Plasmodium falciparum has been previously reported. However, the impact of B19V-infection on the clinical course of malaria is still elusive. In this study, we investigated the prevalence and clinical significance of B19V co-infection in Gabonese children with malaria.High seroprevalence of parvovirus B19 (B19V) coinfection with P. falciparum malaria and 85 healthy controls using polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and direct DNA-sequencing.B19V prevalence was analyzed in serum samples of 197 Gabonese children with P. falciparum malaria patients (14.21%) in comparison to healthy individuals (1.17%) (P<0.001). Notably, the mild-malaria group revealed significantly lower hematocrit levels in B19V/P. falciparum co-infection than in P. falciparum mono-infection (P<0.05). Genetic analysis revealed a predominance of B19V genotype-1 (71.43%) in the studied population. However, B19V-genotype 2 was observed significantly more often in children with severe-malaria than in mild-malaria (P=0.04).B19V was detected in 29/282 (10.28%) of Gabonese children. B19V was observed more frequently in P. falciparum malaria and signifies a possible contribution of B19V on the clinical course of malaria in a genotype-dependent manner. B19V co-infection should be considered as a additional diagnostic measure in malaria patients with life threatening anemia.Our findings reveal that B19V-infection is frequent in Gabonese children with  Plasmodium while Plasmodium falciparum  being the most virulent species [Malaria is one of the major causes of morbidity and mortality in tropical and sub-tropical countries and is caused by the protozoan parasites of the genus  species .P. falciparum infected individuals show different clinical phenotypes, which range from asymptomatic infections to severe forms of malaria. The clinical resolution is influenced by multitude of host and parasite factors [P. falciparum malaria both in adults and in children [ factors . Recent children -8. The Bchildren ,9.Parvoviridae[erythema infectiosum (fifth disease), hydrops fetalis, and aplastic anemia in individuals with underlying hemolysis [B19V belongs to the genus Erythrovirus within the family of voviridae. Parvovivoviridae. An acutvoviridae. B19V caemolysis . Other memolysis ,13, myocemolysis ,15, vascemolysis , neuroloemolysis , and hepemolysis ,19.P. falciparum among young children in the Republic of Nigeria [P. falciparum associated severe anemia  showed an evidence of B19V-infection [B19V-infection has been demonstrated to elevate severe anemia caused by  Nigeria . The B19nfection . Similarnfection ,20,21. Hnfection  and Kenynfection  observedP. falciparum infected Gabonese children from sub-Sahara Africa. We aim to investigate the prevalence of B19V-infection from this malaria endemic area and further sought to determine the relationship of B19V and P. falciparum co-infection on the etiopathogenesis of P. falciparum malaria.In the present case–control study we utilized 282 healthy and P. falciparum with well-characterized clinical profiles  or mild malaria (n=100) based on clinical signs, symptoms and parasite load with clinical profiles as shown in Table P. falciparum infection has been described previously [282 children were recruited at the Albert Schweitzer Hospital, Lambaréné, Gabon, and the Centre Hospitalier de Libreville, Libreville, Gabon. The investigated cohort is from a matched pair, case–control study, to compare severe and mild malaria in Gabon. Details of the study cohort are as described elsewhere ,25. Serues Table . 85/282 ruitment . The heaeviously -29.Severe malaria was defined as severe anemia (hemoglobin <50 g/L) and/or hyperparasitemia  and facultative signs of severe malaria, such as cerebral malaria, convulsions, hypoglycemia, and respiratory distress.Individuals with mild malaria were chosen from patients of the same sex, age, and locality, and admitted as soon as the severe cases were enrolled for this study. For mild malaria, the following criteria were considered: parasitemia between 1,000/l and 50,000/l on admission, no schizonts in the periphery, malarial pigment containing circulating leukocytes <50/μl, hemoglobin >80 g/L, platelets >50/nl, leukocytes <12/nl, and blood glucose >50 mg/dl  according to the manufacturer’s instruction. The detection of B19V-DNA was tested by nested polymerase chain reaction (nPCR) as described previously using primer pairs specific for the VP1/VP2-coding regions . All sam5 IU/ml B19V DNA along with integrated B19V-negative control plasmids. In order to standardize the qPCR, increasing dilutions of B19V plasmid DNA (6.5 × 104 to 6.5 × 101 B19V IU/ml) were included. The lower sensitivity of the B19V-qPCR assay was found to be 2 × 102 IU/ml. Samples were analyzed in duplicates including extraction of nucleic acids.The B19V-DNA load was determined in the serum by B19V-specific quantitative real-time PCR (qPCR) using the GeneAmp 7300 sequence analyser  The primers, probes and program conditions were used as described previously . The speDNA fragments spanning the coding NS1/VP1u region from nt 2355–2690 (nt. position according to AF162273) were amplified by nPCR as described previously for all B19V-DNA positive samples . The seqAnti-B19V-IgM and anti-B19V-IgG (VP1/VP2) were analyzed by enzyme-linked immunosorbent assay (ELISA)  according to the manufacturer’s instructions. An Index Value >1.20 measured of the serum samples in the ELISA assay indicated the presence of B19V IgG or IgM antibodies. The anti-B19V IgM ELISA assay has a reported sensitivity of 89% and specificity of 98% and the anti-B19V IgG ELISA assay a sensitivity of 96% and specificity of 97%, respectively.http://www.ebi.ac.uk/Tools/msa/clustalw2/) and GENEDOC2.5 (http://www.psc.edu/biomed/genedoc). Genetic distances were calculated using the Kimura two-parameter model incorporated into the MEGA5.0 [B19V-sequences were aligned using CLUSTALW ,JMP Statistical Discovery Software, version 5.0.1 (http://www.jmp.com), and Prism5 . A P-value <0.05 was considered to be statistically significant in all statistical computations. We computed Fisher’s exact test and the Mann–Whitney U test and Spearman’s Rho test for our analysis as accordingly.Statistical analysis was performed using Statview 4.57  of Gabonese children. The prevalence of B19V-DNA was significantly higher in patients with P. falciparum malaria compared to healthy subjects .Investigation on the prevalence of B19V in patients with mild, severe P. falciparum were examined for anti-B19V-IgM and anti-B19V-IgG. In total, B19V-IgM and B19V-IgG antibodies were detected in 20/28 (71.43%) and 19/28 (67.86%) serum samples, respectively. Overlapping anti-B19V-IgM and anti-B19V-IgG were detected in 11/28 (39.28%) cases, whereas 9/28 (32.14%) patients were anti-B19V-IgM-positive and anti-B19V-IgG-negative and 8/28 (28.57%) of patients were anti-B19V-IgG-positive and anti-B19V-IgM-negative.Twenty-eight B19V-DNA positive samples of patients with Additionally simultaneous detection of B19V-DNA and B19V-IgM antibodies in 71,4% (20/28) of the serum samples is indicative for an acute or recently acute B19V-infection rather than a persistence of B19V-DNA.P. falciparum co-infections, we examined B19V loads in the serum of the Gabonese children with malaria. The B19V loads in sera of B19V/P. falciparum co-infected patients were 3.08 log10 (range 1.65-4.83) IU/ml. No significant differences of the B19V loads of sera of mild malaria patients  compared to severe malaria patients  were observed, P>0.05  compared to P. falciparum mono-infection .Additionally, the parasitemia of P. falciparum co-infection in comparison to P. falciparum mono-infection  vs. 8 of 28 (28.57%), whilst genotype 3 could not be detected in the study population.Currently, three Erythrovirus genotypes  have been described ,36 whereP. falciparum malaria (mild and severe), we examined the distribution of B19V-genotypes in serum of P. falciparum malaria patients. No significant differences in the levels of parasitemia, hemoglobin and hematocrit in the serum of the malaria patients across the different B19V genotypes were observed (data not shown). Additionally, B19V-DNA loads showed no significant differences .To determine the contribution of B19V-genotypes on the course of Recently, our studies demonstrated that B19V-genotype 1 can be classified into at least two B19V sub-genotypes 1, B19V-1A and B19V-1B . In ordeThe genetic distances among the newly detected B19V- and reference strains were calculated based on the Kimura two-parameter model using MEGA5.0 software. The results of the genetic distance analysis of the sub-genomic B19V NS1/VP1u region of the B19V samples are shown in Figure P. falciparum infection still pose major health problem in developing countries and in many sub-Saharan countries such as Gabon. In regions with high endemicity for P. falciparum infection, the majority of children with malaria present a mild form of the disease. A small proportion of individuals infected with P. falciparum develop severe forms of malaria with a higher risk of mortality and morbidity [P. falciparum malaria is yet controversial. Anemia in malaria is multifactorial and mainly due to haemolysis and dyserythropoiesis [P. falciparum probably resulting in case of co-infection in more severe anemia [Malaria due to orbidity . The assopoiesis . Recentlopoiesis ,40. Thisopoiesis . B19V-inopoiesis ,41. Notae anemia ,21,42-44P. falciparum co-infection in Nigeria [A previous study has shown a high prevalence of B19V and  Nigeria . In cont Nigeria , Ghana [ Nigeria , and Ken Nigeria ,45 foundP. falciparum malaria patients than in healthy children  when compared to mild malaria (12.37%). These findings suggest that B19V may probably play a role in the pathogenesis of P. falciparum malaria following a B19V prevalence profile of healthy control < mild malaria < severe malaria. In accordance with our findings on increased B19V detection in P. falciparum malaria patients, a previous report suggested that depression of cell-mediated immunity in P. falciparum infection might favour co-infection with opportunistic pathogens including B19V [In accordance to the study of Nigeria, we could demonstrate that B19V genomes were detectable in 10.28% of young Gabonese individuals while the frequency of B19V was significantly higher in ing B19V .P. falciparum malaria previous studies have shown a failure of recovery of hemoglobin after effective anti-malarial treatment and delayed recovery of P. falciparum malaria after subsidence of B19V infection [P. falciparum malaria [P. falciparum co-infected patients in comparison to P. falciparum mono-infection , and parasitemia showed no significant differences between mild and severe malaria groups in the studied population possibly postulate that there is no direct or indirect interaction of B19V and P. falciparum patients. Only Erythrovirus (B19V)-genotypes 1 and 2 were observed in the studied population, however genotype 3 cannot be observed in our study population. B19V-genotype 1, and especially B19V-genotype 1A, was the most prevalent genotype in Gabonese children. This is in accordance with previous studies showing a predominance of B19V-genotype 1 worldwide [Different genotypes of viruses, such as human immunodeficiency virus (HIV) and hepatitis B and C viruses, show different susceptibility to disease progression or response to antiviral treatment. We therefore analysed the distribution of B19V-genotypes in the studied orldwide ,35,48,49orldwide ,50. Howeorldwide .P. falciparum malaria patients across the different B19V genotypes 1 and 2. However, genotype 2 was detected significantly more often in children with severe-malaria compared to children with mild-malaria (P<0.05). The detected association of B19V-genotype 2 with severe malaria needs further analysis, although, one can postulate that variability in the host immune response to distinct B19V-genotypes may correspond with the severity of anemia in B19V/P. falciparum patients.We did not observe any significant differences in parasitemia, hemoglobin and hematocrit levels of P. falciparum malaria in Gabonese children. Additionally, our results point towards increased B19V co-infection among young children with P. falciparum malaria. Two of the three Erythrovirus genotypes are detected in these populations while genotype 2 remained more frequent in children with severe malaria. The hematocrit levels of B19V/P. falciparum co-infected mild-malaria patients were significantly decreased as a consequence of co-infection. These findings indicate that the clinical outcome of P. falciparum malaria in Gabonese children is influenced by B19V co-infection and should be considered as a diagnostic measure in patients with worsening anemia and persistent fever in spite of effective anti-malarial therapy. However the precise molecular mechanisms involved in B19V co-infection in the context of pathogenesis of P. falciparum malaria yet needs to be understood and warrants further analysis.Overall to our knowledge this is the first study which supports the hypothesis that an acute B19V infection can modulate the clinical course of B19V: Parvovirus B19; PCR: Polymerase chain reaction; nPCR: nested PCR; qPCR: quantitative real time PCR; ELISA: Enzyme-linked immunosorbent assay.The authors declare that they have no competing interests.PGK, RK, NLT, and CTB conceived the study and designed the research; HVL, NTB, VQB, and TPV participated in developing study instruments; NLT, BTS. LHS, and TPV conducted the research; NLT, BTS, and TPV performed statistical analysis; PGK, RK, NLT, LHS, BTS, TPV and CTB participated in writing and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/375/prepub


Severe malaria remains a major cause of global morbidity and mortality. Despite the use of potent anti-parasitic agents, the mortality rate in severe malaria remains high. Adjunctive therapies that target the underlying pathophysiology of severe malaria may further reduce morbidity and mortality. Endothelial activation plays a central role in the pathogenesis of severe malaria, of which angiopoietin-2 (Ang-2) has recently been shown to function as a key regulator. Nitric oxide (NO) is a major inhibitor of Ang-2 release from endothelium and has been shown to decrease endothelial inflammation and reduce the adhesion of parasitized erythrocytes. Low-flow inhaled nitric oxide (iNO) gas is a US FDA-approved treatment for hypoxic respiratory failure in neonates.This prospective, parallel arm, randomized, placebo-controlled, blinded clinical trial compares adjunctive continuous inhaled nitric oxide at 80 ppm to placebo , among Ugandan children aged 1-10 years of age with severe malaria. The primary endpoint is the longitudinal change in Ang-2, an objective and quantitative biomarker of malaria severity, which will be analysed using a mixed-effects linear model. Secondary endpoints include mortality, recovery time, parasite clearance and neurocognitive sequelae.Noteworthy aspects of this trial design include its efficient sample size supported by a computer simulation study to evaluate statistical power, meticulous attention to complex ethical issues in a cross-cultural setting, and innovative strategies for safety monitoring and blinding to treatment allocation in a resource-constrained setting in sub-Saharan Africa.NCT01255215ClinicalTrials.gov Identifier:  Malaria is the leading parasitic cause of morbidity and mortality worldwide, causing an estimated 240 million clinical cases and 800,000 deaths annually . Childrein vivo by the enzymatic conversion of L-arginine and molecular oxygen to L-citrulline by members of the nitric oxide synthase (NOS) family of proteins. A free radical, NO is a highly labile molecule with a half-life of several seconds that reacts with transition metal or thiol groups of numerous target proteins [NO is a gaseous, lipid-soluble free radical that is produced proteins . NO readproteins . NO reguproteins . A well-proteins .Evidence from a murine model suggests that reduced NO bioavailability contributes to the pathogenesis of experimental cerebral malaria . "FootprThe mechanism of action by which NO might improve outcomes in malaria may involve the vascular endothelium, which plays a central role in the pathogenesis of cerebral malaria. Activation of endothelial cells is characterized by increased surface expression of cellular adhesion molecules, the exocytosis of Weibel-Palade bodies (WPB), and the breakdown of intracellular tight junctions with transudation of intravascular fluid producing end organ dysfunction. In malaria, parasitized erythrocytes (PEs) adhere to the microvascular endothelium, resulting in sequestration and vascular obstruction, impaired perfusion and tissue hypoxia . AutopsyLow-flow iNO at a concentration of 5-80 ppm is approved for use by the US FDA for the treatment of neonates with hypoxic respiratory failure, in whom it reduces requirements for extracorporeal membrane oxygenation (ECMO) and improves survival . After aIn summary, a clinical trial of adjunctive inhaled nitric oxide (iNO) in severe malaria is warranted on the basis of firm proof of concept from animal models  and a clThe study is a prospective, parallel arm, randomized, placebo-controlled, blinded clinical trial of adjunctive continuous inhaled nitric oxide at 80 ppm versus placebo , among children aged 1-10 years of age with severe malaria. Figure The primary objective of this trial is to compare the longitudinal change in Ang-2, an objective and quantitative biochemical marker of malaria severity, among children who are randomized to receive inhaled nitric oxide in addition to standard antimalarial therapy compared to those randomized to placebo in addition to standard antimalarial therapy.• To determine the efficacy of adjunctive iNO in severe malaria based on clinical parameters: mortality, recovery times, length of hospital stay.• To assess the effect of adjunctive iNO on laboratory parameters: parasite clearance, whole blood lactate levels, and other biomarkers of malaria severity.• To determine the efficacy of adjunctive nitric oxide in preventing neurocognitive sequelae after severe malaria.• To assess the tolerability and safety of iNO in severe malaria.The working hypothesis is that young children hospitalized with malaria will benefit from adjunctive iNO, as determined by more rapid improvement in serum Ang-2 levels. We will test this hypothesis by comparing the change in Ang-2 over the hospital admission between the two groups randomized to receive iNO or placebo (room air) using a mixed-effects linear statistical model.The secondary hypotheses are that adjunctive iNO will reduce mortality, accelerate recovery times, and shorten length of hospital stay in severe malaria. Furthermore, we hypothesize that iNO will accelerate improvements in biomarkers of host response to severe malaria, including whole blood lactate, but will not affect parasite clearance. We hypothesize that iNO will reduce the rate of adverse neurocognitive sequelae following severe malaria. Finally, we hypothesize that adjunctive iNO will be safe and well tolerated in children treated for severe malaria.Children will be eligible for the trial if they meet the following inclusion criteria:1. Age 1-10 years2. Positive malaria rapid diagnostic test (RDT)3. One or more features of severe malaria: repeated seizures ; prostration ; impaired consciousness ; respiratory distress: age related tachypnea with sustained nasal flaring, deep breathing or subcostal retractions4. Willing and able to complete follow up schedules for the study - 14 day and 6 months after hospital discharge.P. falciparum antigens, histidine-rich protein-2 (HRP-2) and parasite lactate dehydrogenase (pLDH), and we will require positivity for both antigens for trial inclusion. Thus, our parasitologic criterion is expected to be highly specific, in order to include only patients who are truly parasitemic.The inclusion criteria require timely parasitological confirmation of malaria infection prior to enrolment, which poses logistical challenges at our peripheral centre with limited laboratory resources. For diagnosis, we will use commercially available immunochromatographic RDTs, complemented where possible with microscopy of peripheral smears. Despite well-recognized variability in the test performance characteristics of RDTs under field conditions, one commercially available RDT , Premier Medical Corporation Ltd., India) is highly ranked by the World Health Organisation (WHO), with a 95% detection rate even at low parasitemia and a false positive rate of 0% . This RDThe exclusion criteria for this study are as follows:1. Baseline methemoglobinemia (>2%)2. Known chronic illness: renal, cardiac, or hepatic disease, diabetes, epilepsy, cerebral palsy, or clinical AIDS3. Severe malnutrition, defined as weight-for length or height below -3 standard deviations based on WHO reference charts, or symmetrical edema involving at least the feet .4. Severe malarial anemia  without other signs of severe malaria.The latter exclusion criterion was chosen because of differences in the pathophysiology of SMA  compared to other malaria syndromes characterised by excessive inflammation and endothelial activation. Based on its postulated mechanism of action, it is less clear that iNO would benefit children with SMA.P. falciparum resistance to chloroquine and sulfadoxine-pyrimethamine is widespread in the country (34% to 67%) [The study will be conducted at a single pediatric hospital in Jinja, Uganda. Uganda is a low-income country with a severely resource-constrained healthcare system. Malaria transmission is moderate and seasonal in Jinja, which lies on the northern shore of Lake Victoria in the central area of Uganda near the capital, Kampala. Jinja Regional Referral hospital admits at least 175 children with severe malaria annually , representing over 30% of all admissions.  to 67%) .Participants in the intervention group will receive iNO at a concentration of 80 ppm, in addition to Ugandan standard of care of severe malaria, which includes a potent anti-parasitic agent. In light of the recent AQUAMAT trial that demonstrated a mortality benefit of parenteral artesunate over quinine for the treatment of African children with severe malaria , artesunParticipants in the control group will receive room air (indistinguishable in odour and appearance from the mixture of 80 ppm iNO), in addition to Ugandan standard of care treatment for severe malaria, including parenteral artesunate. An air compressor will be used to deliver continuous flow of vehicle air in both groups.Simple randomization will be employed, using a computer-generated randomization list. Treatment allocation will be recorded on paper and kept in sequentially numbered sealed opaque envelopes, which will be drawn for each randomized participant by an unblinded investigator who is not responsible for patient care, laboratory or data analysis. We will retain all envelopes and records for quality monitoring purposes.2 concentrations [2, and methemoglobin levels. Cylinders containing NO will be attached to the ventilation circuit in all patients, but the flow of NO will be controlled according to treatment arm assignment in a manner blinded to patients, caregivers, and healthcare providers. Only the unblinded study site investigator will administer, monitor, and titrate the delivery of NO or placebo (room air), as well as monitor and record the safety parameters of methemoglobin and NO2 levels. Laboratory analysis for Ang-2 levels (primary outcome) and all other parameter will occur in a manner blinded to treatment allocation.In previous clinical trials using iNO, one of the design and implementation challenges was establishing the blinding procedures while titrating and monitoring concentrations of iNO, as well as anticipated dose-related increases of methemoglobin and NOtrations . The esttrations . Thus, aThe longitudinal change in serum Ang-2 concentration over the first 72 hours of hospital admission will be the primary efficacy endpoint. Elevated Ang-2 levels are associated with poor clinical outcome in severe malaria ,29 and ASecondary outcomes of the trial will include relevant clinical, laboratory and neurocognitive endpoints. We will compare the following clinical endpoints, consistent with other therapeutic trials for malaria ,48,49: mP. falciparum recrudescence, and at 6-months to undergo neurocognitive testing.After admission to hospital for severe malaria, disease survivors will typically be discharged after a week or less. Trial participants will be administered iNO during the first 72 hours of admission (or less if a patient recovers and no longer tolerates the mask), which represents the period of highest mortality. During hospitalization, detailed data on the interim medical history will be collected, with attention to complications like coma, seizures and hypoglycemia that might affect neurocognitive outcome. After discharge, patients will return for a follow-up visit at 14 days to test for NO is approved by the US FDA for the treatment of neonates with hypoxic respiratory failure, where it has been shown to improve oxygenation, decrease pulmonary hypertension, reduce the requirement for extracorporeal membrane oxygenation, and improve survival . In addi2) levels in the inspiratory ventilation circuit are well-recognized, dose-dependent, and reversible adverse effects of nitric oxide administration; however, at doses commonly used in clinical practice, these are not common or clinically important consequences [2 is generated from the gas phase reaction of NO with molecular oxygen, and is a known pulmonary irritant [2 were routinely monitored and elevated levels constituted a criterion for NO dose reduction. Among neonates receiving iNO at 80 ppm, 35% developed methemoglobinemia (>7%) and 19% had elevated NO2 levels (>3%), requiring reduction of the iNO concentration in the ventilation circuit [2 will be monitored and the iNO dose will be titrated downward according to these defined thresholds. Furthermore, elevated baseline methemoglobinemia will be used as an exclusion criterion from study participation as a possible indicator of genetic susceptibility to methemoglobinemia.Methemoglobinemia and elevated nitrogen dioxide  has been convened to supervise the trial. The DSMB is composed of medical and biostatistical experts with representation from Uganda and North America who will meet periodically and as necessary to review trial progress, safety data, and indices of study quality. Severe adverse events, including all deaths in the trial, will be reported in a timely fashion to the DSMB and to the ethics boards that approved the study.Ethical approval has been obtained from the Makerere University Research and Ethics Committee , the Uganda National Council on Science and Technology, and the Research Ethics Board of the University Health Network, Toronto, Canada (REB# 10-0607-B). The research is being conducted in accordance with the Declaration of Helsinki.Informed consent will be obtained from the parent/guardian of all children that participate in the study. Trained study team personnel will seek consent after a comprehensive discussion with the parent/guardian of a prospective participant in the local language (Lusoga) at an education-appropriate level. Assent will also be sought from those children who are alert and able to understand the trial (age 8 and above), and sustained dissent on the part of children will be honoured . SpecifiInternational collaborative research may face complex community challenges ,59. CommWe will use CE to improve awareness of our trial and its findings in the catchment area of the Jinja Regional Referral Hospital. We also hope the CE activities will contribute to key ethical objectives for the trial, including respect for communities, fairness, transparency and accountability of the trial overall. We will focus on the following CE activities and their associated aims: (1) understanding the relevant community by consciously reaching out beyond the hospital to its catchment areas and listening to their issues and concerns; (2) providing information about the trial, including the pre-clinical evidence behind iNO, timeframe, procedures, and what will happen if the trial is successful; (3) building relationships and trust with local frontline healthcare workers; (4) specific educational/training activities, based on consultation with the nurses and/or frontline healthcare workers to ascertain what would be most relevant and beneficial for them; and (5) feedback of trial results, guided by the community itself as to how and what types of feedback activities would be most appropriate. Several levels of community will be targeted, including parents and primary caregivers of children, who comprise the group at highest risk of malaria, as well as healthcare professionals within the hospital catchment area. These activities do not constitute a mechanism for recruitment of participants to the trial, since only children with severe malaria will be eligible. Instead the community engagement process is intended to build trust and avoid misunderstandings through a dynamic exchange of information and ideas between trial scientists and community members.We will enrol 180 patients (approximately 90 in each treatment arm). To arrive at this sample size estimate, we began with a preliminary calculation based on data from a recent clinical data in severe malaria, in which Ang-2 decreased by 2700 pg/mL/day (95% CI 1800-3600 pg/mL/day) . We assutime-by-treatment interaction at the 5% level (two-tailed). Table To validate this preliminary sample size estimate, consistent with our analytic plan (mixed-effects linear model), a simulation study was performed. A number of assumptions needed to be made, such as within-patient correlation and Ang-2 variability. Patient Ang-2 data were simulated using a multivariate normal distribution. A simple autoregressive correlation structure was used with correlations of 0.75, 0.5 and 0.25 for time lags of 1, 2 and 3 days, respectively. In previous studies, variability appears proportional to the mean . SimulatAn interim analysis for efficacy, safety and trial quality is planned at the midpoint of patient enrolment (approximately 45 patients per group). There is no plan to stop the trial prematurely for efficacy or futility based on the primary endpoint (Ang-2), other biochemical parameters, clinical recovery times or parasitological outcomes, given the modest size of the trial and limited statistical power at the midpoint. Data at the time of the interim analysis will be presented to the DSMB members for review, who may advise that the trial continue without modification, continue with changes to the protocol, or be discontinued prematurely. With respect to the statistical interpretation of safety data, the DSMB may recommend termination or modification of the trial if mortality rates exceed statistical thresholds as described above. However, we do not propose that the DSMB be strictly bound by pre-specified criteria, because of the complexity of the trade-offs between safety, efficacy, and the possibility that new information will change considerations. Rather, consideration of stopping guidelines requires a reasoned judgment based on all information that is available at the time of data review.time-by-treatment interaction. Given that we will have repeated measurements, possibly incomplete, over time, a linear mixed-effects model will be used to estimate and test the magnitude of the time-by-treatment interaction. A linear time trend will not be assumed by treating "day" as a categorical variable in the model. The primary analysis will be by intention-to-treat (ITT). That is, patients will be analysed in the group to which they were randomized, regardless of deviations from study protocol. A secondary per-protocol analysis may be considered if important deviations from the protocol compromise the validity of the ITT analysis.The primary focus is whether or not the rate of reduction in Ang-2 differs between the treatment groups. In statistical terms, this is a 2 test or Fisher's exact test. In addition, we will present Kaplan-Meier survival curves comparing patients treated with iNO and placebo. Time to death will be analysed using survival analysis (log-rank test for difference between treatment arms).Analysis will follow standard methods in other clinical trials for malaria ,48,49. MAmong survivors (a subgroup of randomized participants), recovery times will be analysed by survival analysis (log-rank test). Time to sit unsupported, time to coma resolution (in the subset of patients with coma at study admission) and time to discharge will be documented by treating clinicians blinded to treatment allocation. Time to fever resolution, defined as the time required to achieve a temperature <38°C and the time to maintenance of temperature <38°C, will be determined from frequent vital sign monitoring. The time required to achieve a reduction in parasite density of 50%, 90% and to undetectable levels will be determined from daily blood smears. Results will be expressed as the median time to each event, with 95% confidence intervals.Additional biomarkers  will be analysed using mixed-effects linear models, with the raw value or log-transformed value of the biomarker level as the dependent variable, as appropriate.2 test or Fisher's exact test. Differences in cognitive areas affected in the two age groups  will be assessed by comparing frequency of individuals with deficits in each area by χ2 test or Fisher's exact test. This will also serve as the best surrogate of impairment in a particular area in one age group versus another, since the type of testing for each cognitive area will be different for the two age groups, so no direct comparison of level of impairment will be possible across age groups. For individual tests, age-adjusted z-scores  will be analyzed by means of mixed-effects models to examine study group differences in relation to neurocognitive outcomes. The models will provide estimated mean differences between children treated with iNO to placebo controls.As in previous studies involving children in sub-Saharan Africa , standarAlthough our sample size is modest, we will explore subgroup effects by examining interaction terms in the mixed-effects linear model. Subgroup analyses related to important prognostic factors will be performed: age < 5 years or ≥5 years; HIV seropositivity; and bacterial co-infection.Quality management (QM), both quality control and assurance, is a continuous, ongoing process of evaluation of the quality of the conduct and documentation of studies. The first step in QM will be training/re-training of the research staff to ensure consistency in clinical management, sample processing and data collection. Standard operating procedures (SOPs) have been developed for all study related procedures and protocols, and study personnel will document any deviation from SOPs together with the reason for the deviation. The next step for QM will be monitoring of collected data on a prospective basis, with daily review of source documents for completeness, accuracy and consistency. Next, data entry will be verified periodically, and discrepancies will be reviewed with the nurses and medical officers to discover the reason for errors, take corrective measures and prevent future errors. Collection, storage and transport of clinical samples will also be monitored on a regular basis. The ethical conduct of the study will be monitored through initial training in research ethics for study staff, and documentation, mediation and resolution of any perceived violations of ethical standards by participants, their parents/guardians, or members of the community at large. Measures to minimize bias in the trial will be subjected to formal evaluation. Quality of randomization and allocation concealment will be evaluated by keeping sequentially numbered opaque envelopes containing the randomization code, which will be opened, signed and dated at the time of randomization. Quality of trial blinding will be evaluated by asking key trial persons  to guess patients' treatments at the end of their trial participation, and compare the answers with the actual treatments, as previously described . ExternaNitric oxide is an attractive candidate for an adjunctive therapeutic agent for severe malaria given pre-clinical data on its efficacy in animal models and an established track record of safety in clinical practice and previous trials. Unlike other NO donor molecules, iNO has not been reported to cause systemic vasodilation and hypotension . FurtherOutside the existing patent, iNO is relatively inexpensive , and canOther adjunctive treatment strategies for severe malaria previously tested in randomized controlled trials include immunomodulation, iron chelation, reduction of oxidative stress, anti-coagulation, volume expansion, reduction of intracranial pressure, and prevention of seizure activity . Only ona priori power calculation provides further refinement on a crude sample size calculation and provides additional evidence that the planned number of patients is adequate to test our hypothesis. This sample size validation is particularly important in light of a review highlighting that numerous previous trials of adjunctive treatments for cerebral malaria had insufficient statistical power to detect even large treatment effects [Our hypothesis that children with severe malaria will benefit from adjunctive iNO can be answered using a modest sample size (n = 180). This parsimonious design was made possible by selecting a quantitative biomarker of malaria severity, Ang-2, as the primary endpoint and by using a powerful statistical technique (mixed-effects linear model). In contrast, a study using mortality (dichotomous variable) as the primary endpoint would require a total of over 1,000 patients to detect a 30% reduction from the baseline mortality of 20% with 80% power, likely necessitating multicentre recruitment over several years and monumental resources. Ang-2 is a surrogate but well validated measure of malaria severity, appropriate for an early efficacy trial in a human population. Repeated longitudinal measurement of Ang-2 allows for increased precision in the quantitative outcome variable, thereby reducing the necessary sample size. The analytic plan, a mixed-effects linear statistical model, includes random-effect terms, appropriate for representing clustered and therefore dependent data arising when data are gathered over time on the same individuals . We perf effects .2 and methemoglobin monitoring) need to be introduced to the facility for the trial. A commercially available portable biochemistry instrument  and a pulse CO-oximeter for non-invasive methemoglobin monitoring  will allow for onsite monitoring of critical investigations, with outsourcing of other clinical testing to reference laboratories in Kampala. Objective qualitative determination of parasitemia at presentation for the purposes of trial enrolment using commercially available lateral flow immunochromatographic tests for parasite antigen detection (rapid diagnostic tests) will be used to supplement local microscopy, which may be subject to error in the absence of rigorous laboratory quality control [Some unique aspects of our trial relate to its setting in a peripheral, resource-constrained pediatric hospital in sub-Saharan Africa. Standard clinical investigations including quality-controlled microscopy, biochemistry and microbiology, as well as equipment to monitor gas delivery . On the other hand, Ang-2 has been extensively validated as an objective and quantitative biomarker of malaria severity ,29,76,77In summary, based on compelling data supporting the efficacy of iNO in experimental cerebral malaria in animal models, coupled with the documented safety of iNO in clinical practice and trials for other diseases, we have outlined a protocol for a randomized clinical trial of iNO for the adjunctive treatment of severe malaria in Ugandan children. If our study demonstrates a significant treatment effect, this would represent a major and important advance in the treatment of severe malaria with broad potential for global public health impact.2:nitrogen dioxide; NOS: nitric oxide synthase; PE: parasitized erythrocyte; pLDH: parasite lactate dehydrogenase; QM: quality management; RDT: rapid diagnostic test; sGC: soluble guanylate cyclise; SMA: severe malarial anemia; SOP: standard operating procedure; vWF: von Willebrand Factor; WPB: Weibel-Palade bodies.Ang-1: Angiopoietin-1; Ang-2: Angiopoietin-2; CE: community engagement; CONSORT: Consolidated Standards of Reporting Trials; DPTA/NO: dipropylenetriamine NONOate; DSMB: Data and Safety Monitoring Board; ECMO: extracorporeal membrane oxygenation; HRP-2: histidine-rich protein-2; ICAM-1: intercellular cell adhesion molecule-1; iNO: inhaled nitric oxide; NO: nitric oxide; NOCM is Chief Scientific Officer of Nitric Solutions Inc., developer of nitric oxide (NO) based medical products.KCK, WCL, and ALC are listed as inventors on a patent owned by University Health Network (Toronto) related to Ang-2 as a biomarker for infectious diseases that compromise endothelial integrity.All other authors: no conflicts.MH designed the study and wrote the manuscript. ROO participated in study design and logistical planning, and obtained ethical approval from the Ugandan bodies. SN participated in study design and logistical planning. CM participated in the study design and provided expertise related to iNO administration and safety monitoring. KET provided statistical input, designed the primary statistical analysis, and performed the computer simulation experiment for validation of the sample size. JVL provided expertise in ethics and community engagement. ALC participated in the study design and helped to draft the manuscript. WCL participated in the study design and helped to draft the manuscript. CCJ participated in the study design and helped to draft the manuscript. KCK conceived the study, participated in the study design and helped to draft the manuscript. All authors read and approved the final manuscript.Details of a process control chart to monitor mortality. This Microsoft Word file provides details of the statistical thresholds and the performance characteristics of a statistical control chart to monitor mortality in this trial of critically ill children.Click here for file


Despite some problems related to accuracy and applicability of malaria rapid diagnostic tests (RDTs), they are currently the best option in areas with limited laboratory services for improving case management through parasitological diagnosis and reducing over-treatment. This study was conducted in areas with declining malaria burden to assess; 1) the accuracy of RDTs when used at different community settings, 2) the impact of using RDTs on anti-malarial dispensing by community-owned resource persons (CORPs) and 3) adherence of CORPs to treatment guidelines by providing treatment based on RDT results.Data were obtained from: 1) a longitudinal study of passive case detection of fevers using CORPs in six villages in Korogwe; and 2) cross-sectional surveys (CSS) in six villages of Korogwe and Muheza districts, north-eastern, Tanzania. Performance of RDTs was compared with microscopy as a gold standard, and factors affecting their accuracy were explored using a multivariate logistic regression model.2 = 367.7, p < 0.001), while the specificity was significantly higher  when compared to the longitudinal study. As determinants of sensitivity of RDTs in both studies, parasite density of < 200 asexual parasites/μl was significantly associated with high risk of false negative RDTs , while the risk of false negative test was significantly lower among cases with fever (axillary temperature ≥37.5°C) . The risk of false positive RDT (as a determinant of specificity) was significantly higher in cases with fever compared to afebrile cases . Using RDTs reduced anti-malarials dispensing from 98.9% to 32.1% in cases aged ≥5 years.Overall sensitivity and specificity of RDTs in the longitudinal study  were 88.6% and 88.2%, respectively. In the CSS, the sensitivity was significantly lower (63.4%; χAlthough RDTs had low sensitivity and specificity, which varied widely depending on fever and parasite density, using RDTs reduced over-treatment with anti-malarials significantly. Thus, with declining malaria prevalence, RDTs will potentially identify majority of febrile cases with parasites and lead to improved management of malaria and non-malaria fevers. Prompt and correct diagnosis is important for effective management of malaria cases in order to reduce morbidity and mortality caused by delayed or poor management of patients particularly among under-fives and pregnant women. Malaria diagnosis has for a long time, and particularly at community level, depended on clinical diagnosis (based on patients' history and symptoms), which has low specificity leading to over-diagnosis and over-treatment . Thus, rMicroscopic examination of Giemsa-stained blood smears confirms the presence of malaria parasites and remains the gold standard for malaria diagnosis despite its technical challenges and demand for trained personnel. However, most peripheral health facilities in malaria endemic countries lack the capacity to carry out parasitological diagnosis of malaria by microscopy -6. In TaPlasmodium falciparum histidine rich protein 2 (PfHRP-2), Plasmodium lactate dehydrogenase (pLDH) or aldolase  were neither treated with ALu nor referred to the nearest health facility by CORPs. Using a cut-off of parasite density of 5,000 asexual parasites/μl, only 36(0.2%) cases [including 5(0.1%) under-fives and 31(0.2%) aged ≥5 years] were not treated with anti-malarial drugs and thus put at risk of developing a severe disease in case they did not return to CORPs or go to find treatment elsewhere.The WHO has recently recommended that treatment with anti-malarial drugs should be given to patients with malaria parasites confirmed by laboratory tests or RDTs in areas lacking the capacity for detection of malaria parasite by microscopy . The Tanet al. [et al (unpublished data). The low sensitivity observed in the CSS might mainly be due to large number of cases with low level parasitaemia, which was below the detection limits of RDTs giving rise to high rate of false negative results. The low sensitivity of RDTs observed in the current studies was similar to what has been reported in other field studies conducted in South-eastern Tanzania [The findings of this study showed that both RDT brands (Paracheck and ParaHIT) had low sensitivity but relatively high specificity depending on fever status, parasite density, malaria parasite prevalence and transmission intensity. The overall sensitivity of RDTs was significantly higher in the longitudinal study compared to CSS and decreased with declining malaria parasite prevalence in both studies corresponding to the declining burden of malaria in the study areas as recently shown by Mmbando et al.  and IsheTanzania ,32 and oTanzania -35. HoweTanzania ,36 and aTanzania and UgandTanzania -39, and Tanzania ,40,41).Despite the differences in parasite density in the two studies  which increased with decreasing parasite prevalence, the sensitivity of RDTs was higher in febrile cases, and this was similar to what has been reported in studies conducted elsewhere in Africa ,37,42. IDespite significantly higher specificity in the CSS compared to the longitudinal study , the specificity observed in both studies was higher than what has been recently reported with similar RDTs (based on HRP-2) in Malawi . False pBefore introduction of RDTs, almost all cases attended by CORPs were treated with SP based on presenting symptoms leading to a significant level of over-treatment, since only 20.1% of all cases treated with SP during this period had malaria parasites confirmed by microscopy. However, this approach was cost-effective due to low cost of SP and was also safe since most of the febrile cases (regardless of parasite infection status) were promptly treated with anti-malarials thus reducing the risks associated with severe malaria, which often occurs when a patient is left without treatment. Despite the observed low accuracy of RDTs, which was lower than the sensitivity and specificity of ≥95% recommended by WHO , deploymAdherence to treatment guidelines by CORPs as stipulated in the SOPs was high (> 95%) in cases of all age groups. The level of adherence to treatment guidelines shown in this study was higher than what was reported by previous studies conducted in Zanzibar  and BurkThe current study utilized two brands of HRP-2 based RDTs (Paracheck and ParaHIT) which showed variable results regarding their accuracy, whereby both brands had similar sensitivity in the longitudinal study while Paracheck showed higher sensitivity in the CSS. In both studies, ParaHIT had higher specificity compared to Paracheck. Although these differences could be a true reflection of their performance under field conditions in an area where malaria has remarkably declined , this stin-vivo efficacy study of anti-malarials and in-vitro monitoring of drug resistance (which were stopped due to lack of enough samples). Unlike in health facilities and community studies where febrile patients are commonly attended and RDTs are intended to detect patients with parasites, the tests cannot be similarly used in CSS due to low number of patients with fever, low parasite density and therefore low sensitivity as shown by the findings of the CSS. However, these findings provide important information which will potentially guide future applicability of RDTs when fully introduced in many endemic countries for malaria diagnosis in the health facilities and communities; emphasizing that the tests have more relevance for case management than for disease surveillance.Furthermore, the performance of RDTs was compared to expert microscopy whose quality cannot be attained under health facility settings as recently shown by studies conducted in Tanzania  and otheet al. personal communication). Based on the setting of the longitudinal study where medical services were made available in the community through CORPs, cases not given anti-malarials had an opportunity to get treatment whenever they felt unwell, thus making this strategy safe and rational for testing the implementation of malaria case management based on RDT results.In the longitudinal study, some cases with malaria parasites (confirmed by microscopy) were neither treated with anti-malarials  nor given a referral to be attended at nearby health facilities as stipulated in the guidelines given to CORPs. While it would intuitively be important to know what happened to such cases, these issues are beyond the scope of this study since only passive case detection of fever episodes was performed, and follow-up of cases after treatment or those with negative RDT results who were not given anti-malarials was not done. However, other studies conducted in northern Tanzania showed that only 1% of children with negative RDT results developed parasitaemia during the 28 days of follow-up and only 1 out of 816 children developed a severe malaria condition demanding hospitalisation (Reyburn This study showed that RDTs used in the two community studies had varying accuracy (low sensitivity but relatively higher specificity) indicating that diagnosis of malaria using these HRP-2 based RDTs particularly in this era of declining burden of malaria remains a problem. Patients presenting in the health facilities from these communities for diagnosis of malaria are likely to have low parasite density which the current tests may fail to detect due to low sensitivity. High specificity of the RDTs indicates that most of the patients without malaria were correctly detected. However, introduction of RDTs in the longitudinal study reduced the number of cases without malaria who were treated with antimalarial drugs during the pre-RDTs as compared to the post-RDT period. Therefore, deployment of RDTs, coupled with supportive supervision can potentially reduce over-treatment and provide an opportunity for improved malaria diagnosis, and proper management of both malaria and non-malaria fevers. However, continued search and eventually introducing other alternative and sensitive malaria diagnostic methods should be explored.The authors declare that they have no competing interests.DSI, JPA, MA, TGT, IBC and MML conceived of and designed the study, DSI and PM conducted the field work and supervised the laboratory analyses. JPA and MML supervised field and laboratory work in Korogwe and Muheza districts. DSI, FF and BPM participated in data management and analysis, and DSI wrote the initial draft of the manuscript. All authors contributed during writing, read and approved the manuscript.


Plasmodium vivax malaria in Bikaner (western India). Patients exhibited cerebral malaria, renal failure, circulatory collapse, severe anemia, hemoglobinurea, abnormal bleeding, acute respiratory distress syndrome, and jaundice. Peripheral blood microscopy, parasite antigen–based assays, and parasite 18s rRNA gene–based polymerase chain reaction showed the presence of P. vivax and absence of P. falciparum.We report 11 cases of severe  Plasmodium vivax malaria is prevalent in many regions of the world. It accounts for more than half of all malaria cases in Asia and Latin America. Despite the high prevalence of disease caused by this parasite, research into its effects has lagged disproportionately  involvement with P. vivax, in which the diagnosis was confirmed by polymerase chain reaction (PCR) studies. Beg et al. reviewed the P. vivax cases with CNS involvement reported before 2002; however, most were diagnosed by examination of peripheral blood films (PBF) , acute respiratory distress syndrome (ARDS), and renal involvement caused by P. vivax. In most cases, the diagnosis was made by PBF examination without molecular diagnostic confirmation, thus allowing for potential errors in species diagnosis , and PCR. Our findings are shown in We searched available literature and could find only isolated reports of severe All patients were admitted to an intensive care ward dedicated to malaria control. Clinical, biochemical, and radiologic examinations were conducted to establish the diagnosis. Severe malaria was categorized and a treatment regimen of intravenous quinine was instituted according to World Health Organization guidelines  5′GCTCAAAGATACAAATATAAGC 3′–P. falciparum specific  and 18S rRNA–based PCR test, apart from PBF examination.The essential pathologic feature of severe malaria is sequestration of erythrocytes that contain mature forms of the parasite in the deep vascular beds of vital organs, thus producing cerebral malaria, renal failure, hepatic dysfunction, or ARDS. However, severe anemia and thrombocytopenia that causes bleeding diathesis is produced by hemolysis, reduced cell deformity of parasitized and nonparasitized erythrocytes, increased splenic clearance, reduction of platelet survival, decreased platelet production, and increased splenic uptake of platelets, and can be produced by P. vivax malaria. We cannot comment on a pathogenic mechanism causing multiple organ dysfunction and the characteristics of host-parasite interrelationship responsible for it. A detailed prospective study is required to address these issues.This is the first detailed report of severe


A randomized trial reported by Diadier Diallo and colleagues shows that intermittent preventive treatment for malaria in children who are protected from mosquitoes using insecticide-treated bednets provides substantial protection from malaria. Intermittent preventive treatment of malaria in children (IPTc) is a promising new approach to the control of malaria in areas of seasonal malaria transmission but it is not known if IPTc adds to the protection provided by an insecticide-treated net (ITN).p<0.001). There was a 69% (95% CI 6%–90%) reduction in incidence of severe malaria (p = 0.04) and a 46% (95% CI 7%–69%) (p = 0.03) reduction in the incidence of all-cause hospital admissions. IPTc reduced the prevalence of malaria infection at the end of the malaria transmission season by 73% (95% CI 68%–77%) (p<0.001) and that of moderately severe anaemia by 56% (95% CI 36%–70%) (p<0.001). IPTc reduced the risks of wasting  (p = 0.05) and of being underweight  (p = 0.03). Children who received IPTc were 2.8 (95% CI 2.3–3.5) (p<0.001) times more likely to vomit than children who received placebo but no drug-related serious adverse event was recorded.An individually randomised, double-blind, placebo-controlled trial of seasonal IPTc was conducted in Burkina Faso in children aged 3 to 59 months who were provided with a long-lasting insecticide-treated bednet (LLIN). Three rounds of treatment with sulphadoxine pyrimethamine plus amodiaquine or placebos were given at monthly intervals during the malaria transmission season. Passive surveillance for malaria episodes was established, a cross-sectional survey was conducted at the end of the malaria transmission season, and use of ITNs was monitored during the intervention period. Incidence rates of malaria were compared using a Cox regression model and generalized linear models were fitted to examine the effect of IPTc on the prevalence of malaria infection, anaemia, and on anthropometric indicators. 3,052 children were screened and 3,014 were enrolled in the trial; 1,505 in the control arm and 1,509 in the intervention arm. Similar proportions of children in the two treatment arms were reported to sleep under an LLIN during the intervention period (93%). The incidence of malaria, defined as fever or history of fever with parasitaemia ≥5,000/µl, was 2.88  per child during the intervention period in the control arm versus 0.87 (95% CI 0.78–0.97) in the intervention arm, a protective efficacy (PE) of 70% (95% CI 66%–74%)  involves administration of antimalarial drugs at defined time intervals to individuals, regardless of whether they are known to be infected with malaria, to prevent morbidity and mortality. IPT was initially recommended for pregnant women and recently this strategy was extended to include infants (IPTi). Now, there is also IPT of malaria in children (IPTc), which is designed to protect against malaria during the high malaria transmission season.Large clinical trials have shown that IPTc involving the administration of two to three doses of an antimalarial drug (sulphadoxine pyrimethamine [SP] and artesunate [AS] or amodiaquine [AQ]) during the high malaria transmission season effectively reduces the incidence of malaria. However, these studies were conducted in countries where the use of insecticide-treated bednets—an intervention that provides at least 50% protection against morbidity from malaria and is the main tool used for malaria control in most of sub-Saharan Africa—was relatively low. Therefore, it is unclear whether IPTc will be as effective in children who sleep under insecticide-treated bednets as has been previously shown in communities where insecticide-treated bednet usage is low. So to determine the answer to this important question, the researchers conducted a randomized, placebo-controlled trial of IPTc with SP + AQ (chosen because of the effectiveness of this combination in a pilot study) in children who slept under an insecticide-treated bednet in an area of seasonal malaria transmission in Burkina Faso.The researchers enrolled 3,014 eligible children aged 3–59 months into a randomized double-blind, placebo-controlled trial during the 2008 malaria transmission season in Burkina Faso. All children were given a long-lasting insecticide-treated bednet at the start of the study with instructions to their family on the correct use of the net. Children were then randomized into two arms—1,509 were allocated to the intervention group and 1,505 to the control group—to receive three courses of IPTc with SP plus AQ or placebos given at monthly intervals during the peak malaria transmission season. The researchers monitored the incidence of malaria throughout the malaria season and also monitored the use of long-lasting insecticide-treated bednets throughout the study period. In addition, researchers conducted a cross-sectional survey in 150 randomly selected children every week and in every child enrolled in the trial 6 weeks after the last course of IPTc, to measure their temperature, height and weight, and blood hemoglobin and parasite count levels.P. falciparum per microliter) was 2.88 in the control arm versus 0.87 in the intervention arm—giving a protective efficacy of 70%. There were 13 cases of severe malaria in the control arm and four in the IPTc arm—a 69% reduction in incidence. Additionally, all-cause hospital admission rate was reduced by 46%. At the end of the malaria transmission period, IPTc reduced the proportion of children infected with malaria parasites by 73% and reduced anemia by 33%. In addition, IPTc appeared to reduce the risk of wasting (risk ratio  = 0.79) and of being underweight (risk ratio  = 0.84). However, children who received IPTc were almost three times more likely to vomit than children who received placebo but there were no drug-related serious adverse events.The number of children who slept under their long-lasting insecticide-treated bednet was similar in both arms. During the intervention period, the researchers found that the incidence of clinical malaria  provides information on insecticide-treated bednetsRoll Back Malaria has an information sheet on  Significant efforts have been made in recent years to improve malaria control. However, malaria still remains a major public health problem in sub-Saharan Africa, responsible for about 800,000 deaths annually IPTc involves the administration of two to three doses of antimalarial drug during the high malaria transmission season. IPTc with artesunate (AS) plus amodiaquine (AQ) given to children aged 3–59 mo old on three occasions at monthly intervals during the malaria transmission season led to an 86% reduction in the incidence of clinical episodes of malaria in Senegal Insecticide-treated bednets (ITNs) provide at least 50% protection against morbidity from malaria The successful trials of IPTc described above were conducted in areas with relatively low coverage of ITNs. Thus, it is not known whether IPTc will be as effective in children who sleep under an ITN as has been found in communities where ITN usage is low. To determine this difference, we have conducted a randomised, placebo-controlled trial of IPTc with SP + AQ in children who slept under an ITN in an area of seasonal malaria transmission in Burkina Faso. A parallel study has been conducted in Mali employing a very similar protocol SP + AQ combination was chosen because this drug combination is cheap and remains highly efficacious for malaria treatment in Burkina Faso The protocol of the trial , protocoAn individually randomised, double-blind, placebo-controlled trial was carried out during the 2008 malaria transmission season to evaluate the efficacy of IPTc in children who slept under an LLIN. All children enrolled in the trial (aged 3–59 mo) were given an LLIN at the start of the study and their family instructed in the use of the net. Children were then randomised to receive three courses of IPTc with SP plus AQ or placebos given at monthly intervals during the peak malaria transmission season. The combination of SP plus AQ was chosen for the trial following a pilot study conducted in the study villages in 2006, which showed that this combination was effective at treating uncomplicated falciparum malaria . The incEthical clearance for the trial was obtained from the Health Ethics Committee of Burkina Faso and from the London School of Hygiene and Tropical Medicine ethics committee. The study's objectives and methods were explained to each study community prior to the start of the trial and written informed consent was obtained from care givers of children before enrolment in the study. The trial was monitored by an independent Data Safety and Monitoring Board (DSMB).Anopheles gambiae s.s., An. Arabiensis, and An. funestusPlasmodium falciparum is responsible for more than 95% of malaria infections in the study area.The study was carried out in four villages  in Boussé health district, Kourweogo province, approximately 40 km northwest of Ouagadougou. The climate in the study area is characteristic of the Sudanese savannah with a dry season from November to June and a rainy season from July to October. The main malaria vectors are P. falciparum per microlitre. This threshold has previously been shown to be of value in differentiating symptomatic malaria from other causes of fever with coincidental parasitaemia P. falciparum asexual parasites at any density; (2) the incidence of severe malaria defined according to WHO criteria The primary endpoint of the study was the incidence of clinical malaria defined as the presence of fever (axillary temperature ≥37.5°C) or a history of fever in the past 24 h, the absence of any other obvious cause of fever, and the presence of at least 5,000 asexual parasites of z-scores (WAZ) <−2 were classified as underweight and children with height-for-age z-scores (HAZ) <−2 were classified as stunted. Children with weight-for-height z-scores (WHZ) <−2 were classified as wasted.Hospital admission was defined as any event that involved a child staying at a hospital or health centre for at least 24 h for medical care. Anaemia was defined as a haemoglobin (Hb) concentration <11 g/dl, moderately severe anaemia as an Hb <8 g/dl, and severe anaemia as an Hb <5 g/dl. Wasting , stunting , and underweight  were assessed using the WHO child growth standards We first calculated the sample size required to provide the trial with 90% power to detect a 20% reduction in the incidence of clinical malaria in the IPTc arm of the study at the 5% level of significance. We assumed that the incidence of clinical malaria in children under 5 y of age in the study area, in the absence of any intervention, would be in the range of 1.0–2.0 episodes per year. We further assumed that in children protected by an ITN, the incidence of malaria would be reduced by 50% to 0.5–1.0 episodes per year. To detect a 20% reduction in the incidence of malaria, from 0.5 to 0.4 episode per year, in children who received IPTc, and allowing for a 20% loss to follow-up, we estimated that approximately 1,000 children would be required in each study arm. We further estimated that a study with this sample size would have at least 90% power to detect a 20% reduction in the prevalence of malaria infection (from 50% to 40%) at the end of the malaria transmission season With a sample size of 1,000 children per arm, the study was not powered to detect an impact on the incidence of severe malaria or hospitalization with malaria. We estimated that the rate of hospital admissions with malaria would be in the range of 20 to 60 per 1,000 children under 5 y of age. Assuming a rate of 40 per 1,000, approximately 3,000 children would be required per treatment arm to detect a 50% reduction in hospital admissions with severe malaria , assuming 20% loss to follow-up. A protocol amendment was made to increase the sample size after the site in Ghana was dropped from the trial . TherefoStudy villages were located in an area in which a demographic surveillance system (DSS) was implemented until 2002. We used the DSS database to identify and select four villages, each with a health centre and with a combined population of 3,600 to 3,800 children aged 3–59 mo. The aim was to sample about 500 children from the DSS database for the baseline prevalence of genetic markers of resistance to SP and AQ and screen the remaining children for enrolment in the trial, allowing for the possibility that a fraction of these children would not meet the inclusion criteria. Therefore, all children aged 3–59 mo in the four selected study villages were enumerated before the start of the intervention and screened for eligibility to participate in the study. The child's weight and height were measured. To be eligible, a child had to fulfil the following inclusion criteria: body weight of at least 5 kg, resident in one of the study villages with no plan to move out of the study area during the intervention period, no signs or symptoms of severe chronic illness, absence of signs of severe malnutrition, and signed informed consent obtained from the care giver. Children with a history of sensitivity to any antimalarial drug were excluded. Children diagnosed with clinical malaria during screening were randomised and then treated according to local guidelines.Eligible children were randomised to the intervention or control group using sealed envelopes. The randomisation list was prepared in three strata for children weighing 5–9 kg, 10–18 kg, and ≥19 kg by a statistician, and treatment group was assigned in each stratum in a 1∶1 ratio in permuted blocks of size 10. Blocks were not split across villages. Envelopes in each stratum were assigned in strict numerical order by the research team. When the child was enrolled, the child's name and randomization code were written on the outside of the envelope before the envelope was opened. Each child was given an identification (ID) card to facilitate follow-up.www.vestergaard-frandsen.com) regardless of their eligibility status. LLINs were marked to differentiate them from already existing nets and care givers were advised on how to use them.All screened children were given an LLIN  for malaria was performed for children with fever or a history of fever in the previous 24 h. If the RDT was negative, the child was given IPTc and appointments were made for subsequent IPTc visits before referral to the health centre to check for illnesses other than malaria. If the RDT was positive, a blood film was prepared and the child was treated with Coartem by the research team; children with severe malaria were referred to the health centres for treatment with quinine according to local guidelines. In such situations, IPTc was not given but the care giver was invited to bring the child back for the next round of IPTc.Passive surveillance for malaria and other illnesses was established soon after the enrolment of children. Health centres were supplied with lists of children enrolled in the study and with equipment and consumables for the diagnosis of malaria and anaemia. Care givers were asked to bring any child with fever to the health centre together with the child's ID card. The child's identity was verified against the list of enrolled children. Clinical examination was then conducted and signs and symptoms of any illness were recorded. An RDT was performed and if the test was positive, thick and thin blood films were prepared and sent to the CNRFP laboratory for microscopy. Coartem was not available at the health centres, so on the basis of RDT test results, treatment with AS+AQ was given if the child was suspected to have uncomplicated malaria. If a child had signs or symptoms of severe malaria, s/he was treated with quinine at the health centre or referred to the district hospital according to local guidelines. Children whose RDT was negative were examined for other causes of fever and an appropriate treatment was administered if required. A medically qualified member of the research team was based at the district hospital with responsibility for follow-up of study children who were referred to the district hospital. Full treatment costs, and the cost of transportation to the district hospital, were covered by the project.From mid-September to the end of November 2008, 150 enrolled children were randomly selected and surveyed each week to monitor the prevalence of malaria infection. These children were visited at home by nurses who measured the child's temperature, prepared thick and thin blood films for all children, and completed data collection forms. Care givers were asked if the child had had a fever within the last 24 h and whether the LLIN allocated to them was still in the house. If the LLIN was present, the care giver was asked if the child had slept under the net the previous night. Children with measured or reported fever were referred to the health centre for clinical examination and treatment as described above.A cross-sectional survey of all children enrolled in the study was carried out 6 wk after the last course of IPTc had been given (November 2008). Fever within the last 24 h was documented, and axillary temperature, weight, and height were measured. Thick and thin blood films and filter paper blood spots were prepared and Hb concentration was measured. An RDT was performed if a child had fever or a history of fever before referral to the health centre for clinical examination and appropriate treatment.Clinical personnel participating in the trial were trained to identify serious adverse events, including Stevens-Johnson syndrome, and to report them immediately to the local principal investigator. Adverse events were monitored on the day of administration of each dose and on the day after the last dose of each treatment course by members of the research team who were not involved in giving treatment. Questions were asked specifically about the occurrence of any of the following adverse events: fever, vomiting, diarrhoea, drowsiness, skin rash, coughing, loss of appetite, and any other events that the child had experienced were recorded. The duration and severity of adverse events was noted. If a serious adverse event was observed or reported, the Data Safety and Monitoring Board (DSMB) was notified of this event within 72 h. Detailed information was collected on a separate case report form and sent to the DSMB within 2 wk.P. falciparum parasitaemia during this survey were randomly selected from the intervention and control arms for PCR analysis. This sample size was based on results obtained from the monitoring of resistance markers in an IPT trial in Senegalese children 412 children not included in the main trial were randomly selected from the census list to assess the baseline prevalence of genetic markers of resistance to SP and AQ. Children were clinically examined, and thick and thin blood films and filter paper blood spots were prepared for malaria diagnosis and molecular testing for drug resistance. These children were given an LLIN but were not enrolled in the IPTc trial. Filter paper blood spots were also collected 6 wk after the third course of treatment from children involved in the trial to assess whether treatment with SP + AQ had led to an increase in the prevalence of genetic markers of resistance to these drugs. 312 of the filter papers collected from 746 children with Thick and thin blood films were air dried and stained with 5% Giemsa. Each slide was read by two laboratory technicians. Asexual and sexual parasites were counted separately and species differentiated. Malaria parasites were counted against 200 white blood cells (WBC). A slide was declared negative only after reading against 2,000 WBC without observation of a malaria parasite. The parasite count was converted to a parasite density per microlitre of blood by multiplying the number of parasites counted by 8,000 and dividing by the number of WBC counted. In the event of a discrepancy between the two readers in terms of the presence or absence of malaria parasites, or if parasite densities differed by more than 30%, the slide was reexamined by a third laboratory technician. The arithmetic mean of the two final readings was used as the final parasite density. If there was no agreement after the third reading, the arithmetic mean of the three parasite densities was used. External quality control of slide reading was performed by the Malaria Diagnosis Centre of Excellence (MDCoE) of the Walter Reed/Kenya Medical Research Institute, in Kisumu (Kenya). The results showed that the quality of slide reading was good with higher than 90% concordance on parasite detection and species identification .Hb concentration was determined using a Hemocue 321 (Hemocue AB). The measurement was repeated for any Hb value outside the manufacturer's range (<5 g/dl and >18 g/dl). The performance of the Hemocue was checked weekly with samples of known Hb concentration.dhfr gene and at codons 437 and 540 of the dhps gene as described previously P. falciparum chloroquine transporter gene (pfcrt-76) and at codon 86 of the P. falciparum multidrug resistance gene one (pfmdr1-86) DNA was extracted from filter papers collected before and after the intervention to look for the presence of genetic markers of resistance to SP and AQ. Nested PCR was performed to detect the presence of mutations at codons 51, 59, and 108 of the www.stata.com). Analyses were performed on the basis of intention to treat (ITT).Double data entry by two independent data entry clerks was performed. Data were entered using Microsoft Access. Consistency checks and audit trail programs were developed and the database was cleaned and validated before analysis. The trial analysis plan was approved by the DSMB. Data analysis was performed using STATA version 10  for the effect of IPTc on the incidence of malaria was estimated using Cox regression models with robust standard errors to account for repeated episodes within the same child. In secondary analyses, age, sex, and village were included in the regression model as covariates to obtain adjusted IRRs. Protective efficacy (PE) was derived from the IRR as follows: PE = (1 − IRR) ×100. Kaplan-Meier survival curves were plotted to compare the times to the first episode of malaria between children who received IPTc and those allocated to the placebo arm.Incidence rates of all-cause hospital admissions were estimated as the number of all hospital admissions divided by the number of child-days at risk computed as described above.Malaria parasitaemia was defined as the presence of malaria parasites irrespective of the developmental stage or species. The presence or absence of malaria infection was coded as a binary variable. Proportions of children with malaria parasites during weekly visits and at the cross-sectional survey carried out 6 wk after the last dose of IPTc were estimated for placebo and intervention arms. The presence of anaemia or moderately severe anaemia 6 wk after the last dose of IPTc was also coded as a binary variable. The risk ratios (RRs) for malaria infection, anaemia, and moderately severe anaemia among IPTc children compared with controls were estimated using generalized linear models. Age, sex, and village were included as covariates to obtain adjusted RRs. The PE of IPTc was calculated as follows: PE = (1 − RR) ×100.z-scores for weight-for-age , height-for-age , and weight-for-height . Wasting, stunting, and being underweight were defined according to the WHO child growth standards Anthropometric data collected 6 wk after the last course of IPTc were transferred from STATA version 10 to WHO's anthropometric software 3,052 children were screened and 3,014 were enrolled in the trial ; 1,509 wSimilar proportions of children in the intervention and control groups received at least the first dose of the first treatment course (77% versus 78%). However, a higher proportion of children in the intervention than in the control group received at least the first dose of the second and the third courses of treatment . The main reason for not receiving treatment was an acute illness. The numbers of children who received a full course of treatment (three doses) at each round were 1,055 (70%), 1,102 (73%), and 1,104 (73%) in the control group and 1,084 (72%), 1,213 (80%), and 1,272 (84%) in the intervention group for the first, second, and third rounds of treatment, respectively. Use of LLINs was monitored in a subsample of children through weekly home visits. Similar proportions of children in the control and in the intervention group slept under an LLIN (92.7% versus 92.8%).p<0.001), indicating a PE of 70%. Adjustment for age, sex, and village did not alter this estimate. There was strong evidence that the IRR varied with age (p<0.0001) with the effect of IPTc being strongest in the youngest children (3–23 mo). IPTc with SP + AQ was effective in reducing clinical malaria in the four study villages (p = 0.06); the smallest protective effect was observed in the village of Sao and in the remaining villages, the protective effect was similar. Very few children were reported as not having used a net the previous night and so whether the effect of IPTc on clinical malaria varied with ITN use could not be examined.169 (8.2%) children who had a positive RDT test were parasite negative by microscopy. Incidence of malaria defined as fever or history of fever with asexual parasitaemia ≥5,000/µl (primary endpoint) was estimated at 1.92 (95% CI 1.73–2.14) per child during the study period in Toeghin; 2.12 (95% CI 1.92–2.36) in Niou; 1.62 (95% CI 1.44–1.81) in Laye; and 1.65 (95% CI 1.47–1.84) in Sao. Overall the incidence of malaria was 1.3 (95% CI 1.11–1.53) in children aged 3–11 mo; 2.50 (95% CI 2.26–2.76) in children aged 12–23 mo; 2.56 (95% CI 2.31–2.83) in children aged 24–35 mo; 1.36 (95% CI 1.19–1.56) in children aged 36–47 mo; and 1.27 (95% CI 1.10–1.47) in children aged 48–59 mo. The incidence of clinical malaria was highest in children aged 12–35 mo . 982 epivillages . There wP. falciparum asexual parasitaemia at a density of 5,000 parasites/µl or more (p<0.0001)  .p<0.001) to the reduction observed when clinical malaria was defined as fever or history of fever with the presence of at least 5,000 asexual parasites/µl. Severe malaria was observed in 13 children in the control group and in four children in the intervention group  (p = 0.04) (p = 0.03)  showed a similar reduction  ( = 0.04) . The inc = 0.03) .p<0.001) (p<0.001).The prevalence of malaria parasitaemia among children visited at home during the intervention period was lower in the intervention than in the control group (18.6% versus 45.8%)  (p<0.001) . A lowerP. falciparum and P. malariae, respectively, the remainder being mixed infections. The prevalence of malaria infection increased with age (p<0.001) and varied between villages (unpublished data). 6 wk after the last course of IPTc treatment, 11.4% (164) of children in the IPTc arm had malaria parasitaemia compared to 41.5% (594) in the control group  (p = 0.001). There was strong evidence that the effect of IPTc on malaria infection varied with age (p = 0.003) with the effect being strongest in children aged 3–23 mo. The proportion of children who carried gametocytes postintervention as assessed by microscopy was also lower in children from the intervention arm than in the control arm (2.0% versus 9.3%) indicating a 79% (95% CI 68%–86%) reduction in the risk of gametocytes carriage in children who had received IPTc (p<0.001).The overall prevalence of malaria parasitaemia was 26.4% during the survey conducted at the end of the malaria transmission season (6 wk after the last course of IPTc treatment); 94.2% (714) and 1.2% (9) of these infections were single infections with p<0.0001) but there was no evidence that it varied between village (p = 0.42).The prevalence of anaemia (Hb<11 g/dl) overall was high but decreased with age, from 70% (772) in children aged 3–23 mo to 45% (809) in children aged 24–59 mo (p<0.001). Anaemia (Hb<11 g/dl) was less common in children in the IPTc arm than in the control arm  (p<0.001). 89 (6.2%) children in the control group had moderately severe anaemia (Hb<8 g/dl) versus 39 (2.7%) children in the intervention group  (p<0.001). There were too few cases of severe anaemia (Hb<5 g/dl) to perform any meaningful comparative analysis between the intervention and control groups .Hb concentration was higher in the intervention group than in the control group at the end of the malaria transmission season (mean Hb 11.01 g/dl [95% CI 10.94–11.08 g/dl] versus a mean of 10.35 g/dl [95% CI 10.27–0.42 g/dl]) (p = 0.05) in the risk of wasting (p = 0.03). The prevalence of stunting was similar in the two groups (p = 0.5). Comparison of the mean weight gain in the two treatment arms using data from children who were present both at the baseline and postintervention survey indicated greater weight gain in the intervention than in the control group (0.72 kg versus 0.57 kg) (p<0.0001). The effect of IPTc on clinical malaria varied in stunted and nonstunted children (p = 0.0001) with PEs of 62% (95% CI 54%–69%) and 78% (95% CI 73%–81%), respectively. No evidence of an effect modification was observed for underweight (p = 0.18) or wasting (p = 0.07).At the end of the malaria transmission season, IPTc was associated with a 21% reduction (95% CI 0%–35%) , associated with resistance to sulphadoxine, were estimated at 32.6% and 25%, respectively  and triple dhfr plus a single dhps (codon 437) mutation (p = 0.001). The proportions of children harbouring parasites with the pfcrt-76 or pfmdr1-86 mutations were similar in the control and intervention arms and did not increase postintervention as compared to baseline.The prevalence of genetic markers of resistance to SP and AQ was assessed before the intervention in children 3–59 mo old who were not enrolled in the trial but who lived in the same communities. The proportions of children carrying parasites with triple ectively . No chilin vivo study conducted in asymptomatic children resident in the study area in the year after the intervention study confirmed the efficacy of the SP + AQ combination in clearing asymptomatic P. falciparum infections  were associated with malaria, three with acute diarrhoea and malnutrition, two with diarrhoea only, and that one death was due to pneumonia.No drug-related serious adverse events were observed during the follow-up period. The risk of itching, skin rash, diarrhoea, drowsiness, or loss of appetite did not differ between children who received SP plus AQ and those who received placebo . The proTo the best of our knowledge, this is the first study to test if IPTc provides protection against malaria in children who are already protected by an ITN. Our results show conclusively that IPTc does provide substantial protection against clinical malaria episodes, severe malaria, and all-cause hospital admissions in children using an ITN. The primary role of ITNs is to prevent mosquito bites, thus reducing the risk of malaria infection, whereas IPTc clears existing infections and has a prophylactic effect preventing new blood stage infections. The high PE of IPTc in children using ITNs may partly reflect protection from infections acquired because of exposure to mosquitoes at night outside sleeping hours. Coverage of ITNs was low among older children and adults in the study area; the absolute reduction in malaria incidence due to IPTc may have been less if coverage of ITNs in the population as a whole had been higher. A similar additional effect of combining chemoprevention with ITNs was observed in a community randomised study of ITNs and chemoprophylaxis with Maloprim (dapsone-pyrimethamine) given every 2 wk in Sierra Leone The efficacy of IPTc against clinical attacks of malaria was a little less in our study than that seen in Senegal The reduction in the prevalence of anaemia seen in our study is consistent with the results of the study conducted in Hohoe, Ghana The study was not powered to detect an impact on cases of severe malaria, hospital admissions, or deaths. However, encouraging results were found with a reduction in deaths, overall hospital admissions, and severe malaria in children who received IPTc with differences in cases of severe malaria and hospital admissions being statistically significant. Thus, it seems likely that if IPTc was widely deployed in areas with a similar pattern of malaria transmission to that of the study area it would have a significant impact on severe morbidity and mortality from malaria. This supposition is supported by the results of an earlier study conducted in The Gambia We monitored adverse events over 4 d during each course of treatment and detected no serious adverse events related to the study drugs, which were reasonably well tolerated and safe. However, we observed a higher risk of vomiting in the intervention group, as has been reported in previous IPTc studies that used drug combinations containing AQ dhfr resistance markers seen at the end of the transmission season and sustained implementation of IPTc would inevitably increase drug pressure, a risk that would need to be balanced against the marked benefit that can result from this intervention. This risk can be reduced by using a drug combination rather than a single drug for IPTc and by using a different drug combination for IPTc than the one used for first-line treatment of symptomatic malaria.A number of concerns have been raised about the adoption of IPTc as a malaria control strategy. These include the possibility that IPTc will encourage the spread of resistance to the drugs used for IPTc. We observed an increase in the proportion of children harbouring genetic mutations associated with resistance to SP at the end of the malaria transmission season compared with the preintervention period but not in the prevalence of genetic mutations associated with resistance to chloroquine. However, in contrast to the findings of a study conducted in Senegal Another concern is that IPTc might interfere with the development of naturally acquired immunity to malaria, leading to an increase in the incidence of malaria in children after they move out of the age range in which IPTc is given. Administration of IPTc for 1 y did not lead to an increase in the incidence of clinical attacks of malaria in the following year in children in Senegal Another major concern is whether IPTc could be delivered on a large scale. As this was an efficacy trial, medications were given under the control of project staff and this study did not address the issue of the implementability of IPTc. This issue has been addressed in a previous study undertaken in Ghana kdr-mediated resistance to pyrethroids was found  were obtained from a well-established manufacturer (Vestergaard). Checks on the deltamethrin content of ITNs obtained at the end of the malaria transmission season showed protective levels of deltamethrin in most samples and this was confirmed by bioassays . Howeveras found . AlthougIPT of malaria with SP plus AQ was safe and provided substantial additional protection against severe and uncomplicated malaria to children who slept under an LLIN. There is now strong evidence to support the integration of IPTc into malaria control strategies in areas of seasonal malaria transmission. Further research is needed to identify alternative drugs as the long-term use of SP for IPTc is uncertain.Text S1Study protocol.(0.19 MB PDF)Click here for additional data file.Text S2Protocol amendment.(0.11 MB PDF)Click here for additional data file.Text S3CONSORT checklist.(0.09 MB PDF)Click here for additional data file.Text S4In vivo efficacy of the SP + AQ combination. Results of a pilot study of in vivo efficacy of SP plus AQ in children with uncomplicated malaria in the study area.(0.51 MB PDF)Click here for additional data file.Text S5In vivo efficacy of the SP + AQ combination. Results of an in vivo study of the efficacy of SP plus AQ in clearing malaria infection in children with asymptomatic malaria. The study was conducted 1 y after the intervention.(0.44 MB PDF)Click here for additional data file.Text S6External quality assurance of malaria microscopic diagnosis. Report of external quality control of malaria microscopic diagnosis.(0.10 MB PDF)Click here for additional data file.Text S7Entomological investigations. Reports on malaria transmission intensity estimation, bioassays, vector resistance to insecticide, and concentration of insecticide on LLINs.(0.46 MB PDF)Click here for additional data file.