Interventions that reduce exposure to malaria infection may lead to delayed malaria morbidity and mortality. We investigated whether intermittent preventive treatment of malaria in children (IPTc) was associated with an increase in the incidence of malaria after cessation of the intervention.An individually randomised, trial of IPTc, comparing three courses of sulphadoxine pyrimethamine (SP) plus amodiaquine (AQ) with placebos was implemented in children aged 3–59 months during the 2008 malaria transmission season in Burkina Faso. All children in the trial were given a long lasting insecticide treated net; 1509 children received SP+AQ and 1505 received placebos. Passive surveillance for malaria was maintained until the end of the subsequent malaria transmission season in 2009, and active surveillance for malaria infection, anaemia and malnutrition was conducted.On thousand, four hundred and sixteen children (93.8%) and 1399 children (93.0%) initially enrolled in the intervention and control arms of the trial respectively were followed during the 2009 malaria transmission season. During the period July 2009 to November 2009, incidence rates of clinical malaria were 3.84  and 3.45  episodes per child during the follow up period in children who had previously received IPT or placebos, indicating a small increase in risk for children in the former intervention arm  (P = 0.003). Children who had received SP+AQ had a lower prevalence of malaria infection (adjusted PR: 0.88 95%CI: 0.79–0.98) (P = 0.04) but they had a higher parasite density (P = 0.001) if they were infected. There was no evidence that the risks of moderately severe anaemia (Hb<8 g/dL), wasting, stunting, or of being underweight in children differed between treatment arms.IPT with SP+AQ was associated with a small increase in the incidence of clinical malaria in the subsequent malaria transmission season.NCT00738946ClinicalTrials.gov  Intermittent preventive treatment (IPT) of malaria is a promising strategy for malaria control in infants and young children. Meta analysis indicates a 30% (95%CI: 20-39) protective efficacy in infants against clinical malaria Interventions which reduce exposure to malaria could interfere with the development of acquired immunity which could, in turn, lead to a shift of morbidity and mortality from malaria from younger to older age groups (“rebound effect”) One of the rationales underlying the development of IPT as a malaria control tool is that by allowing some infections to occur between drug administrations, it is less likely than chemoprophylaxis to interfere with the development of naturally acquired immunity to malaria. Therefore, a number of studies have examined the malaria experience of infants in the year after they had received IPT (IPTi). In the initial study of IPTi undertaken in Tanzania Only limited information is available as to whether administration of IPT to older children is associated with an increased risk of malaria in the period following the intervention. No overall increase in risk was observed in Senegal and Mali The original protocol for this trial , the ameEthical approval to conduct the trial was obtained from the health ethics committee of Burkina Faso and from the London School of Hygiene and Tropical Medicine ethics committee . MeetingThe study was conducted from August 2008 to November 2009 in Boussé health district, in central Burkina Faso. Four villages  which each have a health centre were included. Malaria transmission in the study area is high and seasonal with most of the transmission occurring between July and October. Children aged 3–59 months residing in the study villages were enrolled. Details of the study area have been published previously www.vestergaard-frandsen.com); 1509 children were randomly assigned to receive IPT with sulphadoxine/pyrimethamine (SP) plus amodiaquine (AQ) and 1505 were randomised to receive placebos. SP was given as single dose of 25 mg per kg of sulphadoxine plus 1.25 mg of pyrimethamine and AQ was given for 3 days as a daily dose of 10 mg per kg. Three rounds of IPTc were given starting in August 2008 with a one-month interval between treatments. Details of the eligibility, randomisation and intervention procedures have been presented elsewhere An individually randomised, double-blind, placebo-controlled trial of intermittent preventive treatment of malaria was conducted during the malaria transmission season of 2008; 3014 children were enrolled after screening. All children were given a long lasting insecticide treated net (LLIN)  was performed and thick and thin blood films were prepared if a child had fever or a history of fever.Children with a positive RDT who were thought to have uncomplicated malaria were treated with artesunate plus amodiaquine (AS+AQ) or artemether lumethantrine (Coartem®) at the health centre. If a child had signs or symptoms of severe malaria, treatment with quinine was provided and, when required, the child was referred to the district hospital in keeping with local guidelines. Children with an illness other than malaria were given appropriate treatment free of charge. Hospital admissions due to malaria and other causes were recorded and children's condition was monitored until release from the health centre.From July 2009 to November 2009, weekly home visits were made to monitor the prevalence of malaria infection. Each week, 150 children per treatment arm were randomly selected for a home visit. A history of fever in the previous 24 hours was recorded, axillary temperature was measured and thin and thick blood films were prepared for all children. During home visits, caregivers of children were asked if the LLIN allocated to the child was still in the house and whether the child had slept under the net the previous night.At the end of the 2009 malaria transmission season, we performed a cross-sectional survey of all children from the original trial who were available. Children had a clinical examination performed by study medical staff. Caregivers were asked if their child had fever within the previous 24 hours and axillary temperature was measured. Thick and thin blood films and filter paper blood spots were prepared and haemoglobin concentration was measured. Children's weight and height were recorded.During these surveys, a rapid malaria diagnostic test (RDT) was performed on the spot if a child had fever or a history of fever. If the RDT test was positive and the child was suspected of having uncomplicated malaria, s/he was referred to health centre for appropriate treatment with AS+AQ or Coartem®. Children with severe malaria or an illness other than malaria were treated as described in the previous section. Details of the procedures used during these surveys have been presented previously Thick and thin blood films were air-dried and stained with 5% Giemsa and read independently by two laboratory technicians. The parasite count was measured against 200 white blood cells (WBC) and converted to a parasite density per µl assuming that 1 µL of blood contains 8000 WBC per µL. In the event of a discrepancy between the two readers, the slide was re-examined by a third laboratory technician. The arithmetic mean of the 2 readings was used as the final parasite density if there was agreement between two of readers. If there was no agreement after the third reading, the arithmetic mean of the three parasite densities was used.Haemoglobin concentration was determined using a Hemocue® 321  as described previously The sample size for the first phase of the study was set to detect a 20% reduction in the incidence of clinical malaria and a 50% reduction in the incidence of admissions to hospital for severe malaria in children who had received intermittent preventive treatment. Details regarding sample size calculation for the main study have been published elsewhere . Based on the requirements set out above, at least 1500 children were needed per treatment arm for the study to have a 90% power to detect reductions in the endpoint mentioned above. Thus, 1509 and 1505 children were enrolled and randomized to receive SP+AQ or placebos respectively. It was anticipated that there would be a 10%–20% of loss to follow-up in the second year. Therefore, it was estimated that with this sample size, the study would have greater than 90% power to detect a 20% increase in the incidence of clinical malaria and greater than 90% power to detect a 20% increase in parasite prevalence in subsequent high malaria transmission season post intervention in children who received IPT with SP+AQ.www.stata.com). The intervention period was defined as the period from the first dose of IPT administration to the end of November 2008 (42 days after the third dose of IPT). The post intervention high malaria transmission period was defined as the period from July to November 2009 and the whole post intervention period was defined as the period from December 2008 to November 2009, which included the low malaria transmission season dry season (December 2008 to June 2009). Children were grouped in two age categories for adjusted analyses: children aged 3 to 23 months and children aged 24 to 59 months at the start of the intervention in August 2008.Data entry was performed by two independent data clerks using Microsoft ACCESS and analyses were performed using STATA version 11  of malaria infection, anaemia (Hb<11 g/dL) and moderately severe anaemia (Hb<8 g/dL) were estimated using a generalized linear model. Parasite density and Hb concentrations were compared using Student's t-test.Anthropometric data were analysed as described previously Baseline characteristics of the 1509 and 1505 children randomized at the beginning of the trial to receive IPTc or placebos are reported elsewhere During the 2009 malaria transmission season (July 2009 to November 2009), 3493 episodes of malaria were recorded in study children; 1834 in children who had received IPTc in 2008 and 1659 episodes in those who had received placebos . IncidenKaplan-Meier survival plots  for timeThere was no evidence of an increased incidence of malaria post-intervention in the IPT arm when a malaria episode was defined as fever or history of fever and the presence of any parasitaemia  (P = 0.29).From July 2009 to November 2009, 37 study children who had received IPTc the previous year were admitted to hospital, including 19 hospitalisations due to malaria compared with 39 children, 16 with malaria, among those who had previously received placebos . There wWeekly home visits were carried out from July 2009 to November 2009 to 1495 children in each treatment arm; blood films were available from 1457 children (97.4%) who had previously received IPTc and from 1442 (96.4%) children who had previously received placebos. Five hundred and forty-eight children (37.6%) in the original IPTc group and 607 (42.1%) children in the original placebo group were parasitaemic . There wOf the children who were present at the beginning of the high malaria transmission season of 2009 (1416 in the former intervention and 1399 and the control group) 1336 children in the former intervention arm and 1321 children in the former control arm participated in the cross-sectional survey at the end of the 2009 malaria transmission season. Thick and thin blood films were available from 1324 (99.1%) of children who had previously received IPT and from 1302 (98.6%) children who had previously received placebos. Overall, the prevalence of malaria infection increased with age with children aged 24 months or more being 1.72-fold (95%CI 1.49–2.00) (P<0.001) more likely to carry malaria infection than their younger siblings. The prevalence of malaria was 40.4% in children from the former IPTc group and 40.1% in children in the control group   (P = 0.54) The prevalences of anaemia (Hb<11 g/dL) and moderately severe anaemia (Hb<8 g/dLl) were similar in children in the two study arms 678 (51.2%) compared with 631 (48.4%) and 49 (3.7%) compared with 57 (4.7%) respectively (RR: 1.06 95%CI: 0.95–1.18) (P = 0.29) and  (P = 0.40) .Weight and height were measured at the end of the post-intervention period in 1336 and 1321 children in former intervention and control arms respectively; z scores for weight for age, height for age and weight for height were obtained from 999 children in former intervention arm and from 987 children in the former control arm . The preChildren previously enrolled in a trial of IPTc with SP+AQ were followed up for a further 12 months to investigate whether children who had received IPTc were at increased risk of malaria  in the subsequent malaria transmission season as a consequence of impaired development of acquired immunity due to reduced exposure to malaria. The study found evidence that children who had received IPTc during the previous year experienced a small increase in the incidence of clinical malaria in the subsequent malaria transmission but there was no shift in the age at which these children had malaria. In a parallel study in Mali, which used very similar methods, a similar small increase  was observed Previous IPT studies that examined whether IPT was followed by a ‘rebound’ in malaria were conducted in areas with relatively low use of ITNs and low malaria transmission intensity. In our study area malaria transmission is now moderate and 93% of the children slept under an ITN. This could also interfere with the development of acquired immunity to malaria The effect of IPTc on clinical malaria during the post intervention year appeared slightly higher in older children who were likely to have been more exposed to malaria infection in their lifetime than in their younger siblings. This observation is consistent with previous reports from Senegal The small excess of clinical malaria in the post intervention period in children in the former intervention arm was not accompanied by an increase in all-cause hospital admissions or hospital admissions due to malaria. However, our study was not powered to detect a rebound effect in hospital admission, and caution is required in the interpretation of these results.Children who had received IPTc during the previous year were not at increased risk of harbouring a malaria infection at the end of the subsequent malaria transmission season nor did they have a higher prevalence of malaria infection during weekly surveys, despite their slightly increased susceptibility to clinical malaria. Although children who had received IPT in 2008 appeared to have lower prevalence of malaria infection in the subsequent malaria transmission season than control children, they had a reduced ability to control parasite density which is important for the development of clinical malaria, and may partially explain the slight increase in clinical malaria. Similar findings have been observed previously in children A limitation of this study, and previous studies that examined the role of IPTc in causing rebound malaria morbidity, is that the intervention was given during only one malaria transmission season. This may not be sufficient to significantly impair the acquisition of immunity and induce a large increase in malaria morbidity. In addition, most of these children enrolled in the studies undertaken so far had experienced significant exposure to malaria infection prior to the intervention and may, therefore, have developed some level of immunity to malaria before receiving IPTc which may have been sufficient to counterbalance the potential negative effects of reduced exposure to malaria on malaria morbidity in the subsequent transmission season. If IPTc is to be deployed on a major scale and given annually for the first few years of a child's life, it will be essential that the impact of several years of IPTc on the development of immunity to malaria is investigated carefully.Protocol S1Trial protocol(DOC)Click here for additional data file.Protocol S2Protocol amendment(DOC)Click here for additional data file.Checklist S1Consort Checklist(DOC)Click here for additional data file.Ethics S1(PDF)Click here for additional data file.


Human immunodeficiency virus (HIV)-1 infection increases the burden of malaria by increasing susceptibility to infection and decreasing the response to malarial treatment. HIV-1 has also been found to suppress the immune system and predispose to severe forms of malaria in adults. There is still a paucity of data on the association between HIV-1 infection and cerebral malaria in children. The aim of this study was to determine whether HIV-1 infection is a risk factor for cerebral malaria in children.We conducted an unmatched case-control study, in which 100 children with cerebral malaria were compared with 132 with uncomplicated malaria and 120 with no malaria. In stratified analyses we estimated odds ratios (ORs) and 95% confidence intervals (CIs) adjusted for age.HIV-1 infection was present in 9% of children with cerebral malaria compared to 2.3% in uncomplicated malaria (age-adjusted odds ratio (aOR) 5.94  1.36-25.94, p = 0.012); and 2.5% in children with no malaria . The age-adjusted odds of being HIV-positive among children with cerebral malaria compared to the control groups  was 4.98 (95% CI 1.54-16.07), p-value = 0.003.P.falciparum malaria and associated complications such as cerebral malaria. Other malaria preventive measures such as use of insecticide-treated mosquito nets should also be emphasized during counselling sessions.HIV-1 infection is associated with clinical presentation of cerebral malaria in children. Clinicians should ensure that children diagnosed with HIV infection are initiated on cotrimoxazole prophylaxis as soon as the diagnosis is made and caretakers counselled on the importance of adherence to the cotrimoxazole towards reducing the risk of acquiring  Malaria and Human immunodeficiency virus (HIV)-1 are two of the most common global health challenges today and the two infections commonly overlap in distribution in most countries especially in sub-Saharan Africa-6. HIV-1HIV-1 infected adults have a greater percentage of severe malaria episodes with more frequent hospitalizations compared to their uninfected counterparts and are at a greater risk of cerebral malaria ,3,7,9. IP. falciparum malaria infection and has a case fatality ranging from 5 to 40%  for more than 30 minutes after termination of a seizure or correction of hypoglycaemia, detection of asexual forms of All children recruited in this study were of unknown HIV status at time of enrolment and therefore were not on cotrimoxazole prophylaxis. We required 100 cerebral malaria cases to determine the association between HIV infection and cerebral malaria, in a case to control ratio of 1:1, with a power of 80% and 95% confidence interval according to Fleiss formula for case control studies . We contP. falciparum parasite density, HIV serology and CD4 cell count, blood glucose and serum electrolytes which were done in different laboratories. A finger prick was done for children with no malaria for the blood smear and rapid HIV test and a blood sample for CD4 count taken if the child was found to be HIV positive. The HIV tests were rapid and the children who were HIV positive had blood sent for CD4 cell count immediately. These were used to stage level of immunosuppression according to the WHO guidelines[3 and a CSF protein of ≤40 mg/dl. Patients with cerebral malaria and those with uncomplicated malaria received standard care according to the Mulago Hospital Department of Paediatrics guidelines. Those without malaria were seen by the surgeons who offered appropriate treatment for the surgical condition. HIV positive children were referred to the Paediatric Infectious Disease Clinic (PIDC) where they were evaluated, managed and followed up. The number of asexual parasites per 200 white blood cells (WBCs) was counted and parasite densities computed by assuming a mean WBC count of 8,000/litre (WHO)[A questionnaire was used to collect information such as the socioeconomic, demographic characteristics, clinical history, and physical examination as well as laboratory findings. We consecutively enrolled patients who fulfilled the inclusion criteria for the three study groups until the sample size was achieved. Cases and controls were recruited at the same time. Patients were examined by a qualified medical officer who documented all the findings. A trained counsellor was available to do both pre- and post-test counselling for HIV. Five millilitres of blood were drawn from each child with cerebral malaria and 2-3 millilitres in children with uncomplicated malaria for a malaria smear, uidelines. No repetre (WHO).The primary objective of this study was to assess HIV-1 as an independent risk factor for cerebral malaria. The secondary objective was to determine the prevalence of HIV-1 infection in children with cerebral malaria and the correlation between the level of immunosuppression (CD4+ T-cell count) and parasite density.Data was entered into EPI-DATA 3.1 software package and analysed using the statistical program SPSS version 13 software packages. Categorical variables were summarized as proportions while means and standard deviation were used for normally distributed continuous variables. Medians with corresponding inter-quartile ranges were used for skewed data. The Chi- square test was used to compare categorical variables between those cerebral malaria and those without. Multivariate analysis (logistic regression by backward method) was then used to tease out whether HIV infection is independently associated with clinical cerebral malaria.Informed consent to participate in the study was obtained from the caretakers after explanation of the study objectives and relevance, risks and benefits. A separate consent for HIV testing was also obtained. Laboratory results were available for patient management. Approval was obtained from the Makerere University Faculty of Medicine, Research and Ethics Committees, Mulago Hospital, and the Uganda National Council of Science and Technology.The study was conducted from November 2006 to March 2007 during which 352 children were recruited. One hundred had cerebral malaria (CM), 132 had uncomplicated malaria (UM) and 120 had no malaria (NM). Thirty two patients with suspected cerebral malaria were excluded, 28 of whom we could not do complete assessment and 4 had incomplete data. Ten percent (13/133) of children with no malaria were found with asymptomatic parasitaemia and these were also excluded from the analysis Figure Children with cerebral malaria also had median serum sodium of 137.0 mmol/L (range 131-142 mmol/L). One patient had severe hypoglycaemia at the time of enrolment (blood sugar below 2.2 mmol/L). The median blood sugar was 7.4 mmol/L (range 5.9-9.5 mmol/L). Thirty eight percent of patients with CM had severe anaemia (Hb < 5 g/dl). No microbiological culture or virological tests were done on the CSF since the white blood cell count was not raised.Children with cerebral malaria who were also HIV positive children were more likely to have had fever for more than three days, been on treatment prior to this admission, present with higher temperatures (>38°C) and have a higher parasite density Table . HIV posSixty seven (56%) of the 120 controls with no malaria recruited from SOPD had inguino-scrotal hernias, 26 (22%) presented with hydroceles, and the others had congenital anorectal malformations, umbilical hernias, undescended testis, rectal prolapse and different types of cysts. None of these controls required urgent intervention but only an appointment for elective surgery. Ideally, these controls  would have been selected from similar communities as the cases but this was not the case due to limited funds for this. This could have potentially caused bias in the HIV prevalence. The prevalence of HIV was 9% among children with cerebral malaria compared to 2.3% and 2.5% in the uncomplicated malaria and no malaria groups respectively.There was an association between HIV infection and clinical cerebral malaria, HIV infected children were more likely to have cerebral malaria than the HIV negative ones, unadjusted OR = 4.05 95%CI 1.24-14.19), p = 0.006 and age-adjusted odds ratio (aOR) was 4.98 (95%CI 1.54-16.07) p = 0.003. Among children below the age of 5 years, the HIV prevalence in cerebral malaria cases was 8.6% (7 of 81) compared to 1.2% (2 out of 166) among all controls. The odds of presenting with clinical cerebral malaria below the age of five was higher among children who were HIV positive compared to the HIV negative counter parts OR = 7.76 ; p-value 0.003  p = 0.037. The aOR was 5.94 (95% CI 1.36-25.94) when comparing children with cerebral malaria to children with uncomplicated malaria, p-value 0.012 ; p-value = 0.336). However, the numbers of HIV positive children in this sample was very small.Our results are in agreement with studies conducted in Kwazulu Natal, South Africa and in Kenya where researchers in those countries also found similar association with HIV infection and severe malaria in children ,24,25. IP. falciparum malaria and severe forms of malaria [The prevalence of HIV was lower than expected in this study population probably because of early HIV testing and diagnosis and initiation of cotrimoxazole prophylaxis for children found to be HIV positive which would also be protective against  malaria ,26,27. NHIV infected children in this study were more likely to have hyperparasitaemia  which was consistent with what was found in studies in the Kenya y and Zimbabwe studies in which HIV infection was associated with higher parasite density ,25. PresDue to the low number of HIV infected children in our controls, we found no association between cerebral malaria and low CD4 cell count. A longitudinal study in Malawi has already indicated that lower CD4 cell counts were associated with higher incidences of pneumonia, sepsis, and tuberculosis but not of malaria(.Other factors found to be associated with an increased risk of presenting with clinical cerebral malaria were age below five years and not using a mosquito net. These findings are also consistent with previous studies ,34,35.P. falciparum malaria in children and are of both clinical and public health importance.Our findings support the hypothesis that HIV infection is a risk factor for cerebral malaria in children. The findings from this study add to the existing knowledge of interaction between HIV infection and P.falciparum malaria and associated complications such as cerebral malaria. Other malaria preventive measures such as use of insecticide-treated mosquito nets should also be emphasized during counselling sessions.HIV-1 infection is associated with clinical presentation of cerebral malaria in children. Clinicians should ensure that children diagnosed with HIV infection are initiated on cotrimoxazole prophylaxis as soon as the diagnosis is made and caretakers counselled on the importance of adherence to the cotrimoxazole towards reducing the risk of acquiring There are sources of potential sources of bias that cannot be eliminated in this study. These inherent sources include selection of no malaria controls from a hospital setting which may have affected our results. We however tried to limit this by selecting hospital controls from similar communities. We also excluded children with conditions clearly documented to be associated with HIV such as pneumonia, severe malnutrition, persistent diarrhoea, and chronic otitis media. The design of the study may have also affected the results. The case control design may have led to exaggeration of the risk because cases and controls were not matched on characteristics such as age, maternal characteristics and others. The parasite density may not be a true reflection of the level of parasitaemia since the individual WBC count was not available to estimate the parasite density but instead assumed 8,000/μl. There is a possibility that the WBC count was different in HIV+ and HIV- children.Children with cerebral malaria were not followed up to determine whether the outcome depended on HIV serostatus. The strength of the study is that there were more than one control groups and given that cerebral malaria is a relatively rare complication of malaria, it was an appropriate study design to conduct given the time.The authors declare that they have no competing interests.PDI conceived and designed the study, performed analysis and interpretation of data and drafted the manuscript. PM, JB and JKT assisted with the design, interpretation of data and the critical review of the manuscript. All authors read and approved the final manuscript and participated in its revision. JKT is the guarantor of the work.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/11/5/prepub


Few data are available about malaria epidemiological situation in Niger. However, implementation of new strategies such as vaccination or seasonal treatment of a target population requires the knowledge of baseline epidemiological features of malaria. A population-based study was conducted to provide better characterization of malaria seasonal variations and population groups the most at risk in this particular area.From July 2007 to December 2009, presumptive cases of malaria among a study population living in a typical Sahelian village of Niger were recorded, and confirmed by microscopic examination. In parallel, asymptomatic carriers were actively detected at the end of each dry season in 2007, 2008 and 2009.Among the 965 presumptive malaria cases recorded, 29% were confirmed by microscopic examination. The incidence of malaria was found to decrease significantly with age (p < 0.01). The mean annual incidence was 0.254. The results show that the risk of malaria was higher in children under ten years (p < 0.0001). The number of malaria episodes generally followed the temporal pattern of changes in precipitation levels, with a peak of transmission in August and September. One-thousand and ninety subjects were submitted to an active detection of asymptomatic carriage of whom 16% tested positive; asymptomatic carriage decreased with increasing age. A higher prevalence of gametocyte carriage among asymptomatic population was recorded in children aged two to ten years, though it did not reach significance.In Southern Niger, malaria transmission mostly occurs from July to October. Children aged two to ten years are the most at risk of malaria, and may also represent the main reservoir for gametocytes. Strategies such as intermittent preventive treatment in children (IPTc) could be of interest in this area, where malaria transmission is highly seasonal. Based on these preliminary data, a pilot study could be implemented in Zindarou using IPTc targeting children aged two to ten years, during the three months of malaria transmission, together with an accurate monitoring of drug resistance. Malaria is the principal public health problem in this country, well ahead of meningitis, respiratory infections and diarrhoeal diseases. It is the leading cause of morbidity and mortality in children under the age of five years (more than three million subjects) and pregnant women  [Niger  and the implementation of intermittent presumptive treatment (IPT) in pregnant women . ArtemetPlasmodium falciparum could, therefore, be envisaged in this part of Niger, which is also the most heavily populated part of the country [In line with this new approach, seasonal malaria chemoprevention (SMC), previously described as intermittent preventive treatment in children, has proved effective for preventing malaria in children in zones of seasonal malaria transmission -12. The  country . HoweverThe epidemiological features of malaria have been well described in Mali, Senegal and Sudan, but few data are available from Niger. With the aim of exploring the feasibility of, and conditions required for improved interventions against malaria in Niger the population of a village in an environment representative of the Sahelian zone of Niger was surveyed. The exposed population was parasitologically and clinically followed for a period of over two years, taking into account short-term meteorological variations. The results presented here are complementary to the entomological data acquired previously in the same region ,15. TogeAnopheles gambiae sl. and Anopheles funestus[This study was carried out in the village of Zindarou (13°26.09 N/2°55.23 E), in a rural area in the district of Birni N’Gaoure about 100 km southeast of Niamey, in Niger, West Africa. The village may be considered representative of the Sahelian ecological zone of Niger. The climate consists of three seasons: hot and rainy in June to October, warm and dry from November to January, and hot and dry from February to May -16. Acco funestus,15. AcceThe study was conducted from July 2007 to December 2009. All inhabitants were invited to participate to the survey, and those giving informed consent, including minors whose parents or legal guardians gave consent, were included. There were 522 inhabitants in Zindarou , and a total of 441 of these individuals consented to participate in the survey. The study population was thus 441 subjects, with a mean age of 20.1 ± 17.8 years (range: 0.3 to “100 years”), 7.0% (31/441) of whom were under the age of two years, 14.6% (64/441) of whom were between two and four years of age, 14.0% (62/441) of whom were between five and nine years of age, 13.1% (58/441) of whom were between ten and 14 years of age and 51.3% (226/441) of whom were adults, defined as 15 years old or older for this population  consulting at the Zindarou health centre with symptoms suggestive of malaria, including fever, was recorded as a presumptive case of malaria and was immediately treated with an ACT, in line with Ministry of Health policy. Fever was defined as an axillary temperature exceeding 37.5°C. Registration code number, name, age, sex, and temperature were noted. Thick and thin blood smears were prepared from finger-prick blood samples for each patient with presumptive malaria. The site was visited once a week by a medical supervisor for the collection of clinical information and its entry into the database. The blood smears obtained in this study were not processed locally, but were brought to laboratory facilities at Niamey on a weekly basis, for an a posteriori at the laboratory, by microscopy examination. Individuals with a positive blood smear and an axillary temperature <37.5°C were considered to be asymptomatic carriers.Asymptomatic carriers were detected upon cross-sectional surveys performed in 2007, 2008 and 2009 on all individuals participating in the study, at the end of the dry seasons, when malaria transmission rates were considered to be the lowest. Among the 441 inhabitants of Zindarou enrolled at the start of the study, 358, 378 and 354 were available for malaria screening in June 2007, June 2008 and June 2009, respectively. For these patients, the female/male ratio was 1.01 . Axillary temperature was recorded, and thick and thin blood smears were systematically prepared for all the inhabitants of the village. The presence of the parasite was confirmed Plasmodium species.The blood smears were fixed and stained with Giemsa and examined by an experienced microscopist, according to standard procedure. The slide was considered positive if malaria parasites were detected in the blood smear. A blood smear was considered negative if no parasite was detected after the examination of 200 oil-immersion fields on the thick smear. When malaria parasites were detected in the blood film, parasite density was determined by counting the number of parasites present per 200 white blood cells (WBC) in the thick smear and multiplying by 40, to obtain an approximate parasite count per ml of blood. This calculation was based on the assumption of a mean WBC count of 8,000/μl of blood, considered as a representative standard value allowing calculation of the parasite count . The thiA standard meteorological station was installed in Zindarou to collect data on temperature, rainfall, humidity, radiation, soil moisture, and wind. The observations were collected at high resolution (~30 minutes) and were stored using an automatic data logger. The data on temperature and rainfall are presented to shed light on the role of these meteorological variables in explaining observed seasonal variability in concurrent epidemiological data describing malaria incidence in Zindarou. The same dataset has been used to force a mechanistic model of the hydrology, entomology and malaria transmission over this village .t-tests. Values of p < 0.05 were considered significant. The positivity rate was defined as the percentage of patients with suspected malaria (“presumptive case”) proved positive by microscopy (“confirmed case”). The relative risk of malaria in the various age groups was calculated as the ratio between the proportions of subjects testing positive in two groups, using the group of under five years old as the reference group [The data collected during both longitudinal and transversal surveys were entered into two separate Excel files and processed for statistical analysis with Excel or Open Epi. Percentages, means and standard deviation were calculated. Differences in proportions were assessed in Chi-squared tests. Sample means were compared in unpaired Student’s ce group .This study was authorized by the “Health Department Direction of Dosso”, and ethical clearance was obtained from the National Ethical Review Committee of Niger. A rally was also organized, during which the purpose of the study was clearly explained to the population. Participation in the study was voluntary. All diagnosed cases of malaria were treated free of charge, in accordance with the recommendations of the Niger National Malaria Control Programme.vs 41.6% (401/965); p < 0.0001, Chi-squared test). The proportion of febrile patients testing positive by microscopy significantly varied according to age . Patients under the age of five years were more frequently diagnosed with uncomplicated malaria than patients from older age groups; the rate of malaria confirmation then decreased with age from 56.7% for children under the age of five years to 15.8% for individuals over the age of 15 years. The mean number of malaria cases per person also varied with age group, the youngest children displaying more malaria episodes than older individuals, 0.9 and 0.38, respectively. In particular, the number of malaria episodes decreased sharply after the age of ten, most likely due the development of immunity to malaria. P. falciparum was found to be responsible for 99.6% of infections and Plasmodium ovale was detected only once (0.4% of cases). No Plasmodium vivax or Plasmodium malariae cases were observed. Mean asexual parasite densities detected in confirmed malaria cases  varied significantly according to the various age group  but did not show any specific trend. A significant difference was however observed between mean parasite densities in individuals less than 5 years old  and those above 5 years old  . High parasite densities are associated with more severe infection. The mean annual malaria incidence estimated from the collected data was 0.254. The incidence of malaria was found to increase with age until the age of five years, then gradually decreased to the age of 15 years and remaining stable in adults thereafter. Table The study was carried out from July 2007 to December 2009 to assess the incidence of malaria among the 441 inhabitants of Zindarou enrolled in the study. Incidence was estimated by recording malaria cases, defined here as fever and/or clinical signs sufficiently severe for the patient to seek care at the local health unit, and being positive for asexual malaria parasites. The relationships between parasite density, fever incidence, seasonal variations, and age in the Zindarou area are not fully detailed here but presented in detail elsewhere . During the passive survey, 965 presumptive malaria cases were recorded . They included 280 (29.0%) that tested positive for malaria by microscopic examinations of stained blood smears  and only rare specimens, mostly of An. funestus, are caught throughout the dry season [vs 29.4°C), but the difference was not significant . By contrast, monthly cumulative rainfall was 15.5 times higher during the rainy season (899 mm) than during the dry season (58 mm), and this difference was highly significant. The incidence of malaria was higher during the first than second year of follow-up, with a monthly maximum of 46 cases of infection in September 2008; the incidence was lower in 2009, with the largest number of malaria cases in any given month being 30, in September. It is difficult to determine whether this difference reflects a study effect or short-term fluctuations of meteorological conditions. The mean number of malaria cases recorded monthly was 2.4 times higher in the rainy season than in the dry season. Most cases occurred between July and October, which can therefore be considered the target period for the implementation of preventive strategies in the Sahelian fringe of Niger. The mean parasite densities tended to increase as the incidence increased, i.e. mainly during the rainy season between July and October (data not shown). Indeed, changes in the numbers of malaria episodes broadly followed the temporal pattern of changes in precipitation, with a lag of 15 to 30 days between major rainfall episodes and malaria cases. This pattern presumably reflects longitudinal fluctuations of the entomological parameters associated with rainfall, and led to a peak of transmission in August in both 2008 and 2009 (data not shown). In Zindarou, fluctuations in ground water level follow the seasonality of rainfall with a lag of about one to two months [In the Sahelian part of Niger, the dry season extends from November to May, and the rainy season from June to October. At the Zindarou site, malaria is transmitted by y season . Figure o months .P. falciparum. The exception, in 2009, was due to P. malariae . Asexual forms were the most frequently detected , and a small number of individuals were found to carry either sexual forms only  or both asexual forms and gametocytes . Age had a significant effect on the asymptomatic carriage of asexual forms of the parasite . Asymptomatic carriage tended to decrease with age, with the parasite prevalence being 36.2, 29.4, 18.1 and 5.8% for the two to four, five to nine, ten to 14 and >15 years age groups, respectively. The exception to this general trend was that only 11.1% of children under the age of two years tested positive. There was no significant relationship between age and gametocyte carriage in the asymptomatic population. However, our data for gametocyte carriage should be treated with caution due to the small number of observations: the proportion of individuals carrying gametocytes was generally low. There was a trend for subjects aged 2 to 10 years old to have higher absolute gametocyte counts than other age groups, suggesting that individuals in this age group are the major reservoir at the end of the dry season.Table SMC requires the acquisition of up-to-date data relative to the regional epidemiological features of malaria prior to implementation, such that target populations can be identified and protocols for treatment delivery optimised. Here, we report longitudinal follow-up of clinical cases of malaria and active screening for asymptomatic carriers in the village of Zindarou for this purpose.Malaria diagnosis and patient follow-up in Niger is based almost exclusively on a clinical examination focusing on non-specific symptoms. This presumptive diagnosis is imprecise and over-estimates the number of cases, by confusing malaria and other fevers, such as those caused by arboviruses. In Niger, the proportion of positive slides is strongly associated with the bioclimatic context. The frequency of positive slides is negatively correlated with latitude during the rainy season, whereas no relationship to longitude is found, irrespective of the transmission rate . At ZindIncidence decreased with age at Zindarou. This has also been observed in other Sahelian settings, indicating that this village is indeed representative of the Sahelian zone of Niger ,20. The Changes in the number of malaria cases broadly follow short-term variations of meteorological conditions. The lag time in Zindarou between an excess risk of malaria, linked to precipitation, and a significant increase in the number of cases, is about 30 days. A previous study in Magaria  estimated the lag time between rain and a malaria surge to be 22 to 39 days . HoweverActive screening for asymptomatic carriers at the end of the dry season revealed that parasite carriage was more frequent in children (36.2% for children aged two to four years and 29.4% for children aged five to nine years) than in adults , again confirming the epidemiological relevance of the village chosen. The youngest individuals were asymptomatic carriers not only of asexual stages, but also of gametocytes. These sexual forms are responsible for transmission restarting after the end of the dry season. Asymptomatic carriers of gametocytes were found in all age groups, but were clearly over represented in the two- to ten-year age group, identifying these children as the principal reservoir for the transmission restarting with the first rains of May. A similar pattern has been found in Burkina Faso .SMC involves the administration of anti-malarial drugs at pre-determined times, regardless of whether or not the treated subjects are infected. It therefore seems to conflict with WHO guidelines recommending that suspected cases of malaria should be confirmed biologically before treatment. However, by considering specific regional features when developing preventive strategies, the two approaches  could prove not only to be complementary, but also synergic, increasing the efficacy of anti-malarial programmes. Since 2012, the WHO has been recommending, through its global anti-malarial programme, SMC for the seasonal transmission in the Sahelian zone . SMC tarex vivo study with fresh isolates collected in south Niger showed that P. falciparum had retained its susceptibility to conventional antimalarials, including AQ, and to new molecules, including artemisinin derivatives [falciparum malaria, with adequate clinical and parasitological response rates in 98.5 and 95.5% of cases, respectively (unpublished data). By contrast, the current extent of resistance to SP in Niger is only poorly documented. Before complete, intermittent treatment with AQ + SP can be considered, additional observations are required, including more recent data, providing a better description of the susceptibility of the parasite to these anti-malarial drugs. The establishment of a reliable system for monitoring drug resistance at national scale and the continuing operation of a point-of-care network to document cases and possible adverse effects of treatment are pre-requisites for SMC implementation.Over the last 30 years, the malaria control strategies implemented have come up against the problem of the emergence and rapid propagation of parasites resistant to anti-malarial drugs, and especially artemisinin . The medivatives . SimilarMany studies have shown that SMC in young children is an effective way of preventing malaria in regions of seasonal malaria transmission -12. The On the basis of the results presented here, a pilot study of the administration of a complete AQ + SP treatment to children between the ages of two and five years, during the months of July, August and September, could be envisaged at Zindarou. The clinical and parasitological follow-up of a selected cohort, with, in particular, close monitoring of responses to anti-malarial drugs and the observation of possible rebound effects in the following years, would make it possible to explore the technical feasibility of this approach. If feasible, and if the consequences in terms of morbidity and mortality are satisfactory, the approach could then be extended to the entire Sahelian zone in Niger.The authors declare no conflict of interest. None of the authors have financial or personal conflicts of interest related to this study. The corresponding author has full access to all data in the study and final responsibility for the decision to submit this publication.JG and TF designed the study and coordinated its execution, analysed the data and wrote the first draft of the paper. PD and JBD provided guidance and field expertise, and reviewed the design of the study and the report drafts; they selected the Zindarou study site and conceived the initial protocol for the follow-up. AM supervised field work and was responsible for the supervision of data collection. HZ, MK, IA, MIL, and RL contributed to the data collection, and to field and laboratory works. AABE contributed to meteorological data collection. All authors have read and approved the final version submitted.


In 2000/2001, the Australian Defense Forces (ADF), in collaboration with SmithKline Beecham and the United States Army, conducted a field trial to evaluate the safety, tolerability and efficacy of tafenoquine and mefloquine/primaquine for the prophylaxis of malaria amongst non-immune Australian soldiers deployed to East Timor  for peacekeeping operations. The lack of a concurrent placebo control arm prevented an internal estimate of the malaria attack rate and so the protective efficacy of the study regimens was not determined at the time.Plasmodium vivax attack rate was estimated during the prophylactic phase of the deployment by adjusting the observed P. vivax relapse rate during post-deployment to account for the known anti-relapse efficacies (or effectiveness) of the study medications (determined from prior studies). The all species malaria attack rate  was then determined by adjusting the P. vivax attack rate based on the ratio of P. falciparum to P. vivax observed during prior ADF deployments to Timor Leste. This estimated all species malaria attack rate was then used as the ‘constant estimated attack rate’ in the calculation of the protective efficacy of tafenoquine and mefloquine during the prophylactic phase of the deployment.In a retrospective analysis of the trial results, the all species malaria attack rate was estimated for the prophylactic phase of the study which was defined as the period between administration of the first prophylactic dose and the first dose of post-deployment medication. First, the The estimated attack rate during the prophylactic phase of the study was determined to be 7.88%. The protective efficacies of tafenoquine and mefloquine, with corresponding 95% confidence intervals (95% CI), were determined to be 100% (93%-100%) and 100% (79%-100%) respectively.The protective efficacy of tafenoquine  is similar to that of the weekly standard of care . Tafenoquine is a long elimination half-life  primaquist Battalion, Royal Australian Regiment (1RAR) deployed on peacekeeping operations to Timor Leste and who volunteered to participate in a Phase 3 study  was then calculated as follows (equation 1):The number of Pv = total number of P. vivax relapses and N = total ITT sample sizeWhere RP. falciparum to P. vivax was estimated as described below and summarized in Table th Battalion, Royal Australian Regiment (6RAR), was deployed in the six months prior to 1RAR in which conditions were drier, and in which the incidence of malaria is usually lower 100%). Note that this represents the effectiveness of primaquine in this context because administration of the drug in many of the studies analysed by John et al. (2012) was not directly observed.ARETq) was based on data reported by Walsh et al.100%).The assumed anti-relapse efficacy of tafenoquine .The estimated all malaria attack rate during the six-month prophylactic phase of Study 033 was calculated as the sum of the estimated six month attack rates for P. vivax and P. falciparum were excluded.The calculations are further summarized in Table P. vivax attack rate (ARPv) during the prophylaxis phase was estimated by adjusting the total observed relapse rate of 1.229% (8/651) during the post-deployment period to take into account the combined anti-relapse efficacy of 82.1% in the tafenoquine and mefloquine/primaquine arms  was determined using data from the CMR as described earlier.It was assumed that all P. falciparum attack rate during the six-month prophylaxis was based on the estimate of the P. vivax attack rate during the prophylaxis phase  and the ratio of P. falciparum relative to P. vivax  based on epidemiological data from the 1999/00 ADF deployments to Timor Leste  during the prophylaxis phase as follows (equation 9):The usual definition of the protective (prophylactic) efficacy (PE) in a placebo-controlled trial was used to determine the PE for tafenoquine (PETq and ARPl are the cumulative risk estimates (cumulative attack rates) for the period of prophylaxis in those receiving tafenoquine compared to those receiving a hypothetical placebo. A similar definition applies to the protective efficacy of mefloquine (PEMef). Since Study 033 had no placebo control arm, conventional calculation of protective efficacy and corresponding confidence interval (CI) was not possible. Because in Study 033 there were no cases of malaria it should be noted that, for any estimated attack rate, the point estimate PETq and PEMef would be 100%. However, standard methods for calculating a confidence interval (CI) for PE (Tq or Mef) depend on the placebo attack rate and sample size. To determine the PE and CI it was assumed that the attack rate (ARPl) would have been the estimated all malaria attack rate of 7.88% calculated above (equation 6) during the prophylactic phase of Study 033. In addition a constant ARPl was (= 7.88%) used for calculating corresponding 95% CIs for PE. A sensitivity analysis of this assumption was carried out (Table Pl).Where ARTq) and mefloquine (ARMef) during the prophylactic phase was determined based on the ITT results of study 033  and were computed as follows (equation 10):The attack rate for tafenoquine (ARTq (PEMef) was obtained by first computing the lower and upper limits  for a proportion (ARTq or ARMef) using the Wilson (score) method, and then converting these limits into corresponding limits for PE (assuming ARPl = 7.88%). When there are 0 cases, AR = 0 (0/n) and the lower confidence limit for AR is always 0 and the corresponding upper limit for PE is 100%. Given lower and upper Wilson confidence limits for the attack rates  the corresponding limits for PE (Tq or Mef) are therefore given as follows (equation 11):The 95% CI for PETq)]. Hypothetical estimated attack rates (ARPl) from 1% -10% and sample sizes (nPL) from 10–1000 were considered. Combinations of ARPl and nPl were selected to give integer values for the corresponding number of placebo cases (nPL × ARPL). All calculations assumed no observed cases in the tafenoquine arm and a sample size (ITT) of n = 490 (Study 033 results). Over the range of ARPl considered (1%-10%) the estimate of the lower limit of the 95% confidence interval (one-sided) for PETq did not vary substantially with changes in sample size  is because there were no observed malaria cases and the sample size in the tafenoquine arm employed in Study 033 was large (ITT n = 490). For a given ARPl and nPl, Koopman's (score test) method was used for computing the lower 95% limit for PE. StatXact (vs. 9.0) was used and the confidence level was set to 90% .The above method for determining a confidence interval for PE assumes a constant estimated AR (7.88%). This assumption does not take into account additional variability had the malaria exposure during Study 033 been estimated based on results from a placebo control arm (with a given sample size). A sensitivity analysis was conducted Table  to deterP. falciparum and P. vivax are tabulated for each ADF deployment in Table P. falciparum and P. vivax attack rates during the 1999/2000 deployments were 7.88% and 1.15%, yielding a P. falciparum to P. vivax species ratio of 0.146 . Thus, the observed post-deployment P. vivax relapse rate during Study 033 was 1.23% (8/651). The number of P. vivax relapses observed (8) was higher than reported previously [All eight  1.23% 8/51. The nIn Study 033, of 490 ITT subjects given tafenoquine and 161 ITT subjects given mefloquine, no symptomatic cases of malaria were observed during the prophylactic phase ([vs 490). These calculations are based on a retrospective estimate of an attack rate of 7.88%. That attack rate was calculated based on the following assumptions: (i) that the P. falciparum:P. vivax species ratio in the Study 033 was the same as that directly observed in Australian soldiers in the 1999/2000 deployment, (ii) that the anti-relapse effectiveness of primaquine at the dose used in Study 033 was 69.5%, and (iii) that the anti-relapse efficacy of the tafenoquine dose used in Study 033 was 86.3%. These assumptions are conservative for the reasons outlined in the following paragraphs. Therefore, the attack rate reported in this study is likely to be an under-estimate.This paper describes methodology for calculating protective efficacy when there is no concurrent placebo control arm data. Using this methodology, it was possible to demonstrate that tafenoquine exhibited a protective efficacy of 100% with a 95% lower confidence limit of 93% while mefloquine exhibited a protective efficacy of 100% with a 95% lower confidence limit of 79%. The lower value of the lower limit for mefloquine is due to the smaller sample size  or the ~2:1 ratio observed for malaria cases originating amongst Australian soldiers in Timor Leste during the 1999–2000 deployment [P. falciparum would not have been reasonable given that there was clearly transmission of P. falciparum amongst the civilian population living close to the 1 RAR garrison positions [P. falciparum:P. vivax ratios of 1:1 or 2:1 would imply attack rates of 14% and 22% respectively. However, this would have assumed either parity in malaria exposure between unprophylaxed civilians and soldiers, or that compliance with doxycycline and primaquine prophylaxis could be estimated in the absence of data (remember that in Study 033 compliance with medications was recorded whereas in the 1999–2000 deployment it was not). While these assumptions may be defensible, the more conservative of the reasonable approaches was taken.The utilization of a ployment ,12 couldositions . Assuminet al.[et al. provide data. It was decided to use effectiveness data for low dose primaquine (2.5 - < 5 mg/kg) because there are only two studies in the literature which have compared relapse rates for a blood schizonticidal drug with and without high dose primaquine (> 5 mg/kg). Neither of these is directly analogous to Study 033. Baird et al.[P. vivax in Irian Jaya (recurrences may be recrudescences not true relapses) are confounding. Leslie et al.[Using data from John et al., it was rd et al. reportedie et al. reportedet al., unpublished observations). The calculations also assume similar P. vivax relapse rates and susceptibility to tafenoquine in Thailand and Timor Leste. This is reasonable given the long follow-up times in both the Walsh study and Study 033, and the lack of evidence suggesting any regional differences in susceptibility to 8-aminoquinolines in the western Pacific.The assumed anti-relapse efficacy of tafenoquine of 86.3% was calculated using data for a prophylactic regimen of tafenoquine that was different from that used in Study 033 . This isP. falciparum strains were chloroquine-resistant, the protective efficacy (95% CI) of mefloquine prophylaxis relative to chloroquine and chloroquine/proguanil prophylaxis was 94% (86-97%) and 86% (67-94%) respectively [Mefloquine is an effective anti-malarial drug and remains the standard of care for weekly malaria prophylaxis where this is justified by the risk:benefit context. The reported efficacy of mefloquine prophylaxis in malaria naïve individuals is broadly similar to the efficacy of tafenoquine reported in this and prior clinical reports. In U.S. Peace Corps volunteers stationed in East Africa, where the majority of ectively . Presumaectively .The protective efficacy of tafenoquine and mefloquine during the deployment period in Study 033 was 100% since no symptomatic cases of malaria were observed. This was lower than the level of prophylactic efficacy of the same drugs (~86% for both drugs) observed amongst semi-immune residents of Ghana in one of the Phase II studies . This seP. falciparum malaria from a late deployment exposure is managed through administration of additional weekly doses of the drug for four weeks, while the risk of P. vivax relapses is managed through administration of primaquine (as in Study 033). In the case of tafenoquine, it is known that symptomatic failures occur when plasma levels of the drug fall below 80 ng/ml [P. falciparum cases, although the precise regimen has not been determined. Based on open-label clinical studies [P. vivax relapses of approximately the same magnitude as primaquine can be anticipated.The conclusion that prophylactic efficacy of tafenoquine and mefloquine was 100% in Study 033 applies only to the period of time between the first dose of the drug and the first dose of post-deployment medication. In the case of mefloquine the persisting risk of a post-deployment case of 80 ng/ml . Therefo studies , a residP. vivax relapse rate during follow-up to account for the anti-relapse efficacy of the study drugs, is an alternative approach that could be considered.The population that may benefit the most from drugs for malaria prophylaxis are non-immune individuals deployed or travelling to areas with endemic malaria, although in some cases prophylaxis may also have considerable utility for malaria control efforts in endemic countries. In the context of a clinical study to determine the protective efficacy of a new prophylactic anti-malarial in non-immune individuals, it may be very challenging to directly determine an attack rate if the use of a placebo is not feasible. During a military engagement, placebo control arms are generally not used because of their possible impact on mission effectiveness. As argued elsewhere , the useThis study retrospectively determined a conservative estimate of 7.88% for the all species malaria attack rate to which Australian Defence Forces personnel on peacekeeping duties in Timor Leste were exposed during their participation in a Phase III prophylaxis study to evaluate the prophylactic efficacy of tafenoquine and mefloquine. The prophylactic efficacy of mefloquine and tafenoquine during the period between administration of the first prophylactic dose and the first dose of post deployment medication was 100% (93-100%) and 100% (79-100%) respectively. The methodology employed to determine an attack retrospectively in the absence of placebo control arm should be considered when planning future prophylactic studies involving malaria naïve subjects in which it is not feasible to include a placebo control arm.The authors have a personal and professional interest in ensuring the regulatory approval of tafenoquine for malaria prophylaxis but no competing interests.GSD led the technical team that performed the analysis and wrote the manuscript. MR extracted records from the Central Malaria Registry and conducted the analysis of attack rates from prior deployments. WFM and DT performed the statistical analyses. BS and DS provided valuable perspective on the clinical aspects of malaria, methodological aspects of clinical trial and product history with tafenoquine. All authors read and approved the final manuscript.


Plasmodium falciparum isolates from countries in central and west Africa were assessed for allelic diversity. Samples were collected on filter paper from 600 P. falciparum-infected symptomatic patients in Cameroon, Republic of Congo, Burkina Faso, Ghana, and Senegal and screened for class-specific amplification fragments. Genetic diversity, assessed by mean heterozygosity, was comparable among countries. We detected a clinical increase in eba 175 F-allele frequency from west to east across the study region. No statistical difference in msp-3 allele distribution between countries was observed. The ama-1 3D7 alleles were present at a lower frequency in central Africa than in West Africa. We also detected little to no genetic differentiation among sampling locations. This finding indicates that, at least at the level of resolution offered by restriction fragment length polymorphism analysis, these antigens showed remarkable genetic homogeneity throughout the region sampled, perhaps caused by balancing selection to maintain a diverse array of antigen haplotyes.The malaria vaccine candidate antigens erythrocyte binding antigen 175 (EBA-175), merozoite surface protein 3 (MSP-3), and apical membrane antigen (AMA-1) from  Plasmodium falciparum malaria is a major public health challenge and a serious impediment to socioeconomic development, particularly in sub-Saharan Africa, where 85% of the global burden occurs. An estimated 0.9 million deaths caused by malaria occur each year, and 89% of deaths occurr in children less than five years of age and in pregnant women living in malaria-endemic areas.P. falciparum malaria is caused in part by the development of anti-malarial drug resistance, primarily against those drugs targeting the parasite asexual erythrocytic stages. This stage is also responsible for pathologic and clinical manifestations of the disease.P. falciparum stage-specific antigens have been characterized as vaccine candidates through molecular epidemiology. Characterization of the distribution of malaria parasite antigen genotypes circulating in a wide geographic area may provide important genetic information helpful for malaria vaccines design. We have analyzed the genetic diversity of three antigens with promise as vaccine candidates: erythrocyte binding antigen 175 (EBA-175), apical membrane antigen (AMA-1), and merozoite surface protein 3 (MSP-3).The extensive genetic diversity of the malaria parasite constitutes major drawbacks to the development of a successful malaria vaccine.Plasmodium falciparum EBA-175 antigen plays a central role in erythrocyte invasion by sialic acid–dependent binding to glycophorin A. It is well known that antibodies against EBA-175 inhibit binding to glycophorin A and thus prevent merozoite invasion of erythrocytes.Apical membrane antigen is an 83-kD type I integral membrane protein with a 55-amino acid cytoplasmic segment and a 550-amino acid extracellular region that can be divided into three domains on the basis of intra-domain disulfide bonds. Although its function is still not well characterized, it is expressed in the late schizont stage of the parasite and is required for merozoite invasion of erythrocytes and sporozoite invasion of hepatocytes. Antibodies against AMA-1 have been shown to block parasite invasion of human erythrocytes.18Plasmodium flaciparum ama-1 is highly polymorphic,22Plasmodium falciparum MSP-3 is encoded by a single locus on parasite chromosome 10, and is an important target for protective immunity because antibodies against it could prevent erythrocyte invasion by merozoites.msp-3 gene from P. falciparum isolates showed that polymorphism in the gene is predominantly confined to sequence in the N-terminal extremity within and flanking the heptad-repeat previously identified as a site of antigenic diversity among MSP-3 polypeptides.msp-3 alleles, but the sequence polymorphism define 2 major allele classes (K1 like and 3D7 like), which show only limited recombination.27P. falciparum candidate vaccine antigens  across varied eco-epidemiologic zones in central and West Africa. Field isolates were obtained from symptomatic persons in five countries and were typed with respect to vaccine antigen candidate major allele classes as defined by RFLPs. Knowledge of the distribution of polymorphic malaria antigens across this broad geographic range may contribute to the rational development of a malaria vaccine.The purpose of this study was to determine the distribution of major allele classes for three The study was conducted in different sites in five countries : Cameroo®; Roche, Basel, Switzerland) during a one-year longitudinal follow-up study. Malaria transmission is perennial and seasonal in the district, and there is a peak during the rainy season (May–October). The entomologic inoculation rate was estimated to be 200 infected/bites/person/year in the study area.Samples were collected from the Saponé Marché and Kounda communities health clinics located within an 8-km radius in the Saponé Health District situated approximately 50 km southwest of Ouagadougou, the capital of Burkina Faso. The Saponé Health District is located at 12°13′N, 1°48′W and has approximately 80,000 inhabitants, most (> 95%) from the Mossi ethnic group. The number of children less than five years of age in the health clinics area of the two communities was approximately 465. All malaria cases were freely managed by using artemisinin-based combination therapy  in Yaounde. The three localities have equal malaria transmission intensities. Yaounde is located at 3°52′0″N, 11°31′0″E. The population is comprised of heterogeneous ethnic backgrounds from every region of the country. Malaria is the primary cause of morbidity in these localities. Maximal transmission (EIR > 200 infected bites/person/year) occurs during and immediately after the two rainy seasons (March–June and September–November).Anopheles gambiae. Other vectors, such as An. moucheti, An. funestus, and An. nili, are also found.30The study was conducted at the Tenrikyo Integrated Health Center, which is located within the Makélékélé Health District in Brazzaville, Republic of Congo. Brazzaville is located situated between 4°13′S and 4°18′S and 15°13′E and 15°18′E. The rainy season lasts eight months (October–May), and the dry season last four months (June–September).The study was conducted at the Navrongo War Memorial Hospital in the Kassena-Nankana District (northern Ghana) and the Begoro District Hospital in the Fanteakwa District (southern Ghana). The Kassena-Nankana District is located in the Upper East Region along the Ghana–Burkina Faso border. The district is hot and dry and is located in the Guinea Savannah Belt. Navrongo, the district capital, is located at 10°53′5″N, 1°5′25″W. Estimated malaria attack rates in the district are approximately 4.7 and 7.1 infections/person/year for the dry and wet seasons, respectively.31The Fanteakwa District is located in the Eastern Region of Ghana. Begoro, which is the district capital of the Fanteakwa District, is located at 6°22′N, 0°23′W, approximately 150 km north of Accra, the capital of Ghana. Malaria is known as the most prevalent disease reported at the Begoro District Hospital.The study took place in the rural community of Keur Socé located approximately 17 km south of Kaolack. Keur Socé is situated at 14°33′46.14″N, 15°35′30.53″W. Children less than five years of age represent approximately 20% of the population. Malaria transmission in this locality is seasonal. Entomologic inoculation rates vary between 9 and 12 infected bites/person/night during the high transmission season.Ethical clearances and administrative authorizations were obtained by the competent Ethical Review Committees and issuing bodies for the respective studies with approval including consent to use samples for malaria related studies. These committees were the Health Research Ethics Review Committee in Burkina Faso, the National Ethic Committee in Cameroon, the Ministry of Health and the Ministry of Scientific Research in the Republic of Congo, the Institutional Review Board of the Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana, and the Health Ethical Committee based at the Ministry of Health in Senegal. The purpose of the study and all anticipated risks and benefits were clearly explained to the participants. Written informed consent and assent were obtained from the participants or their guardians, and participation in the studies was strictly voluntary.P. falciparum-infected, symptomatic, voluntary children or infants with malaria living in the five study countries irrespective of parasite density. Symptomatic malaria includes uncomplicated malaria and severe malaria as per the World Health Organization malaria definition. To limit any bias related to data collection, samples were collected between during two consecutive years during 2007–2009. In Ghana, samples were obtained in 2007 and 2008 from children 6–60 months of age who were enrolled in a study to assess the therapeutic efficacy of anti-malaria drugs.Sampling was conducted at clinics and hospitals. Samples collection criteria were P. falciparum-infected symptomatic children with malaria living in five countries in central and west Africa: 231 isolates from Saponé Health District (Burkina Faso), 105 isolates from Keur Socé , 122 isolates from Yaounde (Cameroon), 100 isolates from the Republic of Congo, and 101 isolates from Begoro (southern Ghana) and Navrongo District Hospitals (northern Ghana).Samples were obtained at clinics or hospitals from 659 P. falciparum. Whole blood from P. falciparum-positive persons was blotted on sterile, Whatman  III filter paper, stored dry at room temperature and protected with a silica gel desiccant, and archived for later P. falciparum DNA isolation.Thick and thin blood smears were prepared for microscopic identification of P. falciparum. Slides were examined by experienced microscopists in each study site. A person was considered positive if malaria parasites were detected in the blood smear and negative if parasites were not detected after examining 200 oil-immersion fields of a thick blood smear.Giemsa-stained blood smears were microscopically examined to identify mono-infections with Plasmodium falciparum DNA was isolated from archived blood spots by using two methods: Tris-EDTA (TE) with heateba-175, nested PCR products were analyzed for amplification fragment length polymorphisms.msp-3, semi-nested PCR was used to produce DNA fragments. The ama-1 haplotypes were analyzed by using PCR-RFLP.For 2, 200 μM dNTP (Invitrogen), and 5 μL of DNA template extracted from blood. The second round amplification reactions were performed similarly to the first with 2.5 μL of the first PCR product as DNA template in 25 μL total reaction volume (eba-175 and msp-3). Second-round reaction mixtures for ama-1 were performed in a final volume of 50 μL that contained 5 μL of 10× PCR buffer (Invitrogen), 2 units of Taq DNA polymerase (Invitrogen), and 5 μL of DNA template. All reactions were performed using 96-wells plate on a classic 96-well thermocycler (Eppendorf).The first-round PCR amplifications were performed in a final volume of 25 μL that contained 1 μM of each primer, 1 unit of Taq DNA polymerase , 2.5 μL of 10× PCR buffer (Invitrogen), 2.0 mM MgCleba-175 and msp-3 were 94°C for 30 seconds, 54°C for 30 seconds, and 68°C for 2 minutes, followed by a final extension at 68°C for 5 minutes, for a total of 30 and 40 cycles respectively. For ama-1, the amplification cycles were 95°C for 2 minutes 30 seconds, 51°C for 30 seconds, and 68°C for 45 seconds, followed by a final extension at 68°C for 5 minutes for 30 cycles for the first nested PCR and 35 cycles for the second PCR.Cycling conditions for the first and second PCR cycles of P. falciparum 3D7 (MRA-102G), HB3-B2 (MRA-149G), Dd2 (MRA-152G), 7G8 (MRA-153G), W2 (MRA-157G), K1 (MRA-159), V1/S (MRA-176), and FCR3 (MRA-731) strains was provided by the Malaria Research and Reference Reagent Resource Center, American Type Culture Collection  and used as positive controls during the amplification reactions.Purified DNA from eba-175 and msp-3 were directly separated by electrophoresis on a 2.0% ethidium bromide agarose gel and visualized on a GEL LOGIC 220 UV-Transillumination Imaging System . The second ama-1 amplification product was digested by using three restriction enzymes , which generated digestion fragments specific for the D7, K1, and HB3/7G8 ama-1 allele classes, respectively.The second amplification products from msp-3 K1 allele was identified as a single approximately 500-basepair fragment, and the msp-3 3D7 allele was identified as a single approximately 400-basepair fragment. Any other msp-3 fragments of different sizes were also reported. The CAMP allele was identified as a single approximately 714-basepair fragment, and the FCR3 allele was identified as a single approximately 795-basepair fragment. Mixed infections were defined as the simultaneous presence of K1 and 3D7 msp-3 alleles or the F and C eba-175 fragments in the same sample. The ama-1 K1, 3D7, and HB3/7G8 alleles were identified as single fragments of 285, 400, and 335 basepairs, respectively, when subjected to digestion with Mse I, Ssp I, and BfuC I, respectively.Positive controls were used to scored and interpreted fragment sizes. The P < 0.05. Allele frequencies were estimated in GenAlEx (http://www.anu.edu.au/BoZo/GenAlEx/). Genetic diversity was estimated by determining the heterozygosity based on the three antigens combined, where heterozygosity (h) = 1 − ∑ pi2 ranged from 0 to 1, where pi is the frequency of the thi- allele. The t-test was used to compare mean heterozyosity among the different sites.Allelic data were analyzed by using GenAlex 6,To compare allele frequencies along an east–west transect, data were pooled into three geographic groups: Cameroon and Republic of Congo (east), Burkina Faso, southern and northern Ghana (middle) and Senegal (west).We used Nei's standard genetic distance, and the Mantel test to measure the genetic differentiation of genes across the different study sites. The option of the Mantel test we used enables a statistical relationship between the elements of any two distance matrices with matching entries. This typical application includes testing for isolation-by-distance for which one might compare Nei genetic distance matrix with the geographic distance matrix for the respective populations. Typically, it is reported as an XY graph with log-distance versus linearized Fest (in our case using binary data it is linear zed-Phi). We examined how genetic variation was partitioned among geographic units by using analysis of molecular variance.37The main goal of this study was to analyze separately and in combination three polymorphic antigen genes across west and central Africa to identify differences in allele frequency and genetic diversity and to determine whether geographic distance contributes to genetic differentiation across our study region.P. falciparum samples obtained from the five countries, 418 samples were successfully scored for eba-175, 535 for msp-3, and 318 for ama-1. Two hundred sixty-six samples were used in the combined datasets analyses. To minimize the confounding effects of missing data, inclusion in the combined dataset was limited to samples with missing data for no more than one marker. Failure to amplify domains from a fraction of the patient samples  was attributed to variance in parasitemia, quality of the filter paper used, and extraction efficiency from the paper. All these PCR failures were excluded from further analysis.Of the 659 confirmed eba-175 or msp-3 alleles. This number is likely an underestimation because all markers were not successfully scored for all isolates. Most of the mixed infections (> 40%) were from rural villages in Burkina Faso. Mixed infections were included in all further analyses.A total of 97 mixed infections, or 15% of all infections sampled, were identified on the basis of multiple eba-175 alleles in Burkina Faso, Republic of Congo, and Cameroon, where it was twice as common as the C-loop allele  than in Burkina Faso, Cameroon, and Republic of Congo (< 2%)  . Despitern Ghana .msp-3 allele classes (K1 and 3D7 types) showed comparable prevalence in all countries   in the ama-1 3D7 allele prevalence and showed a lower prevalence in the eastern portion of the study region (Republic of Congo and Cameroon) (The ountries . In conte = 11%) . The oth country . In addiameroon) .h) was calculated from allele frequencies to compare genetic diversity in the different study sites. For the combined dataset, the number of alleles ranged from 8 to 10. One msp-3 allele, (msp3 > 500bp) was exclusively present in Cameroon  and a corresponding spike for eba-175 (P < 0.001).Mean heterozygosity  and for ama-1, eba-175, and msp3 individually. However, because the southern Ghana sample size (5.6% combined [15 of 266]) was small , our heterozygosity estimate for this site is probably not an accurate reflection of the true value.The overall heterozygosity was significantly higher in Senegal . Most genetic variation was captured within individual populations (97%), and there was little variance among a population within a region (3%) and no partitioning among geographic regions . Nei's SPlasmodium falciparum is known to exhibit extensive genetic diversity, particularly among surface antigens that have been under selective immune pressure and historically considered as the main targets of subunit vaccines. Unfortunately, this extreme genetic diversity poses a major challenge for effective vaccine development because it could lead to vaccine-resistant malaria with non-vaccine type parasites growing in frequency within vaccinated populations.P. falciparum infection, multiple allelic forms of an antigen need to be incorporated in a vaccine formulation.38The present study was designed to assess the distribution of major allele classes for three malaria vaccine candidates in a number of study sites across a geographic region spanning central and West Africa. Although collection protocols were not standardized across study sites, active case detection was used at all sites, and samples were collected within the same three-year time frame. In addition, all molecular methods and gel interpretations were standardized and conducted in the same laboratory at the same time. The markers used in this study did not have the resolution to measure the extensive genetic diversity known to exist for these surface antigens, but rather provides a pre-vaccine, baseline assessment of the distribution of major allelic classes. Our results address the feasibility of deploying a single vaccine throughout this large geographic region, and highlight the challenges posed by the considerable genetic diversity of these surface antigens.eba-175 F-loop allele clearly predominates in the eastern portion of our study region (Cameroon and Republic of Congo), there is no one allele that is most common throughout the rest of the study area. The predominance observed in F-loop allele was similar to previous observations made in other parts of Africa.40Although the msp-3 allele classes (K1 and 3D7 type)msp-3 heterozygosity values observed across sites. We observed additional but less common, msp-3 alleles (350 bp and 500 bp), which highlights the existence of significant alternative polymorphisms, as suggested by Polley and others.msp-3 dimorphic allele classes were maintained intact at a low frequency of recombination.41The P. falciparum in our study belonged to three of the four ama-1 allele classes previously identified based on the hypervariable region. The fourth allelic class was not observed in our study and is presumed absent in sub-Saharan Africa. Polymorphism analysis of ama-1 in the Amazon Basin in Peru identified three of the four ama-1 allelic classes,ama-1 3D7-like allele was less common in the eastern region, which may have been caused by a directional selection for humoral immune response.ama-1 diversity was high and comparable across the study sites even where there were regional differences in the prevalence of the different allelic classes and within-class variability, which may pose a problem for a vaccine based on this antigen. Although a remarkable conservation of the AMA-l molecule has been previously reported,ama-1, which we were not able to detect by using RFLPs in the present study, may compromise vaccine development. It is well known that the total repertoire of circulating ama-1 alleles is probably large.20The isolates of eba-175, msp-3 and ama-1 at high and comparable frequencies throughout the study region. The lack of genetic differentiation or isolation-by-distance is probably caused by balancing selection. It is likely that immune selection for maximal diversity has overwhelmed any geographic partitioning we might otherwise have observed by using neutral markers.Most of the observed genetic variance was not related to regional difference but attributed to variation within study sites. In addition, geographic distance did not contribute to antigen genetic distance. It is highly unlike that unrestricted gene flow across such a large geographic region is responsible for these patterns. Our data showed the presence of the main allele classes of Each of the target loci is currently in vaccine trials intended for populations in sub-Saharan Africa where the number of malaria cases identified in the past 10 years shows little evidence of decreasing.