The commonly accepted gold standard diagnostic method for detecting malaria is a microscopic reading of Giemsa-stained blood films. However, symptomatic diagnosis remains the basis of therapeutic care for the majority of febrile patients in malaria endemic areas. This study aims to compare the discrepancy in malaria and anaemia burdens between symptomatic diagnosed patients with those diagnosed through the laboratory.Data were collected from Western Kenya during a follow-up study of 887 children with suspected cases of malaria visiting the health facilities. In the laboratory, blood samples were analysed for malaria parasite and haemoglobin levels. Differences in malaria prevalence between symptomatic diagnosis and laboratory diagnosis were analysed by Chi-square test. Bayesian probabilities were used for the approximation of the malaria and anaemia burdens. Regression analysis was applied to: (1) determine the relationships between haemoglobin levels, and malaria parasite density and (2) relate the prevalence of anaemia and the prevalence of malaria.P. falciparum (92.3%), which occurred in higher density in children aged 2‒5 years. Fever, high temperature, sweating, shivering, vomiting and severe headache symptoms were associated with malaria during presumptive diagnosis. After conducting laboratory diagnosis, lower malaria prevalence was reported among the presumptively diagnosed patients. Surprisingly, there were no attempts to detect anaemia in the same cohort. There was a significant negative correlation between Hb levels and parasite density. We also found a positive correlation between the prevalence of anaemia and the prevalence of malaria after laboratory diagnosis indicating possible co-occurrence of malaria and anaemia.The prevalence of malaria and anaemia ranged from 10% to 34%, being highest during the rainy seasons. The predominant malaria parasite was Symptomatic diagnosis of malaria overestimates malaria prevalence, but underestimates the anaemia burden in children. Good clinical practice dictates that a laboratory should confirm the presence of parasites for all suspected cases of malaria. Plasmodium, which lives in the erythrocytes of the host, is the causative agent of malaria in humans. In many areas of SSA with stable malaria, infestations with Plasmodium remain asymptomatic, undetected and untreated [Plasmodium will not only lead to proper treatment of malaria, but also augments the detection of anaemia in the same cohort.Despite global efforts to eradicate malaria, the disease burden is increasing worldwide, with almost two million estimated deaths annually ,2. The sntreated -6. Only ntreated ,8. Anaemntreated . TherefoPlasmodium parasites  using a microscope. It is also possible to identify patients with anaemia based on the haemoglobin counts during laboratory analysis. Electing to use laboratory analysis, however, has its challenges in SSA, since most deaths due to malaria occur at home. Furthermore, functional microscopes are not available in most health facilities frequented by patients [Plasmodium parasites [There are now two commonly used methods of malaria diagnosis, especially in SSA: laboratory diagnostic methods and symptomatic diagnosis. In the laboratory diagnostic methods, the blood samples are analysed for patients . For thearasites ,12. The arasites -16. Symparasites ,18.In SSA, there is a limitation of finance for proper health care and low number of qualified medical personnel; there are also fewer hospitals that can adequately cover the population . SurprisThe study participants were obtained from Nandi and Uasin Gishu County, located in the Western part of the Kenya. Nandi County is situated at latitude 34°48’51.70”E- 35°26’04.52” E and longitude 0°06’50.90”S- 0°33’17.78”N. The Uasin Gishu County is located at latitude 35°07’41.63”-35°30’14. 52”E and longitude 0°00’34.73”N- 0°55’39.76”N. The rainfall ranges from 1200 mm to 2000 mm. The area has a moderate malaria prevalence (13-25%) [This study was conducted between January to December 2010. The epidemiological data on patients were collected from 15 hospitals located in Nandi and Uasin Gishu Counties. They included hospitals in Turbo (n = 1), Kapkangani (n = 1) Kapsabet (2), Ndalat (n = 1), Chepkoilel (n = 1), Kesses (n = 2), Kapseret (n = 2) and Eldoret Town (n = 5). We stationed two research assistants in each of the 15 health facilities to identify the patients attending the facilities for malaria diagnosis and treatment. There were a total of 2022 children initially enrolled in the study based the number of visits to the hospitals. However, we managed to successfully follow 887 who had sought treatment after symptomatic diagnosis during the one year period of the current study. Following the slide examination by the project technician, confirmed malaria cases were referred for treatment with malaria drugs provided by the research team.Each person selected for the current study population was clinically examined by two project physicians. The entry into the study population was made on the basis of observed elevated temperature (>37.6°C). Other symptoms that are associated with malaria that were considered were: fever, profuse sweating, shivers, vomiting, severe headache, dehydration, nausea, diarrhoea, convulsions, jaundice, myalgia, backache, and joint pains. A clinical history of symptoms was taken, including length of illness. The duration, combination, and sequence of symptoms were noted. Other personal data were collected from the respondents using structured questionnaires.All the laboratory-based analyses of the study participants were conducted Moi Teaching and Referral Hospital (Accreditation number NCST/NBC/AC/0612). Microscopic analysis was done to identify the types of malaria parasite and their respective densities. For malaria parasites analyses; blood samples were collected by the standard finger-prick method and thick and thin smears prepared on labelled slides. The smears were allowed to air dry, then fixed in methanol and stained in 4% Giemsa for 30 minutes. The stained smears were afterwards examined using the magnification of × 1,000 (oil immersion) to identify and count the parasite species. For quality control, 10% of the negative samples and 20% of the positive samples were re-examined by at least two microscopist during the study. Results showing large discrepancies  were referred to the third expert for analysis. Parasite density was scored against 300 leukocytes in positive slides. Parasite densities were converted to a number of parasites per microliter of blood, assuming a leukocyte count of 8,000 cells/μL .The WHO definition of anaemia  was usedχ2). Bayesian probabilities were used to estimate the malaria and anaemia burden in the presumptively and laboratory diagnosed malaria cases. The relationships between: haemoglobin levels and malaria parasite density as well as between the prevalence of anaemia and prevalence of laboratory diagnosed malaria in children were assessed by regression analysis. For all statistical tests, p < 0.05 was considered significant.Data was entered and validated using Epi-Info™ version 6.04d software . The datThe study protocol was reviewed and approved by the institutional review boards of the Kenya Medical Research Institute  and the Moi University Ethical Review Committee . All survey participants provided written informed consent. For children, the parents provided written informed consent.Of 2022 children initially enrolled; 887 finished the full follow-up and were included in the follow up survey. Pertinent characteristics of the study subjects who completed the follow up study are presented in Table 2 = 0.7821, p = 0.0001). The predominant species of infection were P. falciparum (92.3%), 4.6% of all infections were P. malariae, 3.1% were mixed infections . Age specific mean density of P. falciparum and prevalence of different parasite densities among study respondents is presented in Figure P. falciparum among study respondents (p = 0.0008). The highest mean density of P. falciparum occurred in children aged 2‒5 years (516.4 parasite/μL blood) followed by those aged 5‒12 years (489.2 parasites/μL blood) while those aged < 6 months had the least parasite density in blood smears (162 parasites/μL blood). In terms of P. falciparum densities, the study area had discernable age-specific variations in malaria density (p = 0.0004)  of the study subjects reported severe headache, 855 individuals (97.5%) reported high temperature, 820 (92.4%) had fever, 819 (92.3%) had sweated, 818 (92.2%) shivered while 798 (90%) reported vomiting as signs of malaria (Table p = 0.0002).Up to 60.4% of the study population did not have anaemia. However, 29.8% of the sample had mild anaemia, 7.0% moderate anaemia, while 2.8% of the sample reported severe anaemia. During our follow up study of the study population who apparently had malaria after symptomatic malaria diagnosis, between 31.2-41.0% were confirmed positive for malaria through microscopic analysis, and 20.7-35.3% were found to be anaemic (Table p = 0.0019) negative correlation between Hb levels and parasite density among the study subjects, which exhibited an exponential decrease in Hb levels with increasing malaria parasite density. Additionally, there was a significant (p = 0.0021) positive linear association between prevalence of anaemia and prevalence malaria in the laboratory diagnosed children based on the age groups of the study subjects . The remaining 41.7% were symptomatically diagnosed and treated for malaria without seeking any intervention from the hospital. Few and mostly inconsistent records are available for laboratory confirmation of the malaria prevalence among the presumptively diagnosed patients without any evidence that diagnoses of anaemia among the patients were ever conducted in patients seeking malaria treatments. Therefore, the aim of this study was to determine the variance between the prevalence of malaria and anaemia in patients following presumptive diagnosis and treatment compared to those seeking laboratory diagnosis and detection of the staining  ensured In Kenya, malaria transmission patterns are classified based on transmission intensity as endemic, and unstable endemic zones and epidemic zones ,26. FromPlasmodium causes severe complications as cerebral malaria, severe anaemia, acute renal failure, hypoglycaemia and pulmonary infection. In the current study, microscopic examination of blood smears was used to confirm P. falciparum as the most prevalent (92.3%) while P. malariae was 3.1%, similar to previous findings [P. falciparium from the other human malaria is the ability to attack erythrocytes of all ages, causing high parasitaemia and enhanced growth and the capability to adhere to vascular endothelium through sequestration [P. falciparum among children aged 2‒5 years, perhaps explain why this age bracket is highly affected by the occurrence of malaria in many countries of SSA. The lowest mean parasite density among children aged < 6 months may be due to higher frequency of insecticide treated mosquito bed nets usage usually provided for the mothers immediately after birth [findings -30. The stration . The higer birth  or may ber birth .P. falciparum parasites. These observations may suggest that the P. falciparum parasites are undetected during symptomatic diagnosis and are causing increased haemolysis of parasitized erythrocytes leading to a higher prevalence of anaemia [During the study, most of the study subjects reported presence of fever, high temperature, sweating, shivering, vomiting and severe headache as a precursor of malaria, despite the obvious sign that these symptoms may also indicate the presence of other diseases. This method is likely to underestimate the occurrence of other diseases in the human populations. In the present study, patients who suspected they were suffering from malaria did not seek medical laboratory confirmation for malaria parasites, and neither did a test for the presence of anaemia. However, overall mild anaemia had been about 30%, moderate anaemia was 7.0%, while severe anaemia was 2.8% indicating that 40% of the patients had anaemia, which would remain undetected through symptomatic diagnosis. We also established that children who were symptomatically diagnosed, higher percentage had anaemia, with the highest differences being observed among children aged between 2‒5 years due to the high density of Plasmodium infection [We confirmed that indeed there was a negative correlation between Hb levels and parasite density among the study subjects, suggesting direct depression or haemolysis of the erythrocytes by nfection . Again, Symptomatic diagnosis is imprecise, but remains the basis of therapeutic care for most of the febrile patients in malaria endemic areas, where laboratory support is often out of reach ,18. HoweThis study provides clinical evidence that symptomatic diagnosis of malaria overestimates prevalence of malaria, but underestimates the prevalence of anaemia in children. Despite primary health care strategies for the reduction of mortality and morbidity in children resulting from malaria, malaria induced anaemia still occur frequently. This perhaps suggests that additional and urgent measures are needed to in combating malaria and its complications of which anaemia forms a major part. Although symptomatic diagnosis and treatments of malaria have been considered reasonable in resource-poor settings with high malaria transmission where laboratory infrastructure is inadequate, the contemporary level of misdiagnosis is unsustainable particularly resulting in overestimation of malaria burden while increasing the incidences of anaemia among the patients. So, sound clinical practice dictates that a laboratory should confirm the presence of parasites for all suspected cases of malaria, which can be achieved through RDTs and/or microscopic analysis.All authors declare that they have no competing interests.JKC conceived and designed the study; MAGN, JKC and WA collected data from the field. MMN, FE provided logistical support and vital equipment during research design and data collection. JKC, EO-O, FA, WA, JK, CK-W, EJK and OO participated in drafting and revising the manuscript. WA also provided most of the research materials. EO-O analysed the data. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/332/prepub


OnSite (Pf/Pan) antigen test, a new RDT introduced by CTK Biotech Inc, USA for malaria diagnosis in a reference setting.Accurate diagnosis of malaria is an essential prerequisite for proper treatment and drug resistance monitoring. Microscopy is considered the gold standard for malaria diagnosis but has limitations. ELISA, PCR, and Real Time PCR are also used to diagnose malaria in reference laboratories, although their application at the field level is currently not feasible. Rapid diagnostic tests (RDTs) however, have been brought into field operation and widely adopted in recent days. This study evaluates OnSite (Pf/Pan) RDT was performed on preserved whole blood samples.Blood samples were collected from febrile patients referred for malaria diagnosis by clinicians. Subjects were included in this study from two different Upazila Health Complexes (UHCs) situated in two malaria endemic districts of Bangladesh. Microscopy and nested PCR were considered the gold standard in this study. Plasmodium infection detected by microscopy and nested PCR. OnSite (Pf/Pan) RDT was 94.2% sensitive  and 99.5% specific  for Plasmodium falciparum diagnosis and 97.3% sensitive  and 98.7% specific  for Plasmodium vivax diagnosis. Sensitivity varied with differential parasite count for both P. falciparum and P. vivax. The highest sensitivity was observed in febrile patients with parasitaemia that ranged from 501–1,000 parasites/μL regardless of the Plasmodium species.In total, 372 febrile subjects were included in this study. Of these subjects, 229 (61.6%) tested positive for OnSite (Pf/Pan) RDT is both sensitive and specific for symptomatic malaria diagnosis in standard laboratory conditions.The new  While the number of malaria cases has declined in recent times, 50% of the world’s population living in 106 countries is still at risk of malaria infection. In 2010, 216 million malaria cases were recorded worldwide. Africa has the highest burden of malaria with 81% of the cases and 91% of the deaths due to malaria globally [Plasmodium falciparum infection in Bangladesh and transmitted by a wide variety of malaria vectors [Bangladesh is a hypo-endemic area for malaria transmission with 13 endemic districts. In 2011, 52,598 malaria cases were reported in Bangladesh [vectors .Accurate diagnosis of malaria is the key factor not only to prevent morbidity and mortality but also to restrict the use of anti-malarial drugs to minimize the spread of drug resistance . DiagnosAlthough clinical diagnosis of malaria is paramount in resource-limited areas, at times it raises doubt due to the overlap of similar symptoms of other tropical diseases and co-infection . FurtherIn 1904, Gustav Giemsa introduced of a novel mixture of methylene blue and eosin stain .P. falciparum infection. Forms of lactate dehydrogenases (pLDH) are used to identify both P. falciparum and Plasmodium vivax infection and a variant can be used to detect all species (Pan-specific). The third target antigen is aldolase, which is Pan-specific for all malaria species. Combining any two of these three antigen detection tests into malaria RDTs allow detection of P. falciparum alone, P. vivax alone, or mixed infection [Different approaches, including ELISA, PCR and Real Time PCR have been utilized to improve malaria diagnosis. Although these methods improve sensitivity and specificity and are applicable in reference settings where laboratory facilities are specialized available, these methods are difficult to implement at field level and can only be used as a standard method to validate other methods . Immunocfection . Researcfection . World Hfection . FollowiOnSite (Pf /Pan) , was evaluated compared to microscopy adjusted with nested PCR as the gold standard.A number of malaria RDTs are available in local markets and are being used for diagnosis throughout Bangladesh, but their clinical proficiency needs to be evaluated. In this study, a newly available RDT, Whole blood samples were obtained from two different sub-districts of the south-eastern part of Bangladesh: Matiranga Upazila in Khagrachhari district and Ramu Upaliza in Cox’s Bazar district. Subjects were enrolled into the study at the Upazila Health Complexes (UHCs). Febrile patients identified by clinical symptoms and referred to laboratory investigations from May 2009 to December 2010 were included in this study. Samples were excluded from the study in the case of mixed infection or differential diagnosis by the standard methods (microscopy and nested PCR).Approximately 5 mL of blood was taken through venipuncture from adult subjects by an expert medical technologist. In the case of children or minor subjects, approximately 3 mL of blood was taken. Thick and thin smear slides were prepared in duplicate using two drops of blood for each sample. The remaining blood was preserved at −20°C in EDTA tube and transported to the icddr,b Dhaka laboratory in cool boxes, maintaining the temperature below 4°C using ice bags.Microscopy was conducted at the field site by experienced microscopists following the standard procedure  in the cOnsite (Pf/Pan) RDT device, produced by CTK Biotech Inc, USA, was evaluated in this study. This device has the ability to detect Pan-specific lactate dehydrogenase (pLDH) and P. falciparum specific HRP-2.P. falciparum positive. Conversely, only LDH positive samples were diagnosed as non- falciparum, exclusively P. vivax in this study. These RDTs were stored between 20-25°C at the icddr,b Dhaka laboratory.All RDTs were performed on six months to two years preserved samples at the Parasitology Laboratory, icddr,b Dhaka. If both LDH and HRP line or only HRP line is found then the experimental sample is considered as DNA extraction was done using QiaAmp blood mini kit from Qiagen, Germany according to the manufacturer’s instructions from preserved whole blood.et al. [A modified version of the nested PCR approach described by Snounou et al.  was usedAll data analysis was conducted in Stata version 11.0 . Sensitivity, specificity, predictive values and accuracy were calculated with their corresponding 95% confidence intervals (95% CI) by McNemar test and exact McNemar test using the ‘diagt’ command.The study was approved by the Research Review Committee and Ethical Review Committee of icddr,b. Informed consent was obtained from all adult subjects, and assent was obtained from the legal guardians in the case of minor subjects before the collection of blood sample. Complete anonymity was maintained in all stages of the study. Good clinical and laboratory practices were followed in all the procedures.After adjusting microscopy with nested PCR, blood samples from 372 febrile patients were included in this evaluation. Of these subjects, 52.8% were male and 47.2% were female and the age of the subjects ranged from 1.5 years to 82 years with a median of 19.4 years.P. falciparum and 31.9% (73/229) by P. vivax  with median value of 19,960 parasites/μL. Only 3.8% (6/156) of samples had less than 100 parasites/μL while 89.1% (139/156) of samples contained 1,000 parasites/μL or more. For P. vivax infection, the minimum count was identical to P. falciparum (16 parasites/μL) whereas the maximum parasite density observed was 25,120 parasites/μL  with a median parasitaemia of 1,950 parasites/μL. The majority of P. vivax samples had a parasitaemia greater than 1,000 parasites/μL .Malaria cases were confirmed by both microscopy and nested PCR. There were 229 positive malaria cases 61.6%). Among these cases, 68.1% (156/229) were infected with .6%. AmonOnsite (Pf/Pan) RDT was 94.2% sensitive  and 99.5% specific  for P. falciparum diagnosis. The sensitivity and specificity for P. vivax diagnosis were 97.3%  and 98.7% , respectively. Sensitivity, specificity, positive predictive value (PPV) and negative predictive values (NPV) are shown in Table P. falciparum (50%) and P. vivax (80%). The maximum sensitivity (100%) was observed in samples where the parasitaemia was between 501–1,000 parasites/μL for both species. It was evident that sensitivity increased with increasing parasitaemia (Table OnSite (Pf/Pan), a newly available RDT for malaria diagnosis, performed well in this study. OnSite (Pf/Pan) RDT was 96.7% sensitive and 98.6% specific with varying performance for P. falciparum and P. vivax detection. OnSite (Pf/Pan) RDT has inherent capability to detect all types of non-falciparum malaria but only P. vivax was considered in this study due to the absence of Plasmodium malariae and Plasmodium ovale samples in the study areas. In the case of P. falciparum, the sensitivity ranged from 50% to 100% depending on the parasitaemia.OnSite (Pf/Pan) failed to achieve this level of sensitivity. However, only six P. falciparum and five P. vivax samples had parasite loads below 100 parasites/μL in this study. Therefore, the low sensitivity observed in low parasitaemia samples may have been driven by the lack of sample size and it cannot be concluded that OnSite (Pf/Pan) does not perform well in low parasitaemia samples.The World Health Organization recommends at least 95% sensitivity of RDTs for low parasite counts [P. vivax, the sensitivity consistently increased as the parasite load increased. This supports the idea of the intra-species conserved nature of pLDH [P. falciparum, the sensitivity did not always increase as the parasite load increased  RDT which might be due to cross reactivity of other infections, such as rheumatoid factor, heteropheline antibodies [P. falciparum samples tested positive in Pan line but HRP2 line did not appear. This might be due to inherent limitation of the device, intra-species variation in the gene encoding the HRP gene or deletion of HRP gene.In the case of of pLDH . For P. d Table . This ocibodies . Histidiibodies , which cOnSite (Pf/Pan) RDT was also incorporated into the WHO RDT evaluation programme in Round 3 and evaluated with good score[P. falciparum and P. vivax, respectively, when parasitaemia were 200 parasites/μL. Furthermore, it had 100% detection rate/score for species in the cases of parasitaemia at 2,000 parasites/μL or more in WHO Panel. This study also reveals an almost similar level of sensitivity especially for higher parasite load, denoting the accuracy of this RDT in identifying symptomatic malaria cases.od score. This RDOnSite (Pf/Pan) performed better than some similar RDTs developed and widely adopted for malaria diagnosis. Parascreen Pf/Pan RDT was evaluated in different studies [P. falciparum detection. For Pan detection, it showed a maximum sensitivity of 82.5% and specificity of 78.2% for symptomatic patients [studies -26 whereP. falciparum and 60.7%-100% for P. vivax malaria while specificity was close to 95% or above [OptiMAL (Pf/Pan) is another similar RDT that has been implemented in both the field and the laboratory and has been evaluated several times -30. Sensr above ,28.P. falciparum diagnosis was observed at 99.4% in Sierra Leone [P. vivax diagnosis, the highest sensitivity was 92.3% in a study in Madagascar [P. falciparum detection sensitivity was lowest [P .vivax detection tests were always very good, above 95% in recent studies.CareStart™ (Pf/Pan) was also evaluated in several experimental conditions ,27,30-35a Leone  while tha Leone  with higagascar  in the s lowest . SpecifiOnSite (Pf/Pan) can be considered an effective diagnostic tool for detecting both P. falciparum and P. vivax malaria. This test has high positive predictive value and negative predictive value which indicates that it is capable of detecting true malaria cases as well as excluding non-malaria cases with overlapping symptoms to reduce treatment burden, which happens to be the major concern since artemisinin-based combination therapy is the last oral option presently used in these areas against P. falciparum.Compared to the performance of various other RDTs with similar characteristics, OnSite (Pf/Pan) RDT, which may not be controlled in field settings. Thus, a slight variant result may be possible in the case of fresh blood samples in ambient field settings.This study used frozen blood samples and well preserved devices for evaluating OnSite (Pf/Pan) RDT, newly manufactured by CTK Biotech Inc, USA, performed satisfactorily in standard experimental conditions. It can be utilized for diagnosis of symptomatic malaria as well as to discriminate falciparum malaria infection from vivax malaria in endemic areas and during outbreaks. However, a field evaluation is required to assess its applicability for routine diagnosis in resource-limited settings where the feasibility of microscopy is limited. Careful monitoring should be maintained at the level of manufacturing and post-marketing surveillance to assure quality in different lots.The authors declare that they have no competing interests.ANM and MSA conceptualized and designed the study, collected and identified samples, analysed the data, drafted the manuscript and made final revisions. ANM, RE. MPP, KM, WAK, RH and MSA did sample analyses and made critical revision of the manuscript. MSH conducted data analysis. ANM, RE and MSA drafted the manuscript. All the authors read the final version of the manuscript and approved.


Plasmodium falciparum and anaemia and their association with cognitive and education outcomes on the Kenyan coast, an area of moderate malaria transmission.Studies have typically investigated health and educational consequences of malaria among school-aged children in areas of high malaria transmission, but few have investigated these issues in moderate transmission settings. This study investigates the patterns of and risks for P. falciparum infection and anaemia and the associations between health status and measures of cognition and educational achievement. Results are presented for 2400 randomly selected children who were enrolled in the 51 intervention schools.As part of a cluster randomised trial, a baseline cross-sectional survey assessed the prevalence of and risk factors for P. falciparum infection and anaemia was 13.0% and 45.5%, respectively. There was marked heterogeneity in the prevalence of P. falciparum infection by school. In multivariable analysis, being male, younger age, not sleeping under a mosquito net and household crowding were adjusted risk factors for P. falciparum infection, whilst P. falciparum infection, being male and indicators of poor nutritional intake were risk factors for anaemia. No association was observed between either P. falciparum or anaemia and performance on tests of sustained attention, cognition, literacy or numeracy.The overall prevalence of P. falciparum is strongly associated with anaemia, but there is no clear association between health status and education. Intervention studies are underway to investigate whether removing the burden of chronic asymptomatic P. falciparum and related anaemia can improve education outcomes.The results indicate that in this moderate malaria transmission setting,  Plasmodium falciparum infection  . Children  found to be RDT-positive are treated with artemether-lumefantrine , an artemisinin-based combination therapy. The current investigation uses baseline cross-sectional data collected between February and March 2010 in the 51 intervention schools that were allocated to receive the intermittent screening and treatment for malaria. No baseline data were collected on P. falciparum infection for the 50 control schools not receiving the malaria intervention owing to ethical considerations about screening for malaria and not providing treatment. Results reported here on P. falciparum infection are based on expert microscopy. The reliability of the RDTs employed for the IST will be evaluated in future analyses.The study design and methods of the intervention trial have been previously detailed  checklist.The surveys were conducted in Kwale and Msambweni districts on the south Kenyan coast, where malaria transmission is seasonal following the two rainy seasons (April-July and September-November) . In 2009A school census of all 101 schools included in the trial was conducted by trained personnel and was used as a sampling frame from which 25 children from class 1 and 30 children from class 5 were randomly selected using random number tables. Fewer children were selected from class 1 because of the extra educational assessments undertaken with these children and the feasibility of conducting the tests in a single day.Finger prick blood samples were obtained from all children to assess haemoglobin concentration (Hb) using a portable haemoglobinometer . Children with severe anaemia (Hb < 80 g/l) were referred to the nearest health facility for iron therapy as per the national guidelines. Height and weight were measured and axillary temperature was digitally recorded. Thick and thin blood smears, prepared for malaria microscopy, were stained with 2% Giemsa for 30 minutes Parasite densities were determined from thick blood smears by counting the number of asexual parasites per 200 white blood cells, assuming a white blood cell count of 8000/μl. A smear was considered negative after reviewing 100 high-powered fields. Thin blood smears were reviewed for species identification. Two independent microscopists read the slides, with a third microscopist resolving discrepancies.These have been detailed elsewhere  receiver . Elevation of schools was recorded and used as a geographical marker of distance from the coast.Data were double-entered using customized data entry screens in Microsoft Access . Consistency checks were performed and all discrepancies and queries were verified against the original paper forms. Health data were linked by school location and mapped using ArcGIS 9.3.1 .Plasmodium falciparum infection was defined on the basis of duplicate slide readings. Anaemia was defined using age- and sex-corrected WHO thresholds , based on the entire trial population. The principal component analysis (PCA) approach proposed by Filmer and Pritchett  and scores for spelling, number identification, numeracy, comprehension, code transmission and pencil-tapping tasks (continuous outcomes). Univariable associations between the health-related outcomes and risk factors were assessed using multilevel logistic regression, accounting for school-level clustering (a priori risk factors and retained in multivariable models. A priori interactions between net use with age and sex and between school feeding and elevation (distance from the coast) were investigated.Analyses were performed using STATA version 11.0 . The outcomes of interest examined were prevalence of ustering . VariablP. falciparum infection and with anaemia, additionally accounting for age and sex as a priori risk factors. For the pencil-tap assessment of sustained attention in class 1 children, the analysis was split into two because of the significant proportion of children who were disengaged and scored zero. The proportion of children engaged in the task was examined by different variables using multilevel logistic regression accounting for school-level clustering. For each of the spelling assessments in classes 1 (score 0–20) and 5 (score 0–43); the numeracy in classes 1 (score 0–20) and 5 (score 0–38); the Ravens assessment in class 1 (score 0–20); the sentence comprehension in class 5 (score 0–40); the code transmission assessment of sustained attention in class 5 children (score 0–20); and the analysis of children who were engaged in the pencil-tap task (score 1–20), the effect of explanatory variables was quantified by mean differences in test performance using linear regression. Bootstrapping was used to account for non-normality of the scores, whereby schools were resampled to account for school-level clustering (P < 0.1) variables identified in univariable analysis were considered for the multivariable model which employed stepwise elimination.Analysis of the cognitive and education outcomes was stratified by class and focused on associations with ustering . Bias-coThe study was approved by the Kenya Medical Research Institute and National Ethics Review Committee (SSC No. 1543), the London School of Hygiene and Tropical Medicine Ethics Committee (5503), and the Harvard University Committee on the Use of Human Subjects in Research (F17578-101). Meetings were held in participating schools to explain the nature and purpose of the trial to parents or legal guardians, and written informed consent was obtained.Of the 3850 children randomly selected to participate in the study, 2400 (1160 in class 1 and 1240 in class 5) were included in the analysis , with a P. falciparum was 13.0% ; only 11 infected children had documented fever. Infection prevalence varied markedly by school, ranging from 0 to 75.0%  for children with high intensity infection vs. those with no infection, and for children who were stunted. Significantly lower odds of anaemia were associated with children who were female, and with being aged 10–12 years old vs. 5–9 years old. The effect of a school feeding programme on anaemia was modified by elevation of school (distance from coast) and thus is presented by stratum-specific odds ratios. School feeding was associated with lower odds of anaemia in schools closest to the coast , with no evidence of an association for schools positioned further from the coast.In univariable analysis, anaemia was significantly associated with male sex, younger age, P. falciparum infection, irrespective of parasite density, nor anaemia was found to be associated with any cognitive or educational outcome. Interestingly, poor engagement in the attention task for class 1 was associated with eating breakfast and attending a school with school feeding, and better spelling performance in class 5 was found among children who were classified as thin on the basis of BMI.Results from the univariable analysis of the associations between cognition and educational achievement and health and other factors are presented in online-only A number of household factors were also associated with the scores. Higher household socio-economic status was associated with higher scores in the comprehension task in class 5 and the spelling in both classes. Lower scores were associated with living in a house with a high number of children for the class 5 comprehension task. Higher parental education levels were associated with higher scores in the class 5 comprehension and class 1 numeracy tasks. School environment and educational administrative zones were found to be associated with several of the tasks, with lower literacy and numeracy scores associated with children learning in classrooms without desks, and significantly higher spelling and numeracy scores in class 5 as well as attention and cognitive scores in class 1 found in children schooling in coastal Diani zone.P. falciparum, reaching 75% in some schools. There was also evidence that infection is strongly associated with anaemia, with the odds higher with increasing density of infection. The results also show potentially important variation in the malaria burden between the sexes and age groups and by school. The scale of observed health problems strongly supports the need for school health programmes aimed at reducing the health burden of malaria in school children. Despite this health burden, the analysis of educational data suggested no association between current health status and measurements of sustained attention and educational achievement.The evidence presented here shows that in this moderate malaria transmission setting, there is marked variation in the prevalence of P. falciparum infection is likely to reflect a complexity of factors that influence vector distribution and density as well as vector–human contact and human infection (Anopheles gambiae s.l. and An. funestus, which in our study area have been shown to exhibit strong spatial and temporal heterogeneity related, in part, to variation in rainfall (P. falciparum infection, but is also likely to be due to differences in food availability and the prevalence of helminth infection, important aetiological factors for anaemia.The geographical heterogeneity observed in the prevalence of nfection . The priP. falciparum infection and anaemia has been observed in other school-aged populations in East Africa (P. falciparum infection on haemoglobin levels is attributed to increased red blood cell destruction and decreased red blood cell production (P. falciparum is bound to further increase the stress on the haemoglobin status in individuals who are already anaemic (The protective effect of sleeping under a mosquito net is consistent with previous cross-sectional findings ( anaemic .P. falciparum can persist for over three months and as children may be constantly re-infected, it is probable that infection has a cumulative effect on cognitive function over an extended period of time. Thus, the single time point of our cross-sectional design may not sufficiently capture the effects of recurrent, chronic infection over an extended period (The lack of observed association between health status and sustained attention and education may not necessarily reflect an absence of effect of malaria on education. First, asymptomatic d period . Second,The association found in both classes between higher literacy and attention scores and indicators of SES is supported by previous findings where SES has been found to be strongly related to psychometric and education test scores in school children (P. falciparum infection, underscoring the need for geographical targeting of malaria interventions. The observed strong association between infection and anaemia provides evidence of the, presumably cumulative, negative effects of asymptomatic P. falciparum infection on the haemoglobin status of school children. The ongoing trial of IST will provide an indication of how much of this effect can be reversed and whether malaria control can also improve the cognitive and educational performance of children in the current setting. The trial will also help understand how much of the observed poor performance is attributed to health factors and how much is attributed to teaching quality and household factors.In conclusion, we have found a strong geographical variation in the prevalence of


Plasmodium falciparum infections at delivery are themselves more susceptible to malaria attacks or to infection in early life.Infants of mothers with placental To assess the impact of either the timing or the number of pregnancy-associated malaria (PAM) infections on the incidence of parasitemia or malaria attacks in infancy, we followed 218 mothers through pregnancy (monthly visits) up to delivery and their infants from birth to 12 months of age (fortnightly visits), collecting detailed clinical and parasitological data. After adjustment on location, mother’s age, birth season, bed net use, and placental malaria, infants born to a mother with PAM during the third trimester of pregnancy had a significantly increased risk of infection  or of malaria attack . PAM during the first and second trimesters had no such impact. Similarly significant results were found for the effect of the overall number of PAM episodes on the time to first parasitemia and first malaria attack  respectively.This study highlights the importance of protecting newborns by preventing repeated episodes of PAM in their mothers. Plasmodium falciparum in endemic areas ) or malaria attacks (AHR 3.2 [1.6–6.4]) in infancy . PlacentThe effect of the number of PAM episodes on first parasitemia and malaria attack was also significant with a 2.3-  and a 2.2-  fold increase in the risk for these two outcomes for mothers who experienced a malaria infection twice or more.Previous studies demonstrated that infants born to mothers with placental infection at delivery were at increased risk of parasitemia or malaria attacks in the first year of life, but in none of the published studies were the women followed during pregnancy, and hence the analyses could not take account of maternal infection histories prior to delivery. The STOPPAM project, of which the study described here was a part, included longitudinal follow-up as an integral component. Importantly, almost all participating women (97.4%) had an early ultrasound examination, the reference method, resulting in very accurate determination of gestational ages ,26. Coupin utero exposure to soluble parasite-derived antigens, leading to altered immune responses – frequently characterized as ‘tolerance’ – to these antigens that are detectable at birth but also later in life [in utero or early in infancy following exposure during pregnancy to environmental immunotoxicants [From a biological point of view, the increased susceptibility to malaria of the infant is thought to result from  in life ,27-30. Boxicants , urban aoxicants , infectioxicants , or undeoxicants ,35. The P. falciparum during the third trimester of pregnancy, characterized by poor birth outcomes such as low birth weight or maternal anemia at delivery [P. falciparum infections detected long after the last dose of IPTp was given [Our results indicate a major role of maternal parasitemia present late in pregnancy, emphasized by the association with a more than 4-fold increased risk of the child suffering a malaria attack during the first year of life. This is consistent with our own previous reports of the deleterious effects of infection with delivery ,36. Stridelivery . In areaas given . The firas given ,36. Howeas given . In marked contrast with previous studies, and although parasitemia or malaria attacks were more common in infants born from infected placentas, the association did not reach significance. Until now, placental malaria has been considered a surrogate for infections arising earlier, and a 10-fold increased risk of placental infection in women parasitized after 7 months of pregnancy has been reported . The recThis study is one of the few where clinical symptoms were systematically recorded during the children’s follow-up. Probably reflecting the immaturity of infants’ immune responses, nearly 80% of parasitemias were associated with fever. Interestingly, we found very similar results using both variables as main endpoints , with increased risks between 4 and 5-fold for PAM during the third trimester, and between 2 and 3-fold for a number of PAM episodes ≥2. This finding is partly explained by the high proportion of symptomatic children, but also confirms the importance of the number of gestational infections in terms of consequences for the baby.This study was primarily designed for immunological investigations of infants’ first infections, rather than for epidemiological purposes, and thus has limitations. First, the mother-infant pairs selected for follow-up may not be fully representative of the overall population of STOPPAM women and their offspring. Although most variables did not differ, the rate of placental infection was higher in the 218 pairs than in the remaining 601 (19.7% vs 7.5%), which may be explained by a higher rate of recruitment in the second and third trimesters of the year, at STOPPAM’s mid-term, when malaria transmission was highest. Nevertheless, as our main purpose was to study the relationship between PAM and parasitemia in infancy, representativeness of the sample is not obligatory, and a potential selection bias towards more heavily infected mothers, although unlikely, should not have modified the associations we observed.Second, more frequent parasitemia during pregnancy suggests greater exposure to transmission, with infants sharing the same environment equally exposed, giving a possible alternative explanation to the associations we found. This emphasizes the importance of carefully assessing malaria exposure in such studies . No entoThird, RDTs were used to diagnose parasitemias during pregnancy. The sensitivity and specificity of this method is roughly comparable to thick smear examination. By identifying circulating plasmodial antigens, RDTs may detect parasites sequestered in the placenta, but more sensitive PCR methods detect submicroscopic parasitemias. RDTs do not detect such small parasite loads that may also play a role in the susceptibility of infants to malaria.In conclusion, the present study showed that both maternal parasitemias during the third trimester, and the number of malaria infections during pregnancy were associated with an increased risk of infections and malaria attacks during the first year of life. This highlights the importance of improving malaria prevention strategies during pregnancy to optimally protect the infant. In this respect, the recent recommendations of the WHO to give SP-IPTp at each scheduled antenatal visit with an advised number of four visits, will probably facilitate the systematic use of a third dose of IPTp. This should help to decrease infants’ susceptibility to malaria by shifting the last IPTp dose into the latter part of pregnancy. The development of a PAM-specific vaccine, obviating the need for IPTp, should similarly lower the burden of malaria in early life.


Knowledge of appropriate reference intervals is critical not only to provide optimal clinical care, but also to enrol populations in medical research. The aim of this study was to generate normal ranges of laboratory values for haemoglobin among healthy Ethiopian adults and children and to determine if anaemia is a possible indicator of malaria in women and children in this area of Ethiopia.This study was carried out from January 2008 to May 2010. The reference sample population with malaria-negative consisted of 454 individuals, divided women, men and children. The malaria-infected sample population consisted of 117 individuals. The reference ranges were based on the guidelines from the Clinical and Laboratory Standards Institute. Haemoglobin concentration was determined by Hemo-Control EKF Diagnostic Analyser on whole blood. Testing for malaria-positive and negative infection was done by microscopy and by PCR.The lower limits for adult haemoglobin range obtained from this population were slightly higher than those derived from other African populations, but were equal to those established by other studies in Ethiopia and the World Health Organization (WHO). Regarding children, the minimum values were lower than those obtained from different African populations and those established by WHO. The malaria-negative group had anaemia in 35.6% of cases and in the malaria-positive group in 70.9%. There was a stronger, statistically significant association between anaemia and malaria-positive samples than between anaemia and malaria-negative samples in women and both groups of children.The results from this study are a contribution in the definition of the haemoglobin parameters in African populations, which could be taken as standards for interpretation of laboratory results. The haemoglobin indices in adults from Gambo tended to be higher than other African populations and in children were lower than other studies in Africa. The results also suggest that anaemia is not useful as a supportive diagnostic criterion to monitor and evaluate malaria in women and children from Ethiopia, because a 29.1% of malaria cases will be not detected, because of not having anaemia. Locally determined reference values are of immense clinical importance when taking into account intra- and inter-population variations, so that the correct clinical interpretation of laboratory results can be made. The refReference values for various blood parameters can be derived from healthy volunteers, from subjects attending health screening clinics or having annual medical examinations. Such paClinical diagnosis is widely used for the diagnosis of malaria, especially in resource-poor countries. Haematological changes in malaria, such as anaemia, thrombocytopaenia and leucocytosis or leucopaenia are well recognized. The WorAnaemia is an important contributor to malaria-attributable deaths in hospitals, with severe anaemia accounting for between 17 and 54% of malaria-attributed deaths in children under five years of age-20. TherThe aim of this study was to generate normal ranges of laboratory values for haemoglobin in Gambo village. To obtain the results of this study were assessed the haemoglobin parameter in healthy adults and children, and contrasted with that previously reported from African and Caucasian populations. In addition, this study suggests anaemia as a possible indicator of malaria in women and children in this area of Ethiopia as recommended by WHO and RBM.All haemoglobin values were taken at the Gambo General Rural Hospital, from patients attending health screening. Gambo is 2,200 m above sea level and lies 245 km southeast of Addis Ababa. The prevalence of malaria for the country is 8%. The refPatients were enrolled in this study from January 2008 to May 2010 at Gambo General Rural Hospital. Oral informed consent in local languages (Oromo and Amharic) was obtained. In the case of children, their parents or a head of household permitted the inclusion in the study. Clinical histories and samples were included in the study by code and the data were analysed anonymously. The research protocol had been reviewed and approved by the Ethical Committee of Gambo General Rural Hospital.Haemoglobin concentration (g/dL) was determined by a Hemo-Control EKF Diagnostic analiser on whole blood. This analysis was performed within five min of collection. Testing for malaria-positive and negative infection was by microscopy and polymerase chain reaction (PCR). Haemoglobin concentration and microscopy was performed at the laboratory of the above-mentioned hospital and PCR were performed at the University Institute of Tropical Diseases and Public Health of the Canary Islands (IUETSPC). All experiments were performed according to good laboratory practice using appropriate standard operating procedures by well-trained laboratory staff.Plasmodium species was performed using thick and thin blood smears[Plasmodium species were confirmed by microscopy and nested PCR targeting the small subunit ribosomal RNA gene[Microscopy for detection of d smears, with twRNA gene.Statistical analysis was based on the guidelines of the Clinical and Laboratory Standards Institute . Median and 95% reference ranges were determined for haemoglobin parameters and analysed by age and gender for adults. All calculations for determining reference ranges were based on the guidelines found in CLSI, document C28-A2. The norP value) of <0.05 was considered significant. All statistical calculations were performed with SPSS19 statistical and Resampling Stats for Microsoft Excel 2007 software.An error probability  and nine years old (six to 12 years of age).vs 13.5 g/dL p < 0.05). Box and whisker plots for haemoglobin values by gender and age are shown in Figure All haematological values were normally distributed. The analysis of haemoglobin values of these patients showed a mean, median and 95% reference range values stratified by age and gender, which are shown in Table The means and 95th reference intervals for the Gambo General Rural Hospital population evaluated in the present study were compared with parameters obtained from other African populations and with parameters set by WHO. In general, the lower limits for adult haemoglobin range obtained from this population were slightly higher than those derived from Kenya, UgandaPlasmodium vivax, 40.2% (n = 47) were Plasmodium falciparum and 8.5% (n = 10) were mixed .In the 117 malaria-positive samples, asexual malaria parasites were observed in the blood smears. The mean of haemoglobin values for the malaria-positive individuals group was determined and stratified by sex and age Table . Of the P < 0.05) lower for infected groups than for the uninfected groups among women, men and children.Haemoglobin values of the malaria-positive individuals were compared with those of the sex and age-specific malaria-negative groups using Mann-Whitney U test. For the different groups, the mean values of haemoglobin were significantly . In men, the haemoglobin value mean in malaria produced by P. falciparum, or P. vivax was not calculated due to the low number of malaria-positive samples.If the malaria-positive group with anaemia is analysed, it observed in women that 45% infected by Clinical laboratory values provide important data to help assess the health of an individual. In the absence of locally derived reference values for African populations, clinicians and researchers have had to use reference values from Caucasian populations. Differences in haematological values among distinct populations have been known for a long time and are discussed by several authors. Previous studies have shown that such values vary with age, ethnic origin, sociodemographic characteristics, genetic differences and environmental context,9,12,29.WHO has designated haemoglobin, among other values, as essential to be measured by laboratory services in sub-Saharan Africa. This stThe haemoglobin indices in women and men from Gambo tended to be similar to Caucasian population intervals or higher than other African populations. This finding is consistent with previous studies in healthy and HIV-uninfected populations in other Ethiopian regions,32. ThesHowever, haemoglobin values for children between one and five years of age 8.6-9.6 g/dL) and children between six and 12 years of age (9.7-10.8 g/dL) were lower than those reported from other studies in Africa and among Caucasian populations . The development of this reference range may provide guidelines to be used by local health practitioners in patient management within this region and for the design, performance and evaluation of clinical research. The findings obtained in this study were comparable to those of other studies within Africa,14,26-28Finally, WHO defines anaemia as a haemoglobin concentration of <12.0 g/dL, <13.0 g/dL in females and males, and 11.0 g/dL and 11.5 g/dL in children between one and five years old and those between six and 12, respectively. AnaemiaThe results from this study are a contribution in the definition of haemoglobin parameters in African populations, which could be taken as standard for interpretation of laboratory results. The haemoglobin indices in adults from Gambo tended to be higher than in other African populations and in children were lower than other studies in Africa.The results also suggest that anaemia is not useful as a supportive diagnostic criterion to monitor and evaluate malaria in women and children from Ethiopia, because 29.1% of malaria cases will be not detected, because of not having anaemia.The authors declare that they have no competing interests.MASM carried out the sample collection in Gambo (Ethiopia), the microscopy and molecular study, the analysis and interpretation of data and prepared the manuscript. MAQ, RNAL and JLM helped with the statistical analysis and interpretation. PB and AB helped with the performance of molecular studies. FR and GT helped with the collection of samples and analysed haemoglobin values. BV helped to draft the manuscript and EMC coordinated and funded the study and drafted the manuscript. All authors read and approved the final manuscript.


Recently, there has been mounting interest in scaling-up vector control against malaria in Africa. It needs to be determined if indoor residual spraying (IRS with DDT) will provide significant marginal protection against malaria over current best practice of long-lasting insecticidal nets (LLINs) and prompt treatment in a controlled trial, given that DDT is currently the most persistent insecticide for IRS.Anopheles gambiae species and sporozoite infection. Study children will be surveyed at the end of each transmission season to estimate the prevalence of Plasmodium falciparum infection and the prevalence of anaemia.A 2 armed cluster-randomised controlled trial will be conducted to assess whether DDT IRS and LLINs combined provide better protection against clinical malaria in children than LLINs alone in rural Gambia. Each cluster will be a village, or a group of small adjacent villages; all clusters will receive LLINs and half will receive IRS in addition. Study children, aged 6 months to 13 years, will be enrolled from all clusters and followed for clinical malaria using passive case detection to estimate malaria incidence for 2 malaria transmission seasons in 2010 and 2011. This will be the primary endpoint. Exposure to malaria parasites will be assessed using light and exit traps followed by detection of Practical issues concerning intervention implementation, as well as the potential benefits and risks of the study, are discussed.ISRCTN01738840 - Spraying And Nets Towards malaria Elimination (SANTE) In 20 Africa' . A recenIn contrast there is much stronger evidence that ITNs, and by implication LLINs, are protective with a Cochrane Review concluding that in areas of stable malaria they reduce the incidence of uncomplicated malaria in children by 50% compared to no nets . There iSpecifically it is key to determine if IRS with DDT will provide marginal protection against malaria over current best practice of LLINs and prompt treatment in a controlled trial, given that DDT is currently the most persistent insecticide for IRS . Since L• Primary objective: To assess whether IRS with DDT plus LLINs provide added protection against clinical malaria in children compared with LLINs alone.• Secondary objective: To assess whether IRS with DDT plus LLINs provide added protection against anaemia and/or parasite prevalence in children compared with LLINs alone.• Primary objective: To assess whether IRS with DDT plus LLINs reduces vector density inside houses when compared with LLIN alone.2 and is bisected by the river into the north and south banks. The population of the region was 182,586 in 2003, the most recent census, with the majority living in small rural villages in houses made with mud or cement walls and thatched or metal roofs. The study's field station is based at Basse Santa Su town  which is the only sizeable urban area.The study is situated in the far east of The Gambia, in URR, extending approximately 240 to 320 km inland from the mouth of the Gambia River on the Atlantic coast, an area of open Sudan savanna. The climate consists of a single rainy season from May to October followed by a long dry season. This defines the highly seasonal malaria transmission with most malaria episodes experienced during or immediately following the rainy season. This region covers an area of 1995 kmVillages or groups of neighbouring villages, will be selected for the study that have > 110 children aged 6 months -13 years and that are ≥2 km from another study village to reduce likelihood of movement of mosquitoes from one cluster to the next. The study cohort of children, aged 6 months - 13 years old in June in Year 1, who are village residents, will be enumerated and an average of 110/cluster will be selected randomly and invited to participate in the clinical surveys and passive case detection (PCD). In order for the results from this study to be as generalizable as possible, no distinctions will be made regarding gender, ethnic group, medical condition or physical health. Children will be included providing their carers/parents give witnessed informed written consent and, in the case of older children, assent is provided. Assurance will also be sought from carers/parents that they expect that the child will remain resident in their village during the transmission season and will not be sent away for example for live stock herding or schooling. Subjects and households are free to withdraw from participating in the study at any time without giving a reason. If consent is not provided then replacement children will be randomly selected from those remaining. In year 2, overage children will leave the study and infants aged 6 to 18 months will be recruited; any other children who have left the study, for whatever reason, will be replaced.Study subjects will be enrolled in June 2010 in Year 1. Enumeration, recruitment and survey of study subjects and application of the interventions will take place over 2 months, before the start of the transmission season. As is customary in The Gambia, sensitisation will start by discussions with community elders and then the whole study community in order to explain the nature of the study and what will be required during the interventions and investigations. We will seek verbal consent before IRS and verification of positioning of mosquito traps, and written informed consent from the parents or caregivers of all enrolled study children. During these procedures it will be made clear that people will be able to leave the study at any time.Informed consent will be sought at the village level after sensitization meetings attended by village community leaders and health staff (community level information sheet attached). The key attendees names and roles will be documented for each village. Witnessed verbal consent will be sought from each household head, or representative, before IRS and before population, net and house surveys.A cohort of 8,000 children will be enrolled from all the clusters to assess the impact of the intervention on malaria. In approximately 50% of clusters, equally divided between the two study arms, 6 rooms will be selected, each with a single male sleeper, to monitor the impact of the intervention on the density of malaria vectors and their infection rate with malaria. These will be randomly selected from households that have consented to join the study, stratified by housing types in the cluster. Amongst the selected households, potential rooms will be randomly selected by numbering, the numbers being placed in a hat and withdrawn blind. If the room owner does not consent or withdraws from the study or moves away, the process will be repeated.A cluster-randomized controlled study design will be used, as IRS is a community-level intervention and the village cluster as a suitable unit for randomization. All households in all the clusters will be offered LLIN at no cost to the recipients to enable all residents to benefit from this intervention. The clusters will then be randomized into two equal groups; all households in the villages in one group will be offered IRS and those in the other group will not. It is not possible to conduct the IRS in a blinded manner since a placebo would quickly be identified by the community as non-protective. To assess whether DDT IRS and LLINs combined provide better protection against clinical malaria in children than LLINs alone, 75- 125 children aged from 6 months to 13 years old will be sampled according to cluster size enrolled into a study cohort. These children will be followed during the malaria transmission season in 2010 and 2011. Clinical malaria will be recorded by study staff using PCD in close collaboration with government health workers both at the village and health facility levels. Parasites will be detected by a rapid diagnostic test  and treatment will follow government treatment guidelines.Plasmodium falciparum parasitaemia cut-off giving optimum sensitivity and specificity for malaria cases in this setting [The study cohort will be surveyed for malaria indices  at the end of both transmission seasons to generate data for the secondary endpoints and also at baseline before the year 1 transmission season. Baseline data will be used to compare the 2 groups before the interventions and end of season survey data will be used to attempt to define a  setting .Anopheles gambiae species, parity status and sporozoite infection. A schematic representation of the trial is shown in Figure Exposure to malaria vector mosquitoes and parasites indoors will be assessed using CDC light traps and window traps every 4 weeks from June to December and every 8 weeks from January to May in at least 6 randomly selected households in 16 village clusters in each arm of the study. This will be followed by detection of An equal number of clusters will be randomized to receive IRS, in addition to the LLINs provided to all enrolled households, and in these all households will be treated. In cluster-randomised controlled trials it is particularly important to minimize imbalance for factors known to be highly correlated with the disease outcome; in this case age and access to health facilities and location. At the village level we will stratify by presence of a primary health care centre (PHC) and geographical location by dividing the total area into four with two of equal area on each bank of the river. Stratification for PHCs will also ensure that the larger villages are evenly distributed in the experimental groups. In addition, balanced randomization will be used to ensure that children of similar ages are enrolled at the village level.Stratified randomisation of villages to the study groups, as outlined above, will reduce the likelihood of chance imbalances between study arms but as only relatively small number of units can be randomized in such a cluster design, both groups cannot be assumed similar for all factors. Baseline data on spleen rates, parasite prevalence and anaemia will be collected on a representative sample of enrolled children in each village at the beginning of the main transmission season in 2010 to assess imbalances in malaria at village level.Observer bias will be reduced where feasible. Blood films will be read by microscopists blinded to the identity and intervention status of the subjects. Mosquito collector bias will be reduced by using standard light traps and exit traps, which do not rely on the ability of the fieldworker to collect specimens. Trap catches will be examined by a different person to the trap collector and they will be blinded to the trap location. The processes of the interventions, both for LLINs and IRS, will be closely monitored not only for quality but also to document any bias between the villages.2), in May/June, at the start of the main malaria transmission season, in 2010 and 2011. DDT-WP 75, contains 500g of DDT per 670g of product and is packaged in 670g/sachets for making up to 10L with water. The sachets are clearly labelled following WHOPES guidelines (WHO/CDS/WHOPES/2003.7) and are delivered 25/heavy duty carton.In study clusters randomized to IRS, indoor residual spraying will use a wettable powder of DDT (DDT- WP 75) manufactured to WHO specifications and supplied by Hindustan Insecticide Ltd., New Delhi, India. Indoor surfaces will receive DDT  and in accordance with the Gambian National Environmental Agency's regulations. Thus the study DDT will be stored in a similar manner to DDT being used currently by the NMCP. The cartons containing individual sachets will be clearly marked with the study name (SANTE) and stored in a locked designated area in the Central Medical Stores until the week before the IRS campaign, each year, when they will be moved to a locked designated area at the Regional Store in Basse. While in the Central and Regional Stores the DDT will be under the control of the NMCP Study Investigators, who will supervise the interventions. The product will be supplied with an expiry date 2-years post-manufacture which will extend beyond June 2011.During the week before spraying, the sensitization team will inform villages that their village has been selected for IRS, the purpose of spraying, what is required of householders and the excepted IRS date. On the day of IRS, trained spray personnel will inform householders of the spraying schedule and repeat the purpose of spraying and what is required of householders. They will then allow the household time to prepare and vacate the house. Occupants must leave houses before spraying and any rooms occupied by sick people who cannot be moved will not be sprayed. The householder should remove all household items, including water, food, cooking utensils and toys from the house. When possible furniture should be moved outside; if this is not possible, it should be moved to the middle of the room to allow easy access for spraying walls and covered. All wall hangings should be removed. When water-jars cannot be removed they should be covered. Pets and domestic animals should be tethered away from the house. Rooms will not be sprayed if people or animals are present, if household items are not correctly removed or positioned or if the walls are covered with gloss paint. Thatch ceilings also will be sprayed but not tin or hardboard ones. Data will be collected on interior wall surfaces at baseline. Householders will be allowed back into their houses after spraying and will be asked to mop any excess solution from the floors and wash the floors with water before they allow children back into the house. They are requested not to wash, paint or re-plaster sprayed walls at least until the end of the malaria transmission season.http://www.who.int/whopes/Long_lasting_insecticidal_nets_Aug09.pdf) with permethrin at 2% w/w incorporated into polyethylene fibres giving adequate release of permethrin for up to five years. Recipients will be offered a choice between the "large" conical  and the "family" rectangular (160 cm wide × 180 cm l × 150 cm h) models. Olyset nets are supplied in individual sachets labelled by the manufacturers and they will be distributed to recipients in pre-opened individual sachets in May 2010, at the beginning of the main transmission season.LLINs distributed during this project will be Olyset Nets, Sumitomo Chemicals. These meet WHO specifications  who run the health posts will use RDTs in their diagnosis of malaria and treat malaria in children who are enrolled into the study. Nurses placed in strategic health facilities will also perform a RDT in children who present directly to them who are enrolled into the study. Project field workers or nurses will visit each VHW regularly, at least once a week, to record children with malaria episodes.The parents of any child treated for malaria at a clinical survey or during the passive surveillance will be asked to bring their child to the nearest health facility if the child does not show improvement within 48 h. When such study subjects report to a health facility government nurses will treat the child following Gambian government guidelines and inform the study nurse stationed in that facility. The study nurse will inform the study physician of the case within a minimum of delay. If such study subjects report to the VHW, he/she will advise them to report to the nearest health facility. Referred study subjects will be compensated for transport to the health facilities.The end of transmission season survey in December 2011 will be the final study visit for all enrolled children and also the final study visit to the villages. The final survey will be similar to the baseline and end of the first transmission season survey in the previous year.P. falciparum parasites detected by RDT in the absence of other detectable cause of fever. The parent/carer of any child we treat for malaria will be asked to bring their child to their nearest health post or facility if the child does not make a recovery within 48 hours. Children with severe malaria will be referred to Basse Health Centre for treatment.During the course of the study participants are free to receive medication from health personnel outside the study team. During the clinical surveys, iron supplementation will be given to any enrolled child with anaemia (Hb < 9 g/dL). Artemether-lumefantrine (Co-Artem) will be given to any child with a temperature of ≥37.5°C or a history of fever in the past 48 h, and During the passive surveillance for malaria, parents/carers of study children will be encourage to take their child to the nearest VHW or Health Facility if the child is unwell. In both cases children with symptoms suggestive of malaria will be tested for the presence of parasites using RDTs. Treatment for other conditions will be carried out in accordance with national guidelines and/or referral to Government health facilities.Exposure to mosquitoes will be measured by surveillance with light traps and house exit rates estimated using window traps from June to November each year. To obtain the primary entomological endpoint light traps will be used. They are routinely used in The Gambia and are unaffected by the presence of treated nets . A CDC mThe main morbidity outcome will be incidence of clinical disease assessed by PCD; data from this will provide the primary endpoint of the study. PCD is considered to generate information of more direct relevance to public health than active case detection and is thus more suitable for evaluating population level malaria interventions . Mild ma®) in the field.All children present in their villages at the surveys will be clinically examined for obvious symptoms and signs of illness, temperature and spleen enlargement. A sample of all the study children, at least 50/village cluster will be randomly selected stratified by age, and these as well as those reporting fever in the last 48 hours and/or with a temperature of ≥37.5°C will be finger pricked for immediate measurement of anaemia and presence of parasites by RDT. Anaemia will be assessed by measuring haemoglobin concentration using a spectrophotometer , flat fan pattern, 80° angle, flow rate 0.76L/min at 45psi pressure). They will be rigorously trained in the use of these and their maintenance. A 2-day refresher training will be provided before the spray campaign in year 2. Any remnant in the sprayers at the end of daily spraying will be collected following the "Progressive Rinse" method and recycled to minimize waste .In advance of the spray campaign in year 1, spray operatives and supervisors will be trained on the WHO methods for IRS with DDT for two weeks by the NMCP vector control officer seconded to the project and an internationally recognized IRS consultant. They will be equipped with personnel protective clothing, including boots, long-sleeved cotton overalls, hat, gloves, mask and boots and trained in the maintenance and use of these. Spray operatives will use spray pressurized canisters made to WHO specifications WHO/VBC/89.970 , and the products will be used in compliance with their recommended use and guidelines.There are no apparent risks to the safety of individuals or communities in this study. Permethrin-treated long lasting nets and DDT IRS has been fully evaluated by the WHO Pesticide Evaluation Scheme and approved for vector control  during the course of the study this will be captured on the CRF for PCD if it is associated with a confirmed or suspected malaria episode. When a study child consults with the VHW with a severe illness that is not malaria, this will be captured in the VHW daily log. The CRF for PCD or the details from the VHW daily log will be submitted to the study physician by the nurse or fieldworker responsible for the village-cluster on a weekly basis. If it is a serious adverse event (SAE) the study physician will record and manage the SAE in accordance with Good Clinical Practice (GCP).The study physician will check all CRF regularly, at least once a week, and will inform the IDMC of the numbers of malaria attacks and SAE by village as required by the ID. Suspicion of potential harm to participants or the environment caused by the interventions may lead to discontinuation of the study. Excessive clustering of SAE by village(s) will be reported to the study's Data Monitoring and Ethics Committee.If an individual wants to terminate his/her participation, no further follow-up will be performed. In the case of a study child leaving during year 1, they will be replaced in year 2 of the study (June 2011). There will be no replacement during the surveillance period either year. If a household withdraws consent, no further follow-up will be made and there will be no replacement. If a village opts out of the study before July in either year replacement by a neighbouring village will be considered.P. falciparum parasites of any density detected by microscopy and/or RDT in the absence of other detectable cause of fever.Primary: Incidence of clinical episodes of malaria presenting at health facilities defined as a child with an axillary temperature of ≥37.5°C or a history of fever in the past 48 h, together with the presence of Secondary: (i) mean haemoglobin concentration in children in the two study arms measured in the end of the transmission season survey, (ii) parasite prevalence in children in the two study arms measured at the end of the transmission season.and a P. falciparum parasitaemia cut-off to give optimum sensitivity and specificity in this setting, and number of children with enlarged spleens at the end of the transmission season.In addition we will also calculate the incidence of malaria defined by history of fever or temperature of ≥37.5°C Transmission parameters in the two arms of the study will be estimated from measurements made throughout the transmission season.An. gambiae s.l./light trap/night inside sleeping rooms.Primary: The mean number of female Secondary: The entomological inoculation rate (EIR) in each study group will be estimated as the mean number of sporozoite infective bites/child/season)An. gambiae complex in light trap & exit traps collected in each arm of the trial, (iii) the relative abundance of An. gambiae s.l. collected outdoors (iv) the parity of An. gambiae, to measure mean population longevity.Tertiary: (i) The proportion of mosquitoes exiting houses (since DDT has repellent properties), (ii) the relative abundance of different members of the Anopheline house entry and contact with LLINs). To cover these possibilities we expect that LLINs will reduce incidence by 50%, with LLINs and IRS combined reducing the residual incidence by 30-60% . A study of 3-60 month old children during the end of the rainy season (Sept-Dec) in the URR in 2007 found a seasonal incidence of 0.79 (95% CI: 0.58-1.08) cases/child-years by PCD (Bojang pers. comm.); ITN prevalence was 50%. We estimate conservatively that increasing LLIN provision to > 80% and inclusion of older children will reduce incidence rates by about 60-80%, so incidence rates of 0.30 (LLINs) to 0.15 cases/child-years (LLINs & IRS) have been included in the sample size estimations. The study will also record PCD over two high transmission seasons, not 1 as in the Bojang study, further enhancing our ability to detect differences between study arms. Estimations for k, the corrected coefficient of variation to adjust for between cluster variation (based on within and between cluster variation) [Both LLINs and IRS alone reduce malaria morbidity by approximately 50% . The comriation) , from a Data from the same cluster trial quoted above was used to explore sample size considerations for the parasitological and anaemia, endpoints. Considering parasitaemia as a proportion and haemoglobin as a concentration with 35 clusters of 110 children each suggested that sampling half the child cohort would have 80% power to detect a 30% reduction in parasitaemia and a 0.5 mg/mL reduction in haemoglobin at 95% confidence.An. gambiae s.l. per trap will be 4.8 . In order to demonstrate a 60% reduction in hungry indoor mosquitoes (i.e. unfed An. gambiae) associated with DDT IRS, with 90% power and at the 5% level of significance, we would require 8 houses in each cluster and 12 clusters in each arm of the trial over 2 years [Previous studies with experimental huts indicated that DDT IRS deterred 60-70% of mosquitoes from entering houses . LLINs w 2 years . Since tThe demographic data will be recorded by fieldworkers and the clinical data by study nurses on standardised data forms. Each participant will have a unique identification (ID) number. All data recorded on individuals will be made by recording the anonymous ID.Data forms will be scanned to produce electronic copies and into the data set using Snap Survey  software and verified against electronic copies of the original forms by data entry clerks to produce 2 independently verified versions. These will be combined and errors corrected to produce a single dataset. This will be submitted to consistency checking by generic and study specific algorithms designed to identify sources of error. When inconsistencies are found, these will be checked against the original forms and subsequently amended in the dataset. Errors will be corrected when possible, with checking in the field when necessary and possible, to produce final datasets.All forms with subject names and/or clinical data will be kept in a locked cabinet, when not in use, and the key kept by the study coordinator or delegate. These datasets will be, password protected. Data will be stored for at least 10 years. The clinical data will be kept separately from that containing personal information. All forms and datasets, apart from those for enumeration, will identify subjects by their subject specific ID and names will not be collected or entered. After the collection and verification in Snap Survey data will be moved to MRC's SQL server 2008 SWIFT. These datasets will be, password protected and only accessed by senior study staff and the study Data Manager. Data will be stored for at least 10 years.The Principal Investigator (PI) will maintain appropriate medical and research records for this study in compliance with GCP and regulatory and institutional requirements. Authorised representatives of the sponsor, the ethics committee(s) or regulatory bodies may inspect all documents and records required to be maintained by the investigator. The PI or designee will ensure the access to facilities and the records.The PI or designee will document and explain any deviation from the approved protocol on the CRF, where appropriate, and record and explain any deviation in a file note that will be maintained as an essential document. Deviations from the protocol, GCP or study specific requirements that might have an impact on the conduct of the study or the safety of participants will be reported within 15 working days to the sponsor and relevant Ethics Committee, as appropriate. Any other protocol deviation will be reported to the local EC together with the annual report.The primary endpoint is a comparison of the incidence of clinical episodes of malaria in children in the 2 intervention groups, measured by PCD. After a case of malaria is treated, time of observation will be censored for 3 weeks and further attacks of malaria during this period are not considered. History of travel away from the village for prolonged periods will be captured by monthly surveys and time at risk will be censored for such periods. In addition, malaria cases in children who resided outside their study village for more than half the elapsed study period at the time of illness will be censored. An initial unadjusted analysis will be based on comparisons of the incidence rates between the 2 groups. Incidence rates will be calculated for each cluster and the ratio of their un-weighted means will give an estimate of the intervention effects. Confidence intervals for the intervention effect will be obtained using the approximations given by Bennett  and testDifferences in malaria transmission experienced in the 2 groups will be made by comparing the mean number of mosquitoes caught indoors in houses between the intervention groups. Generalised estimating equations will be used to estimate differences in numbers of indoor-resting mosquitoes, adjusting for repeated measures within clusters and possible covariates.P. falciparum parasitaemia cut-off defined to give optimum sensitivity and specificity in this setting, (iii) parasite prevalence in children at the end of the transmission season, (iv) number of children with enlarged spleens at the end of the transmission season. In general, all quantitative outcomes will be compared using appropriate summary statistics, such as mean differences or risk ratios. Hypothesis testing will be based on a two sample unpaired t-test, adjusting for possible co-variates. If the data do not satisfy normality and constant variance assumptions, an appropriate non-parametric test will be used. Both linear regression (for continuous variables such as haemoglobin levels) and logistic regression (for dichotomous variables such as presence or absence of parasitaemia) will be used to quantify the group differences allowing for individual level covariates  using random effects to allow for clustering.For the secondary clinical endpoints we will compare: (i) mean haemoglobin concentration in children at the end of the transmission season, (ii) incidence of malaria defined by history of fever or temperature of ≥37.5°C and a The timetable of activities is shown in Table This study is conducted in accordance with the principles set forth in the ICH Harmonised Tripartite Guideline for GCP and the Declaration of Helsinki in its current version, whichever affords the greater protection to the participants. It was approved by the Gambian Government/MRC Laboratories Joint Ethics Committee first approved on the 12th August 2008  and the London School of Hygiene and Tropical Medicine Ethics Committee approved on the 16th September 2009 (ref: 5592).As enthusiasm for malaria control in Africa gathers pace, there is an urgent need to evaluate the effectiveness of multiple interventions. This study evaluates the efficacy of the 2 most common vector control tools used for malaria control: IRS and LLINs. This study sets out to examine whether there is an additional benefit of using IRS in combination with LLINs compared to current best practise, the use of LLINs alone. It is designed to measure whether the double intervention will provide greater protection against malaria incidence by substantially reducing transmission.The main morbidity outcome will be incidence of clinical disease assessed using PCD. PCD is considered to generate information of more direct relevance to public health than active case detection and is thus more suitable for evaluating population level malaria interventions . It is eThere are no apparent risks to the safety of individuals or communities in the use of these interventions. Permethrin-treated long lasting nets and DDT-WP have been fully evaluated by the WHO Pesticide Evaluation Scheme and approved for vector control . The bigLLIN are similarly beneficial at the individual level, and at the community level where high coverage is attained. However, in The Gambia no community protection has been found with ITNs -39 as itIt is also important that the correct normal practice of parents/carers in the case of their child becoming sick is not altered in a detrimental fashion by their child being enrolled in the study. For example a parent/carer may delay taking a child to the nearest health facility/post if they think that a study nurse is visiting their village the next day. Both the provision of fares when a children reports sick at a health facility and emphasis of the vital role of parent/carers during enrolment and the conduct of the study will help to mitigate against this.http://www.rollbackmalaria.org). The trial will benefit our collaborators at the NMCP in The Gambia by providing capacity building: developing management systems, resistance monitoring strategies and providing training on IRS according to the principles laid down by WHO [There is currently a rush for DDT IRS in many African countries, yet we lack the evidence of the protective efficacy of this intervention against clinical malaria. To date, most trials have been non-randomised and, often non-controlled, making it difficult to evaluate protective efficacy . The pron by WHO . This isCDC: Centers for Disease Control and Prevention; CRF: Case report form; CV: coefficient of variation; DDT: Dichloro-diphenyl-trichloro-ethane; EC: Ethical committee; EIR: Entomological inoculation rate; GCP: Good clinical practice; GLLAMM: Generalized Linear Latent and Mixed Models; ICH: International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use; ID: Identification; IRS: Indoor residual spraying; ITN: Insecticide Treated Net; LLINS: Long lasting impregnated nets; LSHTM: London School of Hygiene and Tropical Medicine; MRC: Medical Research Council; NMCP: National malaria control programme; PCD: Passive case detection; PCR: Polymerase chain reaction; PI: Principal Investigator; PHC: Primary Health Care; RDT: Rapid Diagnostic Test; SAE: Serious Adverse Event; URR: Upper River Region; VHW: Village health worker; WHO: World Health Organisation; WHOPES: Who Pesticide Evaluation SchemeThe authors declare that they have no competing interests.SWL and MP conceived and designed the study, and drafted the manuscript. MJ and LJ contributed to the design of the entomological collections and IRS application. LJ and BK advised on the interventions and the study communities. DJ provided the statistical input to the study. KB advised on clinical aspects of the trial. DC contributed to the design and management of the study. ML advised on the design and management of the IRS. JM reviewed the protocol. All authors read and approved the final manuscript.