Plasmodium falciparum-infected erythrocytes (IE) sequester in deep vascular beds where their adhesion is mediated by an array of endothelial surface receptors. Because parasite adhesion has been associated with disease, antibodies that block this activity may confer protective immunity. Here, levels of plasma anti-adhesion activity and surface reactivity against freshly collected IEs from malaria-infected children were measured in a Malian birth cohort and related to child age and malaria infection history.Plasma samples from children enrolled at birth in a longitudinal cohort study of mother–infant pairs in Ouelessebougou, Mali were collected at multiple time points during follow-up visits. Anti-adhesion antibodies  and reactivity with surface IE proteins were measured using a binding inhibition assay and by flow cytometry, respectively.3β1 and laminin increased with age. The breadth of antibodies that inhibit ICAM-1 and laminin adhesion (defined as the proportion of IE isolates whose binding was reduced by ≥ 50%) also significantly increased with age. The number of malaria infections prior to plasma collection was associated with levels of plasma reactivity to IE surface proteins, but not levels of anti-adhesion activity.Levels of antibodies that inhibit the binding of children’s IE to the receptors ICAM-1, integrin αAge is associated with increased levels of antibodies that reduce adhesion of children’s IE to three of the ten endothelial receptors evaluated here. These results suggest that anti-adhesion antibodies to some but not all endothelial receptors are acquired during the first few years of life.The online version of this article (10.1186/s12936-019-2764-4) contains supplementary material, which is available to authorized users. Plasmodium falciparum is able to sequester in deep vascular beds of various tissues. Infected erythrocytes (IE) have been demonstrated to bind a number of receptors expressed on the endothelial cell surface, including thrombospondin (TSP), CD36, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin (ELAM-1), P-selectin, PECAM-1/CD31, EPCR, members of integrin family, extracellular matrix proteins like laminin and cellular fibronectin [P. falciparum . Aniewed in –19, but iewed in , 21. PfEiewed in ).In nonhuman primate studies, treatment of malaria-infected animals with antibodies developed against the infecting parasite  rapidly reversed IE cytoadhesion, resulting in the release of the parasite’s mature forms into the peripheral blood circulation . The samHere, in the context of a longitudinal birth cohort, antibodies that inhibit IE adhesion to several endothelial receptors (anti-adhesion antibodies), and antibodies reacting with IE surface proteins of fresh parasites, were related with child age and prior malaria infection. The study was designed to evaluate plasma antibody to diverse surface proteins by assaying IE collected from children in the same cohort, described here as heterologous parasites.> 2 convulsions in the past 24 h; prostration (inability to sit unaided or in younger infants inability to move/feed); haemoglobin < 5 g/dl; respiratory distress ; coma (Blantyre score < 3). The presence of severe malaria symptoms due to other diseases were clinically ruled out.Evaluation of samples collected during a longitudinal cohort study of newborns and children aged 0–3 years conducted in Ouelessebougou, Mali. The study site is located 80 km south of Bamako, an area of intense but highly seasonal malaria transmission. Prior to enrollment, written informed consent was obtained from the parents/guardians on behalf of their children after receiving a study explanation form and oral explanation from a study clinician in their native language. The protocol and study procedures were approved by the institutional review board of the National Institute of Allergy and Infectious Diseases at the US National Institutes of Health , and the Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry at the University of Bamako, Mali. An intensive follow-up included monthly scheduled clinic visits during the malaria transmission season, and every 2 months during the dry season, as well as ad hoc visits when symptoms occurred. Clinical information was collected by project clinicians on standardized forms. Malaria infections were treated with artemether–lumefantrine or quinine as clinically indicated. Severe malaria was defined as parasitaemia detected by blood smear microscopy and at least one of the following World Health Organization criteria for severe malaria: P. falciparum parasites in assays. Of 390 parasite-infected samples assayed, 15 samples were collected from children presenting with severe malaria. Parasite samples were collected from children aged (median (range)) 27.6 months (1.3–59.8 months), with parasite density of (median (range)) 72,325 parasites/µl . Each parasite sample was used for assays of 12–18 plasma samples, and plasma samples were assayed against an average (range) of 2.7 (1–9) parasite samples.Blood samples collected from malaria-infected children were used as the source of 3β1, integrin α5β1, integrin αvβ3, JAM-B, from R&D Systems Minneapolis, MN, cellular fibronectin, and laminin from placenta from Sigma, St. Louis, MO) at a concentration of 10 µg/ml were immobilized by adsorption on Petri dishes. The parasite suspension was preincubated with plasma diluted 1:5 for 30 min at room temperature then allowed to bind to the immobilized receptor for 30 min at room temperature. Plates were washed three times with PBS to remove unbound cells. Bound cells were fixed in 0.5% glutaraldehyde for 10 min, stained with 10% Giemsa for 2 min and quantified by counting the number of parasites in 20 high-power fields. Level of inhibition was defined based on the level of IE binding in the presence of a pool of plasma samples collected from malaria-naïve adults in the USA (negative control) as follow (100 − (Ntest/Ncontrol) × 100)). A minimum IE count of 20 in the presence of naïve plasma was required for the results to be accepted.Ring stage parasite samples were allowed to mature to the trophozoite/schizont stages during in vitro culture for 16–24 h, then mature parasite forms were enriched by gelatin flotation. Parasite binding inhibition assay was performed using a parasite suspension at 2–20% parasitaemia and 0.5% haematocrit . 20 µl otest/%IEcontrol.Mature IE were incubated with plasma samples diluted 1:10 followed by incubation with anti-human IgG conjugated to PE (eBioscience) and stained with SYBR green. A pool of hyperimmune adult plasma was used as a positive control and a pool of plasma samples collected from malaria-naïve adults was used as a negative control. Reactivity of antibodies with the IE surface is expressed as a ratio of the proportion of IE recognized by test and a pool of naïve plasma donors, as follows: Ratio = %IEData were collected on standardized forms and optically scanned into the database using DataFax . Linear mixed effects models accounting for multiple measurements per child were fitted to relate anti-adhesion antibodies and reactivity with IE surface with age and number of previous infections. Breadth of anti-adhesion antibody activity and surface IE recognition were analysed using mixed effects beta binomial logistic regression models, accounting for the number of parasite isolates tested. Separate models were fitted for age and the number of previous malaria infections. P value was corrected for multiple comparisons using the Holm method. Analyses were carried out in R (version 3.3.2).P. falciparum infections documented before sample collection  identified in a binding phenotype survey of IE collected from children in this study [The level of anti-adhesion activity and the breadth of anti-adhesion activity were analysed in relation to age and number of prior infections. Plasma samples were tested for inhibiting IE adhesion to the following receptors: CD36, ICAM-1, P-selectin, platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), integrin αis study . Level ois study . In the 3β1 and laminin were positively associated with age  based on the median (1.4) was used to define positive recognition of a parasite isolate. The breadth of reactivity with IE surface proteins trended towards a positive association with the number of prior malaria infections, but the relationship did not achieve statistical significance  based on homology in the flanking region and chromosome location [var genes in children with severe malaria [var genes (Group 2) more readily recognized by infant’s plasma than other PfEMP1s [Over years of exposure, residents of endemic areas acquire immunity that prevents severe disease and limits blood stage parasite growth , 40. Extlocation . Multipl malaria . Further malaria , with DC malaria –45. Anal malaria , 47, wit PfEMP1s . Antibod PfEMP1s , 49.Infection induces variant-specific agglutinating antibodies and antibodies that recognize the IE surface by flow cytometry , 51. Somvβ3 [3β1 and laminin. These results suggest that it may take longer to acquire anti-adhesion antibodies to common receptors like CD36 and integrin αvβ3, consistent with a previous report describing low levels of anti-adhesion antibodies to CD36 in children aged 3–4 years compared to children aged 10–11 years [In the current study, the development of anti-adhesion antibodies to several endothelial receptors with fresh isolates collected from children participating in the same longitudinal cohort was examined. The two most common endothelial receptors for adhesion of IE collected from children in this cohort were CD36 and integrin αvβ3 . Neither11 years . In the Although levels or breadth of anti-adhesion antibodies was not significantly associated with the number of prior malaria infections after correcting for multiple comparisons, antibody levels and breadth of antibodies that inhibit IE binding to the receptor laminin were associated with an increased number of prior malaria infections before correction. Similarly, a trend for prior malaria infection to be associated with breadth of anti-adhesion antibodies to the receptors ICAM-1 and JAM-B was not statistically significant  and interquartile range (box), the whiskers indicate the 5th and 95th percentiles.


In a screening program, we detected submicroscopic malaria in 8.9% of recent migrants to Spain from sub-Saharan Africa. Hemoglobinopathies and filarial infection occurred more frequently in newly arrived migrants with submicroscopic malaria than in those without. Our findings could justify systematic screening in immigrants and recent travelers from malaria-endemic areas. Plasmodium infection detected only by molecular methods  is defined as low-density Anopheles atroparvus mosquitoes can transmit strains of P. vivax  during October 2004–December 2016. This hospital’s protocol comprised a series of complementary tests to screen for imported diseases, including chest and abdominal radiographs; blood count; liver and renal function tests, iron metabolism tests; serologic screening for syphilis, HIV, hepatitis B virus, hepatitis C virus, P. vivax, P. falciparum, P. ovale, and P. malariae) in 2 consecutive multiplexing amplifications. The first reaction amplifies Plasmodium DNA from blood samples. The second reaction enables identification of the infecting species of Plasmodium . We excluded from the study patients <14 years of age and patients for whom no smear and/or rapid diagnostic test for malaria was available. All SMM patients were treated according to World Health Organization guidelines (The study population comprised patients who had lived in Europe for <1 year (newly arrived migrants [NAM]) or who had visited their home country  within the previous year who sought care for any reason other than patent malaria and were screened for SMM using the conventional nested multiplex malaria PCR. The nested multiplex malaria PCR can identify 4 human malaria species .We conducted 3 statistical analyses. First, we compared SMM patients with non-SMM patients. Then, within the SMM patient group, we compared those with and without filarial co-infection. We conducted a descriptive statistical analysis in which continuous variables were expressed as medians and interquartile ranges. Categorical variables were described as frequencies and percentages. We analyzed differences in continuous data between groups using nonparametric Mann-Whitney U test and used the Fisher exact test or χOf 2,719 sub-Saharan Africa patients seen, 370 (13.6%) were included in the study . SMM wasPlasmodium species most frequently found was P. falciparum (26 [78.8%] patients), followed by P. malariae (4 [12.1%]), P. ovale (2 [6.1%]), and 1 mixed parasitization by P. falciparum and P. malariae (1 [3.0%]). Patients with and without SMM did not differ in baseline laboratory data, except for the presence of hemoglobinopathies, which occurred more frequently among SMM patients .The Mansonella perstans nematodes were responsible of all filarial infections; in addition, 3 patients were infected by Loa loa eyeworms.When we analyzed other associated infections , we founAmong SMM patients, all filariasis was found in NAMs. These co-infected patients had higher IgE values  and higher total eosinophil counts  than those who had only SMM. The co-infected group also tended to have higher platelet levels .>1 hemoglobinopathies increased the odds by 3.93. Time after leaving a malaria-endemic area correlated inversely with risk for SMM (p = 0.038)  . For NAMScreening for SMM in patients from sub-Saharan Africa in a reference unit in Spain showed a prevalence of 8.9%. The presence of filarial infection or hemoglobinopathies and a shorter time since leaving malaria-endemic areas were associated with a higher risk for SMM.SMM is usually asymptomatic. Infrequently, it produces acute disease, especially in children  and for pregnant women. The diagnosis and treatment of SMM also can prevent future reactivations and the existence of an occult malaria reservoir in countries to which it is not endemic. Our results suggest that the presence of filariasis, hemoglobinopathies, or both should also prompt a search for SMM because these patients are at higher risk.


Accurate and timely diagnosis of malaria is essential for disease management and surveillance. Thin and thick blood smear microscopy and malaria rapid diagnostic tests (RDTs) are standard malaria diagnostics, but both methods have limitations. The novel automated hematology analyzer XN-30 provides standard complete blood counts (CBC) as well as quantification of malaria parasitemia at the price of a CBC. This study assessed the accuracy of XN-30 for malaria detection in a controlled human malaria infection (CHMI) study and a phase 3 diagnostic accuracy study in Burkina Faso.Plasmodium falciparum-infected mosquitoes. Blood was sampled daily for XN-30, blood smear microscopy, and malaria qPCR. The accuracy study included patients aged > 3 months presenting with acute febrile illness. XN-30, microscopy, and rapid diagnostic tests (HRP-2/pLDH) were performed on site; qPCR was done in retrospect. The malaria reference standard was microscopy, and results were corrected for sub-microscopic cases.Sixteen healthy, malaria-naive CHMI participants were challenged with five R2 = 0.91; p < .0001). The XN-30 accurately monitored treatment and allowed detection of recrudescence. Out of 908 patients in the accuracy study, 241 had microscopic malaria . The sensitivity and specificity of XN-30 compared to microscopy were 98.7% and 99.4% . Results were corrected for qPCR-confirmed sub-microscopic cases. Three microscopy-confirmed cases were not detected by XN-30. However, XN-30 detected 19/134 (14.2%) qPCR-confirmed cases missed by microscopy. Among qPCR-confirmed cases, XN-30 had a higher sensitivity  and similar specificity  as microscopy. The accuracy of XN-30 for microscopic malaria was equal to or higher than HRP-2 and pLDH RDTs, respectively.All CHMI participants became parasitemic by qPCR and XN-30 with a strong correlation for parasite density  and NCT02669823 (diagnostic accuracy study).Both trials were registered on clinicaltrials.gov with respective identifiers The online version of this article (10.1186/s12916-019-1334-5) contains supplementary material, which is available to authorized users. Plasmodium falciparum accounting for 99% of estimated cases [Malaria remains a major cause of morbidity and mortality around the world. Sub-Saharan Africa is most affected with ed cases . Timely ed cases . Thin aned cases . Both teed cases . Consequed cases . WHO thePlasmodium parasites  in blood using violet laser technology [Plasmodium falciparum malaria. First, we assessed its performance in healthy participants in whom low-density parasitemia was induced in a controlled human malaria infection (CHMI) study. To establish the performance under field conditions, we then performed a phase 3 diagnostic accuracy study among febrile patients in Burkina Faso.The XN-30  is a novel automated hematology analyzer and malaria diagnostic that directly detects and quantifies chnology –8. Our sClinicalTrials.gov, NCT02836002). Details and results of the primary objectives of this study have recently been published [P. falciparum parasites. Both techniques are described in detail elsewhere [The ability of the XN-30 to detect low-density sexual and asexual parasitemia and monitoring of treatment was studied in a CHMI trial  was performed at the Clinical Research Unit of Nanoro (CRUN) [This prospective, double-blinded, phase 3 diagnostic accuracy study (o (CRUN) . The priPlasmodium falciparum, though Plasmodium ovale and Plasmodium malariae are sporadically found [Nanoro is a rural area of Burkina Faso which is hyperendemic for ly found . ParticiData was collected on case report forms (CRFs) and entered into a secure database  after conformity check by a medical doctor. Entered data were checked against the CRFs by a data manager. Approximately 10 % of participant study files were checked by an independent monitor. Results from qPCR were entered into an Excel  database and merged with the principal database. The researchers were blinded to the XN-30 until the clinical database was locked. Interpretation of the index test was done by blinded researchers after inclusion was completed. Laboratory procedures were standardized; quality controls were performed according to good clinical and laboratory practices (GCLP) guidelines.The XN-30 is an automated hematology analyzer  which can be used for malaria detection as described in detail elsewhere . The anaThe analyzer aspirates approximately 60 μL whole blood per analysis; results are available within 1 min. XN-30 will be made for global distribution, and the price of a measurement is expected to be comparable to regular hemocytometry [Plasmodium parasites according to WHO procedures [Plasmodium species and presence of gametocytes were also recorded. Slides were viewed by two independent microscopists. Presence of one or more Plasmodium parasites was considered positive. In case of discrepancies, the result of a third microscopist was decisive.Both RDT and microscopy were performed using EDTA-anticoagulated blood. Thick and thin blood films were examined for the presence of ocedures . ResultsPlasmodium falciparum histidine-rich protein-2 (HRP-2) and pan-Plasmodium species parasite-lactate dehydrogenase (pLDH) were scored, whereby any visible test line was considered positive. Results for pLDH and HRP-2 were recorded separately. Quantitative PCR (qPCR) targeting the multicopy 18S rRNA gene was performed in retrospect at the Department of Microbiology of Radboud University Medical Centre, the Netherlands, using 200 μL blood per patient using previously described methods [SD Bioline Malaria Ag-P.f (HRP2/pLDH)  was performed according to the manufacturer’s instructions. Five microliters of venous blood was inoculated on the RDT cassette. After 5 and 15 min, the test line reactivity of the  methods . The lowMalaria parasitemia was defined as the presence of one or more parasites in the asexual stage in malaria microscopy or a malaria qPCR result over 0.05 p/μL in the diagnostic accuracy study. In case only gametocytes were observed, microscopy was considered negative. Severe malaria was defined according to WHO criteria 2014 [U test in case of not normally distributed data. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were assessed using the STATA diagt-package. A receiver operation characteristics (ROC) curve analysis was done to assess the area under the curve (AUC). Linear regression analyses were done to assess the correlation in parasite densities between qPCR, malaria thick smear, and XN-30. Comparative analyses of sensitivity and specificity were done using a McNemar test and reported as test ratio with significance level. A significance level of 5% was used for all analyses.A statistical plan was made before data inspection. Analyses were done using Stata 14 . Patients with missing data on either reference test, index test, or qPCR were excluded from the analysis. In the case of inconclusive index test results due to an abnormal scattergram distribution, the result was reported as “inconclusive.” Differences in proportions, medians, or means were compared using respectively the chi-square test, two-tailed Fisher’s exact test, or Mann-Whitney R2 = 0.91; p < .0001)  whereas specificity did not differ between both tests . The cutoff value of best fit to qPCR in the current dataset was a parasite density of 26 p/μL, which gave similar results to the proposed cutoff value  , while specificity was equal . Conversely, XN-30 had higher specificity  than HRP-2, while the sensitivity of HRP-2 was higher than that of XN-30 . The difference in sensitivity between HRP-2 and XN-30 was mostly caused by sub-microscopic parasitemia cases , most of whom (64.0%) had been pre-treated with antimalarials. This suggests that at least part of the cases detected by HRP-2, but missed by XN-30, may have been based on remaining presence of antigens after a recent malaria infection. Among microscopically confirmed cases, XN-30 had an equal or higher sensitivity and higher specificity than HRP-2 and pLDH.A secondary objective was to compare the diagnostic performance of XN-30 to diagnose malaria parasitemia against two RDTs. Table r = 0.82, p < .0001; qPCR r = 0.79, p < .0001). Follow-up samples were available in 125 patients (51.9%) with XN-30-confirmed malaria. The median parasite density dropped from 19,138 p/μL  at inclusion to below the limit of positivity (20 p/μL) at follow-up, confirming that XN-30 can be used for treatment follow-up in a clinical setting. A total of 15 cases of non-falciparum malaria parasitemia were identified by microscopy, all of which were picked up by XN-30, though they were not differentiated from P. falciparum.We compared parasite densities as recorded by XN-30 with densities quantified by microscopy and qPCR. Figure Plasmodium falciparum parasitemia was superior to that of microscopy in qPCR-confirmed cases, whereas the specificity was comparable. XN-30 had an equal or higher sensitivity than HRP-2 and pLDH respectively, and a higher specificity than both for microscopy-confirmed malaria. Our results are in line with results from a recently published retrospective study on the performance of the XN-30 from South Africa [P. falciparum, as found in Central Africa, is present that prevents the detection of this strain [This is the first prospective clinical study to present the diagnostic performance of XN-30. The sensitivity of XN-30 to detect h Africa . The acch Africa . The reph Africa , 18, excs strain .In case of signs of severe malaria, follow-up of admitted malaria patients, or RDT-confirmed malaria cases with persistent fever despite anti-malarial treatment, the WHO recommends the use of malaria microscopy . Our datAutomated hematology analyzers to diagnose malaria have been studied in the last two decades, applying different techniques such as abnormal depolarization in the granularity of phagocytes, abnormalities in the size of blood cells, or blood cell differentiation patterns –27. HoweThe limit of detection as observed in the CHMI study was lower than that of the diagnostic accuracy study (8.1 versus 20p/μL). We have no obvious explanation for this difference; previous studies demonstrate that malaria negative samples with low hemoglobin, high reticulocyte count, thrombocytopenia or hemoglobinopathies had no significant influence on the reliability of the malaria result [Plasmodium species prevalent in Burkina Faso. In addition, this study was performed in a malaria hyperendemic setting; therefore, the population-level effect of the sensitivity and specificity could vary by different settings e.g. low-endemicity countries.In the present study, XN-30 had an inconclusive result due to an abnormal scatter gram distribution in 71 cases (7.8%). This implies that when used in clinical settings, approximately one in 12 patients would need an additional malaria diagnostic when the XN-30 is inconclusive. Second, the XN-30 currently does not reliably distinguish In conclusion, the XN-30 holds promise as a rapid and sensitive test for malaria detection and subsequent treatment monitoring. Since XN-30 provides a CBC with each analysis, it provides critical information for malaria management at a price comparable to CBC. Its higher sensitivity compared to microscopy in low-density parasitemia also makes it a useful tool for mass screening in control programs.Additional file 1:Table S1. Characteristics of patients with inconclusive XN-30 result. (DOCX 18 kb)Additional file 2:Table S2. ROC analysis of cutoff value of best fit, XN-30 compared to qPCR. (DOCX 20 kb)Additional file 3:Figure S1. CHMI study participant inclusion flow. (TIF 73 kb)Additional file 4:Figure S2. Correlation of the XN-30 with microscopy and qPCR, in cultured parasites. (TIF 660 kb)Additional file 5:Figure S3. Stability data of cultured ring-stage samples. (TIF 181 kb)Additional file 6:Figure S4. ROC curve at cutoff value of best fit, XN-30 compared to qPCR. (TIF 1006 kb)


Injectable artesunate (AS) is the World Health Organization (WHO) recommended medication for the treatment of severe malaria followed with an oral artemisinin-based combination therapy (ACT). There are few studies indicating how physicians prescribe injectable AS, injectable quinine (Q) or injectable artemether (AR) and ACT for severe malaria. This study was undertaken to evaluate prescription compliance to the WHO recommendation in 8 public health facilities in Ghana and Uganda. This was a modified cohort event monitoring study involving patients who were administered with injectable anti-malarial for treatment of presumed or confirmed severe malaria. Patients prescribed at least one dose of injectable artesunate, artemether or quinine qualified to enrol in the study. Patients were recruited at inpatient facilities and followed up in the hospital, by phone or at home. Following WHO recommendations, patients are to be prescribed 3 doses of injectable AS, Q or AR for at least 24 h followed with oral ACT. Compliance rate was estimated as the number of patient prescriptions that met the WHO recommendation for treatment of severe malaria divided by the total number of patients who completed the study by end of follow up. Log-binomial regression model was used to identify predictors for compliance. Based on the literature and limitations of available data from the patients’ record, the diagnosis results, age, gender, weight, and country were considered as potential predictors of prescriber adherence to the WHO recommendations.A total of 1191 patients completed the study, of which 93% were prescribed injectable AS, 3.1% (injectable AR or Q) with 32.5% prescribed follow-on oral ACT and 26% on concomitant antibiotics. 391 (32.8%) were in Ghana and 800 (67.2%) in Uganda. There were 582 (48.9%) women. The median age was 3.9 years  and median weight was 13 kg . Of the 1191 patients, 329 of the prescriptions complied with the WHO recommendation . Diagnostic results  = 4.56; 95% = ; p < 0.0001) and weight  were identified as factors independently associated with compliance.Injectable AS is the most commonly prescribed medicine in the management of severe malaria in Ghana and Uganda. However, adherence to the WHO recommendation of at least 3 doses of injectable anti-malarial in 24 h followed by a full course of ACT is low, at less than 30%. Plasmodium falciparum. Poorly managed P. falciparum infections can lead to severe malaria which is associated with extremely high case fatality rates averaging 16–20% though extreme ranges as low as 2% and high as 100% have also been reported [Over the past 10 years, there has been a huge decline in malaria cases as well as deaths from malaria. According to the World Malaria Report, there has been a 22% reduction in malaria cases between 2010 and 2017  aim to rreported  these exThe World Health Organization (WHO) recommendation for severe malaria is the appropriate use of injectable artesunate . This reIn a study in Swaziland covering the period 2011–2015, where patients were diagnosed either by rapid diagnostic tests (RDTs), microscopy or both , less thIn view of the high case fatality rate associated with severe malaria, it is important to understand the routine management of patients with severe malaria and to assess whether prescribing patterns are in line with recommended guidelines. The focus of this manuscript is on prescribers’ compliance to the WHO recommendations for treating patients with severe malaria especially considering the paucity of publications on prescription adherence to recommended treatment for severe malaria.This study sought to describe medicine prescription in public health facilities in Ghana and Uganda regarding WHO guidance on the treatment of severe malaria to find out the level of compliance to the recommendations. It also examined factors independently associated with compliance to the WHO recommendation. The findings provide insight into what pertains in real-world settings in relation to WHO guidance on treatment of severe malaria in particular in the use of the WHO recommended approach of prescribing injectable anti-malarials  followed by a full course of oral ACT for the treatment of severe malaria.Plasmodium of any species) presumed or diagnosed as per national policies and health facility practice/protocol [This was a modified cohort event monitoring study involving patients who were prescribed injectable anti-malarial for treatment of presumed severe malaria in 8 sites (4 each in Ghana and Uganda) between May and December 2016. The full details of the methods have been previously published . In shorData on medicine prescription were extracted from patients’ records kept in paper “folders” in each health facility together with physician clerking notes, nurses’ medicines administration records and pharmacy medicine supply details. Since the criteria for enrolment in this study was the prescription of injectable AS, AR or Q, all patients in the study had at least one dose of these medicines prescribed in addition to any concomitant medications prescribed. Data was extracted from the patient medical files on method of diagnosis, diagnosis or test results, date of treatment, age, gender, dose and frequency of anti-malarial as well as the prescribed route of administration. Data on laboratory results was important as the WHO recommendations require 12-hourly microscopy to monitor response to treatment.The sample size calculation was based on the primary outcome of compliance to WHO recommendation for treatment of severe malaria. The main analysis estimated the compliance rate and its 95% confidence. Therefore, the sample size was estimated using precision approach for one proportion in PASS 16 . Although previous studies reported approximately 90–95% prescription of 3 doses of AS, no study reported appropriate prescription in terms of 3 doses of AS followed by ACT. However, anecdotal evidence from routine hospital data showed that this was low. Therefore, a conservative compliance rate of 27% was assumed. Based on this assumption, a sample size of 1212 produces a two-sided 95% confidence interval with a margin of error equal to ± 2.5%  when the sample proportion is 0.27.Data on patient demography, the method used to test for malaria parasitaemia as part of the diagnosis of severe malaria as well as the overall management of the severe malaria episode itself were extracted from the patient records contained in paper “patient folders” in each facility. All these data were then transcribed onto the study Case Report Forms (CRFs). There were individual CRFs for collecting Demography, Medical History, Study and Concomitant Medication data, Laboratory data, the various Follow-Up information as well as individual CRFs for collecting Adverse Drug Reactions and Serious Adverse Drug Reactions data. All these CRFs formed what was called a CRF book per patient. Upon completion of a patient CRF book, the Site Coordinator as well as the Principal Investigator signed off on the book indicating that the book is complete. Data were entered, managed and stored in a specially created version of MedSpina™, an in-house electronic health records system that allows clinicians and other health workers to collect patients’ data, including laboratory results, to facilitate patient care. The Data Manager performed first pass quality control on all data points per patient CRF book by making sure data on the CRF matched the data in the database. All discrepant data points were queried for clarification/correction. Upon completion of the data management process, all data points for all patients were extracted for statistical analysis.The primary outcome was compliance to the WHO recommended treatment guidelines for severe malaria  defined Based on the literature and limitations of available data from the patients’ record, the diagnosis results, age, gender, weight, and country were considered as potential predictors of prescription. Use of concomitant medications was defined as any other medicines taken in addition to the WHO recommended anti-malarials. Thus, medicines being taken by patients during the course of the study such as antibiotics, haematinics, and analgesics among others was considered as covariates.The prescribed medicines were re-coded into seven categories namely: injectable AS, AR or Q, ACT, antibiotics, analgesic or antipyretic, haematinic or vitamin, and others. The primary outcome was calculated as the number of patient prescriptions that met the WHO recommendation for treatment of severe malaria divided by the total number of patients who completed the study by end of follow up.The log binomial regression model was used to identify factors independently associated with compliance to the WHO recommendation. To construct a parsimonious model using all the potential predictors, a model for each potential predictor was first fitted. In this model, each variable were candidates for inclusion in the full model if the p-value for association with prescription was 0.2 or less when considered individually. Variables were then removed from the model if the p-value for the likelihood ratio test was more than 0.2, provided removal did not change coefficients of variables in the model by more than 10%. Standard errors were adjusted for clustering of patients within health facilities. Missingness in the potential predictors were assumed as having occurred at random and not related to the outcome and, therefore, were not imputed. In a secondary analysis, the proportion of patients who were diagnosed with different malaria diagnostic methods were estimated. All analyses were performed using Stata 15 MP .A total of 1262 patients were screened but 46 declined to participate giving 1216 patients treated with AS and or other anti-malarials and 25 having been lost to follow up leaving 1191 patients who were included in the analysis  of the patients were prescribed injectable artesunate, 3.1% (injectable artemether or quinine), and 32.5% oral ACT  were tested for parasitaemia using microscopy only whereas 353 (29.6%) were tested using RDT only. For each setting, 56.8% (222/391) of patients in Ghana were tested using microscopy only compared to 43.4% (347/800) in Uganda whereas 16.6% (65/391) of the Ghanaian cohort were tested using RDT only compared to 36.0% (288/800) in Uganda  followed by oral ACT. There was high differences between countries with compliance being 20 times higher in Ghana compared to Uganda where only 4.8% of patients had a prescription for an injectable anti-malarial followed by a co-prescription of oral ACT. This finding contrasts with that of Achan et al.  where 42In this study, prescribers appeared to pay particular attention to children under 5 years in respect of compliance with the WHO recommendations. The findings showed a 20% increase in compliance among prescribers in relation to children under 5 years of age compared to older patients, suggesting a predilection towards more careful management of these children considered as high risk group. Although not statistically significant at 5% level in this study, compliance when prescribing for those who were negative for malaria parasites was about 15% higher than those positive for malaria parasites. Prescribing anti-malarials for patients without parasitaemia is well known, both for severe as well as uncomplicated malaria and in one study in Kenya in 2016, 69% of patients with negative malaria tests were still given parenteral treatment . An earlThe main medicines co-prescribed with the anti-malarial treatments were analgesics (37%), antibiotics (26%) and haematinics (15%). Analgesics and haematinics are routinely prescribed for both uncomplicated and severe malaria to treat fever and anaemia, respectively. As explained above, the use of antibiotics may be justified by the fact that attending physicians may be treating for other conditions which may be co-existing with the severe malaria. The drug prescription pattern in this study can be compared with those of a similar study on prescribing for patients with uncomplicated malaria where 31% and 26% received antibiotics and vitamins, respectively . Such prIn relation to testing for malaria itself, 71.8% of the patients in Ghana were tested using RDT or microscopy with a further 19.7% being tested with both RDT and microscopy. Just under 80% of the patients in Uganda were tested for presence of malaria parasites using either RDT or microscopy with less than 1% being tested using both RDT and microscopy. Nearly 20% of patients in Uganda and 10% of patients in Ghana were diagnosed clinically without parasitological confirmation by the use of RDT or microscopy. The WHO guidelines for the treatment of malaria as well as the practical handbook for Severe Malaria Management recommend the use of microscopy as part of the procedure for the diagnosis of severe malaria with a further recommendation that microscopy should be undertaken every 12 h during the 2–3 days of parenteral treatment to monitor response. None of the patients had 12-h microscopy to monitor treatment response.Limitations of this study include the fact that the findings relate to prescriptions for patients in in-patient settings with no evidence that patients had been administered these medicines. Patients whose medications may be changed due to other considerations are still included in the analysis. Prescriptions that patients may already have but are unrelated to the current episode and admission may be missed. A drug utilisation study which examines availability of medicines in the treating facility as well as drug administration and its appropriateness in relation to age, weight and dose will be very helpful. In addition, the inclusion criteria for patients in this study was the prescription of parenteral anti-malarials  for treating presumed or confirmed severe malaria even though it may just represent overuse of injectable anti-malarials as found in previous studies. Further, this study did not look at provider related factors such as training on guidelines and health system related factors such as drug stock-outs which may influence the prescription patterns.Inj AS is the most commonly prescribed medicine in the management of severe malaria in Ghana and Uganda. However, adherence to the WHO guidelines which recommends the prescribing of injectable anti-malarial for at least 24 h followed by a full course of oral ACT is low.


Arterial stiffness independently predicts cardiovascular disease. However, few studies have evaluated the associations of central and peripheral pulse wave velocity (PWV) with biomarkers of both myocardial stress (natriuretic peptide [NT-proBNP]) and damage (high-sensitivity cardiac troponin-T [hs-cTnT]) among persons without cardiac disease.We examined 3,348 participants (67–90 years) without prevalent cardiac disease in the Atherosclerosis Risk in Communities (ARIC) Study (2011–13). The cross-sectional associations of PWV quartiles for central arterial segments  and peripheral artery  with NT-proBNP and hs-cTnT were evaluated accounting for potential confounders.Most PWV measures demonstrated J- or U-shaped associations with the two cardiac biomarkers. The highest (Q4) vs. second lowest (Q2) quartile of central PWV measures  were associated with higher levels of NT-proBNP independently of demographic characteristics. The associations were less evident for hs-cTnT. These associations were attenuated after adjusting for traditional cardiovascular risk factors, but the heart-carotid PWV-NT-proBNP relationship remained borderline significant . Peripheral PWV demonstrated inverse associations. Higher values of NT-proBNP were seen in the lowest vs. second lowest quartile of all PWV measures.Central stiffness measures showed stronger associations with cardiac biomarkers (particularly NT-proBNP) than peripheral measures among older adults without cardiac disease. Our findings are consistent with the concept of ventricular-vascular coupling and suggest that central rather than peripheral arterial hemodynamics are more closely related to myocardial stress rather than damage. Arterial stiffness indicates increased rigidity and decreased elasticity of the arterial wall in response to fluctuations in pulsatile pressure  and is cSome studies have demonstrated an association between arterial stiffness and cardiac biomarkers including natriuretic peptides –8 and caTherefore, we examined the associations of segment-specific PWV measures with biomarkers of both myocardial stress (natriuretic peptide) and damage (cTnT) in a large cohort of community-dwelling older adults without clinical cardiac disease in the Atherosclerosis Risk in Communities (ARIC) Study.https://www2.cscc.unc.edu/aric/pubs-policies-and-forms-pg. ARIC data can be also obtained from the NHLBI BioLINCC repository (https://biolincc.nhlbi.nih.gov/home/).Data availability and detailed policies for requesting ARIC data can be found at S1 Fig).ARIC is a community-based cohort study that recruited 15,792 participants aged 45–64 years from Forsyth County, NC, Jackson, MS, suburbs of Minneapolis, MN, and Washington County, MD at baseline (visit 1) during 1987–1989 . The ARIWe further excluded 14 non-white/non-black participants and 673 participants with any missing values of covariates. We also excluded 153 participants without N-terminal pro-B-type natriuretic peptide (NT-proBNP) values and 2 participants without high-sensitivity cTnT (hs-cTnT) values. Finally, we excluded 879 participants without any PWV measures; 494 participants with clinical conditions that impair the quality of the PWV measurement such as body mass index >40 or missing (n = 191), severe arrhythmia like atrial fibrillation at visit 5 (n = 166), self-reported aortic surgery (n = 55), history of peripheral revascularization (n = 25), aortic aneurysm (n = 3), aortic stenosis and aortic regurgitation (n = 48), and LV ejection fraction <30% (n = 6); and 255 participants with any of PWV measures deviating 3 standard deviations from their respective mean. The final analytical sample included 3,348 participants.PWV was defined as the distance between two arterial sites divided by the time the wave transmits that distance, and its higher values indicate greater arterial stiffness . Using aBlood samples were drawn at visit 5 and laboratory tests were performed according to a common protocol by trained technicians at each of the ARIC field centers. NT-proBNP as a biomarker of myocardial stress (or cardiac overload)  and hs-c2). Total cholesterol concentration was determined via an enzymatic method [2 [2 for male and >95 g/m2 for female [2 [All variables were collected at visit 5 except education level (high school or lower vs. college or above), which was recorded at visit 1. Age, sex/gender, race, current smoking status and current alcohol habit were self-reported. Body mass index was calculated by dividing body weight (kg) by height squared  and ANOVA for normally distributed data, median  and Kruskal-Wallis test for non-normally distributed data, and frequency (percentage) and Pearson’s chi-squared test for categorical data.To graphically examine the association of each PWV measure with NT-proBNP and hs-cTnT, we visualized the average levels of each cardiac biomarker according to PWV measures adjusting for demographic variables  using linear regression models. To allow for potentially non-linear associations, each PWV measure was modeled with its spline terms (knots placed at the thresholds of its quartiles).Since, indeed, we observed some non-linear relationships (often J- or U-shaped) in several associations, each PWV measure was modeled as quartiles, with the second lowest quartile as a reference. We adjusted for three sets of covariates. Model 1 adjusted for demographic variables . Model 2 further adjusted for other cardiovascular risk factors . Model 3 additionally adjusted for the echocardiographic parameters of LV . NT-proBNP and hs-cTnT were log-transformed in linear regression models and dichotomized based on clinical cutpoints (NT-proBNP ≥300 pg/ml  and hs-cWe further examined whether the associations with NT-proBNP (based on stronger relationship than hs-cTnT as shown below) were modified by age (≥75 vs. <75 years), gender, race (white vs. black), systolic blood pressure (≥140 vs <140 mmHg), diabetes, smoking status (current vs. former), drinking status (current vs. former), kidney damage, or diastolic dysfunction based on a priori hypothesis by performing stratified analysis adjusting for age, sex, race, and study center. We tested for the interaction between cfPWV  or hcPWV (based on its consistent positive association with NT-proBNP) and the subgroups using a likelihood ratio test.All analyses were performed with Stata version 14 , and a P-value <0.05 was considered nominally statistically significant.Table 1). The median values of NT-proBNP and hs-cTnT were 109  pg/mL and 10  ng/L, respectively. Individuals in the higher quartiles of cfPWV were more likely to be older, black, and more educated, and to have comorbidities including hypertension, diabetes, reduced kidney function, and kidney damage. The prevalence of LVH and concentric remodeling were greater with higher cfPWV. Those with higher values of the other PWV measures were consistently older and had higher systolic blood pressure compared to their counterparts with lower values, but showed varying patterns for other factors . Specifically, the prevalence of diabetes, reduced kidney function, and kidney damage were positively correlated with central PWV (hcPWV and hfPWV). In contrast, the prevalence of diabetes and reduced kidney function were inversely associated with peripheral PWV (faPWV) but the prevalence of kidney damage was similar across the quartiles of faPWV. The prevalence of LVH and diastolic dysfunction were also inversely correlated with faPWV, whereas at least one of three cardiac echo parameters showed positive relationships to the other PWV measures.The median age of the 3,348 participants was 74  years, 39.2% were males, and 77.5% were white (S4 Table). Overall, hcPWV showed weak correlations with other PWV measures. There was no evident correlation between faPWV and any of three central stiffness measures.Among the PWV measures, the highest correlation was seen between the two measures reflecting central stiffness, cfPWV and hfPWV (correlation coefficient of 0.841) . faPWV demonstrated an inverse relationship. In the higher values of PWV, the slope was the steepest for hfPWV .For NT-proBNP, the demographic-adjusted associations with central PWV measures were J- or U-shaped . faPWV again demonstrated an inverse relationship with hs-cTnT. In unadjusted models, the associations were more evident especially for cfPWV and hfPWV . faPWV was consistently inversely associated with hs-cTnT.The demographic-adjusted associations of hs-cTnT with PWV were observed to be flatter at each arterial segment (Table 2). The second highest quartiles (Q3) did not reach statistical significance. Further adjusting for traditional cardiovascular risk factors (Model 2) showed that only the top quartile vs. the second lowest quartile of hcPWV remained borderline significant . Peripheral artery stiffness (faPWV), showed an inverse relationship with NT-proBNP in both Model 1 and Model 2. Significantly higher values of NT-proBNP in the lowest quartile were observed in all PWV measures except in hcPWV in Model 2. Additional adjustment for cardiac echocardiographic parameters slightly attenuated the associations, but general patterns remained similar (Model 3).Adjusting for demographic variables (Model 1), the highest quartile (Q4) of PWV measures reflecting central arterial stiffness were statistically significantly associated with higher NT-proBNP values (Table 3). Specifically, statistically significant associations were observed for the highest quartile (Q4) of cfPWV and hfPWV vs. the reference (Q2) in Model 1, adjusting for demographic factors . These associations were no longer statistically significant after adjusting for cardiovascular risk factors (Model 2). The highest quartile (Q4) of hcPWV showed positive but weaker and non-significant associations. faPWV was generally inversely correlated with hs-cTnT but statistical significance was only seen in its lowest quartile in Model 1.Overall the associations of hs-cTnT with PWV measures were less evident (Tables 5). Nonetheless, the general patterns were similar, with positive associations between greater central stiffness measures and NT-proBNP in Model 1. Significantly higher odds of NT-proBNP elevation in the lowest vs. second lowest quartile were seen in some PWV measures, particularly in Model 2 (significant for hcPWV and borderline significant for hfPWV).As anticipated given the limited number of participants above the clinical threshold for each of NT-proBNP (n = 469) and hs-cTnT (n = 958), the results of the logistic regression models were less evident than the linear regression results (Table 5). Higher odds of hs-cTnT elevation were seen in several PWV parameters but statistical significance was only seen in the highest quartile of cfPWV in Model 1.Again, overall less evident associations were seen for hs-cTnT (S5 Table). The association between hcPWV and NT-proBNP was also consistent within categories of the subgroups, without significant interactions (S6 Table).There was no statistically significant effect modification in the association between cfPWV and NT-proBNP (based on Model 1) by age, sex, race, high systolic blood pressure, diabetes, smoking status, drinking status, kidney damage, or diastolic dysfunction  were associated with higher levels of NT-proBNP, independently of demographic characteristics. Although these associations were considerably attenuated once adjusting for traditional cardiovascular risk factors, the relationship between the highest hcPWV quartile and higher levels of NT-proBNP remained borderline significant. faPWV, which is representative of peripheral stiffness, was generally inversely associated with both cardiac biomarkers. Of interest, higher values of NT-proBNP in the lowest quartile than in the second lowest quartile were seen for most PWV measures. Overall, the associations were weaker for hs-cTnT than for NT-proBNP.The present study is one of the first to comprehensively examine central and peripheral PWV measures with both NT-proBNP and hs-cTnT in older adults without prevalent cardiac disease. Our main findings of a positive association between measures of central arterial stiffness and cardiac biomarkers are largely consistent with previous studies , 8; howeWe found that the overall associations between central PWV measures  with NT-proBNP or hs-cTnT were stronger than peripheral arterial stiffness (faPWV). This result is consistent with previous studies that investigating similar associations , 8, 9, aS7 Table). Residual confounding may also contribute to the observed inverse association, as some risk factors were differentially associated with faPWV compared to central PWV measures. Nonetheless, our multivariable models adjusted for a wide range of potential confounders.The inverse associations between faPWV, a measure of peripheral stiffness, and both cardiac biomarkers are of interest. Although this is the first study, to our knowledge, to demonstrate this inverse relationship, two studies similarly reported an inverse association for carotid-radial PWV  among adults without CVD , 8. The In our study, the associations with PWV measures were more evident for NT-proBNP than for hs-cTnT, which is largely consistent with the only previous study assessing both NT-proBNP and hs-cTnT . This obWe found higher values of NT-proBNP in the lowest quartile than in the second lowest quartile for most PWV measures, resulting in overall J- or U-shaped associations. Although the reasons for such associations are unclear and previous studies did not report a similar pattern, a J-shaped association between clinical characteristics and CVD has been shown for various factors, like blood pressure , blood gOur findings may have important clinical and research implications by suggesting the importance of focusing on arterial stiffness measurements incorporating a central artery component. Furthermore, our results of similar or sometimes stronger relationships of hcPWV and hfPWV over cfPWV suggest the potential usefulness of central arterial stiffness measures other than cfPWV. In particular, hcPWV and hfPWV may have some technical advantage over cfPWV, as cfPWV requires probes on both neck and groin, which can be cumbersome to technicians and the subject .In addition to a cross-sectional design described above, there are a few limitations in our study. As with any observational study, we cannot rule out the possibility of residual confounding although we included several important confounders in our models. Also, as our study population includes mainly older white participants and most black participants were from Jackson, MS, generalization of our findings to younger population or other ethnic groups should be done carefully. Moreover, this study population may seem highly selected with participants who have survived and are healthier than those who died. However, given high retention rate in ARIC over 30 years, it seems likely that our study population is less selected as compared to a scenario of establishing a cohort of older adults de novo.In conclusion, among older adults without prevalent cardiac disease, central PWV measures were associated with higher levels of NT-proBNP but less so with hs-cTnT. These findings are consistent with the concept of ventricular-vascular coupling, whereby central rather than peripheral arterial hemodynamics are more directly related to myocardial stress rather than damage. Our study further supports the pathophysiological importance of central arterial stiffness over peripheral arterial stiffness in subclinical cardiac stress.S1 Fig(PDF)Click here for additional data file.S2 FigUnadjusted associations of central (A-C) and peripheral (D) pulse wave velocity (PWV) measures with NT-proBNP.(PDF)Click here for additional data file.S3 FigUnadjusted associations of central (A-C) and peripheral (D) pulse wave velocity (PWV) measures with hs-cTnT.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.


Human malaria is a complex disease that can show a wide array of clinical outcomes, from asymptomatic carriage and chronic infection to acute disease presenting various life-threatening pathologies. The specific outcome of an infection is believed to be determined by a multifactorial interplay between the host and the parasite but with a general trend toward disease attenuation with increasing prior exposure. Therefore, the main burden of malaria in a population can be understood as a function of transmission intensity, which itself is intricately linked to the prevalence of infected hosts and mosquito vectors, the distribution of infection outcomes, and the parasite population diversity. Predicting the long-term impact of malaria intervention measures therefore requires an in-depth understanding of how the parasite causes disease, how this relates to previous exposures, and how different infection pathologies contribute to parasite transmission. Here, we provide a brief overview of recent advances in the molecular epidemiology of clinical malaria and how these might prove to be influential in our fight against this important disease. Plasmodium spp. There are five species known to infect humans:P. falciparum,P. vivax,P. malariae,P. ovale, andP. knowlesi, of whichP. falciparum malaria is the most virulent form, causing the most morbidity and mortality in humans, and thus will be the focus here. Despite continual control efforts over the last 50 years or more, the burden of disease is still substantial, and recent estimates put the annual number of malaria cases in the region of more than 200 million, leading to over 400,000 deaths, predominantly in young children living in sub-Saharan Africa1. The first licensed malaria vaccine  has shown limited efficacy2 and currently is introduced as only a pilot scheme in a few settings in sub-Saharan Africa. The main disease intervention therefore still relies on drug treatment of patients and prevention of exposure by (insecticide-treated) bed nets, spraying of houses with insecticides, and other general mosquito control measures. Following a large scale-up of vector control in combination with artemisinin combination therapy, there has been an overall reduction in the number of malaria cases over the last decade3. However, this reduction does not always correspond well with known control measures4 and shows significant geographic variations. For example, big reductions have been achieved in Southeast Asia and the Western Pacific, whereas several regions in the Americas and Africa have experienced no change or even an increase in cases in recent years5.Malaria is caused by the mosquito-transmitted protozoan parasite8. Predicting the epidemiological outcome of control measures therefore requires a more in-depth knowledge of the factors responsible for severe malaria as well as a better understanding of who is currently infected and who contributes to transmission.The challenges in malaria control are manifold, and, even in regions where a drastic reduction has been achieved and where local elimination is theoretically possible, maintaining a disease-free state without achieving a similar reduction in neighboring regions will be difficult. Underlying these challenges is a lack of understanding of the basic biology of malaria transmission and its relationship to the epidemiological patterns of infection and disease in different transmission settings. In particular, not all infections cause severe clinical symptoms, and most infections contributing to transmission in a given location cause only mild illness or are classified as asymptomatic  that leads, both directly and indirectly, to considerable cell destruction. In addition to anemia as a direct result of RBC loss, splenic clearance of uninfected RBCs, and reduced RBC production, malaria pathology is often caused by parasites sequestering in the deep vasculature, leading to local inflammation, hemorrhages, tissue damage, and obstruction of blood flow. Sequestration itself is the result of infected RBCs (iRBCs) adhering to a number of different host endothelial cell receptors18.Despite its diversity, PfEMP1 plays a central role as a target of naturally acquired immunity (NAI). Over years of repeated infections, individuals living in malaria-endemic areas acquire a repertoire of PfEMP1 variant-specific immune responses through repeated infections that are believed to confer protection from life-threatening disease 20. The fact that this protein appears unusually conserved is now being exploited in the design of the first placental malaria vaccines that are currently undergoing clinical tests22.So far, the high diversity of PfEMP1 has precluded this family of molecules from being considered a serious vaccine target. However, the discovery that certain disease manifestations are associated with the expression of restricted subsets of PfEMP1 variants has opened up the debate of whether an anti-disease or anti-virulence vaccine in fact might be a feasible optionvar genes can be extended to those that are involved in childhood malaria. For example, based on upstream promoter sequence (Ups),var genes can be divided into three groups—UpsA, UpsB, and UpsC—of which UpsA genes are frequently found to be upregulated during severe infection, particularly in young children. Although sequence diversification within this UpsA group of genes appears to be more restricted than others, they are still too diverse as a whole to be considered potential vaccine targets. As such, the recent discovery that a much smaller gene subset, those containing specific domain types called CIDRα1, and their binding to the endothelial protein C receptor (EPCR) appeared to be associated with cerebral malaria caused great excitement26. In fact, confidence in the importance of this interaction underlying severe infection outcomes is such that it is now being promoted as a potential anti-disease vaccine target19.This functional subdivision ofet al., who found thatin vitro iRBC binding to endothelial cells is often independent of EPCR and can easily be interrupted under flow conditions27. Approaches to gain a better understanding of the host–parasite interaction in capillaries include (1) improved understanding of the parasites actually responsible for pathology through direct sampling of sequestered parasites in different tissues by either using skin biopsies in patients28 or sampling from tissues post-mortem29; (2) improved assessment of parasite sequestration in tissues through direct observation of parasites within capillaries through mucosal surfaces to correlate capillary congestion with disease outcome30; (3) seeking associations between peripheral parasite gene expression levels and direct measures of sequestration through malarial retinopathy31; and (4) improved understanding of the role of NAI in shaping the infecting parasite population by seeking associations between peripheral parasite gene expression levels and pre-existing antibody responses in controlled human infections of volunteers with differing levels of natural exposure to infection32.However, the crux of the problem is that, in most cases, findings are based on observed associations between pathological outcomes and the proportional expression of gene variants within the infecting parasite population sampled from peripheral blood rather than directly from parasites that are sequestered in tissues. The issues with these kinds of studies are further compounded by the enormous technical challenge of fully taking into account individuals’ exposure histories and therefore their immune status at the time of infection. This means that caution has to be exercised when trying to infer causality, which was recently re-emphasized by Azasi33. Paradoxically, this appears to be the case regardless of transmission setting or exposure/immunity, and a higher fraction of submicroscopic infections occur in low-transmission settings. Indeed, it has been estimated that in areas with less than 10% prevalence by PCR, 88% of infections would not be detected by microscopy33. Recent ultrasensitive PCR techniques34 have lowered this detection limit even further to 22 parasites per milliliter, which has led to researchers confirming the substantial reservoir of low-density infections. In the absence of molecular methods for routine surveillance, understanding the relationship between clinical cases (which form the basis of surveillance in most endemic countries) and overall prevalence of infection remains a key challenge.As the outcome of an infection is partially determined by an individual’s exposure history to the parasite, elucidating the clinical epidemiology of malaria requires an understanding of a region’s  transmission intensity and therefore knowledge of who is currently infected, who contributes to transmission, and how much. However, measuring disease prevalence and relating this prevalence to transmission remain important challenges that can be severely hampered by the relatively high proportion of clinically silent and low-parasite density infections, especially in highly endemic settings. With the improvement of molecular methods for parasite detection, it has become increasingly clear that microscopy—still the gold standard for diagnosis in many places—systematically misses a large number of low-density infections. Microscopy detects parasite densities in the blood of greater than about 100 parasites per microliter, a detection threshold similar to that of rapid diagnostic tests, but misses an average of half of all malaria infections compared with standard polymerase chain reaction (PCR)35. One result of these findings is a move toward the concept of using the molecular force of infection (molFOI), which measures the number of new genotypes acquired by individuals over time36, to define transmission settings. An added value of these highly sensitive diagnostic tools is that they allow tracking of the genetic relatedness between parasites or infections and, with it, the identification of transmission chains and focal transmission areas. The latter will be particularly important for regions that are nearing malaria elimination and where the monitoring and characterization of residual transmission will be key for sustained malaria control.The prevalence of infection is not the only consideration for defining transmission, however. Many infections are composed of multiple parasite clones, and new infections often occur and cause new episodes of disease against the backdrop of ongoing asymptomatic parasitemia. This means that even with accurate estimates of the fraction of infected people, we are still unable to describe the incidence or force of infection, which is related to the number of new infections over time. The enormous genomic diversity of the parasite, coupled with the frequency of low-density infections, makes it difficult to detect how many clones each infection is composed of, and there are few robust strain markers with which to follow chains of transmission. Recently, new sequencing methods and accompanying analytical tools have shed light on the extent of superinfection, revealing substantial “complexity of infection”, particularly in high-transmission settings like Uganda where individuals can harbor up to 20 clones37. Furthermore, early notions that directly relate asexual parasitemia with infectivity, which implied that young individuals suffering from severe disease are by far the highest contributor to malaria transmission, have also been put into question by revealing that asymptomatic infections contribute significantly more to transmission than previously thought40. For example, a recent study in Ethiopia used mosquito blood-feeding experiments to establish that only 15% ofP. falciparum-infected individuals were infectious and that asexual parasitemia was not correlated with infectiousness40. Earlier findings that gametocytes are not homogeneously distributed within the blood and may cluster under the skin to promote transmission42, plus the considerable uncertainties associated with the determinants of parasite fitness in the mosquito as it transitions from sexual gametocytes to infectious sporozoites, mean that there are still some outstanding difficulties in quantitatively linking standard measures of prevalence with transmission intensity.The surveillance issues described above are all designed to measure asexual parasites in the blood, which cause the clinical manifestations of malaria but cannot be transmitted to mosquitoes. This means that quantifying the infectious reservoir requires different approaches. Only a small fraction of blood-stage parasites develop into male and female gametocytes, the sexual parasite stages that are taken up during a blood meal and underlie infectiousness. The molecular pathways responsible for the switch to sexual development and the dynamics of gametocytes in the body, as well as their relationship to transmission, are still mysterious44 and that host immune responses have an influence on what variants are expressed during infection45. However, this alone cannot explain why an infection causes cerebral malaria in one child but severe malarial anemia in another. Furthermore, the observed hierarchical expression ofvar genes may be due simply to the existence of alternative molecular strategies used by the parasites to evade immune responses in individuals of different levels of immunity. There are reported examples of asymptomatic infections that exhibit high levels of expression of group A-likevar genes previously found to be associated with severe malaria, which suggests that the PfEMP1 antigens they encode can play a role in the maintenance of chronic infections46. At this point, more integrated  approaches should be able to offer more detailed information about the specific immunological and parasitological processes involved in the progression toward disease, especially when taking into consideration the composition of the infecting parasite population in relation to the host’s immune history  a clear definition of what constitutes protection and (2) robust and measurable correlates of protection, neither of which are straightforward. As mentioned above, NAI has to be considered as a multi-stage process or even a continuum whereby infection severity generally attenuates with cumulative exposure to infection. Numerous studies have tried to find correlates of protection by means of prospective cohort studies in which individuals’ immune responses to predefined panels of antigens are correlated with the incidence of clinical episodes. One of the main problems with these studies is the often small effect size, leading to contradictory findings and poor reproducibility (reviewed in50. In another study, Helbet al. used a predictive framework based on machine learning to estimate recent exposure to the malaria parasite51. However, these powerful methods crucially rely on detailed and robust datasets that permit appropriate cross-validation and verification of research findings. One important step forward in that direction is the use ofensemble datasets across a wide range of studies, as was recently advocated in order to better define the infectious reservoir and measure transmission more accurately52.In that respect, it is also imperative to embrace more sophisticated methods to analyze increasingly complex datasets. Machine learning approaches offer a number of advantages over more traditional, univariate analyses in their ability to extract non-linear relationships and interactions from high-dimensional data in a hypothesis-free manner. For example, in a recent study, we used a machine learning approach to identify predictive signatures of clinical protection from protein microarray data containing thousands of measured immune markers53, together with more detailed investigations into the behavioral and ecological factors underlying this part of the transmission cycle, will have a central role to play in developing a fine-grained and holistic understanding of malaria epidemiology that incorporates thePlasmodium parasite, the human host, and the mosquito vector.Finally, a more improved understanding of the biology of mosquito–human and human–mosquito transmission needs to include better knowledge of local vector ecologies. Surprisingly, we still know relatively little about how changes in mosquito abundance and species distributions over the last few years and decades—some of the most important determinants of malaria epidemiology—might have not only influenced but actively shaped some of the observed changes in malaria incidence. Unfortunately, detailed and long-term surveillance data on vector distribution are scarce and are available for only a small number of vector species and epidemiological settings. Therefore, large-scale vector sequencing initiatives, such as the malariaGEN 1000 genomes project


Asymptomatic malaria parasites are significant sources of infections for onward malaria transmission. Conventional tools for malaria diagnosis such as microscopy and rapid diagnostic test kits (RDT) have relatively low sensitivity, hence the need for alternative tools for active screening of such low-density infections.var acidic terminal sequence-based (varATS) quantitative polymerase chain reaction (qPCR) for screening asymptomatic Plasmodium falciparum infections among dwellers of a sub-urban community in Lagos, Nigeria. Clinically healthy participants were screened for malaria using microscopy, RDT and varATS qPCR techniques. Participants were stratified into three age groups: 1–5, 6–14 and > 14 years old.This study tested varATS qPCR. Participants aged 6–14 years had the highest prevalence of asymptomatic malaria, with geometric means of ~ 116 parasites/µL and ~ 6689 parasites/µL as detected by microscopy and varATS, respectively.Of the 316 participants screened for asymptomatic malaria infection, 78 (24.68%) were positive by microscopy, 99 (31.33%) were positive by RDT and 112 (35.44%) by varATS detecting additional sub-microscopic infections. The highest concentration of asymptomatic malaria was observed among school-age children between 6 and 14 years old. A large-scale screening to identify other potential hotspots of asymptomatic parasites in the country is recommended.This study has revealed high prevalence of asymptomatic malaria in the study population, with  Malaria remains a major cause of morbidity and mortality in sub-Saharan Africa. The prevalence of malaria has remained static in recent years unlike the success recorded in early 2000s . ConsequAlthough there have been some success stories of malaria reduction following rapid diagnostic test kits (RDTs) and microscopy-based diagnosis , 10, botvar gene family present in the sub-telomere of the parasite. Each parasite isolate comprises about 50–150 var genes, which possess acidic terminal sequences (ATS) with well-conserved domains [varATS of the parasite has a high sensitivity with a limit of detection of 0.03 parasites/μL blood [varATS quantitative PCR (varATS qPCR) for diagnosing asymptomatic Plasmodium falciparum infections in the country. This study evaluated the effectiveness of varATS qPCR against conventional microscopy and RDTs for the assessment of asymptomatic P. falciparum carriage among sub-urban settlers in Lagos, Nigeria.A relatively recent technique for the ultra-sensitive detection of low parasitaemia has so far yielded promising outcomes . This pr domains . PCR tarμL blood . While mμL blood –22, therA cross-sectional, community-based study was carried out in July 2018 to screen residents of Bayeku for asymptomatic malaria. Bayeku is a rural community in Ikorodu Local Government, an outskirt of Lagos, Nigeria. Malaria is meso-endemic in Lagos, with peak transmission during the rainy season between April and September of every year , 24. TheP. falciparum; CAT NO: G0141, Access Bio Inc., USA). Thick and thin blood films were prepared on a glass slide for parasite identification and speciation using Giemsa technique. The slides were stained and viewed using 100× oil immersion objective lens of a compound microscope. At least 100 high power fields were examined before a thick smear was reported as negative. For positive slides, parasite species and stages were assessed and parasitaemia (parasite density) was determined by counting only the asexual stages against 300 white blood cells (WBC) and then multiplying by 25, assuming the mean total WBC count of individuals is 7500 cells/μL of blood [Blood samples were tested for malaria parasites using CareStart™ Malaria HRP2 . The var gene acidic terminal sequence (varATS) quantitative PCR was used to detect multi-copy genomic sequences of low-density infections [P. falciparum 3D7 standard control (with medonic read of 3.74 × 106 erythrocytes/µL and thin film parasitaemia of 1197 parasites/µL of blood). The serial dilution procedure is described in Additional file fections . The pri2) test and P-value < 0.05 was considered statistically significant. Student’s t-test was used for comparison of means. Kappa (K) coefficient was used to test the measure of agreement between RDT and varATS as well as microscopy vs varATS. K values < 0.20, 0.21–0.40, 0.41–0.60, 0.61–0.80, and 0.8–1.0 depicted poor, fair, moderate, good, and very good strengths of agreement, respectively [Participants were stratified into three age categories: 1–5 years, 6–14 years and > 14 years. Data from this study were coded, entered and analysed in GraphPad Prism 8. The difference in parasite densities between age groups was analysed using Chi-square  were positive by at least one of microscopy, RDT and varATS diagnostics. The demographic profile of participants is summarized in Table A total of 316 asymptomatic participants were screened for varATS PCR counts were used to ascertain the prevalence of asymptomatic malaria in the study population. Prevalence of asymptomatic infections was 78 (24.68%) and 99 (31.33%) for microscopy and RDT, respectively, while varATS qPCR detected additional 10% sub-microscopic infections in the population 112 (35.44%). The geometric mean parasite density by varATS was higher in male participants (5409.86/µL)  was higher than microscopy versus varATS (Kappa = 0.67). When the sensitivity and specificity of RDT and microscopy were benchmarked against varATS , benchmarked against varATS in this study, is consistent with the assertion that the detection limit of RDTs is typically around 100 parasites/µL [Analysis of the relationship between RDT and microscopy sensitivity revealed higher numbers of asymptomatic infections detected by RDT relative to microscopy. This challenges existing knowledge, which implies less sensitivity of RDT than microscopy  even thosites/µL . Meanwhisites/µL , 35, an varATS group, however, there was no major difference across age groups. This provides additional evidence of the effectiveness of varATS to detect malaria parasites across all age groups. Older individuals are expected to acquire immunity from several episodes of malaria during the early part of their lives [Microscopy and RDT analyses showed that participants who were positive for asymptomatic malaria were significantly younger than those who tested negative. In the ir lives , 37, henir lives –40. A plvarATS PCR technique has only been developed for P. falciparum diagnosis [varATS, the deployment of the molecular tool in field diagnosis will facilitate malaria elimination.Meanwhile, there could be other factors contributing to the prevalence of sub-microscopic infections that were not described in this study. Treatment inefficacy, for instance, may influence residual carriage of infections , 42. Oyeiagnosis . TherefovarATS detecting additional sub-microscopic infections. The reports suggested that older children were at increased risk of asymptomatic malaria in Bayeku community. A large-scale screening to identify more hotspots of asymptomatic parasite reservoirs in the country is recommended.This study revealed high cases of asymptomatic malaria in the study population, with Additional file 1. Calculation of varATS qPCR standards.Additional file 2. Additional tables.Additional file 3. varATS/microscopy vs varATS/RDT raw values.


Despite recommendation from the World Health Organization that all malaria suspected patients undergo a parasitological confirmation using rapid diagnostic test or light microscopy prior to treatment, health facilities in remote malaria endemic settings sometimes resort to presumptive diagnosis of malaria for clinical management for various reasons. Following observation of this practice, we undertook a cross-sectional study aimed at comparing presumptive diagnosis based on axillary temperature, SD Bioline™ rapid test, and light microscopy as strategies for malaria diagnosis in the coastal region of Mutengene in the South West of Cameroon with the overall goal of supporting improved malaria diagnosis at local levels. Plasmodium sp following standard procedures. The axillary temperature of each participant was also measured. The sensitivity, specificity, and predictive values and their confidence intervals were determined for each of the methods with PCR as the reference. The area under the curve was used to estimate accuracy of diagnostic method and compared between test method using the X2 test with P<0.05 considered significant. Venous blood from 320 participants was used to detect the presence of malaria parasite using SD Bioline™ mRDT and Giemsa stained microscopy or spotted on filter paper for PCR amplification of the 18s rRNA gene ofμl of blood but a sensitivity of 100% for parasite counts above 500 parasites/μl of blood. The predictive values of the positive test were highly comparable between light microscopy  and SD Bioline™ mRDT , P=0.98 with kappa values of 0.898 but lower for presumptive diagnosis , P<0.0001, and kappa value of 0.277. Perfect agreement was observed between SD Bioline™ mRDT and light microscopy (Cohen kappa= 0.924). The overall diagnostic sensitivities of presumptive diagnosis using axillary temperature, light microscopy, and SD Bioline™ were observed to be 74.30% (95%CI: 67.90-80.01), 94.86% (95%CI: 90.99-97.41), and 95.33% (95%CI: 91.57-97.74), respectively, and their respective diagnostic specificities were 53.77% (95%CI: 43.82-63.51), 94.34% (95%CI: 88.09-97.87), and 94.34%(95%CI: 88.09-97.89). SD Bioline™ had a diagnostic sensitivity of 91.80% [95%CI: 81.90-97.28] at a parasitaemia of less than 500 parasites/ Plasmodium ovale in this study area indicate that treatment guidelines may require revision if same trend is proven in several other areas of same ecology. The study showed that SD Bioline™ was as good as light microscopy in the diagnosis of malaria in remote areas of perennial transmission in South West Cameroon. This study equally revealed the limitations of presumptive diagnosis of malaria (as opposed to the use of RDTs or microscopy). Efforts should be made in such areas to promote parasitological confirmation of malaria using quality assured rapid tests or light microscopy for case management of malaria. The presence of nonnegligible levels of Although global reports show declining trends of malaria since 2010, the world malaria report of 2017 shows that the WHO African region still holds the record of 90% of malaria cases and deaths worldwide  and the diagnostic specificity, the PPV, and NPV for falciparum malaria alone were 95.24%  [95%CI: 89.24-98.44], 97.14%  [95%CI: 93.58-98.77], and 92.59%  [95%CI: 86.38-96.1], respectively. Using light microscopy as the “gold standard” test, the diagnostic sensitivity, specificity, PPV, and NPV of SD Bioline™ mRDT were observed to be 97.62%  [95%CI:94.53-99.22], 95.45%  [95%CI:89.71-98.51], 97.62%  [95%CI:94.57-98.98], and 95.45%  [95%CI:89.82-98.04], respectively.The diagnostic sensitivity for the detection ofHighly sensitive diagnostic methods are important in endemic areas where the febrile illness can become rapidly fatal while high specificity is essential in all settings to reduce unnecessary treatment with antimalarial drugs thereby improving on diagnosis of other febrile illnesses. This study was designed to evaluate the diagnostic accuracy of rapid antigen detection test adopted by the national malaria control program for diagnosis of malaria in Cameroon and to compare this with conventional methods in use. hrp-2/hrp3 such that the parasite no longer produces the antigen or produces a mutant antigen that is not recognized by antibodies on the test strip [ hrp2 gene will still be in circulation. Such samples will test positive by SD Bioline™ RDT but microscopically they will show negative. Some studies have shown that HRP2 antigens could still remain in circulation for as long as 31 days following treatment [ hrp2/hrp3 gene fragment because the target sequence recognized by the oligonucleotide primers is deleted or mutated [In this study, SD Bioline™ mRDT was unable to detect 5 samples microscopically confirmed positive in which parasite DNA was later detected by PCR. Such false negative results produced by SD Bioline™ mRDT could be accounted for by either intraspecies HRP2/HRP3 sequence variation in field isolates , deletiost strip , 15. Samreatment , 21. PCR mutated . Six sam mutated . Such sa paracheck in an area of similar climatic conditions and transmission intensity. The diagnostic specificity of presumptive diagnosis on the contrary was observed to be low implying there will be high chances of treating people who are not sick of malaria . Presumptive treatments based on axillary temperature remain challenging as the World Health Organization strongly recommends confirmation of parasites in body fluids for all suspected cases and treatment on the basis of clinical suspicion only considered when a parasitological diagnosis is not accessible [P>0.05). The two test methods were not very much different from each other in their diagnostic performance with AUC >0.90 and perfect agreement between the test methods. The overall sensitivity of SD Bioline™ was higher than obtained for other forms of rapid diagnostic tests such as the CareStart™ Malaria HRP-2/pLDH, (Pf/pan) Combo, [ OptiMAL and ICT [The results showed that the diagnostic sensitivity of presumptive diagnosis of malaria was higher compared to results obtained by Batwala et al.,  who carrcessible . Despitecessible . This co) Combo,  and Opti and ICT . Despite and ICT , the dia Plasmodium species prevalence within the study area showed that most of the infections were due to monoinfection with Plasmodium falciparum (82.7%) and Plasmodium Ovale (5.1%). Plasmodium falciparum still remain economically the most important malaria causing parasite within the study area. The results are similar to those obtained in a study carried out by Bigoga et al. [ falciparum malaria. Contrary to results obtained by Cho-Fru et al. [ Plasmodium vivax cases in the same study area, no cases of vivax malaria were observed. The results differ from those obtained by Achonduh et al. [ Plasmodium malariae was observed compared to Plasmodium Ovale. This shows an inverse relationship between the prevalence of ovale and malariae in the Guinea savanna region of Bangolan and the littoral forest region of Mutengene. While mixed infections of Plasmodium falciparum and malariae were the most common cause of malaria in Bangolan, monoinfection with Plasmodium falciparum was the main cause of malaria in the littoral forest region of Mutengene. The presence of Plasmodium Ovale in the study area necessitates the review of the current treatment protocol to cater for relapses common with Plasmodium ovale and Plasmodium vivax infections. The world health organization in its 2015 guidelines for malaria treatment recommends the addition of primaquine in the treatment protocol for patients with ovale and vivax infections so as to prevent relapses from hypnozoites, a phenomenon common with these species of Plasmodium [ Plasmodium ovale in the study area necessitates the inclusion of primaquine in treating non-falciparum malaria. Plasmodium malariae was found only coinfecting with falciparum malaria. Although malariae alone is a mild infection and never life-threatening, coinfecting with falciparum greatly changes the manifestation dynamics through nonspecific and cross-specific responses [Molecular analysis ofa et al.  in Tiko u et al.  that reph et al.  in Bangoasmodium . In suchesponses  that canesponses . Plasmodium ovale in this study area requires a review of the current treatment protocol to cater for relapses.The study showed that SD Bioline™ can be an addition to or alternative to light microscopy in the diagnosis of malaria in the South West Region of Cameroon. With a diagnostic sensitivity above 90% as required for confident diagnosis of malaria, SD Bioline™ could replace light microscopy in remote areas with difficult access to reagents and electricity. The study equally highlighted the inefficiency of presumptive diagnosis with sensitivity far below the recommendations for confident diagnosis of malaria. Efforts should therefore be made to replace such practices with parasitological confirmation of the parasite before ACT therapy. The presence of


Plasmodium species infections is critical for a country with an elimination agenda. Namibia is targeting malaria elimination by 2020. To support decision making regarding targeted intervention, we examined for the first time, the foci of Plasmodium species infections and regional prevalence in northern Namibia, using nested and quantitative polymerase chain reaction (PCR) methods.Knowledge of the foci of Plasmodium species.We used cross-sectional multi-staged sampling to select 952 children below 9 years old from schools and clinics in seven districts in northern Namibia, to assess the presence of Plasmodium infection were asymptomatic (63.4%), presenting a challenge for elimination. The respective parasite prevalence for Plasmodium falciparum (Pf), Plasmodium vivax (Pv) and Plasmodium ovale curtisi (Po) were ; with Kavango East and West  and Ohangwena (4.5%) having the most prevalence. Pv was localized in Ohangwena, Omusati and Oshana, while Po was found in Kavango. All children with Pv/Pf coinfections in Ohangwena, had previously visited Angola, affirming that perennial migrations are risks for importation of Plasmodium species. The mean hemoglobin was lower in those with Plasmodium infection compared to those without  indicating that quasi-endemicity exists in the low transmission setting.The median participant age was 6 years (25–75%ile 4–8 y). Participants had a median hemoglobin of 12.0 g/dL (25–75%ile 11.1–12.7 g/dL), although 21% of the cohort was anemic, with anemia being severer in the younger population (p<0.002). Most of children with Pv and Po species are present in northern Namibia. Additionally, the higher number of asymptomatic infections present challenges to the efforts at elimination for the country. Careful planning, coordination with neighboring Angola and execution of targeted active intervention, will be required for a successful elimination agenda.We conclude that  Plasmodium falciparum as the primary parasite species. Foci of transmission is found in the northern border with Angola and Zambia, which also carries the highest population density. Recently as part of the elimination efforts to predict areas likely to have rebound epidemics, three regions Ohangwena, Kavango and Zambezi were identified. In order to affirm these findings and decision-making process for intervention, we assessed the parasite prevalence in 7 northern regional sites for four Plasmodium species. We identified Pv and Po curtisi parasites in Omusati, Ohangwena and Kavango, as well as a significant number of asymptomatic Pf and Pv infections, part of which may be due to importation from neighboring Angola. As Namibia is targeting elimination by 2020, careful thought and planning will be required to reach the goal.Namibia is a member of the SADC elimination 8 (E8) group with a target to eliminate malaria by 2020. This target stems from years of aggressive interventional strategies that has led to significant reductions in morbidity and mortality. The focus of this strategy is mainly on  Plasmodium species infections with sustained surveillance efforts, for targeted intervention and prevention of epidemic outbreaks , 3. This meutbreaks , 8], ..PlasmodiWHO, World Malaria Report, 2017). It has a population of 2.2 million over an area of 0.83 km2 . F. F17]. Fferences , 20]. S. SPf, Pvferences ,  and positive controls . The qPCR procedure for the abTES Malaria qPCR II Kit was done according to the manufacturer’s instructions.The qPCR assay for Plasmodium infection), continuous covariates were compared using linear regression or the student t-test and categorical variables were compared using logistic regression, the Chi square test, or Fisher’s exact test. For logistic regression analyses, odds ratios (ORs) were provided. For all point estimates, 95% confidence intervals (CIs) were provided. Anemia was defined as Hb < 11.0 g/dL. Statistical significance for all comparisons was set at p<0.05 and adjustment was not done for multiple comparisons in this exploratory study. P-values smaller than 0.001 were reported as p<0.001.Data were entered in an Excel data sheet and STATA v11.2  was used for analysis. Descriptive statistics and appropriate measures of central tendency were provided for relevant demographic covariates. To describe differences between study sub-populations . Participants recruited in Kunene , Ohangwena , and Omusati  were all significantly younger than those participants recruited in Kavango East . Overall, 52.6% were female. Participants had a median hemoglobin of 12.0 g/dL (25–75%ile 11.1–12.7 g/dL); 21% of the cohort was anemic. There were significant differences in mean participant hemoglobin levels between different regions; those from Kunene  and Ohangwena  had lower levels than children from Kavango East.A total of 952 children from 7 districts . were asPlasmodium infections were Pf (n = 41), with 8 Pv infections, 3 Po infections and no Pm infections  were afebrile. All three children with Pv/Pf coinfections and two with Pf infection in Ohangwena, had previously visited Angola. The mean hemoglobin was lower in those with Plasmodium infection as compared to those who did not . There were no differences in the age or gender distributions for those that did and did not have Plasmodium infections. There were clear differences in the proportions of participants infected in the different regions; the highest prevalence was seen in Kavango East (10.4%) and there was a statistically lower prevalence seen in the regions of Kunene , Ohangwena , Omusati , and Oshana . There was no statistical difference between the prevalence of mixed infections in the different regions. Kavango region interestingly, is the most endemic for malaria from the 2015 incidence Map. Pv was localized within Omusati, Ohangwena and Oshana regions, while P ovale was seen in Kavango. The precise number and type of Plasmodium species for each region is presented in Pv and Po accounting for 0.84% and 0.31% respectively. The two species therefore account for 1.1% of the parasite prevalence.The majority of fections . CoinfecPv foci of infection in Northern Namibia encompasses three regions: Omusati, Ohangwena and Oshana, while Po focus is in Kavango. The study affirms Kavango as remaining a focus for Pf infections in northern Namibia, as shown in the incidence map in 2015 and epidemic outbreaks in 2016 . S. SPv foculocytes , so undenfection , will innfection , 27], s, sPv focnfection , 29]. I. IPv focic state  to sustafections . The Pf arasites . The eliPv and Po hypnozoite forms add to the complexity of dealing with asymptomatic infections with their sequestration in the liver and or bone marrow , . C. CPv andPv asymptomatic infections and Po are present in northern Namibia as are asymptomatic infections of Pf. These introduce new paradigms in the elimination agenda for Namibia, that requires careful planning and thought for blocking transmission and aggressive targeting of all populations and species affected.We conclude that S1 Checklist(DOC)Click here for additional data file.