Plasmodium knowlesi are increasingly diagnosed in South East Asia and have also been reported in travellers. A severe case of imported P. knowlesi infection in a 73-year old German is presented, who had been travelling through Myanmar and Thailand for three weeks. Microscopy showed a parasitaemia of 3% and different parasite stages including band-forms resembling Plasmodium malariae. Due to the clinical picture of severe malaria and the microscopical aspect , P. knowlesi was suspected. The patient was treated with intravenous quinine; he was put on mechanical ventilation and catecholamines due to cardiorespiratory failure. Parasitaemia was cleared rapidly but renal function deteriorated resulting in intermittent haemodialysis. The patient was hospitalized for six weeks but he recovered completely without any physical sequelae. Plasmodium knowlesi mono-infection was confirmed by molecular methods later on.During the last two decades human infections with Plasmodium knowlesi infection has to be taken into account in feverish travellers returning from Thailand/Myanmar. Moreover this species can cause life-threatening or even lethal complications. Accordingly severe P. knowlesi infection should be treated like severe P. falciparum infections. Plasmodium knowlesi infection in a 73-year old man who had been travelling three weeks through Burma and southern Thailand in November/December 2013 is described.A severe case of P. knowlesi to Germany and the most severe described so far. The severe course of the disease was likely due to delayed medical presentation by the patient and thus diagnosis could only be established seven days after onset of symptoms.According to published cases, this is the third imported infection with Plasmodium knowlesi is known since the early 1930, it affects macaques endemic in Southeast Asia and it has been named in honour of Robert Knowles . Knowles first described this Plasmodium species in 1930 and he even showed the capacity of artificial human infection with subsequent malaria symptoms. During the last two decades, human P. knowlesi cases are found more and more often in many countries in Southeast Asia.The 73-year old German patient had been travelling in December 2013 for three weeks for diving and rainforest excursions; he visited the island of Mecleod (Myanmar) and in Thailand Khura Buri, the island Koh Ra, Khao Lak, and Phuket. The day after his return, he developed high fever and chills. He had not taken anti-malarial chemoprophylaxis. After five days with high fever at home (six days after returning from Southeast-Asia), the patient presented to his general practitioner who referred him immediately to the nearest hospital.2 74%). Due to symptoms of shock, decreased gas exchange  and pulmonary distress (tachypnoea), the patient was put on mechanical ventilation. Catecholamines had to be administered to maintain sufficient perfusion and circulation. Initially, sepsis due to a urinary tract infection was suspected and antibiotic therapy with ciprofloxacin was initialized. However, the patient’s condition deteriorated rapidly and piperacillin/sulbactam were added to the antibiotic regimen. As the thrombocyte count dropped dramatically , malaria infection was suspected. The Department of Infectious Diseases and Tropical Medicine at Schwabing Hospital in Munich was contacted and the patient was transferred to its ICU via helicopter.On admission, the patient was already hypotonic and in respiratory distress with signs of somnolence  and small bilateral pleural effusions. The chest x-ray at the first day showed beginning ARDS , chest x-ray four days later showed bilateral basal infiltrations.On a moderate dosage of catecholamines (0.4 μg arterenol/min), vital parameters were stable. Laboratory parameters are displayed in Tables Plasmodium falciparum and Plasmodium malariae. Band-forms and gametocytes reminded of P. malariae  in combination with doxycycline (200 mg per day) immediately upon arrival at the intensive care unit (ICU) of Schwabing hospital. The patient showed a quick response to quinine and was completely cleared from parasites within 48 hours. A subsequently developed ventilator-associated pneumonia was treated successfully with meropenem. Creatinine and glomerular filtration rate (GFR) were already impaired on admission and acute anuric kidney failure developed within the first day . Two P. knowlesi-specific nested PCRs based on the 18S rRNA gene showed the expected amplicons [P. knowlesi strains from Thailand and Malaysia.Definite diagnosis was confirmed by PCR (DITM). A mplicons . Direct Written informed consent of the patient was procured for publication.Plasmodium knowlesi is known since the early 1930 as a parasite of macaques endemic in Southeast Asia [et al. recently detected a high prevalence of P. knowlesi in humans in East Malaysia but it still seems to be under-diagnosed due to diagnostic constraints and possibly also due to a lack of awareness concerning this Plasmodium species [ast Asia . Retrosp species , 5–7.P. knowlesi was as high as 50%, whereas in Thailand it was around 1% [P. knowlesi infections were observed so far in Malaysia, Thailand, Burma, Vietnam, the Philippines, Singapore, and Indonesia [In certain areas of Malaysia , the prevalence of human round 1% –11. Humandonesia . As a trndonesia , 12. Thindonesia , 5, 11.P. knowlesi infections in humans during the last two decades is discussed and might be attributable to a reduction of the natural habitat of macaques, the invasion of humans into rainforests  and an increasing number of the Anopheles vectors feeding on both humans and monkeys [Along with better diagnostic tools and awareness raising, an increase of  monkeys .P. knowlesi, Anopheles cracens and Anopheles lateens, feed on macaques as well as on humans [Anopheles stephensi which is widely distributed in Southeast Asia, can be easily infected with P. knowlesi, and were found in rural as well as urban areas [P. knowlesi had visited rainforest regions prior to manifestation of malaria [P. knowlesi infection without prior rainforest excursions [P. knowlesi. So far, there is no evidence of a stable human-Anopheles-human transmission [P. knowlesi cases published so far [The main vectors for n humans . Also, tan areas . Most re malaria . So far cursions . This insmission . The patd so far , 16.Plasmodium knowlesi may take a severe, even fatal course, such as described for P. falciparum infections, in about 7%, as P. knowlesi can lead to hyperparasitaemia [P. falciparum, P. knowlesi can invade all erythrocytes, not only very young erythrocytes as in Plasmodium vivax and Plasmodium ovale, or very mature erythrocytes as in P. malariae[P. knowlesi infection [sitaemia , 17. Likmalariae. Moreovenfection , 13.P. knowlesi has no liver hypnozoite stage the infection does not relapse [P. knowlesi with renal failure and respiratory distress in patients with low parasitaemia [Therefore, immediate initiation of treatment and vigilant clinical observation is essential. As  relapse . Neverthsitaemia . On the sitaemia , 6.The presented patient faced a severe, life-threatening course of the disease with 3% parasitaemia.P. knowlesi infection was based on the detection of intra-erythrocytic ring forms which resembled P. falciparum as well as band forms and schizonts indistinguishable from P. malariae in the patient’s thin blood smear. This is a typical aspect of P. knowlesi infection and, therefore, P. knowlesi is prone to misidentification and needs further confirmation by means of PCR [He developed complete anuric kidney failure, impaired consciousness, respiratory distress and circulatory deficits, and therefore needed mechanical ventilation and catecholamines shortly after hospital admission. In this case, initial suspicion of a s of PCR , 6.P. knowlesi parasitaemia and platelet count is assumed and it seems that the degree of the thrombocytopaenia could be even worse than in other types of malaria [The laboratory results often reveal a significant to severe thrombocytopaenia, signs of haemolysis with anaemia, and elevated liver enzymes, like as in this patient. A close correlation between  malaria . However malaria , 19.P. knowlesi infection are: acidosis, rapid drop in erythrocyte count, jaundice, renal impairment or even renal failure, respiratory distress, and hypotonia [P. knowlesi malaria were similar to those found in P. falciparum malaria with multiple petechial haemorrhages and sequestration of pigmented parasitized red blood cells in capillaries and venules. However, unlike in falciparum malaria ICAM 1 (intracellular-adhesion-molecule) was not found to be up-regulated, therefore the pathophysiological aspects seem to be different [Other important signs of a life-threatening situation in a ypotonia , 18, 20.ifferent .Undoubtedly the patient suffered from severe malaria corresponding with the WHO classification and his altered mental status (GSCS 7-8) suggests cerebral involvement. The patient had anuric kidney and lung failure, severe haemolysis and cerebral impairment qualifying him for severe malaria with multiorgan failure (apart from the hyperparasitaemia).P. knowlesi malaria suggest that lung injury occurs in non-falciparum-malaria and ARDS has to be taken into account [Autoptic changes in the lung tissue of a patient who had died from  account .Moreover a case series of five patients who died from knowlesi malaria in Malaysia showed that pulmonary problems and respiratory distress were quite common in this infection .P. knowlesi malaria based on RCTs but it seems that most of the anti-malarial drugs are effective [P. knowlesi malaria [So far there is no standard treatment protocol for ffective , 13. How malaria . Current malaria , 22–24. P. knowlesi has to be taken into account in feverish patients returning from Southeast Asia , especially when the parasites in malaria blood slides have close resemblance to P. malariae and/or P. falciparum. PCR diagnosis is crucial for definite characterization of P. knowlesi as microscopic differentiation is hardly possible. As a travel related disease P. knowlesi malaria is still highly exceptional with only a small number of cases reported every year from non-endemic, western countries. Plasmodium knowlesi may cause a rapid clinical deterioration as the intra-erythrocytic cycle is only 24 hours and in almost 7% it has a severe, life-threatening course with multi-organ failure . Correspondingly, severe P. knowlesi malaria should be treated like severe P. falciparum malaria.Infection due to


Plasmodium spp. parasitemia (n=8390) and Plasmodiumfalciparum HRP-2 (PfHRP-2)-related antigenemia(n=6121) following vitamin A supplementation and standard vaccination. BacilleCalmette Guerin-vaccinated children were more likely to be PfHRP-2positive . No association was identified with parasitemia. Measlesand polio vaccination were not associated with malaria. Children receiving vitamin Awere less likely to present with parasitemia  and antigenemia . Future studies focusing on climate seasonality, placentalmalaria and HIV are needed to characterize better the association between vitamin Aand malaria infection in different settings.Recent studies, partly based on murine models, suggest childhood immunization andvitamin A supplements may confer protection against malaria infection, althoughstrong evidence to support these theories in humans has so far been lacking. Weanalyzed national survey data from children aged 6–59 months in foursub-Saharan African countries over an 18-month time period, to determine the risk ofDOI:http://dx.doi.org/10.7554/eLife.03925.001 More than half of the world's population is at risk of malaria, with an estimated 198million clinical cases each year. A vaccine that fully prevents it has not yet beendiscovered. Most cases of malaria occur among children living in sub-Saharan Africa,a region where many receive routine vaccinations designed to prevent other diseases;for example, 75% of children in sub-Saharan Africa receive measles vaccines. Manyalso receive vitamin A supplements, which have been linked not only to the protectionof a child's vision, but also to a lower risk of death and an improved ability tofight off infections.Plasmodium parasites that cause malariainfections. However, this effect depended on several factors, including how thevaccine was administered and whether the vaccination was given before or after themouse developed malaria.Some researchers have suggested that vitamin A supplements and routine childhoodvaccinations for other diseases may also provide some protection against malaria. Forexample, some studies performed in mice have shown that a commonly used tuberculosisvaccine may eliminate Plasmodium parasites.It is less clear whether vaccines or vitamin A have antimalarial effects in humans.To address this, Hollm-Delgado et al. analyzed national survey data collected fromthousands of children aged between 6 months and 5 years old who lived in fourdifferent countries in sub-Saharan Africa. The surveys contained information aboutthe vaccines and supplements the children received, and whether their blood showedsigns of infection with malaria-causing Schistosoma, producing false-positive results for malaria.Hollm-Delgado et al. found that routine vaccinations did not affect the likelihood ofmalaria parasites being detected in the child's blood. However, children vaccinatedagainst tuberculosis were more likely to have a specific type of protein releasedwhen malaria infects the blood. Hollm-Delgado et al. suspect that the tests mayactually have inadvertently detected other parasitic infections in the children, suchas In contrast, Hollm-Delgado et al. found that children who received vitamin Asupplements were less likely to become infected with malaria. The benefits of thesupplements appeared to be affected by several conditions, including the time of yearwhen the children received their supplements or when they were tested for malaria,and whether their mother had malaria when pregnant. Clinical trials are now needed toconfirm these results and investigate how effectively vitamin A prevents malaria.DOI:http://dx.doi.org/10.7554/eLife.03925.002 Malaria remains a major public health challenge, with more than 7% of deaths amongchildren under 5 years in the world attributable to the disease . Many ofPlasmodiuminfection  vaccine may confer sterilizing protection against nfection . Howevers tested. Converss tested. Epidemis tested.Plasmodiumfalciparum-infected erythrocytes, by way of its active metabolite, retinoicacid   and deatoicacid . Lessceoicacid  and Tanzoicacid , a clustoicacid . On the oicacid . The undoicacid .Plasmodium parasitemia and P. falciparum-specificantigenemia following vitamin A supplementation and standard vaccination in childrenunder 5 years of age in sub-Saharan Africa.Based on this evidence, the purpose of this study was to determine the risk ofP. falciparumhistidine rich protein-2 (PƒHRP-2) tests, 3356 (28%) weretested only with blood films, and 30 (0.2%) were tested with only rapidPƒHRP-2 tests. Among those tested, we identified 3544 (30%)with positive blood films for Plasmodium spp. and 3131 (36%) withpositive PƒHRP-2 antigenemia. Results from both tests were 87%concordant. Complete confounder information was available for 8390 (70%) subjects whowere tested for parasitemia and 6121 (70%) tested for antigenemia. Of 20,984 children who presented health cards during survey interviews, 18,413 wereeligible for blood screening from which 12,058 provided capillary blood for malariatesting . From thPlasmodium parasitemia, it was linked to anincreased risk of PƒHRP-2 antigenemia. This effect remainedafter controlling for patient characteristics associated with BCG use in the inverseprobability weighted (IPW) model. DTP was associated with a lower risk ofPlasmodium parasitemia and PƒHRP-2, butonly in the weighted models. Measles and poliomyelitis vaccination were notassociated with malaria antigenemia or parasitemia. Plasmodiumparasitemia and PƒHRP-2 antigenemia , the more timepassed since supplementation (ptrend<0.001) or if supplementation wasgiven during the wet season (ptrend=0.04). PƒHRP-2 antigenemia was stronger if children had a negativemalaria blood film, were 36–59 months of age, primigravidae, or had not takenan antimalarial recently. Vitamin A was more protective againstPƒHRP-2 among children with low birth weight but was lessprotective against parasitemia. The association between vitamin A andPƒHRP-2 was stronger among children tested during the dryseason or who lived in communities with a longer rainy season, and in communitieswith a lower annual mean for enhanced vegetation index (EVI). Associations betweenvitamin A and parasitemia were stronger among communities with a higher meanpredicted sickle cell hemoglobin (HbS) allele frequency or median predictedglucose-6-phosphate dehydrogenase deficiency (G6PDd) allele frequency. We hadinsufficient data to identify cross-interactions between vaccines and vitamin A usingan adjusted model, but in unadjusted models we found no significant interactionsbetween vaccination and vitamin A supplementation.Vitamin A supplementation appeared protective against igenemia . These ePlasmodiumparasitemia and PƒHRP-2 antigenemia among children aged6–59 months. None of the vaccines examined in our study  appeared to be linked with Plasmodium parasitemia inunweighted models but we did observe a threefold to fourfold elevated risk ofPƒHRP-2 among BCG vaccinated children. Controlling for factorsrelated to the utilization of vaccines, this association remained significant for BCG.Measles vaccination did not appear to be associated with eitherPlasmodium parasitemia or PƒHRP-2 antigenemiasuggesting that the vaccine may not incur any long-term differences in the risk ofmalaria, as previously suggested in the literature.In this large, population-based study within sub-Saharan Africa, vitamin Asupplementation was associated with a negative risk of Our study extends previous randomized clinical trial evidence from Burkina Faso, Papua NDifferences in the impact of vitamin A on malaria infection by birth weight, andhaving a mother who used antimalarials during the child's gestational period suggestsexposure to placental malaria may play a critical role in vitamin A's protectiveeffect mechanisms later on in childhood. Published evidence regarding placentalmalaria, antenatal vitamin supplementation, and infant health are limited. However,Plasmodium parasitemia and PƒHRP-2antigenemia may be due to another factor. In 2012, the tropical diseases research(TDR) programme reported false positivity caused by schistosomiasis and Chagas in onelot of Paracheck Pƒ rapid diagnostics . However, due to geography, blood filmswere likely to be detecting P. falciparum due to geography , HIV is an established risk factor for malaria infection . To addrData were extracted from Macro International Demographic and Health Surveys (DHS), a databhttp://reverb.echo.nasa.gov/), 42 of which were required to createeach 8-day mosaic of Africa for 2000 through 2012. EVI was then calculated foreach mosaic using the equation defined by To estimate seasonality, an EVI dataset was used as it relates to seasonalprecipitation, albeit lagged in time, and the emergence of vector mosquitospecies. This dataset was created from moderate resolution imagingspectroradiometer (MODIS) bidirectional reflectance distribution function(BRDF)-corrected composites (MCD43B4) , which hPredicted allele frequencies for HbS  and G6PDInformation regarding children's vaccination and vitamin A supplementation historywas ascertained from health card data recorded during the survey interview. Thisincluded a child's date of vaccination using BCG, DTP, measles and polio as well asthe number of vaccine doses received (for DTP and polio only). We used these datathen to calculate age in months at the moment of vaccination, and time in monthssince vaccination. Children whose mother/guardian responded in the affirmative tovaccination (e.g. child vaccinated during campaign) but did not provide health cardsto document this information were excluded from the analysis. For vitamin Asupplementations, data were recorded regarding the date of last dose received and useof the vitamin supplement during the 6 months prior to the survey interview.Plasmodium spp. to assess parasitemia and the presence ofPƒHRP-2 to assess antigenemia. The microscopic presence ofPlasmodium spp. was determined by reading Giemsa-stained thickblood films. Species of Plasmodium spp. were not recorded. Capillaryblood was collected during household interviews and tested onsite for malariaantigenemia. Pƒ-specific HRP-2 antigens were detected usingthe rapid diagnostic Paracheck Pf  for Burkina Faso and Senegal surveys and first response malaria antigen HRP-2 for the Rwanda survey. Thick blood smearswere prepared by trained technicians for all children, and sent for gold standardtesting of malaria to the Centre National de Recherche et de Formation sur lePaludisme (Burkina Faso), Centro de Investigações em Saúde deManhiça (Mozambique), TRAC/Plus Malaria Unit in Kigali (Rwanda) and Departmentof Parasitology at the Université Cheikh Anta Diop . Internal qualitycontrol was conducted using standard laboratory protocol and procedures, includinghaving slides randomly read by different laboratory technicians or the chieftechnician/supervisor for internal quality control measures. External quality control(Mozambique only) included selecting samples by a computerized system developed byICF Macro and sending them for external quality control testing at the Muraz Center(Bobo-Dioulasso).Primary study outcomes included the microscopic detection ofCapillary blood was collected during household interviews from all women aged15–29 years. Samples were collected on filter paper, dried for 24 hr and thensent for testing to the Centre Regional de Transfusion Sanguine de Ouagadougou(Burkina Faso), National Reference Laboratory (Rwanda) and National ReferenceLaboratory of Bacteriology and Virology at A Le Dantec Hospital . Viralantibodies were detected using the enzyme-linked immunosorbent assay (ELISA)-basedassay VironostikaÒ HIV Uni-Form II plus O . Positive samples and a portion of negative samples were tested using theELISA assay EnzygnostÒ Anti-HIV ½ plus .Discordant samples were further tested using the Pepti Lav assay  or InnoLia (Burkina Faso and Rwanda). Internal qualitycontrol measures  included using control wells(positive and negative) provided with manufacturer's screening kit on each testplate. All positive samples and 10% of randomly selected negative samples were alsotested using more than one assay, with discordant samples further tested using athird assay. External quality control measures (Burkina Faso only) consisted ofsending all positive samples and 80 randomly selected negative samples to the CenterMuraz (Bobo-Dioulasso) for additional HIV testing.Multivariable logistic regression models were used to estimate associations betweenmalaria and childhood vaccination/supplementation. The impact of age at vaccination,time since vaccination, and malaria season on the date of vaccination were alsoexamined. To avoid statistical inference errors in our results due to clustering andstratification from the sampling process, we defined country and PSU as strata, andhouseholds as clusters using logistic regression models that accounted for thecomplex survey data structure. To test for linear trends, covariates were included ascontinuous terms in the regression model. We adjusted models for continuous variablesof age and wealth index score, and categorical variables of gender (boy vs girl),malaria treatment during previous week (yes vs no), ownership of bed net (yes vs no),proportion of household members under 5 years old using bed net during previous night, indoor household insecticide spraying (yes vs no),malaria transmission season (dry vs wet), community setting ,mother's highest educational level , and access to antenatalcare during last pregnancy (yes vs no). Complete subject analysis was used. Effect modifiers for each vaccine/vitamin model were identified by individuallytesting cross-product terms between type of immunization/supplement received andindividual-based characteristics including age, gender, pregnancy order of child,birth weight, receipt of antimalarial during previous week, receipt of intestinalworm treatment during previous 6 months, if mother took antimalarial during child'sgestational period, family owns bed net, or type of community setting;climate-related factors including malaria transmission season at time ofimmunization, length of rainy season, length of time that EVI was above annual mean,range of EVI per year, or annual mean for EVI; and malaria genetic factors includingthe predicted allele frequency in a community for HbS (mean estimate) and G6PDd(median estimate). To account for uncertainty in spatial prediction values for HbSand G6PDd, we conducted a sensitivity analysis by excluding from models anyprediction value with degrees of uncertainty (i.e. interquartile range) greater than20%.Due to the observational nature of the data (not randomized), we controlled fordifferences in patient characteristics associated with vaccine or vitamin use, byinverse probability weighting of models using the propensity score for receiving avaccine or vitamin supplement. Predictors of vaccination/supplementation includedin propensity score models were: age, gender, low birth weight, presence ofradio/television in home, urban versus rural setting, breastfeeding status, wealthindex score, as well as mother's age, highest education level, antenatal careduring last pregnancy, and tetanus status during last pregnancy. Standardized biasmeasures were used to assess numerical balance in propensity score covariatedistributions, before and after IPW weighting .FactorsAll statistical analyses were completed using SAS v.9.3 and R v.3.0.0 .Prior to enrolling in the survey, the parent/adult responsible for the child had toprovide written informed consent to participate, along with additional informedconsent for blood sample collection and testing. If a child tested positive duringthe survey using malaria RDT, their parent/guardian was informed of results and thechild was immediately given treatment according to the current treatment guidelines.HIV tests for adults were anonymous, with survey data only being linked to testresults after respondent identifiers were deleted from the database. Although HIVtest results were not available to study participants, patients were offered cardsenabling them to obtain free HIV testing and counselling at Voluntary TestingCenters. The Institutional Review Board of ICF Macro reviewed and approved theMEASURE Demographic and Health Surveys Project Phase III, in compliance with UnitedStates (U.S.) Department of Health and Human Services (DHHS) regulation 45 Code ofFederal Regulations (CFR) 46 for ‘Protection of Human Subjects’research. Protocols for blood specimen collection, HIV and malaria testing were alsoapproved by ICF Macro International Institutional Review Board (IRB) [all countries]in compliance with U.S. DHHS regulation 45 CFR 46 for ‘Protection of HumanSubjects’ research, National Ethics Committees  and U.S. Centers for Disease Control [Rwanda only]. For Senegal, supervisoryvisits were also organized by the National Ethics Committee to ensure fieldcompliance with ethical regulations. Geographical waypoints were randomly displacedto ensure confidentiality . DHS provided anonymized data to study authors. As a secondary analysisof publically available de-identified data, the study was determined exempt fromethics review by Johns Hopkins Bloomberg School of Public Health IRB Office accordingto U.S. DHHS regulation 45 CFR 46.102 for non-human subject research. reviewprocess). Similarly, the author response typically shows only responsesto the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of thepublished articles . An edited version of theletter sent to the authors after peer review is shown, indicating the substantiveconcerns or comments; minor concerns are not usually shown. Reviewers have theopportunity to discuss the decision before the letter is sent , a Reviewing editor, and 4 reviewers.Thank you for sending your work entitled “Vitamin A supplements, routineimmunization, and the subsequent risk of malaria among children 6 to 59 months ofage” for consideration at The following individuals responsible for the peer review of your submission have agreedto reveal their identity: Stephen Tollman (Reviewing editor) and John Pettifor (peerreviewer). Please note that a further three reviewers conducted detailedassessments.The Reviewing editor and the reviewers discussed their comments before we reached thisdecision, and the Reviewing editor has assembled the following comments to help youprepare a revised submission.Schistosomiasis. Due to the low malaria burden in twoof the four countries, the study is driven by data from Burkina Faso and Mozambique. Ifconfirmed, the findings are of importance to the many children living in high malariainfection zones—with potential policy impact.The study aimed to assess the effects of both vitamin A supplementation and immunizationon malaria parasitemia and antigenemia in sub–Saharan children between the agesof 6 and 59 months, in order to verify the findings in experimental animals in whichvitamin A supplementation and immunizations have been shown to confer some protectionagainst malaria infection. The authors conclude that BCG vaccination did not influencemalaria parasitemia but did increase PƒHRP2 antigenemia, while vitamin Asupplementation reduced the prevalence of both parasitemia and antigenemia. The BCGeffect on antigenemia was thought to be due to an increase in false positivity due toChagas disease or Substantive comments are detailed below. However, the major concern of all reviewersrelates to limitations of the DHS dataset, ensuring that comparisons and analyses of theobservational work are valid and adequately explained/justified, gaining a betterunderstanding of country–level effects, and concerns about ethical practice inglobal health survey work.Major/substantive comments:1) The four populations studied differ in malaria burden, climate and vitamin A use. Itis unclear whether some of the differences between the blood film and RDT assessmentsare due to the omission of Mozambique  from the latter. Further, amidst the range ofcorrecting/confounding variables it is not clear how 'country' as ageopolitical unit was considered.2) Because the populations are not the same for blood film and antigenaemia readings, itis hard to interpret the findings from the two analyses . This makes it difficult to single out the take–homemessage; or have confidence that the antigen findings are generalisable.3) Clearly there is value in capitalising on the cross–country comparisonspossible from DHS data. But the Methods section could be much clearer on whetherchildren vaccinated were compared with those unvaccinated . Since this is observational rather than trialsdata, adequate explanation of this design/analysis issue is essential. 4) Quality of National Data: The authors are silent on country–level qualitycontrol activities for malaria diagnosis, especially on the expertise and methods usedby those responsible for blood smear readings.5) Ethical approval: The authors state that “the study was determined exempt fromethics review by Johns Hopkins Bloomberg School of Public Health”. It is notappropriate to have such approval from Johns Hopkins only; an understanding of theethical status of the work in countries where the data were collected is needed.Furthermore, it would be helpful to know how children who tested malaria positive weremanaged.6) It is mentioned that 20,948 children presented health cards during the surveyinterviews. It is stated that 12,028 were tested using thick blood films and 8702 usingRDTs, but were the tests on each child limited to either thick film examination or RDTor both? If only one, how was concordance determined; and if both, what was the overlap or RDT(8702) to sum to 20,948)?Note: numbers in the text and 7) Please provide information on selection of confounders. And consider:a) HIV status and vertical HIV exposure, as these may influence a child's immuneresponse to both vaccination and vitamin A supplementation;b) Number of doses of each of the immunizations or vitamin A supplements received (it islikely that older children had more immunizations and A supplements than younger). Wereimmunization schedules and supplementation programmes similar in the four countries?8) Third paragraph in the Results section: the authors indicate that vitamin A was moreprotective the greater the time from the last dose. The last panel of 9) Fourth paragraph in the Discussion section: the authors suggest that the increase inantigenemia associated with BCG vaccination might be due to false positivity from otherparasitic infections; also that false–positivity has been reported in Paracheckassays. However, given low numbers and wide CIs, a type I error could be a possibility.Also, since different assays were used, can the authors advise whether the increase inantigenemia was similar in the subsamples when using either one of the assays?Schistosomiasis.Please note that Chagas disease is not found in these countries. As most children testedwere very young, they are unlikely to have significant exposure and antibodies to 1) The four populations studied differ in malaria burden, climate and vitamin Ause. It is unclear whether some of the differences between the blood film and RDTassessments are due to the omission of Mozambique  from the latter. Further, amidst the range ofcorrecting/confounding variables it is not clear how 'country' as ageopolitical unit was considered.We thank the reviewers for their insightful comment and recognize their concerns. Toaddress the issue of study population differences, we ran a sensitivity analysis usingblood film data including and excluding Mozambique from our analysis. This analysis(Author response table 1) showed that when excluding Mozambique data, vitamin A not onlyremained protective but the magnitude of its protective effect was amplified. In otherwords, including data from Mozambique in the blood film model, provided a moreconservative estimate of vitamin A effects. We have included the more conservativeresults in our paper. Unfortunately, RDT testing was not conducted among Mozambiquesubjects making it impossible to conduct similar sensitivity analysis for RDTtesting.Our models did adjust for factors that could explain differences between the fourpopulations (including vitamin A use and climate). This is indicated in the seventhparagraph of the Materials and methods section of the manuscript. Furthermore, to avoidstatistical inference errors in our results due to clustering and stratification fromthe sampling process, we used logistic regression models that accounted for complexsurvey data structure and defined country and primary sampling unit (∼district)as strata, and household as cluster. We recognize that this was not fully clear in ouroriginal paper and have now incorporated this information in the seventh paragraph ofthe Materials and methods section of the manuscript.Author response table 1. Adjusted PS-IPW relative risk of malaria, by group of childrenanalyzedAbbreviations: PS IPW, Propensity-score inverse probability weighting2) Because the populations are not the same for blood film and antigenaemiareadings, it is hard to interpret the findings from the two analyses . This makes itdifficult to single out the take–home message; or haveconfidence that the antigen findings are generalisable.We apologize for the confusion. As previously mentioned, our models was adjusted forfactors that could explain population differences. More importantly, our analysis inAuthor response table 1 shows that when Mozambique data was excluded from the blood filmanalysis , themagnitude of protective vitamin A effects increased causing results in both the bloodfilm and antigenemia models to be nearly identical.Plasmodium spp., whileantigenemia is testing for one particular type of Plasmodium. We recognize the confusion caused by using these two terms,and have tried to make the distinction between these tests clearer by re-labeling tablesand figure using taxonomic terms for malaria, restructuring malaria sp. detection throughout the manuscript. However, it shouldbe noted that due to geography, blood films were likely detectingP.falciparum. This would explain the similar results identifiedbetween blood film and antigenemia results when excluding Mozambique data from ouranalysis.Technically speaking, blood films and antigenaemia readings are used to identify twodifferent outcomes. Blood films test for That said, we have chosen to include Mozambique data for blood films in our manuscriptsince these results provide more conservative estimates of Vitamin A effects. All ofthis discussion is now included in the fifth paragraph of the Discussion section in themanuscript.3) Clearly there is value in capitalising on thecross–country comparisons possible from DHS data. But theMethods section could be much clearer on whether children vaccinated were comparedwith those unvaccinated . Since this is observational rather than trials data, adequate explanationof this design/analysis issue is essential. We fully agree with the reviewers regarding the value of cross-country comparisons. Infact, during the initial stages of our analysis we had hoped to presentcountry-stratified models, but were unable to do so due to issues with modelconvergence. A similar problem occurred when including country as a covariate in‘overall’ models investigating effect modification. As for theobservational nature of the study, we recognized early on that vaccination uptake initself might introduce a selection bias. To account for this, we used inverseprobability weighting by propensity scores. The propensity score was based on knownpredictors for use of vaccination. We recognize that this may not have been fully clearin our manuscript, and have now included this information under the header“Avoidance of Selection Bias” in the Materials and methods section of themanuscript. Please note that the unweighted models  provided more conservative estimates, and for this reasonare included in the paper.4) Quality of National Data: The authors are silent oncountry–level quality control activities for malariadiagnosis, especially on the expertise and methods used by those responsible forblood smear readings.We are grateful for the reviewers’ comment. Information on both internal andexternal quality control measures for malaria testing is now included in the fifthparagraph of the Materials and methods section. We have also included quality controlmeasures used for HIV testing in the same section.5) Ethical approval: The authors state that “the study was determinedexempt from ethics review by Johns Hopkins Bloomberg School of Public Health”.It is not appropriate to have such approval from Johns Hopkins only; an understandingof the ethical status of the work in countries where the data were collected isneeded. Furthermore, it would be helpful to know how children who tested malariapositive were managed.A specific section entitled ‘Ethical Considerations’ has now been added tothe paper, describing detailed information on ethical procedures related to the study.Please note that, aside from ethics review by JHSPH IRB, additional review was conductedduring the original DHS survey by the ICF Macro International IRB ,the National Ethics Committees (three countries), and by the U.S. Centers for DiseaseControl (one country). Also, if a child tested positive during the survey using malariaRDT, his/her parent/guardian was informed of the results and the child was immediatelygiven treatment according to the current treatment guidelines. This information is nowincluded in the “Ethical Considerations” paragraph in the manuscript.6) It is mentioned that 20,948 children presented health cards during the surveyinterviews. It is stated that 12,028 were tested using thick blood films and 8702using RDTs, but were the tests on each child limited to either thick film examinationor RDT or both? If only one, how was concordance determined; and if both, what wasthe overlap (and please explain how children were accounted for as either thick film or RDT (8702) to sum to 20,948)?Note: numbers in the text andare not all in agreement. Since microscopy is the stated 'goldstandard' it might be clearer if the analysis inincluded only those children with microscopy results.We apologize for the confusion. From 20,948 children who presented health cards duringsurvey interviews, 18,413 were eligible for blood screening from which 12,058 providedcapillary blood for malaria testing. 7) Please provide information on selection of confounders. And consider: a) HIVstatus and vertical HIV exposure, as these may influence a child's immuneresponse to both vaccination and vitamin A supplementation;We appreciate the potential impact of HIV on associations between vitamin A and malaria.Unfortunately, data on children’s HIV status was not available. However, 2540mothers of children participants in Burkina Faso, Rwanda and Senegal were tested by theDHS for HIV status. Using this data, we found less than 1% of mothers tested positivefor HIV, suggesting a low potential for vertical transmission in our study population.This information is included in the first paragraph of the Results section. Adjustingfor maternal HIV status in models using subjects whose mothers were tested, we foundthat the association between vitamin A and malaria remained protective (Author responsetable 2). Still, the low prevalence of maternal HIV in our study population  makes it difficult for us to rule out potential vitaminA*HIV interactions. We fully recognize the limitations of this indicator and havenow included a discussion of these concerns in the Discussion section of themanuscript.Author response table 2. Adjusted PS-IPW relative risk of malaria, by mother’sHIV statusb) Number of doses of each of the immunizations or vitamin A supplementsreceived (it is likely that older children had more immunizations and A supplementsthan younger). Were immunization schedules and supplementationprogrammes similar in the four countries?Our study models controlled for age both through the use of propensity scores and in theactual model. As for immunization and vitamin A schedules, Author response table 3 showsthat they were fairly similar across different countries. The main distributionmechanism was also similar for vitamin A. We have included this information in the firstparagraph of the Results section of the revised manuscript.1Author response table 3. Overview of Immunization Schedules for eachcountry*Guidelines report immunization schedule in months . Converted intoweeks for this table.1Adapted from: Unicef and WHO. Immunization Summary: The 2007 Edition.(published February 2007), Vaccination Information Management System: www.vimsdata.org and personal communication with E. Alden (UNICEF) onDecember 8, 2014.8) Third paragraph in the Results section: the authors indicate that vitamin Awas more protective the greater the time from the last dose. The last panelofdoes not seem to show this, nor does the statement tally with the firstparagraph of the Discussion.We apologize for the confusion. The increasingly protective effect occurs with malariaantigenemia but not malaria parasitemia. To avoid any confusion, this phrase has nowbeen removed from the Discussion.9) Fourth paragraph in the Discussion section: the authors suggest that theincrease in antigenemia associated with BCG vaccination might be due to falsepositivity from other parasitic infections; also that false–positivity hasbeen reported in Paracheck assays. However, given low numbers and wide CIs, a type Ierror could be a possibility. Also, since different assays were used,can the authors advise whether the increase in antigenemia was similar in thesubsamples when using either one of the assays?Please note that Chagas disease is not found in these countries. As mostchildren tested were very young, they are unlikely to have significant exposure andantibodies to Schistosomiasis.Schistosomiasis, there is growingevidence to suggest it may be an overlooked endemic disease among infants and youngchildren in Africa. We have included a statement and reference to this material in thesame section of the text. Finally, in terms of examining increases in antigenemiastratified by type of RDT, regrettably, the small sample size particularly in theBCG-negative, malaria-positive cells (2x2 table) led to problems in model convergencewhen stratifying results by diagnostic used.We thank the reviewers’ for their observations. We have now included an explicitstatement in the fourth paragraph of the Discussion section that Chagas is not endemicin the countries we studied. As for


Plasmodium infection, and its relations with the malaria disease, especially when comparing dry and rainy seasons in a hyperendemic area of West Africa.To study the manifestations of The study was carried out in an area where malaria transmission is high, showing important seasonal variations. One thousand children, representing the total child population (1–12 year old), were observed transversally at the end of three consecutive seasons (dry/rainy/dry). The usual indicators, such as parasite density, splenomegaly, anaemia, or febrile disease were recorded and analysed.Plasmodium falciparum was high in all age groups and seasons, constantly around 60%. The high transmission season (rainy) showed higher rates of anaemia and spleen enlargement and, in the youngest children only, higher parasite densities. There were also differences between the two dry seasons: in the first one, there was a higher rate of fever than in the second one (p < 0.001). Low parasite density  was never associated with fever during any season, raising some concern with regard to the usefulness of parasite detection. The possible origins of fever are discussed, together with the potential usefulness of analyzing these indices on a population sample, at a time when fever incidence rises and malaria is one potential cause among others. The distinction to be made between the Plasmodium infection and the malaria disease is highlighted.The prevalence of These data confirm previous hypotheses of a strong difference in malaria infection and disease between dry and rainy seasons. The most relevant seasonal indicator was not mainly parasite rate and density but anaemia, spleen enlargement, prevalence and possible origin of fever.Plasmodium infection and not necessarily of a malaria disease. In such a situation, it seems suitable to obtain, through national malaria teams, a well-defined situation of transmission and prevalence of Plasmodium infection following zones and seasons, in order to adapt control strategies. For researchers, a systematic management of data separately for dry and rainy season appears mandatory.In any situation (i.e. fever or not) and especially during the dry season, one must consider that detection of parasites in the blood is only evidence of a  Plasmodium infection in the population, prevalence of other diseases, genetic background, age, season, or nutrition [Plasmodium infection is high [In regions where malaria is highly prevalent, malaria-related morbidity and mortality are high and mainly affect subjects whose immunity is considered to be low, mostly young children  and pregnant women, especially during their first pregnancy ,2. Many utrition ,4. Becauutrition . To makeutrition . Moreove is high .Within a given area, there are often differences between the patterns of malaria indices between high and low-transmission seasons : parasitAmong other signs, anaemia is strongly linked to the malaria disease in areas where malaria transmission occurs ,11, and In order to explore further the manifestations of the malaria disease and the dynamics of malaria indices during dry and wet seasons in a hyperendemic area, about one thousand children were observed in a three times repeated cross-sectional study.The study aimed at following most common malaria indices during three consecutive seasons in children from 1 to 12 years in the rural village of Mendela . The main criteria for the choice of the village were based on the need of a population size of about 1,000 children aged from one to 12 years of age and the absence of a permanent pond throughout the year.The rainy season lasts six months (May – October) with a peak of precipitations  occurring from July to September. To check the possibility of an early malaria transmission during the dry seasons, daily rainfall records were obtained retrospectively from Sikasso meteorological station  through the U.S. National Climatic Data Center and the average daily rainfall was calculated on the period comprised between day 15 and day 45 prior to the medical and biological measurements . BecauseThe study was cross-sectional with three surveys in the same village. A total of 709 children were observed during the first dry season, 702 at the end of the rainy season, and 728 at the end of the second dry season Table : althougFollowing acceptation of the study from National Ethical Committee, Regional Direction of Health and Village Authorities, the purpose of the study was presented to the villagers. The family head of each individual participant signed an informed consent form.Axillary body temperature was measured using an electronic thermometer (Terumo®). Fever was defined as a temperature exceeding 37.5°C. PCV (packed cell volume or haematocrit) was chosen as the index to estimate anaemia, resulting from previous observations in the same region where PCV is strongly linked to haemoglobin concentration, leading mostly to normochromic anaemia. PCV was immediately assessed twice using a micro haematocrit centrifugator (Hettich®). According to WHO standards, severe anaemia (SA) was defined as PCV <30%. In this analysis, 32% was the threshold used for moderate anaemia (MA). Blood films were prepared from capillary blood and read by an experienced parasitologist from the Malaria Research and Training Centre, Faculty of Medicine, Pharmacy and Odontostomatology, University of Sciences, Techniques and Technology, P.O. Box 1805, Bamako, Mali, in order to estimate blood parasite density. The parasite count was calculated on the basis of an average concentration of 7,500 leukocyte/μl after observing 300 leukocytes in blood films. In the text, the different classes of parasite densities are referred as to: “very low” (1 to 199 p/μl), “low” , “moderate”  and “high” . Splenomegaly was evaluated by palpation and classified according to the criteria of Hackett, by MDs from the national team.Analyses were performed using the R statistical software. The agreement between both PCV measurements was checked by the method described previously, analysing the differences of pairs of measurements against their means . During The average daily rainfall, calculated as described in the Methods section, was estimated at 1.6, 5.2 and 2.7 mm prior to the first dry season, rainy season and second dry season respectively. During the three surveys, 58% of children included in the study were aged between 3 and 9 years, while the children below 3 years of age represented about 16% of the sample. Females and males were equally represented during each survey , significant differences were observed in the prevalence of P. falciparum, between the rainy season and the dry seasons (p < 0.05).The prevalence of The definition of mild and severe anaemia is based on cutoff values chosen from the distribution of haematocrit (PCV). The haematocrit level was subject to variations between age classes  of positive subjects varied according to age group, season and haematocrit level , and this was also true within all age classes taken separately. The GMPD from the first dry season was significantly higher than in the second dry season  and in the youngest children only (aged 1-3y.), this difference was not significant (see Discussion section).Fever prevalence between seasons was first compared in univariate analyses between seasons using a low (37.5°C) or higher (38°C) threshold. Using the 37.5°C threshold, fever prevalence in the first dry season  was higher, but not significantly different from the rainy season , but it was significantly higher than in the second dry season . If the threshold was set at 38°C, fever prevalence was significantly higher in the rainy season  than in the second dry season , but not significantly different from the first dry season . In any case, the high prevalence of fever observed in the first dry season suggests that an outbreak of unknown origin could have occurred around that period of time.never associated with fever during any season. This observation is common when endemicity is high, raising some concern with regard to the usefulness of parasite detection in febrile children, especially if parasite density cannot be estimated due to physiological dynamics and technical limitations .When stratifying children into five classes of parasite density, there was a frequent, though not systematic, association between moderate or high parasite density  and fever: it is worthy of note that moderate parasite density  was not associated with fever during the first dry season, although a high GMPD was found (p = 0.089). Finally, very low (<200 p/μl) and low parasite density  was Although higher percentages of febrile children were observed among those with severe anaemia, when adjusting for age, the prevalence of fever (38°C or 37.5°C) was not significantly higher among children with severe anaemia compared to those without severe anaemia, during any of the three seasons studied. Therefore, age appeared as a confounding factor when measuring the association between anaemia and fever. The same pattern was observed for the association between splenomegaly and fever for a threshold set at 38°C, but not for a threshold set at 37.5°C: in this case, these two variables were linked during the rainy season (p < 0.002), the second dry season (p < 0.005), but not significantly during the first dry season (p = 0.073).P. falciparum is endemic, other factors can contribute to it . In the village where this study was carried out, previous analyses showed that these factors were present but at a level which cannot explain a significant effect beside falciparum malaria. Therefore, anaemia and/or levels of PCV can be used, at least at a population level, to detect a rise in malaria transmission and malaria disease.Although malaria is considered to be the main factor responsible for anaemia where If parasite density is a parameter to consider in a febrile child, it is not sufficient for diagnosing the malaria disease for two main reasons: first, even when malaria parasite discovery is concomitant with fever, it is not necessarily causal due to the high proportion of asymptomatic cases. For example, it has been shown that a certain number of patients living in a hyperendemic area carried bacterial infections . Second,Although these results were obtained in a series of three cross-sectional surveys, they highlight classical seasonal patterns of malaria indices: they show a seasonal rise of severe anaemia/moderate anaemia and splenomegaly during the high-transmission season, and found a strong association between anaemia and age, splenomegaly and moderate-to-high parasite density. This latter association, as well as the absence of link with low or very low parasite density confirms previous findings -25.P. falciparum did not follow any significant change: in these particular epidemiological conditions (high transmission), there is a discrepancy between the presence of malaria parasites and malaria-related morbidity.It should be noted that the observed important variations of malaria indices between seasons occurred while the prevalence of Plasmodium in the blood appears as necessary, but not as sufficient to assert malaria as the origin of fever.The fact that fever was associated only with a few of the malaria indices can be explained by the presence of other diseases, and by age being a confounding factor, the youngest children being the most affected by morbidity signs. It is to note however that low or very low parasite densities  were never associated with fever, even if not adjusting for age, suggesting a high proportion of asymptomatic carriers within this range of parasite densities. This observation argues in favour of a diagnosis using indicators supplementary to the presence of parasites: the detection of Interestingly, these data from two consecutive dry seasons show a striking difference in the prevalence of fever. In general, the observations of the first dry season appear unusual and those of the second dry season showed the more expected “low” values for the following indices: fever prevalence, parasite density, rate of severe anaemia and rate of splenic enlargement . Two hyp. Therefore, the observation of high parasite densities during that first dry season could suggest that malaria transmission had abnormally increased, explaining a rise in clinical malaria cases. Nonetheless, parasite density cannot be seen as the unique risk factor: many studies attempted to estimate a cutoff parasite density supposed to trigger fever and clinical malaria (pyrogenic threshold) but there is no consensus around this theoretical value, which instead appears to vary with age and epidemiological conditions [The first hypothesis finds its stronger argument in the fact that the mean parasite density was particularly high, and as high as in the rainy season. Statistically, high parasite densities are associated with an increased risk of fever -28. Thernditions -34.middle-term indices, because they vary with a delay of several days or weeks after the malaria disease is declared, in contrast to parasite density or body temperature . Therefore, despite the fact that those parameters are expected to be more sensitive to variations in a low-transmission season , it remains also possible that this survey happened at the very beginning of a malaria epidemic, when anaemia and splenomegaly had not yet increased significantly.The second hypothesis - most febrile children suffered from an infection that is not malaria - is supported by several arguments: first, the observation that most fevers from this first dry season were below 38°C may suggest a disease other than malaria, as this latter is generally associated with higher body temperatures . Second,in fine the disease affecting each child. This work did not include such a follow-up ; this may be needed in further studies. Nonetheless, these data show important seasonal changes in the pattern of malaria indices, reflecting classical or particular conditions in which “stable” indicators  reflect much better the differences between high or low transmission seasons. The main argument to explain the difference between the two dry seasons is not based only on haematocrit or parasite density, but on a robust agreement between all the indices recorded in this study.With the data available here, it is not possible to go further this discussion and choose one hypothesis or the other. In particular, the cure or relapse of symptoms in children within the days after treatment would have been useful to determine Although determining the origin of fever and the appropriate treatment will remain a probabilistic approach whatever the diagnosing strategy is, a better understanding of the malaria disease would help health workers by clarifying the most useful clinical signs with which to assess patients. Fever incidence sometimes suddenly increases in regions where malaria can be present, and relying only on the detection of parasites in patients can be misleading. Routine surveys of malaria indices over several seasons can usefully increase the comprehension of malaria with regard to regional conditions: in return, it could foster greater confidence in the identification of subsequent epidemics.Plasmodium origin [Moreover and possibly the most important, the knowledge of malaria endemicity in a given area is probably the best information to decide whether or not a systematic laboratory detection of malaria parasites in febrile children would provide a better “proof” of its m origin .Finally, these results are a strong argument to advocate the necessity, in any case, to analyse research and routine data on malaria separately for the dry and the wet seasons in areas where an important seasonal variation occurs in the transmission of malaria.PCV: Packed cell volume (haematocrit); SA: Severe anaemia; MA: Moderate anaemia; GMPD: Geometric mean of parasite density; RDT: Rapid diagnostic test.The authors declare that they have no competing interests.DLA performed data analysis and manuscript preparation. OKD and AM managed the teams for data collection; TS supervised the collection of clinical data. AR developed the concept and the design of the study. AR, BG, JF and SM directed the database analysis. All authors read and approved the final manuscript.Anaemia among different strata of children. Mean haematocrit values (PCV), severe anaemia (SA) and moderate anaemia (MA) rates and odds ratios (OR) showing their associations with various variables in children. (n) Number of subjects in each class. (‡) p-value obtained by comparing values from different strata with the first line as the reference, using Wilcoxon tests (PCV means) or Chi-square tests (SA and MA rates). (*) Statistical significance was set at 0.05. (**) p-value less than 0.001.Click here for file


Grant Dorsey and colleagues investigate the efficacy of three antimalarial drugs for preventing malaria in children living in Uganda, an area of high transmission intensity.Please see later in the article for the Editors' Summary Chemoprevention offers a promising strategy for prevention of malaria in African children. However, the optimal chemoprevention drug and dosing strategy is unclear in areas of year-round transmission and resistance to many antimalarial drugs. To compare three available regimens, we conducted an open-label randomized controlled trial of chemoprevention in Ugandan children.p<0.001) for DP, 28%  for TS, and 7% for SP . PQ levels were below the detection limit 52% of the time when malaria was diagnosed in the DP arm, suggesting non-adherence. There were no differences between the study arms in the incidence of serious adverse events during the intervention and the incidence of malaria during the 1-y period after the intervention was stopped.This study was conducted between June 28, 2010, and September 25, 2013. 400 infants were enrolled and 393 randomized at 6 mo of age to no chemoprevention, monthly sulfadoxine-pyrimethamine (SP), daily trimethoprim-sulfamethoxazole (TS), or monthly dihydroartemisinin-piperaquine (DP). Study drugs were administered at home without supervision. Piperaquine (PQ) levels were used as a measure of compliance in the DP arm. Participants were given insecticide-treated bednets, and caregivers were encouraged to bring their child to a study clinic whenever they were ill. Chemoprevention was stopped at 24 mo of age, and participants followed-up an additional year. Primary outcome was the incidence of malaria during the intervention period. During the intervention, the incidence of malaria in the no chemoprevention arm was 6.95 episodes per person-year at risk. Protective efficacy was 58%  every year. Malaria parasites, which are transmitted to people through the bites of night-flying mosquitoes, cause a characteristic fever that needs to be treated promptly with antimalarial drugs to prevent anemia and organ damage. Prompt treatment also helps to reduce malaria transmission and is a component of the Global Malaria Action Plan, which aims to control and eventually eliminate malaria. Other components of this plan include the provision of insecticide-treated bednets for people to sleep under to avoid mosquito bites and indoor residual spraying with insecticides. Widespread deployment of these preventative tools and the increased availability of effective antimalarial drugs have greatly reduced malaria-related deaths worldwide over the past decade, but new strategies are still urgently needed to reduce the burden of malaria among those most at risk—young children living in Africa.One promising strategy for the prevention of malaria in African children is the use of antimalarial drugs to prevent rather than treat malaria. In trials, giving infants sulfadoxine-pyrimethamine (SP) alongside routine vaccinations, for example, reduced the incidence of malaria (the number of new cases in the population in a year) by about 30% during the first year of life (a protective efficacy of 30%). However, the optimal chemoprevention drug and dosing strategy for children living in African regions where there is year-round transmission of malaria and where resistance to antimalarial drugs is common remains unclear. Here, the researchers undertake an open-label randomized controlled trial (RCT) of chemoprevention in infants in the Tororo District of eastern Uganda, an area with intense year-round malaria transmission. RCTs compare outcomes in groups of people chosen to receive different interventions through the play of chance; in open-label RCTs, both the researchers and the participants know which treatment is being administered.The researchers assigned 393 six-month-old infants to receive no chemoprevention, monthly SP, daily trimethoprim-sulfamethoxazole (TS), or monthly dihydroartemisinin-piperaquine (DP) until they were 24 months old. SP and TS block the production of folic acid, which malaria needs for survival, whereas DP is a newer artemisinin-based combination therapy (ACT). All the drugs were given at home without supervision, and caregivers were asked to bring their children to a study clinic whenever they were ill. During the intervention, the incidence of malaria was 6.95 episodes per person-year at risk in the no chemoprevention arm but only three episodes per person-year at risk in the DP arm. That is, the protective efficacy of DP was 58%. By contrast, the protective efficacies of TS and SP were 28% and 7%, respectively. However, for SP the protective efficacy was not statistically different compared to the no chemoprevention arm. Notably, piperaquine levels on the day that malaria was diagnosed were below the detection limit in half of the malaria episodes in the DP arm, which suggests that a complete dose of DP had not been given to the infant in the previous month, despite caregivers reporting that they had administered virtually all the assigned doses. Finally, the incidence of serious adverse events was similar in all the study arms during the intervention, as was the incidence of malaria during the year after the intervention, which suggests that the chemoprevention strategies did not affect the development of naturally acquired immunity.These findings show that, for children living in an area of intense malaria transmission, monthly DP was the most efficacious strategy for malaria chemoprevention but that adherence to the strategy may have been a problem. These findings also suggest that monthly SP and daily TS may not be appropriate chemoprevention strategies in areas of high transmission intensity, particularly those where resistance to antifolate drugs is common. The accuracy of these findings may be affected by drug administration being self-reported and by the number of comparisons included in the trial, which may have increased the risk of false-positive results. Moreover, the results of this trial may not be generalizable to other regions of sub-Saharan Africa. Overall, however, these results suggest that monthly DP is a strategy worth considering in regions in need of improved malaria control measures, with the important caveat that widespread ACT use for chemoprevention could compromise the efficacy of ACT when used for treatment.http://dx.doi.org/10.1371/journal.pmed.1001689.Please access these websites via the online version of this summary at malaria , including information on malaria chemoprevention; the World Malaria Report 2013 provides details of the current global malaria situation, including information on malaria in UgandaInformation is available from the World Health Organization on malaria, including information on ways to reduce malaria cases and deaths; it also provides a selection of personal stories about malaria, including a story about malaria in a child in AfricaThe US Centers for Disease Control and Prevention provide information on Roll Back Malaria Partnership on the global control of malaria and on the Global Malaria Action Plan (in English and French); its website includes fact sheets about malaria in Africa and about children and malariaInformation is available from the malaria (in English and Spanish)MedlinePlus provides links to additional information on trial is availableMore information about this  Malaria remains one of the most important infectious diseases worldwide, with an estimated 219 million cases and 660,000 deaths in 2010 The use of antimalarial drugs for the prevention of malaria in children at high risk has recently received widespread attention. Most studied is intermittent preventive therapy in infants (IPTi) with sulfadoxine-pyrimethamine (SP) at the time of routine vaccinations. A pooled analysis of six randomized controlled trials reported that IPTi with SP was safe and was associated with a 30%  protective efficacy against malaria in the first year of life To compare available drug regimens for the prevention of malaria in an area with high antifolate resistance and intense, year-round transmission, we conducted a randomized controlled trial evaluating three regimens for the prevention of malaria in young children in Tororo, Uganda: monthly SP, daily TS, and monthly dihydroartemisinin-piperaquine (DP), an ACT that is especially attractive because of its excellent treatment efficacy and prolonged post-treatment prophylactic effect Ethical approval was obtained from the Uganda National Council for Science and Technology, the Makerere University School of Medicine Research and Ethics Committee, and the University of California, San Francisco, Committee on Human Research.We performed a randomized, controlled, open-label trial from June 28, 2010, to September 25, 2013, comparing the efficacy and safety of three regimens versus no therapy for the prevention of malaria in Tororo District, eastern Uganda, an area with intense year-round malaria transmission and an entomological inoculation rate estimated at 562 infectious bites per person-year in 2002 Convenience sampling was used to enroll a cohort of 400 infants of 4–5 mo of age from the Tororo District Hospital Maternal and Child Health Clinic between June 28, 2010, and February 24, 2011. Eligibility criteria included the following: (1) born to HIV-uninfected mothers, (2) residency within 30 km of the study clinic with no intention of moving outside the study area, (3) agreement to come to the study clinic for any illness and to avoid medications outside the study protocol, (4) provision of informed consent by parent/guardian, (5) no history of allergy or sensitivity to any study drugs, (6) absence of active medical problem requiring in-patient evaluation or chronic medical conditions requiring frequent attention, and (7) absence of clinically significant electrocardiogram (ECG) abnormalities, family history of long QT syndrome, and current use of drugs that prolong the QTc interval. Only one eligible child was enrolled per household, and at enrollment each household was given two long-lasting ITNs. The purposes and benefits of sleeping under the ITNs were explained to the primary caregivers, and ITN use was encouraged throughout the study.A randomization list using permuted variable-sized blocks of 4, 8, and 12 was computer generated by a member of the study not directly involved in patient care. Study participants were randomized to their assigned treatment group at 6 mo of age using premade, consecutively numbered, sealed envelopes. Treatment allocation was performed by nurses not involved with patient care. Study drugs were dosed as follows: TS , single dose once daily; SP , single dose each month; and DP , once daily for three consecutive days each month; each drug was provided for administration at home according to weight-based guidelines. All study drugs were in compliance with local Good Manufacturing Practices. At the time of treatment allocation, parents/guardians were given a 2-mo supply of drugs and a diary with dates for dosing and check-offs to indicate administration. Parents/guardians were instructed to re-administer drugs if children vomited within 30 min of administration and to bring children to the clinic if they vomited again. During each visit to the study clinic, parent/guardians were questioned about study drug use and resupplied to maintain a 2-mo supply.Participants received all of their medical care at a designated study clinic open every day. Parents/guardians were encouraged to bring their children to the clinic any time they were ill. Children who presented with a documented fever (tympanic temperature ≥38.0°C) or history of fever in the previous 24 h had blood obtained by finger prick for a thick blood smear. If the smear was positive, the patient was diagnosed with malaria, and a complete blood count and thin blood smear for parasite speciation were performed. Episodes of uncomplicated malaria were treated with artemether-lumefantrine (AL), the recommended first-line treatment in Uganda. AL was administered twice a day for 3 d, with the first daily dose given directly observed in the clinic and the second daily dose administered at home. Episodes of complicated malaria  Chemoprevention was stopped at 24 mo of age, and study participants were followed-up one additional year until they reached 36 mo of age. Study participants were prematurely withdrawn from the study for (1) movement out of the study area, (2) failure to be seen in the study clinic for >60 consecutive days, (3) withdrawal of informed consent, or (4) inability to comply with the study schedule and procedures.Thick and thin blood smears were stained with 2% Giemsa for 30 min. Parasite density was estimated by counting the number of asexual parasites per 200 white blood cells and assuming a white blood cell count of 8,000 per microliter. A smear was deemed negative if no parasites were seen in 100 high-powered fields. Microscopy quality control included rereading all blood smears and resolution of any discrepancies by a third microscopist.5 was used as the internal standard because its retention time (1.17 min) was the closest to that of PQ (1.21 min) among several compounds screened. The extract (10 µl) was directly injected onto a liquid chromatography–tandem mass spectrometry system API5000. Separation was achieved on a PFP column  eluted with aqueous 0.14% trifluoroacetic acid, 10 mM ammonium formate, and acetonitrile. The PQ assay demonstrated a lower limit of quantitation of 10 ng/ml with a calibration range of 10–100 ng/ml. Inter- and intra-day accuracy ranged from 97.6% to 106% and from 93.9% to 112%, respectively, and inter- and intra-day variation ranged from 7.4% to 12% and from 4.4% to 14%, respectively.Piperaquine (PQ) drug levels were measured from capillary blood collected on filter paper on the day malaria was diagnosed among study participants randomized to monthly DP. Levels of drugs from the other treatment arms were not measured because of resource constraints. Briefly, dried blood spots were punched from Whatman cards and extracted with 100 µl of a 1∶1 mixture of 20% trichloroacetic acid and acetonitrile. Nevirapine-dThis study was part of a protocol that included two randomized controlled trials to evaluate the protective efficacy and safety of antimalarial chemopreventive regimens in distinct populations of HIV-unexposed (HIV-uninfected children born to HIV-uninfected mothers) and HIV-exposed (HIV-uninfected children born to HIV-infected mothers) children. Here we only present the findings from the trial conducted in HIV-unexposed children. The other results will be presented elsewhere. The study presented here was designed to test the hypotheses that chemoprevention lowers the incidence of malaria compared to no chemoprevention, and that the optimal chemoprevention regimen is DP. The sample size was calculated to detect at least a 32% lower incidence of malaria in the DP arm compared to that in the TS arm. We assumed that the incidence of malaria would be 1.85 episodes per person-year with TS chemoprevention based on a prior cohort study in the same area Data were double-entered and verified in Microsoft Access, and statistical analyses were performed using Stata, version 12 (StataCorp). All analyses used a modified intention-to-treat approach, including all study participants randomized to therapy and including all follow-up time until the study participant reached a study end point or early study termination, regardless of whether the intervention was stopped due to an adverse event. Descriptive statistics included means and standard deviations for continuous variables and proportions for categorical variables. The primary outcome was the incidence of malaria, defined as the number of incident episodes per time at risk, during the period the intervention was given (6–24 mo of age). Treatments within 14 d of a prior episode were not considered incident events. Time at risk was from the day following the initiation of study drugs to the last day of observation, minus 14 d after each treatment for malaria. Secondary outcomes included the incidence of complicated malaria, all-cause hospitalizations, diarrheal illnesses, respiratory tract infections, and serious adverse events or adverse events of moderate or greater severity (grade 3–4); the prevalence of moderate–severe anemia (hemoglobin<8 gm/dl) measured at the time of each episode of malaria and at the time of routine testing done every 4 mo; and the prevalence of parasitemia and gametocytemia measured at the time of monthly routine blood smears in study participants who were asymptomatic. Response to antimalarial therapy among children diagnosed with symptomatic malaria was also a secondary outcome, and will be presented separately. To assess the impact of chemoprevention on the development of naturally acquired immunity, the incidence of malaria, complicated malaria, and hospitalizations was compared between treatment arms for children 24–36 mo of age after the intervention was stopped.p<0.05 was considered statistically significant.Incidence outcomes were compared using a negative binomial regression model, and prevalence outcomes and measure of use of ITNs at the time of monthly assessments were compared using generalized estimating equations with adjustment for repeated measures in the same study participant. For all analyses, only the assigned study arm was included as a covariate, with the exception of a multivariable analysis performed for the primary outcome of malaria incidence, which also included other covariates that were thought to be potential independent risk factors for malaria. Measures of association were expressed as protective efficacy  during the intervention and incident rate ratios after the intervention was stopped. p = 0.36).400 infants were enrolled, 393 (98%) of whom reached 6 mo of age and were randomized to one of the four study arms . Baselinp = 0.57), daily TS was moderately protective , and monthly DP provided the greatest protection . Results were very similar when using multivariable analyses to adjust for household wealth index, level of education of primary caregiver, whether the child was parasitemic at enrollment, residence in village or town, nutritional status at enrollment (defined as length-for-age z-score), and type of materials used for house construction (p<0.001).During the intervention, the incidence of malaria in the no chemoprevention group was 6.95 episodes per person-year at risk . Comparetruction . In all truction , and thetruction . When coThe proportions of assigned doses reportedly administered at home based on diaries completed by primary caregivers were 99.2% for SP, 98.5% for TS, and 99.2% for DP. To further explore adherence among study participants randomized to monthly DP, PQ blood concentrations were measured at the time of each episode of malaria. Among 366 episodes, 348 samples were available for analysis. For ten episodes, PQ levels were >100 ng/ml, and the primary caregivers reported giving the DP within the prior 24 h, suggesting the coincidental onset of malaria just before the scheduled time for monthly DP dosing. For the remaining 338 samples there was no relationship between PQ levels and the number of days since the last dose of DP was reported given . In addiPlasmodium falciparum), including 2,460 for incident episodes and 27 for episodes occurring within 14 d of a prior treatment. A total of 21 (0.8%) treatments were for complicated malaria, 12 for danger signs , and nine for severe malaria . A total of 58 hospitalizations occurred in 34 different children, most commonly for anemia without complicated malaria (16), complicated malaria (14), gastrointestinal illness (seven), respiratory tract infection (six), measles (four), and septicemia (four). There were no significant differences in the incidence of complicated malaria, hospitalization, diarrheal illnesses, and respiratory tract infections between any of the intervention arms and the no chemoprevention arm (p = 0.04) and significantly lower in the DP arm    . The meactively) .Considering grade 3–4 adverse events regardless of relationship to study drugs, the overall incidence and the incidence of elevated temperature, anemia, and thrombocytopenia were significantly lower in the DP arm, but not the SP or TS arms, compared to the no chemoprevention arm . Only 19p = 0.004)  . There wWe evaluated the antimalarial protective efficacy of monthly SP, daily TS, and monthly DP in a high transmission region of Uganda where, despite use of ITNs, the incidence of malaria in those not receiving chemoprevention was almost seven episodes per person-year at risk for children 6–24 mo of age and almost 11 episodes per person-year at risk for children 24–36 mo of age. This finding underlines the fact that ITNs are not a panacea for the control of malaria in high transmission areas. Monthly SP had no protective efficacy against malaria and was associated with an increased prevalence of moderate–severe anemia. Daily TS reduced the incidence of malaria by 28% , but offered no protection against moderate–severe anemia. In contrast, monthly DP was highly effective, reducing the incidence of malaria by 58% , the prevalence of moderate–severe anemia by 47% , the prevalence of asymptomatic parasitemia by 76% , and the prevalence of gametocytemia by 89% . In terms of measures of impact, approximately 3 mo of chemopreventive therapy with monthly DP was needed for each episode of malaria prevented. There were no differences between the study arms in the incidence of malaria during the 1-y period after the intervention was stopped, suggesting that none of the chemoprevention regimens had a significant impact on the development of naturally acquired immunity.The regular use of drugs for the prevention of malaria has been studied in African children for over 60 y. Earlier studies focused on continuous chemoprophylaxis, the frequent sub-therapeutic dosing of drugs to prevent infection or its clinical manifestations. In several randomized controlled trials chemoprophylaxis reduced the incidence of malaria and increased hemoglobin concentrations pfdhps K540E mutation of ≤50% pfdhps mutation is very high The most studied strategy for IPT in African children is the administration of SP at the time of routine vaccinations, usually three treatments in the first year of life (IPTi). In a pooled analysis of six randomized controlled trials, IPTi with SP was associated with a protective efficacy of 30% against malaria, 21% against anemia, and 23% against all-cause hospitalizations In parts of west Africa—where malaria is seasonally restricted, the main burden is in older children, and the prevalence of SP resistance is low—a different approach, SMC, has been studied pfdhfr/pfdhps mutations that together have been strongly correlated with antifolate resistance In this study, chemoprevention interventions were tailored to an area of east Africa with intense, year-round malaria transmission, with regular administration from 6 to 24 mo of age. Two intensive antifolate regimens were studied, monthly SP and daily TS, providing much more consistent drug exposure than the infrequent IPTi regimens previously studied in east Africa. Despite the more intensive dosing, SP offered no significant protection against malaria, but an excess risk of moderate–severe anemia, and TS was associated with only 28% protective efficacy. Although noncompliance with study drugs likely contributed to a reduction in the protective efficacy, these results and others The impact of chemoprevention on the development of naturally acquired immunity to malaria was also of interest in this study. Results from prior studies of the impact of chemoprevention on the incidence of malaria in the period after the intervention was stopped have been mixed. In a study from Tanzania, chemoprophylaxis with weekly pyrimethamine plus dapsone in children 8–48 wk of age was highly efficacious, but associated with a “rebound” effect leading to an increased incidence of malaria in the year after the intervention was stopped, suggesting that continuous chemoprophylaxis impaired the development of naturally acquired immunity There were several limitations of this study. Administration of study drugs was not directly observed. Rather, study drugs were administered at home in a way that could be standardized across study drug regimens. Although attempts were made to measure compliance through the use of adherence calendars, pharmacokinetic data from the DP arm suggested that compliance was much lower than what was reported by parents/guardians. The study also had multiple study arms with multiple primary and secondary outcomes. Given the number of comparisons made, caution should be taken in interpretation of significance because of the increased risk of type-1 errors. Finally, this study was conducted in an area of high transmission intensity and widespread antifolate resistance; therefore, results may not be generalizable to other settings.Our results lead to some key questions. First, considering the long exposure to PQ after monthly dosing pfmdr1 genotypes associated with decreased sensitivity to AL, while DP selected for opposite genotypes The excellent efficacy of DP for the treatment of malaria in multiple countries Checklist S1CONSORT checklist.(DOC)Click here for additional data file.Protocol S1Promote–Chemoprevention protocol.(PDF)Click here for additional data file.Text S1Institutional review board approval from Makerere University School of Medicine Research and Ethics Committee.(PDF)Click here for additional data file.Text S2Institutional review board approval from the Uganda National Council for Science and Technology.(PDF)Click here for additional data file.Text S3Institutional review board approval from the University of California, San Francisco.(PDF)Click here for additional data file.


In spite of the World Health Organization recommendations for the treatment of malaria, febrile patients are still infrequently tested and erroneously treated for malaria. This study aimed to investigate the adherence to malaria national protocol for the management of malaria among under five years old children.A cross sectional hospital-based study was conducted during the period from September through December 2013 among febrile children below the age of five years attending the outpatient department of Omdurman Children Hospital, Sudan. Demographic, clinical and laboratory data  and anti-malarials and/or antibiotics prescription were recorded.A total of 749 febrile children were enrolled. The mean (SD) age was 37.51 (41.6) months. Less than a half,  of children were investigated for malaria using microscopy , RDT  or both . Malaria was not investigated for more than a half,  however investigations targeting other causes of fever were requested for them. Malaria was positive in 72 (22%) of the 327 investigated children. Five (1.6%) out of 255 with negative malaria tests were treated by an anti-malarials. Quinine was the most frequently prescribed anti-malarials  then artemisinin-based combination therapy (ACT) . The majority of the 749 children  were prescribed an antibiotic.There is a poor adherence to malaria management protocol in Sudan among physicians treating children below five years of age. There was a high rate of antibiotic prescription needs. Plasmodium falciparum resistant strains in the majority of malaria endemic countries, the World Health Organization (WHO) recommended artemisinin-based combination therapy (ACT) for the treatment of uncomplicated P. falciparum malaria, which is adopted in most of the African countries including Sudan . Table Around two fifth  of these children were investigated for malaria using microscopy , RDT  or both . Other investigations were requested beside malaria for 96 (12.8%) children, these were; white blood cell count (53), haemoglobin (41) and chest X-ray (2). Malaria was not investigated for over half 422 (56.3%) of the children however, haemoglobin concentration, white blood cell count and chest X-ray were requested for , 415/422  and  of these 422 children, respectively. Malaria was positive in 72 (22%) of the 327 investigated children, 63 of them were positive by microscopy, four by RDT and five were by both.The majority of 67, 93.1%) of malaria positive 72 children received anti-malarials whereas, five (6.9%), were not. Malaria was negative in 255 (78%) of the 327 tested children however, 5 (2%) of them received anti-malarials. Among children who were not tested for malaria (n = 422) but tested for other causes of fever, 10 (2.4%) were presumptively treated as malaria  for 65 (72.2%) cases, followed by AS+ SP and AL for 25 (27.8%) cases. Forty-two (64.6%) out of 65 patients who were treated by quinine, had no any sign of severe malaria.In a logistic regression, while none of the demographic and clinical characters associated with physicians’ anti-malarials prescription, investigating malaria (via blood film/RDT) was the only variable associated with physicians’ anti-malarials prescription Table . The varThe majority of the children, 87.4% (655/749) were prescribed an antibiotic. Antibiotic prescription was regardless of the status of malaria testing.In malaria endemic areas including Sudan, the WHO and the National Malaria Programme recommended parasite-based malaria diagnosis by light microscopy or RDTs. Positive result is the only indication for anti-malarials treatment whereas negative cases “should be reassessed for other common causes of fever” ,6,16,17.et al. two years ago in a cluster-sample survey including all age groups in Sudan [This study investigated physicians’ adherence to malaria management guidelines in a reference setting in Khartoum, Sudan. Less than a half of all febrile children were tested by a parasite-based test for malaria though all should have been tested according to the management protocol . Testingin Sudan . This rain Sudan -21. The et al., one of the favourable reports [Although the majority of tested positive children were treated for malaria, almost 7% of them were not owing to the reluctance of physicians to treat possibly because of the low specificity of microscopy at hospitals in Sudan and their reliance on clinical judgment . A bette reports . The var reports . Further reports ,27.et al. as 36% used injectable artemether monotherapy rather than quinine [The most striking was that injectable quinine was unduly used in this cohort for the treatment of 72.2% of children with non-severe malaria instead of the recommended first-line therapy, ACT. This highly discordant practice was almost double the figure reported by Abdelgadir  quinine . Neverth quinine . In one  quinine .et al. and Manyando et al. [Almost 2.5% of patients not tested for malaria were treated by an anti-malarial in this cohort. This rate of presumptive treatment of malaria was less than the 19.2% rate reported by Zurovac o et al. ,29. Thiset al. found no significant difference in antibiotics co-prescription with anti-malarial between those tested positive for malaria and not tested in febrile patients in Tanzania [Near to 88% of children in this study were prescribed an antibiotic whether or not tested positive for malaria. Njozi Tanzania . HoweverTanzania . DiffereThis study however has numerous limitations. First, the hospital setting is a site of referral for sick children and thus results cannot be generalized as for all settings in the country. Second, prior antibiotic and anti-malarial treatment were not identified in this cohort as it may affect the practitioner’s prescription decision. Third, follow-up and patients outcome data was not assessed. Finally, observer bias cannot be ruled out although an expert clinician had collected the data.Despite a decade adoption of “test and treat” policy for malaria in this country, there is still poor adherence to malaria management protocol in Sudan among physicians treating children below five years of age. The poor adherence materializes in low testing rate, faulty prescription of anti-malarial to test-negative children and ominously about three quarters of children with malaria were treated with non-recommended anti-malarials. There is a high rate of antibiotic prescription, which needs further studies to clarify the drive behind such clinical behaviour. The ambiguity of different guidelines  may contribute to the confusion of prescription among health workers.