There is a need to investigate the effectiveness and appropriateness of antibiotics prescription within the Integrated Management of Childhood Illness (IMCI) strategy in the context of routine outpatient clinics.Making use of a passive case detection system established for a malaria prevention trial in outpatient clinics in Papua New Guinea, the appropriateness and effectiveness of the use of antibiotics within the IMCI was assessed in 1605 young children. Main outcomes were prescription of antibiotics and re-attendances within 14 days for mild pneumonia, mild diarrhoea and uncomplicated malaria whether they were managed with or without antibiotics (proxy of effectiveness). Appropriateness was assessed for both mild and severe cases, while effectiveness was assessed only for mild diseases.A total of 6975 illness episodes out of 8944 fulfilled inclusion criteria . Clinical incidence rates  were 0.85 (0.81–0.90) for pneumonia, 0.62 (0.58–0.66) for malaria and 0.72 (0.65–0.93) for diarrhoea. Fifty three percent of 6975 sick children were treated with antibiotics, 11% were not treated with antibiotics when they should have been and in 29% antibiotics were prescribed when they should not have been. Re-attendance rates within 14 days following clinical diagnosis of mild pneumonia were 9% (126/1401) when managed with antibiotics compared to 8% (56/701) when managed without (adjusted Hazard Ratio (aHR) = 1.00 (0.57–1.76), p = 0.98). Rates for mild diarrhoea were 8% (73/874) and 9% (79/866) respectively (aHR = 0.8 (0.42–1.57), p = 0.53).Non-adherence to IMCI recommendations for prescription of antibiotics is common in routine settings in Papua New Guinea. Although recommended, the use of antibiotics in young children with mild pneumonia as defined by IMCI criteria did not impact on their outcome. Better tools and new strategies for the identification of bacterial infections that require antibiotics are urgently needed. Worldwide, the leading causes of death in children under five years  are pneumonia, diarrhoea and malaria. These three diseases are responsible for an estimated 5 million deaths yearly, over 90% of which occur in Africa and other developing countries with limited resources. For decades, a high importance was given to malaria as a cause of fever, which has led to an overestimation of its burden and at the same time an underestimation of other diseases. Although the IMCI strategy has been used for more than 20 years, few studies have investigated its effectiveness and appropriateness. Specifically, there is limited information on how well health workers comply with IMCI guidelines in routine practice, and on the impact of IMCI recommendations on health outcomes. Papua New Guinea (PNG) has a high burden of malaria in coastal areas, while pneumonia is the leading cause of admission in children under five years nationally. As part of a trial on malaria prevention, a passive case detection system was set up to record illness episodes in all study participants aged 3 to 27 months. All illness episodes were managed in accordance with the PNG IMCI guidelines, except for malaria, where treatment was guided by RDT instead of using presumptive diagnosis. www.ClinicalTrials.gov: NCT00285662) This work was carried out alongside a 3-arm trial of Intermittent Preventive Treatment for malaria in infants (IPTi) that included 1605 children 3–27 months old from June 2006 to June 2010 (The study took place in two regions of PNG: Mugil (Madang Province) with 1125 study participants and in the Wosera (East Sepik Province) with 480 study participants. Each study site included approximately 20 villages, serviced by four major health centres: Mugil and Alexishafen in Madang area, and Kunjingini and Kaugia in Wosera. Several aid posts were also used for the morbidity surveillance. All IPTi preventive treatments were given at time of routine vaccination at 3, 6 and 9 months or of Vitamin A supplementation at 12 months following the PNG national expanded programme of immunization.The study was carried out in accordance with Good Clinical Practice (GCP) and monitored by an independent external monitor. The study was approved by the PNG Medical Research Advisory Committee (MRAC number 05.20). Parents or carers were asked to fill a written consent form prior to enrolment of their child in the trial.Children enrolled in the trial were randomly allocated to receive placebo or two different antimalarial drug regimes [sulphadoxine/pyrimethamine (single dose) with either 3 days of amodiaquine (SP-AQ3) or 3 days of artesunate (SP-AS3)]. The primary objective of the trial was the determination of the protective effect of the two IPTi regimens on malaria episodes.Throughout the trial, a passive case detection system was maintained at the four study health centres’ outpatient clinics as well as at four aid posts to assure a continuous morbidity surveillance of study participants. This means that parents of study participants were asked to attend the study clinics free of charge for clinical management whenever their child was sick. They were seen by study nurses, often in collaboration with the health centre’s staff. No active follow-up of study participants was performed for acute diseases (such as regular home visits). In that perspective, this approach is more likely to reflect routine practice in PNG and thus avoid overestimation of the burden of diseases. The passive surveillance records collected were used to perform the present analysis.Each illness episode was assessed by study staff using a standard case report form. Main signs and symptoms were systematically recorded using tick boxes. In case of history of fever in the past 48 hours or an axillary temperature >37.5°C, a RDT for malaria  was performed and haemoglobin (Hb) level measured using a portable Hemocue 201® machine . Malaria treatment decision was based on the RDT result, and only children with a positive result were treated with artemether/lumefantrine, irrespective of the species. The children with moderate to severe anaemia (Hb<8 g/dl) received iron supplementation at the dose of 5 mg/Kg of ferrous sulphate for 6 weeks. Pulse oximetry was implemented in December 2007 in Mugil study clinic (Madang site) and used systematically to assess sick study participants from that date onwards. It was recommended to clinical staff that they should consider admitting the child if oxygen saturation dropped below 94%. All other illnesses were treated according to the standard treatment guidelines of PNG, which is based on IMCI. A severe disease was defined as an illness episode with potential life-threatening features such as respiratory distress , impaired consciousness or severe pallor. At least one danger sign or symptom needed to be present for an episode to be classified as severe disease. A child with criteria for severe disease was not necessarily admitted for the following reasons: parents refused admission; health staff decided not to admit the child according to their personal judgment or because they did not identified properly danger signs and symptoms.The medical staff was composed of about 20 nurses and community health workers (CHW). They were specifically trained for malaria and anaemia management in line with the trial’s requirements (diagnosis and treatment). A finger prick blood sample was used to perform a rapid diagnostic test (RDT) for malaria on site  and to record haemoglobin concentration. A blood slide was prepared for expert microscopy that was performed elsewhere.Using records of signs and symptoms (tick boxes) of the passive case detection forms, objective diagnoses were generated by computer according to IMCI definitions of diseases (e.g. mild pneumonia = cough and/or difficulties in breathing+respiratory rate >50 if <1 year or respiratory rate >40 if >1 year without sign/symptom of severity). For the present study, three diseases were explored with different levels of severity according to IMCI: 1) mild and moderate-to-severe pneumonia, 2) mild and severe diarrhoea and 3) uncomplicated and severe malaria. Ten distinct treatment categories were created: 1) artemether/lumefantrine, 2) iron+folic acid, 3) albendazole, 4) amoxicillin, 5) co-trimoxazole, 6) paracetamol, 7) tinidazole, 8) other antibiotics , 9) other antimalarial drugs  other drugs.Illness episodes were included in the study if they fulfilled the following inclusion criteria:IPTi study participantNo illness episode in the past 14 days (to avoid counting the same episode twice)Full clinical records (signs and symptoms available)The appropriateness of the prescription of antibiotics by health staff was assessed for main diagnoses according to the IMCI recommendations, including both severe and mild disease episodes.The effectiveness of antibiotics was investigated for mild diseases only, by calculating the rate of re-attendance within 14 days and the outcome upon re-attendance  for specific syndromes depending whether they were treated with antibiotics or not. Severe cases were excluded from this analysis for several reasons: i) severe cases (i.e. with danger signs) had to be admitted according to recommendations and managed systematically with intravenous antimalarial and antibiotics irrespective of the symptoms and signs; ii) the low prevalence of severe cases, which are almost all treated with antibiotics, prevent the investigation of the effectiveness because of the absence of a valid control group; iii) re-attendance rates was used as a proxy for effectiveness, but this is not a valid measure for assessing effectiveness in severe cases, as they should be all admitted in the first place and would therefore not re-attend; iv) there is no debate yet on the benefit of treating all severe cases with antibiotics.This analysis was possible as syndromes were not necessarily correctly identified by health staff and therefore some children did not receive antibiotics when they should have.All data were double entered using FoxPro software. Data analyses were performed using STATA software (version 12.0). More than one diagnosis was possible for each illness episode. Incidence rates were calculated using a negative binomial regression model. Cox regression model was used to calculate hazard ratios for the impact of antibiotics on pneumonia, diarrhoea and malaria (excluding severe diseases). The model was adjusted for potential confounders (markers of severity of respiratory infections): respiratory rate (continuous), temperature (continuous) and pulse oximetry (continuous). Kaplan-Meier survival curves were also calculated and displayed as time to first re-attendance over a 14-day observation period following an initial pneumonia, malaria or diarrhoea. All rates are displayed with a confidence interval of 95%. Differences between rates were assessed using a chi square test.Out of 1605 children 1512 (94%) presented at least 1 illness episode (Madang = 1053 and Maprik = 459). The morbidity surveillance recorded a total of 8943 illness episodes (Madang = 5977 and Maprik = 2966). Three hundred seventy one severe illnesses and 13 deaths occurred during the study. Overall, 2903 illness episodes occurred in the placebo group, compared to 3038 in the SP-AQ group and 3002 in the SP-AS arm.Inclusion criteria were fulfilled for 6975 illness episodes . Out of the 6975 illness episodes, 76.5% (5335) were ARI, including 44.7% (3116) URTI, 30.1% (2102) mild pneumonia and 1.7% (117) severe pneumonia. 25.6% (1788) had diarrhoea, including 24.9% (1740) mild diarrhoea and 0.7% (48) severe diarrhoea. 25.1% (1750) had malaria, including 23.6% (1643) uncomplicated malaria and 1.5% (107) severe malaria. 10.2% (710) could not be classified in one of these syndromes. In this category, there were mainly children with running nose as single symptom (which cannot be classified as URTI according to IMCI), skin infections and fever without any other accompanying symptom.The incidence rates  were 0.85  for mild pneumonia, 0.72  for mild diarrhoea and 0.62  for uncomplicated malaria. No differences were found in the incidence of non-malarial illnesses between the placebo and IPTi intervention arms. Indeed, incidence rates for all pneumonia in children 3–15 months old were very similar in all arms with 0.97  in the placebo group, 1.00  in the SP-AQ3 group and 0.98  in the SP-AR3 group, p-values for incidence rate ratios (IRR) were 0.63 and 0.87 respectively. For diarrhoea (severe included), the incidence rates from 3–15 months were similar in all arms with: 0.94  for placebo, 0.92  for SP-AQ3 and 0.89  for SP-AS3, p-values for IRR were 0.85 and 0.51 respectively. By contrast, the IPTi has proven to be effective on malaria as described elsewhere. Main results showed that the incidence of clinical malaria between the first dose at 3 months and 15 months was 0.85 per year-at-risk overall and 0.22 and 0.68 for P. falciparum and P. vivax, respectively. In the intention-to-treat analyses, the protective efficacy against all cases of malaria was 29%  in the SP-AQ3 group and 12%  in the SP-AS3 group Out of the 6975 illness episodes, 53.6% (3741) were treated with antibiotics  and 25.1% (1750) received antimalarial drugs (97.6% of whom had a positive RDT result). Among the children who were treated for mild pneumonia with antibiotics, 69% received amoxicillin only, 9% received co-trimoxazole only, 16% received other antibiotics and 6% received a combination of amoxicillin and other antibiotics.The overall appropriateness of antibiotics use was evaluated in 6969 illness episodes for which clear recommendations exist in the IMCI whether to prescribe or not antibiotics. According to these data, 40% of children were not treated appropriately, of whom 11% did not receive antibiotics when they should have and 29% received antibiotics when they should not have. Figure 1Among the 2102 children with an objective diagnosis of mild pneumonia, 66.7% (1401) received (appropriately) antibiotics. Of 532 children who presented with criteria for both mild pneumonia and uncomplicated malaria (based on positive RDT), only 40% (213) were prescribed antibiotics. In contrast, when they had criteria for pneumonia only , 75.7% (1188/1569) were prescribed antibiotics (p<0.001). Among 117 children with a diagnosis of severe pneumonia, 96.6% (113) received antibiotics, of whom 75.2% (88) were treated with parenteral antibiotics as per WHO guidelines and 49.6% (58) were admitted.Re-attendance rate within 14 days following an objective diagnosis of mild pneumonia (IMCI criteria) was 9% when treated with antibiotics compared to 8% when they did not receive any antibiotics, unadjusted Hazard Ratio (HR) was 1.09 . For uncomplicated malaria, re-attendance rates were 4% when they received antibiotics vs 6% when they did not [HR 0.66 ]. For diarrhoea, re-attendance rates were 8% when they received antibiotics and 9% when they did not [HR 0.85 ]. When adjusting for potential confounders including severity parameters , the adjusted HR (aHR) for mild pneumonia was 1.00 , for mild malaria 0.70  and for mild diarrhoea 0.81. The aim of this prospective study was to explore the appropriateness and effectiveness of prescription of antibiotics in routine outpatient settings of Papua New Guinea in the frame of the IMCI recommendations.A high rate of antibiotic prescription was observed in the cohort, with 54% of cases treated (including mild and severe cases) when a maximum of 36% should have received antibiotics according to IMCI criteria, This is high compared to other settings, where it is usually reported that approximately 20% of patients require antibiotics when the diagnosis is made by highly skilled clinicians. A poor compliance to guidelines was observed as 40% of patients were not treated according to the recommendations (29% received antibiotics when they should not have and 11% did not receive antibiotics when they should have). Apart from the inaccurate clinical judgment of health staff, other factors might influence their decision. In our study, we observed that the prescription of antibiotics was strongly influenced by the result of the RDT for malaria in feverish patients. Indeed, we observed that in children presenting with criteria for mild pneumonia (who should receive antibiotics), only 40% were treated when the RDT was positive, compared to 76% when the test was negative. On one hand, it is reassuring to observe that clinicians are influenced in their clinical judgment and treatment attitude by an objective test. On the other hand, considering only one diagnosis may have potential serious consequences for children, as it might leave children with bacterial infections untreated. This may be the downside of the introduction of RDT in routine clinics. Indeed, improving the diagnosis of malaria may have adverse effects for the management of other diseases, which is in line with findings by Bisoffi et al. in Africa It is finally reassuring to observe that almost all children (97%) with criteria for a severe pneumonia received antibiotics.One might worry that such a high rate of non-compliance to standard IMCI recommendations might leave a lot of children with potential bacterial pneumonia untreated and might re-attend with a more severe clinical presentation associated with poor outcomes. However, our data indicate that it is in fact not the case. Indeed, the rate of re-attendance following a visit for mild pneumonia, which can be considered as a proxy of the effectiveness of the antibiotics, was similar, irrespective of antibiotic prescription . This is well in line with recent findings in Pakistan where the use of amoxicillin was not found to provide any benefit for non-severe pneumonia as defined by WHO. Adenovirus and Respiratory Syncytial Virus . Similar findings have also been published in other countries. The absence of difference in the re-attendance rates is probably due to the fact that the vast majority of mild pneumonia episodes have a viral aetiology rather than bacterial, highlighting the lack of specificity of the clinical criteria to identify bacterial pneumonia. This was confirmed by a microbiological investigation made on nasal swabs in a sub-sample of these children. The rapid antigen diagnostic tests for viral pathogens showed that more than half of children with mild clinical pneumonia were positive for at least one of the four most common respiratory viruses: Influenza A&B, Influenza need to be investigated. Beyond the aetiology of respiratory infections, it would be also valuable to develop tests able to identify children with potentially severe (invasive) diseases, which require specific supportive care. In that regard, inflammatory markers such as pro-calcitonin (PCT) can be valuable tools. Recent reviews by Schuetz et al. The diagnosis of pneumonia is more complex than that of malaria as no reliable and easy-to-use tests are available to identify respiratory infections that require antibiotics. It has been recognized that pneumonia is often associated with virus detection and that there is a complex and poorly understood interplay between viruses and bacteria in the development of pneumonia. Similarly to what was observed with mild pneumonia, antibiotics did not provide any benefit for diarrhoea or malaria in our cohort, which supports the current WHO recommendations of not treating mild gastroenteritis and uncomplicated malaria with antibiotics.The main limitation of this study is that the clinical management of malaria episodes was performed in ideal conditions because it was conducted alongside a malaria prevention trial (i.e. treatment of only RDT positive cases). While RDT-based malaria treatment was not part of the PNG malaria treatment guidelines when the study was conducted, there was rapid roll-out and rapid up-take of RDTs This study shows that adherence to IMCI recommendations for prescription of antibiotics was poor in routine outpatient settings of Papua New Guinea and was strongly influenced by RDT results for malaria, independently of the clinical presentation. However, the management of mild pneumonia with antibiotics based on IMCI guidelines did not change health outcome in terms of re-attendance and complications when compared to no antibiotics. This absence of effectiveness of antibiotics is certainly due to the lack of specificity of the present recommendations to differentiate children with true bacterial diseases and those with viral infections. It is urgently needed to develop better strategies to improve the identification of pneumonia and other syndromes that require antibiotics or more intensive clinical care.We would like to warmly thank all study nurses of the IPTi trial who looked after the data collection as well as after the study participants. We would like also to thank all the laboratory staff of the PNG IMR who looked at some many slides and analysis.Finally our deep gratitude goes to all the study participants and their parents for agreeing to participate to this study. We also would like to thank Dr Amanda Ross, who is a post-doctoral biostatistician at the Swiss TPH, for her guidance to perform the survival analysis.


P. falciparum infections are common and may predict host protection. Here, we have investigated the effect of clearing asymptomatic infections on the risk of clinical malaria.Protective immunity to malaria is acquired after repeated infections in endemic areas. Asymptomatic multiclonal P. falciparum merozoite surface protein 2 using fluorescent PCR and capillary electrophoresis. Following the second survey, asymptomatic infections were cleared with a full course of dihydroartemisinin.Malaria episodes were continuously monitored in 405 children (1–6 years) in an area of moderate transmission, coastal Kenya. Blood samples collected on four occasions were assessed by genotyping the polymorphic Children who were parasite negative by PCR had a lower risk of subsequent malaria regardless of whether treatment had been given. Children with ≥2 clones had a reduced risk of febrile malaria compared with 1 clone after clearance of asymptomatic infections, but not if asymptomatic infections were not cleared. Multiclonal infection was associated with an increased risk of re-infection after drug treatment. However, among the children who were re-infected, multiclonal infections were associated with a shift from clinical malaria to asymptomatic parasitaemia.P. falciparum infections is associated with protective immunity, but there appears to be an additional effect in untreated multiclonal infections that offsets this protective effect.The number of clones was associated with exposure as well as blood stage immunity. These effects were distinguished by clearing asymptomatic infection with anti-malarials. Exposure to multiple  Plasmodium falciparum malaria is gradually acquired after repeated infections and largely dependent on exposure intensity Immune protection against P. falciparum infections is the presence of several genetically distinct parasite subpopulations, i.e. clones, within a single individual. The number of infecting clones varies with age A characteristic of P. falciparum infections on host immunity, we have investigated how clearance of asymptomatic infections affects the subsequent risk of clinical malaria in children (1–6 years) living on the coast of Kenya. A short acting anti-malarial drug (dihydroartemisinin) was given at the beginning of the malaria transmission season P. falciparum infections was investigated by genotyping the highly polymorphic gene encoding the merozoite surface protein 2 (msp2) using fluorescent PCR and capillary electrophoresis To clarify the importance of persistent multiclonal The study included samples collected during a randomized controlled trial of a candidate malaria vaccine. The study and extended follow up has been described in detail elsewhere Scheduled venous blood samples were collected in EDTA at initial screening in February 2005 and post-vaccination in May 2005. All children were then treated (independently of being parasite positive or not) with direct observed dihydroartemisinin monotherapy for seven days to clear asymptomatic parasiteamias, beginning on the day of the blood sample taken in May. Parasite clearance was confirmed by microscopical analysis of blood films one week after ended treatment. Additional blood samples were taken at cross-sectional surveys in August 2005 and January 2006 followed by an additional 9 months follow-up. To calculate parasite density, the number of asexual-stage parasites per 200 leukocytes was counted by conventional light microscopy, and parasiteamia was estimated on the basis of an assumed uniform white cell count of 8000 leukocytes/µl blood. Hemoglobin (Hb) levels were measured at all cross-sectional surveys.Clinical malaria was monitored weekly by home visits during the total 18 months follow-up. Fieldworkers were resident in the villages where children were recruited and readily accessible for unscheduled visits if the children developed fever. When the temperature was greater than 37.5°C, a blood film and a rapid diagnostic test  for malaria were conducted. The blood films were reviewed within 24–48 hours, but the immediate treatment decisions were based on the rapid test result.P. falciparum parasites/µl. This parasite threshold was set to increase the specificity of the endpoint since asymptomatic parasiteamias are common among children in this endemic area Clinical malaria was defined as fever (axillary temperature >37.5°C) and presence of >2500 msp2. Blood smear positive and negative samples were included, so as to include submicroscopic infections. DNA was extracted using ABI Prism 6100 Nucleic Acid PrepStation (Applied Biosystems) (n = 858) or PUREGENE™ DNA Isolation Kit (Gentra systems) (n = 484). P. falciparum parasite populations were characterized by msp2 genotyping using fluorescent PCR and capillary electrophoresis (CE) as described previously msp2 (block 3) was amplified in a primary reaction, followed by nested reactions where the respective allelic types of msp2, FC27 and IC, were targeted with fluorescently labeled primers in separate reactions. Fragment analysis was performed in 96-well format in a 3130xl DNA sequencer and the results were analyzed using GeneMapper® Software version 4.0 (both from Applied Biosystems). A 150 relative fluorescent unit (rfu) cut off was set to distinguish true fragment peaks from background fluorescence and other artefacts. Microscopy positive samples that were negative by PCR were concentrated by ethanol precipitation before repeating the PCR reaction.Blood samples (n = 1342) collected at the four cross-sectional surveys were genotyped for Analysis was performed using STATA (v10). Only strictly asymptomatic children were included in the analysis, therefore all children with a clinical episode at survey or in the period 28 days before until 7 days after survey were excluded. Each child contributed up to 4 separate periods of three months follow-up after the respective cross-sectional bleeds.Association between number of clones (no infection/1 clone/2 or more clones) and village of residence (high/low transmission), age , Hb levels, vaccine i.e. malaria or control (rabies vaccine) and insecticide treated bednet (ITN) use (yes/no) were investigated by a Poisson model adjusted for transmission season (dry/rainy) and corrected for repeated observations per child by the robust cluster estimator. A single multivariable model was produced. Correlation between parasite density and age and number of clones was estimated using Spearman rank correlation coefficient. This method was also used to calculate the correlation of number of clones between surveys.P = 0.38). MOI was fit in categories (0 clones/1 clone/≥2 clones).For survival analyses, data from the respective follow-up periods was analyzed individually followed by pooled analysis for the surveys without treatment . The follow-up after survey 2, with treatment, was analyzed separately. Cox regression was used to assess the time to the first or only clinical episode during follow-up (without/with treatment) in relation to number of clones . Hazard ratios (HR) were adjusted for ITN use, age and village of residence (high/low transmission) and transmission season i.e. dry/rainy . Schoenfeld residuals for non-proportionality were analysed for each covariate. The linear fit of the model was tested by examining multiple fractional polynomials and was not rejected in favour of polynomial fits for age  during follow-up, (2) no clinical malaria but presence of asymptomatic parasiteamia at the cross-sectional survey three months later (survey 3), or (3) remaining uninfected i.e. having no clinical episode during follow-up nor any detectable parasites at the following cross-sectional survey. Factors associated with the different outcomes, i.e. number of clones, age, village of residence (high/low transmission) and ITN use, were investigated by logistic regression. When the outcome was re-infection , the analysis was performed excluding children who remained uninfected as these children were considered less exposed.In an analysis taking exposure into account, survey 2 was used as baseline and the outcome during the three months follow-up after parasite clearance was classified into three categories; (1) clinical malaria . The vaccine groups, i.e. malaria/control were therefore pooled for further analysis.Vaccination had no effect on the incidence of clinical malaria episodes or on prevalence of asymptomatic parasiteamias P. falciparum by PCR. Parasite prevalence and densities were lower at the next survey just before drug treatment (May 2005) as well as after the two consecutive surveys three (Aug 2005) and nine months (Jan 2006) after treatment .At the first cross-sectional survey (Feb 2005), 263 (78%) children were positive for reatment . P. falce by PCR . The preP. falciparum infections were mainly composed of ≥2 clones with up to 8 clones at the most (P<0.001) for survey 1 and 2, and r = 0.53 (P<0.001) for survey 3 and 4, respectively (the most . The proectively .P<0.001) with an overall lower number of clones in the survey after treatment , village of residence  and Hb levels , however not with ITN use . There was no systematic difference in the results obtained with the two different DNA extraction methods (data not shown). The number of clones was correlated with age  (P<0.001). This correlation was non-linear indicating that at high parasite densities, further increases in density are no longer associated with increases in number of clones  . Parasitf clones .P. falciparum (>2500 parasites/µl) were reported in 337, 360 and 288 children in the follow-up periods without treatment , while 59 episodes were reported in 360 children during the three months after treatment (after survey 2). Age was associated with a decreased risk of clinical malaria both without and with parasite clearance before follow-up (P = 0.7) after adjusting for MOI, village and age.In total 36, 13 and 12 clinical episodes with fever (>37.5°C) and ollow-up . Childreollow-up . Anemia P = 0.28), ITN use (P = 0.59), number of clones (P = 0.65) or age (P = 0.07). Data from these different follow-up periods without treatment  were therefore pooled.When time to the first or only clinical episode was assessed for the respective follow-up periods, the number of clones was significantly associated with disease risk only after treatment in survey 2. There were fewer episodes in the surveys without drug-treatment, but no statistical evidence of interactions between survey number and village (P = 0.045 by Cox regression) and HR 0.52 , respectively. The number of clones at the surveys not followed by treatment was not associated with risk compared to one clone  (P = 0.026)  . However= 0.026) .P = 0.36 by Cox regression). However, the interaction term was HR = 3.54  for the effect of ≥2 clones and the effect of treatment. This confirms that the number of clones acts significantly differently on the risk of clinical malaria depending on whether not treatment was given after the survey.The interaction term between the number of clones and the effect of treatment on subsequent clinical malaria was analyzed in a model using data from all follow-up periods (without and with treatment). There was no significant interaction with the effect of 0 clones, HR = 0.63 . In those re-infected, being parasite negative or infected with ≥2 clones at survey 2 were both associated with a reduced risk of clinical malaria compared with 1 clone   compared with those remaining uninfected. In those re-infected, the risk of clinical malaria was compared with asymptomatic infections. The risk of re-infection when comparing 0 clones with 1 clone was not significantly different, however ≥2 clones was associated with an increased risk of re-infection with borderline significance (OR = 1.97 95% CI 0.99–3.93 by logistic regression) . When clctively) .P. falciparum infections with one full course of an effective anti-malarial drug affected the genetic diversity of infections during follow-up and the subsequent risk of clinical malaria. Without treatment, we did not observe any significant effect of the number of clones on the risk of infection. However, when the infections were cleared, children with 2 or more clones just prior to treatment had significantly lower risk of subsequent febrile malaria than children with 1 clone. We tested the interaction term between number of clones and time of survey, and confirmed that multiclonal infections act differently depending on whether anti-malarial treatment had been given or not. Children who were parasite negative at the cross-sectional surveys had a lower risk of subsequent malaria in the following three months periods irrespective of whether the child had received anti-malarial treatment.Clearance of asymptomatic We analyzed the outcomes following clearance of asymptomatic infection to distinguish lack of exposure from immunity, as previously described per se are important or it they just represent a marker of previous exposure. In two other areas in Kilifi, infections with 2 clones were associated with an increased risk of subsequent malaria in an area of low-moderate transmission, while in a low transmission village the diversity did not predict the risk of clinical malaria The present study provides with interesting insights into the importance of multiclonal infections. Previous studies have shown conflicting results, with the number of clones being related to risk as well as to protection msp2 gene has proven to be the most informative marker of parasite population diversity Several factors can affect the number of clones in an individual at a certain time point The number of clones was associated with age, village of residence and hemoglobin levels, however not with ITN use. There was a substantial variance in the number of clones which was not explained by age. The number of infecting clones at a certain time point might reflect the individuals' level of acquired immunity as a result of previous exposure. Children with asymptomatic, presumably chronic multiclonal infections, persisting through the preceding low transmission season may have mounted immune protection against super-infecting parasite clones while children with previous single clone infections are more susceptible when infected with novel parasite clones as the transmission season starts.P. falciparum infections after 6 months of IPT given monthly or bimonthly to children in an area of high seasonal transmission in Ghana resulted in a temporary reduction of the number of clones and higher susceptibility compared to the placebo group in which ≥2 clones conferred a reduced risk Repeated clearance of asymptomatic parasitemias is the basis of intermittent preventive treatment (IPT), a new strategy for malaria control. Genotyping of P. falciparum are indeed more long lived in the presence of asymptomatic infections Long periods of malaria chemoprophylaxis have been followed by increased incidence of clinical malaria P. falciparum infections and risk of subsequent clinical malaria. Why being infected with multiple clones is only associated with protection following clearance of parasites in this setting remains unclear. Possibly the presence of parasites is partly immunosuppressive during some stage of immune acquisition. However, multiclonal infections also represent a marker of acquired immunity and memory. Understanding how immunity to multiclonal P. falciparum infections, develops and how it is affected by different interventions will be important in the development and evaluation of future strategies for malaria control.In conclusion, a course of treatment of a short acting anti-malarial drug affected the diversity of Figure S1Correlation between age and number of clones.(TIFF)Click here for additional data file.Figure S2Correlation between parasite densities and number of clones.(TIFF)Click here for additional data file.Table S1A–C. Number of children infected with different number of clones in consecutive pairs of surveys.(DOCX)Click here for additional data file.


Malaria is a major health burden in sub-Saharan African countries, including Mali. The disease is complex, with multiple genetic determinants influencing the observed variation in response to infection, progression, and severity. We assess the influence of sixty-four candidate loci, including the sickle cell polymorphism (HbS), on severe malaria in a case-control study consisting of over 900 individuals from Bamako, Mali. We confirm the known protective effects of the blood group O and the HbS AS genotype on life-threatening malaria. In addition, our analysis revealed a marginal susceptibility effect for the CD40 ligand (CD40L)+220C allele. The lack of statistical evidence for other candidates may demonstrate the need for large-scale genome-wide association studies in malaria to discover new polymorphisms. It also demonstrates the need for establishing the region-specific repertoire of functional variation in important genes, including the glucose-6-phosphatase deficiency gene, before embarking on focused genotyping. Malaria is a life-threatening parasitic disease transmitted by mosquitoes. Despite the concerted and renewed efforts to control the disease, it still persists as a major health burden, being responsible for 655,000 deaths in 2010, mainly children in Sub-Sahara Africa This study was approved by the Faculty of Medicine, Pharmacy and Dentistry (University of Bamako) Ethics Review Committee. All clinical and biological samples were collected and DNA was genotyped following approval by this committee. Written informed consent was obtained from the next of kin, carers or guardians on the behalf of the minors/children participants involved in this study.Patient samples were collected as part of ongoing epidemiological studies of severe malaria at the Centre Hospitalier Universitaire Gabriel Toure, Bamako, Mali ; healthy controls 393 (42.1%)). They had a median age of ∼3 years, and were predominantly from the Bambara ethnic group (53%) see .P. falciparum on blood film and clinical features of uncomplicated and severe malaria All cases were children admitted to hospital with evidence of www.malariagen.net, and a list of SNPs typed can also be found in Genomic DNA samples underwent whole genome amplification through Primer Extension Pre-amplification (PEP) http://www.r-project.org). Performing multiple statistical tests leads to inflation in the occurrence of false positives and using a permutation approach that accounted for correlation between markers and tests, we estimated a p-value cut-off of 0.002 to be statistically significant. Allele frequency differences were estimated using the Fst metric, with values potentially varying from zero (no population differentiation) to one (complete differentiation) Genotypic deviations from Hardy-Weinberg equilibrium (HWE) were assessed using a chi-square statistical test. SNPs were excluded from analysis if they had at least 10% of genotype calls missing or there was significant deviation from HWE (p<0.001). Case-control association analysis using SNP alleles/genotypes was undertaken by logistic regression and included the covariates: ethnic group and the HbS polymorphism. In this approach we modelled the SNP of interest assuming several related genotypic mechanisms  and reported the minimum p-value from these correlated tests. All analyses were performed using the R statistical package , deviated from HWE in controls  or had high rates of missing genotype calls (rs1801274). Allelic-based tests revealed potential associations of HbS polymorphism , the O blood group (and its components rs8176746 and rs8176719), and rs1126535 (CD40L+220) with severe malaria (P<0.002) . Figure In our study, we set out to investigate the role of candidate malaria polymorphisms on severe malaria risk in a predominantly Bambara population in and around Bamako. To minimise errors we standardised procedures using case report forms, pre-defined definitions of severe malaria and sub-clinical phenotypes, and ensured all samples underwent genotyping on the same Sequenom MassArray platform with resulting low rates of missing data (<5%). In addition, all analyses were adjusted for ethnicity, minimising the confounding effects and potential false positives arising from population stratification.http://www.map.ox.ac.uk/) in West  and East  Africa. The frequency of the HbC allele was 5% in cases and 6% in controls, both greater than the HbS allele, but there was no strong evidence of association (P>0.3). The higher frequency of the HbC allele has been observed in other West African populations Data analysis confirmed the known (∼90%) reduced severe malaria risk from the sickle cell AS genotype P. falciparum rosetting Our analysis also confirms the known protective effects of the blood group O on life threatening malaria, which is thought to act on malaria pathogenesis through the mechanism of reduced Our analysis revealed a marginal susceptibility effect for the CD40 ligand (CD40L)+220C allele. CD40L is a glycoprotein involved in B cell proliferation, antigen presenting cell activation, and Ig class switching, and therefore important in the immune response to infection P. falciparum has shaped our genome. These approaches could expose new candidate protective polymorphisms, resulting in functional work to understand underlying molecular mechanisms, leading to the development of therapies and vaccines for malaria control.In conclusion, our work reinforces in a Malian (mainly Bambara) population the importance of the sickle cell polymorphism and ABO blood group on severe malaria susceptibility. It may also demonstrate the need for establishing the region-specific repertoire of functional variation in important genes such as G6PD, before embarking on focused genotyping. Proposed large-scale genomewide association studies and resequencing of important genes in a number of Malian (and other African) populations is likely to generate insights into how Table S1List of all candidate polymorphisms considered in our work.(DOC)Click here for additional data file.


Plasmodium vivax malaria clinical outcomes are a consequence of the interaction of multiple parasite, environmental and host factors. The host molecular and genetic determinants driving susceptibility to disease severity in this infection are largely unknown. Here, a network analysis of large-scale data from a significant number of individuals with different clinical presentations of P. vivax malaria was performed in an attempt to identify patterns of association between various candidate biomarkers and the clinical outcomes.P. vivax-infected individuals who developed different clinical outcomes  as well as 176 non-infected controls, was performed. Plasma levels of liver transaminases, bilirubins, creatinine, fibrinogen, C-reactive protein, superoxide dismutase (SOD)-1, haem oxygenase (HO)-1 and a panel composed by multiple cytokines and chemokines were measured and compared between the different clinical groups using network analysis.A retrospective analysis of 530 individuals from the Brazilian Amazon, including P. vivax malaria infection were associated with significant interactions between TNF ALT, HO-1 and SOD-1.Non-infected individuals displayed several statistically significant interactions in the networks, including associations between the levels of IL-10 and IL-4 with the chemokine CXCL9. Individuals with asymptomatic malaria displayed multiple significant interactions involving IL-4. Subjects with mild or severe non-lethal malaria displayed substantial loss of interactions in the networks and TNF had significant associations more frequently with other parameters. Cases of lethal P. vivax malaria is associated with multiple significant interactions in the network, mostly involving IL-4, while lethality is linked to a systematic reduction of complexity of these interactions and to an increase in connections between markers linked to haemolysis-induced damage.The findings imply that clinical immunity to  Plasmodium parasite itself, malaria is a consequence of multiple host molecular and genetic alterations compounded by environmental factors [Plasmodium vivax, which has been relatively neglected for many years and recently re-emerged as a potentially lethal disease [The vast majority of human diseases do not result from single molecular changes, and malaria is certainly included in this group. Besides factors related to the  factors . Most of factors -6. Recen factors -9. Howev disease -13.The use of a comprehensive approach to exploring the relationship between groups of molecules likely to be related to the severity of the disease manifestations may contribute to a better understanding of the patterns of susceptibility to disease severity or disease tolerance. In this study, network analysis of a large dataset from a significant number of individuals presenting with different clinical manifestations of vivax malaria was performed in an attempt to identify patterns of association between various candidate biomarkers and the clinical outcomes. The findings from this exploratory study demonstrate a new level of complexity connecting several mediators involved in the immunopathogenesis of vivax malaria.P. vivax used in the project were published previously [Plasmodium falciparum infection documented by both microscopy and nested polymerase chain reaction (PCR), documented viral hepatitis, chronic alcoholism, human immunodeficiency virus type 1 infection, yellow fever, leptospirosis, cancer and chronic degenerative diseases, sickle cell trait and the use of hepatotoxic or immunosuppressant drugs. The malaria diagnosis was made in a reference centre from the National Foundation of Health in the endemic area and confirmed with nested PCR as previously described [Plasmodium within this period of 30 days and presented no malaria-related symptoms, such as fever (axillary temperature >37.8°C), chills, sweats, myalgia, arthralgia, strong headaches, nausea, vomiting, jaundice, and severe asthenia during this period were considered as asymptomatic malaria cases. During the first clinical visit (previously to the results of the diagnostic tests) all the asymptomatic individuals received counselling and were oriented to seek for health care in a malaria reference centre in case symptoms appeared during the period between the 2 clinical visits. After the second clinical visit, all the patients that had positive malaria screening with microscopy and/or nested PCR were treated following the treatment guidelines of the Brazilian Ministry of Health. The plasma samples used to assess the biomarkers were collected during the second clinical visit, before the initiation of anti-malarial drugs. Symptomatic individuals were promptly treated. Data from a total of 530 individuals were analysed, and the individuals were stratified into the following categories: non-infected (n=176), asymptomatic vivax malaria (n=148), symptomatic vivax malaria (n=187), severe non-lethal vivax malaria (n=13), and severe vivax malaria associated with mortality (n=6). The plasma samples obtained from patients with severe forms of malaria, including the cases that eventually died during the follow up were collected at the hospital admission and the procedures are described elsewhere [The present study is a retrospective analysis of a databank containing clinical, epidemiological and immunological data from 530 individuals from the Brazilian Amazon  recruited between 2006 and 2007, as part of a project aimed at describing determinants of susceptibility to vivax malaria that was finalized on 2010. In this project, both active and passive malaria case detection were performed. These included home visits in areas of high transmission (active case detection), and study of individuals seeking care at the diagnostic centres of Brazilian National Foundation of Health (FUNASA) or in a municipal hospital in Buritis, Rondônia, Brazil (passive case detection). Individuals of both sexes, ranging in age from five to 70 years, who had resided in the endemic area for more than six months, were invited to participate. The details of the recruitment, diagnosis approach and clinical definitions of asymptomatic infection or severe malaria caused by eviously ,14-17. Tescribed ,17. IndiWritten informed consent was obtained from all participants or their legally responsible guardians, and all clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. The project was approved by the institutional review board of the Faculdade de Medicina, Faculdade São Lucas, Rondônia, Brazil, where the study was performed.Several mediators were selected based on the assessment of the overall inflammatory status and immune responses in malaria. All the biomarkers that were measured in all the patients, and were contained in the databank, were included in the analysis and no pre-selection was done. Plasma measurements of aspartate amino-transferase (AST), alanine amino-transaminase (ALT), total bilirubin, direct bilirubin, creatinine, fibrinogen and C-reactive protein (CRP) were made at the clinical laboratory of Faculdade São Lucas and at the Pharmacy School . The cytokines IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12p70, IFN-γ, TNF and the chemokines CCL2 (MCP-1), CCL5 (RANTES), CXCL9 (MIG) and CXCL10 (IP-10) were measured using the cytometric bead array (CBA) . The flow cytometric assay was performed and analysed by a single operator, and standard curves were derived from cytokine standards. The experiments were performed according to the manufacturers’ instructions. ELISA kits were used to measure the soluble TNF receptor I , transforming growth factor (TGF)-β (R&R Systems), SOD-1  and HO-1  according to the manufacturers’ protocols. In order to compare the distribution of the biomarkers according to the clinical groups, the laboratory parameters were categorized in cytokines Table  and chemThe inferential network was generated from the values of each mediator measured in the plasma samples, in which it was observed that patterns of the concentrations and clinical classification of the subjects were based on the disease outcomes. The systemic levels of each mediator were input in the DimReduction software . As a reIn order to analyse the structure of the biomarker networks, the network density was adopted, which is, in the context of this work, the ratio of the number of edges inferred in the network over total number of possible edges between all pairs of nodes. The density measure is defined as follows: density = L/(N (N-1)/2), in which L is the number of observed edges and N is the total number of the nodes in the network. The density is normalized, ranging between 0 (no edges in the network) and 1 .In the exploratory analysis of the data, frequency tables were constructed and the Chi-square test was applied to evaluate the association between qualitative variables. The quantitative variables were tested for Gaussian distribution within the total sample using D’Agostino and Pearson omnibus normality test. The variables with normal distribution were compared between the groups by one-way ANOVA with linear trend or Bonferroni’s multiple comparisons post-test. Further analysis were based on non-parametric tests only, considering the small number of individuals located in the two groups with severe vivax malaria. In this context, Kruskal Wallis test was used to assess the differences between the clinical groups and Mann-Kendall test was used to estimate liner trends. Only results from the non-parametric analyses are shown. Correlations between parasitaemia and the biomarkers from the networks were assessed using the Spearman rank test. For each analysis, P<0.05 was considered statistically significant. The graphics for the network analysis were customized using the Ingenuity Systems Pathway Analysis software . The statistical analyses were performed using the programs GraphPad Prism 5.0 , STATA 9.0 , and JMP 9.0 .The plasma concentrations of most mediators measured were statistically different among the clinical groups, and many markers displayed linear trend to increase or decrease according to the degree of disease severity, as detailed in the Tables P. vivax infection. In those individuals in which infection did not lead to the onset of symptoms, the number of significant interactions was similar to the group of non-infected people , but the number of associations involving mainly IL-4 and to a lesser extent IL-1β was greatly increased in these individuals, leading to a totally different pattern of connectivity in the network , free iron and biliverdin. Higher HO-1 levels were described in patients with symptomatic vivax malaria and this enzyme may contribute (both very high and low levels) to disease severity [P. vivax malaria [2O2 and the production of more free radicals by Fenton’s reaction [2 and TGF-β from mononuclear cells via SOD-1 [While networking the immune response in lethal cases of severe severity ,33. Lowe malaria , displayreaction . Indeed,reaction  and highia SOD-1 . Thus, SPlasmodium antigens by downregulating TNF, and IFN-γ levels by dampening the release of IL-12p70, thus resulting in an impaired immune response that may lead to lethality [Furthermore, TNF, IFN-γ and IL-10 also had important interactions in individuals who succumbed to malaria infection. IL-10 seems to modulate Th1-type responses to ethality ,36. Howeethality . These fPlasmodium yoelii suggest that IL-4 is also required for the development of CD8+ T lymphocytes [P. vivax and further investigations should address in detail the importance of IL-4 in symptomless malaria. A summary of the results with regard to the complexity of the network connectivity in the context of the natural evolution of vivax malaria is shown in Figure A major observation from the analysis of the groups of patients with mild or severe malaria was that the overall complexity of the networks by means of a number of significant interactions was reduced while compared to what was seen in healthy subjects. Intriguingly, in the group of individuals with asymptomatic malaria, the complexity of the network was restored and several significant interactions were detected, but with a clear central role for IL-4. The potential anti-inflammatory effect of IL-4 in the immune response during malaria is not yet fully understood . IL-4 sephocytes  and for phocytes . Furtherphocytes . The higP. vivax parasitaemia displayed several positive interactions with previously described inflammatory mediators that participate in the immunopathogenesis of malaria, such as TNF, IFN-γ and IL-10 [P. vivax.In the groups of patients with severe malaria, nd IL-10 ,7 in addP. vivax malaria according to disease severity and clinical outcome. This approach also revealed other levels of complexity of the disease, involving intricate associations between unique markers, such as TNF, IFN-γ, IL-4, HO-1 and SOD-1. IL-4, despite being at low systemic levels, seems to be a central mediator with several significant interactions in individuals with asymptomatic malaria. In addition, connectivity involving CXCL9 and IFN-γ was more prevalent in the groups of individuals non-infected or with mild manifestations of the disease while associations involving SOD-1 and HO-1 were more evident in more severe and lethal cases. In severe malaria, parasitaemia seems to positively associate with pro-inflammatory mediators, and the simultaneous negative correlations involving the regulatory cytokines IL-10 and TGF-β argue that there is a deregulated balance of the host immune response that is skewed towards amplification of inflammation. Networking analysis may represent a potential tool to understand the interactions between several mediators in the context of malarial disease severity. The identification of critical factors driving malaria pathogenesis can guide future therapeutic approaches.In summary, the systematic analysis of several mediators of inflammation measured simultaneously was able to characterize the overall pattern of immune response of patients with (SOD)-1: Superoxide dismutase;(HO)-1: Haem oxygenase;FUNASA: Brazilian national foundation of health;PCR: Polymerase chain reaction;AST: Aspartate amino-transferase;ALT: Alanine amino-transaminase;CRP: C-reactive protein;CBA: Cytometric bead array;sTNF-RI: TNF receptor I;(TGF)-β: Transforming growth factor;CO: Carbon monoxideThe authors declare that they have no competing interests.VRRM helped interpreting the data and to write the manuscript together with BBA and MB-N. AQ and FML performed the network analysis. BBA conceptualized the study, coordinated the clinical assessments and performed the laboratory experiments, analysed the data and wrote the first draft of the manuscript. MB-N supervised the clinical study and helped with data interpretation and the writing of the manuscript. All authors have read and approved the final version of the manuscript.


In areas of declining malaria transmission such as in The Gambia, the identification of malaria infected individuals becomes increasingly harder. School surveys may be used to identify foci of malaria transmission in the community.The survey was carried out in May–June 2011, before the beginning of the malaria transmission season. Thirty two schools in the Upper River Region of The Gambia were selected with probability proportional to size; in each school approximately 100 children were randomly chosen for inclusion in the study. Each child had a finger prick blood sample collected for the determination of antimalarial antibodies by ELISA, malaria infection by microscopy and PCR, and for haemoglobin measurement. In addition, a simple questionnaire on socio-demographic variables and the use of insecticide-treated bed nets was completed. The cut-off for positivity for antimalarial antibodies was obtained using finite mixture models. The clustered nature of the data was taken into account in the analyses.19, 11.6% (364/2997) [95%CI = 9.4–14.5] for MSP2, and 20.0% (593/2973) , with males and females evenly distributed. The prevalence of malaria infection as determined by PCR was 13.6% (426/3124) [95% CI = 12.2–16.3] with marked variation between schools , while the seroprevalence was 7.8% (234/2994) [95%CI = 6.4–9.8] for MSP1This survey shows that malaria prevalence and seroprevalence before the transmission season were highly heterogeneous. Malaria is currently endemic in 104 countries and territories around the world, and is a major health problem in many countries in sub-Saharan Africa School surveys are attractive, as both their location and target population are easily accessible, with often high compliance In the past few years, a reduction of the burden of malaria has been described in some African settings In areas of declining transmission such as The Gambia, where the identification of malaria infected individuals becomes increasingly harder, school surveys may represent an economic and easy method to identify communities where malaria transmission remains substantial. We hypothesise that school surveys can be used to detect foci of malaria transmission in the community.P.falciparum infections. In 2007–2008, the incidence of malaria in children in this region was estimated at 0.79 per child year Anopheles gambiae is the main vector, with increased vector density between August-September and an estimated annual entomological inoculate rate of 2.27 The study was conducted in the Upper River Region (URR) in The Gambia, West Africa, where malaria transmission is seasonal and occurs mainly during the “wet” months (July-December). Most of the malaria burden is due to The survey was carried out before the beginning of the transmission season, in May/June 2011, and lasted for two weeks. Children were selected using a two-stage sampling process; first, primary schools in URR were selected with probability proportional to size (according to the size of the school), and then secondly, in each school, about 100 children were randomly chosen. The sampling process was done using the random generator on the Microsoft Excel package. All children attending the selected schools were eligible, regardless of the age, though it was expected that the majority of them would fall in the 5–14 years age group. Children involved in another research project at the time of the survey were excluded.An information sheet, explaining the objectives of the survey and the study procedures, was distributed, prior to the survey, to the children’s parents who were asked to provide a written informed consent on the day of the survey.The study proposal was reviewed and approved by the Scientific Coordinating Committee of the Medical Research Council Unit, The Gambia, and subsequently ethical approval was granted by The Joint Gambia Government/Medical Research Council Ethics committee.P.falciparum antigens was performed and, if positive, the child was treated with AL.Data on demographic characteristics and on reported use of bed nets, including long-lasting Insecticidal nets (LLIN), were collected using a structured questionnaire on the case report form (CRF). This was done by interviewing the parent or carer of each child. Weight and height were measured, the former using a bathroom scale and the latter a stadiometer (Leicester portable Height Measure). For each child, a finger prick blood sample was collected for thick smear, haemoglobin (Hb) measurement, and two blood spots on filter paper (Whatman 3 MM) for later serology and molecular analysis. Filter paper blood spots were air dried and stored in plastic bags containing a desiccant. In case of fever (body temperature ≥37.5°C), a rapid diagnostic test  for 19 (MSP119), MSP2, and apical membrane antigen-1 (AMA1), were detected by indirect ELISA on eluted filter paper samples. Discs (3 mm), two for each sample, were punched and placed at the bottom of 96 well plates. Serum was eluted from dried blood spots (DBS) following overnight (18 h) incubation at room temperature in 150 µL of reconstitution buffer  Tween20/0.05% (w/v) NaN3) with gentle mixing (rotary shaker at 200 revs per min). The reconstituted solution was equivalent to a 1/100 dilution of whole blood, corresponding to 1∶200 dilution of plasma or serum antibodies, if the blood were at 50% haematocrit. For ELISA, antigens were coated on Immunolon HBX plate at a concentration of 5 µg/ml and each reconstituted sample was further diluted fivefold  and analysed in duplicate wells. Standard ELISA procedures for blocking unbound sites, washing of unbound antigens and antibodies, and incubation were followed as per described DBS protocols Antimalarial antibodies against merozoite surface protein-1Molecular diagnosis for malaria parasites and speciation of Plasmodium spp. employed DNA extracted from DBS on the QIA X-tractor robot following the manufacturer’s protocol (Qiagen). Briefly, 3 mm discs punched from each sample were placed at the bottom of 96 well plates and treated with tissue digest buffer at 60°C for 1 hour. DBS digest eluates were then applied on capture plates, washed and DNA eluted with 100 µL of elution buffer. Diagnostic PCR followed amplification of the multi copy Plasmodium ribosomal RNA gene sequences using genus and species specific primers, as previously described Blood slides corresponding to all PCR positive samples were read by microscopy in order to determine the parasite density. In addition, 30 randomly chosen PCR negative samples were included to assess the agreement between microscopy and PCR. Thick films for microscopy were prepared by placing two drops of peripheral blood on the centre of the slide. Films were air dried horizontally, and left to dry overnight and then stained with Giemsa(1∶10 dilution for 10 minutes). Two hundred high power fields (HPF) were read before declaring the slide negative. If positive, the number of parasites against HPF was counted until 200 HPF were screened. The parasite density per µl was determined assuming 500 HPF per µl Haemoglobin (Hb) was measured with the Hemocue 301 machine  following the manufacturer’s instructions.The lowest seroprevalence of antimalarial antibodies in a previous survey carried out in the same area in January/February 2008 was around 20% Data from the CRFs was double-entered into a database . After cleaning, the data was analysed with STATA version 12 . Means and standard deviation were estimated for quantitative variables. The variable’s distribution was examined using histograms, and in case this was skewed, the variable was log transformed for the estimation of the geometric mean. Kernel distribution curves were also used to study the distribution of quantitative variables, particularly the antibody titres. In case of categorical variables, proportions in tables were used to describe the variables. Anthropometric measurements for height and weight were used to estimate the weight-for-age (wfaz), height-for-age (hfaz) and Body Mass Index-BMI for age (bfaz) z scores, by comparing the survey data to WHO growth reference data for school-age children and adolescents The cut-off for positivity for antimalarial antibodies was obtained by modelling their normalized value (log transformed), using finite mixture models - Gaussian distribution with two components Seroprevalence rates were analysed according the following age groups: <8 years, 8–9, 10–11, ≥12 years so there would be a similar number of children in each group. The seroprevalence in each age group was obtained using proportions in contingency tables. Regression models (logistic) were also used with age as a continuous variable. A line of best fit, using the quadratic estimation, was used to obtain the predicted probability of being seropositive for a particular parasite antigen. A similar model was applied to obtain the age-specific PCR parasite prevalence.The distribution of parasite, antimalarial antibodies  and anaemia prevalence were estimated across schools, taking into account the clustered nature of the data. The data was set for survey design taking into account the two stage sampling with the number of schools and the total number of children in the sampling frame. The participants were arranged according to the geographical coordinates of the schools in order to determine spatial heterogeneity. The designed-based F test was used to determine if the difference across schools was significant .The correlation between parasite prevalence and seroprevalence and the correlation between parasite prevalence across schools was examined by scatter plots. The correlation coefficient was estimated with the Spearman non parametric test, and a linear regression line of best fit was used to estimate the relationship between the two variables.Among the 77 primary schools in URR attended by 16,554 children, 32 were selected and from these 3,277 children were enrolled into the survey. The mean age was 10 (SD = 2.7) years [range 4–21], with males and females evenly distributed, and a large majority of the children  used a bed net . At the Plasmodium falciparum while for the remaining samples no other Plasmodium species  was found. Given the low parasite density in the survey samples, this discrepancy could have resulted from the greater sensitivity of the genus-specific PCR. Prevalence of infection varied with age, with the lowest values found in 8–9 years old children , and the highest in the 10–11 year old . Malaria prevalence tended to be higher in febrile  than  afebrile children but the difference was not statistically significant (p = 0.44). No infection was detected by microscopy in 15.3% (65/424) of PCR positive samples, while in 2 out of 29 PCR negative samples (for one sample the blood slide was not found), microscopy identified a malaria infection. The mean parasite density in these 2 discrepant samples was extremely low (32 parasites per µl). Overall, there was 84.7% concordance between PCR and microscopy in the subset of samples screened by both methods.PCR analysis was carried out on 3,124 filter paper blood samples; 426 (13.6%) [95% CI = 12.2–16.3] were found to be positive for malaria infection, though only 278 (8.9%) were confirmed to be 19, to 11.6% (364/2997) [95%CI = 9.4–14.5] for MSP2 and 20% (593/2973)  for MSP1for AMA1 . The proicipants . A similicipants . The ageicipants .19, 1% to 50% for MSP2 and 4% to 76% for AMA1. Differences between schools were statistically significant for MSP2 and AMA1 (p<0.001) but not for MSP119 (p = 0.12), with the highest variability with AMA1. Though seroprevalence against the individuals antigens was correlated, this was stronger between MSP2 and AMA1 (r = 0.74) (p<0.001) than between MSP119 and the other two antigens (r = 0.3).Malaria prevalence varied substantially (range 4–26%) between schools (p<0.001), and was spatially heterogeneous, with low (<10%) and high (>20%) prevalence schools geographically contiguous . SimilarParasite prevalence and seroprevalence for each of the antigens were weakly correlated . Similarly, parasite prevalence and seroprevalence for at least one antigen were weakly correlated .19, MSP2 or AMA1 seroprevalence did not differ according to bed net use (data not shown).Malaria prevalence tended to be lower in children using a bed net  than in the others  (p = 0.10) while the risk of anaemia  (p = 0.17) and severe anaemia  (p = 0.46) was similar between these two groups. The MSP1There was no association between the anthropometric indices  and the risk of malaria infection (data not shown). However, anaemia was significantly associated with hfaz; its prevalence was significantly higher in children with low  than in those with medium  and high  hfaz (p = 0.004).The prevalence of malaria infection in school children in URR was higher than expected as the survey was carried out before the beginning of the malaria transmission season. The large majority of children were asymptomatic and carried infections of low parasite density. Since in The Gambia there is little or no malaria transmission during the dry season, between January and June, the infections detected in this survey were probably acquired during the previous transmission season and then carried for several months. These infections, mostly asymptomatic and therefore not treated, are probably responsible for restarting the transmission at the beginning of the rains, in June-July, when the vector density increases. Although higher than expected, the parasite prevalence in this study is still consistent with the recently reported decline in malaria transmission in The Gambia 19, MSP2 and AMA1 antibodies have been associated with protection against clinical malaria Anti-MSP1Prevalence of malaria infection and of antimalarial antibodies was substantially heterogeneous, showing that this is not a feature limited to the later phases of malaria elimination Although the anthropometric indices wfaz, hfaz and bfaz, are related, each indicates some specific underlying processes or outcomes. In general, a low wfaz, commonly termed underweight, indicating a child is not gaining sufficient weight relative to the age, could be explained by wasting/stunting In conclusion, the prevalence of asymptomatic malaria infection in school children at the end of the dry season was heterogeneous, with some schools having a relatively high prevalence. Such infections have probably been acquired during the previous transmission season and carried for several months. In areas where transmission has decreased substantially over a relatively short period, such as The Gambia, these asymptomatic and low density infections will restart the transmission at the time the vector density increases, after the onset of the rains. Unless targeted by specific interventions, they will continue to defeat any attempt of interrupting malaria transmission. These may be represented by either mass drug administration or mass screening and treatment. However, for the latter a sufficiently sensitive and field-adapted diagnostic test able to detect sub-patent infections would be needed. The choice of the approach to be taken will depend on its feasibility according to the local conditions and the percentage of infected people. Above a given malaria prevalence, mass drug administration may be more appropriate and cost-effective than mass screening and treatment.


Mosquito vectors of malaria in Southeast Asia readily feed outdoors making malaria control through indoor insecticides such as long-lasting insecticidal nets (LLINs) and indoor residual spraying more difficult. Topical insect repellents may be able to protect users from outdoor biting, thereby providing additional protection above the current best practice of LLINs.Plasmodium falciparum and P. vivax cases were actively identified by monthly rapid diagnostic tests. Intention to treat analysis found no effect from the use of repellent on malaria incidence . A higher socio-economic score was found to significantly decrease malaria risk . Women were also found to have a reduced risk of infection . According to protocol analysis which excluded participants using the lotions less than 90% of the time found similar results with no effect from the use of repellent.A double blind, household randomised, placebo-controlled trial of insect repellent to reduce malaria was carried out in southern Lao PDR to determine whether the use of repellent and long-lasting insecticidal nets (LLINs) could reduce malaria more than LLINs alone. A total of 1,597 households, including 7,979 participants, were recruited in June 2009 and April 2010. Equal group allocation, stratified by village, was used to randomise 795 households to a 15% DEET lotion and the remainder were given a placebo lotion. Participants, field staff and data analysts were blinded to the group assignment until data analysis had been completed. All households received new LLINs. Participants were asked to apply their lotion to exposed skin every evening and sleep under the LLINs each night. This randomised controlled trial suggests that topical repellents are not a suitable intervention in addition to LLINs against malaria amongst agricultural populations in southern Lao PDR. These results are also likely to be applicable to much of the Greater Mekong Sub-region.NCT00938379This trial is registered with number  Anopheles gambiae typically feeds at night and indoors, making LLINs a perfect intervention tool against this species. However the major vectors in Southeast Asia and South America commonly feed outdoors At present global malaria prevention efforts are focused on the distribution of long-lasting insecticidal nets (LLINs) and indoor residual spraying N,N-toluamide (DEET) concentrations of 15–20% decrease mosquito biting by over 83% Limonia acidissima) mixed with DEET or thanaka alone. Although a 29% reduction in P. falciparum was observed, the transmission level was too low for this to be statistically significant P. vivax) observed in people using a repellent soap containing 20% DEET and 5% permethrin was non-significant P. falciparum infection was found in Pakistan amongst households using repellent soap compared to those using a placebo, although no effect was found for P. vivax infections p-Menthane-3,8-diol  in Bolivia had an 80% lower incidence of P. vivaxP. falciparum but case numbers were too low to reach significance. A number of common problems have affected the results of these trials. Lower than expected malaria rates have resulted in insufficient sample sizes and non-significant reductions. Compliance is also very important, since repellent requires application every few hours it is easy to forget, lose and even apply in insufficient doses. The inconsistency of these results means that it is not yet established whether the use of insect repellent can reduce malaria infection.Topical repellents have the potential to reduce vector contact in this setting. Field trials in Thailand and Malaysia show di-ethyl-Plasmodium falciparum causes almost 97% of cases and P. vivax the remainder Anopheles dirus which is strongly associated with forests and is frequently found biting outdoors Anopheles minimus and An. maculatus are also important vectors and are similarly found biting outdoors in the early evening The highest malaria incidence rates in Lao PDR are found in Attapeu and Sekong provinces along the southern borders with Cambodia and Vietnam The aim of this trial was to determine whether using a topical 15% DEET repellent, established by landing catches to reduce mosquito biting by 98.9%, would reduce malaria incidence against exophagic vectors amongst rural populations in southern Lao PDR using LLINs.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see P. falciparum parasite rate in Attapeu and Sekong about twice as high as the national average Plasmodium falciparum is found in about 80% of cases, and P. vivax in most of the rest An. dirus, An. minimus and An. maculatusAttapeu and Sekong are the most south-eastern provinces in the Lao PDR, sharing borders with Vietnam and Cambodia . The AnnHouseholds were recruited from 126 villages; 72 in Attapeu Province and 54 in Sekong Province. Participants were primarily rural agricultural workers that often work and sleep overnight away from the village during the wet season. Eligible households needed five volunteers aged 6–60 years. Study households also had to be separated by at least 10 m to prevent diversion of biting from repellent users to placebo users. District health staff obtained written informed consent from all participants or the carers of participants aged under 18 years. Baseline information was collected on age, gender and household wealth indicators.P. falciparum from village surveys P. vivax and a 44% reduction in P. falciparum infection P. falciparum supporting the assumption that cases were not over-dispersed by household. Therefore a coefficient of variation of 0.25 was used. The spatial heterogeneity at village level howerer, underlined the importance of stratifying randomisation by village. Sample sizes were recalculated and a sample size of 633 households per arm was found necessary to detect a 50% reduction in malaria incidence with 95% level of significance at 80% power. This was adjusted to approximately 800 households per arm to account for non-compliance and loss to follow up.A household-cluster randomised design was chosen so that members of the same household were assigned to the same treatment. This avoided accidental mix-up of treatments, but also prevented potential diversion of biting from repellent users to placebo users in close proximity. This effect has been demonstrated over 1 m and could lead to the overestimation of the protection given by repellents Households were randomised to one of the two treatment arms using equal groups allocation which was stratified by village. Heads of households picked treatment codes out of a bowl. A 15% DEET lotion was provided to households in one treatment arm and the remainder received a placebo lotion . Adult participants were provided with three 100 ml bottles of lotion to last one month (approximately 10 ml per day). Children under 12 years were provided with two bottles per month, corresponding to approximately 7 ml per day. This amount was considered sufficient to apply the treatments to arms and legs as demonstrated by trial staff. Participants were instructed to use the lotion every evening. Full USA compliant consumer product information were given verbally in the local language. Any contraindications or side effects were recorded and reported at each monthly follow-up for appropriate action to local District Health departments.Although previous repellent trials have used 20% DEET, a 15% DEET formulation was selected because this was the lowest concentration shown to be effective against mosquito biting in the study area 2, mesh 25 holes/cm2), defined as one net for every 1.5 persons in the household, plus another for use away from home. Participants were instructed to sleep under a net every night, particularly when away from the village. At monthly follow-up visits participants reported how many nights they had slept under the nets in the village and also when sleeping in the forest or rice fields.All study households were provided with sufficient LLINs  every month during active case detection. Follow-up surveys finished in December in both years to ensure testing throughout the wet season and into the following transition/dry season when previous surveys had found high parasite rates P. falciparum and P. vivax. Positive RDTs, paired with a negative RDT matched by age and village, were confirmed by polymerase chain reaction at the London School of Hygiene & Tropical Medicine.The primary outcome was malaria incidence measured by monthly RDTs for P. vivax positive result for each participant. However, in the event no participants presented with multiple P. vivax infections and all positive cases were therefore assumed to be new infections. The low malaria rates also meant that the original analysis using Poisson regression adjusted for repeated measures and household clustered was unnecessarily complicated and a simpler model chosen here.The analytical plan was written prior to data collection. To avoid the risk of including relapse infections, the plan was to only include the first P. falciparum and P. vivax infection were tested by Mantel-Cox regression adjusted for intra-cluster household variation by robust methods. Variables maintaining their association in the multivariable model at p<0.05 were kept in the final model. All analyses were carried out using STATA version 12 .An intention-to-treat analysis was performed on all data, followed by a per protocol analysis which excluded participants who slept under an LLIN and used the lotion in the evenings less than 90% of the month. Subsidiary analyses were also carried out at 75% and 50% compliance cut-offs. A principal component analysis (PCA) using data on education, house construction materials, type of electricity supply, ownership of motorbikes, tractors and televisions and animal ownership was carried out to establish overall socio-economic scores for each household. The PCA scores along with the age, gender, nights slept under an LLIN and nights spent away from the village were considered for inclusion in the regression. All variables except treatment group were first tested by non-parametric univariate methods and those with a significant association with the outcome at p<0.2 were considered for inclusion in the final model. Outcomes of time to first malaria, Lower than expected malaria rates during the first year of the trial led to a revision of the sample size required. Therefore 1300 households were recruited in the second year rather than the previously planned 700. This increase meant the sideline serological work on arbovirus infections had to be dropped due to insufficient funding, and the trial focused solely on malaria infections the major vector borne disease in the area.th June to 4th July 2009 enrolled 300 households to the trial and a further 1,297 households were recruited between 24th April and 18th May 2010. A total of 7,980 participants were initially recruited but 40 (0.5%) were excluded after the baseline survey as they were outside the 6–60 years age limit. Almost half of households  were randomised to the repellent arm and the remaining 802 allocated to the placebo lotion. Baseline household-level socioeconomic scores were derived from principle components analysis on data about the occupation and education of household heads, house construction materials, possessions and animal ownership. Baseline malaria rates were slightly higher in the placebo group, but not significantly so . Men had a slightly higher rate of malaria, but this did not reach significance . Children aged 6–10 years had the highest infection rates although differences were again not significant. Overall, the households in the two treatment groups were very similar despite wide variation between households . Health staff also observed the volume of lotion that was returned and found less than half of participants had used all the lotion . A comparison of full compliance from these two measures showed the false positive rate, self-reported full compliance with non-lotion use was 46.7%, was much higher than the false negative rate, complete lotion use with self-reported non-compliance 28.5% .The most common reason for non-compliance in both repellent and placebo users was forgetting to use the lotion, although allowing for household clustering there was no significant difference between treatment arms . Other reasons for no compliance included disliking the smell , and allergies which were slightly higher in repellent users .2 = 316.1, p<0.001).Compliance with LLIN use was much higher than for lotion use regardless of treatment group and accounting for household clustering . But a relationship was found between compliance with LLIN use and compliance with lotion use, and those participants who did not sleep under their LLIN every night were much less likely to use the lotion every day  and P. vivax infection .Mean time to first malaria episode was 4.0 months (range: 0.9–7.5) in the placebo group and 3.9 months (range: 0.7–7.5) in the repellent group . After aAccording to protocol analyses including only those participants who had both reported and observed compliance with lotion use above 50%, 75% and 90% also found no effect on malaria from the use of repellent . A higheA randomised placebo controlled trial of 15% DEET repellent lotion used by agricultural communities in southern Lao PDR was carried out over two wet seasons in 2009–2010. The trial was powered to detect an intervention effect of 50% on malaria incidence. No significant reduction in malaria incidence was found from the use of the topical repellent.The regression analysis identified socio-economic scores as being the most important risk factors for malaria. This score was derived from data on household possessions and house building materials. An increase of 1 in this score could represent having a tiled roof compared to a thatched one, ownership of 1.5 more motorbikes or 1 more television and corresponded with a 20–45% reduction in the risk of malaria. Although malaria researchers in the Lao PDR have not previously looked for a link between malaria risk and wealth, our results are consistent with the findings of other studies where lower socio-economic status is associated with increased malaria risk There was a sustained drop of over 50% in monthly malaria prevalence from baseline when all households were provided with LLINs. Although this effect could be a result of changes in malaria as there was no control group, the fact that lower prevalences continued to be recorded throughout the wet season when they would have been expected to increase supports the view that LLINs and repeated treatment of malaria patients was effective at reducing overall malaria transmission. However, the baseline rates in 2009 and 2010 were similar suggesting there had been no overall drop in malaria in the area between the two years.An. minimus and An. maculatus, were very rare in the pilot study, so the level of protection by 15% DEET from these species was not tested. Anophelines show less response to repellents than other genera, including Stegomyia and Culex mosquitoes that made up the majority of catches in the local area The use of 15% DEET was chosen based on human landing catches in a village in rural Lao PDR While this gap in the efficacy testing of the intervention should be acknowledged, it is probably not as important as the variation in the dosage of DEET applied to the skin that would result from variation between user applications. A participant applying only 5 ml of the repellent lotion, would achieve the same DEET dosage as 10 ml of a 7.5% DEET lotion. Even two participants applying the same volume of lotion would end up with slightly different dosages depending on their relative body surface area. This variability is a major limitation with topical insect repellents as an intervention tool, but this does not rule out other forms of repellent such as impregnated fabrics that can be better standardised.Our pilot study found a reduction in mosquito biting of over 95% when individuals used the trial repellent compared to the placebo, indicating repellent users might easily be able to distinguish which group they had been assigned to after a short period of use. All households in one treatment arm from a particular village withdrew from the trial after three months because they believed they had the placebo rather than repellent lotion, and unblinding found that they were correct. This meant that although the allocation of treatments and analysis was carried out blind, it is unlikely that all participants remained successfully blinded throughout the trial. An alternative that could prevent the two lotions being directly compared would be to assign separate batch numbers to each household's supply meaning there was no easy way for participants to separate the two treatment groups. This method was not implemented in this trial as distribution of lotion was made each month by village health workers with limited education and it was judged that the system should work as simply as possible to avoid confusion.In order to reduce the possibility of artificially increasing malaria rates in the placebo group through diversion of biting from repellent users, a maximum of 25% of any village were recruited to the trial. However although all members of a household were randomised to the same study arm, it was not feasible to enforce repellent use by all members of a household at all times. Therefore, diversion of mosquitoes from participants using the repellent to participants within the same household not using the repellent could increase malaria risk for those individuals. Individual compliance could have a large impact on this household randomised trial, and the ability to accurately measure this could also have an important impact on the outcome. Participants were not compelled to use repellent during this trial, a demonstration of how much, where and when to apply the lotion was given at the start of the trial. Participants self-reported the number of evenings per month they used the lotion and as a second measure the amount of lotion returned was recorded. Random checks were also carried out in the evenings on a small sample of villages. Self-reported data on compliance is notoriously unreliable, so these data were verified by the volume of lotion used, as reported by interviewers, in order to filter non-compliers out of the ATP analysis. However uncertainty remained over the actual daily use of the lotion, in particular whether all members of a single household had used the lotion supplied equally.A small number of RDT tests were verified by PCR and a single false negative was recorded. This raises the possibility that some infections were missed by the pLDH based tests used in this trial. Sub-microscopic infections have been shown to be of greater importance for malaria transmission in low-transmission settings such as were found in southern Lao PDR Compliance was lower in this trial than in previous repellent trials. Self-reported and observed data gave estimates of full compliance from 48–60%, other trials have reported compliance levels from 68–98% This trial was focused on agricultural populations and the results may not be applicable to one important malaria risk group within the GMS. Forestry workers spend much more time in the forest potentially without access to good healthcare, and live in more temporary accommodation meaning they may be more exposed to vector biting. They often come from elsewhere in the region and their movement between endemic and non-endemic areas has been linked to the spread of antimalarial resistance in the region Southern Lao PDR shares similarities in malaria vectors, environment and the human population with much of the GMS and the results of this trial are likely to be applicable across this region. Topical repellents are not likely to be a suitable intervention for agricultural populations in this region already using LLINs who require long-term protection throughout the wet season.Protocol S1Can the insect repellent N,N-diethyl-m-toluamide (DEET) provide additional protection against clinical malaria over current best practice? A cluster-randomised controlled trial.(DOC)Click here for additional data file.Checklist S1CONSORT 2010 checklist of information to include when reporting a randomised trial.(DOC)Click here for additional data file.Analysis S1Poisson Regression Analysis.(DOC)Click here for additional data file.Analysis S2Principal Components Analysis.(DOC)Click here for additional data file.