Malaria is a public health problem in Uganda; affecting mainly women and children. Effective treatment has been hampered by over-diagnosis and over-treatment with anti-malarial drugs among patients presenting with fever. In order to understand the effect of drug pressure on sulfadoxine-pyrimethamine (SP) resistance in pregnancy, a sample of pregnant women presenting with fever in out–patient clinics was studied. The main objective was to assess prescription patterns and drug use in pregnancy especially SP; and draw implications on the efficacy of SP for intermittent preventive treatment of malaria in pregnancy (IPTp).A total of 998 pregnant women with a history of fever were interviewed and blood samples taken for diagnosis of malaria and HIV infections. Data were captured on the drugs prescribed for the current febrile episode and previous use of drugs especially SP, anti-retroviral drugs (ARVs) and cotrimoxazole.P.falciparum; and of these, 72 (56.3%) received first-line treatment with Artemether-lumefantrine (Coartem®) 14 (10.9%) SP and 33 (25.8%) quinine. Of the parasite negative patients , 186 (21.4%) received Coartem, 423 (48.6%) SP and 19 (2.1%) cotrimoxazole. Overall, malaria was appropriately treated in 35.5% of cases. Almost all febrile pregnant women, 91.1%, were sleeping under a mosquito net. The majority of them, 911 (91.3%), accepted to have an HIV test done and 92 (9.2%) were HIV positive. Of the HIV positive women, 23 (25.0%) were on ARVs, 10 (10.9%) on cotrimoxazole and 30 (32.6%) on SP. A significant proportion of women, 40 (43.5%), were on both SP and cotrimoxazole. Age and occupation were associated with diagnosis and treatment of malaria and HIV infections.Few pregnant women, 128 (12.8%) were parasitaemic for There is inappropriate treatment of malaria and non-malarial fevers among pregnant women in these facilities. This is due to non-adherence to the guidelines. Over-prescription and use of anti-malarial drugs, especially SP may have implications on resistance against SP for malaria prevention in pregnancy. The policy implications of these findings are to evaluate SP efficacy as IPTp; and the need to enforce adherence to the current clinical treatment guidelines. Uganda has a high burden of malaria that contributes greatly to morbidity and mortality ,2. MalarP. falciparum parasites resistant to SP in malaria endemic areas poses a major threat to prevention of malaria in pregnancy[Malaria in pregnancy is a major public health problem in malaria endemic areas -7. The cpregnancy-11. Therpregnancy-14. Howepregnancy,16. Thuspregnancy. In ordeP. falciparum, HIV-positive women are recommended to receive monthly doses of SP as IPTp rather than the 2-dose regimen [In countries with a high HIV prevalence, IPTp with SP has been found to be less effective thus leading to a recommendation of increasing the number of doses of IPTp in pregnancy. Thus in areas of high transmission of  regimen ,16. The  regimen . It is p regimen -21. In UPrevious studies in Uganda have assessed prescription patterns of drugs in the public and private facilities. In one study, only 34% of the recommended doses of chloroquine followed the guidelines . It has Understanding treatment patterns for febrile illness among pregnant women in countries endemic for malaria is of public health importance for three reasons: 1) to gain an understanding of factors associated with treatment seeking among this vulnerable group and design interventions to increase access to effective treatment and prevention of malaria and other non-malarial fevers; 2) drugs given in pregnancy may have direct and indirect effects on the baby; 3) to understand the level of appropriate treatment since inappropriate drug use may lead to drug resistance. The main objective of this study was to assess prescription patterns and drug use among pregnant women with fever. Specifically the study assessed use of anti-malarial drugs during the current and previous febrile episodes; and use of SP and cotrimoxazole among HIV positive women. This article presents results on drug use among a sample of pregnant women presenting with fever at out-patient clinics in Uganda. Implications of the results and further areas of research are discussed.The study was conducted in the malaria endemic district of Mukono, central Uganda. The total population of the district is 850,900 with an annual growth rate of 2.3% and consists predominantly of subsistence farmers of the Baganda ethnic group. The majority of the population, 88%, lives in the rural areas.A survey targeting pregnant women presenting with fever in out–patient clinics was carried out from January to November 2011. Inclusion criteria were: i) Actual fever or history of fever during the last 14 days, ii) temperature at least 37.5°C; this was taken using a digital thermometer iii) menstrual history and a clinical examination by a trained midwifes and clinical officers to confirm a pregnancy. A structured questionnaire was administered to all eligible consenting pregnant women with fever attending outpatient clinics over a period of 10 months. The questionnaire captured data on demographic characteristics, history of febrile illness, treatment received, HIV counselling and testing practices, history of taking SP, antimalarial drugs, ARVs and other drugs. Midwives working in two maternity units at Kawolo hospital and Mukono health centre IV conducted the interviews and patients who consented to give a blood sample were sent to a laboratory. The midwives were trained for 2 days on study procedures and they participated in the pre-testing and reviewed the tools before the study. Questionnaires were initially developed in English and translated into the local language (Luganda). The field coordinator and the research team supervised all aspects of data collection.Blood (4 mls) was taken by venepuncture from every patient who consented to the study and stored in EDTA tubes. Haemoglobin, HIV and malaria infections and were assessed. Smears were stained with Giemsa and malaria parasites were counted against 200 leukocytes and expressed as number of parasites per μl of blood assuming a standard leukocyte count of 8000/μl of blood. A blood smear was regarded negative after examining a minimum of 100 high power fields with no parasites seen. This is because the study area is known to be highly endemic for malaria thus the selection of this cut-off point. All buffers, solutions and stains were stored appropriately and cleanliness and sterility of equipment maintained. For quality assurance, all malaria positive samples examined in the field laboratory were re-examined at the reference laboratory at the Uganda Virus Research Institute. Discrepancy between the two readings was less than 5%.HIV test was performed following the national algorithm on HIV testing. A blood sample undergoes three tests: the first is Determine® and if positive, it is confirmed by Statpak® if still positive by Unigold®. Results were recorded on forms and clients told the results.Data was entered and verified using Microsoft Access 2007  and analysed using STATA version 11.0 . Univariate analyses were carried to get the proportions of drugs prescribed to women with malaria, non-malaria fevers and those who were HIV positive. Bivariate analysis was carried to assess factors associated with diagnosis and treatment of malaria and HIV infections. Proportions were compared using a chi-Square test and P-values <0.05 were considered significant. Appropriate treatment was calculated as the number of parasite positive pregnant women who received Coartem® plus parasite negative women who received no antimalarial drug divided by the total number of febrile cases. The sample size calculation was based on the proportion of pregnant women appropriately treated for malaria estimated at 25% . The stuEthical approval for the research was granted by review boards at the Uganda National Council of Science and Technology (Reference HS. 747). Written consent was obtained from all participating women.A total of 998 pregnant women presenting with fever were included in the study. The mean age was 23.9 years with a range of 14–42 years. The majority of women, 64.9%, were aged between 20–29 years, 33.7% had attained secondary education and 78.2% were married; while 47.7% were peasants earning from household agricultural activities  had parasitaemia. Of these, 72 (56.3%) received the first-line antimalarial drug, Coartem®. A large proportion 33 (25.8%) received quinine while 14 (10.9%) received SP (Fansidar®). Among pregnant women with no malaria parasites, 186 (21.4%) were given Coartem® and 423 (48.6%) Fansidar® (SP); 113 (13.0%) were given antibiotics. Appropriate treatment of malaria  and patients without malaria receiving no antimalarial drug) occurred in 35.5% of pregnant women. The majority of patients 909 (91.1%) were sleeping under a mosquito net (Table P = 0.0001 (data not shown). Among the pregnant women who had an HIV test, 92 (9.2%) were HIV positive. A quarter, (25.0%) of HIV positive women were on ARVs, while 30 (32.6%) of them were taking cotrimoxazole. Forty HIV positive women (43.5%) were taking both cotrimoxazole and SP  ; and access to ARVs . Education, marital status and religion were not associated with diagnosis and treatment of malaria and HIV infections in this population.We explored factors associated with diagnosis and treatment of malaria and HIV. Younger women were likely to test positive for malaria, mean age 22.3 years versus 24 years for negative women, This study shows that there is inappropriate treatment of malaria among pregnant women with fever attending outpatient clinics. Although ACT are the first-line anti-malarial drug, many patients with confirmed malaria were not given ACT but SP. This may lead to parasite resistance to SP. Similarly, inappropriate use of ACT in pregnant women with non-malarial fevers may also lead to resistance against these drugs. These results will contribute to the design of a further study to evaluate the efficacy of SP as IPTp in Uganda.A recent study in health facilities in Uganda found out that there were constraints to parasite diagnosis like lack of microscopes and RDTs, inadequate personnel and infrastructure. This scenario impacts on appropriate treatment of malaria and other diseases. Similarly a study in Jinja, a neighbouring district in Eastern Uganda showed that 85% of self-reported febrile illnesses were treated with antimalarial drugs .The present results compare with those from a study carried out in South-Western Uganda in an area of low malaria endemicity which found high prescription rates for antibiotics in febrile patients, the majority of whom were children aged less than 5 years, and concluded that testing negative for malaria increases the use of antibiotics . The preOur study shows that access to ACT is high, consistent with findings from another Ugandan study that the most commonly prescribed antimalarial drugs are artemether-lumefantrine and quinine with a relatively high adherence to the new antimalarial treatment policy . This coIn interpreting the results it is worth noting that this study targeted pregnant women with fevers reporting in outpatient clinics thus the drug use and prescription patterns in other groups were not included. Clinical examination was relied to estimate the gestation period and thus could have missed pregnancies in the first trimester. In addition, the study was limited in the capacity to identify the causes of non-malaria fevers. A study carried on Thai-Burmese border found that non-malaria fevers in pregnancy were mainly scrub and typhus fevers . Since sThe implications of these results are to ensure that health workers adhere to laboratory test results and prescribe according to the treatment guidelines. This will ensure appropriate treatment and avoid indiscriminate drug use potentially leading to drug resistance. Printing and distributing treatment guidelines supported by technical supervision are urgently needed to address this situation. Further areas for research include evaluating the efficacy of SP as IPTp and the review of the guidelines for IPTp among HIV positive women since interaction of SP with cotrimoxazole could hamper the effectiveness of SP. There is also need to strengthen pharmacovigilance of antimalarial drugs to enable timely review of policies.There is inappropriate treatment of malaria and non-malarial fevers among pregnant women in these facilities. This is due to non-adherence to the guidelines. Over-prescription and use of anti-malarial drugs, especially SP may have implications on resistance against SP for malaria prevention in pregnancy. The policy implications of these findings are to evaluate SP efficacy as IPTp; and the need to enforce adherence to the current clinical treatment guidelines.The authors declare that they have no competing interests.AKM conceived the study; AKM, JB, SY and PM participated in the design, implementation, coordination of the study and drafting the manuscript. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/237/prepub


Individual malaria interventions provide only partial protection in most epidemiological situations. Thus, there is a need to investigate whether combining interventions provides added benefit in reducing mortality and morbidity from malaria. The potential benefits of combining IPT in children (IPTc) with home management of malaria (HMM) was investigated.During the 2008 malaria transmission season, 1,277 children under five years of age resident in villages within the rural Farafenni demographic surveillance system (DSS) in North Bank Region, The Gambia were randomized to receive monthly IPTc with a single dose of sulphadoxine/pyrimethamine (SP) plus three doses of amodiaquine (AQ) or SP and AQ placebos given by village health workers (VHWs) on three occasions during the months of September, October and November, in a double-blind trial. Children in all study villages who developed an acute febrile illness suggestive of malaria were treated by VHWs who had been taught how to manage malaria with artemether-lumefantrine (Coartem™). The primary aims of the project were to determine whether IPTc added significant benefit to HMM and whether VHWs could effectively combine the delivery of both interventions.The incidence of clinical attacks of malaria was very low in both study groups. The incidence rate of malaria in children who received IPTc was 0.44 clinical attacks per 1,000 child months at risk while that for control children was 1.32 per 1,000 child months at risk, a protective efficacy of 66% . The mean (standard deviation) haemoglobin concentration at the end of the malaria transmission season was similar in the two treatment groups: 10.2 (1.6) g/dL in the IPTc group compared to 10.3 (1.5) g/dL in the placebo group. Coverage with IPTc was high, with 94% of children receiving all three treatments during the study period.Due to the very low incidence of malaria, no firm conclusion can be drawn on the added benefit of IPTc in preventing clinical episodes of malaria among children who had access to HMM in The Gambia. However, the study showed that VHWs can successfully combine provision of HMM with provision of IPTc.ClinicalTrials.gov NCT00944840 Although the incidence of malaria appears to be declining in a number of African countries, it remains an important cause of mortality and morbidity among young children and pregnant women. Malaria control strategies deployed in Africa include prompt treatment of clinical attacks of malaria with an effective anti-malarial drug combination, vector control using insecticide-treated nets (ITNs) or curtains or indoor residual spraying (IRS) and intermittent preventive treatment (IPT). However, individually these interventions provide only partial protection in most epidemiological situations . Thus, tStudies have shown that IPT with sulphadoxine/pyrimethamine (SP) given on two or three occasions during pregnancy provides significant protection against maternal anaemia  and low Home-based management of malaria (HMM) is now considered an important strategy for improving treatment practices ,13 and fAlthough a number of trials of IPTc and HMM in children, given as individual interventions, have been conducted, only one trial has formally addressed the added value of IPTc in children who have access to HMM. In this study conducted in Ghana, IPTc with amodiaquine and artesunate was given three times during the course of the year to children aged 6 - 60 months who also had access to HMM. The prevalence of malaria parasitaemia was significantly lower at the end of the intervention than at baseline but, as no control group was included in the study, it is not possible to define the exact role played by IPTc in achieving this improvement . An addiThe study, based at the MRC Field Station, Farafernni in the North Bank Region of The Gambia, was conducted between May 2008 and January 2009. The trial was undertaken in a group of 24 villages near Farafenni which are part of the rural Farafenni demographic surveillance system (DSS), described in detail elsewhere . There aMalaria in The Gambia occurs almost exclusively during the rainy season and immediately afterwards (July to November) with peak transmission occurring during October and November. The annual entomological inoculation rate varies enormously across the country and estimates have been reported in the range of 1-177 .The study was designed as a double-blind, randomized placebo controlled trial. Children were individually randomized to receive monthly treatment with a single dose of SP plus three doses of amodiaquine (AQ) or SP and AQ placebos from a VHW at monthly intervals on three occasions during the months of September, October and November. All study children had ready access to a VHW who had been trained to administer HMM with Coartem™ to children who developed symptoms compatible with malaria during the malaria transmission season.Prior to the start of the trial, meetings were held in all potential study villages to explain the purpose and methods of the study and to answer questions from parents and guardians of eligible children. During these meetings, agreement of a village to participate in the study was obtained from the villagers. A list of children living in the 24 villages, which agreed to join the study who would be aged between six and 59 months at the time of the first treatment with IPTc in September 2008 was obtained from the DSS database. These children and their parents were invited for screening for potential participation in the trial between July and August 2008. Written informed consent was obtained from parents or guardians of all potentially eligible children before screening. The age and identity of these children were checked, and they were examined to rule out any clinically significant disease that might interfere with the outcome of the trial. Children were considered eligible if they had no clinically significant acute or chronic disease. Exclusion criteria included known allergy to any anti-malarial drug or the presence of acute or chronic, clinically significant pulmonary, cardiovascular, hepatic or renal disease. A finger-prick blood sample was collected from all children who met the inclusion criteria for preparation of a thick blood smear and measurement of haemoglobin (Hb) concentration. If a child had symptoms suggestive of malaria, the blood film was examined shortly after collection, whilst remaining blood films were examined at a later date. Children with acute malaria were treated with Coartem™ and excluded from the study and those with anaemia were treated with oral iron.A reporter based in each village was asked to keep records of all deaths, births and immigrations and emigrations that occurred during the study period. Village reporters were visited fortnightly by a project field worker and their records collected and checked. Deaths were investigated using a modified version of the INDEPTH post mortem questionnaire .One thousand two hundred and seventy-seven eligible children whose parents consented were individually randomized into either the SP plus AQ or placebo group in a 1:1 ratio. The randomization list was generated using permuted blocks of 12 by the trial statistician, using the random number generator of STATA version 10. One tablet of SP plus three tablets of AQ or one tablet of SP placebo plus three tablets of AQ placebo were placed in sealed opaque envelopes labelled with the randomization number. For each study subject, a set of three envelopes with the same randomization number, one for each month were prepared. Each child enrolled was assigned the next study number in sequence. Neither the VHWs who administered the treatment, the field or laboratory staff who collected data nor the families of study children knew which was the active drug and which placebo.A recording system for drug administration that could be used by VHWs, many of whom were illiterate, was devised. This consisted of a treatment card held by the mother or guardian of each child and an IPT register held by the VHW. The treatment card and the IPT register were labelled with the child's, mother's and village's names and compound, demographic surveillance and study numbers. The nature of the treatment and the number of tablets of that treatment that each child should receive was indicated on the treatment card using a colour code to indicate the treatment (white for SP or SP placebo and yellow for AQ or AQ placebo) and full circles, half circles and quarter circles to indicate the number of whole, half or quarter tablets that the child should receive. Dosage was based on the child's age. Children aged 3-11 months received half a tablet of SP or SP placebo and a whole tablet was given to children aged 1-5 years. One quarter, half or whole tablet of AQ or AQ placebo was given daily for three days to children aged 3-11 months, 1-2 years and 3-5 years respectively. SP tablets (500 mg sulphadoxine/25 mg pyrimethamine) and amodiaquine (AQ) tablets (200 mg base tablets) were obtained from Kina Pharma Ltd, Accra, Ghana and their solubility and active drug content confirmed at the London School of Hygiene and Tropical Medicine. The first dose of treatment was given under direct observation, the second and third doses by the parent or guardian at home.Passive surveillance for malaria was carried out during the 2008 transmission season. Parents or guardians were encouraged to take their child to the VHW at any time their child became unwell. VHWs were instructed to presumptively treat febrile illnesses suggestive of malaria with Coartem™.One field worker was attached to two to three VHWs during the surveillance period. The role of the field worker was to prepare a thick blood smear from children who had been diagnosed as a case of malaria by the VHW and scheduled for treatment with Coartem™ for subsequent confirmation of the diagnosis. Details of children treated by the VHWs were recorded in books with help from field workers. VHWs referred children who failed to improve on treatment and those with danger signs  to the nearest health centre or to the AFPRC Hospital in Farafenni for further evaluation and management. Project staff members were based at each of these health facilities to identify children in the trial when they presented for treatment and to ensure that they were properly investigated and treated promptly. At each clinic visit, axillary temperature was recorded using a digital thermometer and Hb concentration measured using a Hemocue machine. A Rapid Diagnostic Test for malaria (RDT)  was done if a child had fever (axillary temperature of ≥ 37.5°C) or a history of fever within the previous 48 hours. In such cases, a thick blood smear was also collected for subsequent confirmation of the diagnosis. Children with documented fever (axillary temperature of ≥ 37.5°C) or a history of recent fever and malaria parasitaemia or a positive RDT were treated with Coartem™. Children with severe malaria were treated with intramuscular quinine. The treatment of study subjects seen at the health centres for conditions other than malaria was carried out in accordance with national guidelines.A cross-sectional survey of all available study children was undertaken at the end of the malaria transmission season. Children were examined by a project clinician and anthropometric data were collected and a finger-prick blood sample was obtained for preparation of thick blood smears and determination of Hb concentration.Thick smears were prepared in duplicate. If a child had symptoms of malaria, one smear was stained with Field's stain and read promptly to guide treatment. The other smear was stained with Giemsa and 200 high power fields (HPF) were examined before a smear was declared negative. Only the Giemsa-stained slide readings were used for the trial analysis. Parasite density was expressed per μl with the assumption that 1 parasite per high-powered field (hpf) equals a density of 500 parasites per μl. All slides were read by two laboratory technicians. If there was disagreement between their readings on parasite positivity or if the difference of the log-densities recorded was more than 1.5, slides were read by a third technician. Agreement was reached among the three technicians after the slides had been re-checked. When a laboratory technician was not immediately available to read a thick blood smear the result of the RDT was used to guide treatment, and a thick blood smear collected for subsequent confirmation of the diagnosis. Hb concentration was measured at recruitment, during morbidity surveillance and at the end of malaria transmission season surveys using a portable haemoglobinometer .Data from participants were recorded on paper forms and were checked by field supervisors, study physician, and data manager for consistency and accuracy. All data were entered twice into a SQL database using MS Access software. The accuracy of data input was checked and validated using customized validation programmes. The cleaned data were converted to STATA version 10 file  prior to analysis.The primary study endpoint was the incidence of clinical malaria  among study subjects seen at one of the health centres or at the district hospital during the surveillance period. Secondary endpoints included incidence of a febrile illness with parasitaemia at any density among children seen by a VHW or at a health centre or hospital, incidence of anaemia among children seen at a health centre or hospital, the prevalence of parasitaemia at the end of malaria transmission season cross-sectional survey, the prevalence of anaemia at the end of malaria transmission season cross-sectional survey, the proportion of children who received three IPTc treatment courses and the proportion of children who received no IPTc treatment course.The incidence of malaria seen at a health centre or hospital in children who had access to HMM and who received placebo was estimated to be 0.2 episodes per child per transmission season. To detect a 40% reduction in the incidence of malaria with a power of 90%, using a significance level of 5% and assuming 20% loss to follow-up in children who received IPTc, 625 children per group were needed. For the cross-sectional survey, assuming a parasite prevalence of 20% at the end of transmission season in children who had access to HMM and received placebo, 557 children in each study group were needed to show a 40% reduction in the prevalence of malaria in children who received IPTc in addition to HMM with 90% power at the 5% level of significance assuming a 20% loss to follow-up. A reporting and analysis plan (RAP) was developed prior to breaking the code. The primary analysis included all children who were randomized, regardless of the number of treatments received. Characteristics of all children enrolled in the study were tabulated by study arm. For the primary outcome of malaria incidence during the study period, analysis was by intention to treat. Time at risk was calculated from date of enrolment until the date of cross-sectional survey or until date of death or emigration from the study area with no return. No child had more than one episode of malaria so no adjustment for clustering was required. The protective effect of IPTc in addition to HMM against malaria during the surveillance period was calculated as 100(1-R), where R is the incidence rate ratio for the IPTc versus the placebo group, calculated using Cox regression. The effects of IPTc on incidence of anaemia and other complaints were calculated in the same way.Prevalences of anaemia and parasitaemia in the cross-sectional survey at the end of the malaria transmission season were compared between the two groups using logistic regression. Hb concentrations at the end of the transmission season were compared using linear regression, adjusting for Hb at enrolment. Coverage with IPTc was evaluated from records kept by VHWs of treatment administration. The proportions of children who had received each monthly treatment course, those who received three treatment courses, and those who received no treatment were tabulated.The study was approved by the London School of Hygiene & Tropical Medicine Ethics Committee and by the joint MRC/Gambia Government Ethics Committee.Plasmodium falciparum parasitaemia was found infrequently at enrolment; only 0.5% of study subjects in either treatment group had parasitaemia; 93% of the study subjects slept under an ITN.One thousand three hundred and thirty-four children aged 6-59 months were selected from the Farafenni DSS and screened; 1,277 children who met the entry criteria were enrolled and allocated to receive monthly IPT with SP plus AQ (639) or SP placebo plus AQ placebo (638). The remaining 57 were not enrolled because they did not meet the inclusion criteria, no consent was given or because they had one of the exclusion criteria  in the control group (p = 0.73). The prevalence of splenomegaly at the end of malaria transmission season was also very low in the two groups . The proportions of children with a Hb concentration of < 8 g/dL at the end of the malaria transmission season were similar in the two groups of children, 44/513(8.6%) in children who received SP/AQ and 45/533 (8.4%) in those who received placebo . The mean (standard deviation) Hb concentration at the end of the malaria transmission season was similar in the two treatment groups; 10.2 (1.6) g/dL in the SP/AQ group compared to 10.3 (1.5) in the placebo group, difference adjusted for baseline Hb 0.04 .One thousand and forty-six study children  were seen at the end of the malaria transmission season. Three (0.67%) children in the SP/AQ group had asexual stage There was only one death during the surveillance period among the 1,277 study children. This child, who was in the placebo group, died at home in December; a verbal autopsy suggested a diagnosis of pneumonia. Fourteen children were admitted to one of the health facilities in the study area during the surveillance period, two of them twice. The 16 admissions were equally distributed between treatment arms, nine children were diagnosed with pneumonia  and with acute gastroenteritis (2 and 1 in the intervention and control groups respectively). Remaining admissions were for anaemia, malnutrition, upper respiratory tract infection and severe helminth infection. One child in the SP/AQ group developed a drug eruption which presented as multiple areas of macular hyperpigmentation of different sizes mainly on the trunk with sharply demarcated outlines.This study was carried out to determine whether IPTc with SP plus AQ provided additional benefit in reducing malaria morbidity in children who received HMM in the context of high ITN coverage. Very few cases of malaria were detected in either arm of the trial so the study was not able to determine its primary objective. In addition, IPTc with SP plus AQ did not have any measurable impact on anaemia or splenomegaly.The very low incidence of malaria encountered in the Farafenni area of The Gambia was surprising but consistent with other recent data from The Gambia that have indicated a dramatic fall in the incidence of malaria in The Gambia in recent years . Why sucIn recent years, significant reductions in the incidence of malaria have been reported in several countries in Africa where malaria was previously moderately endemic . This suNo serious adverse events which could be directly attributed to the study medication were observed. One skin rash was observed but this did not have features of Stevens Johnson syndrome.IPTc would not be a useful intervention in The Gambia if the incidence of malaria persists at its currently very low level. However, recent studies in Burkina Faso and Mali ,24 have The authors declare that they have no competing interests.KAB, BG conceived and designed the experiments. KAB, SS performed the experiments. EW, KAB, PM, SS analysed the data. ET, MS contributed reagents/materials/analysis tools. KAB PM, EW, BG wrote the paper. All authors read and approved the final manuscript.


In Cambodia, malaria transmission is low and most cases occur in forested areas. Sero-epidemiological techniques can be used to identify both areas of ongoing transmission and high-risk groups to be targeted by control interventions. This study utilizes repeated cross-sectional data to assess the risk of being malaria sero-positive at two consecutive time points during the rainy season and investigates who is most likely to sero-convert over the transmission season.Plasmodium falciparum Glutamate Rich Protein (GLURP) and Plasmodium vivax Merozoite Surface Protein-119 (MSP-119) were detected using Enzyme Linked Immunosorbent Assay (ELISA). The force of infection was estimated using a simple catalytic model fitted using maximum likelihood methods. Risks for sero-converting during the rainy season were analysed using the Classification and Regression Tree (CART) method.In 2005, two cross-sectional surveys, one in the middle and the other at the end of the malaria transmission season, were carried out in two ecologically distinct regions in Cambodia. Parasitological and serological data were collected in four districts. Antibodies to P. falciparum and 7.9% and 6.0% for P. vivax in August and November respectively). P. falciparum force of infection was higher in the eastern region and increased between August and November, whilst P. vivax force of infection was higher in the western region and remained similar in both surveys. In the western region, malaria transmission changed very little across the season (for both species). CART analysis for P. falciparum in the east highlighted age, ethnicity, village of residence and forest work as important predictors for malaria exposure during the rainy season. Adults were more likely to increase their antibody responses to P. falciparum during the transmission season than children, whilst members of the Charay ethnic group demonstrated the largest increases.A total of 804 individuals participating in both surveys were analysed. The overall parasite prevalence was low (4.6% and 2.0% for In areas of low transmission intensity, such as in Cambodia, the analysis of longitudinal serological data enables a sensitive evaluation of transmission dynamics. Consecutive serological surveys allow an insight into spatio-temporal patterns of malaria transmission. The use of CART enabled multiple interactions to be accounted for simultaneously and permitted risk factors for exposure to be clearly identified. Malaria transmission is often focal, particularly in low endemic areas. Entomological and parasitological measures are traditionally used to estimate its intensity, though in areas of low transmission the required sample sizes for entomological surveys increase exponentially because of the difficulty of finding infected mosquitoes. In addition, parasitological surveys using microscopy can be extremely time consuming and may not detect sub-patent infection . SerologWhere malaria transmission is seasonal, the characterization of its dynamics requires the longitudinal collection of the variables of interest . As antibodies remain in the blood longer than parasites, they are less subject to seasonal variations. Whilst individual responses may fluctuate -12, prevPlasmodium falciparum and Plasmodium vivax malaria compounds the difficulties of estimating transmission intensity and controlling the disease [Cambodian malaria control programmes have historically focussed on the border area between Thailand and Cambodia, which was one of the sites of emergence of chloroquine and, more recently, of artemisinin resistance, increasing the urgency for containment and/or elimination. One of the long-term challenges Cambodia faces is forest-related malaria and the residual transmission amongst high-risk populations  in remote areas ,15. Mala disease -20.The CAMALFOR project was a joint collaboration between the Institute of Tropical Medicine in Antwerp, Belgium and the National Center for Malaria Control, Parasitology and Entomology (CNM) in Phnom Penh, Cambodia. In 2005, two surveys, one in the middle and the other at the end of the transmission season, were carried out in four districts: Borkeo and O'Chum in the east and Pailin and Veal Vang in the west Figure . EntomolP. falciparum and P. vivax malaria were present and Anopheles dirus sensu stricto was found to be the major malaria vector in these study villages, with Anopheles minimus s.s and Anopheles barbirostris representing minor vectors [Two malariometric cross-sectional surveys were performed in four districts of Cambodia: two in the eastern region (Borkeo and O'Chum in Rattanakiri Province) and two in the western region , in August and November 2005 Figure . In each vectors .The study was originally designed to evaluate malaria transmission intensity in order to plan targeted interventions. In each of the four "forested" districts, three villages  were selected for their representativeness of forest coverage in the area. Each village consisted of approximately 200 inhabitants, of which 100 were randomly sampled for the survey. However, in Veal Vang district  bloodspot filter papers from one of the villages (VX) were stored incorrectly and therefore could not be analysed for the serological survey. Hence, this village was not included in the analysis. For direct comparison of antibody responses and associated risks across the second part of the rainy season (August to November), only individuals who took part in both surveys were analysed in this paper. Analyses for each of the two regions have been kept separate throughout the paper.A full census of the study population collecting information on age, gender, ethnicity, education, housing conditions, socio-economic level, forest-related activities and malaria prevention measures was carried out in July 2005. The cross -sectional surveys (August and November) included a random sample of 100 individuals per village - this sample size was chosen to provide a precision of 3% for a minimum estimated parasite index of 5%. Each participant had thick and thin slides taken for microscopic examination, as well as having a filter paper blood spot collected for malaria antibody detection. Everyone was examined for clinical signs of malaria and a rapid diagnostic test (RDT) was administered to participants with symptoms. Malaria treatment was based on the RDT results and followed the National guidelines. A new variable combining ownership of bicycle, TV and motorbike was generated using principal component analysis in order to estimate each family's income level , as described previously . Bed netParasite prevalence was determined by microscopy. Slides were stained for 15 minutes with a 10% Giemsa solution. Thick films and thin films were taken from each participant and parasite density was computed based on the examination of 200 white blood cells and assuming a white blood cell count of 8,000/μl. Microscopy was performed at the National Center for Entomology, Parasitology and Malariology (CNM) in Phnom Penh, Cambodia, and quality control was done on 10% of randomly chosen slides by a second reader. Any discordant slides were re-read by both readers until an agreement was found.P. falciparum GLURP antibodies and P. vivax MSP-119 antibodies were detected using ELISA. Briefly, from each blood spot, a disc of 5 mm diameter was punched out and eluted overnight at 4°C in 2 ml of PBS-Blotto-Tween . Two hundred μl of the eluate was added in duplicate to blocked ELISA plates coated separately with P. falciparum GLURP R2 [P. vivax MSP-119 [P. falciparum or P. vivax infected patients and from five non-infected control group were diluted at 1:400 in PBS-Blotto to serve as positive and negative control respectively. Goat anti-human IgG(H + L) peroxidase  was diluted to 1:20,000 in PBS-Tween and incubated for 1 h before development of the ELISA using 200 μl ABTS substrate-chromogen solution. Optical Densities (ODs) were read at 415 nm . Corrected OD values were obtained by subtracting the mean OD of the antigen negative control wells from the mean OD of the corresponding antigen containing wells. Subsequently, the percent positivity (PP) of each specimen was calculated using the positive control serum' OD as 100%. This ensured the sample results were standardized across ELISA plates. A mixture model was used to generate a cut off for positivity as previously described [A blood spot was taken on filter paper (Whatman grade 3) and allowed to dry before being individually stored in a zipped plastic bag containing silica gel and kept at -20°C until ELISA assays were performed at ITM, Antwerp. GLURP R2  and P. v MSP-119 . Pooled All data were double entered, validated and cleaned in Epi Info 6 . The dataset was analysed using STATA v.11  and CART  software.A simple reversible catalytic conversion model was used to fit the dichotomized serological results, using maximum likelihood methods ,13. The P. falciparum results from the eastern surveys as this area experienced the biggest serological differences between the surveys. Wilcox paired rank tests were used to test for differences in antibody responses between August and November (p < 0.05 considered significant).To evaluate changes in antibody responses during the rainy season, analysis focussed on The use of Classification and Regression Trees (CART) is described extensively elsewhere ,25. ThisPermission was received from village leaders after explaining the study aims and objectives. Oral informed consent was obtained from all adults and parents/guardians. Ethical clearance was obtained from the CNM, Phnom Penh and the IRB of the ITM, Antwerp (n° OG018). Permission was also obtained from the Cambodian Ministry of Health.Individuals who participated in both surveys (N = 804) constituted approximately 50% of the population originally surveyed . No bias in terms of census information was detected in this sample, although the Pore ethnic group in the western region  did not participate in both surveys, so were not included in this analysis. Baseline data of individuals who participated in both surveys is summarized in Table P. falciparum was twice as prevalent as P. vivax, and both parasite prevalences decreased by more than half between August and November (from 8.1% to 3.4% (p = 0.014) and from 4.4% to 2.1% (p = 0.077), for P. falciparum and P. vivax respectively). During this same period, P. falciparum sero-prevalence increased more than two-fold (from 23% to 49% (p < 0.001)) while P. vivax sero-prevalence remained similar (12.8% and 11.2% in August and November respectively (N.S)). In the western region, P. vivax parasite prevalence was almost 10-fold higher than P. falciparum (10.1% versus 1.2%) and did not decrease significantly between August and November (from 10.7% to 9.1% (N.S)) whereas P. falciparum prevalence decreased by more than half to reach 0.5% (N.S) in November. P. falciparum sero-prevalence also decreased by half, from 14% to 7% (p < 0.001) and P. vivax sero-prevalence decreased from 20% to 13% (p < 0.001).Malariometric indices indicated clear differences in malaria transmission patterns between the east and west regions Table . In the There was no correlation between individuals' antibody responses to the two species (p > 0.1), indicating an absence of cross-reactivity between the two antigens and suggestive of separate transmission patterns for the two species.1 and λ2) were detected for P. falciparum (LR test p < 0.05) in August and November , although the confidence intervals overlapped with those in the east. A single force of infection was detected for P. vivax in August  between August and November, although no differences were detected for P. vivax responses in this area. In the west, P. falciparum responses did not change significantly over season; however a significant decrease was detected in P. vivax responses (p < 0.001).An increase in a specific serological response is indicative of exposure to the parasite - therefore increases in antibody responses between August and November were used as a proxy for exposure. Individual responses (as measured by percent positivity (PP)) to P. falciparum data was analysed, as the largest differences in sero-prevalence were seen in this region. CART was used to divide PP responses into four homogenous categories: negative (PP < 3.3%), low positive (PP more than 3.3% and less than 10.3%), medium positive (PP more than 10.3% and less than 24%) and high positive (PP more than 24%). Individuals were then classified depending on whether they remained negative or decreased a PP category (indicating no exposure), remained in the same positive category (indicating possible exposure) or on the number of categories they increased between August and November (indicating exposure). A total of 55% of the study population (n = 210) either remained negative or decreased a serological category between August and November whilst 8% (n = 30) remained in the same positive category, 18% (n = 70) increased one serological category and 19% (n = 74) increased two or more serological categories. Figure In order to further investigate the changes in transmission dynamics during the rainy season, the eastern ive PP < .3%, low P. falciparum between August and November  were analysed using CART . The villages of BY and BZ were further split at the age of nine  where 71% of under nine year olds (27/38) remained negative, compared to only 10% (7/72) of over nines. The other villages were subsequently split at age six. The under sixes remained mostly negative between the two surveys, while among the over six year olds there was another split by village with OZ (fully forested) remaining predominantly negative , and only approximately 40% (50/128) of the remaining villages (BX (deforested), OX (rubber plantation), OY (scattered forest)) remained negative. In these villages, the lowest income group was more likely to increase at least one serological category (58%) compared to medium and high income groups (29%). The latter was further split by ethnicity, first separating the Tumpurn (with 36% (15/42) remaining in a positive category), and then a further split separating the Khmer  and the Charay (with 65% increasing at least one category (13/20)). The table highlighted village (100%), ethnicity (73%) and age (50%) as the three most important predictor variables, while roof type, wall type, income and working in the forest had less importance (below 20%).The risk factors associated with exposure to T Figure . VillageIn areas of low malaria transmission such as Cambodia, parasitological indicators of transmission intensity using microscopy can be insensitive. Serological measures allow for more sensitive detection of malaria exposure and can be used as a proxy of transmission intensity ,4,6,26. P. falciparum dominating in the east and P. vivax dominating in the west. This is likely to be due to the ecological differences between the two areas, such as a more intensive deforestation and lower rainfall in the western region, but also to differences in educational levels and the use of bed nets (both of which were lower in the east). P. vivax has been reported to become more prevalent as transmission decreases [P. falciparum infections may also have underlying P. vivax infections [P. falciparum infections are cleared through treatment. In addition, P. falciparum infections can sometimes mask other infections when diagnosis is performed by microscopy, meaning mixed or other malarial infections remain unrecorded. Importantly, the life cycle of P. vivax differs from P. falciparum. In P. vivax infections dormant liver forms (hypnozoites) can remain present for long periods of time, causing relapses months or years later. Consequently, P. vivax can be more difficult to eliminate and therefore may become more prominent in areas where P. falciparum has been successfully reduced .Malaria parasite prevalence in the eastern and western regions was substantially different, with ecreases  and somefections , which bP. falciparum exposure over the rainy season. Interestingly, the village OZ, characterized as a forested village, was highlighted as having little malaria exposure between August and November. It would be expected that a more forested village would be subject to higher malaria exposure. According to the entomological results obtained in the study villages, the vector densities in OZ were similar to the other villages in the eastern part of the country  however, there was a particularly high usage of bed nets in this village (71% reported sufficient bed net use compared to an average of 40% in the other villages), suggesting that this may have reduced exposure successfully. Fifty percent (55/110) of the participants in villages BY and BZ (both of which are forested) increased two or more P. falciparum serological categories, suggesting high transmission in these villages between August and November. The serological differences between villages highlight the heterogeneity in transmission at a micro level, as has been reported previously [CART analysis indicated that individuals in different villages in the eastern region experienced varying levels of  percent 5/110 of P. falciparum in the eastern sites. This may be a consequence of highly seasonal yet low transmission meaning that the rarity of exposure to malaria parasites results in the loss of antibodies out of season, and any new exposure causes a large boost of antibody responses. This is demonstrated by the substantial increase in overall sero-prevalence and age sero-prevalence curves between August and November  between adults and children. A step in the sero-prevalence curve can be indicative of a recent reduction in transmission intensity [P. falciparum seropositivity in children was extremely low and deforestation in this area has resulted in dramatic reductions in malaria transmission. In this area, the two forces of infection may represent current transmission intensity (extremely low) and previous transmission intensity (low). Future studies in the west would allow further interpretation depending on whether the step in the seroprevalence curves remains at the same age  or shifts with time (indicating a drop in the force of infection).The sero-prevalence curves for ntensity . HoweverP. falciparum in children in the western region was extremely low, suggesting they had experienced very little exposure. SCRs increased above the age of 10 years old, which is a plausible age at which children begin to accompany their parents to work in the forest. However, the sero-prevalence data indicated that SCR in children in the more forested eastern region also increased over season albeit to a lesser extent . Both the Tumpurn and Charay ethnic groups appear to be at higher risk of exposure, compared to Khmer individuals. Traditionally, the ethnic minorities in this area are poorer than Khmer individuals. In the population sampled in the eastern region, 60% of Charays were classified in the poorest income group, compared with 45% of Khmer and 42% of Tumpurn. Poorer status may mean these individuals spend longer periods in the forest, and are therefore exposed for longer time periods. However, CART highlighted that ethnicity was also important in the higher income groups. This may be a reflection of the type of work undertaken by the different ethnic groups, as Khmer traditionally spend less time in the forest. Moreover in this study, the use of bed nets is much lower in the minority groups with over 50% of Charays reporting no bed nets in their household, compared with less than 1% of Khmers.It is important to acknowledge that the serological data used in this study is based on a single antigen for each species. Although both the antigens used have been demonstrated to be relatively immunogenic ,24,37-39Traditional multi-variate techniques were not suitable for the analysis of this dataset as multiple interactions and collinearity resulted in a lack of power. By using CART it was possible to investigate the data further and to clearly identify risk factors associated with malaria exposure. This was further enhanced by the analysis of consecutive serological surveys. In order to establish risk factors and to discern whether adults are experiencing a higher force of infection, only one survey would be necessary. However, to determine whether transmission is decreasing over time and eventually to determine whether elimination has occurred, more frequent surveys would be required to ensure antibody fluctuations do not result in unreliable evidence for elimination. In order to investigate antibody dynamics, samples need to be collected at more regular intervals and analyses of changes in sero-response over season, such as performed in this study, will become increasingly required.In areas of low transmission intensity, serological indicators can be used to infer risk factors for exposure and to assess changing transmission dynamics across season and over time (and space). In areas where elimination protocols have been implemented, serological measures, in combination with molecular parasitological measures, can help to confirm areas where infection is no longer present.The authors declare that they have no competing interests.JC analysed the data and wrote the paper; NS contributed to the data analysis and paper review; TS supervised all the field work activities; HS contributed to the study design and fieldwork supervision; MS carried out and coordinated the fieldwork; FC supervised the sample processing with ELISA; KL processed all blood samples with ELISA; MT and IS contributed antigen to the project; UDA contributed to the study design, data analysis and reviewed the manuscript; MC contributed to the study design and coordination of all field and laboratory activities and reviewed the paper; AE contributed to the study design, the supervision of the laboratory activities, the data analysis, and reviewed the paper. All authors read and approved the final manuscript.


Malaria elimination will require that both symptomatic- and asymptomatic-infected persons be identified and treated. However, well-characterized, individual-level risk factors for malaria may not be valid in regions with declining malaria transmission. Changes in individual-level correlates of malaria infection were evaluated over three years in a region of declining malaria transmission in southern Zambia.Malaria surveys were conducted in two study areas within the catchment area of Macha Hospital, Zambia in 2007 and 2008/2009. A random sample of households was identified from a digitized satellite image of the study areas. Cross-sectional surveys were conducted approximately five times throughout the year in each of the two study areas. During study visits, adults and caretakers of children were administered questionnaires and a blood sample was obtained for a rapid diagnostic test (RDT) for malaria.In the 2007 study area, 330 individuals were surveyed. 40.9% of participants lived in a household with at least one insecticide-treated bed net (ITN); however, only 45.2% reported sleeping under the ITN. 23.9% of participants were RDT positive. Correlates of RDT positivity included younger age, the presence of symptoms, testing during the rainy season, using an open water source, and not sleeping under an ITN. In the 2008 study area, 435 individuals were surveyed. 77.0% of participants lived in a household with at least one ITN; however, only 56.4% reported sleeping under the ITN. 8.1% of participants were RDT positive. RDT positivity was negatively correlated with the presence of symptoms within the last two weeks but positively correlated with documented fever. In 2009, 716 individuals were surveyed in the same area as 2008. 63.7% of participants lived in a household with at least one ITN; however, only 57.7% reported sleeping under the ITN. 1.5% of participants were RDT positive. Only self-reported fever was significantly correlated with RDT positivity.With declining malaria prevalence, few individual-level characteristics were correlated with RDT positivity. This lack of correlation with individual characteristics hampers identification of individuals infected with malaria. Strategies based on ecological or environmental risk factors may be needed to target control efforts and achieve further reductions and elimination. Globally, 225 million cases of malaria and 781, 000 deaths were estimated to occur in 2009, with the majority in sub-Saharan Africa [In regions that have achieved low levels of transmission, further malaria control and elimination will require that interventions are not only incorporated into national control programmes and accepted and used by individuals, but that symptomatic- and asymptomatic-infected persons be identified and treated. Numerous prior studies identified individual-level risk factors for malaria, but many were conducted in areas of high endemicity  and amonZambia is one of eleven countries in sub-Saharan Africa that achieved a greater than 50% reduction in the number of malaria cases between 2000 and 2009 . The preAnopheles arabiensis is the primary vector responsible for malaria transmission [2 region to the east of Macha Hospital and the Malaria Institute at Macha  and imported into ArcGIS 9.2 . Structures of appropriate size and shape were identified as potential residences and locations of households were identified and enumerated manually. Simple random sampling was used to select households for the surveys, which were carried out throughout the year.® ear thermometer. A blood sample was collected by finger prick for a rapid diagnostic test (RDT) for malaria and haemoglobin determination using a Hemocue® photometer. The RDT  was used to detect P. falciparum histidine-rich protein 2. This RDT was shown to detect 82% of test samples with wild-type P. falciparum at a concentration of 200 parasites/μL and 98% of test samples with a concentration of 2, 000 parasites/μL, with false positives in 0.6% of negative samples [®).Study procedures began with community mobilization activities, including approvals from local chiefs and headmen. A field team was provided with images and coordinates of the randomly selected households. Households consisted of one or more domestic structures where members of a family resided. All individuals within a household were eligible to participate. After obtaining permission from the head of household and individual written informed consent, a questionnaire was administered to each participant older than 18 years of age residing within the household and to parents or guardians of those younger than 18 years of age. Data collected included demographic information, history of recent malaria and anti-malarial treatment, knowledge of malaria transmission and prevention, and the use of ITNs. Each participant's temperature was measured using a Braun Thermoscan samples . IndividThis study was approved by the University of Zambia Research Ethics Committee and by the Institutional Review Board at the Johns Hopkins Bloomberg School of Public Health.All participants in the cross-sectional surveys and the first visit for participants in the longitudinal cohort were included in this analysis. The rainy season was assumed to occur from January 1 to June 30 and November 1 to December 31, and was defined based on rainfall and hospitalization data from Macha Hospital during the study period. Fever was defined as a temperature greater or equal to 38°C. Anaemia was defined by age and sex according to World Health Organization criteria . IndividCharacteristics hypothesized to be associated with malaria were compared between individuals who were RDT positive and negative using the chi-square test for binary characteristics and the Wilcoxon ranksum test for continuous characteristics. For years in which multiple correlates of RDT positivity were found, crude and adjusted prevalence ratios were calculated using log-binomial regression with generalized estimating equations to account for clustering by households. Characteristics that were found to be correlated with RDT positivity in the crude analysis (p < 0.10) were included in the adjusted model.In 2007, 72 heads of household were approached to participate and 52 (72%) agreed to enroll their households. All of the 330 individuals within the households who were approached agreed to participate. In 2008, 81 heads of household were approached to participate and 76 (94%) agreed to enroll their households. Of the 466 individuals within households who were approached, 435 (93%) agreed to participate. In 2009, 122 heads of household were approached to participate and 119 (98%) agreed to enroll their households. Of the 730 individuals within the households who were approached, 716 (98%) agreed to participate. In all years, the majority of participants were young children and adolescents, and approximately half were male (Table The majority of adults (≥ 18 years) were familiar with the symptoms, causes and prevention of malaria Table . HoweverThe majority of adults reported learning about malaria from health workers . Others learned from posters in health centres  and schools . Less common sources of information included non-governmental organizations, friends, the radio, and the study team. Some participants reported never having learned about malaria . These participants were more likely to be older and to have less than a primary school education.® bed nets .Less than half of participants in 2007, but the majority of participants in 2008 and 2009, lived in a household with a bed net Table . Most hoOnly 1.8% of participants in 2007, 1.4% in 2008 and 2.0% 2009 reported that their house had ever been sprayed with insecticide to control mosquitoes.The prevalence and correlates of RDT positivity differed by study location and year. In the 2007 study area, 79 (23.9%) participants were positive for malaria by RDT Figure . 51.9% oIn the 2008 study area, 35 8.1%) participants were positive for malaria by RDT  participants were positive for malaria by RDT  of individuals infected with malaria to be asymptomatic -28. ThisThis study was subject to several limitations. First, the study was conducted over two geographic areas. Participants surveyed in 2008 and 2009 were from the same geographic area, while those surveyed in 2007 were from an area further east. Consequently, it is possible that the different risk factors found between the two areas, was not due to the decline in malaria transmission but to differences in the ecology or characteristics and behaviours of the participants surveyed. When participants and households were compared, however, there did not appear to be many differences based on measured characteristics. Second, we did not collect information on behaviors, occupation, assets, migration, and travel that might be associated with malaria. Finally, as previously discussed, the power to detect associations was low in 2008 and 2009 with declining malaria transmission.In an area with high malaria prevalence, several established, individual-level characteristics were correlated with RDT positivity. With lower malaria prevalence, however, few individual-level characteristics were correlated with RDT positivity and the majority of infected persons were asymptomatic. This lack of correlation with previously established individual-level characteristics hampers identification of asymptomatic-infected individuals critical to the development of a malaria elimination programme in Zambia . StrategEIR: entomological inoculation rate; IQR: inter-quartile range; ITN: insecticide-treated net; RDT: rapid diagnostic test.The authors declare that they have no competing interests.CGS performed the data analysis and drafted the manuscript. TK participated in the coordination of the study and reviewed the manuscript. HH participated in the design and coordination of field aspects of the study and reviewed the manuscript. TS participated in the design and coordination of the study and reviewed the manuscript. AK participated in the design and coordination of field aspects of the study and reviewed the manuscript. SM participated in the design and coordination of the study and reviewed the manuscript. PET participated in the design and coordination of the study and reviewed the manuscript. GG participated in the design and coordination of the study and reviewed the manuscript. WJM conceived of the study, participated in its design and coordination, and participated in the preparation of the manuscript. All authors read and approved the final manuscript.Correlates of RDT positivity, by study year. a among participants 18 years of age or older b self-reported symptoms include fever, headache, chills, nausea/vomiting, cough, diarrhea c anemia defined by age and sex: < 11 g/dL for children 0-4.9 yrs; < 11.5 g/dL for children 5-11.9 yrs; < 12 g/dL for children 12-14.9 yrs; < 12 g/dL for women ≥ 15 yrs; < 13 g/dL for men ≥ 15 yrs d open water source: unprotected well or river/stream.Click here for file


Plasmodium falciparum infection to severe malaria (SM) are still incompletely understood. Altered antibody and cellular immunity against P. falciparum might contribute to increase the risk of developing SM.The factors involved in the progression from P. falciparum lysate, merozoite antigens , a Duffy binding like (DBL)-α rosetting domain and antigens on the surface of infected erythrocytes were measured by ELISA or flow cytometry. Plasma concentrations of IL-12p70, IL-2, IFN-γ, IL-4, IL-5, IL-10, IL-8, IL-6, IL-1β, TNF, TNF-β and TGF-β1 were measured using fluorescent bead immunoassays. Data was analysed using McNemar’s and Signtest.To identify immune responses associated with SM, a sex- and age-matched case–control study was carried out in 134 Mozambican children with SM  or uncomplicated malaria (UM). IgG and IgM against P < 0.001), IgM against MSP-119 (P = 0.050) and AMA-1 (P = 0.047), TGF-β1 (P <0.001) and IL-12 (P = 0.039). In addition, levels of IgG against P. falciparum lysate and IL-6 concentrations were increased . Anti-DBLα IgG was the only antibody response associated to reduced parasite densities in a multivariate regression model (P = 0.026).Compared to UM, matched children with SM had reduced levels of IgG against DBLα (P. falciparum in the SM group. IgM against P. falciparum and specific IgG against a rosetting PfEMP1 domain may play a role in the control of SM, whereas an imbalanced pro-inflammatory cytokine response may exacerbate the severity of infection. A high overlap in symptoms together with a limited sample size of different SM clinical groups reduced the power to identify immunological correlates for particular forms of SM.The lower levels of antibodies found in children with SM compared to children with UM were not attributable to lower exposure to  Plasmodium falciparum, severe malaria (SM) was responsible for approximately 0.7 million deaths in 2010, predominantly children under five years of age ) than in children with UM .Among 142 children recruited, four tested negative for P = 0.032 and P < 0.001, respectively), whereas no differences were found for IgG against merozoite antigens  and placental isolates. A similar seroprevalence was found for IEs isolated from SM patients  or UM patients , and a similar but not significant trend was found for DBLα . In general, different antibody responses correlated weakly  and IgM against EBA-175 and AMA-1 .Prevalence and levels of IgG against DBLα were significantly lower in children with SM compared to their matched controls (P = 0.031), ARD (P = 0.015) and prostration (P = 0.002) compared to their respective matched controls  and 0.11 including only SM cases , but the effect was not observed among UM controls .A linear regression model including all antibody responses showed that IgG against DBLα was the only antibody independently associated to lower P = 0.047, Figure P = 0.013), although no significant differences were found for IL-1β concentrations. Conversely, children with SM had lower levels of TGF-β1 and IL-12 . No significant correlations were found between antibodies and cytokines.Children with SM had significantly higher IL-6 concentrations in plasma than children with UM (P = 0.032) and IL-6 (P = 0.002) and lower levels of TGF-β1 (P = 0.002) compared to their controls .Stratification by severe clinical presentations showed that children with ARD had higher levels of IL-10 P = 0.03 and IL-6P = 0.006) and including only children with SM , but the association was not found in children with UM . On the other hand, higher concentrations of IL-10 were associated with an increase in parasite densities in all groups .IL-12 concentrations were associated with a reduction of parasite densities when all children were included in the analysis . IgG antibodies to several DBLα domains have already been linked to natural acquisition of immunity ,41 and t from SM . Convers from SM , suggestA PfEMP1 , to whicA PfEMP1 . On the A PfEMP1 .Plasmodium infection through early limitation of parasite growth [This study also reports a reduction of IgM responses to blood stage antigens in children with SM compared to UM, in agreement with findings from previous studies -14. IgM e growth  or throue growth .P. falciparum in SM cases, since children with SM had similar levels of IgM and even higher IgG against total P. falciparum antigens compared to children with UM. Moreover, differences could not be explained by a longer progression of the disease in SM cases because the number of days with symptoms of malaria prior to enrolment was similar to that in the UM group. Importantly, a higher number of previous SM episodes were reported among cases. This observation may indicate that host genetic factors can predispose children to develop SM [in utero exposure to P. falciparum antigens, producing immunological tolerance and increasing susceptibility to subsequent malaria infections [The differences in antibody levels between children with SM and UM observed in this study were not attributable to a lower exposure to velop SM  or that fections . Also, afections ,53 whichet al. in Mali [SM was also found to be associated with alterations of the fine balance between pro-inflammatory and anti-inflammatory cytokines . Childre in Mali , TNF did in Mali ,57. It i in Mali ,20. Chil in Mali ,57, a cy in Mali ,17,22. C in Mali . Overall in Mali , lower T in Mali ,61 and a in Mali . TherefoThe present study is limited by a number of factors. First, antibodies and cytokines measured in peripheral blood at a single time point may not reflect the course of infection or the response in specific organs where parasite sequestration is occurring. Second, it was not possible to control for the effects that co-infection with HIV or helminthes may have on anti-malarial immunity ,63. NeveP. falciparum and IgG to rosetting PfEMP1 domains in controlling SM. Also, imbalanced pro-inflammatory cytokine responses may exacerbate the severity of infection in children. This information is of relevance to understand how immunity to SM is acquired in early childhood and also for the design of strategies to control life-threatening malaria. In particular, subunit vaccines containing DBLα epitopes from rosetting PfEMP1 might potentially elicit functional antibodies that reduce the risk of SM in children. Prospective cohort studies assessing IgG isotypes and functionality of antibodies, together with detailed phenotype analysis of cytokine-producing cells will help to understand the mechanisms underlying natural acquisition of immunity to malaria severe disease.These data supports a role for specific IgM to AMA: apical membrane antigen; ARD: acidosis and/or respiratory distress; CM: cerebral malaria; DBL: Duffy binding like; EBA: erythrocyte binding antigen; IEs: infected erythrocytes; IFN: interferon; IgG: immunoglobulin G; IgM: immunoglobulin M; IL: interleukin; MFI: mean fluorescence intensity; MOI: multiplicity of infection; MS: multiple seizures; MSP: merozoite surface protein; OD: optical density; PfEMP: Plasmodium falciparum erythrocyte membrane protein; SA: severe anaemia; SM: severe malaria; TNF: tumor necrosis factor; TGF: transforming growth factor; UM: uncomplicated malaria.Authors declare that they have no competing interests.ERV and GM carried out the antibody and cytokine measurements, the analysis and interpretation of data and wrote the manuscript. QB and SM performed the clinical diagnosis and management of malaria patients. RA coordinated data collection and sample processing. LP contributed to sample processing and antibody measurements. LQ designed statistical analysis and helped to draft the manuscript. CC produced the recombinant proteins. CM and PA contributed to the study design and drafting of the manuscript. AM and CD conceived and coordinated the study, participated in the analysis and interpretation of the data and contributed to the preparation of the manuscript. All authors read and approved the final manuscript.P < 0.05.Spearman’s rank correlation coefficients between IgG and IgM levels in the study population. * Click here for fileMatched comparisons of antibody levels and cytokine and chemokine concentrations in children with different severe malaria clinical presentations.Click here for fileP < 0.05.Spearman’s rank correlation coefficients between cytokine and chemokine concentrations in the study population. * Click here for file


Background. In malaria-endemic settings, asymptomaticparasitemia complicates the diagnosis of malaria. Histidine-rich protein 2 (HRP2) isproduced by Plasmodium falciparum, and its plasma concentration reflectsthe total body parasite burden. We aimed to define the malaria-attributable fraction ofsevere febrile illness, using the distributions of plasma P. falciparumHRP2 (PfHRP2) concentrations from parasitemic children with differentclinical presentations.Methods. Plasma samples were collected from andperipheral blood slides prepared for 1435 children aged 6−60 months in communitiesand a nearby hospital in northeastern Tanzania. The study population included childrenwith severe or uncomplicated malaria, asymptomatic carriers, and healthy control subjectswho had negative results of rapid diagnostic tests. The distributions of plasmaPfHRP2 concentrations among the different groups were used to modelsevere malaria-attributable disease.Results. The plasma PfHRP2concentration showed a close correlation with the severity of infection.PfHRP2 concentrations of >1000 ng/mL denoted a malaria-attributablefraction of severe disease of 99% , with a sensitivity of 74% , whereas a concentration of <200 ng/mL denoted severefebrile illness of an alternative diagnosis in >10%  of patients. Bacteremia was more common among patients in thelowest and highest PfHRP2 concentration quintiles.Conclusions. The plasma PfHRP2concentration defines malaria-attributable disease and distinguishes severe malaria fromcoincidental parasitemia in African children in a moderate-to-high transmissionsetting. Children <5 years old have the highest burden of malaria and malaria-associatedmortality in sub-Saharan Africa –4. In thCommonly used case definitions of malaria rely on the presence of fever and detection ofmalaria parasites on peripheral blood films and thus lack specificity. In addition, symptomsof severe malaria are nonspecific and can have different etiologies –13.More-accurate case definitions for clinical or severe malaria are required for clinicalmanagement and research purposes. The specificity of a malaria case definition can beimproved by using a parasite density threshold based on peripheral blood parasitemia , 14,15.Plasmodium falciparum histidine-rich protein 2 (PfHRP2)is a parasite-derived water-soluble protein and is released in discrete amounts into theplasma, predominantly during schizont rupture ) is then determined bythe probability (jkm) that this individual belongs todiagnostic group k.Analysis of the observed PfHRP2-basedmalaria-attributable fraction in the unselected group of parasitemic patients with aclinical diagnosis of severe malaria (group 5). It differentiates severe malaria from thepopulation with asymptomatic parasitemia and the population with uncomplicated malaria,who have severe disease of a different origin. The proportion of malaria-attributabledisease (y), according to PfHRP2 concentration(h), is given byPfHRP2 concentration(h), the malaria-attributable fraction of severe disease ism54Wdivided by the total number of individuals with the same PfHRP2concentration (h) predicted by the model asTwo different groups with clinical severe malaria were included in the model, of whichone was partly selected on the basis of the concomitant presence of bacteremia . The model was used to define the plasma WinBUGS . Three cP < .0001) orasymptomatic parasitemia (P < .0001) and also had lower hemoglobinconcentrations (P < .0001). Admission characteristics and outcomes ofpatients with severe malaria (groups 4 and 5) are summarized in Table We analyzed data from 49 healthy RDT-negative controls group 1), 172 children withasymptomatic parasitemia (group 2), 285 patients with uncomplicated malaria (group 3), and226 patients (group 4) and 703 patients (group 5) with clinical severe malaria , 156 of 172 asymptomatic patients (91%), 269 of 285 patientswith uncomplicated malaria (94%), and 222 of 226 patients  and698 of 703 patients  with severe malaria ,with a sensitivity of 74% . Theproportion of malaria-attributable disease declined at lower PfHRP2concentrations. Below 200 ng/mL, an alternative diagnosis than malaria was suggested in>10%  of patients, whereas thisproportion increased to >50%  atconcentrations of <50 ng/mL. The observed PfHRP2 quintiles had positive blood culture results. Blood culture results were positive among 83 patients with severe malaria Table , and bass Figure. Of 90 pPfHRP2 concentrationsaccording to disease severity from asymptomatic parasitemia, to uncomplicated malaria, tosevere malaria. There was substantially less overlap in the distributions of plasmaPfHRP2 concentrations between groups as compared to the distributions ofperipheral blood parasitemia. The distinct distributions between diagnostic groups enabledus to model the proportion of malaria-attributable disease on the basis of plasmaPfHRP2 level at admission and to distinguish this from patients withcoincidental peripheral blood parasitemia in whom severe disease is caused by an alternativedisease. The PfHRP2-based model performed better than a previouslydescribed model that was based on peripheral blood parasitemia [PfHRP2concentrations of <50 ng/mL, in which case additional diagnostic tests are indicated toidentify alternative diseases. The current model also accurately identified patients with avery high probability of severe malaria, with acceptable sensitivity. A threshold of 1000ng/mL defined a population of patients with severe malaria not diluted by patients withcoincidental parasitemia (<1%), which is mainly useful for defining a studypopulation in a research setting, but is also useful for the treating clinician. A lowplasma PfHRP2 concentration in a parasitemic patient with severity signsshould not result in withholding of treatment with antimalarials, but should prompt thetreating physician to look for other possible diseases, depending on the clinicalpresentation and resources . In African settings, were diagnostic facilities arescarce and treatment stock-outs occur, the PfHRP2 concentration can alsohelp to prioritize these resources.This study shows a clear stepwise increase in plasma asitemia . The proPfHRP2 level for death in a large cohort of African children with severemalaria and modelled the malaria attributable fraction in fatal cases [PfHRP2 concentrations byincorporating children with asymptomatic parasitemia and uncomplicated malaria. It isreassuring that the identified plasma PfHRP2 thresholds denoting high orlow probabilities of alternative disease were highly consistent between these studies, whichused different modeling techniques.A previous study reported the strong prognostic significance of plasmaal cases . The curPfHRP2 value of 1008 ng/mL inpatients with cerebral malaria, compared with a PfHRP2 concentration of 443ng/mL in patients with uncomplicated malaria [PfHRP2 concentrationin pediatric cerebral malaria was also confirmed in a Malawian study, where the presence ofmalarial retinopathy was used as the reference test [PfHRP2 concentration does not reflect severity inchildren. In Papuan children, the median PfHRP2 concentrations inuncomplicated and severe malaria were similar (584 vs 456 ng/mL) [PfHRP2 concentrations reported. A small study involving 22 Kenyanchildren with severe malaria reported low median PfHRP2 concentrations of63 ng/mL with absence of decay over 48 hours, which could be related to problems in thePfHRP2 assay [Our findings are supported by 2 recent studies involving African children. A small studyamong Tanzanian children showed a mean  malaria . The diance test . In cont6 ng/mL) . HoweverP2 assay .PfHRP2 concentration is in line withprevious reports involving adult populations. A study among Thai adults showed a similarstepwise increase in plasma PfHRP2 levels that was associated with diseaseseverity [PfHRP2 value among patients with severe malaria was 1863 ng/mL, comparedwith 314 ng/mL among patients with moderately severe malaria [PfHRP2concentration was prognostic for a fatal outcome.The prognostic usefulness of plasma severity . In Indo malaria . In bothPfHRP2 concentrations in healthyasymptomatic children in a moderate-to-high transmission area. Parasite densities that canbe tolerated without causing symptoms vary substantially between individuals of differentage groups, transmission intensities, and seasons [PfHRP2 concentrationthresholds for defining malaria-attributable disease will vary with the level of acquiredimmunity in the population, because this factor determines the relative sizes of populationswith asymptomatic parasitemia, compared with populations with uncomplicated or severemalaria, and thus determines the corresponding overlap of plasma PfHRP2distributions. The model prediction as a function of transmission intensity will be exploredin a separate study. In addition, the prevalence of bacteremia will also affect the size ofthe population of individuals who have asymptomatic parasite infections or uncomplicatedmalaria but present with severe illness. Indeed, in the current study, selection of patientswith a positive blood culture result (group 4) resulted in a relatively higher proportion ofparasitemic patients with severe illness due to diseases other than malaria.This is the first study to assess  seasons , 35, 36. seasons , 35. ThePfHRP2 concentration is positively associated with the entire clinicalseverity spectrum of P. falciparum infection. In this study, plasmaPfHRP2 was assessed by a quantitative ELISA. Our findings call for thedevelopment of a low-cost semiquantitative rapid test for the detection of plasmaPfHRP2 at suitable thresholds.Detection of malarial retinopathy by fundoscopy is an alternative diagnostic tool that hasbeen evaluated for its ability to distinguish children with cerebral malaria fromencephalopathic children with coincidental parasitemia –40. In tPfHRP2concentration quintiles. Blood cultures are known to have a limited sensitivity (around40%) for detecting bacteremia [PfHRP2 concentration of <200 ng/mL (2.5 times the observed proportionof 18%). This would be consistent with results from a Malawian autopsy series, inwhich invasive bacterial infection was reported as the cause of death in 4 of 7 parasitemicpatients (64%) with an alternative diagnosis [PfHRP2concentrations indicate concomitant bacteremia during severe malaria. There are severalmechanisms that may explain this high rate of concomitant bacteremia, including a reductionin gut barrier function due to intense sequestration [Salmonella bacteremia [P. falciparum infection predisposes togram-negative bacteremia and can account for more than half of invasive bacterial disease inmalaria-endemic areas [Positive blood culture results, particularly those for gram-negative organisms, wereoverrepresented among patients within the lowest and highest cteremia . Theactiagnosis . Positivstration , which fstration –46. Sevecteremia . P. falcic areas . Our datPfHRP2 concentration. Patients with severemalaria in group 4 were partly selected on the basis of blood culture positivity. However,patients were selected on the basis of clinical criteria and RDT results and not on thebasis of PfHRP2 concentrations, and the PfHRP2distributions in both severe malaria groups were similar. In patients with low parasitemia,the sensitivities of the peripheral blood slide and the RDT are relatively low, which couldhave affected the composition of the clinical groups.The current study has several limitations. This is a retrospective analysis of pooled datasets. Patients with severe malaria in group 5 were also included in a previous publicationon the prognostic value of PfHRP2 concentration can beused to estimate the proportion of malaria-attributable disease in African children inmoderate-to-high transmission settings and can distinguish severe malaria from severefebrile illness with coincidental peripheral blood parasitemia. Bacteremia is prominentamong patients with severe illness and low plasma PfHRP2 concentrations,suggesting that malaria may not be their primary diagnosis. Bacteremia is also more frequentamong patients with high plasma PfHRP2 concentrations, denoting concomitantsepsis with severe malaria, which implies that administration of antibiotics is warrantedfor all patients with a clinical diagnosis of severe malaria.In conclusion, our study shows that the plasma Supplementary materials are available at The Journal of InfectiousDiseases online . 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P. falciparum (Pf Sch07/03).Programmes of pre-elimination of malaria have been implemented in Senegal since 2010, and the burden of malaria has decreased substantially. These changes in the epidemiology should be monitored with effective tools that allow changes in patterns of transmission to be estimated. In Dielmo and Ndiop, two villages of Senegal with different malaria endemicity, infections have been followed longitudinally for 20 years, during which time there have been several control interventions leading to substantial decreases of transmission. This study aimed to compare malaria antibody responses of the inhabitants of these two villages, between 2000 and 2010, using schizont crude extracts of a local strain of Pf Sch07/03. Three ages groups were defined: [5-9] yrs, [10-14] yrs and [15-19] yrs. Statistical comparisons were performed. Seroprevalence and the magnitude of antibody responses were compared between age groups, villages and periods.Sera collected from inhabitants of the two villages  were used to assess the prevalence of antibodies against crude schizont extracts of P.fSch07/03 decreased between 2000 and 2010 in both villages: from 94.4% to 44.4% in Dielmo and from 74.4% to 34.6% in Ndiop. The difference between Dielmo and Ndiop was highly significant in 2000 (p < 0.001) but not in 2010 (p >0.20). The decrease in seroprevalence was larger in younger (more than 40%) than older (less than 19%) inhabitants. Longitudinal monitoring of the younger group showed that seroprevalence decreased between 2000 and 2010 in Dielmo from 98.7 to 79.3, but not in Ndiop from 67.6 to 66.7. The magnitude of antibody responses in seropositive individuals was significantly higher in 2000 than 2010 for both villages.Overall seroprevalence to P. falciparum are appropriate tools for evaluating malaria prevalence at different periods, and in both low and high endemic area. Using crude extracts from local strains to assess transmission may allow efficient evaluation of the consequences of control programs on malaria transmission.Crude extracts of  There has been significant progress over the last ten years in the fight against malaria, a major threat to public health. The World Health Organization (WHO) reported that of 102 countries in which transmission continues, 52 recorded a decrease of more than 50% in the number of malaria cases between 2000 and 2010. In parallel, malaria mortality has also decreased by more than 45% globally and, in particular, by 47% in Africa. These results have been obtained by a combination of interventions, including artemisinin-based combination therapy (ACT), extensive coverage of exposed populations with long-lasting insecticide-impregnated bed nets (LLITN), malaria diagnosis by rapid diagnostic tests (RDTs), and intermittent treatment strategies .Various methods have been used since the 1960s to measure humoral responses to malaria and thereby evaluate malaria seroprevalence and transmission intensity. However, the variability of sources of antigen and subjectivity of some of the detection methods led to these methods falling out of favour . SensitiThe objectives of this study were to assess the value and feasibility of testing immune responses to parasite crude extracts. This approach was used to evaluate changes in malaria transmission over a ten year period in Dielmo and Ndiop, two villages of Senegal where there has been longitudinal follow-up since 1990. Various malaria interventions were implemented between 2000 and 2010, in accordance with the recommendations of the National Malaria Control Programme  yrs age group in 2000 and that ten years later (2010) in the age group ten years older  and 2010 (222 subjects). The prevalence of anti-P. falciparum Abs differed between Dielmo and Ndiop in 2000. The seroprevalence to PfSch07/03 was high for all age groups studied in Dielmo, and highest in young adults (around 100%) in 2000. This high seroprevalence to PfSch07/03 is consistent with the high levels of exposure in this region of perennial transmission and holoendemicity [et al.[p > 0.05). These findings are in agreement with previous epidemiological studies [Seropositivity to demicity . The sery [et al.-25. Study [et al. and Westy [et al.. The sery [et al.. There w studies .PfSch07/03 IgG response in the youngest age group in 2000 (corresponding to the 15–19 year-old group in 2010) revealed another interesting feature of malaria transmission and seroprevalence: the percentage of positive subjects in Dielmo was significantly lower 10 years later, suggesting that adults had, by 2010 lost the immune response to malaria antigens that they had had when they were ten years younger. By contrast, in Ndiop the seroprevalence was unchanged after 10 years in the same group. This observation might reflect the low intensity of transmission, especially among adults, in Ndiop.Differences in the increases and decreases of antibody prevalence between age groups were also considered. Seroprevalence decreased further and more significantly in the youngest group (by more than 40%) than the oldest group (less than 19%). There is probably a delay before acquiring an immune response in younger individuals, whereas adults display a ‘memory effect’ and their immune response can persist as a consequence of a history of contact with the parasite ,30. ThisPfSch07/03 Abs decreased substantially between 2000 and 2010 in both Dielmo and Ndiop. These declines were such that the magnitude of antibody responses did not differ between the two villages in 2010. Although seroprevalence appears to have remained constant over 10 years in Ndiop, the mean levels of Abs responses were highly significantly different between the younger group in 2000 and the corresponding older group in 2010. The decrease of the antibodies levels was comparable in Dielmo and Ndiop suggesting that a similar mechanism was operating to reduce malaria prevalence in the two villages. Trape et al. reported a longitudinal follow up of inhabitants of Dielmo, showing that the incidence density decreased after LLITN distribution [P. falciparum in assessments of the overall anti-blood-stage immune responses would make this approach even more useful for monitoring exposure to malaria.The mean levels of anti- ribution . Recent ribution . The preThe authors declare that they have no competing interests.AT conceived and coordinated this study. FD and AT conceived the manuscript. FD VR, GD and AT designed the methodology, conducted the analyses and wrote the manuscript and BD, MF, CS, JT, ND and AdT contributed to developing the concepts presented in this paper and provided valuable insights during the revision and editing stages. All authors read and approved the final manuscript.