Seasonal malaria chemoprevention (SMC) is now widely deployed in the Sahel, including several countries that are major contributors to the global burden of malaria. Consequently, it is important to understand whether SMC continues to provide a high level of protection and how SMC might be improved. SMC was evaluated using data from a large, household-randomised trial in Houndé, Burkina Faso and Bougouni, Mali.The parent trial evaluated monthly SMC plus either azithromycin (AZ) or placebo, administered as directly observed therapy 4 times per year between August and November (2014–2016). In July 2014, 19,578 children aged 3–59 months were randomised by household to study group. Children who remained within the age range 3–59 months in August each year, plus children born into study households or who moved into the study area, received study drugs in 2015 and 2016. These analyses focus on the approximately 10,000 children  under observation each year in the SMC plus placebo group. Despite high coverage and high adherence to SMC, the incidence of hospitalisations or deaths due to malaria and uncomplicated clinical malaria remained high in the study areas : 11.2, 14.1] and 871.1  cases per 1,000 person-years, respectively) and peaked in July each year, before SMC delivery began in August. The incidence rate ratio comparing SMC within the past 28 days with SMC more than 35 days ago—adjusted for age, country, and household clustering—was 0.13 , P < 0.001 for malaria hospitalisations and deaths from malaria and 0.21 , P < 0.001 for uncomplicated malaria, indicating protective efficacy of 87.4%  and 78.3% , respectively. The prevalence of malaria parasitaemia at weekly surveys during the rainy season and at the end of the transmission season was several times higher in children who missed the SMC course preceding the survey contact, and the smallest prevalence ratio observed was 2.98 , P < 0.001. The frequency of molecular markers of sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) resistance did not increase markedly over the study period either amongst study children or amongst school-age children resident in the study areas. After 3 years of SMC deployment, the day 28 PCR-unadjusted adequate clinical and parasitological response rate of the SP + AQ regimen in children with asymptomatic malaria was 98.3%  in Burkina Faso and 96.1%  in Mali. Key limitations of this study are the potential overdiagnosis of uncomplicated malaria by rapid diagnostic tests and the potential for residual confounding from factors related to adherence to the monthly SMC schedule.Despite strong evidence that SMC is providing a high level of protection, the burden of malaria remains substantial in the 2 study areas. These results emphasise the need for continuing support of SMC programmes. A fifth monthly SMC course is needed to adequately cover the whole transmission season in the study areas and in settings with similar epidemiology.NCT02211729.The AZ-SMC trial in which these data were collected was registered at clinicaltrials.gov:  Matthew Cairns and colleagues show additional courses of seasonal chemoprevention are needed in Burkina Faso and Mali to control malaria. Seasonal malaria chemoprevention (SMC) is recommended for children under 5 years of age in countries of the Sahel and sub-Sahel. Many countries in West and Central Africa now have large-scale SMC programmes.The malaria burden remains high in several countries that have introduced SMC, including Burkina Faso and Mali, which are amongst the most important contributors to the global burden of malaria cases and deaths.It is important to understand whether SMC retains a high level of protection in these areas and how its impact might be improved.In this study, the level of protection provided by SMC was investigated using data from a large but closely supervised clinical trial in 2 districts in southern Burkina Faso and Mali.SMC was delivered 4 times per year over 3 years, reaching a very high percentage of children. All the daily doses of SMC were supervised by the study team. Specific substudies showed that molecular markers of resistance to the combination of antimalarials used for SMC were rare amongst malaria parasites and that the SMC combination was highly effective in curing infections detected at the end of the rainy season.Malaria incidence was markedly reduced in the period immediately after each SMC course. In the first 4 weeks after SMC, malaria cases were reduced by 78%, and malaria hospitalisations and deaths from malaria were reduced by 87%.Despite the benefits of SMC, the number of malaria cases, hospital admissions for malaria, and deaths from malaria remained very high in the study areas. There was a large peak in July each year, coinciding with the beginning of the rainy season, before SMC delivery began in August.SMC is likely to be averting a very large number of malaria cases, hospitalisations, and deaths in the study areas, but malaria has still not been brought under control.At least one additional monthly course of SMC is needed to address the high burden of malaria, including malaria deaths, that currently occurs outside the peak transmission season. This is likely to be the case in other areas of the Sahel that have a longer transmission season than can be covered by a 4-month SMC programme.Additional new tools are needed urgently to further reduce malaria in these districts and areas with similar epidemiology. Seasonal malaria chemoprevention (SMC) comprises monthly courses of sulfadoxine-pyrimethamine plus amodiaquine (SP + AQ) given to children 3–59 months of age. After encouraging results from trials in a range of settings, including in the context of high coverage of long-lasting insecticide-treated nets (LLINs) ,2, SMC bEvaluation of SMC at scale suggests that this intervention has an important impact on uncomplicated malaria, hospital admissions for malaria, and malaria deaths  –11. HoweAnother concern that must be carefully monitored is the potential effect of drug resistance. Parasites resistant to SP or AQ could be at a selective advantage in areas where SMC is used widely, and this could lead to a reduction in the efficacy of SMC over time. Large-scale surveys of molecular markers have been conducted in several SMC countries . Case–coIn this study, data were reanalysed from a large-scale, closely supervised trial of SMC in southern Burkina Faso and Mali . High coThis analysis uses data collected during the azithromycin (AZ)-SMC study, a household-randomised, placebo-controlled trial conducted in Houndé District, Burkina Faso and Bougouni District, Mali between August 2014 and December 2016 , undertaBetween August and November each year, study children received 4 monthly courses of SMC with SP + AQ, with either AZ or a matching AZ placebo . SMC had not been deployed in the study area prior to the trial. In 2014, SMC delivery began in late August; in 2015 and 2016, delivery began on the first of August. Although there was no evidence of a benefit of AZ against all-cause hospitalisations and deaths, hospital admissions or deaths due to malaria, or uncomplicated clinical malaria, to avoid any possible influence of AZ on the results presented in this paper, only children who were randomised to SMC plus placebo are included. In total, 108,176 courses  of SMC plus placebo were administered as directly observed therapy by the study team. All doses of study medication were recorded using tablet PCs. Scanning of the Quick Response (QR) code on the drug package and a child’s photo ID card were used to ensure linkage of treatment to the correct child, and the date and time stamp of each drug administration was recorded by the tablet PC.Plasmodium falciparum parasitaemia. The child’s photo ID card was used to ensure that morbidity episodes were recorded for the correct study child. The following passively detected outcomes were included in this study: 1) incidence of malaria hospital admissions or deaths from malaria (as a combined outcome), defined as hospital admission for malaria combined with a positive blood slide or rapid diagnostic test, or deaths for which malaria was listed as the primary diagnosis; and 2) uncomplicated clinical malaria episodes, defined as history of fever or measured axillary temperature ≥37.5 °C combined with a positive RDT. Histidine-rich protein 2 (HRP2)-based RDTs were used to diagnose uncomplicated malaria and to guide treatment. Blood smears were taken from a systematic sample of study children for quality control of the RDT . All blood smears, both those used to diagnose malaria in hospital and those used for QC of the RDT, were double read, with discrepancies resolved by a third reader using a standardised algorithm [Morbidity episodes were detected passively throughout the study period at the hospital and health centres in each study area  Figs. Inlgorithm . For dealgorithm . To assiP. falciparum infection was determined amongst randomly selected study children at cross-sectional surveys . Surveys were timed to occur at least 1 month after the final SMC administration and thus took place later in the first year of the study because of the later start in 2014. Two infection outcomes were defined: P. falciparum infection of any density and P. falciparum infection with a density ≥5,000 per microlitre. Blood samples were also taken through school-based surveys from school-age children resident in the same areas as the study children (500 per country in each year) who were not eligible to receive SMC because of their age.At the end of each rainy season, the prevalence of P. falciparum to SP and AQ was determined amongst all study children who carried P. falciparum infection at the end-of-season surveys and from 50 school-age children with P. falciparum infection at the same time point. DNA was extracted from dried blood spots as described previously [The presence of molecular markers of resistance of eviously ,21 . A target sample size of 100 per week (50 per randomisation group) was used in each country. Blood smears were taken and read later, so a positive result did not result in the child being treated. If a child had symptoms of malaria, they were tested with an RDT and, if positive, referred for treatment at the health centre.Each week during the malaria transmission season, The AZ-SMC trial was approved by the ethics committees of the London School of Hygiene and Tropical Medicine, London ; the Malaria Research and Training Center, University of Bamako, Bamako, Mali ; the Ministry of Health, Ouagadougou, Burkina Faso ; and the national regulatory authorities of Burkina Faso and Mali. Written informed consent was obtained from the parent or guardian of all study children.Person-time at risk was calculated from the first dose of SMC in 2014 (or from time of entry into the study for children born in study household or who migrated into the study area) until the study ended in December 2016 or the child died or exited the study area or became too old to receive SMC. For children lost to follow-up, the date of the last contact was used. Multiple events were included. Person-time was not adjusted after a malaria episode , but repThe incidence of 1) malaria hospital admissions or deaths from malaria and 2) uncomplicated malaria was calculated by age in the 2 centres combined and by country. The incidence of these 2 outcomes was also calculated in each calendar month of the study, allowing comparison of periods when SMC was distributed (August–November) with periods when SMC was not deployed. For uncomplicated clinical malaria, incidence when SMC was deployed was further stratified according to whether a child had received SMC in the past 28 days (‘recent SMC’) or had missed the previous SMC course (‘no recent SMC’). To limit the potential for confounding by factors related to access to SMC, the incidence rates amongst children with ‘no recent SMC’ in each calendar month was restricted to children who otherwise received all SMC courses that year .The detailed SMC history obtained for each child was used to further stratify person-time at risk according to the number of days since the most recent SMC course . Person-time and events within these strata were then pooled over the study period to calculate incidence rates by time since SMC. Poisson regression with a gamma-distributed random effect fitted at the household level was used to calculate incidence rate ratios comparing incidence within the first 28 days post-SMC with incidence in the period beyond 35 days post-SMC , accounting for household clustering and adjusting for age of the child in years.P. falciparum infection was calculated for study children and for school-age children resident in the study areas. Prevalence was also calculated separately for study children who had and had not received SMC at the most recent round of SMC administration before the survey. Prevalence ratios were calculated comparing children with and without recent SMC using modified Poisson regression [For the end-of-season surveys, the prevalence of gression , with a P. falciparum prevalence was calculated overall and according to receipt (or not) of the most recent round of SMC administration prior to the weekly survey. As for the end-of-season surveys, prevalence ratios were calculated comparing children with and without recent SMC using modified Poisson regression [For the weekly surveys during the transmission season, gression , with a The frequency and prevalence of resistant mutants were calculated as 1) the number of resistant mutants/the total number of clones and 2) the number of resistant mutants/the number of study children, respectively  were investigated to determine whether the infection was caused by a recrudescence or by reinfection. DNA extracted from dried blood spots using a previously published method  rates were recalculated both unadjusted for reinfection and adjusted for reinfection by limiting the cases of failure to true recrudescent parasites [At the end of the study period, after 3 years of SMC deployment in each of the study areas, a treatment efficacy study was undertaken amongst children with asymptomatic arasites .P. falciparum infection were prespecified in the statistical analysis plan for the main trial [Analysis of the frequency and prevalence of molecular markers of SP and AQ resistance amongst study children and school-age children and the treatment efficacy of SP plus AQ amongst children with asymptomatic in trial . AnalyseIn the first year of the study (August 2014–July 2015), 9,603 children were under observation in the placebo group. The cohort under observation grew to 10,760 and 11,047 children in the second and third years, respectively, with similar numbers in each country throughout the study. Demographic information is presented in Adherence to the monthly SMC schedule and 3-day SMC regimen were high in both countries . The perIn total, 23,817.2 person-years of follow-up were recorded amongst children in the SMC plus placebo group . One hundred thirty-five nonfatal malaria hospital admissions, 28 deaths from malaria, and 10,329 uncomplicated clinical malaria episodes occurred amongst children in the SMC plus placebo group in Burkina Faso; the corresponding number of these events were 109, 26, and 10,419, respectively, in children randomised to SMC plus placebo in Mali. The overall incidence of malaria hospital admissions or death from malaria and uncomplicated malaria was 12.5  and 871.1  cases per 1,000 child-years at risk, respectively. The rates were slightly higher in Burkina Faso: 13.8  malaria hospital admissions or deaths from malaria and 877.4  uncomplicated malaria episodes per 1,000 person-years at risk, whereas in Mali, the corresponding rates were 11.2  and 865.2  per 1,000 person-years. Amongst 2,626 episodes of uncomplicated malaria for which a blood smear was available in addition to the RDT result, 31.3%  were slide-negative in Burkina Faso and 31.3%  in Mali.Incidence of both hospital admissions and deaths from malaria and uncomplicated malaria peaked in children 2 years of age in both countries . MalariaIn each year of the study, both malaria hospitalisations and deaths from malaria  and uncoThe incidence of malaria hospitalisations and deaths from malaria was 13.6 per 1,000 child-years at risk  in the first 28 days after SMC treatment  but incrA similar pattern was observed for the incidence of uncomplicated clinical malaria , with a P. falciparum amongst study children was below 10% at all 3 end-of-season cross-sectional surveys in both countries  did not increase amongst study children between 2014 and 2016  and sulfadoxine resistance  did not increase over the study period. In particular, the pfdhps-K540E mutation, associated with high-grade resistance to sulfadoxine, remained at low frequency, with just 8 out of 254 samples resistant in 2016. Similar results were observed by country or when the prevalence of resistance markers amongst study children was calculated . Two children experienced early treatment failure, one in each country  with asymptomatic  country . In bothP. falciparum was markedly lower in children with recent SMC at both the end-of-season surveys and the weekly surveys carried out during the rainy season. The remaining burden amongst study children therefore reflects the exceptionally high infection rates to which study children were exposed, illustrated by a prevalence of P. falciparum parasitaemia in excess of 50% amongst school-age children resident in the same areas.Despite the very high level of malaria transmission in the 2 study districts, SMC appeared to provide a high level of protection against both malaria hospitalisations and deaths from malaria and uncomplicated clinical malaria; this was demonstrated by 3 key findings. Firstly, the incidence of malaria hospitalisations and deaths from malaria and episodes of uncomplicated clinical malaria was markedly lower during the SMC period (August to November) than in the month immediately preceding it, with evidence that children who received SMC in a particular month were very well protected. Secondly, incidence of malaria hospitalisations and deaths from malaria and clinical malaria was substantially lower during the first 4 weeks after an SMC course than in subsequent time periods. The rate ratios obtained and the profile of protection over time were compatible with results in earlier trials and with observational studies . ThirdlyThe observed high level of protection from SMC against malaria is compatible with the current low prevalence of key molecular markers of SP and AQ resistance observed in the cross-sectional surveys, the very high curative efficacy of the SMC drug combination in treatment, and results from other studies that have evaluated SMC in the Sahel region during the period of the trial . A serieStrengths of this study include the large sample size, the careful documentation of all SMC courses (and daily doses) and morbidity episodes, and the use of tablet PCs  to link all contacts to the correct child with a very high degree of accuracy. Because the main trial was powered to investigate hospital admissions and mortality as a primary outcome, these analyses have high statistical power to investigate uncomplicated malaria and severe malaria by time since SMC in more detail than has been possible in previous  trials.However, there are a number of limitations to this study. Given the large scale of the study and the need to manage children who were unwell promptly, it was not possible for blood smears to be prepared for all suspected malaria cases. The subset of children for which both an RDT and blood smear were available indicate that around a third of children positive by RDT may no longer carry parasites; this was similar in both countries. Low specificity is likely to be a consequence of parasite antigens remaining in the circulation of children whose infection has been cleared by SMC or treatment, as observed in a previous SMC study in Senegal . OverdiaDeaths that occurred in the community were diagnosed by verbal autopsy, and thus parasitological confirmation of malaria was not usually possible. Comparison of the intervention effect over the study period is made difficult by the variability in transmission intensity each year in these and similar study areas and by the differences in the timing of SMC and end-of-season surveys . In the analyses of malaria incidence, children with and without recent SMC were compared, but since children without SMC in a particular month were those who missed their scheduled SMC, this was not a randomly allocated exposure. It is possible that children with poor SMC coverage live in more remote parts of the community and have higher malaria risk. When comparing children on the basis of their adherence to SMC, the analysis strategy attempted to minimise confounding through this mechanism by including only those children who received at least 3 of the 4 scheduled SMC courses in that calendar year .Prevalence ratios were calculated as a simple way to show the large differences in carriage of parasites between those with and without SMC. However, the specific values of the prevalence ratios may not be widely generalisable .Although the prevalence of resistance markers was low in 2014 and in 2016, it will be important to monitor longer-term changes on molecular markers of resistance. The timing of the treatment efficacy study towards the end of the malaria transmission season is likely to have limited the number of reinfections to which study children were exposed. It is also unclear whether curative efficacy would differ in children with higher-density infections, although these children would be more likely to have symptoms and thus be treated with artemether-lumefantrine, the first-line treatment regimen in both study areas, rather than SMC.Despite the positive findings with respect to the benefit of SMC in the study areas, these results show that more must be done to protect young children from the consequences of malaria in areas of intense seasonal transmission. It is clear that missed SMC courses result in a markedly elevated risk of both uncomplicated and malaria hospitalisations and deaths from malaria, and thus, it is important to ensure that delivery reaches as many children as possible each month. This may require a mixture of delivery strategies .Our findings indicate that 4 courses of SMC are not sufficient to cover the entire malaria transmission season in the southern part of Burkina Faso and Mali, where rainfall peaks over a longer period than in the northern parts of the Sahel region. In each year of the study, a large number of cases of uncomplicated malaria, malaria hospitalisation, and deaths from malaria were recorded in July, prior to SMC delivery in August. Providing additional SMC courses may, therefore, have an important impact on the malaria burden. SMC programmes using 5 or 6 courses should be evaluated in these areas and other areas at similar latitudes, such as northern Ghana, that have similar epidemiology ,28 and lAdditional interventions are also needed to reduce the malaria risk when SMC courses are inevitably missed. Seasonal vaccination with a malaria vaccine  may be oIn summary, SMC remains highly protective in the trial areas, and our results emphasise the need for continued support of SMC programmes. However, the high incidence of malaria in July each year emphasises the need for a fifth monthly course of SMC to adequately cover the whole transmission season. Additional measures beyond SMC and LLINs are needed to further reduce malaria burden in these areas and areas with similar epidemiology.S1 Methods(DOCX)Click here for additional data file.S1 Fighttps://doi.org/10.1371/journal.pone.0136919.s001.Base map adapted from digitised boundaries of health districts in Burkina Faso and Mali. Noor AM, Kibuchi E, Mitto B, Coulibaly D, Doumbo OK, Snow RW. Sub-National Targeting of Seasonal Malaria Chemoprevention in the Sahelian Countries of the Nouakchott Initiative. PLoS One. 2015;10(8):e0136919. (DOCX)Click here for additional data file.S2 Fighttps://doi.org/10.1371/journal.pone.0136919.s001. GPS, Global Positioning System.Data points from study GPS. Study health centres are shown by blue circles. The study hospital is shown by a red square. Base map adapted from digitised boundaries of health districts in Burkina Faso. Noor AM, Kibuchi E, Mitto B, Coulibaly D, Doumbo OK, Snow RW. Sub-National Targeting of Seasonal Malaria Chemoprevention in the Sahelian Countries of the Nouakchott Initiative. PLoS One. 2015;10(8):e0136919. (DOCX)Click here for additional data file.S3 Fighttps://doi.org/10.1371/journal.pone.0136919.s001. GPS, Global Positioning System.Data points from study GPS. Study health centres are shown by blue circles. The study hospital is shown by a red square. Base map adapted from digitised boundaries of health districts in Mali. Noor AM, Kibuchi E, Mitto B, Coulibaly D, Doumbo OK, Snow RW. Sub-National Targeting of Seasonal Malaria Chemoprevention in the Sahelian Countries of the Nouakchott Initiative. PLoS One. 2015;10(8):e0136919. (DOCX)Click here for additional data file.S4 FigP. falciparum infection or deaths for which malaria was listed as the primary diagnosis. Clinical malaria was defined as attendance at study health facility with a history of fever or measured temperature ≥37.5 °C, with malaria infection confirmed by RDT. Incidence rates are presented per 1,000 person-years and include repeat events in the same child, provided the healthcare contact occurred more than 7 days apart. Vertical bars show 95% CIs. CI, confidence interval; RDT, rapid diagnostic test.Incidence of malaria hospitalisations and deaths from malaria (left panel) and clinical malaria (right panel) by age group over the study period. Malaria hospitalisations and deaths from malaria were defined as hospital admission with a diagnosis of malaria and blood-slide–or RDT-confirmed (DOCX)Click here for additional data file.S5 FigP. falciparum infection or deaths for which malaria was listed as the primary diagnosis. Clinical malaria was defined as attendance at study health facility with a history of fever or measured temperature ≥37.5 °C, with malaria infection confirmed by RDT. Incidence rates are presented per 1,000 person-months at risk rather than per 1,000 person-years and include repeat events in the same child, provided the healthcare contact occurred more than 7 days apart. Vertical bars show 95% CIs. Note that in Burkina Faso, in 2015, one-third of the study cohort received SMC 1 month late because of the global shortage of SP-AQ in 2015. AQ, amodiaquine; CI, confidence interval; RDT, rapid diagnostic test; SMC, seasonal malaria chemoprevention; SP, sulfadoxine-pyrimethamine.Incidence of malaria hospitalisations and deaths from malaria (a) and clinical malaria (b) by calendar month over the study period in Houndé District, Burkina Faso. (c) shows the incidence of uncomplicated malaria during the period when SMC was delivered  amongst children who had received SMC within the previous 28 days or who had not received recent SMC (no SMC in the previous 35 days). The analysis of children with ‘no recent SMC’ was restricted to children who received 3 courses of SMC during that intervention year . Malaria hospitalisations and deaths from malaria were defined as hospital admission with a diagnosis of malaria and blood-slide–or RDT-confirmed (DOCX)Click here for additional data file.S6 FigP. falciparum infection or deaths for which malaria was listed as the primary diagnosis. Clinical malaria was defined as attendance at study health facility with a history of fever or measured temperature ≥37.5 °C, with malaria infection confirmed by RDT. Incidence rates are presented per 1,000 person-months at risk rather than per 1,000 person-years and include repeat events in the same child, provided the healthcare contact occurred more than 7 days apart. Vertical bars show 95% CIs. CI, confidence interval; RDT, rapid diagnostic test; SMC, seasonal malaria chemoprevention.Incidence of malaria hospitalisations and deaths from malaria (a) and clinical malaria (b) by calendar month over the study period in Bougouni District, Mali. (c) shows the incidence of uncomplicated malaria during the period when SMC was delivered  amongst children who had received SMC within the previous 28 days or who had not received recent SMC (no SMC in the previous 35 days). The analysis of children with ‘no recent SMC’ was restricted to children who received 3 courses of SMC during that intervention year . Malaria hospitalisations and deaths from malaria were defined as hospital admission with a diagnosis of malaria and blood-slide–or RDT-confirmed (DOCX)Click here for additional data file.S1 TableSMC, seasonal malaria chemoprevention.(DOCX)Click here for additional data file.S2 TableSMC, seasonal malaria chemoprevention.(DOCX)Click here for additional data file.S3 TableResults from both countries are combined. SMC, seasonal malaria chemoprevention.(DOCX)Click here for additional data file.S4 TableP. falciparum infection at the end-of-season surveys in both study centres overall, in Houndé District, Burkina Faso and in Bougouni District, Mali. Prevalence is calculated as number of resistant mutations/number of children, with mixed mutations at a single locus counted amongst those resistant. Samples in which both mutant and wild type were detected at 2 or more codons were excluded. AQ, amodiaquine; SMC, seasonal malaria chemoprevention; SP, sulfadoxine-pyrimethamine.Prevalence of SP and AQ resistance mutations amongst study children with (DOCX)Click here for additional data file.S5 TableACPR, adequate clinical and parasitological response; PCR, polymerase chain reaction.(DOCX)Click here for additional data file.S1 STROBE ChecklistSTROBE, Strengthening the Reporting of Observational Studies in Epidemiology.(DOCX)Click here for additional data file.


Plasmodium falciparum and Plasmodium vivax are present. Acute febrile patients were enrolled from July 2018 to April 2019 in Quibdó, Colombia. Malaria diagnoses were obtained from MI and RDT in the field and later confirmed by qPCR. Venous blood samples in EDTA were processed with an XN-31p in the field. Sensitivity, specificity, positive/negative predictive values, and the likelihood ratios of positive and negative tests were calculated with respect to the results from qPCR, MI and RDT. The intraclass correlation coefficient (ICC) and Bland–Altman plot were used to evaluate the concordance in the parasitemia with respect to MI. A total of 1,754 subjects were enrolled. The mean age was 27.0 years (IQR 14–44); 89.6% were Afro-Colombians, 94.3% lived in urban areas and 0.91% were pregnant. With respect to qPCR, the XN-31p showed a sensitivity of 90% (95% CI 87.24–92.34) and a specificity of 99.83% (95% CI 99.38–99.98) in detecting Plasmodium spp.; both parameters were equivalent to those for MI and RDT. Using MI as the reference, the XN-31p showed a sensitivity of 98.09% (95% CI 96.51–99.08), a specificity of 99.83% (95% CI 99.4–99.98), an ICC of 0.85 (95% CI 0.83–0.87) and an average difference of − 3096 parasites/µL when compared with thick-smear MI and an ICC of 0.98 (95% CI 0.97–0.98) and an average difference of − 0.0013% when compared with thin-smear MI. The XN-31p offers a rapid and accurate alternative method for diagnosing malaria in clinical laboratories in areas where P. falciparum and P. vivax cocirculate.Early and accurate diagnosis is critical in reducing the morbidity and mortality associated with malaria. Microscopy (MI) is the current diagnostic gold standard in the field; however, it requires expert personnel, is time-consuming, and has limited sensitivity. Although rapid diagnostic tests for antigen detection (RDTs) are an alternative to diagnosis, they also have limited sensitivity and produce false positive results in detecting recent past infection. The automated hematology analyzer XN-31 prototype (XN-31p)  is able to identify plasmodium-infected erythrocytes, count parasitemia and perform complete blood-cell counts within one minute. The performance of the XN-31p in diagnosing malaria was evaluated and compared with real-time polymerase chain reaction (qPCR), MI and RDT in an endemic area of Colombia where  Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and the zoonotic parasite Plasmodium knowlesi. The most prevalent and fatal species worldwide is P. falciparum, accounting for 99.7% of estimated malaria cases in Africa. Globally, P. vivax causes 3.3% of estimated cases, with 53% of the P. vivax burden being in the South-East Asia region. P. vivax is the predominant parasite in the Americas, causing 75% of malaria cases1.Malaria infection remains a significant public health problem worldwide. In 2018, an estimated 228 million cases of malaria infection and 405,000 associated deaths were reported. Africa has the largest burden of malaria morbidity (93%), followed by South-East Asia (3.4%) and the Eastern Mediterranean region (2.1%). Five species of the genus Plasmodium cause human infection: 2. Early and accurate diagnosis is one of the most important strategies for malaria control and elimination; therefore, the WHO recommends that all patients with suspected malaria have their diagnoses confirmed by microscopy (MI) or a rapid diagnostic test (RDT) before treatment2.Although there has been a decrease in malaria morbidity and mortality rates since 2010, significant efforts are needed to approach the WHO global targets by 20303.Malaria diagnosis is performed routinely by morphologic identification of the parasite through MI, which is considered the gold standard test for diagnosis in clinical settings. This diagnostic method is low cost, allows quantification of parasitemia, is useful during follow-up of parasitemia clearance and allows the possible identification of other pathogens. However, MI is time consuming, requests trained personal and has an operator-dependent limit of detection, as expert microscopists can identify up to 10–50 parasites/μL, while nonexperts have detection levels in the range of 100–500 parasites/μL4. In addition, HRP2-based RDTs are not useful for following up patients on treatment, since this antigen can circulate for weeks after parasite clearance5.Alternatives to MI include rapid diagnostic tests (RDTs), which are point-of-care tests that can be performed quickly, are easy to use and do not require trained personnel. Although RDTs have improved access to malaria diagnoses outside health facilities, they have important limitations, such as the inability to quantify parasitemia and differentiate parasite stages and a high limit of detection (100–200 parasites/μL)8. Parasite quantification is possible, but it is not available in clinical setting. They present disadvantages for implementation in the field since they are expensive and time-consuming, require trained personnel, yield results that have little use during follow-up after treatment, and do not allow differentiation of parasite stages. However, PCR is very useful in reference laboratories3.Molecular methods such as nucleic acid amplification techniques (NAATs), including polymerase chain reaction (PCR), provide superior sensitivity over other methods, with reported detection thresholds < 5 parasites/μL and the highest specificity11. The automated hematology analyzer XN-31 prototype (XN-31p)  is a novel analyzer model that has been optimized to detect Plasmodium-infected red blood cells (iRBCs) circulating in human blood using a combination of optical fluorescence techniques, flow cytometry and laser-optical recognition by a violet semiconductor 405 nm laser beam. In this study, an XN-31p was used that can perform a complete blood count (CBC) and classify different blood cell populations, as well as identify iRBCs and count parasitemia within one minute with a limit of quantification of 20 iRBCs/μL10.The search for alternative diagnostic methods for the detection and quantification of Plasmodium infections is ongoing. Automated hematology analyzers can offer fast, sensitive and cost-effective assessments of all suspected malaria infections, as they reduce analytical time and improve accuracy12.While the XN-31p was designed for in vitro diagnosis use, the XN-30, which had been developed for research use, had been previously evaluated in several studies. Both analyzers have the same physical characteristics, hardware configuration, and reagent compositionP. falciparum parasites cultured in vitro, which permitted the identification of parasite stages and the sensitive and reproducible calculation of parasitemia13. Later, the XN-30 was applied in the screening new antiplasmodial compounds14. In addition, the XN-30 system permitted the simultaneous measurement of iRBCs in mouse blood samples infected with rodent malarial parasites and hematological evaluation15.Tougan and colleagues reported the use of the XN-30 in recognizing 11. Pillay and colleagues validated the parameters of the XN-30, such as the limit of blank (LoB), limit of detection (LoD), limit of quantitation (LoQ), carryover, precision and stability; in addition, they used 191 samples with malaria diagnoses previously confirmed by MI and RDT to assess the identification of iRBCs by the XN-3011. Post and colleagues evaluated the performance of the XN-30 in diagnosing malaria and compared it to the performance of MI and qPCR in 908 clinical samples from an endemic region of Burkina Faso, which is hyperendemic for P. falciparum; the sensitivity and specificity of XN-30 were, respectively, 98.7% and 99.4% relative to MI and 70.9% and 99.6% relative to qPCR10. Previous studies have shown the potential application of the XN-30 for malaria diagnosis in the clinical laboratories of endemic regions; however, no evaluation has been performed for the identification of Plasmodium species by the XN-30 in a clinical setting with cocirculating P. falciparum and P. vivax. In addition, the concordance in the parasite count with respect to MI has not been evaluated.Two previous studies evaluated the performance of the XN-30 in a clinical settingThe primary aim of this study was to evaluate the performance of the XN-31p in diagnosing malaria using venous blood and compare it with that of qPCR, MI and RDT in an endemic region of Colombia. Additionally, a pilot study was performed to evaluate the concordance in the qualitative and quantitative detection of Plasmodium infection between the XN-31p with finger-prick blood and with venous blood.16.An observational cross-sectional study of diagnostic accuracy was carried out to evaluate the performance of the XN-31p in the detection of Plasmodium infections in a clinical setting. Sociodemographic and clinical data were collected from each subject before conducting the diagnostic test . Samples were tested by the XN-31p, RDT and MI simultaneously at a field site, and a reference standard test (qPCR) was performed later in the reference laboratory at the University of Antioquia, Medellin city. The Standards for Reporting Diagnostic Accuracy Studies (STARD) guidelines were used in this studyP. falciparum (57.5%), P. vivax (38.3%) and mixed infection  (4.1%)17. Quibdó is the capital of Chocó state, and it reported 6.7% of all malaria cases in Colombia in 2018 from periurban areas18. The participants of this study were enrolled at the Local Hospital Ismael Roldán Valencia (HLIRV), which is the reference hospital for patients with suspected uncomplicated malaria from Quibdó and other nearby municipalities.The study was conducted in the municipality of Quibdó, Chocó, Colombia , which is located in the Pacific coast region  and 30 uninfected, according to the MI diagnosis made in the field. These subjects were selected among those enrolled in the framework for assessing the performance of the XN-31p using venous blood.The sample size was estimated assuming an expected sensitivity and specificity for the XN-31p of 80% and 90%, respectively, relative to qPCR (gold standard test) for the diagnosis of malaria. The sample size was calculated assuming an expected malaria prevalence of 15% in patients from Quibdó with acute febrile illness, with a 95% confidence interval and 5% error, corresponding to 1,640 participants. A pilot study for evaluating the performance of the XN-31p with finger-prick samples included a convenience sample of 60 participants, 30 with Plasmodium infection  or history of fever 72 h prior to admission, less than 7 days of fever evolution, no other apparent localized cause of fever, and residence in any malaria transmission area. The participants were enrolled consecutively as they were admitted to the emergency and outpatient services of the HLIRV from July 2018 to April 2019.All eligible participants were invited to participate and signed informed consent forms. At enrollment, a clinical evaluation was performed by a physician; information including demographic characteristics, current disease, history of malaria episodes, and use of preventive measures for malaria was recorded. A finger prick blood sample (20 µL) was obtained to determine the malaria diagnosis by MI. Venous blood samples (4 mL) were collected from each subject in EDTA tubes and analyzed by the XN-31p; the same sample was used for the malaria RDT test and analyzed by an XN-450 Sysmex analyzer, which had been approved for clinical use, to obtain subject hematological parameters. An aliquot of this sample (500 µL) was frozen in liquid nitrogen and sent to the reference laboratory at University of Antioquia in Medellín for later confirmation of the malaria diagnosis by qPCR (within the next month). When a participant was selected for the pilot study using finger-prick blood, an additional capillary sample (100 µL) was obtained using a high-blood volume lancet and microtubes containing EDTA.This study included an XN-31p automated hematology analyzer . The EDTA venous blood samples were processed directly by the analyzer within 4 h after venipuncture. Briefly, 60 µL of the venous blood sample was used for analysis by the XN-31p in Low Malaria (LM) mode. The EDTA capillary sample (20 μL) was diluted with CELLPACK DCL reagent (120 μL) within 30 min after the finger prick and run by the analyzer immediately after dilution in Pre-Dilution (PD) mode.P. falciparum if the “Malaria?(P.f.)” flag is displayed, on other non-P. falciparum species if the “Malaria?(others)” flag is displayed, and on unclassified species if the “Malaria?(UNC)” flag is present. Mixed infections according to the index test were considered when both P. falciparum and other non-P. falciparum species were flagged simultaneously in a sample. All the results were automatically downloaded from the instrument as an Excel file that was exported and sent weekly to the data manager at the University of Antioquia.The samples were aspirated and diluted automatically by the analyzer, and staining for nucleic acids was performed. Subsequently, infected red blood cells (iRBCs) and white blood cells (WBCs) were detected by a violet semiconductor 405 nm laser beam in the M channel. The data were automatically determined by analyzing the plotted scattergrams with forward-side scattered light and side fluorescent light, which allowed visualization of the iRBC population separate from the RBC population . The microscopist in the field had 2 years of demonstrated expertise in malaria diagnosis and the two microscopists at the reference laboratory had 8 years and were classified as level expertise 1 according to WHO19.Two thick blood films and one thin film were prepared from capillary samples from all participants and stained with Giemsa according to international guidelines20. For positive slides, the stained thin smear was used to confirm species diagnosis, and parasite density was calculated from both thick and thin films as previously described19. The thick films were used to count the number of asexual and sexual parasites per 500 leukocytes (when count was > 500 parasites/µL) or 1,500 leukocytes (when count was < 500 parasites/µL); parasite density was estimated using WBC count obtained by the XN-450 for each participant. The parasitemia from the thin film was calculated by establishing the percentage of infected red blood cells (iRBCs) among 10,000 counted erythrocytes. The parasitemia obtained by the second microscopist was used for comparison with the iRBC% and iRBC# calculated by the XN-31p.A stained, thick blood smear was used to establish positive and negative judgment; the smear was considered negative if, after completing examination of 500 fields at 100 × magnification, no parasites  were found. A second thick smear was used as a backup for cases where there was any problem with staining or doubts with judgment. Mixed infection was determined according to WHO criteriaPlasmodium falciparum histidine-rich protein-2 (HRP2) and Plasmodium vivax parasite-lactate dehydrogenase (LDH) was scored. Any visible test line was considered positive, and the results were reported as P. falciparum, P. vivax, mixed infection (Pf/Pv) or negative.Rapid diagnostic tests (SD BIOLINE Pf/Pv (HRP2/LDH), Ref 05KF80) were performed on EDTA-anticoagulated venous blood within 30 min after venipuncture, according to recommendations from the manufacturer. The RDTs were performed simultaneously with the sample processing by the Sysmex analyzers and before the MI. The test line reactivity of 21. Reactions were run on an Agilent AriaMx Real-Time PCR System. A positive result was determined for samples with a cycle threshold (Ct) value > 35.1, which corresponds to a parasitemia of 0.5 parasites/µL (limit of quantification (LoQ)). A negative result was reported when the Ct value > 39.5. Samples with Ct values ≥ 35.1 and ≤ 39.5 were classified with a final diagnosis of inconclusive; DNA was detected, but it was under the LoQ (very low parasitemia); qPCR genus detection was subsequently repeated for these samples to confirm the final classification. Qualitative results were reported since quantitative results do not represent the count of iRBCs.Plasmodium genus detection: Molecular diagnosis was carried out in the reference laboratory in a blinded manner. DNA was extracted from a 160 µL aliquot of EDTA-venous blood after defrosting using a Qiagen QIAamp DNA Mini Kit . An aliquot of 5 µL of DNA was used to amplify a region of the multicopy 18S rRNA gene from the genus Plasmodium by qPCR, as described by Rougemont et al., using Platinum Quantitative PCR superMix-UDG TaqMan Universal Master Mix (Invitrogen Thermo Scientific), primers and probes as previously reportedP. falciparum and P. vivax species amplification protocols using two independent qPCR assays with primers described by Sing et al.22 and 5 × Hot FIREPol EvaGreen qPCR Supermix. Only samples that had not been amplified by P. falciparum or P. vivax protocols were additionally processed by qPCR for P. malariae using primers described by Sing et al.22 and detected by EvaGreen. If the Plasmodium species result persisted as inconclusive, nested PCR (nPCR) was carried out for P. ovale detection using a protocol reported by Sing et al.22. A positive result for P. falciparum, P. vivax or P. malariae species was defined according to the Ct, last relative fluorescence unit (ΔR last) and temperature melting (Tm) values, and a positive result for P. ovale was defined by the presence of bands on the agarose gel. Mixed infection was determined by the presence of two or more positive results from independent species amplification protocols. Samples with positive and inconclusive results from the genus protocol but negative results from the four species protocols were classified as Plasmodium spp.Plasmodium species detection: All samples with a final diagnosis of positive and inconclusive were processed with 3/μL and severe thrombocytopenia as PLT < 50*103/μL24.The EDTA venous blood samples (25 μL) were processed by the automated hematology analyzer XN-450  in WB mode. The samples were processed within 4 h after venipuncture, with a maximum interval of 30 min for processing between the XN-31p and XN-450. The data for the parameters white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), hematocrit and platelet count (PLT) were collected from the XN-450 analyzer to describe the study participants. Anemia was defined as Hb < 11 g/dL, thrombocytopenia as PLT < 150*10Demographic, clinical, and epidemiological information was registered in a Microsoft Access 2010 database in the field. The database was sent weekly to the data manager at the University of Antioquia, who completed it with reference laboratory results (MI and qPCR) as well as results from the XN-31p.P. falciparum and P. vivax species. For the last item, detection was assumed when the flagging mechanism for non-P. falciparum species was positive.Descriptive analysis was carried out for the participant characteristics. Independent analysis of the diagnostic accuracy of the XN-31p was performed for genus Plasmodium, P. falciparum and P. vivax detection was assessed after excluding mixed infections from the results of the reference method. The intraclass correlation coefficient (ICC)25 and Bland–Altman plot26 were used to evaluate the concordance in the parasitemia between the XN-31p and MI among positive samples for both methods. The 95% confidence interval (95% CI) was estimated for each parameter.For the performance of the XN-31p (index test) with venous blood samples, sensitivity (SE), specificity (SP), positive/negative predictive values  and likelihood ratio of positive and negative tests (LRP and LRN) were calculated in reference to qPCR (gold standard test). In addition, the performance of the XN-31p was compared with that of MI and RDT because these are the currently available tests in the field. The McNemar test was applied to compare the SE and SP of the XN-31p, MI and RDT, all in reference to qPCR. Inconclusive results from genus qPCR were considered positive since these samples had Plasmodium DNA at very low concentrations (sometimes the result was not reproducible), and the strictest result was assumed to evaluate the performance of the analyzer. Independent analysis for genus detection was carried out assuming three scenarios for the MI-RBC Abn Scattergrams (index test inconclusive): positive, negative or excluded from analysis. The performance of the XN-31p for For the performance of the XN-31p with finger-prick blood samples, SE, SP, PPV, NPV, LRP and LRN and their 95% CIs were calculated in reference to the qPCR results. Concordance in the malaria diagnosis and parasitemia between venous blood and finger-prick samples was evaluated by Cohen's kappa coefficient and the ICC/Bland–Altman plot method, respectively.27 for calculating SE, SP, PPV, NPV, LRP and LRN; DTComPair version 1.0.328 for comparing SE and SP with the McNemar test; blandr29 for generating the Bland–Altman plot; irr30 for calculating the intraclass coefficient; and psych31 for calculating Cohen's kappa coefficient.Descriptive analysis was carried out using IBM SPSS statistics program version 24 licensed by University of Antioquia, and performance analysis of the diagnostic tests was carried out using R version 3.6.3 and RStudio cloud programs with the following packages: epiR version 1.0–14The study was reviewed and approved by the Facultad de Medicina Ethics Committee at the Universidad de Antioquia, Medellín, Colombia , and Sysmex Corporation received approval from the Ethics Review Committee of the Japanese Association for the Promotion of State of the Art in Medicine . This study complied with the principles stated in the Declaration of Helsinki , the Belmont report  and resolution 8430/1993 of the Ministry of National Health of Colombia . Before starting any study procedure, written informed consent was obtained from all participants. For participants < 18 years old, additional informed consent from their parents or legal guardian was also obtained. All the methods were carried out in accordance with the approved guidelines. Recorded data from participants were anonymized using a code. Participants were diagnosed by microscopy and treated according to the malaria guidelines from the Health Ministry of Colombia. The results from the XN-31p were not used to manage patients since this was the exact instrument under evaluation in this study. The information obtained from the questionnaires and clinical evaluations was stored and guarded in the Malaria Group of the University of Antioquia in Medellín.A total of 1,967 eligible participants were enrolled, and 1754 blood samples were analyzed by the XN-31p. No samples were excluded from analysis due to inadequate sample storage, sample mismatch, hemolysis or a shortage of sampled blood. Only one sample had missing MI data.Baseline demographic and clinical characteristics and data on malaria history are presented in Table The results of the malaria diagnosis test for the enrolled febrile participants are presented in Table A total of 1,754 samples from febrile participants were included for the evaluation of the performance of the XN-31p with respect to the qPCR results in a clinical setting from a malaria-endemic region in Colombia ; the demographic characteristics and history of malaria episodes of these participants were similar to those with valid results (positive or negative). However, some differences were observed among a number of hematological parameters  . On the other hand, 82.5% (47/57) of the false negative samples detected by the XN-31p had submicroscopic infections, and 76.6% (36/47) of them had a Ct level less than the LoQ from qPCR. The false-negative samples with microscopic infections  had parasite densities ranging from 16 to 261 parasites/µL; only three of these samples had parasitemia higher than the LoQ of the XN-31p (20 iRBCs/µL). In addition, the percentages by species among these samples were 57.9% (33/57) P. falciparum, 15.8% (9/57) P. vivax, 1.7% (1/57) mixed Pf/Pv and 24.6% (14/57) Plasmodium spp.The parasite densities of the two false positive samples detected by the XN-31p were 30 and 40 parasites/µL. Here, the XN-31p detected iRBCs, but the flag mechanisms for detecting species were not indicated; in addition, both samples had high WBC counts , while the SP was similar for the three methods  infection). The false negative rate for P. falciparum was 3.6% ; of these samples, 83.6% (51/61) had iRBCs with < 100/μl, and iRBCs were detected (> 20 iRBCs/µL) among only 45.9% (28/61).The SE and SP of the XN-31p with respect to qPCR in the detection of P. vivax infections were 91.94 and 98.91, respectively. Both values were similar to those for MI (SE 92.31% and SP 100%), and the SE was better than that for RDT (85.94) in detecting P. vivax . The false negative rate for P. vivax was 0.59% (10/1686); all samples had iRBCs with < 100/μL, but iRBCs could not be detected among 90.0% (9/10) of the samples (< 20 iRBCs/µL).The SE and SP of the XN-31p with respect to qPCR in the detection of The concordance in the parasitemia between the XN-31p (MI-RBC#/µL) and the thick blood smear was good, with an ICC of 0.85 and an average difference of − 3096 parasites/µL. In addition, there was excellent concordance in the parasitemia between the thin blood smear and the XN-31p (MI-RBC%), with an ICC 0.98 and an average difference of − 0.0013% iRBCs Table . Only 1.The results of the malaria diagnosis test for the capillary samples are presented in Table The concordance in the parasitemia between the XN-31p with finger-prick samples (PD mode) and with venous samples (LM mode) for Plasmodium detection was very good . On the other hand, the concordance in the parasitemia according to MI-RBC #/µL and MI-RBC% was excellent, with an ICC of 0.995 (95% CI 0.989–0.997), an average difference of 1,085.3 parasites/µL  an ICC of 0.957 (95% CI 0.913–0.980) and an average difference of − 0.05% (SD 0.15%), respectively  of 34.1 and 49.4 per 1000, respectively18. This condition could have favored the high percentage of malaria among our febrile participants (approximately 30%) and makes this study similar to others in high-transmission areas34. This study evaluated the diagnostic accuracy of the XN-31p in a prospective cohort of acute febrile patients who consulted a physician for medical care and were tested for malaria. This was an advantageous scenario for testing the performance of the XN-31p, since there was a chance to enroll patients with a wide range of clinical presentations. However, the clinical spectrum of participants in this study was ultimately very homogenous and typical of those with uncomplicated malaria, reflecting the expected percentage of severe malaria in Colombia of approximately 2%18.Although Colombia is classified as a low and unstable malaria transmission region11, the SE and SP were high , and the false positive rate was 0.12%, less than the limit of the WHO criteria for recommending the procurement of an RDT procurement (< 10%)35. The XN-31p requires mains electricity and is not portable; therefore, it is not considered a point of care test. However, it could be very useful for malaria screening among acute febrile individuals who seek care at health centers in malaria endemic areas, offering a malaria diagnosis and automated blood count simultaneously.The XN-31p showed good performance with respect to qPCR in diagnosing malaria (Plasmodium genus), comparable to that MI and RDT, conventional methods used in the field. Using a previously defined cutoff of 20 iRBCs/μL36, the XN-31p may not be the best option for diagnosis, as a higher false positive rate is expected.A false negative rate of 3.3% was found for the XN-31p, which may not be of concern for febrile individuals informed about potential warning signs and the need for serial diagnostic tests if their symptoms persist. On the other hand, in settings of active surveillance in low-transmission areas where low parasitemia is common (< 100 parasites/µL)11. It is necessary to evaluate the performance of the XN-31p for participants with severe diseases to confirm the possible MI-RBC Abn Scattergrams caused by hematological alterations.Approximately 2% of samples tested by the XN-31p had inconclusive results (MI-RBC Abn Scattergram); therefore, they would require another test. Hematological alterations in these participants could contribute to the difficulty of the analyzer in accurately classifying these samples. Pillay et al. 2019 reported a MI-RBC Abn Scattergram flag from an XN-31p in LM mode in 36% of samples with hematological alterations 19, explaining the excellent performance of MI with respect to qPCR, in contrast to other studies37. It is possible that the performance of the XN-31p could be different than that reported here if compared with MI performed in field conditions.Current study employed expert microscopists to read all slides (Level 1)P. vivax was equivalent to that of expert MI and RDT, P. falciparum detection was possible but with a lower performance than MI and RDT. The critical point for discriminating Plasmodium species based on the flagging mechanisms in this study was the level of parasitemia. The XN-31p can discriminate species when the parasitemia is higher than 100 iRBCs/µL; in the current study, the parasitemia for P. falciparum infections were lower than those for P. vivax infections. On the other hand, some P. vivax stages are larger in size than P. falciparum, which also favors the detection of P. vivax by the XN-31p. Additional effort is necessary to study the performance of the XN-31p with samples with ultralow parasitemia, mainly with P. falciparum infections. Moreover, these flagging mechanisms have not yet been fully validated for differentiating every species by the manufacturer due to a lack of data on rare species such as P. malarie, P. ovale or P. knowlesi. It is important to differentiate these species to determine appropriate treatment; therefore, further evaluation including these less frequent species should be performed to improve the species flagging mechanism.When the results were evaluated by species, the performance of the XN-31p was variable; while the detection of 10, which may be because the Post et al. study was carried out in a setting with a higher percentage of complicated malaria (approximately 35%), a predominance of P. falciparum infections (99%) and a higher reporting of antimalarial uptake in the past two weeks; all conditions offer disadvantages for diagnosing malaria with a hematological analyzer.The performance of the XN-31p in this epidemiological setting in Colombia is better than that reported by Post et al. 2019 in Burkina Faso (SE 90.% vs 70.9%)The concordance in the parasitemia between the XN-31p and MI was very good, showing an important strength of the former because MI requires a longer reading time for the accurate estimation of parasitemia. It is important to consider that thin smear parasitemia estimates can be closer to XN-31p values since both methods count complete erythrocytes.On the other hand, the XN-31p had malaria detection capabilities equivalent to those of qPCR and MI, even with finger-prick blood. At present, the XN-31p is intended to be used with venous blood, and it is important to accumulate positive results such as those obtained here to expand its use with finger-prick blood as well.The fact that there were virtually no severe malaria cases represented in our cohort constitutes a limitation of this study since the XN-31p results cannot be generalized to this group of patients. It was not possible to determine the performance of the XN-31p for mixed infections, as the percentage of coinfections was low in this study and the number of samples with this condition was insufficient to guarantee the accuracy of the results. The pilot study for evaluating the concordance in the malaria diagnosis between venous blood and finger-prick blood from the XN-31p included a limited number of samples, which were selected according to different levels of parasitemia; this could represent a potential source bias.P. falciparum. The performance of the XN-31p in the discrimination of coinfections needs to be evaluated in future studies.The XN-31p analyzer offers a rapid and accurate alternative method for malaria diagnosis in clinical laboratories located in endemic areas, although an economic evaluation study should be carried out. This analyzer is comparable to expert MI in the detection of Plasmodium infections and the quantification of parasitemia; in addition, it performs a complete blood-cell count and produces a results report in one minute. The XN-31p is able to discriminate parasite species; however, it shows some room for improvement in detecting Supplementary Information


P. falciparum endemic regions such as western Kenya. Carriage of different sickle cell genotypes may influence haematological parameter during malaria. Children resident in malaria holoendemic regions suffer more from malaria-related complications and this is moderated by the presence of the SCD. In the current study, we determined the association between sickle cell genotypes and haematological parameters in children with P. falciparum malaria resident in Kisumu County in Western Kenya.Sickle cell disease (SCD) is a monogenic disorder due to point mutation in the β-globin gene resulting in substitution of Valine for Glutamic acid. The SCD is prevalent in n = 217, aged 1–192 months) with acute febrile condition were recruited at Jaramogi Oginga Odinga Teaching and Referral Hospital. Chi-square (χ2) analysis was used to determine differences between proportions. Differences in haematological parameters were compared across groups using Kruskal Wallis test and between groups using Mann Whitney U test. Multivariate logistic regression analysis controlling for infection status was used to determine the association between sickle cell genotypes and haematological parameters.Children (P = 0.041], reduced haematocrit , reduced RBC count , reduced MCHC , increased leucocytosis  and reduced monocytosis . However, carriage of HbAS was only associated with increased micro-platelets .Using HbAA as the reference group, multivariate logistic regression analysis revealed that carriage of HbSS was associated with reduced haemoglobin [OR = 0.310, 95% CI = 0.101–0.956, Results show that carriage of HbSS in children influence the levels of haemoglobin, haematocrit, RBC, MCHC, WBC and Monocytes. Therefore prior knowledge of HbSS should be considered to improve clinical management of haematological alterations during malaria in children.The online version contains supplementary material available at 10.1186/s12879-020-05625-z. The sickle cell haemoglobin (HbS) is a genetic disorder due to a single nucleotide polymorphism (SNP) or a point mutation substituting thymine for adenine at the sixth codon of β gene, (6GAG > 6GTG). This leads to valine incorporation, rather than glutamine and results in haemoglobin tetramers (HbS) that aggregate into arrays upon deoxygenation in the tissues . The resPlasmodium of which the main species is falciparum  were older compared to those in non-infected group . Otherwise age did not differ between infected and non-infected groups both in HbAS  and  and HbSS  and , respectively. Haematological parameters were compared in the infected and non-infected children (controls) of the equal genotype (within the group) and in the infected children of different genotypes (across the group) i.e. HbAS versus HbSS. As shown in Table P = 0.001), haematocrit (P = 0.001) and RBC (P = 0.004) count were relatively lower in the infected group compared to non-infected group within each genotype. The RDW (P = 0.007) was moderately higher in the infected group compared to non-infected group in each stratum. Haemoglobin, haematocrit, RBC count and RDW were the most significantly different red cell parameter across the genotypes . Furthermore, MCV (P = 0.332), MCH (P = 0.784) and MCHC (P = 0.179) were higher in the non-infected group relative to infected group in each genotype.Children (IQR); 16 , P = 0.5P = 0.817) but higher in the infected group within both HbAS (P = 0.433) and HbSS (P = 0.569) genotypes, these differences were not statistically significant. However, the WBC count was statistically significant across the genotypes (P = 0.004). Lymphocyte was lower in the infected group relative to non-infected group within the HbAA genotype , respectively. Both monocytes (P = 0.866) and granulocytes (P = 0.833) were comparable within the genotypes. Among the platelet parameters and its indices, platelet count was more exceptional in its comparability within the HbAA genotype. Infected group had lower platelet count relative to non-infected group , respectively. This significance was equally simulated in the comparison across the three genotypes (P = 0.001). The MPV (P = 0.710) and PDW (P = 0.611) were comparable across the genotypes. The PCT was only statistically significant within HbAA genotype with  for infected and non-infected groups, respectively. All the determined demographic and laboratory characteristics are summarized in Table Even though the WBC count were numerically lower in the infected group relative to non-infected group in HbAA genotype P = 0.87 but higP. falciparum infected children were modelled using logistic regression model while controlling for the infection status. Infection status was controlled for since sickle gene has an influence of the haematological parameters regardless of infection, however, malaria infection can further worsen the levels of these parameters. Using the homozygous wild-type genotypes (HbAA) as the reference group, results revealed no association between HbAS and haemoglobin , haematocrit , RBC count  and RDW . However, there was a significant association between HbSS and haemoglobin , haematocrit , RBC count  and RDW    was not associated with HbAS, however, association between HbSS and increased MCV did not run in the regression model. Additional analyses revealed that reduced MCH did not show any association with HbAS . Moreover, reduced MCH did not show any association with HbSS  while reduced MCHC did not reveal association with HbAS . However, reduced MCHC showed association HbSS . Total reduction in white blood cell count (leucocytopaenia) was not associated with HbAS . However, leucocytosis showed increased association with HbSS . Both elevated and reduced lymphocyte and granulocyte counts did not reveal association with either HbAS or HbSS in P. falciparum infected children. However, increased monocyte count (monocytosis) revealed association with HbSS .Considering the distribution of haematological parameters in the three stratified study groups, their association with sickle cell genotypes in P = 0.512]. Furthermore, increased thrombocyte count (thrombocytosis) equally showed no association with HbAS . Unfortunately, the model could not determine association of either thrombocytopenia or thrombocytosis with HbSS genotype in children infected with malaria. Micro-platelet was defined as a platelet with reduced mean platelet volume (MPV) to below 6 fL [P = 0.012] but was no associated with HbSS . Furthermore, PDW did not show any association with HbAS , however, its association with HbSS was undetermined. Finally, PCT did not show any association with HbAS  and with HbSS   did not show association with HbAS  and HbSS , was not associated with P. falciparum infection  of asymptomatic and symptomatic P. falciparum infection of variable severity  hrocytes . In addimonoxide  and thatmonoxide  transloclocation . Nonethelocation –49. Incrlocation . HoweverLeucocytosis has previously been reported in children infected with malaria , 50 and 12 μL-1) with regard to age, sex and altitude [P. falciparum infection [P. falciparum were less likely to have reduced MCHC. The mean corpuscular haemoglobin concentration (MCHC) was defined as a measure of the concentration of haemoglobin in a given volume of packed red blood cell with a reference range of 28–36 gdL-1 [P. falciparum were nine times more likely to have leucocytosis. Increased association of leucocytosis with carriage of HbSS in the current study may still be attributed to increased erythropoiesis and consistent haemolysis [To determine the associations of haematological parameters and sickle cell genotypes, anaemia was defined as a condition where the haemoglobin level was below the lower reference (< 10 gdL-1) and RBC count , and alpha thalassaemia which is known to have epistasis effects on sickle cell gene with respect to P. falciparum malaria [Numerous studies have demonstrated protective effect of HbSS from on occur , 63, 64.outcomes . The dif malaria . A longiP. falciparum malaria. Moreover, our study revealed that carriage of HbAS is associated with lower MPV in children infected with malaria. Results from this study can help in the differential diagnosis of malaria infection in HbSS genotype based on haematological parameters in resource limited setting where sickle cell genotyping remains a challenge. Therefore, for proper management of P. falciparum malaria, sickle cell genotyping should be considered to guide on the administration of antimalarial prophylaxis to HbSS patients.HbSS genotype is associated with low haemoglobin, haematocrit, RBC, MCHC, WBC and monocytes. In addition we show that, carriage of HbAS is associated with lower MPV in children infected with malaria. HbSS genotype is association with low haemoglobin, haematocrit, RBC, MCHC and moncytopenia. However, it is associated with increased leucocytosis in children with Additional file 1.Additional file 2.


Plasmodium knowlesi and Plasmodium cynomolgi, which can both cause malaria in humans, and Plasmodium inui, which can be experimentally transmitted to humans. A significant number of zoonotic malaria cases have been reported in humans throughout Southeast Asia, including Thailand. There have been only two studies undertaken in Thailand to identify malaria parasites in non-human primates in 6 provinces. The objective of this study was to determine the prevalence of P. knowlesi, P. cynomolgi, P. inui, Plasmodium coatneyi and Plasmodium fieldi in non-human primates from 4 new locations in Thailand.Certain species of macaques are natural hosts of Macaca fascicularis, Macaca leonina and Macaca arctoides were collected from four locations in Thailand: 32 were captive M. fascicularis from Chachoengsao Province (CHA), 4 were wild M. fascicularis from Ranong Province (RAN), 32 were wild M. arctoides from Prachuap Kiri Khan Province (PRA), and 25 were wild M. leonina from Nakornratchasima Province (NAK). DNA was extracted from these samples and analysed by nested PCR assays to detect Plasmodium, and subsequently to detect P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi.A total of 93 blood samples from Plasmodium-positive by nested PCR assays. Among wild macaques, all 4 M. fascicularis at RAN were infected with malaria parasites followed by 50% of 32 M. arctoides at PRA and 20% of 25 M. leonina at NAK. Only 2 (6.3%) of the 32 captive M. fascicularis at CHA were malaria-positive. All 5 species of Plasmodium were detected and 16 (59.3%) of the 27 macaques had single infections, 9 had double and 2 had triple infections. The composition of Plasmodium species in macaques at each sampling site was different. Macaca arctoides from PRA were infected with P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi.Twenty-seven of the 93 (29%) samples were Plasmodium varied among the wild and captive macaques, and between macaques at 4 sampling sites in Thailand. Macaca arctoides is a new natural host for P. knowlesi, P. inui, P. coatneyi and P. fieldi.The prevalence and species of  Plasmodium and over 250 Plasmodium spp. have been described in mammals, birds, rodents and reptiles [Plasmodium cynomolgi, Plasmodium knowlesi and Plasmodium inui from Old World monkeys, Plasmodium brasilianum and Plasmodium simium from New World monkeys and Plasmodium schwetzi from chimpanzees [Malaria is caused by the protozoan parasite members of the genus reptiles –3. More mpanzees –7.Plasmodium knowlesi was first reported as a significant cause of human malaria in Malaysia in 2004 [P. knowlesi were documented in several other countries in Southeast Asia including Thailand [P. knowlesi was reported in 2004, in a patient who had visited the forest in Prachuap Kiri Khan Province, Southern Thailand near the Myanmar border [P. knowlesi infected patients have been reported in Tak, Chantaburi, Yala, Narathiwat, Prachuap Kiri Khan and Ranong Provinces [P. knowlesi, naturally acquired human P. cynomolgi infections have recently been reported in Peninsular Malaysia [P. inui have not been described, P. inui can cause malaria in humans by blood passage [ in 2004 . SubsequThailand , 10, IndThailand , PhilippThailand , SingapoThailand , VietnamThailand , CambodiThailand , Laos [1Thailand  and MyanThailand . FurtherThailand , 19. In rovinces . These arovinces –25 and Frovinces  with knoMalaysia , MalaysiMalaysia , 29 and Malaysia  and in aMalaysia . Althoug passage .P. knowlesi, P. cynomolgi and P. inui are long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (Macaca nemestrina) which are found in nature in Southeast Asia [P. knowlesi infection in a banded leaf monkey  in Peninsular Malaysia [Semnopithecus obscurus) in Thailand [P. cynomolgi are Macaca radiata, Macaca cyclopis, Macaca sinica, Macaca mulatta, Presbytis cristasus and Presbytis entellus [Plasmodium inui also naturally infects many monkey species, including M. cyclopis, M. mulatta, M. radiata, Presbytis cristasus, P. obscurus and Cynopithecus niger [P. knowlesi and P. cynomolgi. Experimental hosts of P. knowlesi described to date have included Callithrix jacchus, Cebus spp., Cercocebus fuliginosus, C. cephus, Cynochepalus papio, Hoolock hoolock, M. radiata, M. arctoides, Papio doguera, Papio jubilaeus, Papio papio, Presbytis cristatus, Saimiri sciureus and Semnopithecus entellus [P. cynomolgi experimental hosts have included M. mulatta, Cercopithecus aethiops, Cebus capucinus and Papio papio [The main natural hosts of ast Asia . There hMalaysia  and a duThailand . The othentellus . Plasmodus niger . Besidesentellus . For P. io papio .M. fascicularis, M. nemestrina, the northern pig-tailed macaque (M. leonina), the rhesus macaque (M. mulatta), the stump-tailed macaque (M. arctoides) and the Assamese macaque (Macaca assamensis) [P. inui and P. coatneyi were identified in wild-caught M. fascicularis in Ranong Province, near the Myanmar border [P. inui, P. coatneyi and P. knowlesi were described in wild-caught M. fascicularis and M. nemestrina in Yala and Narathiwat Provinces, in Southern Thailand near the Malaysian border [In Thailand, six species of macaques have been identified in nature; amensis) –39. Therr border . Then inn border . It is iM. fascicularis at the Krabok Koo Wildlife Breeding Center, Chachoengsao Province (CHA), 32 samples from wild M. arctoides at the Pa La U waterfall, Huahin district, Prachuap Kiri Khan Province (PRA), 25 samples from wild M. leonina at the Khao Yai National Park, Nakornratchasima Province (NAK) and 4 samples from wild M. fascicularis at Chang Island, Mu Ko Ranong National Park, Ranong Province (RAN). The Department of National Parks, Wildlife and Plant Conservation, Thailand, approved the protocol for study in a protected area, collection of the blood samples and released of wild macaques (Permit Number: 0909.204/14187). All procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, U.S.A., and were approved by the Animal use and care committee of Kasetsart University Research and Development Institute, Kasetsart University, Thailand (ID: ACKU59-SCI-011).A total of 93 blood samples were collected from captive and wild macaques in 4 areas of Thailand from 2017–July 2019  and Xylazine hydrochloride (0.5–2 mg/kg body weight). The blood samples  were collected using a syringe from the femoral vein of the macaques into a tube with EDTA. From the EDTA tubes, three 40–50 μL of blood from each sample were transferred to Whatman 3 MM filter paper. The samples were initially kept at room temperature and were transported to the Department of Zoology, Faculty of Science, Kasetsart University until molecular analysis. Blood spots on filter papers were transported to the Malaria Research Centre, Universiti Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia for DNA extraction and molecular analysis.Plasmodium with the aid of genus-specific primers  as described previously [Plasmodium-positive samples were then examined by nested PCR assays using species-specific primers to detect P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi, as described previously [DNA was extracted from blood spots on filter papers with the use of InstaGene  as described previously . The DNAeviously . Plasmodeviously . The proPlasmodium species between locations and among troops of macaques. The statistical analysis was undertaken using software R (version 3.5.2) and statistical significance was set at P < 0.05.The Fisher-Freeman-Halton exact test was used to compute the exact probabilities of the prevalence of Plasmodium-positive  were ve Table . Among wPlasmodium parasites were detected; P. knowlesi, P. inui, P. cynomolgi, P. coatneyi and P. fieldi. The majority of malaria-positive macaques (59.3%) had mono-infections, 33.3% had double and 7.4% had triple infection of Plasmodium spp. Overall, P. inui was the most prevalent species detected, occurring in 35% of the malaria-positive macaques, followed by P. fieldi (30%), P. cynomolgi (20%), P. coatneyi (12.5%) and P. knowlesi (2.5%).All five species of P value for the Fisher exact test was 10–5 indicating that the prevalence of each species of Plasmodium species exhibited bias among different locations. The prevalence of each Plasmodium species infection in M. arctoides was higher than that for the other macaques (P = 0.007). and all five species of Plasmodium that were tested for, were detected in these wild M. arctoides.The prevalence of each parasite species varied significantly among the macaque species and sites of collection Table . The P vPlasmodium spp. in captive M. fascicularis at the Krabok Koo Wildlife breeding centre at CHA was much lower than that observed in wild macaques from the other 3 provinces. These macaques were reported to be trapped and brought to the centre due to human-monkey conflict. It is unlikely that these M. fascicularis acquired their infections at the breeding centre because if there were vectors of malaria in the vicinity and active transmission of malaria, the prevalence of malaria parasites would have been much higher at this location. A more likely explanation is that the 2 M. fascicularis that were infected had acquired their infections before they were transported to the breeding center. Among the infection rates in wild macaques, all the M. fascicularis studied from RAN had malaria parasites, while half the 32 wild M. arctoides studied at PRA and a quarter of the 25 M. leonina at NAK were infected. However, the current study is a relatively small one where only 4 M. fascicularis at RAN were examined, and future surveillance involving a larger number of samples is needed to determine accurately the prevalence of malaria infection among the wild macaques residing in all these areas. In future studies it would also be preferable to sample wild macaques rather than captive monkeys since this would provide more accurate information on zoonotic malaria parasites that are present in that particular ecosystem and could pose a threat to humans visiting these areas for recreation or hunting.The prevalence of Plasmodium spp. infections was observed among the wild macaques studied, which is similar to the results of previous studies on wild macaques in Peninsular Malaysia, Malaysian Borneo, Thailand, Singapore, Laos, Cambodia and the Philippines [Plasmodium present in each host since it has been observed that the parasitaemia of malaria parasites fluctuate over time within a host [P. coatneyi sequester, so in a synchronous infection the parasites would only be detected in the blood if the majority of the parasites were at the early trophozoite or ring stage during sample collection [Plasmodium spp. in macaques and other primates.A complex nature of lippines , 42–46. P. inui was the most prevalent Plasmodium spp. parasite detected in M. leonina, similar with the previous studies conducted on wild M. fascicularis and M. nemestrina in Thailand [Plasmodium species within macaques at each sampling site was different, and this was also observed in the study on M. fascicularis in the Philippines [. of regional populations of M. fascicularis across Southeast Asia [In the current study, Thailand , MalaysiThailand , and PenThailand . Howeverlippines  and in tast Asia . The reaast Asia .Macaca arctoides, or stump-tailed macaques, are found in forested areas in continental Southeast Asian countries including Myanmar, Thailand, Vietnam, Laos, Peninsular Malaysia, and in south-western China and north-eastern India [Macaca arctoides are natural hosts for P. cynomolgi and have been shown experimentally to be susceptible to infection by P. knowlesi and P. coatneyi [. arctoides from RAN in southern Thailand [M. arctoides at PRA were infected with P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi. Since M. arctoides have previously been reported as natural hosts for P. cynomolgi [M. arctoides with P. knowlesi, P. coatneyi, P. inui and P. fieldi.rn India , 38. In rn India , 38. Maccoatneyi . In a prThailand . In the ynomolgi , this isM. fascicularis from the mainland [M. fascicularis at the Wat Khao Takieb temple, while the current study focused on wild M. arctoides at the Pa La U waterfall in Huahin District, 76 km away from the Wat Khao Takieb temple. Therefore, a total of 8 different sites from 6 of the 77 provinces in Thailand have been studied so far to determine the prevalence of malaria parasites in macaques. Further studies, utilizing molecular detection assays and involving more sampling sites and a larger number of monkey blood samples per study site are necessary to determine the geographical range of macaques infected with zoonotic malaria parasites in Thailand and also in other countries in Southeast Asia.Macaques from the following six provinces in Thailand had previously been trapped and studied for malaria parasites by Seethamchai et al. in 2006  and by Pmainland . For PRAM. arctoides with P. knowlesi, P. coatneyi, P. inui and P. fieldi. The presence of macaques infected with malaria parasites, some that are transmissible to humans, presents a potential public health risk to the local population.Macaques sampled from all 4 locations in Thailand were infected with malaria parasites. The prevalence of malaria parasites varied among the species of monkeys and the sites of sample collection. This is the first report of natural infections of


Plasmodium falciparum and the burden of severe, life-threatening disease remains poorly defined. To examine the three most common severe malaria phenotypes from catchment populations across East Africa, we assembled a dataset of 6506 malaria admissions in children aged 3 months to 9 years from 2006 to 2020. Admissions were paired with data from community parasite infection surveys. A Bayesian procedure was used to calibrate uncertainties in exposure  and outcomes . Each 25% increase in prevalence conferred a doubling of severe malaria admission rates. Severe malaria remains a burden predominantly among young children (3 to 59 months) across a wide range of community prevalence typical of East Africa. This study offers a quantitative framework for linking malaria parasite prevalence and severe disease outcomes in children.The relationship between community prevalence of  Plasmodium falciparum causes one of the most deadly, preventable parasitic diseases in Africa. Our understanding of the relationship between parasite exposure, clinical immunity, and malaria mortality is limited by lack of empirical evidence. The attribution of malaria deaths in Africa continues to rely on sparse, outdated information derived from interviews with bereaved relatives , we analysed active surveillance data from 13 hospitals in East Africa. These hospitals served 26 communities over 35 time-site specific periods spanning 2006 to 2020 (PfPR2-10 <1%) at Kabale (Uganda) to high transmission sites (PfPR2-10 >67%) at Bungoma, Busia, Siaya (Kenya), and Muheza (Tanzania)  .The rates of three common severe malaria phenotypes–severe malaria anaemia (SMA), respiratory distress (RD) and cerebral malaria  was favoured over a log-logistic despite an identical DIC, because more complex functional forms with more parameters must be justified by a considerable reduction in the DIC. An intercept-only formulation performed poorly compared with the log-linear structure (ΔDIC = 22) (PfPR2-10 = 0), there would be a background rate of 0.49 (HDI: 0.34 to 0.74) annual admissions of severe malaria phenotypes per 1000 children p.a. Here, PfPR2-10 = 0 was interpreted as a scenario characterized by either low survey power in detecting parasitaemia or instances in which infection was acquired outside the study area.Admission rates of severe malaria increased log-linearly with community parasite prevalence . BecauseIC = 22) . The selIC = 22) . The modPfPR2-10), with a lower DIC than either a log-linear (ΔDIC = 13.4) or an intercept-only function (ΔDIC = 39.7)  . The eff = 39.7) . Admissi = 39.7) , althougPfPR2-10 of 25% to 3.32 months (HDI: 3.05 to 4.02) at 75%. This shift in peak age was concomitant with an approximate four-fold increase  in admissions across the same change in parasite prevalence (25 to 75%).To explore the relationship between admissions rate, age, and parasite prevalence in more detail, a model that described admissions as continuous functions of both parasite prevalence and age was developed . This moWithin our data series, Tororo, Homa Bay, and Kilifi North  were samWe applied a statistical modeling approach to an empirical data series from 35 time-site periods to provide a data-driven analysis of the functional relationships between age, immunity, disease, and exposure. The varying age distributions of severe malaria admissions from low to high intensities of community prevalence have been described previously , 20–22 aGiven the declining rates of SMA and RD admissions with declining community parasite prevalence , A and BPfPR2-10 >75%). The selection of the log-logistic form over the log-linear function (ΔDIC = 13.4) makes it unlikely that this saturation in admissions is solely an artifact of underestimated rates at a few high-prevalence sites. The consistency in rates of CM across transmission settings indicates that associated clinical immunity is acquired in a different way than in other pediatric phenotypes.Our results show that severe, life-threatening malaria remains concentrated predominantly among younger children (3 to 59 months) regardless of transmission intensity, with a slight shift towards older children in low-transmission settings. However, this does not appear to translate to any equivalence or increased lifetime risks. The ecological analysis presented here suggests that few early lifetime infections might still confer some level of functional immunity to severe malaria before age 6 under all endemic settings  25–27).–27.25–27P. falciparum through increased use of LLINs, chemoprevention, and additional vector control efforts with IRS have been shown to rapidly reduce parasite prevalence (Ecological analyses, such as the one presented here, come with unavoidable caveats. The data from each of the sites presented are a product of a diverse range of past transmission intensities, mitigation strategies, and population structures. This can only be resolved through detailed longitudinal data, which are rare in Africa , 29. We evalence –32. In aevalence . SecondaSupplementary material