Plasmodium falciparum is difficult to diagnose accurately in children in high-transmission settings. Using data from 2649 pediatric and adult patients enrolled in four studies of severe illness in three countries , we fitted Bayesian latent class models using two diagnostic markers: the platelet count and the plasma concentration of P. falciparum histidine-rich protein 2 (PfHRP2). In severely ill patients with clinical features consistent with severe malaria, the combination of a platelet count of ≤150,000/μl and a plasma PfHRP2 concentration of ≥1000 ng/ml had an estimated sensitivity of 74% and specificity of 93% in identifying severe falciparum malaria. Compared with misdiagnosed children, pediatric patients with true severe malaria had higher parasite densities, lower hematocrits, lower rates of invasive bacterial disease, and a lower prevalence of both sickle cell trait and sickle cell anemia. We estimate that one-third of the children enrolled into clinical studies of severe malaria in high-transmission settings in Africa had another cause of their severe illness.Severe malaria caused by  Plasmodium falciparum parasites    and othemalaria) , 4. We hmalaria) . To thismalaria) . The diamalaria) . Second,malaria) .The diagnostic value of complete blood counts in severe malaria has great operational utility, because blood counts are widely measured in routine practice at low cost. Moderate thrombocytopenia is a consistent feature of all human malaria infections –8, althoP. falciparum histidine-rich protein 2 (PfHRP2), the basis for most rapid diagnostic tests, is liberated mainly at schizont rupture, such that the amount of PfHRP2 released is proportional to the extent of recent schizogony. Plasma PfHRP2 is a much better discriminant of severe falciparum malaria than the peripheral blood parasite count , which was a randomized comparison of parenteral artesunate versus parenteral quinine in African children clinically diagnosed as having severe falciparum malaria. Overall, artesunate reduced the mortality by about 22% , a randomized comparison conducted in Southeast Asia  an observational study of severe falciparum malaria in Bangladesh , (ii) the Ugandan sites of the Fluid Expansion as a Supportive Therapy (FEAST) trial  (n = 492) (n = 1418)  , (iii) an = 492) , and (iv = 1418) . MalariaPfHRP2 in the enzyme-linked immunosorbent assay (ELISA) but had a parasite density >1000/μl by microscopy. These could have been either assay errors or parasites with HRP2/3 gene deletions. They were removed from the analysis, leaving a total of 2622 samples. A summary of the patient characteristics in the analyzed dataset is shown in In total, 27 patients  had no detectable 10-transformed platelet counts and log10-transformed PfHRP2 concentrations were strongly inversely correlated in both studies of severely ill African children (ρ= −0.54) and in Bangladeshi adults with severe malaria (ρ= −0.36). Patients with platelet counts in the normal range  generally had low to nonmeasurable plasma PfHRP2 concentrations , whereas patients with thromobocytopenia  had a median PfHRP2 concentration of 3031 ng/ml (IQR: 1261 to 6035).LogPfHRP2 concentrations in patients who were clinically diagnosed with severe malaria (n = 2063). This analysis excluded patients from the FEAST trial, which explicitly included nonmalarial causes of severe febrile illness. We fitted a two-component parametric Bayesian latent class model, with the two latent classes representing “severe malaria” and “not severe malaria,” using log10-transformed markers . PfHRP2 concentration. As expected, the platelet count and the PfHRP2 concentration both had high discriminative value, illustrated by the receiver operating characteristic (ROC) curves  showed near-identical results  curves . Overall results .PfHRP2 concentration have approximately the same operating characteristics for diagnosis, so the optimal choice is subjective and can be made based on operational simplicity .For clinical and epidemiological studies, and in contrast to clinical practice, specificity is usually more important than sensitivity. For example, interpretation of effect sizes in randomized trials will depend on the included population and, thus, the specificity of the inclusion criteria. We used the two markers in combination to improve the precision of the definition of severe malaria to achieve low false-positive rates. Under the model, multiple combinations of the platelet count and mplicity . In sevePfHRP2 concentration data from patients from the four studies of severe febrile illness (n = 2622 patients) to estimate the individual patient probabilities that severe malaria was the true cause of their severe illness and to calculate the prevalences of true severe malaria among those diagnosed. The additional third component captured a cluster of patients in the FEAST trial who had no detectable plasma PfHRP2 and normal platelet counts  and a geometric mean PfHRP2 of 3135 ng/ml . The model-based probabilities of severe malaria were highly concordant with our previously published model that used platelet counts and total white blood cell counts . For the subgroup with severe anemia, the false diagnosis rate was 15%, compared to 36% in the subgroup without severe anemia. In the Ugandan cohort, both of these trends were reversed. A false diagnosis of severe malaria was estimated for only 12% of patients with coma but 40% of patients without coma. For severe anemia, these proportions were 36 and 15%, respectively .We compared the estimated false diagnosis rates for cerebral malaria versus severe malaria without coma and for severe malarial anemia versus severe malaria without severe anemia, where severe anemia is defined as a hematocrit ≤15% in the two cohorts of children clinically diagnosed with severe malaria . In the Mortality rates varied substantially as a function of the estimated probability of severe malaria and across the studies . In AfriWe explored the relationship between the model-estimated probability of severe malaria and the admission parasite densities, admission hematocrit, the total white blood cell counts, and the blood culture positivity rates . In the three African studies, parasite densities were between 12 and 16 times higher in patients with a high probability of severe malaria versus those with a low probability of severe malaria . After aThe admission hematocrits also highly correlated with the model-estimated probability of severe malaria , A to D.P = 0.0002) for a positive culture in patients likely to have severe malaria versus those unlikely to have severe malaria. This difference in blood culture positivity rates was also reflected in the total white blood cell counts (P = 10−12).Blood cultures were done for all 1400 Kenyan children and for 298 of the 332 (90%) Ugandan children in the FEAST study who had malaria parasitemia on admission. Overall, 51 and 35 patients, respectively, had positive blood cultures after removing likely contaminants. The probability of having severe malaria was highly predictive of the blood culture result, with an adjusted odds ratio of 0.43 , and for patients with HbSS relative to patients with HbAA, the odds ratio was 0.08 . Under an additive model of association, each additional hemoglobin S (HbS) allele was associated with an odds ratio for severe malaria of 0.27 . This association between HbS genotypes and the marker-based severe malaria classification was highly concordant across the three studies.In the three studies in African children, the prevalence of both HbAS and HbSS (homozygous sickle cell anemia) was strongly inversely correlated with the model-estimated probability of severe malaria . PoolingP. falciparum infection is the cause of the severe illness. We estimate that fewer than one in five patients with severe malaria will have platelet counts in the normal range . This analysis of data from large prospective studies of severe illness in African children shows that the combined measurement of platelet counts and the plasma concentration of the parasite protein PfHRP2 substantially improve the specificity of the diagnosis of severe falciparum malaria.The diagnosis of severe malaria in African children is imprecise , 4, 26. PfHRP2 concentration by comparing the prevalence of HbS genotypes (HbAS and HbSS) and the blood culture positivity rates across in the inferred subgroups. HbAS is the genotype that provides the strongest known protection against severe falciparum malaria  and may occur in sepsis, although it is much less prevalent in sepsis than malaria . The main goal of the analysis was to use Bayesian latent class modeling to estimate the proportion of misdiagnosed patients in the three severe malaria studies and to assess the operating characteristics of different cutoff values for the platelet count and PfHRP2 concentrations for future clinical studies. For the analysis and reporting, we followed the Enhancing the Quality and Transparency of health Research (EQUATOR) guidelines for the reporting of diagnostic accuracy studies that use Bayesian latent class models .This study is a retrospective analysis of platelet counts and plasma All the clinical studies were prospective studies of severe illness and had appropriate ethical approval. For the Kilifi cohort (Kenya), ethical approval was granted by the Kenya Medical Research Institute/National Ethical Review Committee in Nairobi, Kenya (SCC1192) and the Oxford Tropical Research Ethics Committee (OxTREC) in Oxford, UK (020-06). For the Kampala cohort (Uganda), institutional review boards for human studies at Makerere University and the University of Minnesota granted ethical approval for the study. Additional regulatory approval was obtained from the Uganda National Council for Science and Technology. The FEAST trial protocol was approved by ethics committees at Imperial College, London; Makerere University, Uganda; Medical Research Institute, Kenya; and National Medical Research Institute, Tanzania. For the Bangladesh cohort, ethical approval was given by the OxTREC and the Chittagong Medical College Hospital Ethics Committee. Reuse of existing, appropriately anonymized, human data does not require ethical approval under the OxTREC regulations.We included data from observational studies in severe falciparum malaria conducted by the Mahidol Oxford Tropical Medicine Research Unit in Bangladesh between 2003 and 2019. These pooled data have been described previously . MalariaPfHRP2 concentrations were jointly measured in a total of 172 patients. The majority of these patients received intravenous artesunate.Criteria for severe malaria included coma , pulmonary edema, repeated convulsions (≥2 in 24 hours), severe anemia  or jaundice , renal failure (serum creatinine level >3 mg/dl), hypoglycemia (blood glucose level <40 mg/dl), shock (systolic blood pressure <80 mm Hg with cool extremities), hyperparasitemia , hyperlactemia (venous plasma lactate >4 mM), and/or acidemia (venous plasma bicarbonate level <15 mM). Platelet counts and P. falciparum along with the following: prostration (Blantyre Coma Score of 3 or 4), cerebral malaria (Blantyre Coma Score <3), respiratory distress , and severe anemia (hemoglobin <5 g/dl). The standard-of-care antimalarial treatment during this period was intravenous quinine.The Kenyan case-control cohort has been described in detail previously . Severe n = 3161) that was not specific to severe malaria . The standard-of-care antimalarial treatment during this period was intravenous quinine.Platelet counts and plasma P. falciparum parasites in a blood smear in children with a hemoglobin level ≤5 g/dl. The standard-of-care antimalarial treatment during this study was intravenous quinine.The trial by Brand et al.  was an oBacillus species, coryneforms, Micrococcus species, coagulase-negative Staphylococcus, and citrobacter.For the Kilifi cohort and the Ugandan sites in the FEAST trial, blood samples for bacterial cultures were collected in BD BACTEC Peds Plus bottles and processed with a BD BACTEC automated blood culture instrument (Becton Dickinson) for initial detection of bacteria in the blood. BD BACTEC-positive samples were subcultured on standard media by routine microbiological techniques. Either biochemical test kits , serological tests, or both were used to confirm suspected pathogens. Good Clinical Laboratory Practice was audited by Qualogy, and external quality assurance was provided by the UK National External Quality Assessment service. The following organisms were considered as contaminants: PfHRP2 levels were quantitated using the previously published methodology The marginal distribution of each marker in each latent class is log-normal.2)The marginal distribution of each marker in each latent class is the same across the different studies and countries.3)Informative Bayesian priors on all parameters.We fitted a series of Bayesian parametric latent class models to the available marker data . The keyt distributions, only using data from African children, and using weakly informative priors). We compared models with and without correlation between the markers within each latent class. The models with correlation performed better and were more conservative.Sensitivity analyses relaxed assumptions 1 to 3 . We then used full Bayesian inference with informative priors to determine the distributions of the latent clusters. In the initial exploratory analysis, we also looked at the relationship between the peripheral parasite density and the two markers , the sensitivity is defined as 1 – FSM(x), and the specificity is defined as FnotSM(x), where FSM and FnotSM are the cumulative distribution functions for the platelet count in the severe malaria and not the severe malaria components, respectively.We used the posterior predictive distribution over each class in the two latent class model to estimate the sensitivity and specificity for any given threshold for the platelet count and the plasma N [weakly informative: N] for the severe malaria class and N [weakly informative: N] for the not severe malaria class. The platelet count geometric mean of 100,000/μl versus 200,000/μl in severe malaria versus not severe malaria is informed by the results in . For each marker, the marginal distribution in each latent class  was assumed to be log-normal with the following informative priors (weakly informative): Means and standard deviations (SD) are given on the log 10 scale. For the mean platelet counts in the two latent classes, we used sults in   for severe malaria and N for not severe malaria. The weakly informative prior used an SD of 0.5 instead of 0.2. The mean plasma PfHRP2 values are informed from previous modeling of data from the AQUAMAT trial . We used informative beta priors for the prevalence of severe malaria in the three studies: Beta for Bangladesh, beta for Kilifi, and beta for Kampala .The SD on the log t distribution with 10 degrees of freedom (robustness to assumption 1), (ii) by fitting the model to data only from the studies in African children with severe malaria , and (iii) by using weakly informative priors .To assess the robustness of the model outputs, we performed three sensitivity analyses: (i) by changing the parametric form to a bivariate student-PfHPR2, and a normal platelet count). Thus, the latent class model needed to include a third component to fit the data. Under this model, the marker distributions were also assumed to be log-normal in the severe malaria class but were a mixture of two log-normal distributions for the not severe malaria class. We used the same priors for the second set of models with the addition of the following priors for the third additional component : For the mean platelet count, we used N for the second class of not severe malaria; for the mean PfHRP2, we used N.For the second set of models, we included data from the FEAST trial . FEAST iFor the three-component model, Dirichlet priors were used for the prevalence parameters for each latent class: Dirichlet for the Bangladesh study, Dirichlet for the FEAST trial, and Dirichlet for the Kampala and Kilifi studies. For all Dirichlet priors, the hyperparameters correspond to the severe malaria and the two not severe malaria classes in that order.PfHRP2 concentrations to be included in this analysis, we set all nonmeasurable concentrations to 1 ng/ml . Patients who had nonmeasurable plasma PfHRP2 concentrations but had parasite densities above 1000/μl were excluded from the analysis as these could represent P. falciparum parasites with HRP2/3 gene deletions. The median platelet count in these samples was 142,000/μl .For patients with nonmeasurable plasma PfHRP2 and parasite density). This uses the ordered parameter class in Stan. The Stan language does not support discrete parameters; however, posterior distributions of latent class models can be sampled by using the LogSumExp trick, computing the marginal likelihood over the different class combinations.All posterior distributions were estimated using Monte Carlo methods. All models were implemented in the Stan language . For eacSevere malaria insightsPlasmodium falciparum has been difficult in hightransmission settings because of the high coincidence of malaria with other febrile illnesses. Watson et al. analyzed data from >2000 severely ill children and adults in low-transmission (Bangladesh) and high-transmission (Kenya and Uganda) settings. By fitting Bayesian latent class models using a combination of platelet counts and plasma concentrations of P. falciparum histidine-rich protein 2 (PfHRP2), they estimated that detection of #150,000 platelets/ μl and a plasma PfHRP2 concentration of #1000 ng/ml had a sensitivity of about 74% and a specificity of about 93% in identifying severe malaria. These findings revealed that a proportion of children enrolled in several malaria clinical studies in high-transmission settings have severe febrile illness caused by other types of pathogens.Diagnosis of children with severe malaria caused by infection with Checklist.pdfChecklist.zipFig S1 to S8, Table S1


Malaria is a severe parasitic disease that can progress to complications of the nervous system, respiratory distress, renal problems, metabolic acidosis, and hypoglycemia which can result in death in case of delay or absence of appropriate treatment. Even though health service facilities and vector control strategy in the community are implemented as control measures, variations in temperature and rainfall that can affect the life cycle of parasite are among the factors of malaria prevalence over the years. The aim of this trend analysis was to assess the prevalence and the impact of malaria over the seasons and years.  A cross-sectional study using retrospective information was conducted at two health centers Gorgora and Chuahit in Dembia district. The data was collected from lab logbooks routinely diagnosed and registered for seven years. A systematic sampling technique was used by taking patient results from lab logbooks during the first ten days of every month. Data were entered directly into the EpiData Entry software version 3.1 and analysed with the SPSS software version 20. Moreover, a chi-square test with a level of significance set at less than 5% was used. P. falciparum which accounted for 15.6% of the participants which was threefold higher than P. vivax in the seven-year trend. Moreover, at Gorgora health center, the prevalence which was 15% at the beginning of the study (2012) rose to 33.9% and 30.5% in 2017 and 2018, respectively. In the analysis of the seven years, October and September in which the prevalence of malaria was 32.6% and 27.2%, respectively, constituted the peak months. High malaria prevalence was observed in autumn (September to November) season, and the least was observed in spring (March to May) with the prevalence of (17.8%) (p ≤ 0.001). Malaria attack showed significant variability among different age groups, and the age group 15-29 and males were the most affected (p ≤ 0.001).  From a total of 11,879 clients that participated, 56.6% were males. The overall malaria prevalence in the last seven years was 21.8%, and the dominant parasite was  In this study, malaria transmission remained high, which affected males more than females. Thus, appropriate season-based bed net use, health education, immediate patient treatment, and stagnant water drainage methods are needed to alleviate the problem. Anopheles mosquitoes. Five Plasmodium parasitic species cause malaria in humans; of these, two species, Plasmodium falciparum and Plasmodium vivax, are predominant [Malaria is a life-threatening protozoan disease caused by plasmodium parasites that transmit to people through the bites of infected female dominant –3.Malaria is a severe disease, which even progresses to complications and death if there is a delay or absence of appropriate treatment. Some of the clinical complications and manifestations observed in malaria infection are nervous system involvement, respiratory distress, renal problems, metabolic acidosis, and hypoglycemia .malaria report, there were 219 million malaria cases, of which the 10 highest victims were African countries. It affects the lives of more than 435,000 people, the majority of whom are in Africa each year. Pregnant women and children under five years are among the most vulnerable groups [Malaria infection affects nearly half of the world's population. As per the 2018 World e groups .In Ethiopia, the overall trends of malaria cases and deaths among under-five children decreased by 59.3% and 72.1%, respectively. On the other hand, malaria incidence rate decreased from an average of 43.1 to 29 cases and 2.1 to 1.1 deaths per 1000 people annually between 2001 and 2016 [Malaria transmission control strategies are very complex issues, influenced by various factors that may relate to the host, the parasite, the environment, the vector, and the health system capacity to fully implement the available rapid diagnostic tests and professional skill in microscopic species detection . BesidesTherefore, the prevalence of malaria is expected to shift over the years because of the increment in the number of health service facilities, high vector control strategy coverage by using indoor residual spray and insecticide-treated nets in the community, and the awareness of the rural population about health seeking behavior . BesidesInformation from specific health facility laboratory registers over the long periods of time is valuable for a better malaria control strategy. Therefore, this long-term malaria parasite trend over the years and the variability within a year in laboratories of two primary health facilities have provided valuable information for further action.Dembia district is located in the Amhara region of Ethiopia 35 km from the Gondar town. It was bordered by Gondar town and Takusa, Chilga, and Belesa districts. Dembia has a total population of 307,967 in an area of 148,968 sq. km. Its altitude ranges from 1, 850 to 2, 000 m above sea level with an average annual rainfall of 700 to 1, 160 mm. It has 10 health centers and 40 health posts. Among the ten health centers, Gorgora and Chuahit were chosen for this study. Each health center was 16 km apart, and Gorgora was bordered by Lake Tana. The study participants were all clients who provided blood samples for laboratory blood film microscopic examinations and registered on the lab logbooks of the two health centers [A cross-sectional study using retrospective information was carried out in the two health centers of Dembia district. The data was collected from seven years of registered lab logbook that stained and diagnosed with Giemsa's staining technique. Since taking the whole examined blood films over seven years in the two health centers is cumbersome, we systematically took patient results of the first ten days of every month.Data from the laboratory registered logbook was entered directly into the EpiData Entry software version 3.1 and analysed with the SPSS software version 20. Excel was also used to summarize some figures. Descriptive statistics were used to summarize the data and to display malaria parasite trends over the years and seasonal variations. We compared parasite proportions by gender, age groups, seasons of the year, and trends over the years. Moreover, a chi-square test with a level of significance set at less than 5% was considered.P. falciparum that accounted for 15.6% was the dominant parasitic species, followed by 733 (5.3%) of P. vivax. The rest 101 (0.9%) were mixed or both P. falciparum and P. vivax infections.A total of 11,879 clients were requested for blood film and registered on lab logbooks of the two health centers. Of whom, 6, 724 (56.6) were males and 7, 859 (66.2%) were enrolled from Chuahit health center. The total malaria prevalence over the seven years was 21.8%, of which p ≤ 0.001). The highest prevalence was observed in 2016 (30.2%) followed by 2015 (24%). Of the twelve months of the seven years, October had the highest prevalence (32.6%) followed by September (27.2%). On the other hand, the least prevalence was observed in February (15.1%). As far as malaria prevalence across the different seasons was concerned, the highest prevalence was observed during autumn (September to November) (27.9%), followed by 23.3 and 18.4% in summer (June to August) and winter (December to February), respectively. Spring (March to May) was the season with the least prevalence (17.8%) (p ≤ 0.001)  .p ≤ 0.001). The 15-29-year age group was the most affected followed by the 5-14-year group with the prevalence of 25.5% and 24.6%, respectively. The least malaria prevalence was observed among 50-59 years of the age group (16.3%). In this study, males were more affected than females over the seven years, which ranged from 17.4% to 33.7% as shown in Figures The prevalence of malaria varied among different age groups ranging from 16.3% to 25.5% . Moreover, at Gorgora health center, the prevalence which was 15% at the beginning of the study (2012) rose to 33.9% and 30.5% in 2017 and 2018, respectively. On the other hand, at Chuahit health center, the prevalence decreased from 27.4% in 2012 to 11.6% at the end of 2018, although after high prevalence was seen in 2016  were distributed in proportion to family members every three years. Carbamate group chemicals bendiocarb and propoxur wettable powders were used to control mosquito vectors over the last seven years. Either one of the two or combined composition of bendiocarb and propoxur chemicals was for indoor residual spray to control mosquito landing. The purpose of this trend analysis was to investigate the spread and impact of malaria across the nations of Ethiopia and seasons from 2012 to 2018 in order to highlight priorities for the Ethiopian Vector-borne Disease Control Program intended to free the nation from the disease.According to this study, the trend of the prevalence of malaria among the 11,879 clients who were registered in the lab logbooks of the two health centers and requested for blood films over the seven years was 21.8%. This result was markedly lower than 36.1%, 48%, and 33.8% reported from the nearby localities of Addi Arkay health center, northwest Ethiopia; Woreta town, northwest Ethiopia; and Abeshge, south-central Ethiopia, respectively . On the During the study, the highest prevalence 30.2% was observed in 2016, followed by 2015 (24%). These results were similar to the results of a study on malaria epidemiology and interventions in Ethiopia which reported a decrease in malaria incidence and death rate in Ethiopia from 2001 to 2016. But malaria case number and incidence reported in 2016 exceeded the WHO standard for “pre-elimination strategy” . This miIn the current study, the prevalence of malaria was high in October and the least in February over the seven years of twelve months. Seasonal fluctuations and high prevalence of malaria were observed in autumn (September to December) and the least prevalence in spring (March to May). This was in line with the findings of studies conducted in Ataye, North Shoa, Ethiopia ; Addi Arkay health center (Ethiopia); Abeshge ; and Metema hospital, northwest Ethiopia , 11, 14.The study also showed that the prevalence of malaria was high among males and 15–29 years of age groups over the seven years trend. This was similar to the findings of studies conducted in Addi Arkay health center; Ataye district, North Shoa; and Abeshge, south-central Ethiopia , 11, 14.P. falciparum was more dominant parasite than P. vivax which it exceeded by over threefold. The finding was comparable with the results of studies in Addi Arkay, northwest Ethiopia; Ataye, North Shoa, Ethiopia ; Abeshge, south-central Ethiopia; Woreta town, northwest Ethiopia; and Metema hospital, northwest Ethiopia, which reported that P. falciparum was the dominant parasite over the study periods of each study [P. falciparum parasite can multiply rapidly by involving more than one parasite in a single red blood cell, colonizing all ages of the red blood cells without any selection, parasite-infected red blood cells (RBCs) can accumulate in various organs, and the availability of P. falciparum infected cases in communities [Moreover, ch study –15. The munities , 17.Malaria was more widespread in Gorgora health center compared to Chuahit over the seven years; the prevalence at the beginning of the study was 15% and the peak was 33.9% in 2017. At Chuahit health center, the prevalence declined from 27.4% in 2012 to 11.6% in 2018. This might be due to geographical location as Gorgora is located nearer Lake Tana; the vegetation in the shore may be appropriate for mosquito breeding.In this study area, long-lasting insecticidal nets (LLIN) and IRS were used, though the coverage of IRS was low. This result was similar to the result of a study conducted by the Malaria Epidemiology and Interventions in Ethiopia, and it was reported that IRS coverage was low nationwide with an average coverage of 23% between 2014 and 2016 .Data collection in the two health centers on the whole examined blood films over seven years was cumbersome; we systematically took patient results of the first ten days of every month. Moreover, the collected data relayed on the laboratory logbook which lacks participants' body temperature and clinical presentations were the main limitations of the study.P. falciparum; hence, appropriate vector control methods that target outdoor and indoor transmissions are needed, especially for males during high malaria transmission seasons to avoid factors that affect success of malaria elimination.In this study, malaria transmission remains high, especially in autumn and summer seasons. The most affected were the productive age groups with a high infection rate in males. The dominant parasite was


Malaria diagnosis using microscopy is currently the gold standard. However, malaria rapid diagnostic tests (mRDTs) were developed to simplify the diagnosis in regions without access to functional microscopy.TM with microscopy.The objective of this study was to compare the diagnostic accuracy of mRDT CareStatThis study was conducted in the paediatric primary care clinic of the Federal Medical Centre, Asaba, Nigeria.TM of 95% from published work and 95% level of confidence after adjusting for 20% non-response rate or missing data. Consecutive participants were tested using both microscopy and mRDT. The results were analysed using EPI Info Version 7.A cross-sectional study for diagnostic accuracy was conducted from May 2016 to October 2016. Ninety-eight participants were involved to obtain a precision of 5%, sensitivity of mRDT CareStatA total of 98 children aged 3–59 months were enrolled. Malaria prevalence was found to be 53% , whilst sensitivity and specificity were 29% (95% CI = 20% – 38%) and 89% (95% CI = 83% – 95%), respectively. The positive and negative predictive values were 75% (95% CI = 66.4% – 83.6%) and 53% (95% CI = 46% – 60%), respectively.Agreement between malaria parasitaemia using microscopy and mRDT positivity increased with increase in the parasite density. The mRDT might be negative when malaria parasite density using microscopy is low. The gold standard for malaria diagnosis has been light microscopy examination of Giemsa-stained blood smear for malaria parasites. Microscopy detects the actual parasite and the different species of the plasmodium. However, because of lack of equipment (microscopes and power source) and trained microscopists in most malaria endemic regions, malaria rapid diagnostic tests (mRDTs) were developed to fill this gap. The malaria rapid diagnostic test is an immunochromatographic test relying principally on the capture of the target malaria antigen from the blood specimen of the patient.Malaria is one of the leading causes of morbidity and mortality worldwide, especially in under 5-year-old children and pregnant women in sub-Saharan Africa, where over 80% of cases and at least 90% of malaria deaths occur.The objectives of this study were to determine the sensitivity and specificity of mRDT and factors that affect these parameters using microscopy as the gold standard, to calculate the positive and negative predictive values of mRDT in the study population and to determine the association between parasite density and mRDT.We hypothesized that mRDT has sensitivity and specificity similar to microscopy.The study was conducted in the Children Outpatient Clinic (CHOP) of the Federal Medical Centre, Asaba. The clinic has three consulting rooms that are run by family physicians (Residents and Consultants) in the Family Medicine department. An average of 100 malaria cases are seen monthly amongst under five children presenting to the clinic. Asaba is the capital city of Delta State and shares boundary with Anambra State on the eastern coast of the Niger River.Although some work has been carried out on this topic, there is paucity of published work on the subject in the South – South geopolitical zone of Nigeria, and the adoption of the mRDT is yet to be popular. This study is therefore aimed at bridging this gap and also to find out the parasite density at which the mRDT will become positive with the aim of making recommendations for its adoption during diagnosis of malaria, especially where the microscopic diagnosis is not feasible.TM  and microscopy conducted in the Children Outpatient Clinic (CHOP) of the Federal Medical Centre, Asaba. The kit was preferred as the falciparum species is the predominant species in the west African subregion. Ethical clearance was obtained from the ethical committee of the Federal Medical Centre Asaba (FMC/ASB/T/A81/66).The study was a cross-sectional study, comparing diagnostic accuracy of malaria using mRDT CareStatTM packs. The sample was introduced into the kit chamber and two drops of the buffer solution were introduced and left for 5–10 min. The results were read and recorded as positive or negative for malaria parasite. The microscopist was independent and hence not aware of the mRDT results obtained. This was performed to remove bias in his interpretation of the blood films sent to the laboratory. The results of the microscopy and the mRDT tests were compared using EPI InfoTM 7 (7.1.5) and the data were summarised using proportions, frequency and percentages. The sensitivity, specificity and predictive values of mRDT were compared with that of the microscopy. Regression analysis was used to determine the relationship between malaria parasite density and the positivity of mRDT.Participants were consecutively recruited into the study until the desired sample size was achieved. Finger prick blood sample was obtained, and one thin and one thick blood smears were prepared, stained with 10% Giemsa and read for the presence of malaria parasite by a microscopist. The microscopist was a qualified laboratory scientist with the Federal Medical Centre, Asaba. He has been doing malaria microscopy for 10 years and has been certified as a malaria microscopist. Ten out of the 98 slides were also randomly selected and sent to the parasitology unit of the Medical Microbiology Laboratory of the University College Hospital Ibadan. A drop of blood (about 5 µL) was also taken from the thumb by a dropper that came with the rapid diagnostic test kit. The malaria rapid tests were conducted by the second author, strictly following the instructions on the leaflet in the CareStartThe CHOP of the Federal Medical Centre, Asaba, Delta State, Nigeria, is being run by the Family Medicine Department, and it provides primary care delivery for children attending the hospital. The hospital is situated along Nnebisi road in the west-end area of the town close to Saint Patrick College, Asaba.The study population were children who were under 5 years of age attending the Children Outpatient Clinic of the Federal Medical Centre, Asaba. The inclusion criteria were as follows: children whose parents or guardian provided informed consent, children aged between 3 and 59 months, children with axillary temperature ≥ 37.5 °C at presentation or a history of fever within the previous 48 h, children presenting with symptoms and signs comparable with the clinical picture of malaria. The exclusion criteria were as follows: refusal of the parents or guardians to provide informed consent, children with signs of severe illness or unconscious at presentation and those who were enrolled in other clinical studies.The data were collected using an interviewer-administered questionnaire in English and a translation into pidgin English for parents who did not understand English.3 The parasite density (parasites/µL of blood) was calculated according to the formula below using the World health organization (WHO) recommended assumed WBC count of 8000/µL of blood.The data collected were analysed using the EPI Info Version 7 statistical package. Parasite density was assessed with the thick film, whilst parasite speciation was assessed with the thin film. The slide was considered negative when no parasite was seen or detected after screening 200 high power fields see . AsexualEthical clearance to conduct the study was obtained from the Federal Medical Centre, Asaba, Nigeria  on 16 December 2015. The study was conducted in compliance with International Conference on Harmonisation – Good Clinical Practice (ICH GCP) and the Declaration of Helsinki.A total of 98 children aged between 3 and 59 months were recruited and enrolled in this study. There were 53 males and 45 females, giving a male–female ratio of 1:0.85. Most of the participants were in the age group of 3–24 months, accounting for 50 members (51.02%) of the total participants. The mean age ± standard deviation (s.d.) of the participants was 26.2 months ± 15.7 (range = 3–59 months) as shown in Plasmodium falciparum was the only malaria species in this study, accounting for the entire positive malaria smear.The most common clinical symptom was fever. Eighty-eight of the participants (89.8% [95% CI = 82.03% – 95.0%]) presented with fever. About 74.5% of the participants had raised temperature of ≥ 37.5 °C, with a mean ± s.d. temperature of 38.0 °C ± 0.9 °C (range = 36.0 °C – 39.9 °C) as shown in Both microscopy and mRDT were conducted on every participant. Fifty-two children (53.06% [95% CI = 40.72% – 61.26%]) out of 98 were found positive for the microscopy test, whilst 20 (20.41% [95% CI = 12.93% – 29.74%]) were positive with the mRDT test as shown in Of the 20 participants who tested positive with the rapid diagnostic test, 19 gave a history of fever at the time of presentation. However, there was no significant relationship between fever and positivity of the rapid diagnostic test as shown in There was no significant relationship between the temperature at presentation and the malaria parasite count as shown in r2 = 0.74, p < 0.001). The geometric mean parasite density of P. falciparum was 32 319 asexual parasites/µL.The linear regression plot of the relationship between the natural logarithm of the parasite density and the parasite count of the participants with malaria parasitaemia is shown in The percentage agreement of positive results of mRDT and parasite count using microscopy of +, ++, +++ and ++++ was 14.3%, 45.5%, 80% and 100%, respectively. The percentage agreement of negative results was 10.9%. The proportion is shown in The percentage agreement of positive results of mRDT and parasite count using microscopy is calculated as follows:The sensitivity and specificity of the mRDT were 29% and 89%, respectively, whilst the positive and negative predictive values were 75% and 54%, respectively. The prevalence of malaria using microscopy in this study population was 53%. The false positive and false negative rates were 10.9% and 71.2%, respectively, as shown in This study aimed at comparing the diagnostic accuracy of mRDT with microscopy amongst under five children so as to deploy mRDT for prompt diagnosis and treatment of malaria amongst children presenting to our hospital.4 in Burkina Faso, who had a prevalence of 65.0% amongst the under five children during the rainy season, but higher than that of Oyeyemi et al.5 in a descriptive, cross-sectional study in Ijebu Ode, Western Nigeria, who had a prevalence of 36.8% for mRDT in their study population. The differences in the prevalence results may be because of differences in the endemicity of malaria from the different malaria epidemiological zones where the studies were carried out.The prevalence of malaria in this study using microscopy as a reference diagnostic test was 53% (95% CI = 46% – 60%). This value was lower than that recorded by Samadoulougou et al.P. falciparum. This agrees with a similar study carried out by Oyeyemi et al.5 in South-Western part of Nigeria comparing microscopy and rapid diagnostic test as malaria diagnostic tools where only P. falciparum species of the malaria parasites was identified in all the study participants. However, Agomo et al.6 in Lagos, Nigeria, found that P. falciparum was seen in 95.2% of the cases as either mixed infection with P. malariae (3.6%) or as a mono infection (91.6%). These results agree with literature findings that P. falciparum is responsible for more than 95% of malaria infections in the tropics.8 Therefore HRP2-based mRDTs are more economical and the preferred options for parasitological diagnosis of malaria than the enzyme-based mRDTs in the tropics. In addition, HRP2-based rapid diagnostic tests (HRP2-mRDTs) can withstand the heat and temperature fluctuations of tropical Africa better than the enzyme-based RDTs, where refrigeration and air conditioning are not always feasible.The species of malaria parasite identified in all study participants was The result revealed that 52 children (53.1% [95% CI = 40.7% – 61.3%]) out of the 98 were positive for the microscopy test. Thirty seven (71.2%) out of the 52 children who were positive for the microscopy were found to be negative with mRDT . This gave a high false negative mRDT test when compared with the result of the microscopy in this study. This is very significant in this study, as this may have contributed to the low sensitivity reported. Sensitivity is the proportion of people with disease  who will have a positive result when tested with the diagnostic test kit (mRDT in this case) in the diagnosis of malaria.9 This is below the parasite density threshold  that can be reliably detected by mRDT for malaria diagnosis.10 The low yield of positive results with the mRDT in this study agrees with the fact that the malaria rapid test result positivity is low at low parasite density.10 Amadi et al.11 in Port Harcourt, South – South Nigeria, found that mRDT sensitivity was only 45% when the parasite density was below 100 parasite/µL. These results show that rapid test would not give justifiable results as most of the low parasite density cases could escape detection.The microscopy further showed that the parasite count of (+) made up 67% of the total population of patients with positive microscopy. Using this ‘plus’ system scale of scoring to calculate the parasite density, it therefore means that about 67% of the participants who had positive results with microscopy had a parasite density of between 10 and 90 parasites/µL of blood.TM) used in this study will be capable of detecting correctly (giving a positive result) only 29 out of 100 children with malaria infection and will give a negative result in 89 out of 100 patients without malaria infection. The very low sensitivity recorded in this study as against the WHO recommendations of about 95% may be because of a high false negative rate of 71.2% (37/52 × 100) of mRDT as compared to the microscopy. The high false negative rate is similar to the findings of Berhane et al. where only 10 out of the 50 microscopically confirmed P. falciparum infected specimens were confirmed positive , with all the lots of mRDTs used in the study giving an 80% false negativity proportion.12 The possible explanation for these findings may include a low parasite density below the threshold of mRDT positivity .13 Other studies have also shown some degree of false negative result for mRDT because of hyperparasitaemia, deletion or mutation of HRP-2 gene and the prozone effect . This will eventually affect the sensitivity of the test.15The sensitivity and specificity of the mRDT in this study were 29% and 89%, respectively. This means that the mRDT kit  Nnewi, Nigeria, who reported a sensitivity and specificity of 80% and 93.8%, respectively.17 Xiaodong et al.18 in China also found that the CareStat rapid diagnostic test had a sensitivity of 89.68% and a specificity of 98.26% compared to the gold standard microscopy method for the detection of malaria. Variations in sensitivity between the different studies may be attributed to differences in the types of RDTs used or test methodology and skills of the microscopist. The implication of the low sensitivity in this study is that in areas with low malaria parasitaemia, a negative result should be cross-checked with a microscopy and clinical acumen of the physician to rule out possibilities of false negative results with the mRDT. However, a high specificity of 89% in this study implied that mRDT may be used in primary healthcare centres by community health workers to rule out the absence of malaria where microscopes are hardly seen or where the required human expertise is lacking.The low sensitivity of this study is in agreement with the research conducted by Oyeyemi et al. in Ijebu Ode, western part of Nigeria, who reported a sensitivity and specificity of 42.5% and 87.1%, respectively.19 in rural health centres in Uganda that compared the accuracy of rapid diagnostic tests and microscopy where the overall specificity of Paracheck (a form of HRP2-based mRDTs) was lower than that of the microscopy.It was also noted from this study that five (10.9%) of 46 children whose microscopy results were negative were positive with the rapid test . This may be as a result of persistent antigen of the malaria parasite in the blood even after parasite clearance following adequate anti-malaria treatment of the index cases. The persistent antigenaemia may have contributed to the high specificity recorded in this study. This agreed with the work of Batwala et al.20 The explanation for this may either be as a result of reduced sensitivity of the mRDT at low parasite count as documented by these authors or the over-diagnosis of malaria by the laboratory scientists at low parasite density.22 Kahama-Maro et al.7 in Dar es Salaam found that only 2.1% of the 178 slides that were reported positive by health facility routine microscopy were actually positive by expert microscopy.22 This low percentage (2.1%) may be as a result the possible lack of the rigorous and diligent commitment required to report parasite count of (+) (i.e. 0–10 parasites in 100 high power field) on the part of most microscopists, because of the pressure of work they have to cope with whilst carrying out their routine work. Some of this parasite count of (+) may also be because of artefact resulting from either poor blood film preparation or using reagents with sediments in staining the blood smears.The percentage agreement of positive results of mRDT and parasite count using microscopy was the highest (100%) at parasite count of (++++) and the lowest (14.3%) with parasite count of (+). Many of these (+) using microscopy were missed by the rapid test, thereby giving a low yield in the positivity of the mRDT and, consequently, the sensitivity of the malaria kit at this level of parasite count. This result agreed with the work of Sani et al. in Sokoto, Northern Nigeria, where it was found that the sensitivity of RDT increased consistently from 33% at low parasite density to 93% at high parasite density.23The specificity of this study was comparable with most of other researches. However, the low sensitivity of this study agreed with the work carried out by Kahama-Maro et al. in Dar es Salaam, who found a low sensitivity. The low sensitivity in this study may not be completely explained only by the parasite density of the malaria as documented by researchers like Mawili-Mboumba.7 who had a positive and negative predictive values of 65.6% and 86.1%, respectively. The positive predictive value of 75% means that the kit has the capability of confirming malaria with a precision of 75%, whilst the negative predictive value of 53% means that the mRDT is good in ruling out malaria, thus giving the clinician the confidence that a negative test excluded malaria in about 53% of cases.The positive and negative predictive values in this study were 75% and 53%, respectively. This result is slightly different from the findings of Falade et al.,The false positive and negative rates in this study were 10.9% and 71.2%, respectively. This false negative rate is quite high. Several factors may account for this high rate, which may include low parasite density. According to WHO, false-negative results can be caused by any or a combination of the following:the procurement and use of poor-quality RDTsuse of the wrong comparator for the diagnostic test, such as poor-quality microscopy for cross-checking negative RDTpoor transport and storage conditions for RDTs, with sustained exposure to high temperatureoperator errors during performance and/or interpretation of RDT results (more rarely)HRP-2 gene.deletion or mutation of p < 0.001). This relationship also indirectly affects the positivity of mRDT because the higher the parasite count, the greater the percentage agreement of positive results of mRDT with microscopy. It therefore means that the parasite density is directly related to the mRDT positivity.The linear regression plot  showed tThere was only one microscopist who regularly performed malaria microscopy for clinical care of patients in the study location where the volume of work could sometimes be high. There was no cross-checking of a predetermined percentage of the slides by a second microscopist.p < 0.001). We therefore advocate a more sensitive kit that can detect the malaria parasite at low density for future use, especially to improve the sensitivity of the mRDT kits in malaria management, where trained microscopists for malaria diagnosis are not available.The sensitivity and specificity of mRDT compared with microscopy diagnosis of malaria in this study were found to be 29% and 89%, respectively. There was a significant correlation between parasite count and parasite density (


Plasmodium, the etiological agent of malaria in humans, creating potential risks of zoonotic transmission. Plasmodium brasilianum, a parasite species similar to P. malariae of humans, have been described in NHPs from Central and South America, including Brazil. The merozoite surface protein 1 (MSP1), besides being a malaria vaccine candidate, is highly immunogenic. Due to such properties, we tested this protein for the diagnosis of parasite infection. We used recombinant proteins of P. malariae MSP1, as well as of P. falciparum and P. vivax, for the detection of antibodies anti-MSP1 of these parasite species, in the sera of NHPs collected in different regions of Brazil. About 40% of the NHP sera were confirmed as reactive to the proteins of one or more parasite species. A relatively higher number of reactive sera was found in animals from the Atlantic Forest than those from the Amazon region, possibly reflecting the former more intense parasite circulation among NHPs due to their proximity to humans at a higher populational density. The presence of Plasmodium positive NHPs in the surveyed areas, being therefore potential parasite reservoirs, needs to be considered in any malaria surveillance program.Non-human primates (NHPs) have been shown to be infected by parasites of the genus  The merozoite surface protein 1 (MSP1) is the most abundant protein on the malaria parasite cell surface. It is involved in the erythrocyte invasion process, being one of the most studied targets for a malaria vaccine  and tranPlasmodium malariae MSP1 (pmmsp1) encodes a protein of 1751 amino acids, including a 19 amino acid signal peptide [P. malariae, and in its non-human primate (NHP) equivalent, P. brasilianum, isolated from different geographic regions of Brazil [pmmsp1 gene was performed through the amplification and sequencing of five fragments, F1 to F5 , equivalent to blocks 3 to 17 of the PfMSP1  and covering 60% of the gene [The gene of  peptide . Recentlf Brazil . Analysethe gene . Except the gene , the PmMP. falciparum and P. vivax has allowed for numerous studies revealing whether individuals have been naturally exposed to malaria parasites, by detecting antibodies to the protein in their sera . For thnfection .P. malariae MSP1 recombinant proteins, together with the MSP119 of P. falciparum (PfMSP119) and of P. vivax (PvMSP119), for the detection of anti-MSP1 in the sera of NHPs from malaria endemic regions of Brazil, the Amazon and Atlantic forests, and from a non-endemic region, the Cerrado, in Central Brazil. Using these recombinant proteins, we also aimed to find the prevalence of antibodies against these parasite species in the sera of NHPs and, thus, the potentiality of these animals as malaria reservoirs.In this work, we evaluated the diagnostic capability of the n = 155), collected close to Porto Velho city in Rondônia state, from March 2009 to November 2012, during projects related to environmental management for the construction of hydroelectric power plants [n = 111), from October 1997 to July 2005, in forest fragments around the municipality of São Paulo [n = 107), from April 2000 to March 2001 and January to December 2009, in the canopy of woods in flooded areas of the lake at Porto Primavera dam during wildlife rescue operations [n = 103), 60 being from the São Paulo city Zoo, 31 from the Tietê Ecological Park and 12 from CETAS  in Lorena city (n = 08) and Unimonte in São Vicente city (n = 1), as well as from Bauru city Zoo (n = 1) and the Centre for Biological Research in Indaial city (n = 2). From the Amazon Region (n = 19), samples were collected from animals kept close to areas of human habitation, such as the Ecological Park of Porto Velho city, and animals rescued by IBAMA  which had been kept illegally as pets in rural or suburban areas.Serum samples were collected in two malaria endemic regions in Brazil, the Amazon and Atlantic Forest regions, and in a non-endemic region, the Cerrado region in Central Brazil. A total of 373 samples were collected from free-living animals and 122 from captive animals. Sera from free-living animals A were obr plants ; (ii) inAll procedures were approved by the Ethical Committee in Animal Research (CEUA) of the Institute of Tropical Medicine of São Paulo, University of Sao Paulo (number 2014/281A) and were in full compliance with federal permits issued by the Brazilian Ministry of the Environment .F1, PmMSP1F2, PmMSP1F3, PmMSP1F4, PmMSP119), P. vivax (PvMSP119) and P. falciparum (PfMSP119), representing the polymorphic N-terminal (MSP1F1), the central  and the conserved C-terminal (MSP119) regions [GST and GST-recombinant fusion proteins of PmMSP1 , following the manufacturer’s instructions. Coupled beads were then used for the analyses of the NHP sera as described , with moSera were tested in two replicates and positivity evaluated by the measured MFI values of antibodies binding to the recombinant proteins, minus the MFI value of the same serum to non-fusion GST. The cut-off values are presented as the geometric mean of values obtained with a panel of eight negative control sera plus 3× standard deviations. Average cut-off values are shown in Plasmodium spp. GST-MSP1 fusion proteins coupling to the BioPlex carboxylated beads, multiplex assays were performed using a rabbit anti-GST polyclonal IgG antibody (Biotin)  to detect the fusion protein on the bead. The dilution of the anti-GST antibody was 1:1000 in assay buffer  (50 μL/well). The bound anti-GST antibody was detected with R-phycoerythrin-labelled streptavidin and fluorescence was measured on the BioPlex 200 instrument , as described above.To determine the overall efficiency of the F4), which was unstable and lower in all readings. PvMSP119 presented a similar fluorescence curve but slightly lower, except for Plate 1.Quality control of the coupling was placed on each plate, where the beads that were covalently coated with the different recombinant proteins were tested in the same plate, with biotinylated anti-GST, to evaluate the efficiency of the coupling and verify the reading behavior of each protein. In n = 373); (ii) captive animals (n = 122). Of all samples analyzed, 199 (40.2%) presented IgG antibodies against at least one of the malaria recombinant proteins. Sera that were reactive to the P. malariae PmMSP1F1 were most frequent, with a total of 109 (22%), followed by those reactive to P. vivax MSP119 (19.8%). The least reactive sera were to P. falciparum PfMSP119 (1.6%)  free living animals (9 (1.6%) . The percentage of sera of free-living animals reacting to the recombinant proteins was higher in animals from the Atlantic Forest (63.1%), compared to those from the Amazon and Cerrado regions, which were 49% and 11.2% respectively .F1, PvMSP119 and PmMSP1F3, respectively  were PmMSP1ectively A.F1 and PmMSP1F2, while animals from the Atlantic Forest were most reactive to PvMSP119  and from the Amazon region (31.6%) . The higPvMSP119 . Two serP119  reacted concomitantly to four proteins , while 61 (37%) sera reacted to only one of the proteins, with the lowest positivity to PmMSP1F4 . Of these, 43 (29.1%) were positive to more than one Plasmodium species: P. falciparum and P. malariae (2.7%); P. vivax and P. malariae (25.7%); and P. falciparum, P. malariae and P. vivax (0.7%), yielding evidence of the lifetime exposure of the animals to these Plasmodium species  were found to be positive to the MSP1 C-terminal region of the different malaria parasites  were found positive, in addition to those of the genus Alouatta  in the upper Amazon (Northern Brazil) [P. simium, a tertian malaria parasite genetically and morphologically similar to P. vivax, described in a howler monkey (Alouatta fusca) in the state of São Paulo (southern Brazil) [NHPs have been shown to be infected by parasites from the genus  Brazil)  and P. s Brazil) .P. malariae erythrocytic stage antigens [P. malariae MSP1 , as well as with those of P. falciparum (PfMSP119) and P. vivax (PvMSP119), we found that about 40% of the NHP sera, from different areas in Brazil, were reactive to the recombinant antigens, with PmMSP1F1 being the most frequently detected. This might reflect the potentially high immunogenicity of this region of P. malariae MSP1, like those of P. vivax and P. falciparum MSP1, as described in humans [19) was also high, as expected from studies on the identical regions of P. vivax and P. falciparum MSP1 in humans [P. malariae MSP1 were also found, with the lowest frequency to PmMSP1F4, which might be due to its low binding capacity to the beads, as observed in the coupling efficiency assay.Recently, many studies have been performed to evaluate the presence of malaria parasites in NHPs, both in the Atlantic Forest and Amazon regions, aiming to understand their roles as reservoirs of malaria ,22,23,24antigens ,10,25. Iantigens . With thn humans ,27. Reacn humans . Sera wiPlasmodium circulation among NHPs from this area. This might also reflect the proximity of NHPs to humans at a higher populational density in the Atlantic Forest. The number of positive NHP sera from Cerrado was low, as expected, due to the low level of malaria incidence in Central Brazil. Another expected finding was the higher prevalence of anti- P. vivax and P. malariae antibodies in the sera of NHPs from the Atlantic Forest, as it has been demonstrated that P. simium, the equivalent of P. vivax, and P. brasilianum, the equivalent of P. malariae, are prevalent in this area, with the former being the most prevalent [Serological estimates correlate well with parasitological and entomological measurements in assessing transmission intensity and spatial and demographic risks to malaria ,30. The revalent ,19,31. Alouatta have been found with such antibodies. This singularity has been observed since the description of the first Plasmodium infected animal [It is important to note here the higher diversity of NHPs from the Amazon region that was found to carry the anti-MSP1 antibodies, as compared to the diversity of NHPs from the Atlantic Forest, where only animals of the genus d animal ,18,32,33Plasmodium diagnosis in NHPs in order to contribute to One Health surveillance. We have demonstrated that the MSP1 recombinant proteins used in this study are useful and important candidates to be included in diagnostic tools for the surveillance, and, ultimately, for the control or elimination, of malaria.The seropositive NHPs indicate previous infections as well as the potential presence of infected animals in the surveyed areas, which can play a role in the maintenance of human malarias, making it more difficult to eliminate or even control the disease. Moreover, the illegal wildlife trade, the allocation of species for captivity or the effort to conserve them (translocation or reintroduction), promotes the movement of animals and may favor the transmission of disease ,35. ThusP. vivax and P. malariae  supports the hypothesis that these animals are potential reservoirs of malaria parasites and that NHPs must be considered in any measure for the control or elimination of the disease.This study validates the use of recombinant proteins in multiplex immunoassays to detect IgG antibodies against the MSP1 protein of malaria parasites and demonstrates that this technique is an important tool for making serum-epidemiological surveys of malaria. Lastly, the high prevalence of NHPs with antibodies against


The massive scale-up of long-lasting insecticidal nets (LLINs) has led to major reductions in malaria burden in many sub-Saharan African countries. This progress is threatened by widespread insecticide resistance among malaria vectors. This cluster-randomised controlled trial (c-RCT) compares three of the most promising dual active ingredients LLINs , which incorporate mixtures of insecticides or insecticide synergists to standard LLINs in an area of pyrethroid insecticide resistance.A four-arm, single-blinded, c-RCT will evaluate the effectiveness of three types of dual-AI LLINs (1) Royal Guard, combining two insecticides, pyriproxyfen and the pyrethroid alpha-cypermethrin; (2) Interceptor G2, combining chlorfenapyr and alpha-cypermethrin; (3) Olyset Plus, an LLIN combining a synergist, piperonyl butoxide and the pyrethroid permethrin, compared with; (4) Interceptor LN, a standard LLIN containing the pyrethroid alpha-cypermethrin as the sole AI. The primary outcomes are malaria infection prevalence in children aged 6 months–14 years and entomological inoculation rate (EIR), as a standard measure of malaria transmission at 24 months postintervention and cost-effectiveness.Ethical approval was received from the institutional review boards of the Tanzanian National Institute for Medical Research, Kilimanjaro Christian Medical University College, London School of Hygiene and Tropical Medicine, and University of Ottawa. Study findings will be actively disseminated via reports and presentations to stakeholders, local community leaders, and relevant national and international policy makers as well as through conferences, and peer-reviewed publications.NCT03554616. This study is the first randomised controlled trial (RCT) to evaluate and compare the effectiveness of the next generation of dual-treated long-lasting insecticidal nets (LLINs), Royal Guard (a pyrethoid-pyriproxifen LLIN) and Interceptor G2 (a pyrethroid-chlorfenapyr LLIN) against standard LLIN to prevent malaria infection prevalence and incidence in an area of pyrethroid insecticide resistance.Results of this study will be presented to the WHO for policy recommendations and depending on the study outcomes has the potential to shape malaria vector control strategies across sub-Saharan Africa for the next decade.This is the second RCT to evaluate the new class of pyrethroid-piperonyl butoxide LLINs (Olyset Plus) in the Great Lakes Zone of Tanzania, expanding the evidence basis for deployment of this intervention class in areas of pyrethroid resistance.The trialling of the three main categories of dual active ingredients LLIN against the standard pyrethroid LLIN in the same human cultural community against the same vector complex should offer the best opportunity to unravel relative effectiveness on malaria and effect on selection of insecticide resistance.Some limitations include the size of the cluster buffer areas, which might not prevent all contamination between intervention arms, and the use of malaria rapid diagnostic tests (mRDT) to assess malaria infections rather than double-read blood slides, which may be more sensitive.Long-lasting insecticidal nets (LLINs) are the primary method of malaria control in sub-Saharan Africa. The WHO estimates that over 50% of the population of sub-Saharan Africa now sleep under LLINs.2Several studies have demonstrated that LLINs are becoming less effective at killing mosquitoes in areas of moderate to high pyrethroid resistance compared with those with susceptible vector populations.During the last decade, the WHO has encouraged manufacturers to develop new types of bed nets to control resistant mosquitoes. The chemical industry responded initially by developing a dual active ingredients LLIN , combining a pyrethroid insecticide with the synergist piperonyl butoxide (PBO), which inhibits cytochrome P450 oxidases (CYP) responsible for pyrethroid resistance. Pyrethroid-PBO LLINs (py-PBO LLINs) have been available since 2009,12Anopheles gambiae s.l. of 71% versus 20% in BeninAnopheles arabiensisAnopheles funestus s.l. in Tanzania.Other manufacturers have responded to the WHO call by producing a different kind of dual-AI LLIN that incorporates a mixture of insecticides from different insecticide classes. Mixtures of two insecticides on the same LLIN with differing modes of action have the potential to delay the evolution of resistance and extend the lifespan of both active ingredients on the LLIN. The two latest products are a pyrethroid-pyriproxyfen LLIN  and a pyrethroid-chlorfenapyr LLIN (py-CFP LLIN: Interceptor G2). Both types of product have shown superior efficacy compared with s-LLINs in small-scale experimental hut trials.In Tanzania, insecticide resistance has spread rapidly.Here, we describe the study design and methodology of an RCT, assessing the effectiveness and cost-effectiveness of three novel vector control interventions , compared with the standard best practice of pyrethroid-only LLINs, to prevent malaria in an area of pyrethroid resistance in Tanzania. Each putative new class needs to show high effectiveness versus the s-LLIN. None should select for stronger resistance to pyrethroid. Ideally, none should select for resistance to the other new classes of dual-AI LLIN being tested. A four-arm trial is a highly efficient design, should demonstrate the relative effectiveness of each against malaria, and the merits of each product against the same fauna of vector species and human cultural group. The trial should provide insight into future rotational strategies of deployment, their potential to manage insecticide resistance while controlling malaria.The durability and bio-efficacy of the dual-AI LLIN are also being evaluated as per WHO guidelines32The primary clinical and entomological objectives are to assess the effectiveness of py-PPF-LLINs, py-CFP-LLINs and py-PBO-LLINs compared with s-LLINs:On malaria infection prevalence in children from 6 months–14 years over 2 years postintervention.On the entomological inoculation rate (EIR) of malaria vectors collected indoors over 2 years post intervention.The primary economic objective is to assess the cost-effectiveness of py-PPF-LNs, py-CFP-LNs, py-PBO-LNs and s-LLINs relative to one another.All secondary objectives are detailed in A. gambiae sensu stricto (s.s.) or A. funestus s.s. as the main vectors, insecticide pyrethroid resistance in standard WHO bioassays , and no indoor residual spraying (IRS) planned for the next 3 years. Data from published and unpublished sources  were examined and complemented with data collection in November 2017, as appropriate. Only the district of Misungwi , on the southern border of Lake Victoria in Tanzania met all the criteria.Five potential study sites in Tanzania’s Victoria lake zone were evaluated based on four criteria: report of a minimum of 30% malaria infection prevalence in total population, 2 and includes 27 wards, 78 villages and a population of 351 607 people.34Misungwi covers an area of 2122 kmA. gambiae s.s., A. arabiensis and A. funestus s.s., were found in the area (unpublished data). WHO insecticide resistance tests were also performed and 24-hour mortality in wild caught A. gambiae s.s. exposed to permethrin and deltamethrin was 7% and 19%, respectively. There was also evidence of pyrethroid resistance in A. funestus s.s.  and A. arabiensis . Pyrethroid insecticide resistance has also been observed in the adjacent district of Magu and other lake zone regions, such as Geita and Kagera.28Misungwi has moderate to high malaria transmission. In a study conducted in 2010, prevalence was 52% across all age groups.The main vector control interventions in Misungwi are universal coverage of LLINs and IRS. The last distribution of LLINs and IRS campaign using Actellic 300CS were carried out in Misungwi district in 2015. In 2017, larviciding was done in some parts of the district.An overview of the trial design is given in Mixture py-PPF-LLIN: Royal Guard (intervention 1).Mixture py-CFP-LLIN: Interceptor G2 (intervention 2).Py-PBO-LLIN: Olyset Plus (intervention 3).S-LLIN: Interceptor (control arm).The inhabitants of each cluster are blinded to the type of nets they received, as are the field staff who will collect entomological and clinical data. Nets of each type are similar in appearance apart from a colour-coded loop and a unique identifying number. Only the principal investigator and data manager know which code represents each intervention.Every building of 72 villages comprising 453 hamlets from 17 wards was mapped using a global positioning system handheld unit (Garmin Legend e-trex) and ExpertGPS V.3.8 (TopoGrafix) software. A short Open Data Kit (ODK) programmed questionnaire, including name of the head of the household, number of people living in the house and number of children in each age group  was recorded for each mapped household.Anopheles species composition , assessed during pilot mosquito monitoring from all villages.Wards to map were selected based on both malaria incidence data collected from all health facilities in Misungwi, and As in previous studies,A. gambiae s.s. and for A. funestus s.l. Suitability for each Anopheles species was determined using an ecological niche modelling approach . They differed by study arm as described in the following sections.2, coated onto polyester filaments. Pyrethroids are neurotoxic insecticides, which target the nervous system of insects. Interceptor LN was chosen as a direct comparison to Interceptor G2 LN and Royal Guard LN, as they are all impregnated with the same pyrethroid ; some insecticide resistance mechanisms involving CYPs are specific to type I (permethrin) or type II  pyrethroids.Interceptor LN  is a pyrethroid-only LLIN with alpha-cypermethrin at a target dose of 200 mg/m2 PPF and 216 mg/m2 alpha-cypermethrin. PPF is known to disrupt female mosquito reproduction and fertility of eggs, stopping the production of the next generation of mosquitoes.40Royal Guard LN (Disease Control Technologies) is a mixture LLIN made of polyethylene incorporating 225 mg/m2 chlorfenapyr and 100 mg/m2 alpha-cypermethrin. Chlorfenapyr disrupts cellular respiration and oxidative phosphorylation in mitochondria, and due to this unique mode of action is toxic against mosquitoes that are resistant to standard neurotoxic insecticides like pyrethroids.41Interceptor G2 LN is a mixture LLIN made of polyester coated with a wash-resistant formulation of 200 mg/m2) and the repellent pyrethroid permethrin (800 mg/m2) incorporated into polyethylene fibres. PBO is a chemical synergist which acts by inhibiting mixed function oxidases, preventing detoxification of the pyrethroid insecticide. Two RCTs demonstrated that Olyset Plus LLINs were more effective than Olyset Net LN (the standard of care in Tanzania), in areas with pyrethroid resistance.8 12Olyset Plus LN  is a mixture LLIN combining PBO  in children aged 6 months–14 years old at 24 months postintervention.EIR as a measure for malaria transmission in the primary vector species.Cost-effectiveness of each of the four net types relative to one another.Incidence of malaria cases in children aged 6 months–10 years (measured over 24 months follow-up).Moderate and severe anaemia in children under 5 years old (<8 g/dL) at 12, 18, 24, 30 and 36 months.Malaria infection (by mRDT) in children aged 6 months–14 years at 12, 18, 30 and 36 months.Changes in frequency and intensity of phenotypic and genotypic resistance to pyrethroids , PBO, CFP and PPF.Other entomological outcomes: changes in mosquito resting behaviour indoors and outdoors, species composition and density ratio, host feeding, mosquito ovary development and fecundity.Incremental financial cost to the provider of each of the four net types relative to one another.LLIN coverage will be evaluated 6 months after distribution and during each cross-sectional survey. Three indicators will be usedTo determine infection prevalence, repeated cross-sectional surveys will be conducted at 12, 18, 24, 30 and 36 months after net distribution . At eachInclusion criteria are households with at least one child between 6 months and 14 years old who permanently resides in the selected household and an adult caregiver who can provide written consent. Exclusion criteria include dwellings not found or vacant during the survey, no adult caregiver capable of giving informed consent, or eligible children are severely ill.Information on sex, age distribution, educational status and occupation, SES, house structure, vector control measures used, past malaria cases, net coverage and care seeking behaviour will be collected. Fever or history of fever in the past 48 hours will be recorded for every child selected. Temperatures will be taken, and each child tested for malaria using mRDTs , DiaSys, Wokingham, UK) and haemoglobin levels measured ). When the mRDT is positive, free treatment for malaria will be provided with artemether-lumefantrine , as per the national guidelines. Children with severe malaria or any other diseases that cannot be treated by the team will be referred to the nearest health facility.To assess malaria case incidence, 35 children per cluster, aged 6 months–10 years old, will be randomly selected and followed up every 2 weeks during the high transmission season (October to July) and every month during the low transmission season (August to September), over a 12 months period . To reduAnopheles mosquitoes through dissection.A. gambiae s.l and 10 A. funestus s.l will be randomly picked per household per collection night and preserved for laboratory analysis. This sample of mosquito specimens will be screened for Plasmodium falciparum circumsporozoite protein (Pf-CSP) by ELISA.47Cross-sectional entomological surveys will be carried out in 84 study clusters to monitor the indoor mosquito population density . Each clA. gambiae species complex,A. funestus groupAnopheles collected. A sample of A. gambiae s.s. and A. arabiensis will be genotyped for the L1014F-kdr and L1014S-kdr mutations, associated with pyrethroid and DDT resistance, using TaqMan PCR assays, following the protocol by Bass et al.50PCR TaqMan assays will be used to discriminate members of A. gambiae s.l. and A. funestus s.l. will be assessed using pre-exposure to PBO followed by permethrin resistance intensity assays. All knockdown/dead mosquitoes at 60 min and surviving 72 hours postexposure to insecticides will be stored individually in RNA later and preserved at −20°C for gene expression analysis. RNA will be extracted from pools of A. gambiae s.s. or A. funestus s.s and cDNA synthesised, according to standard procedures. Relative expression of CYPs and other metabolic enzymes, previously identified as being over-expressed in resistant A. gambiae s.s. and A. funestus s.s. populations in Tanzania,54Phenotypic resistance and resistance levels to alpha-cypermethrin, permethrin, PPF and chlorfenapyr, will be characterised at baseline and yearly postintervention, in all four study arms, using WHO cylinder and modified CDC bottle bioassays.Anopheles will be collected using CDC prokopack aspirators. Subsamples of Anopheles will be subjected to the same laboratory tests than routine collection. Additional information about mosquito collection methods is available in Malaria vector abundance, species composition, feeding and resting behaviours , and contribution to outdoor malaria transmission will be assessed at baseline and after implementation of the interventions to assess changes over time in 10 clusters per treatment arm. CDC light trap and adapted Furvela tent trapsData on resource use and unit costs will be collected from primary and secondary sources, including discussions with net manufacturers, donors and experts; review of project, donor and Ministry of Health records; trial survey data on care seeking behaviour; published literature; and WHO-CHOICE unit cost estimates.57 58The sample size was calculated using the method of Hayes and Bennett, taking into account the cluster-randomised design.Sample size calculations for the secondary outcome  were based on the method of Hayes and Bennett.Sample size calculations for the entomological survey were based on the method of Hayes and Bennett.Clinical and entomological measurements in the cohort study and the cross-sectional surveys will be captured on electronic forms using tablets/smartphones installed with ODK and uploaded daily onto the server at London School Hygiene and Tropical Medicine (LSHTM). Other data and clinical measurements during the cross-sectional survey, recorded on paper, will be double-entered into an access database independently by two data clerks. Laboratory data outputs will be available directly from the analyser  and imported into a database. All databases will maintain an audit trail with time-date stamps of data entry and all changes that are made to the data. Anonymised study numbers will be used as unique participant identifiers.The primary analysis will be conducted using the intention to treat (ITT) approach. Secondary per-protocol analyses will also be conducted. The primary outcome, measured at 12, 18, 24, 30 and 36 months after net distribution, will be analysed using mixed effects logistic regression. The unit of analysis will be the individual. The model will include fixed effects for time, study arm, and time by study arm, and will adjust for the baseline prevalence in each community as a covariate, as well as the covariates used in the covariate-constrained allocation procedure. The model will account for within-period and between-period intra-cluster correlations.61Prevalence of malaria infection in each dual-AI LLIN treatment arm will be compared with the prevalence of infection in the reference arm using least square mean differences to assess whether the new LLINs are superior. The primary comparison will occur at the end of the 24 months postintervention period. To adjust for the increased risk of a type I error due to multiple pairwise comparisons, the level of significance will be adjusted using the Bonferroni method. Least square mean differences between each intervention arm versus control will be calculated at each time, together with 98.23% CIs . Secondary analyses will include all possible pairwise comparisons of dual-AI LLIN arms.Anopheles density and EIR between the different arms will be estimated using random effects negative binomial regression taking into account the intracluster correlation. Random effects logistic regression will be used to compare sporozoite rate between study arms.EIR will be estimated as the mean number of sporozoite infected mosquitoes per house per night  and weighted to account for proportion of mosquitoes processed for sporozoites. Differences in Following relevant guidelines,Effects will be presented as disability-adjusted life years (DALYs) discounted at 3% and with no age weighting, calculated as the sum of years of life with disability from malaria-related illness  and years of life lost from malaria deaths.We will plot the costs and effects of each intervention on the cost-effectiveness plane, identify the cost-effectiveness frontier and expansion path, and calculate incremental cost-effectiveness ratios between adjacent points on this path. Deterministic and probabilistic sensitivity analysis will explore the impact of uncertainty and heterogeneity on cost-effectiveness results. Cost-effectiveness acceptability curvesCumulative incidence of malaria infection over the 24 months follow-up (accounting for repeat episodes in the same child but measured on separate cohorts at 12 and 24 months postintervention) will be analysed using mixed effects Poisson or negative binomial regression. The model will include fixed effects for time, study arm, and time by study arm, and will adjust for the covariates used in the covariate-constrained allocation procedure. The model will account for within-period and between-period intra-cluster correlations. Least square mean differences will be obtained from the model to compare each intervention versus the control. Follow-up within 14 days of a previous episode in the same child will be censored and will not be included in the analysis. An offset for duration of follow-up will be included in the model to account for attrition.Prevalence of moderate and severe anaemia in children under 5 at 12,18, 24, 30 and 36 months will be analysed using mixed effects logistic regression similar to the approach outlined for malaria prevalence.Bioassay data will be interpreted according to the updated WHO guidelines: mortality of ≥98% indicates susceptibility at the diagnostic dose, mortality of 90%–97% is suggestive of resistance, and mortality of less than 90% indicates resistance.A. gambiae s.s Kisumu or A. funestus s.s FANG), will be calculated using the 2−ΔΔCT method, incorporating PCR efficiency and normalised relative to the endogenous housekeeping control gene.70For metabolic gene assays, relative expression level and fold change of each target gene from resistant and susceptible field samples, relative to the susceptible laboratory strain , written informed consent  will be 10.1136/bmjopen-2020-046664.supp2Supplementary dataStudy findings will be shared in stakeholder meetings attended by local community leaders, the Ministry of Health, Community Development, Gender, Elderly and Children, the National Malaria Control Programme, the President’s office regional administration and local government representatives. Results will also be shared through peer-reviewed publications, at scientific conferences, and through clinicaltrials.gov.


Intriguingly, this allele did not negatively segregate with any of the clinical symptoms linked to mortality in this cohort. Studies are needed to determine whether PCSK9 inactivation promotes a protective immune response to malaria infection.Recent evidence suggests that proprotein convertase subtilisin/kexin type 9 (PCSK9), a downmodulator of cellular uptake of blood cholesterol, also negatively impacts host immune response to microbial infection. In this study, we investigated whether carrying the loss-of-function (LOF) rs562556  PCSK9 single nucleotide polymorphism (SNP) affected the outcome of severe malaria in children. Archival DNA of a cohort of 207 Malian children suffering from severe malaria was genotyped for the rs562556 SNP. Sixty-four children were either heterozygous or homozygous for the minor G allele (carriers); 143 children were homozygous for the common A allele (noncarriers). Among carriers, there was one mortality case (1.6%), compared to 15 cases (10.5%) among noncarriers ( Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a liver-secreted protein that binds the low-density lipoprotein receptor (LDLR) at the surface of hepatocytes and escorts it inside the cells towards lysosomes where it gets degraded. It thus reduces LDLR-mediated hepatic uptake of plasma LDL-cholesterol (LDL-C), causing its accumulation in the bloodstream. Single nucleotide polymorphisms (SNPs) in the human PCSK9 gene have been associated with either hypercholesterolemia or hypocholesterolemia, depending on whether they increase  or reduce  PCSK9 activity . Recent We wanted to verify whether PCSK9 could influence the course of malaria infection. One step to that end is to determine how common GOF and LOF genetic variations segregate with the severity and the outcome of the disease. We previously reported an association of the GOF PCSK9 SNP rs505151  with severe malaria in Malian children . Using tN = 753) were recruited from June 1999 to December 2003 in and around Bandiagara, a circumscription located 700 km east of Bamako, the capital city of Mali. Each case of severe malaria was paired with a case of uncomplicated malaria as well as with a healthy control of the same gender, age, and ethnicity. As per the criteria specified by the World Health Organization [P. falciparum parasitemia combined with one or several of the following symptoms: impaired consciousness, prostration, multiple convulsions, acidosis, hypoglycemia, renal impairment, severe anemia, jaundice, pulmonary edema, shock, or hyperparasitemia. Inclusion criteria included written informed consent by a participant's adult surrogate, residency in Bandiagara region, and availability for the study duration. Exclusion criteria included participation in a clinical interventional trial, long-term usage of medication with antimalarial activity, known chronic disease or immunosuppressive condition, and any condition which could endanger the participant's safety and infringe his/her rights. All severe malaria cases were treated according to local clinical practice guidelines. The treatment outcome, survival or death, was recorded.Subjects of the present study were 3-month to 14-year-old children diagnosed with severe malaria. They were participants in a previous case-control study investigating physiological factors that increase or decrease the risk for severe malaria . The parnization , severe The Review Board of the Faculty of Medicine, Pharmacy and Dentistry of the University of Mali, and the Research Ethics Committee of the Montreal Clinical Research Institute of Montreal reviewed and approved the study protocol. At recruitment, children's parents or guardians were asked to provide written informed consent.μl of a 10 mM NaOH/200 mM NaCl/0.05% sodium dodecyl sulfate solution and heated for 6 min at 95˚C; it was pulled down as above, and the supernatant was collected.Blood was collected at recruitment. Drops of it were spotted on Whatman FTA cards, dried, and stored at room temperature until use. Leucocyte genomic DNA was extracted as follows: a bloody paper disc, 6 mm in diameter, was punched out of an FTA card and placed into a tube containing 0.4 ml of phosphate buffered saline, pH 7.4; after a 20 min incubation at room temperature, the paper was pulled down the tube by centrifugation, the supernatant was discarded, and the paper was soaked in 40 An aliquot of the supernatant was diluted 12.5-fold in water and used in a TaqMan SNP genotyping assay using a Stratagene Mx 3005P thermocycler instrument . Briefly, an rs562556 SNP-containing DNA fragment was amplified by polymerase chain reaction (PCR); two allele-specific oligonucleotide probes, each conjugated in 5′ to a distinct fluorochrome and in 3′ to a quencher, were allowed to anneal to the PCR amplicon, while concomitant removal of the 3′ quencher generated signature allele-specific fluorescence . The supP value of <0.05 was set for significance in all analyses.Using GraphPad Prism 8.4.3 software package, Fisher' exact tests were applied to compare the frequencies of death/survival outcome or of severe malaria symptoms between carriers and noncarriers of the minor G allele. The strength of genotypic association was expressed as either odds ratio (OR) or relative risk (RR) and 95% confidence intervals (95% CI). A Of 207 children diagnosed with severe malaria, 16 (7.7%) died and 191 (92.3%) survived. As shown in Supplementary Table 1S, age, gender, ethnicity, medication, and use of antimosquito protection were similar between the two groups. However, in survivors, glycemia and hemoglobinemia were higher, while the white blood cell count was lower.p < 0.0001), coma (p < 0.001), severe anemia and repeated vomiting (p < 0.005), lethargy (p < 0.01), convulsion, and prostration and hypoglycemia (p < 0.05). Hyperparasitemia (≥5 × 105 parasites/µL), was associated with lower mortality (p < 0.005) in this cohort.The distribution of individuals symptoms between the groups and their associations with the outcomes are described in p=0.0251), suggesting that the G allele is associated with lower fatality in severe malaria.We examined whether mortality to severe malaria occurred differentially between patients who carried the LOF G allele of the rs562556 PCSK9 SNP (AG and GG genotypes) and those who did not (AA genotype). As shown in p < 0.0001).Surprisingly, none of the symptoms associated with mortality segregated differentially between the two genotypic groups , making Before exploring the association of the rs562556 PCSK9 SNP with malaria mortality, we determined the clinical predictors of fatal malaria in this Malian cohort. Except for severe anemia, they corresponded to those identified in a meta-analysis of a large number of studies on African children . Hyperpan = 304) was recently examined in relation with common PCSK9 SNPs [With a minor allele frequency of 0.203, the rs562556 PCSK9 SNP was relatively common in this cohort. A meta-analysis of previous genetic surveys of populations of different ancestries has established that the minor G allele of this SNP reduces PCSK9 activity, as it is associated with lower cholesterolemia . The serSK9 SNPs . It was SK9 SNPs  and humaSK9 SNPs .Plasmodium-caused systemic inflammation and improves prognosis in severe malaria. Studies are needed to establish whether and how PCSK9 potency influences the expression of cytokines and chemokines in this disease.Elevated plasma TG and inflammation are hallmarks of infectious diseases . ExacerbPlasmodium-infected red blood cells (RBC) as a means of detoxifying the free heme that results from hemoglobin degradation; it is released in circulation following schizont rupture of the RBC and is taken up by leucocytes [The immune response to malaria seems to be modulated by circulating hemozoin . This crucocytes . Incidenucocytes  have shoucocytes ), it shoV474 variant in some of these organs could lead to much presence of LDLR and other scavenger receptors at the surface of their cells resulting in more internalization and accumulation of cholesterol and parasite metabolites , hemozoin) that could cause localized inflammation and abdominal pain. Noteworthy is the fact that, in this Bandiagara cohort, anti-GPI antibodies were higher among severe malaria children who died compared to those who survived [The association of the G allele with abdominal pain remains intriguing. This nonlethal and often transient symptom in severe malaria could originate in the liver, spleen, gallbladder, or kidneys as these organs become inflamed following sequestration of parasite-infected erythrocytes in their microvasculature . We specsurvived , raisingThe missense rs562556 SNP is located in a genomic coding region that has been found to be under evolutionary selection, based on the rates of nonsynonymous versus synonymous variations in the primate phylum . The G aSusceptibility to malaria is a multifactorial phenomenon. It depends on biological, environmental, demographic, and socioeconomic as well as healthcare factors . This stWe have observed a significant association of the minor allele of the rs562556 PCSK9 SNP with reduced mortality in severe malaria. This observation needs corroboration by larger studies taking into consideration immune, lipid, and PCSK9 parameters. Such a corroboration will justify the use of dietary or medicinal anti-PCSK9 interventions to attenuate the clinical course of malaria and reduce its mortality.