Malaria is a major infectious disease and remains the main global cause of death in many endemic areas, including Senegal. Success of malaria control interventions has led to a significant decrease of the disease burden. Between 2000 and 2013 malaria mortality rates decreased by 47% globally, and by 54% in sub-Saharan Africa, the region most affected by the disease [P. falciparum burden resulting from successful malaria control interventions [P. vivax, the most widespread of the Plasmodium species, is fast becoming a recognized cause of different grades of malaria pathologies along with an increasing trend in complicated malaria cases, thus threatening the prospect of malaria elimination in parts of Africa [With decreases in the ventions , attentiventions . P. vivaf Africa –8.P. vivax malaria in sub-Saharan Africa has been largely neglected based on the demonstrated correlation between the lack of expression of the Duffy blood group in sub-Saharan African population and the absence of P. vivax infections [P. vivax infections in Duffy-negative individuals [P. vivax infection. Moreover, P. vivax appears to be more frequent in countries where either it was not present or it was not detected by the available techniques in the past, as is the case of some countries of West and Central [The presence of fections , 10. Howividuals , 8, 11,  Central , becominP. vivax infection has been reported in Senegal despite evidence of its presence in many West African countries [To date, no ountries , 13–15. The study was conducted in the Kedougou region Figure  located Malaria remains highly prevalent in Kedougou region and transmission is highly seasonal occurring during the rainy season (May–November). In 2014, the Senegalese National Malaria Control Programme reported 25.55% confirmed clinical malaria cases of which 2.73% turned into severe disease .P. falciparum isolates in concurrent malaria-arbovirus infections from patients presenting with acute febrile illness (AFI) in Kedougou, a molecular diagnostic was conducted to discriminate P. falciparum from other Plasmodium species. This revealed a unique case of P. vivax infection in a patient from Ninefesha village. Therefore, additional samples from Ninefesha and nearby villages were retrieved from the sera collection and screened to survey P. vivax infections in Kedougou. A total of 263 sera from patients presenting with AFI between July 2009 and July 2013 in the Kedougou region of Senegal were included in this study. Sera were withdrawn from a collection established as part of a monitoring programme of arboviruses in Kedougou region. AFI was defined as ‘any patient older than 1 year with a fever (temperature >38°C) lasting for less than 2 weeks, exhibiting two or more of the following symptoms: headache, myalgia, eye pain, arthralgia, cough, nausea/vomiting, diarrhoea, jaundice, bleeding and neurological signs’.As part of a genetic diversity study of The study objectives, benefits and risks were explained in French language or local dialects to all participants before inclusion. Written informed consent was obtained from all adults participants and from the parents, or legal guardians of children. The study was examined and approved by the Senegalese National Health Research Committee.Plasmodium spp. genomic DNA (gDNA) in frozen serum samples has been reported earlier [Plasmodium parasites was performed using QIamp DNA Mini Kit  according to manufacturer’s instructions. DNA extracted from blood samples of known microscopically confirmed P. falciparum, P. malariae and P. ovale-infected patients were used as positive controls. P. vivax gDNA was kindly donated by Dr Ambroise Ahouidi .The detection of  earlier , 18. GenPlasmodium parasite DNA was based on nested PCR with primers targeting the Plasmodium spp. 18S small sub-unit ribosomal RNA (18S ssrRNA) gene as described previously [Plasmodium genus-specific rPLU5 and rPLU6 primers pairs [Plasmodium species using previously described primers pairs rFAL1 and rFAL2 for P. falciparum, rVIV1 and rVIV2 for P. vivax, rOVA1 and rOVA2 for P. ovale and rMAL1 and rMAL2 for P. malariae [Qualitative detection of eviously . The prirs pairs , 21 and malariae , 21. Themalariae .P. vivax amplified DNA, the specific bands were extracted from the gel and purified using the QIAquick Gel Extraction Kit as described by the manufacturer (Qiagen®). The purified PCR products were then sent to COGENICS for sequencing. For each DNA fragment, sequencing was performed from both the 3′ and 5′ directions (2× coverage). Consensus sequences were generated from the forward and reverse sequences of each sample and used in a BLASTn algorithm against the Genbank database for similarity profile determination.In order to determine the sequences’ identity of the Plasmodium spp. small sub-unit rRNA (SSU RNA) sequences were obtained from Plasmodb version 13.0 and Genbank. Plasmodium vivax: P. vivax Sal1_U030779.1, P. vivax_HF945443.1, P. vivax_HF945441.1; P. falciparum: JQ627152.1; P. ovale: JF894411.1; P. malariae: GU815531.1. Sequences were analysed by the Neighbour Joining method using 100 bootstrap replicates.Sequence alignment and phylogenic trees were performed using Mega 6.06 software. A total of 263 sera from patients presenting with AFI enrolled during arboviruses surveillance in Kedougou were screened in this study for the presence of malaria parasite species. Patients were aged one to 65 years old, the majority originated from the village of Bandafassi Table . The meaP. falciparum, P. vivax, P. malariae, and P. ovale) and sterile distilled water were used as the positive and negative controls, respectively to validate the nested PCR approach. The presence of amplified DNA products in a patient’s specimen corresponding to P. falciparum (205 bp), P. vivax (120 bp), P. malariae (144 bp) and P. ovale (375 bp) was confirmed  of the 263 sera screened in this study  of positive samples or mixed with P. ovale  or P. vivax . Nineteen patients (11.58%) were infected with P. ovale, all of which were mixed with P. falciparum . Four patients (2.43%) were found to be infected by P. vivax, two of which were mixed infections with P. falciparum  matched the Genbank P. vivax isolate SV6 18S ribosomal RNA gene partial sequence (ID: JQ627158.1), P. vivax Sal1 blood stage small sub-unit rRNA gene (ID: PVU03079) and P. vivax isolate SV5 18S ribosomal RNA gene partial sequence (ID: JQ627157.1) by 98, 100, 100, and 97%, respectively . Further investigations with a larger sampling in Kedougou and other Senegalese settings are needed to document the presence and prevalence of P. vivax infections in Senegal before orientated strategies can be deployed. The results presented here indicate that there may be a need for the National Malaria Control Programme to anticipate reviewing the management of malaria, including P. vivax.The present study provides the first molecular evidence of


Lorenz von Seidlein and Jakob Knudsen discuss the changes in malaria incidence recorded at a single site in Africa over 25 years, along with future implications for disease prevention. PLOS Medicine, Polycarp K. Mogeni and colleagues present a unique dataset collected from 69,104 children aged from 3 months to 13 years admitted over the last 25 years to Kilifi County Hospital, Kenya. In 1998, 56% of the children were admitted with malaria, presumably mostly caused by Plasmodium falciparum. By 2009, just 7% of the children were admitted with malaria. After the devastating impact of a very high malaria burden at the end of the last century, malaria-related morbidity and mortality declined dramatically in Kilifi, as it had done in many, but not all, parts of sub-Saharan Africa during the first decade of the 21st century [In this issue of P. falciparum infections meant that fewer children could acquire immunity to this pathogen. Over the following years, these immunologically naïve children in a setting of much reduced but still substantial risk of exposure eventually became infected. This epidemiological shift is shown by the increase in the mean age of children admitted with P. falciparum infections, which increased gradually from 20.2 months in 1990 to 45.3 months in 2014. With a further decrease in malaria incidence, the mean age of malaria patients seems likely to continue to increase.In Kilifi, the downward trend in hospital admission with malaria reached its nadir in 2009 and then slowly started to increase again. This observation, which is not limited to Kilifi , has cauAlthough malaria burden is much reduced, it remains unacceptably high; what can be done to reduce malaria transmission further?Mogeni and colleagues illustrate nicely that the highest incidence of malaria is now in kindergarten and school-age children and no longer in infants and very young children. Malaria control programmes originating from the last century target infants, with a focus on adding malaria-related interventions to the expanded program on immunisation (EPI). Future malaria control initiatives will have to target older children. Seasonal malaria chemoprevention (SMC) is a welcome initiative in this direction . RecommeP. falciparum infections, compared to children in communities with low bednet usage. Following the notion that bednets are a good thing and, therefore, more bednets must be even better, the authors propose universal coverage. The benefits of bednets have been repeatedly highlighted. Lengeler reviewed 18 randomised trials of ITNs and found that ITNs reduce the incidence of uncomplicated malaria by 48% compared to no net, and by 34% compared to non-impregnated nets. ITNs afford a 17% protection against mortality in children under 5 years [Mogeni and colleagues show that in Kilifi County, ITNs were highly protective; children who live in communities with high bednet use were less likely to present with  5 years . With thIn Kilifi, 83% of respondents stated that they already use bednets; can the distribution of ever more bednets be a solution for the malaria problem in Kilifi? Having reached high levels of bednet ownership, the problem is now likely to be the use of bednets, not their availability. The current approach to assessing bednet use is asking respondents in home surveys whether they used bednets during the night before. There is an assumed correlation between the self-reported “have you/your child slept under a bednet last night” and the actual use of the bednet during each night of the malaria season, but there are no reliable data to confirm such an assumption. More likely, the self-reported responses are biased by participants’ inclination to provide what they believe is the desirable response. A more reliable answer could be gained from in-depth interviews, but such time-consuming methods are not suitable for large surveys.There are a number of reasons why people do not use their bednets. Pulford and colleagues found that the negative effect on indoor climate was the most frequently stated reason for not using available bednets . The indWhat measures can be taken to increase the use of bednets for the intended purpose? Our review of factors that improve indoor climate in hot–humid zones found that the single most important factor is proper cross-ventilation. Elevating houses on stilts, using a bamboo or wooden floor, as is standard construction practice in Southeast Asia, and the opening of eaves and windows had the largest positive impact on indoor climate . SurprisMore effort should be made on community engagement and education to discuss novel architecture of new houses being built in Africa, to gradually integrate housing characteristics that improve ventilation while minimising vector entry. Elevating the house off the ground reduces mosquito bites and improves airflow . Adding Housing improvements will benefit all age groups and provide protection against malaria for adults as well as children ,19. Much


Plasmodium falciparum malaria infections are found in many parts of sub-Saharan Africa but little is known about their importance in pregnancy.Non-P. falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale using a nested PCR test. Risk factors for a non-falciparum malaria infection were investigated and the influence of these infections on the outcome of pregnancy was determined.Blood samples were collected at first antenatal clinic attendance from 2526 women enrolled in a trial of intermittent screening and treatment of malaria in pregnancy (ISTp) versus intermittent preventive treatment (IPTp) conducted in Burkina Faso, The Gambia, Ghana and Mali. DNA was extracted from blood spots and tested for P. falciparum infection was detected frequently , with a prevalence ranging from 10.8 % in The Gambia to 56.1 % in Ghana. Non-falciparum malaria infections were found only rarely , ranging from 0.17 % in the Gambia to 3.81 % in Mali. Ten non-falciparum mono-infections and 25 mixed falciparum and non-falciparum infections were found. P. malariae was the most frequent non-falciparum infection identified; P. vivax was detected only in Mali. Only four of the non-falciparum mono-infections were detected by microscopy or rapid diagnostic test. Recruitment during the late rainy season and low socio-economic status were associated with an increased risk of non-falciparum malaria as well as falciparum malaria. The outcome of pregnancy did not differ between women with a non-falciparum malaria infection and those who were not infected with malaria at first ANC attendance.Non-falciparum infections were infrequent in the populations studied, rarely detected when present as a mono-infection and unlikely to have had an important impact on the outcome of pregnancy in the communities studied due to the small number of women infected with non-falciparum parasites. Plasmodium falciparum is the dominant malaria parasite in most parts of sub-Saharan Africa and hence responsible for most infections in pregnancy [Plasmodium vivax is also a significant cause of maternal morbidity during pregnancy and of low birth weight in parts of Asia [P. vivax has been reported from time to time in non-pregnant populations in sub-Saharan Africa, especially when polymerase chain reaction (PCR) assays have been used [P. vivax infections are generally considered to be uncommon in sub-Saharan Africa because of the presence in a high proportion of the population of Duffy antigen negative red cells which cannot be invaded by this malaria parasite. P. vivax infections have been recorded only rarely during pregnancy in East or West Africa [Plasmodium ovale and Plasmodium malariae are relatively common in many parts of East and West Africa causing a significant proportion of febrile malaria episodes [P. malariae and P. ovale in 6.9 and 1.8 % of pregnant Cameroonian women respectively, nearly always in mixed infections with P. falciparum. Mockenhaupt et al. [P. malariae and Coldren et al. [P. ovale in a pregnant women which was probably acquired many years previously in East or West Africa.regnancy . Plasmod of Asia –5 and La of Asia , 7, even of Asia . P. vivaeen used –11, but t Africa , althougt Africa . In contepisodes  and asymepisodes . Howeverepisodes  detectedt et al.  reportedn et al.  describeScreening of pregnant women at ante-natal clinic (ANC) visits with a rapid diagnostic test (RDT) for malaria and treatment of those who are positive with artemisinin-based combination therapy, an approach termed intermittent screening and treatment (ISTp), is being considered as a potential approach to the control of malaria in some epidemiological situations such as low transmission areas where few women are likely to benefit from currently recommended intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP). However, there is concern that this approach may lead to some malaria infections, especially those present at low density, being missed and adversely affecting the outcome of pregnancy. This is especially likely to be the case for non-falciparum infections which are often present at only a low density and for which many RDTs have poor sensitivity. For this reason, the prevalence of falciparum and non-falciparum infections at first ANC attendance was investigated in women enrolled in a trial of ISTp versus IPTp-SP conducted in four West African countries  and determined how many of these infections were detected by an RDT.Details of the way in which the trial of ISTp versus IPTp-SP was conducted have been presented previously . In brie® Malaria Rapid Diagnostic Combo Test) was done and blood films and filter paper blots prepared. Blood films were examined for malaria parasites according to the methods described by Swysen et al. [P. falciparum, P. vivax, P. ovale and P. malariae) and a negative control without DNA. The second PCR reaction detects the presence of Plasmodium infection using the genus specific primers Plasmo-1 and Plasmo-2. The third stage, undertaken on genus specific samples, determines species by running four parallel PCR assays using the following primers . For further differentiation of P. ovale into the two distinct species P. ovale curtisi and P. ovale wallikeri, the P. ovale sp. Tryptophan-rich antigen (PoTRA) nested PCR assay was used [The analysis reported in this paper is based on blood samples obtained at the first ANC visit from the 2526 women in the ISTp-AL group. An RDT , Burkina Faso; Ethics committee of the Ghana Health Service Committee Research & Development Division, Ghana Health Science, Ghana; Navrongo Health Research Centre Institutional Review Board, Ghana; Comité d’éthique de la Faculté de la Faculté de Médecine et d’Odontostomatologie, Mali; The Gambia Government/MRC Laboratories Joint Ethics Committee, The Gambia. Written informed consent was obtained from all participating women. The trial is registered with ClinicalTrials.gov (NCT01084213) and the Pan African Clinical trials Registry (PACT201202000272122).P. falciparum malaria in 981/2526  pregnant women at first ANC attendance . In two of the latter cases, the microscopic diagnosis was P. falciparum. Twenty-three (92 %) of the 25 mixed P. falciparum and non-falciparum infections were detected by RDT, and 18 (72 %) were detected by microscopy, with the microscopic diagnosis being P. falciparum in all cases. Overall, only nine non-falciparum infections were missed by RDT and 14 missed by microscopy in 2526 pregnant women.PCR detected Plasmodium malariae was the most common non-falciparum species detected, being present alone in three women  and in 14 mixed infections . Eleven mixed P. ovale and P. falciparum infections were detected. Both forms of P. ovale were found in roughly equal abundance . P.vivax was found in only four women , all of whom lived in Mali. Because of the unexpected finding of P. vivax parasites in four black, African women resident near Bamako, the identity of these parasites was confirmed using a real-time PCR assay targeting the P. vivax ribosomal RNA gene .There was variation in the prevalence of non-falciparum malaria between study sites, with Mali having the highest prevalence  compared with Ghana , Burkina Faso  and The Gambia .p = 0.045) but there were no differences in older age groups compared to mothers below 18 years of age. Non-falciparum malaria infections were more common among women in the two poorest socio-economic groups; adjusted OR relative to the wealthiest group  in the poor and poorest groups respectively. As found in the previous analysis of P. falciparum infections in study women [p < 0.001  (p = 0.038). There remained no differences between Ghana and Burkina Faso, adjusted OR 0.53  (p = 0.353).There were no major differences in the prevalence of non-falciparum malaria infections overall according to gravidity, gestational age at enrolment, educational level or socio-economic status Table . There wdy women , non-falp = 0.356 (Table p = 0.915). Similarly, there was no evidence of a difference in mean birth weight between women with a mono- or mixed non-falciparum infection and those with no malaria infection during pregnancy: adjusted difference—51.9 g  (p = 0.547), Table p = 0.983).The prevalence of active placental malaria infection at delivery was similar in those with a mono- or mixed non-falciparum malaria infection and those with no infection: 23.1 versus 21.8 % respectively, adjusted OR 1.63 , P. falciparum infection of the four study sites. P. vivax was detected only in Mali, and only in one sub-site in suburban Bamako. This malaria parasite is known to be present in northern Mali [This study confirmed that non-falciparum malaria infections are present in pregnant women in West Africa but that they are uncommon, with an overall prevalence across four countries of 1.4 % at first ANC attendance. It is likely that some additional infections occurred later in pregnancy. However, among the women from Ghana enrolled in this study who were followed throughout pregnancy, at least a half of all malaria infections were detected at the first ANC visit ; the figern Mali , perhapsP. falciparum: only three of the 10 pure non-falciparum infections were detected by microscopy, and two of these were mistaken for P. falciparum despite the fact that study slides were read by two well-trained microscopists and that the quality of microscopical reading was well controlled. In sub-Saharan Africa, microscopists expect malaria infections present in a blood film to be due to P. falciparum and this may have influenced their recording of the results. Rapid diagnostic tests did not perform any better than microscopy, also missing seven of the 10 non-falciparum mono-infections, perhaps because these were present at only a low density. When P. falciparum was also present, RDTs detected all but two of these infections, whereas microscopy missed seven of the mixed infections. These findings suggest that when, in sub-Saharan Africa, detection of non-falciparum malaria infection in pregnancy depends upon microscopy, many non-falciparum infections will be missed. Factors likely to have contributed to the poor performance of microscopy and a RDT in detecting non-falciparum infections are their frequent occurrence as mixed infections with P. falciparum and their presence at only a low density. Why the latter should be the case is uncertain, but it may be due in part to suppression of the minor parasite by P. falciparum in mixed infections and in part due to the fact these may have been more chronic infections than those caused by P. falciparum.The results of this study indicate the difficulty in identifying non-falciparum malaria infections by microscopy when these infections are encountered rarely and often present in mixed infections with Enrolment at the end of the wet season was a risk factor for non-falciparum malaria infection, as was lower socio-economic status. These are both well-established risk factors for malaria infection in general. The suggestion of an association of one specific age group with higher risk of non-falciparum infections does not have a biological explanation and is likely to be a chance finding as the numbers of non-falciparum infections studied was small.There was no association between the presence of a non-falciparum malaria infection at first ANC presentation and an adverse outcome of pregnancy. However, the number of non-falciparum infections was small, 26 of the 35 individuals with non-falciparum infections at baseline were treated with artemether-lumefantrine and it is possible that some women in the uninfected reference group were infected at later time points, diluting differences between the two groups, so this finding is not surprising.P. vivax, are more prevalent and where many more infections might be missed using currently available RDTs.This study was undertaken as part of an evaluation of ISTp because of concerns that a significant number of infections caused by non-falciparum species might be missed during RDT testing, with an adverse impact on the outcome of pregnancy, if this approach to malaria control in pregnancy was to be implemented. Because the majority of non-falciparum infections occurred in conjunction with a falciparum infection which was detected by a RDT, as noted in another recent study in Burkina Faso and Uganda , the majority of women with a non-falciparum infection received appropriate treatment; only seven non-falciparum mono-infections and two mixed non-falciparum and falciparum infections were missed at first ANC visit using a ‘combo’ RDT among 2526 women. For this reason the occurrence of non-falciparum infections is unlikely to be a concern should ISTp be recommended for any population group in sub-Saharan Africa. However, this might not be the case in other parts of the malaria endemic world where non-falciparum infections, particularly those caused by


Plasmodium falciparum malaria and ultimately, the rate of malaria related morbidity and mortality. Potential vaccines being designed to complement current intervention efforts therefore need to be evaluated against different malaria endemicity backgrounds.Differences in parasite transmission intensity influence the process of acquisition of host immunity to The associations between antibody responses to the chimeric merozoite surface protein 1 block 2 hybrid (MSP1 hybrid), glutamate-rich protein region 2 (GLURP R2) and the peptide AS202.11, and the risk of malaria were assessed in children living in malaria hyperendemic  and hypo-endemic  areas. Using the same reagent lots and standardized protocols for both study sites, immunoglobulin (Ig) M, IgG and IgG sub-class levels to each antigen were measured by ELISA in plasma from the children (aged 6–72 months). Associations between antibody levels and risk of malaria were assessed using Cox regression models adjusting for covariates.p < 0.0001) and the Ghanaian  cohorts. MSP1 hybrid IgM was associated  with reduced risk of malaria in Burkina Faso cohort while IgG against AS202.11 in the Ghanaian children was associated with increased risk of malaria .There was a significant association between GLURP R2 IgG3 and reduced risk of malaria after adjusting age of children in both the Burkinabe  contains supplementary material, which is available to authorized users. Plasmodium falciparum isolates was designed and fused with the relatively conserved block 1 sequence of MSP1 to form the MSP1 block 2 hybrid  than in the Ghanaian  cohort  were significantly younger than the Ghanaian children (mean age = 51.8 months) (p < 0.05) with age in children from Burkina Faso (Table p < 0.05) with age. The adjusted R2 values from the univariate linear regression analysis of antibody levels and age was generally low especially for the Ghanaian cohort  , except IgG1 and IgG4 against MSP1 hybrid, increased significantly (p = 0.01) and MSP1 hybrid  were significantly associated with reduced risk of clinical malaria after adjusting for age (Table p < 0.05) in children from Burkina Faso (Table p = 0.01) and MSP1 hybrid  were also significantly associated with reduced risk of clinical malaria in the Burkinabe cohort. In the Ghanaian cohort, only IgG3 against GLURP R2 was significantly  associated with decreased risk of malaria (Table p < 0.05) associated with reduced risk of malaria (Table p = 0.04) in the Ghanaian cohort  and hypo-endemic (Ghana) populations, respectively, using the same study protocols and reagents lots. Overall, high levels of IgG3 against GLURP R2 was strongly associated with reduced risk of clinical malaria in both Burkinabe and Ghanaian children despite the marked differences in malaria transmission intensity between these two populations. In Burkinabe children, IgM responses against MSP1 hybrid contributed significantly to reducing the risk of clinical malaria while in Ghanaian children, increased levels of IgG against the peptide AS202.11 was associated with about 29 % increased risk of clinical malaria Table . In prev2 values observed, particularly for the Ghanaian cohort, suggests in areas of very low malaria transmission, age alone may not adequately explain acquisition of malaria-specific antibodies.In most malaria-endemic populations, intensified transmission prevention measures such as use of ITNs, indoor residual spraying and effective therapeutics are rapidly changing the landscape of malaria epidemiology , 43. MalPlasmodium falciparum diversity at the Ghanaian site is unknown, however, the K1 and MAD 20 allelic families of msp1 have been shown to be the most prevalent in Balonghin, Burkina-Faso [P. falciparum infections. Both GLURP R2 and MSP1 block 2 have been shown to elicit antibodies capable of inhibiting parasite growth in vitro in the monocyte-dependent mechanism, antibody-dependent cellular inhibition (ADCI) [In previous studies, GLURP R2 IgG was associated with protection against malaria in Burkinabe  and Myanina-Faso . Here, tina-Faso . Moreoven (ADCI) , 54. Thin (ADCI) . Antigenn (ADCI)  and Burkn (ADCI)  have foun (ADCI) . Althougn (ADCI) . It has n (ADCI)  or compln (ADCI) . FunctioThis study corroborates findings from previous studies associating GLURP R2 as an important target for protective antibodies against malaria. The results show for the first time, that antibodies against the chimeric MSP1 block 2 hybrid are associated with reduced risk of malaria in a hyperendemic population. The findings support the further development of both GLURP R2 and MSP1 block 2 hybrid as important blood stage vaccine candidates that may be deployed in children living in areas of different malaria endemicity. These may be most effective as a single fused vaccine antigen rather than as individual sub-unit vaccines.


Plasmodium vivax as the most prevalent species. However, only a few studies have been carried out in areas with low and unstable transmission, whereas the clinical profile of malaria has been broadly described in hyperendemic areas. The pattern of clinical manifestations and laboratory findings in low to moderate endemic areas of Colombia is reported here.Malaria transmission in Latin America is generally hypoendemic and unstable, with Plasmodium falciparum, P. vivax or mixed malaria infections attending malaria points-of-care of four malaria endemic-areas with distinct transmission intensities and parasite distribution. Trained physicians recorded clinical symptoms and signs as well as socio-demographic characteristics of study participants. Haematological, biochemical and urine tests were performed at the time of diagnosis.A passive surveillance study was conducted between 2011 and 2013 involving 1,328 patients with P. vivax; 650 (48.9%) were due to P. falciparum; and five (0.4%) patients had mixed infections . Most patients (92.5%) presented with uncomplicated malaria characterized by fever, chills, headache, sweating, myalgia/arthralgia and parasitaemia ≤ 20,000 parasites/μL. Fever, tachycardia, pallor and abdominal pain on palpation were more frequent in P. falciparum patients, whereas mild hepatomegaly and splenomegaly were mostly observed with P. vivax. Non-severe anaemia (Hb 7.0-10.9 g/dL) was observed in 20% of the subjects, whereas severe anaemia (Hb < 7.0 g/dL) was present in four patients. Half of the patients presented thrombocytopaenia regardless of parasite species. Leukopaenia, neutrophilia and monocytosis were frequently observed in patients infected with P. falciparum. Mild-to-moderate biochemical alterations were present in ~25% of the patients, particularly abnormal bilirubin in those with P. falciparum and abnormal transaminases in P. vivax malaria patients. Proteinuria was present in ~50% of the patients regardless of parasite species, whereas haemoglobinuria was more common in P. vivax infections. Only 7.5% of the cases were classified as clinically severe malaria, caused by both P. vivax and P. falciparum.Out of 1,328 cases, 673 (50.7%) were caused by The high prevalence of uncomplicated malaria associated with moderate parasitaemia suggests the importance of timely diagnosis and effective treatment and encourages new activities to further decrease complicated malaria cases and mortality. Plasmodium falciparum infections in sub-Saharan Africa, the region with the highest rates of transmission worldwide. High frequency of severe disease and fatal complications occur primarily during infancy and early childhood in this region  were found mainly in patients infected with 5) Table . Renal fA total of 129/1,323 patients (9.8%), excluding five with mixed infections, presented with one or more signs suggesting severe malaria development  Table 44. TachypP. falciparum infection, microscopic haemoglobinuria (Hb-positive dipstick) and proteinuria (p < 0.010), suggesting an important renal involvement. Microscopic haematuria (>3 RBC) and hyperbilirubinaemia (TB >3 mg/dL) were also observed in 28% and 12% of patients, respectively, with choluria.Choluria (bilirubin in urine) was associated with P. vivax or P. falciparum were: severe prostration (n = 11), respiratory distress (n = 4), hyperparasitaemia (n = 4), abnormal spontaneous bleeding (n = 4), severe anaemia (n = 4), cardiogenic shock (n = 1), and severe thrombocytopenia (n = 1) as well as malaria warning signs including tachypnea in absence of fever and persistent vomiting or diarrhoea. Cerebral malaria was not observed. The remaining 45 patients were diagnosed and treated at the POC as uncomplicated malaria cases; however, when laboratory test were analysed, these revealed abnormalities indicating hepatic and renal dysfunctions according to the WHO and Colombian MoH guidelines [A total of 99 patients 57% male) with a mean age of 27 years  were classified with complicated malaria . This alteration was more frequent in adults than children < 15 years of age (18% vs 4%). In patients with complicated malaria anaemia (23.2%), leukopenia (32.3%), and thrombocytopenia (64.6%) were common haematological alterations. Abnormal transaminases (ALT and AST) and hyperbilirubinaemia (TB > 1 mg/dL) were also observed in 45.5% and 46.5% of patients, respectively , which appear to correlate with the relatively low malaria transmission in the study areas , togetheLikewise, signs associated with severe clinical malaria emphasized in the MoH guidelines (warning signs) have significantly contributed to the early identification of potential malaria complications and to a decrease in the risk of severity and mortality. Warning signs reported here in 129 patients, including choluria, persistent vomiting and diarrhoea, tachypnea in absence of fever, hyperpyrexia and others, have been reported in previous clinical studies conducted in Colombia ,24,31.P. vivax to severe malaria burden, challenging the idea that vivax malaria is ‘benign’ and not lethal [P. vivax-infected erythrocytes adherence and pathophysiology of P. vivax [P. vivax severe cases there have been reported in Colombia [In contrast, in countries with higher malaria transmission such as Papua New Guinea  and Indot lethal ,39. MoreP. vivax -42. In fColombia , Brazil Colombia ,35, PerúColombia  and VeneColombia , in agreP. vivax-infected patients  than in those infected with P. falciparum . This finding is in agreement with previous reports in Colombia [P. vivax (children ≤ 15 years/old), which had a significantly higher parasitaemia than the older age group.Severe anaemia significantly contributes to malaria mortality . As in pColombia  and may Although thrombocytopaenia has been reported in up to 94% of acute malaria cases, frequently with severe intensity , here itMalaria-induced changes in the leukocyte populations are very diverse -54; mostP. vivax sporozoites developed significantly fewer symptoms than naïve volunteers [P. vivax and P. falciparum cases appears to be due to the fact that most of the population where P. vivax is prevalent are mestizos, whereas P. falciparum was more prevalent in Afro-descendants with high prevalence of Duffy negative blood group [Because a significant proportion of the malaria cases (40.3%) occurred in the economically active population (16–30 years of age), and lower prevalence (25.5%) was found in children ≤ 15 years of age, the selection of the study populations was not biased and it is speculated that malaria transmission may occur near to or inside households and schools where children ≤ 15 years old spend most of their time. Although adult members of the family also spend a significant amount of time in the house and surrounding neighborhood, they did not appear to greatly contribute to malaria cases in this study, possibly harboring asymptomatic infections. A recent study in the same sites has indicated a prevalence of ~6% of asymptomatic carriers. In addition, semi-immune young adult volunteers (≥18 years old) from these sites, when exposed to experimental challenge with infectious lunteers . In thislunteers . In low od group .P. falciparum patients would progress to severe and lethal cases at a greater rate as compared to P. vivax [P. vivax or P. falciparum. A prospective study is currently ongoing aimed at a more detailed characterization of malaria complications in hospitalized patients and associated factors, which could contribute to improving MoH policies and strengthening the malaria information system in Colombia.The present study showed a high prevalence of uncomplicated malaria together with moderate parasitaemia, which appear to be associated with low transmission intensity, early diagnosis and effective treatment, as well as to variable degrees of clinical immunity. Mild-to-moderate clinical and laboratory alterations specific to each parasite species were observed. Interestingly, at this transmission intensity patients infected with either parasite species appear to have a similar risk of developing the levels of severe malaria observed. It is likely that in regions with greater transmission intensity and other socio-economic conditions and more limited health infrastructure, P. vivax ,35,56. H


SMC) and community case management with long‐acting artemisinin‐based combination therapies (ACTs) for the control of malaria in areas of extended seasonal malaria transmission.To investigate the effectiveness of seasonal malaria chemoprevention  plus placebo SMC, or (ii) a long‐acting ACT  for case management plus placebo SMC or (iii) AL for case management plus active SMC with sulphadoxine‐pyrimethamine and amodiaquine. SMC or placebo was delivered on five occasions during the rainy season. Malaria cases were managed by community health workers, who used rapid diagnostic tests to confirm infection prior to treatment.Individually randomised, placebo‐controlled trial in the Ashanti Region of Ghana. A total of 2400 children aged 3–59 months received either: (i) a short‐acting SMC during the rainy season. Compared to those given placebo SMC and AL for case management, the adjusted hazard ratio (aHR) was 0.62 , P = 0.020 by intention to treat and 0.53 , P = 0.033 among children given five SMC courses. There were no major differences between groups given different ACTs for case management , P = 0.356).The incidence of malaria was lower in children given SMC may have an important public health impact in areas with a longer transmission season, but further optimisation of SMC schedules is needed to maximise its impact in such settings. Seasonal malaria chemoprevention (SMC) is a highly effective means to control malaria in areas with a short rainy season, where a large proportion of the annual malaria burden occurs within a few months of the year A second form of drug‐based prevention that would be easier for resource limited countries to implement would be to treat malaria patients with long‐acting artemisinin combination therapies (LACTs), such as dihydroartemisinin‐piperaquine (DP) or artesunate‐mefloquine (AS‐MQ), which protect children against further malaria episodes for several weeks To investigate these issues, we undertook a trial in the Ashanti region of Ghana to estimate the protection provided by SMC administered over 5 months and by a long‐acting ACT used for malaria case management. A previous study in this area showed that amodiaquine‐artesunate administered every 2 months reduced fevers presumptively treated as malaria by community‐based health workers (CHWs) by 37% (by intention to treat), and by 61.5% in children who received all three courses over the malaria transmission season The study, registered with ClinicalTrials.gov NCT01651416], was conducted among children aged 3–59 months living in 13 communities in the Kwaso subdistrict, Ejisu‐Juaben municipality, in the Ashanti Region of Ghana . Treatment allocation was held in opaque sealed envelopes labelled only with the study ID number on the front. Upon enrolment to the study, the next envelope in the sequence was opened by the study team member to determine the treatment allocation of the child to be used throughout the study. This was carried out separately from the screening process; individuals screening children for eligibility were unaware of subsequent assignment. The three randomisation groups were (i) placebo SMC, plus a short‐acting ACT (artemether‐lumefantrine) for treatment of malaria episodes (hereafter the ‘AL group’), (ii) placebo SMC plus a long‐acting ACT (dihydroartemisinin‐piperaquine) for treatment of malaria episodes (the ‘DP group’) and (iii) active SMC with sulfadoxine‐pyrimethamine plus amodiaquine, plus artemether‐lumefantrine for treatment of malaria episodes (the ‘SMC group’).Incidence of malaria in the region between 16 and 24 months of age was estimated to be 1.4 per child‐year by active case detection To facilitate community‐based treatment of malaria with the assigned regimen (artemether‐lumefantrine or dihydroartemisinin‐piperaquine), and to ensure that children received the correct regimen if they attended at health centres in the study area, ID cards were colour‐coded according to intervention group and labelled with the regimen to be used for case management. The study was therefore open‐label with respect to the regimen used for case management but blinded with respect to whether seasonal malaria chemoprevention was active or placebo .P. falciparum HRP2/pLDH RDT ), they received a full treatment course of either artemether‐lumefantrine  or dihydroartemisinin‐piperaquine  according to their intervention group. Children without symptoms of malaria, or those who tested negative by RDT, were referred to health centres or the hospitals serving the area. CHWs and health centres serving the study communities were supplied with RDTs and stocks of both ACTs; research staff stationed at health centres recorded illness visits made by study children to the outpatient clinics.The primary outcome was incidence of clinical malaria detected passively by community health workers, and at health centres serving the study area. If children presented with fever, history of fever or other symptoms suggestive of malaria, no other obvious causes of fever, no danger signs and tested positive for malaria by rapid diagnostic test  on five occasions (once per month). SP/SP placebo and the first dose of AQ/AQ placebo were given under observation, and the two remaining AQ/AQ placebo tablets were administered by caregivers at home. Two age‐based dosages were used: children aged 1–4 years received 500/25 mg S/P and 153 mg AQ; infants received half this dose . In the days prior to SMC delivery, community health workers and field supervisors reminded mothers/caregivers that an SMC administration was due to take place; announcements were also made by local FM radio and by town criers. SMC or placebo was delivered by trained members of the study team at a central point in each community. For the final two cycles, community health workers followed up children who did not receive the SMC from the central point to administer the medicine. Children who reported illness or fever at the time of SMC were screened with a rapid diagnostic test, and if positive treated with an ACT according to their intervention group. Caregivers were followed up by CHWs between 4 and 7 days after each SMC administration to ask about adverse events and adherence to the SMC regimen using a standard form.™ analysers . Blood slides were prepared by trained staff and double read at the laboratory of the Centre for Global Health Research, Juaben, Ghana. Children were also asked about any current symptoms of illness using a structured questionnaire.At enrolment, information was collected on basic demographic characteristics, household facilities , durable household assets and ownership, age, condition and use of insecticide‐treated nets. Blood samples were taken for determination of haemoglobin and malaria parasitaemia. Haemoglobin was measured using 301 HemocueTwo further cross‐sectional surveys were undertaken after the period when SMC was administered (January 2013), and at the end of the study period (July 2013). Blood samples were taken for determination of haemoglobin and malaria parasitaemia, and children were asked about any current illness symptoms.A priori, we expected that the malaria incidence would be highest in the AL group and lowest in the group given SMC, with the group given DP for malaria case management intermediate between these two . The AL group was therefore pre‐specified as the reference group for analyses. For the primary endpoint, the hazard ratio for multiple episodes of malaria was estimated by Cox regression models. Robust standard errors (i.e. the Andersen‐Gill extension of the Cox model) were used to account for clustering of failures within individuals; this estimates the total effect of the intervention which is of primary public health importance Plasmodium falciparum parasitaemia and anaemia at the cross‐sectional surveys All data were double entered and any discrepancies verified. Consistency and range checks were also undertaken prior to locking the database for analysis, and linking records with the randomisation code. All analyses were undertaken in Stata version 13 . The study was approved by the ethics committees of the London School of Hygiene and Tropical Medicine, UK, the University of Leeds, UK and the Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.vs. 84.3% and 83.9% in the AL and DP groups, respectively. Finally, there were some imbalances between groups in the number of children recruited from small communities (Table S1).In July 2012, 2400 children were enrolled into the study  and ranged from 13.3% to 57.6% in different communities, with infection generally more common in smaller communities . At baseline, 50.5% of children were anaemic (Hb < 11 g/dl), although most of this was mild, with only 4.98% of children having Hb <8 g/dl. One child had Hb <5 g/dl. The distribution of measured Hb was similar between the three groups .The lowest coverage of SMC occurred at the third cycle of SMC, which was delivered by the study team at a central point in each community Table . Qualitavs. 20.3%: prevalence ratio 0.61 , P < 0.001. Prevalence was similar in the DP and AL groups, 19.5% vs. 20.3%: PR 0.96 , P = 0.727 ; the aHR was 1.18 , P = 0.356 in the DP group .Malaria incidence over the study period is shown in Figure up Table . Among cP = 0.050 and 1.70 , P = 0.045, respectively. However, after adjusting for covariates  aHRs were 1.36 , P = 0.187 and 1.34 , P = 0.199, respectively.During the post‐SMC period, including the start of the following transmission season in 2013, there were 135 episodes of malaria in 916.2 person‐years of follow‐up. Relative to the AL only group, incidence of malaria appeared higher in the SMC group and the DP group: crude HRs 1.73 , P = 0.428 in the SMC group and 1.23 , P = 0.168 in the DP group. There was no evidence of a difference in the interval between recurrent episodes between groups over the whole study period.Over the whole study period, including the SMC period, the dry season and the first few months of the 2013 transmission season, 2143 children never experienced malaria, 198 children had one malaria episode, 40 children experienced two episodes, and 13, 5 and 1 children experienced 3, 4 and 5 episodes, respectively. Compared to the AL only group, the aHR was 0.88 , We investigated an extended programme of seasonal malaria chemoprevention, and case management with a long‐acting ACT in an area of Ghana with a long rainy season with two peaks. Strengths of this study are that a relatively large cohort of children was monitored, the interventions were successfully delivered, and it was possible to ensure a continuous supply of drugs and diagnostic tests to community health workers who documented malaria incidence. Limitations of this study are that not all children were followed over the entire study period, that randomisation did not achieve perfect balance in factors known to be associated with malaria, that for practical reasons it was necessary for the study to be open‐label with respect to the case‐management regimen and that coverage of the full complement of SMC cycles was relatively low.Malaria incidence was lower than expected: 196.4 per 1000 child‐years over the study period in children who received placebo SMC, compared to 700 per 1000 person‐years assumed in the sample size calculation. Several factors may have contributed to this. Firstly, due to a recent distribution campaign, around 80% of children were regularly sleeping under an insecticide‐treated net. Secondly, increased membership of Ghana's National Health Insurance scheme  may have contributed to lower patronage of CHWs, as in a recent study in Burkina Faso The protective efficacy of SMC was lower than expected during the SMC period, at around 38%, with no significant protection over the whole study period, although this is partly diluted by the onset of the 2013 transmission season a few months before the study ended. The lower efficacy may partly be due to relatively low coverage of all five SMC cycles (36.4% in the SMC group), but even among children who received five cycles of SMC, protective efficacy was around 47% during the SMC period, lower than the efficacy of 70‐80% seen in other studies of monthly SP‐AQ Low efficacy may also be a consequence of the timing of administration of the first cycle of SMC. At the baseline survey in July, prevalence was around 26%, suggesting that the transmission season was well underway by this point. Although SP‐AQ is likely to have very good curative efficacy in West Africa Operationally, delivery of a larger number of SMC cycles is more challenging, as the additional drugs needed bring associated issues of cost, supply and storage. There may also be more potential for campaign fatigue where drugs are administered over a longer period, both on the part of parents/caregivers and their children, and on the part of delivery teams. This appeared to be the case here, even in the context of a research study, with decreasing coverage at the second and third cycles. However, the increased coverage at the fourth and fifth cycles, achieved by following up children at home, agrees with previous results showing that door‐to‐door delivery by community‐based health workers can achieve high SMC coverage Five cycles of SMC administered during the peak of the malaria transmission season reduced the burden of malaria by around 50% during the rainy season. Although this is lower than might be expected with monthly SMC, and although the burden of malaria has fallen compared to earlier studies undertaken in this area, this still represents an important public health impact. Lower efficacy may be partly attributable to use of other protective measures and lower SMC coverage. It is also likely that five monthly cycles of SMC were insufficient to adequately address the full malaria transmission season and that an additional number of cycles would be needed to achieve higher levels of protection using SMC in this and similar areas. Further research is needed to consider the role of SMC and other forms of chemoprevention in areas with seasonal malaria that extends over 6 or more months of the year.Figure S1 Prevalence of malaria at baseline by study communityFigure S2. Distribution of haemoglobin by study group in July 2012, January 2013 and July 2013Table S1. Distribution of children by community and study group.Table S2. Malaria incidence during the SMC period according to the number of rounds of SMC/placebo received.Table S3. Malaria incidence during the remainder of the post‐SMC period according to the number of rounds of SMC/placebo receivedTable S4. Malaria incidence over the whole study period according to the number of rounds of SMC/placebo receivedTable S5. Analysis of malaria incidence during the malaria transmission season: results from random effects Poisson regressionTable S6. Symptoms at January surveyTable S7. Symptoms at July SurveyClick here for additional data file.