Background. The World Health Organization recommends that malaria treatment be based on demonstration of the infecting Plasmodium parasite specie. Malaria rapid diagnostic tests (RDTs) are recommended at community points of care because they are accurate and rapid. We report on parasitological results in a malaria study in selected rural communities in 3 African countries.Methods. In Nigeria, community health workers (CHWs) performed RDTs (SD-Bioline) and thick blood smears on all children suspected to have malaria. Malaria RDT-positive children able to swallow received artemisinin-based combination therapy (Coartem). In all countries, children unable to take oral drugs received prereferral rectal artesunate irrespective of RDT result and were referred to the nearest health facility. Thick blood smears and RDTs were usually taken at hospital admission. In Nigeria and Burkina Faso, RDT cassettes and blood smears were re-read by an experienced investigator at study end.Results. Trained CHWs enrolled 2148 children in Nigeria. Complete parasitological data of 1860 (86.6%) enrollees were analyzed. The mean age of enrollees was 30.4 ± 15.7 months. The prevalence of malaria parasitemia in the study population was 77.8% (1447/1860), 77.6% (1439/1855), and 54.1% (862/1593) by RDT performed by CHWs vs an expert clinical research assistant vs microscopy (gold standard), respectively. Geometric mean parasite density was 6946/µL . There were 49 cases of RDT false-negative results with a parasite density range of 40–54 059/µL. False-negative RDT results with high parasitemia could be due to non-falciparum infection or result from a prozone effect. Sensitivity and specificity of SD-Bioline RDT results as read by CHWs were 94.3% and 41.6%, respectively, while the negative and positive predictive values were 86.1% and 65.6%, respectively. The level of agreement in RDT reading by the CHWs and experienced research staff was 86.04% and κ statistic of 0.60. The malaria parasite positivity rate by RDT and microscopy among children with danger signs in the 3 countries was 67.9% and 41.8%, respectively.Conclusions. RDTs are useful in guiding malaria management and were successfully used for diagnosis by trained CHWs. However, false-negative RDT results were identified and can undermine confidence in results and control efforts. Malaria exerts an unacceptably high toll on children in Africa, where 80% of estimated malaria cases and 90% of deaths occur, especially in Nigeria . Early dThere have been major changes in recommendations for malaria management in order to reduce morbidity and mortality , 4, 5. TPlasmodium infection even when the parasites are sequestered in the deep vascular compartments and thus undetectable by microscopic examination of a peripheral blood smear. High-quality RDTs have become available [Plasmodium falciparum is responsible for >95% of malaria infections. In addition, HRP2-based RDTs can withstand better than the enzyme-based RDTs the heat and temperature fluctuations of tropical Africa, where refrigeration and air conditioning are not always feasible. Many have demonstrated their sensitivity, specificity, ease of performance, and reading [Malaria RDTs are more practical at the point of care in communities where community health workers (CHWs) can be trained in their use, as they do not require electricity or special equipment. RDTs may also detect vailable  and are vailable , because reading –9.As part of a large multicenter study conducted in Burkina Faso, Nigeria, and Uganda, evaluating the use of a community-based diagnostic and treatment package for malaria of varying degrees of severity in children aged <5 years in rural communities, we evaluated the performance of an HRP2-based RDT carried out by CHWs. The level of agreement between RDT readings by CHWs vs experienced clinical research staff was also evaluated.The studies were conducted in rural communities of Burkina Faso, Nigeria, and Uganda. These sites are described elsewhere in this supplement .In Burkina Faso, 45 villages were chosen in the health area of Sidéradougou, Mangodara District. The area is a savannah area, with a climate that consists of a rainy season limited to May–October with almost no rain outside this period. This pattern of rainfall defines a very seasonal malaria transmission, with most malaria episodes experienced during or immediately following the rainy season.The Nigerian study was conducted in the Ona-Ara local government area (LGA) of southwest Nigeria between December 2013 and October 2015. The inhabitants are mostly peasant Yoruba farmers and traders. A significant proportion of inhabitants have at least primary school education. Of the 95 villages in 10 wards  that make up the LGA, 33 villages and 4 of the LGA's 6 rural wards, with an estimated total population of 141 017 and 28 203 children <5 years of age , were seSheema District is located in southwestern Uganda and has a relatively wet climate with annual rainfall of 800–2000 mm, and annual temperature range of 12.5°C–30°C. Sheema is considered to be low transmission for Uganda, but malaria endemicity in the Kayunga area is in-between mesoendemic and holoendemic regions. Transmission occurs throughout the year in both Sheema and Kayunga districts, with peaks associated with the 2 rainy seasons, which are September–January and March–May.Children between the ages of 3 and 60 months who presented to CHWs , irrespective of the degree of severity of the illness, were enrolled. Verbal informed consent was obtained from the parents/caregivers of prospective enrollees able to take oral medication, while signed or withnessed verbal informed consent was obtained from the parents/guardians of those unable to take oral drugs —that is, danger signs making them eligible for treatment with rectal artesunate. The history of presenting complaints was obtained and entered into the case report form (CRF) and study register.In Nigeria, CHWs were trained on how to obtain informed consent, the signs and symptoms compatible with symptomatic malaria, recognition of danger signs, and theoretical and hands-on performance and reading of malaria RDTs. Training of CHWs also included preparation of thick blood smears on glass microscope slides using aseptic technique and preparation of blood spots for polymerase chain reaction (PCR) analysis and appropriate storage of the various samples . They wepf RDT, Lot No. 082365 , an HRP2-based immunochromatographic test which detects HRP2 antigen that is specific for P. falciparum, was used in this study. This RDT has very high performance as reported in the most recent annual RDT evaluation program [SD-Bioline malaria  program . All enrPlasmodium on thick blood smears. Parasite density was determined by counting the number of asexual parasites against approximately 200 leukocytes on the thick blood film and converted to parasites per microliter using an assumed total white blood cell count of 8000 cells/µL [All enrollees in Nigeria had thick blood smears prepared on microscope slides from the same finger prick used for RDTs. In Burkina Faso and Uganda, patients treated with rectal artesunate were normally tested with an RDT and a blood smear at the referral hospital. Dried blood smears were stained with fresh Giemsa stain at pH 7.2 using standard procedures , 15. Stacells/µL , 15. BloMalaria RDT results as read by the CHWs were used to determine management of enrollees. Children who tested positive to RDT without evidence of danger signs received 6-dose Coartem at standard dosage based on age of the children. Children who presented with danger signs received rectal artesunate without waiting for the result of the RDT and were referred to the nearest secondary healthcare facility for definitive management. Children who were referred to health facilities were followed up by the referring CHWs or the study team. Data from the subset of children (155/179 with microscopy) with danger signs from the 3 participating countries are also presented in this article. In addition to the referral, the children who presented with danger signs in Nigeria received financial and sometimes logistic assistance in getting to a secondary healthcare facility and accessing treatment.Data were entered into EpiData and transferred to Stata software for analysis. With results of microscopy taken as the gold standard, the sensitivity and specificity of the RDT were calculated as TP/(TP + FN) and TN/(TN + FP), respectively, where TP is the number of true positives, TN the number of true negatives, FP the number of false positives, and FN the number of false negatives. Positive predictive values (PPVs) and negative predictive values (NPVs) were calculated as TP/(TP + FP) and TN/(TN + FN), respectively . The levEthical approval for the main study was obtained from the WHO Ethics Review Committee; the University of Ibadan/University College Hospital Institutional Review Committee and the Oyo State Ministry of Health Institutional Review Board in Nigeria; the National Ethics Committee for the Research on Health in Burkina Faso; and the National Council for Science and Technology in Uganda. Individual informed consent was obtained in accordance with the ethical standards set by the different ethics committees.There are 2 sets of results presented in this report. The first is parasitology data only from Nigerian children aged 3–60 months assessed by CHWs; the second set is parasitology data from all participating countries  for children no longer able to swallow oral drugs and consequently treated with rectal artesunate and referred to the nearest facility.Results of 1860 of 2148 (86.6%) Nigerian children with complete data were analyzed. Parasitology data from 267 children were not available: 20 slides broke, 29 were unreadable, and the identification codes on RDT and microscopy for the remaining 218 enrollees could not be matched. The mean age of the 1860 enrolled children was 30.4 ± 15.7 months  with approximately equal numbers by gender . Details of other patient characteristics are shown in Table Supplementary Figure 1). Prevalence of malaria parasitemia was 77.8% when RDTs were read by CHWs, and 77.6% when the same cassettes were re-read by an experienced clinical assistant . In 2014, wet-season compared with dry-season samples were 92% more likely to be malaria positive  but there was a nonsignificant 30% risk of positivity for the same period in 2015 .The prevalence of malaria parasitemia by microscopy was higher during the rainy-season months than during the dry-season months  enrolled in Burkina Faso, 24 (96%) in Nigeria, and 3 (20%) in Uganda. In Burkina Faso, 74.8% of children had no signs of cerebral malaria, whereas in Nigeria the majority 87.5%) who presented for treatment had repeated convulsions, altered consciousness, or coma  will most likely reduce the challenge of false-negative cases, which is partially responsible for the lack of trust of RDTs among patients and caregivers in Nigeria.Supplementary materials are available at http://cid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.


Plasmodium vivax malaria.The role of repeated episodes of malaria on the cognitive development of children is a relevant issue in endemic areas since it can have a long-lasting impact on individual lifespan. The aim of the current paper was to investigate whether the history of malaria can impair the verbal and performance skills of children living in an endemic area with low transmission of P. vivax malaria in Brazilian Amazon basin. The history of episodes of malaria was used as criteria for inclusion of children in the groups. The cognitive performance was assessed by the Wechsler intelligence scale for children-III edition (WISC-III), which was applied to the participants of study by two trained psychologists.A cross-sectional study was conducted with children living in an endemic area of A total of 17 cases and 26 controls was included in the study. A significant low score of verbal quotient was found in the cases (p = 0.005), however, the performance IQ was similar in both groups (p = 0.304). The full-scale IQ was significantly lower in the cases when compared to the controls (p = 0.042). The factorials index showed significant difference only in the subtest of verbal comprehension with the lower values in the cases (p = 0.0382), compared to the controls. The perceptual organization (p = 0.363), freedom from distractability (p = 0.180) and processing speed (p = 0.132) were similar in both groups.Children with a history of vivax malaria has a significant impairment of verbal and full-scale quotients as well as a significant low index of verbal comprehension. These findings are likely due to the absenteeism caused by malaria and by the low parental education, which impairs an adequate response to the environmental stimulus. Plasmodium vivax is the predominant species accounting for 85% of reported cases. The disease affects equally all ages groups and both genders, and children are considered at risk to develop more serious signals and symptoms due to their relative lack of adequate immune response [Malaria is still an important public health problem in several tropical countries . In Brazresponse –4. In faresponse –5.A relevant issue is the detrimental effect of repeated episodes of malaria on the cognitive development, which can have a long-lasting impact on individual lifespan. In fact, studies have demonstrated a lasting effect on cognition, behaviour and scholar performance of children with history of the disease –9. Most Different tools have been used to evaluate the cognitive impairment of children with malaria, some of them based on psychometric scales, which provide more reliable estimation of the impact of the disease on cognition . There aP. vivax can impair the cognition. The verbal and performance subtests scores of the Wechsler intelligence scale for children-III were measured in children with previous episodes of malaria and then compared to those of a control group with no history of infection by Plasmodium.In endemic areas, the findings of the cognitive performance tests provide rationale base for planning strategies of rehabilitation programmes, however, only a few studies assessed the cognitive impairment of children outside Africa . For thiThe study was submitted to Plataforma Brasil under protocol CAAE 2 07199612.0.0000.0018 and approved by the ethical committee of Health Science Institute of the Para Federal University under the number 261.593/2013.2 and 27,540 inhabitants, of which, 7347 children aged 2–10. The economy is based on agriculture practices, timber extraction, and fishing. The municipality accounts to 17.4% of malaria cases in the state of Para and has a constant annual incidence of cases above 50/1.000 inhabitants. Most cases (86.3%) are caused by P. vivax. The municipality has seven health public facilities, including an ambulatory for the diagnosis and treatment of malaria.This was a cross-sectional study carried out at the municipality of Anajas in the Marajo Island (00º 59′ 21″S and 49º 56′ 24″W). Anajas has an area of 6912 kmP. vivax. The controls were defined as children with negative diagnoses of malaria after microscopic examination of three blood smears for three consecutive days and with no reports by the guardians of previous infection by P. vivax or Plasmodium falciparum. Exclusion criteria for both groups included: children with chronic and other parasitic diseases, obesity and underweight assessed by the body mass index, anaemia, congenital deficit, history of malaria in the last 6 months, and neurological or motor disorders. An additional criterion of exclusion in cases was the occurrence of neurological impairment in the previous episodes of malaria.Children were randomly recruited in two schools of the municipality from January to December, 2014. The guardians of all children gave a written consent to their inclusion in the study and to collect a capillary blood sample for microscopic examination as well as answered a socio-demographic questionnaire. The cases were defined as children with negative diagnoses of malaria after microscopic examination of three blood smears for three consecutive days and with reports by the guardians of previous infection by Parasite counts were performed in Giemsa-stained thick films for three consecutive days. Blood films were examined microscopically using 100× (oil immersion) objectives. Parasite density was expressed as the number of parasites per microliter of blood. This was derived from the number of parasites per 200 white blood cells in a thick film, considering a total white blood cell count of 8000. The limit of detection of parasites was 40/µL .Data were collected by two psychologists trained to administer and score the WISC-III using a set of test materials that require approximately 90 min to complete as well as the use of informed consent forms and the socio-demographic questionnaire.A socio-demographic questionnaire was applied for all guardians of participants of the study, with questions concerning the area of residence , age ranges, scholar degree , gender, familiar stipend, the history of malaria, considering the number of episodes and the time, in months, of the last episode of the disease and the inclusion in the study.The WISC-III was validated in Brazil in 2001 with 801 children from an urban area of the city of Pelotas, RS, with satisfactory values of convergence and discrepancy , 18. TheFurthermore, the WISC-III provides four factorial indexes derived from the verbal and performance subtests, which includes verbal comprehension , perceptual organization , freedom from distractibility (arithmetic and digit span) and processing speed (coding and symbol search). The indexes score also have means of 100 and standard deviations of 15. These factorial indexes provide complementary information to the individual set of subtests , 17, 18.t test were used to compare IQ scores and index scales between the groups of study. Data were analyzed using SPSS software, Release 21 . The significance level accepted was 5%.Data are presented as frequencies of occurrence and as a mean and standard deviation or as median and range. Chi square test, Fischer exact test, and G test were used to compare proportions between case and control groups. Mann–Whitney U-test and Student P. vivax with the standard treatment recommended by World Health Organization [A total of 17 cases and 26 controls met the criteria for inclusion in the study. The mean age of both groups was 8 years. The distribution of gender was similar in both groups p = 0.19), but male predominates in cases and female in controls. The area of residence was similar in both groups with most of children living in the urban area. The mothers were the main guardians of children in both groups (p = 0.119) There is a significant difference in scholar degree of guardians with the prevalence of intermediary degree in the controls (p = 0.011), but the familiar stipend was similar in both groups (p = 0.127) , however, the PIQ was similar in both groups (p = 0.304). The FIQ was significantly lower in the cases, compared to the controls p = 0.042) , compared to the controls. The perceptual organization (p = 0.363), freedom from distractability (p = 0.180) and processing speed (p = 0.132) were similar in both groups (Table 82, compaP. vivax is sustained over the year. Overall, the disease courses with a low parasitaemia and mild signals and symptoms. Frequent and repeated episodes of malaria in children are common in this setting because they are continuously at risk of Anopheles bites. In the casuistic, male predominates in the cases and female in the controls, which may be due to the preference of male children for outdoor recreational activities which generally occurs in the proximity of Anopheles breeding sites. Most of the cases presented more than five episodes of disease with about one episode each year as well as there are no reports of cerebral impairment or other complications of the disease in all cases. The mothers were the main guardians of children in both groups as well as the familiar stipend was similar between groups, but the guardians of children from the control group have a higher scholar degree compared to the cases.The study was designed to evaluate the cognitive performance of children from an endemic area of the Brazilian Amazon basin, where the transmission of The instrument used to evaluate cognitive performance is recognized and used worldwide as a valuable tool to assess several aspects of intelligence based on the definition of Wechsler, that consider intelligence as an individual ability to adapt and constructively solve problems in the environment, that is, intelligence is understood as performance, not as capacity. Therefore, as intellectual capacity cannot be seen nor its existence concretely verified, it cannot be reliably measured; however, the performance may be evaluated. This finding is in line with most of the intelligence tests based on performance measures, such as the Stanford-Binet, the Peabody Picture Vocabulary Test, and the Guilford Intelligence Scales, however, Wechsler scale provide more a complete evaluation of the cognitive performance , 18, 20.The main risk factors of cognitive impairment in children living in Brazilian Amazon basin are anaemia, undernutrition, infestation by parasites and the exposure to environmental pollutants as mercury and pesticides . AmongstThe scores of WISC III revealed significant low VIQ and FIQ in the cases compared to the controls. On the other hand, PIQ were similar in both groups. It is likely that the significant low score of FIQ of the cases was due to the low VIQ saw in this group because FIQ estimation is derived from the sum of VIQ and PIQ scores , 14. TheLarge discrepancies between VIQ and PIQ have been reported in several diseases . In the The verbal and performance subtests used to determine the four factorials index revealed a significant difference only in verbal comprehension index with values significantly low in the cases compared to the controls. The other factorial index, as the perceptual organization, freedom from distractibility and processing speed were similar in both groups.The VIQ has a high correlation with verbal abilities measures while PIQ correlates with nonverbal ones , 14. MorIn the study, the low scores of VIQ in the cases are likely due to the decrease of environmental stimulation that impairs an adequate response to this stimulus, resulting from both low parental education and scholar absenteeism , 15, 24.On the other hand, the data disagrees with the deficit in arithmetic reported in a previous study in the same area , since tThe limitation of the study was the small number of children enrolled in both groups. Several efforts were done to increase the number of participants, however, the high incidence of the disease in the municipality of Anajas imposed difficulties to find children with no history of malaria. However, the data were similar to most studies that evaluated cognitive abnormalities in children with malaria, which can be due to the sensitivity of the Wechsler scale to detect minor change in the cognitive performance in this age group.Finally, the data of the study are relevant to assist public health professionals in the planning of interventions to improve the verbal abilities of children living in endemic areas of malaria.P. vivax has a significant impairment of verbal and full-scale quotients as well as a significant low index of verbal comprehension. These findings are likely due to the absenteeism caused by malaria and to the low parental education, which impairs an adequate response to the environmental stimulus.Children with history of malaria by


Asymptomatic individuals are one of the major challenges for malaria elimination programs in endemic areas. In the absence of clinical symptoms and with a lower parasite density they constitute silent reservoirs considered important for maintaining transmission of human malaria. Studies from Brazil have shown that infected individuals may carry these parasites for long periods.Plasmodium vivax were invited to participate in the study. A standardised pvs25 gene amplification by qPCR was used for P. vivax gametocytes detection. Anopheles aquasalis were fed using membrane feeding assays (MFA) containing blood from malaria patients. Parasitemia of 42 symptomatic and 25 asymptomatic individuals was determined by microscopic examination of blood smears and qPCR. Parasitemia density and gametocyte density were assessed as determinants of infection rates and oocysts densities. A strong correlation between gametocyte densities (microscopy and molecular techniques) and mosquito infectivity (P < 0.001) and oocysts median numbers (P < 0.05) was found in both groups. The ability to infect mosquitoes was higher in the symptomatic group (41%), but infectivity in the asymptomatic group was also seen (1.42%).Patients were selected from three periurban endemic areas of the city of Manaus, in the western Brazilian Amazon. Symptomatic and asymptomatic patients with positive thick blood smear and quantitative real-time PCR (qPCR) positive for P. vivax asymptomatic carriers they are likely to play and important role in malaria transmission in the city of Manaus. The role of asymptomatic infections therefore needs to be considered in future malaria elimination programs in Brazil.Although their infectivity to mosquitoes is relatively low, given the high prevalence of The online version of this article (10.1186/s13071-018-2749-0) contains supplementary material, which is available to authorized users. Plasmodium species and developing the disease   . A repor = 0.09) . In a re × 10-8) . The odd= 0.003) .An. aquasalis when using blood from P. vivax symptomatic patients. In fact, the infection rate has been shown to be variable depending on the study area and Plasmodium species. In Manaus, Amazon, An. albitarsis (s.l.), An. aquasalis, An. darlingi, An. nuneztovari (s.l.) and An. triannulatus (s.l.), respectively, showed infection rates of 44.8%, 44.7%, 18.3%, 24.5% and 8.8% [P. vivax blood samples were infective, i.e. at least one mosquito became infected with oocysts, and nearly 50% of all 4389 An. dirus mosquitoes became infected [An. darlingi/P. vivax [.The infection rate in this study was 41% for and 8.8% . In westinfected . In PeruP. vivax .Anopheles darlingi and An. aquasalis are the most important malaria vectors in Brazil; previous studies have shown differences in infection rates for these species [An. darlingi, An. albitarsis (s.l.), An. nuneztovari (s.l.) and An. triannulatus (s.l.) field populations, and the laboratory-colonised An. aquasalis were shown to be susceptible to P. vivax under laboratory conditions with much higher infection rates than those reported in nature [ species . We obsen nature . Howevern nature . On avern nature –43.Asymptomatic infections showing a higher gametocyte density (transcripts/μl) were as effective as symptomatic ones infecting mosquitoes. The ability to detect low levels of parasitemia is crucial to identifying asymptomatic carriers. Microscopy detected only 10 (40%), asymptomatic infections compared to the PCR-based method that was positive for all 25 samples. This increased sensitivity of molecular methods makes them suitable for identification of asymptomatic malaria , 42–44.Symptomatic patients are promptly treated and remain infectious to mosquitoes only for a few days. Since asymptomatic patients not recognised become the main potentially infectious source for long periods, this can at least partially compensate for the low infectivity rate . Thus, sPlasmodium infection [An. darlingi and An. triannulatus, but not in An. albitarsis (s.l.) and An. aquasalis [An. aquasalis mosquito infectivity was not observed in this study when using an inactive serum  and P. vivax specific qPCR based on detection of 18S rRNA genes and RT-qPCR detecting pvs25 transcripts. For sequences of primers and probes see [obes see , 38. Additional file 3:Table S3. Data from each membrane feeding assay of Anopheles aquasalis performed with samples from symptomatic and asymptomatic individuals of Plasmodium vivax. (DOC 151 kb)Additional file 4:Table S4. Plasmids dilutions containing the sequence of the respective PCR product were used both as assay standards and to determine the limit of detection (LoD) of each assay. Generation of the plasmids is described in [ribed in , 38. (DO


Plasmodium falciparum gametocyte density and the correlation between gametocyte density and parasite density show seasonal variation in chronic (largely asymptomatic) carriers in eastern Sudan.In a markedly seasonal malaria setting, the transition from the transmission-free dry season to the transmission season depends on the resurgence of the mosquito population following the start of annual rains. The sudden onset of malaria outbreaks at the start of the transmission season suggests that parasites persist during the dry season and respond to either the reappearance of vectors, or correlated events, by increasing the production of transmission stages. Here, we investigate whether We recruited and treated 123 malaria patients in the transmission season 2001. We then followed them monthly during four distinct consecutive epidemiological seasons: transmission season 1, transmission-free season, pre-clinical period, and transmission season 2. In samples collected from 25 participants who fulfilled the selection criteria of the current analysis, we used quantitative PCR (qPCR) and RT-qPCR to quantify parasite and gametocyte densities, respectively.P. falciparum may respond to environmental cues, such as mosquito biting, to modulate its transmission strategy. Thus, seasonal changes may be important to ignite transmission in unstable-malaria settings.We observed a significant increase in gametocyte density and a significantly steeper positive correlation between gametocyte density and total parasite density during the pre-clinical period compared to the preceding transmission-free season. However, there was no corresponding increase in the density or prevalence of total parasites or gametocyte prevalence. The increase in gametocyte production during the pre-clinical period supports the hypothesis that  Anopheles mosquitoes are often undetectable or at extremely low density and confined to a few refugia. This occurs in endemic areas across the Sahel [P. falciparum—here denoted the pre-clinical period—is observed in areas with a short transmission season, such as eastern Sudan [In many areas, malaria transmission is seasonal and limited to a short window of time, often a few months, depending on availability of mosquito vector breeding habitats –3, tempehe Sahel , Sudan [he Sahel ,8, Seneghe Sahel , Burkinahe Sahel  and The he Sahel . Followihe Sahel , suggesthe Sahel ,12. Howehe Sahel . The gaprn Sudan .P. falciparum, immature gametocytes remain sequestered in deep tissues for approximately 10 days before appearing as morphologically distinct gametocytes in the peripheral blood [Plasmodium species is thought to be adjusted by parasites in response to the prevailing within-host environment, to balance between-host transmission and within-host survival [P. falciparum including parasite density [Malaria transmission depends on the ability of the parasite to produce the sexual stages (gametocytes) that infect mosquitoes, which in turn develop to produce the stages transmissible to the next vertebrate host (sporozoites). Throughout infections, a variable proportion of asexual stages commit to gametocytogenesis ,15. In Pal blood ,17 with al blood –20. Gamesurvival –23. Seve density ,24,25, m density , anaemia density ,28, dura density , host im density , and ant density –33.P. falciparum modulates gametocyte investment in response to the appearance of vectors at the start of a new transmission season is unknown. Studies on rodent models have yielded contradictory results for whether malaria parasites respond to probing by mosquitoes by increasing gametocyte investment [P. falciparum parasites adjust their transmission strategy in response to the availability of vectors, this could significantly impact malaria transmission dynamics, particularly in seasonal malaria settings, by enabling parasites to maximise transmission opportunities.Whether vestment ,35. A movestment . If P. fP. falciparum in an area of distinct seasonal transmission in eastern Sudan. Our aim is to assess whether gametocyte production shows seasonal variation coinciding with the limited duration for transmission. Specifically, we hypothesise that if parasites respond to, or prepare for, the start of the transmission season, then gametocyte density and the slope of the correlation between gametocyte density and parasite density (i.e. the proportion of parasites that are gametocytes) will increase during the pre-clinical period compared to the preceding transmission-free season.Here, we study naturally occurring human infections of P. falciparum is the predominant malaria parasite, accounting for more than 90% of all malaria infections. Anopheles arabiensis is the main mosquito vector. The entomological inoculation rate is less than one bite per person during the transmission season [The study was carried out in Asar village (longitude 358.300 E and latitude 138.300 N), Gedaref state, eastern Sudan. n season .Anopheline abundance and incidence of malaria cases as described by previous studies at the study site [Anopheles mosquitoes generally appear in late July to early August reaching peak densities in late September/October. Malaria cases are usually reported between late September and November with a peak in October [Anopheles mosquitoes or malaria cases [Anopheles mosquitoes but before clinical cases are reported [Anopheles mosquitoes and clinical cases [We defined four distinct seasons based on a combination of records for temperature, rainfall, humidity, udy site ,37. Anop October ,37. By J October ,37. The  October . Therefoia cases ; (c) prereported ; and (d)al cases  Fig 1B)AnophelinP. falciparum blood films was recruited to the study and treated with chloroquine and/or sulfadoxine/pyrimethamine according to malaria treatment guidelines at the time of the study [The details of enrolment of subjects, sample collection and processing are reported in . Brieflyhe study . Venous he study . Blood she study .Of the 123 recruited participants, 25 individuals with the following infection characteristics were included in the present study:(1) Sustained chronic sub-microscopic parasite densities, determined as at least 3 time points during the study that were parasite-positive by qPCR. Fourteen (56%) participants had 7 to 12 positive time points, 5 (20%) participants had 5 to 6 positive time points and 6 (24%) participants had 3 to 4 positive time points. (2) No evidence of new infections during the pre-clinical period (August and September); in terms of either sudden appearance of high parasite density or appearance of new parasite clone. (3) No self-treatment with anti-malarials during the period between June and September, to rule out any possible effect of drug on gametocyte density during the pre-clinical period. (4) Participants who received treatment (n = 2) or who developed new infections  during transmission season 2 were not excluded from the analysis because their gametocyte density during the transmission-free season and the pre-clinical period could not be affected by future drug treatment.Over the follow up period we analysed 270 samples from the 25 eligible participants. There were 30 missing samples across 12 different time points. For the current analysis, only data from samples collected after drug treatment were described (i.e. samples collected at enrolment in November 2001 were excluded).P. falciparum 18S ribosomal RNA gene copy number was quantified by qPCR as described in [P. falciparum clone 3D7 parasite DNA, a range of 0.014–138938 parasites/μl of DNA. The 18S qPCR amplification efficiency was 97.7% (s.e. 0.01%), the inter-assay variability . The lower limit of detection was 0.139 parasites/μl of DNA corresponding to 0.1 parasites/μl of blood. Total parasite density was quantified in duplicate for each sample and the correlation between duplicate Cq values was significant .The total number of parasites in the samples was quantified from DNA extracted in 2002 (using the Chelex method) from blood spotted onto filter paper . Only 64ribed in , except sitaemia ) betweenpfs25  and pfs230p  genes were quantified by PCR as described in [pfs25 and pfs230p in the cDNA samples were converted to female and male gametocyte counts, respectively, using calibration curves of in vitro DNAs (PCR amplified sequence of DNA spanning the region of the qPCR amplicons) and their relationship to P. falciparum female and male gametocyte counts from in vitro culture [10 male or female gametocyte numbers and Cq values were significantly correlated . The correlation between duplicate quantification of the same samples was strong . Total gametocyte numbers were calculated by summing the male and female gametocytes, with a lower limit of detection of 0.5 gametocytes/μl of blood.To quantify gametocytes, RNA was extracted in 2002 using the High Pure RNA isolation kit (Roche) ,40 and sribed in , with th culture . The fempfs25 RT-PCR, respectively. Between-individual potential differences in GAPHD, were controlled for by using patient identification (ID)  as a random effect in the statistical analysis.Because DNA was extracted from unknown amounts of blood spotted on filter paper and RNA was extracted from 50uL of blood cells , the association to full blood volumes could not be simply determined. Therefore, absolute quantification of parasite- and gametocyte densities per μl of blood was not possible. Instead, parasite and gametocyte counts were normalised against human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH was quantified as described in , with a DNA or /μL bloodRNA to emphasize these are not absolute quantities and are not directly comparable between DNA and RNA samples.Generalized linear mixed models (GLMMs)  were useA variety of error distributions  were tested to describe patterns for total parasite density and gametocyte density. A zero-inflated normal distribution gave the most parsimonious fit and was used for both of the continuous dependent variables. Parasite prevalence and gametocyte prevalence were fitted to binomial distribution models. Models were built using forward stepwise selection, and Log likelihood Ratio Tests (LRT) were used to determine the most parsimonious model. If more than one model fitted the data, the one with the lowest Akaike Information Criterion (AIC) was selected. R program version 3.0.1 and glmmADMB package  were useFor parasite prevalence and density models, fixed effect variables included season , and for gametocyte prevalence and density models fixed effect variables included season , total parasite density, and their interactions. The most parsimonious models included season ; total parasite density ; and season, total parasite density and the interaction between them (for gametocyte density).Parasite prevalence and density data are presented in 10 (s.e. = 0.04) parasites/μl bloodDNA after drug treatment during transmission season 1, i.e. December 2001. Parasite density varied across the seasons (The average estimated parasite density was 1.63 logber 2001 . The ave= 0.024) .Summary data for gametocyte prevalence and density are presented in 10 (se = 0.04) gametocytes/μl bloodRNA. There was significant variation in gametocyte densities across the seasons (The average estimated gametocyte density after drug treatment during transmission season 1 (December 2001) was 0.89 log= 0.003) ; this va= 0.003) .P. falciparum total parasites and gametocytes fluctuated at sub-microscopic levels  in the transmission-free season between January and July 2002. However, following the start of the annual rains in the pre-clinical period (August to September 2002)—prior to the appearance of clinical cases—gametocyte densities increased while total parasite density remained at levels similar to the transmission-free season. This suggests an increase in gametocyte investment, which is further supported by the steeper correlation between gametocyte density and parasite density during the pre-clinical period compared to the transmission-free season.The unique epidemiological setting of eastern Sudan, where malaria transmission occurs over a period of 8 to 10 weeks, and then ceases for 9 to 10 months, provides an opportunity to examine the hypothesis that seasonal variation in gametocyte density coincides with temporal fluctuations in transmission opportunities. This epidemiological setting spans the poor savannah belt of Sub-Saharan Africa ,7–10, whP. falciparum parasites may respond to, or prepare for, the appearance of transmission opportunities by up-regulating investment into gametocytes (i.e. increasing conversion rate). Data from animal models shows that P. relictum responds to mosquitoes biting their avian host by increasing transmission potential [P. falciparum may also respond to mosquito biting or, alternatively, respond to seasonal changes in host physiology. Seasonal changes occur in many aspects of human physiology and behaviour, including immune responses [2+ dependent protein kinases are involved in activation of stage-specific development [The stability of parasite prevalence, density and gametocyte prevalence in the pre-clinical period compared to the transmission-free season suggests that the increase in gametocyte density is unlikely to be due to the acquisition of new infections. Instead, we suggest that otential  but whetesponses ,46. How elopment ,48.pfcrt codons 72–76 and pfmdr1 codon 86) linked to chloroquine resistance showed a significant decrease in the proportion of drug-resistant parasite genotypes and parallel significant increase in wildtype from transmission season to the pre-clinical period among participants treated with chloroquine (data not shown). In the same period, gametocyte production increased and therefore, the pattern we observed cannot be attributed to increased gametocyte production by chloroquine resistant parasites. Anaemia and haemoglobin levels were not measured, and we cannot exclude potential effects of seasonal variation in haemoglobin levels that may occur due to e.g. diseases or physiological changes. However, if only malaria infections were to be considered, it is unlikely that haemoglobin levels would vary significantly between the transmission-free season and pre-clinical period as a result of sub-microscopic asymptomatic malaria infections. With regard to parasite multiplicity, it has been demonstrated that multiplicity among asymptomatic carriers can decrease over time [Variation in gametocyte density during infections has been linked to many in-host environmental and parasite genetic factors, including anti-malarial drug treatment ,49,50, aver time ; we expeIn addition to uncovering the information parasites may use, it is also necessary to test whether they respond in a manner that maximises fitness . For exaP. falciparum gametocytes [Our main observations are supported by epidemiological surveys in other sites where annual rains and malaria transmission are short and highly seasonal ,2,7–10, etocytes ,9,57,58.etocytes . More reetocytes .in vitro studies suggested that P. falciparum isolates could vary significantly in their capacity to produce gametocytes [In our study we have observed similar gametocyte prevalence during the transmission-free and the pre-clinical periods. Indeed, gametocytes must be present to obtain any transmission, however increasing gametocyte densities generally improves transmission success particularly at very low gametocytaemia ,61 and tetocytes ,62. (b) Plasmodium infections, because of overlapping cohorts of gametocytes circulating at the same time, which makes it difficult to establish which proportion of gametocytes originates from a specific asexual replication cycle 10–14 days earlier [Some limitations of our study include first, our sampling method precluded estimates of the gametocyte conversion rate. Estimating conversion rates remains inherently difficult in natural  earlier . Instead earlier . We therP. falciparum sustains chronic asymptomatic infections throughout the transmission-free season, characterised by low parasite densities, but producing gametocytes. Despite, some limitations to our study, we have shown that gametocyte production increases after the transmission-free season. This suggests that in areas of seasonal malaria, the parasite may have evolved to recognise vector abundance, or a proxy for it, and responds in a manner that maximises transmission opportunities. Control measures aiming at reducing asymptomatic carriage, during the transmission-free season, should therefore have a significant impact on control of cyclical malaria in such areas.In summary, in eastern Sudan, an area of distinct seasonal transmission,


Background: The association between the haematological profile  and malaria is not completely understood. There are few published data on haematological profiles of malaria patients in areas with unstable malaria transmission. The current study was conducted to investigate if the haematological parameters, including platelet indices, were reliable predictors for microscopically-diagnosed malaria infection.Plasmodium falciparum  andP. vivax malaria  infections. The controls were aparasitemic individuals. The haematological parameters were investigated using an automated hemo-analyser. Methods: A case-control study with a total of 324 participants (162 in each arm) was conducted at the out-patient clinic of New Halfa hospital during the rainy and post rainy season (August 2014 through to January 2015). The cases were patients with uncomplicated Results: There was no significant difference in the mean (±SD) age between the study groups; however, compared to the controls, patients with uncomplicated malaria had significantly lower haemoglobin, leucocyte and platelet counts, and significantly higher red cell distribution width (RDW), platelet distribution width (PDW) and mean platelet volume (MPV).P. falciparum and P. vivax malaria infection. Conclusions: The study revealed that among the haematological indices, PDW and MPV were the main predictors for uncomplicated Abbreviations: OR: odds ratio. A correct diagnosis is one of the most important tools in the management of malaria. It has been recommended that all persons with suspected malaria should have a parasitological confirmation of diagnosis1. Microscopic examination of malaria consists of the identification of parasite species in thin and/or thick blood films, which is the “gold standard” for malaria diagnosis2. Microscopy requires trained technicians, and well-maintained microscopes with a perfect quality management system. However, acceptable microscopy services are not widely available for the diagnosis of malaria in some areas where malaria is endemic e.g. in communities in Sub-Saharan Africa1.In spite of the preventative measures, malaria remains a major public health concern. Malaria is responsible for 781,000 deaths in a year, the majority of which are in Sub -Saharan Africa3.Previously, measurement of haematological blood parameters was unreliable due to intra and inter-method variation. Nowadays, automated analysers have replaced the traditional methods. Automated analysers are available in most settings and can give reliable results within a short period of time. There is a universal trend toward using these to aid the presumptive diagnosis of malaria infection8. There is no published data on haematological changes in patients infected with malaria parasites in Sudan, where malaria in the major health problem9. The current study was conducted in New Halfa, eastern Sudan to investigate the haematological changes observed during malaria infection and to assess the reliability of the haematological parameters used for diagnosis.Previous studies have reported different results levels of sensitivity and specificity of haematological parameters as predictors of malaria infectionP. falciparum or P. vivax by microscopic examination of Giemsa stained blood smears during the study10. The controls were the patients that presented to the same clinic with symptoms of malaria but were found to have negative blood films for malaria. After the participants  provided written informed consent, a clinical history was gathered using questionnaires. Weight and height were measured and body mass index was expressed as kg/m2.A case-control study was conducted at the out-patient clinic of New Halfa hospital during the rainy and post rainy season (August 2014 through to January 2015). The cases were patients with symptoms and signs of uncomplicated malaria and who were confirmed to be infected with13. The hemogram included measuring the haemoglobin level, leucocyte count and platelet indices, namely platelet count, mean platelet volume (MPV), and platelet distribution width (PDW).2ml of blood was taken from each participant and placed in a container with EDTA, and a complete hemogram was performed using an automated hematology analyser , following the manufacturers' instructions as previously describedThick and thin blood films were prepared and stained with 10% Giemsa to microscopically confirm which participants were infected. If the slide was positive, the parasite density was measured by counting the number of asexual parasites per 200 leukocytes, and multiplied against the participants own leucocytes number/μL. The blood films were considered negative if no parasites were detected in 100 oil immersion fields of a thick blood film.A minimum sample size of 162 participants for each arm of the study was calculated assuming that 10% of participants would have incomplete data. In this way, it would be possible to calculate a significant difference (at α = 0.05) in the means of the proposed variables - mainly haemoglobin, red cell distribution width (RDW), leucocytes, platelets counts and PDW - between the cases and the controls, at 80% power.Statistical analysis was performed using SPSS for Windows, version 20.0 . Proportions of the studied groups were expressed as percentages and compared using the chi-squared test. Continuous data were checked for normality using the Shapiro-Wilk test. The means (±SD) or median (IQR) were used to describe the studied variables, depending if they were normally or non-normally distributed. The t-test  evaluated the differences between the studied groups. Binary regression was calculated, where malaria was the dependent variable and medical and haematological indices were the independent variables. Diagnostic screening tests were used to determine the diagnostic cut-offs of various parameters (based on test sensitivity and specificity) using the receiver operating characteristic (ROC) curve. P < 0.05 was considered statistically significant.P. falciparum andP vivax, respectively. There was no significant difference in the age or BMI between the cases and the controls. Patients had significantly higher body temperature than the controls (107 (66.9%) and 55 (34.0%) of the uncomplicated malaria cases were infected withontrols . Their aCompared with the controls, patients with uncomplicated malaria had significantly lower haemoglobin levels and lower leucocyte, lymphocyte, neutrophil, and platelet counts, but significantly higher RDW, PDW and MPV .A receiver operating characteristic (ROC) curve was used to determine the cut-offs for haemoglobin levels, RDW, leucocytes and platelet counts, PDW and MPV for prediction of malaria infection. The area under the ROC curve is shown inWhen the cut-off levels were evaluated using binary regression analysis, PDW ≥14.550 %  and MPV≥ 9.05fL  were the most important predictors for malaria infection, .P. falciparum andP. vivax were compared  when the cases ofompared .Raw data collected as the basis for this studyP. falciparum.Plasmf = Blood film forClick here for additional data file.Copyright: © 2017 A. Ali E et al.2017Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).P. falciparum andP. vivax malaria infection. This is in line with a recent finding where Al-Salahyet al. reported that patients in Hajjah, Northwest Yemen with malaria parasitemia had significantly lower hemoglobin, hematocrit, leucocytes, lymphocytes, and platelet counts compared to healthy subjects14. Previous studies have shown that patients with complicated malaria had reduced haematological parameters such as platelet, leucocyte, and RBC counts, which provided relatively good predictors for the diagnosis of malaria infection15. On the other hand, the significant differences observed in the haematological parameters between parasitemic Ugandan patients and non-parasitemic Ugandans were only observed in the monocyte and the platelet count16. No significant difference was found between the haemoglobin levels, MCV, MCH, neutrophils, lymphocyte counts or MPV16.According to our present findings, PDW and MPV are the two most important haematological predictors of17. Interestingly, Chandraet al reported that an MPV > 8 fL had a sensitivity and specificity of 70.8% and 50.4% for the diagnosis of malaria, respectively8.In the current study, a PDW ≥14.550% and MPV ≥ 9.05fL were the main predictors for malaria (OR =2.9 and 2.3). Previous studies have reported an increased MPV level in malaria15. A significantly higher level of the key platelet growth factor (thrombopoietin) has been reported in patients with malaria18. Furthermore, the parasitized RBCs could increase in platelet sensitivity to adenosine diphosphate (ADP), prompting secretion of dense granules20.The higher PDW and MPV values in malaria could be explained by bone marrow formation of megakaryocytes to compensate for the low absolute platelet count during acute malaria infection22. Another limitation of the is that we relied on microscopy only for the malaria diagnosis. Some of negative controls may have had undetected parasitemia (submicroscopic parasitemia). We have previously observed that the majority of febrile patients who were parasite negative by microscopy hadP. falciparum infection according to PCR results23. Lastly, other infections that might have an effect on blood parameters were not ruled out in both the cases and controls. In conclusion, the study revealed that a PDW ≥14.550% and MPV ≥ 9.05fL were the main predictors for uncomplicatedP. falciparum andP. vivax malaria infection.Nutritional deficiency and haemoglobinopathies were not investigated in the current study and have to be mentioned as study limitations. Haematological parameters formalaria-infested blood may vary depending on the level of malaria endemicity, presence of haemoglobinopathies and nutritional statusThe data referenced by this article are under copyright with the following copyright statement: Copyright: © 2017 A. Ali E et al.Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Dataset 1: Raw data collected as the basis for this study. Plasmf = Blood film forP. falciparum. DOI,10.5256/f1000research.11767.d16401024The study was approved by the Institutional Review Board of the Medical College, University of Khartoum (3# 2015 1114). This is a nice academic paper that deals with haematological parameters in malaria. Using a case control approach, the authors have identified a mean platelet distribution width and mean platelet volume as possible markers of acute symptomatic malaria in their setting. This is interesting but of limited practical application. Therefore, this paper should emphasise the academic aspects of their findings.   I think this paper can be accepted but with major revisions in two areas, in particular. Sample size calculation – this has been determined using continuous variables yet we do not see any examples. This must be addressed. Discussion – this should be more thorough comparing and contrasting the current data with previously published data. Moreover, readers will want to know important causes of increased RDW, PDW and MPV and where in haematological practice these parameters play an important role. The authors should let us know if their research has led to any new research questions and future avenues of research. The number of malaria related deaths are higher than those reported by the WHO. The authors should obtain the latest data.No mention of the role of malaria rapid diagnostic testsThe second line of the Discussion does not follow. Did the Yemeni study report PDW and MPV ?Page 6 left hand column penultimate paragraph: Remove 'in' in this sentence: Furthermore, the parasitized RBCs could increase in platelet sensitivity to adenosine diphosphate (ADP), prompting secretion of dense granules.  Minor commentsI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The objective of the study should be re-examined in the introduction. The terms "reliability" and "validity" should be differentiated. Reliability refers to the degree to which the measurement procedure can be reproduced.Thus, if the objective is to study the validity of the hematological parameters to make a diagnosis, the methodology must be revised since these studies require a sample size that considers the prevalence and the expected sensitivity. Since this is a case-control study the objective should be consistent with this methodology.It must be specified what a case is; What are the signs and symptoms of uncomplicated malaria?It should be explained why 100 oil inmmersion fields were used as criteria to consider a thick blood film as negative. This does not correspond to international recommendations.The calculation of the sample should be presented with the elements of a case-control study i.e. case-control relation, expected O.R., frequency of outcomes in exposed and unexposed.RESULTS. If the clinical history is available, the time of evolution of the disease could be compared, which may lead to differences in hematological profiles.The results presented in the Tables 2 and 5 are confusing. It is not clear whether the averages or medians are compared and what was the statistical test of comparison. It is not explicitly stated which variables presented normal distribution.The presumptive diagnosis of malaria with haematological analyzers does not seem to be the appropriate way of solving malaria diagnosis problems in endemic areas. In this sense, it is not clear whether the study intends to contribute to the malaria diagnosis using a method that would be non-specific. The study contributes to show hematological differences between cases and controls and this should serve to identify early lesions, but clearly is no a useful method for malaria diagnosis. There is no relevant discussion about the findings. The reference values of the hematological parameters according to age and sex are not presented; the comparisons must take into account this variation. Some aspects related to platelet changes are analyzed but superficially. Leukocyte count and cell line analysis are neglected; no mention is made of the significance of alterations, monocytes and eosinophils are neglected. There are no correlations with parasitemias which is also a variable that can contribute significantly to the hematological alterations as a function of the greater or lesser inflammatory response. REFERENCES. Reference 1.  Who are the authors? Ref 6 and 8 are the same. Ref. 16.  The titlle is incomplete? The journal name?I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. As the authors used several statistical tests to compare the results between cases and controls, it is important to make these tests explicit for each p-value presented in the tables, as a footnote.The title of Table 5 is not clear. In fact, Table 5 compares means (SD), and non-median (IQR).In the last paragraph of the Discussion section the sentence "...parameters formalaria-infested blood..."  should be changed by "... for malaria-infected blood...".Mr Ali and colleagues present a study that revealed the mean platelet volume and platelet distribution width as good parameters to predict uncomplicated malaria parasite infection.The manuscript is well written and presented. The analysis of the results is adequate and correct. Some minor points should be reviewed:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Plasmodium falciparum malaria infection. A commonly used indicator for monitoring and assessing progress in coverage of malaria treatment is the proportion of children younger than 5 years with reported fever in the previous 14 days who have received an ACT. We propose an improved indicator that incorporates parasite infection status (as assessed by a rapid diagnostic test [RDT]), which is available in recent household surveys. In this study we estimated the annual proportion of children younger than 5 years with fever and a positive RDT in Africa who received an ACT in 2003–15.Artemisinin-based combination therapies (ACTs) are the most effective treatment for uncomplicated P falciparum infection assessed by RDT who received an ACT were incorporated in a generalised additive mixed model, including data on ACT distributions, to estimate coverage across all countries and time periods. We did random effects meta-analyses to examine individual, household, and community effects associated with ACT coverage.Our modelling study used cross-sectional data on treatment for fever and RDT status for children younger than 5 years compiled from all nationally available representative household surveys  across sub-Saharan Africa between 2003 and 2015. Estimates for the proportion of children younger than 5 years with a fever within the previous 14 days and P falciparum infection increased across sub-Saharan Africa in 2003–15, but even in 2015, only 19.7% (95% CI 15.6–24.8) of children younger than 5 years with a fever and P falciparum infection received an ACT. In meta-analyses, children younger than 5 years were more likely to receive an ACT for fever and P falciparum infection if they lived in an urban area , had household wealth above the national median , had a caregiver with any education , had a household insecticide-treated net , were older than 2 years , or lived in an area with a higher mean P falciparum prevalence in children aged 2–10 years . In the subgroup of children for whom treatment was sought, those who sought treatment in the public sector were more likely to receive an ACT .We obtained data on 201 704 children younger than 5 years from 103 surveys  across 33 countries. RDT results were available for 40 of these surveys including 40 261 (20%) children, and we predicted RDT status for the remaining 161 443 (80%) children. Our results showed that ACT coverage in children younger than 5 years with a fever and Despite progress during the 2003–15 malaria programme, ACT treatment for children with malaria remains unacceptably low. More work is needed at the country level to understand how health-care access, service delivery, and ACT supply might be improved to ensure appropriate treatment for all children with malaria.US President's Malaria Initiative and Medicines for Malaria Venture. Plasmodium falciparum malaria,1 the most lethal species of malaria parasite. In 2015, P falciparum infections led to an estimated 187 million cases and 398 000 deaths across sub-Saharan Africa.2 Fortunately, P falciparum infection is treatable with available antimalarials. Following the emergence of widespread drug resistance to chloroquine and sulfadoxine–pyrimethamine,3 artemisinin-based combination therapies (ACTs) were introduced as a highly effective treatment for uncomplicated malaria , preventing progression to severe disease and death.5Nearly half of the world's population is at risk of P falciparum malaria in nearly all endemic countries is an ACT.6 Funding for procuring ACTs has greatly increased and they are now widely available from multiple manufacturers and in many formulations. Between 2003 and 2007, nearly all countries in Africa changed their treatment policy to ACTs as the first-line treatment of uncomplicated malaria, and since then, a major rise has been seen in global ACT procurement and distribution.7The WHO-recommended first-line treatment for uncomplicated 8 Although this measure is available from household surveys and gives some indication of the extent of malaria treatment, it has several notable limitations. Most crucially, the proportion of children who have had an antimalarial does not indicate whether the fever was associated with an actual malaria parasite infection. Even if a country achieves a reasonably high level of treatment of fevers with an effective antimalarial, which for the past decade has been an ACT, this measure can be highly misleading because it includes inappropriate treatment of non-malarial fevers. Although an increasing proportion of patients with suspected malaria undergo malaria diagnostic testing, in many settings across Africa, treatment still relies on clinical diagnosis of malaria on the basis of fever without laboratory confirmation. Another limitation of this approach is that household surveys are done intermittently, or not at all in some countries. Because of changes in the availability of drugs or clinical practice, treatment coverage might vary in the years between surveys. Providing an estimate of treatment coverage in non-survey years might help programmes better assess their progress and respond in a more timely manner to deficiencies in service delivery.To date, one of the most commonly used indicators for monitoring global progress in the treatment of malaria has been the proportion of children with a fever in the previous 2 weeks that received an effective antimalarial, as measured by mother's recall during nationally representative household surveys.P falciparum infection in the previous 2 weeks who received an ACT. Since 2006, many national household surveys in Africa have added in assessments using rapid diagnostic tests (RDTs) that detect the P falciparum parasite histidine-rich protein 2 (hrp2), which can be used as a measure of P falciparum parasite prevalence. Because hrp2 circulates for up to several weeks after P falciparum has been cleared,10 RDTs can detect infections up to 42 days after parasite clearance, with almost all infections detected up to 7 days, and about 80% detected up to 14 days after parasite clearance.11 Because of this property, hrp2 RDTs done at the time of the survey provide an approximate measure of 2-week infection period prevalence12 that overlays effectively with the reported history of fever and malaria treatment in the surveys.A more useful indicator for measuring progress in combating malaria is the proportion of children with a fever plus a P falciparum RDT who were treated with an ACT from 2003 to 2015 in all P falciparum-endemic countries in Africa, as measured by national household surveys.In this study, our objective was to estimate the proportion of children younger than 5 years with a recent fever and a positive 13 and a checklist is included in the This study was a modelling analysis that used cross-sectional data obtained from nationally representative household surveys. We used the STROBE guidelines in the reporting of this study,14 In addition to a questionnaire intended to measure household wealth, household insecticide-treated net (ITN) ownership, and caregiver education level, these surveys also ascertain data on occurrence of and care for fever, cough, and diarrhoea from all caregivers of children younger than 5 years, and the MIS and some DHS measure P falciparum infection status of children younger than 5 years in sampled households.We included data from publicly available national population-based surveys that measured the 2-week history of fever and malaria treatment for children younger than 5 years, categorised by type of antimalarial received. These surveys were the Malaria Indicator Surveys (MIS), Demographic and Health Surveys (DHS), and Multiple Indicator Cluster Surveys (MICS) done in 2003–15. The sampling methods for these surveys have been described in detail elsewhere.7 We standardised ACT distributions by dividing by the total population at risk per country  to produce ACT availability per capita (ACTcap), and calculated annual population estimates using growth rates derived from the UN Population Prospects database. We also obtained annual estimates of P falciparum prevalence in children aged 2–10 years (PfPR2–10) from MAP that we extracted at the latitude and longitude for each survey cluster. We excluded countries that had a national mean PfPR2–10 of less than 2% in any year, because the prevalences of P falciparum and fever were too low to provide reasonable sample sizes in these countries.We obtained data on the number of ACTs and other antimalarials distributed by national programmes per year in 2003–15 from the WHO World Malaria Report.P falciparum infection as the proportion of children younger than 5 years with a fever in the previous 2 weeks and a positive RDT test at the time of survey who were reported as receiving an ACT. We defined household ITN ownership as the presence of at least one ITN in the household at the time of the survey, and determined household wealth using principal components analysis of household assets.15 Education level of the child's caregiver was categorised as any or none. Endemicity level was defined for each survey cluster as hypoendemic (<10% PfPR2–10), mesoendemic (10–50%), and hyperendemic or holoendemic (>50%) on the basis of the mean PfPR2–10 at each cluster. Season was determined by whether the median survey date was during the rainy or dry season. We classified the source of treatment for fever as public if treatment was sought from any governmental source, and as private if treatment was sought from any nongovernmental source, including informal care. To examine regional trends, we classified countries on the basis of UN Africa subregion , and whether or not they received funding through the Affordable Medicines Facility malaria (AMFm) scheme.We defined coverage of ACT treatment for children younger than 5 years with fever and a P falciparum infection across countries from 2003–15, we used a three-step modelling approach to predict coverage for surveys without RDT measurement and used approximate Bayesian time series methods that included ACT distribution data to impute data for countries and years with missing data. We used random effects meta-analyses of the survey datasets to assess associations between individual-level ACT treatment and various predictors. All analyses were done using R 3.2.3 and Stata 13.1.In most surveys except the MIS and some recent DHS, parasite prevalence by RDT was not measured. Given the paucity of data available to create a complete time series measuring ACT treatment for children younger than 5 years with fever and a PfPR2–10. To account for uncertainty and obtain predictions of RDT status in all children with a fever from the compiled surveys, we sampled values of logit-transformed PfPR2–10 at each survey location and time, and used the coefficients estimated from logistic model estimations to produce predicted probabilities of RDT status for each child. We then sampled values using the binomial distribution to produce binary predictions for each child , we modelled the propensity of children with a fever to be RDT positive on the basis of a set of predictive factors. We used all available survey datasets with RDT results at the time of survey to parameterise a logistic regression model to predict malaria parasite infection in febrile children. In the regression model, we included the child's age, household wealth quintile, household ITN ownership, urban or rural status, season (rainy or dry), and malaria transmission intensity for the survey year, measured by logit-transformed ch child  p 4. Forcap across countries. National annual ACT coverage was modelled as a function of time, country, UN Africa subregion, and ACTcap. Gaps in the ACT coverage time series for each country were filled on the basis of posterior means of the model's fixed effect, using known values as anchor points  that incorporated the relationship between ACT coverage and ACTr points  pp 11–12Continental-level estimates were weighted by the population at risk for each country. We produced separate estimates of ACT coverage by UN Africa subregion, whether or not they were part of the AMFm scheme, residence , socioeconomic status (above or below country median), endemicity level , and health-care provider (public or private). We also produced estimates of ACT coverage in children younger than 5 years with a fever and a negative RDT result.P falciparum infection and individual, household, and community-level factors using random effects meta-analysis. Urban or rural, wealthy or poor, mean (logit-transformed) PfPR2–10, household ITN ownership, caregiver education (any or none), and child's age (>2 years or ≤2 years) were first included in multivariable models for each survey dataset with a random effect at the primary sampling unit level. The coefficient and SE for each factor from these meta-analyses was then entered into a separate meta-regression for each factor, with a random effect at the survey level. We ran the same models including the health-care provider type, restricted to those children who sought treatment, and additionally a meta-regression model among all children with fever, including only survey-level associations between ACT treatment and RDT status and a covariate for before and after 2012 to assess the effect of RDT status on ACT coverage after policy changes in 2010.Finally, we used survey datasets with both measured and predicted RDT status to assess the association between treatment with an ACT for children with fever and We assessed the models for RDT predictions, ACT coverage, and ACT availability by withholding a sample of the observed values as a testing dataset, and running the prediction process on the remaining training dataset. We assessed predictive accuracy using both individual-level hold-out samples and hold-out samples of entire surveys. We compared predicted values with observed values and assessed the accuracy of predictions using the area under the curve (AUC) statistic for RDT status, and the root mean square error (RMSE), mean absolute error, and mean absolute scaled error for ACT coverage.To assess how a shorter time period (<14 days) between survey and fever date affected our results, we did a sensitivity analysis in which the recall period of a fever was limited to within the previous 2–7 days. This analysis was possible only in a subset of 12 MIS surveys and one DHS survey that measured the days since the fever started.The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. All authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.The final dataset included information on 201 704 children younger than 5 years from 103 surveys  across 33 countries in sub-Saharan Africa from 2003–15. RDT results were available for 40 of these surveys including 40 261 children 20%; , and we PfPR2–10, rural location, and survey being done during the rainy season were all strongly associated with a positive RDT  in 2015  in Somalia to a high of 70.2% (65.6–74.5) in Uganda  of children with malaria did not get a potentially life-saving ACT in 2015. The two biggest drivers of this coverage gap are poor access to and delivery of health services. Only 8.3% of people who did not seek care from a health provider outside the home received an ACT, and even of those who did seek care, only a quarter received an ACT; of these individuals, those who sought treatment in the public sector were far more likely to receive an ACT than those who went to a private health-care provider.In this study we estimated the proportion of children younger than 5 years with a fever and 16 suggesting that higher ACT coverage is linked to overall scale-up of malaria interventions. Greater improvement in coverage was also seen in countries supported by AMFm funding, although we were unable to determine from this analysis whether this effect was directly associated with the total amount or type of funding, or some other factor.Considerable variation was seen in coverage across countries and over time, and a few large countries with low coverage—eg, Nigeria—strongly affected regional and continental estimates. These low-coverage estimates, and especially the low estimates in some large, high-burden countries, show that people most in need in highly endemic areas are often the least likely to get appropriate treatment for malaria. Notably, the greatest improvement in coverage was seen mainly in eastern African countries that have also shown progress in scaling up ITN coverage,17 Although in continental-level estimates, the CIs for ACT coverage stratified by urban or rural residence, wealth, endemicity, and RDT status overlapped, in individual-level meta-analyses we found that poorer children living in rural areas with fever and a P falciparum infection were significantly less likely to receive an ACT than children who lived in urban areas and had a household wealth index above the national median, as were those whose caregiver had no education and whose household did not own an ITN. These variables—area of residence, household wealth, education, and ITN ownership—are all proxies for access to health services and influence the seeking of treatment for fever. Estimates of the proportion of children younger than 5 years with fever taken for any care are between 56% and 69% across sub-Saharan Africa.18 Of children for whom treatment was sought, urban residence and wealth were not significant predictors of ACT treatment. Improving access to care and provision of ACT treatment in poor, rural populations19 is crucial for sustaining efforts to control malaria and progressing towards elimination. Our finding that 8.3% of children received an ACT even when their caregivers did not report seeking treatment outside the home was somewhat surprising, but largely driven by data from countries with very high overall ACT coverage  or where mass drug administration activities have recently been done . In these cases, there might be large quantities of circulating ACTs in the community.Addressing disparities in access to and delivery of ACTs is crucial for preventing severe disease and death in those most in need, and might also contribute to the reduction of transmission when high population coverage is reached.20 documented large differences between the factors that most influenced effectiveness of malaria case management across sub-Saharan Africa, including access to a health-care provider, provider compliance and patient adherence with front-line antimalarial policy, and drug quality. Consistent with our analysis, they found that in many countries effective coverage was low because of poor access to heath-care providers, but in several countries with high access to providers, effective coverage was low because of poor provider compliance with national ACT policy.20 Littrell and colleagues21 similarly found low effective coverage in Zambia due to a combination of low treatment-seeking behaviour by patients and low rates of diagnostic confirmation at health facilities.21 Several similar studies have documented individual provider barriers, including frequent stockouts22 and low availability of diagnostics, and individual patient barriers such as distance to formal facilities, lack of financial resources, and low maternal education.24Our findings are consistent with those of other studies that have explored the combination of patient and provider factors that influence ACT coverage. Galactionova and colleagues26 We found substantially lower coverage for children who sought care at private providers than for those who visited public providers in both continent-level predictions and individual-level regression analyses, which is consistent with previous research.27 Of children with fever and a P falciparum infection who sought treatment in the private sector, less than a fifth received an ACT in 2015. Substantial efforts have been made to increase the availability of ACTs through the private sector in Africa through initiatives like the AMFm, with notable improvements in some areas,30 but the scarce access to ACTs through the private sector remains an important impediment to malaria control and burden reduction, especially for people living in rural areas, who have below-average household wealth.The private sector continues to lag behind the public sector at scaling up RDTs and ACTs, showing a huge unmet need in treatment because in much of sub-Saharan Africa, rural, poor households receive care primarily through the private sector.The traditional indicator of ACT coverage, measured in all children with fever, does not allow for monitoring and assessment of appropriate treatment for those who need it. Instead, the improved indicator of ACT coverage that includes RDT status allows assessment of not only the proportion of children in need who received ACT, but also the proportion without a malaria parasite infection that inappropriately received ACT. We found only minor differences in ACT coverage between children with a malarial fever and those with a non-malarial fever, which points to considerable inefficiencies in ACT allocation. This result is not entirely surprising because RDTs were not widely scaled up across most of Africa until after 2010, and most suspected cases were treated on the basis of clinical diagnosis only. After 2012, our results show that malaria parasite-infected children with fever were more likely to receive an ACT than children with a fever but no malaria infection, suggesting improvements in appropriate treatment. Although we did not examine diagnostic coverage in this study, our results suggest that improvements are happening; however, on a continental scale, scale-up of diagnostics might not yet be having a marked effect on prescription practices and appropriate treatment.P falciparum infection warrant treatment with an ACT, some children who either were not yet infected at the time of their fever or had very low-density infections might have had negative RDT test results or been clinically diagnosed with a different condition. Similarly, a negative RDT at the time of survey does not rule out a P falciparum infection in recent weeks, especially given the relatively low sensitivity of particular RDTs in field settings31 and the possibility of hrp2 deletion in some parasites.32 Third, although predictive models showed good accuracy, we cannot be certain of accurately ascertaining each child's RDT status. However, children predicted to be RDT positive were more likely to be poor, have no household ITN, and live in high-prevalence and rural areas than those who were predicted to be RDT negative, all of which suggest greater need for ACT treatment for fever. Fourth, only a few surveys include the number of days since the fever was reported, which would have allowed us to remove children with very short or long times since fever, and reduced the potential for recall bias. Using those surveys that included this indicator, we did a sensitivity analysis that showed consistency in estimating ACT coverage in children with a fever and P falciparum infection using a shorter, 1 week period of recall. In a previous study,33 we documented high accuracy of caregiver recall for ACT treatment within the previous 2 weeks from a household survey in Zambia, but recall bias could be greater in other settings. Fifth, our predictions were limited by the relatively small sample sizes in many of the surveys, because these surveys are not typically powered to obtain representative estimates for children with fever, and also might not capture subnational heterogeneity in malaria transmission risk. Because the relatively small sample sizes probably influenced our ability to produce precise estimates, we incorporated multiple levels of uncertainty and reflect these in our estimates. Finally, the indicator of ACT treatment for fever and a positive RDT result is only a proxy measure for appropriate treatment for uncomplicated malaria. Although the inclusion of infection status is a notable improvement, appropriate treatment involves additional steps such as ensuring correct dosage and patient adherence, and exploring alternative causes of fever, which are not captured by this indicator. As countries move to improve data collection through the health system, robust routine collection of test results and treatment outcomes for fever is crucial for monitoring and evaluation of malaria case management; periodic health facility surveys that include exit interviews for patients seeking care for fever could provide an additional important datapoint to complement routine and household survey data.The approach used in our study has some limitations. First, because of the time lag between measurement of RDT status and treatment for fever, a positive RDT result at the time of survey does not necessarily indicate that a child had a malaria infection during their recent fever episode and, if they were tested at a clinic, that they would have had a positive RDT result. Although all fevers combined with P falciparum parasite infection) over the past decade of scale-up, and show that even after the large investments in malaria prevention, diagnostic testing, and treatment over the past decade, ACTs are not reaching most children with malaria who need treatment. In addition to investing in new technologies and tools, malaria programmes need to ensure that individuals with malaria receive treatment through greater access to health care and consistent availability and provision of ACTs in areas of greatest need. Improved care in all health sectors, including continued scale-up of integrated community case management, will be essential for improving access to ACTs, especially for rural households with low incomes and low access to health services. This research shows the large gap in appropriate treatment of uncomplicated malaria and the potential to increase the positive impact of malaria control and elimination programmes.We document, for the first time to our knowledge, continental and country-specific trends in ACT coverage in children who need an ACT (ie, those who have fever and a supplement