Early and accurate diagnosis of malaria is a critical aspect of efforts to control the disease, and several diagnostic tools are available. Microscopic assessment of a peripheral blood smear enables direct visualization of parasites in infected red blood cells and is the clinical diagnostic gold standard. However, it is subjective and requires a high level of skill. Numerous indirect detection methods are in use, but are not ideal since surrogate markers of infection are measured. This study describes the first clinical performance evaluation of the automated Sysmex XN-30 analyser, which utilizes fluorescence flow cytometry to directly detect and quantitate parasite-infected red blood cells.Plasmodium species were detected, including Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale. The XN-30 also identified the transmissible gametocytes as a separate cluster on the scattergrams. Abnormal red blood cell indices (low haemoglobin and raised reticulocyte counts), haemoglobinopathies and thrombocytopenia did not interfere with the detection of parasites. The XN-30 also generated a concurrent full blood count for each sample.Residual EDTA blood samples from suspected malaria cases referred for routine diagnosis were analysed on the XN-30. Parasitaemia was reported as a percentage, as well as absolute numbers of infected red blood cells, and scattergrams provided a visual image of the parasitized red blood cell clusters. The results reported by the XN-30 correlated with microscopy and the analyser demonstrated 100% sensitivity and specificity. Measurements were reproducible and storage of samples at room temperature did not affect the parameters. Several Plasmodium species makes it suitable for global use and allows monitoring of the parasite load once therapy has been initiated, thereby providing an early marker of drug resistance. The automated generation of a full blood count for each sample provides an opportunity for detecting unsuspected cases. Asymptomatic carriers can also be identified, which will be useful in blood transfusion centres, and will enable treatment of these individuals to prevent the spread of the disease.The novel technology of the Sysmex XN-30 provides a robust, rapid, automated and accurate platform for diagnosing malaria in a clinical setting. The objective enumeration of red blood cells infected with The online version of this article (10.1186/s12936-019-2655-8) contains supplementary material, which is available to authorized users. Malaria remains one of the most important parasitic diseases globally, with more than 3.2 billion people in 91 countries at risk of infection  and submitted to the local hospital laboratory for routine malaria diagnostic testing were utilized. To be eligible for the study, all samples were required to be at least 1 mL in volume and processed on the analyser by a dedicated operator within 24 h of collection from the patient. This study was approved by the Human Research Ethics Committee of the University of the Witwatersrand (M140995 and M160549) and by the National Health and Education Society (872-14-SK/KS), in RSA and India respectively.In RSA, all adult patients presenting with clinical suspicion of malaria either to Chris Hani Baragwanath Academic Hospital or Charlotte Maxeke Johannesburg Academic Hospital were considered for inclusion. In India, only samples with a microscopic diagnosis of malaria, confirmed at the P. D. Hinduja National Hospital laboratory, were included. At both sites residual blood from specimens collected in EDTA tubes [VacutainerSamples were analysed on the prototype, XN-10 (M), and the XN-30 analysers in whole blood (WB) mode. In addition, samples were processed in low malaria (LM) and pre-dilute (PD) modes on the XN-30. In the LM mode the volume of each sample measured is triple (180 µL) that of the WB mode (60 µL), to obtain improved sensitivity. In contrast, the PD mode requires only 20 µL of WB sample diluted in 120 µL of a standard diluent (Cellpack DCL), to enable analysis of small blood volume samples such as in elderly and paediatric patients. The XN-30 is equipped to perform automatic conversion of results using the appropriate dilution factor.P. falciparum or suspected ‘others’ .Both analysers generate results automatically, which are presented as malaria-negative or malaria-positive, accompanied by a conventional FBC, MI-RBC#, MI-RBC%, an M scattergram and a flag with a suggested species classification, either as suspected For the prototype study, thin peripheral blood smears were prepared and evaluated by routine laboratory staff according to standard procedures . For theP. falciparum-specific HRP2 antigen, and the SureTest MAL malaria antigen test kit  Ltd, India), which detects both P. falciparum and P. vivax species were used. For the XN-30 study, the ICT Malaria dual test kit , which detects P. falciparum, P. vivax, Plasmodium malariae and Plasmodium ovale antigens, was employed.All samples were tested for the presence of malarial antigens and results recorded as either positive or negative. For the prototype study, the MAKROmed malaria rapid test kit (Makromed Manufacturing (Pty) Ltd, RSA) which detects the Plasmodium-specific primers according to a standardized protocol adapted from Hang et al. [In RSA, any discordant results between the analysers and other methods (microscopy and/or RDT) were resolved with qualitative PCR assays using g et al. . For the9/L and raised reticulocyte counts > 5% or equivalent to > 100,000 × 106/L. Additionally in the prototype study, samples from patients with HIV infection (RSA), confirmed acute Dengue infection and an eosinophil count > 1 × 109/L (India) were included. For the performance evaluation of the XN-30, samples from patients with known haemoglobinopathies  were also analysed.To exclude potential interference by non-malarial factors, several samples, confirmed by microscopy and RDT to be malaria-negative, were processed in WB mode on both analysers and additionally in LM mode on the XN-30. These comprised random routine specimens with isolated low haemoglobin < 10 g/dL, isolated low platelet count < 100 × 10The XN-30 was assessed for the following additional performance parameters: limit of blank (LoB); limits of detection (LoD) and quantitation (LoQ); carryover; precision and stability.Blank + 1.645 (SDBlank) [To determine the LoB, samples confirmed by microscopy and RDT to be malaria-negative, were processed. These comprised random routine specimens with normal FBC results. The LoB was calculated using the formula LoB = MeanSDBlank) .Low concentration sample) [To determine the LoD and LoQ, malaria-positive samples, confirmed by microscopy and RDT, were analysed and the MI-RBC# confirmed. Each sample was also typed for ABO blood grouping using a standard kit  and then diluted using ABO-compatible malaria-negative blood obtained from volunteers in the RSA research laboratory. Standards comprising various concentrations of parasites were generated for determining the LoQ  and LoD . Each standard was analysed ten times consecutively and the LoD was calculated using the formula LoD = LoB + 1.645  to a malaria-negative sample was assessed by analysing the positive sample three times consecutively followed by three consecutive analyses of the negative sample. These steps were repeated using Cellpack DCL as a blank instead of the negative sample. Carryover was judged to be clinically significant if the first negative sample or the blank sample demonstrated a MI-RBC# result > LoQ.To assess for precision, malaria-positive samples, confirmed by microscopy and RDT, and representing high (> 5%), medium (1–2%) and low (< 1%) parasite densities were selected, each of which was analysed ten times consecutively.To assess the impact of sample age on malaria classification, malaria-positive samples, confirmed by microscopy and RDT, were initially analysed within 24 h of collection from the patient. Each sample was divided into two aliquots, which were stored at 4–8 °C and room temperature (20–25 °C), respectively, and subsequently analysed after approximately 48, 72 and 96 h post sample collection. If there was insufficient sample volume to test both temperatures, preference was given to samples stored at room temperature.P. falciparum parasites were induced in vitro and purified as previously reported [Gametocytes from NF54 reported . Mature 2), means, standard deviations (SD), and coefficients of variation (CV % = SD÷mean × 100) were calculated using Analyse-it for Microsoft Excel version 5.01 . Confidence intervals  for the sensitivity and specificity data were also determined using this software.Correlation between parasitaemia measured by the XN-30 and microscopy was analysed using regression analysis. The coefficient of determination , 261 confirmed malaria-negative samples from patients with an acute febrile illness in whom malaria had been suspected, 278 malaria-negative interference samples, 128 samples with normal FBC values and a further 100 randomly selected FBC samples from the routine laboratory in India.A total of 1028 samples processed on the prototype analyser gave valid results for further analysis. There were 261 confirmed malaria-positive samples  and non-falciparum in 94.8% of cases .The malaria detection of the XN-10 (M) analyser was evidenced by a sensitivity of 97.7%  and a specificity of 98.7% . In eleven smear-negative cases the XN-10 (M) detected malaria, all of which were subsequently confirmed by PCR to be true positive results. Furthermore, there was one “isolated low platelet count” interference sample that appeared to have given a false positive result on the XN-10 (M) but was subsequently confirmed on smear review to be an unsuspected case of true malaria. All other interference samples, as well as all normal samples with interpretable results (refer to next paragraph), were correctly identified as malaria-negative. There was no difference in malaria detection performance based on The biggest shortcoming observed was the high percentage (14.8%) of indeterminate results. If the particle count in the “M” channel exceeded the lower LoQ, but no distinct cluster was observed in the malaria gating area, then the analyser generated an “abnormal MI-RBC scattergram” flag and did not issue a report on the presence of malaria. Indeterminate results were largely associated with malaria-negative samples , with only three of the malaria-positive cases  being affected. Based on these first performance data, the software was improved, resulting in the XN-30 analyser.P. falciparum: n = 122; P. ovale: n = 2) and 67 were confirmed to be malaria-negative by microscopy and RDT, and where necessary also PCR (n = 5). Figure P. falciparum cases. Examples of RBC flags indicating the malaria species as P. falciparum ‘P. f’ or ‘others’ are provided in Additional file A total of 191 samples were processed on the XN-30: 124 were confirmed to be malaria-positive  and a specificity of 100%  for malaria parasite detection.P. falciparum (98% of cases) or “others” (100% of cases).The LM mode was determined to be most accurate for the correct identification of species as either To exclude potential interference by non-malarial factors, malaria-negative samples with isolated low haemoglobin levels (n = 22), isolated low platelet counts n = 24), haemoglobinopathies  and raised reticulocyte counts (n = 25) were analysed. Examples of M scattergrams generated from samples of patients with thalassaemia, sickle cell disease and reticulocytosis are shown in Additional file , haemoglThe LoB (n = 75), LoD (n = 4) and LoQ (n = 3) are shown in Table Carryover was observed in the LM mode in a single instance whereby an MI-RBC# of 21 parasites/μL , and thus yield valuable data on the spread of artemisinin resistance, which is alarmingly prevalent in the Greater Mekong sub-region , 37.Another important advantage of the XN-30 is that, in addition to the unique property of directly identifying parasites in RBCs, it generates a conventional FBC with each result, which none of the other malaria diagnostic tools offer. This provides clinicians with baseline blood counts and enables them to evaluate the haematological response of the patient during treatment. This feature will also be beneficial in clinical research applications, such as population studies and clinical trials to test new anti-malarial drugs or vaccines. This has recently been exploited in a study of a mouse model of malaria whereby the pharmacokinetics of drugs, as well as therapy-related safety profiles, were investigated on the XN-30 . An addiP. falciparum carriage can range from 2% to more than 55% among healthy blood donors [Transfusion-transmitted malaria (TTM) is a well-recognized entity and is fast emerging as a major public health concern, particularly in malaria-endemic African countries. The prevalence of d donors –40. Currd donors . The XN-P. falciparum culture samples. Gametocytes are critical for parasite survival since they are transmitted to the mosquito vector where they initiate the sexual phase of the parasite life cycle, which results in the production of sporozoites rendering the mosquito infectious and allowing the spread of the disease. The XN-30 may be harnessed to identify asymptomatic carriers who can be treated to prevent transmission of gametocytes and therefore disrupt the parasite life cycle. This strategy is a key component of global initiatives to eliminate the disease. Automated detection of gametocytes may also be useful for further research on gametocytogenesis in patients/asymptomatic carriers and for transmission-blocking drug discovery.A final important benefit of the XN-30 is that it can detect gametocytes as demonstrated here and by a Japanese group  using inA limitation of the current XN-30 is the number of indeterminate results obtained due to the reported “abnormal MI-RBC scattergram” flags. Despite this only occurring in malaria-negative samples, with no false positive results being generated, it may potentially impact on routine workflow, as affected samples would require microscopic review. Whilst the exact cause of the “abnormal MI-RBC scattergram” flag in the malaria-negative samples is currently unknown, the disproportionate occurrence in specimens obtained from patients with reticulocytosis, suggests that it may be related to high red cell turnover. This is currently under investigation by the manufacturer.Timely detection of malaria is life-saving and whilst most health care professionals are trained to recognize the infection, clinical presentation is often non-specific, and diagnosis may initially be overlooked. The XN-30 analyser is an ideal modality for the precise recognition and automated enumeration of malaria parasitaemia. It rapidly detects the actual parasite and not any by-product, such as antigens, phagocytosed parasites or haemozoin, making it more suitable for malaria detection compared to RDTs and other indirect automated methods. Concurrent measurement of haematological parameters is a unique feature that provides valuable data for clinical correlation. Whilst it may not immediately replace microscopy for confirmation of clinical malaria infection, the XN-30 should certainly be considered for utilization as an adjunctive diagnostic tool, because it can facilitate the detection of unsuspected cases. Overall, this technological advance has the potential to significantly impact on malaria diagnostic approaches, therapeutic monitoring and efficacy, anti-malarial drug discovery and clinical trials.Additional file 1: Fig. S1. XN-30 software improvements. a. In the prototype software, the M scattergram plot area was divided into 3 regions classifying signals as WBCs (cluster a), non-infected RBCs, platelets and debris (cluster b) and MI-RBCs (cluster c). In brief, the gating strategy sequence was to first identify cluster a and then cluster b. In the prototype, all remaining signals were then assigned to cluster c. Upon further investigation, it was identified that the specified MI-RBC area (cluster c) was too broad, giving rise to false positive MI-RBC results. b. The software was subsequently improved for the XN-30 by narrowing the MI-RBC area (cluster c) and incorporating the recognition of one or more distinct clusters  within an appropriate shape and position as a prerequisite for generating an MI-RBC result. Fig. S2. XN-30 malaria classification algorithm. The left side illustrates the algorithm flow if a malaria cluster is not recognized. In such cases, if the MI-RBC# is < LoQ, the sample is reported as malaria negative (MI-RBC green box). However, if the MI-RBC# is ≥ LoQ but signals are detected in the absence of appropriate clustering, or an incorrect cluster shape is generated within the MI-RBC area, then the result is suppressed and an MI-RBC abnormal scattergram flag is generated. An indeterminate result is reported (MI-RBC grey box). An example is presented in M scattergram (a). The right side illustrates the algorithm flow if a malaria cluster is recognized. In such cases, if the MI-RBC# is < LoQ, the sample is reported as malaria negative (MI-RBC green box). However, if the MI-RBC# is ≥ LoQ the sample is reported as malaria positive (MI-RBC red box). An example is presented in M scattergram (b). Fig. S3. XN-30 M scattergrams, MI-RBC values and the species RBC flags for patient samples infected with aP. falciparum (P. f) and bP. ovale (others). Measurements were performed in WB mode. P. ovale parasites were confirmed microscopically. SFL: side fluorescence light; FSC: forward scattered light; blue dots: non-infected RBCs, platelets and debris; red dots: MI-RBCs; green and orange dots: different P. ovale life cycle forms; turquoise dots: white blood cells. Fig. S4. Correlation between P. falciparum parasitaemia obtained from the XN-30 (MI-RBC%) and expert microscopy. Samples were analysed in a LM mode and b PD mode. R2 indicates the coefficient of determination. The diagonal lines represent the regression lines. Fig. S5. XN-30 M scattergrams of malaria-negative patient samples with a thalassaemia, b sickle cell disease and c reticulocytosis of 13.86%. The grey area within the large blue cluster reflects the region where MI-RBCs scatter, but the software correctly triggered an “abnormal MI-RBC scattergram”. Measurements were performed in WB mode. SFL: side fluorescence light; FSC: forward scattered light; blue dots: non-infected RBCs, platelets and debris; turquoise dots: white blood cells. Fig. S6. Stability of MI-RBC parameters (MI-RBC#) at 4–8 °C measured in WB mode on the XN-30. Four samples with P. falciparum parasitaemia ranging from a high (21–107 × 103/µL), to b low (3–4.5 × 103/µL), were stored at 4–8 °C and analysed at various time intervals. The first measurement was performed within 24 h of sample collection from the patient and thus the initial point is different for each sample.Additional file 2: Table S1. WB mode data and M scattergrams. Table S2. LM mode data and M scattergrams. Table S3. All modes precision data.


Although dietary α-linolenic acid (ALA) or linolenic acid (LA) intake was reported to be epidemiologically associated with a lower prevalence of hypertension, recent clinical trials have yielded conflicting results. Comparable experimental evidence for the roles of these two different fatty acids is still lacking and the underlying mechanisms need to be further elucidated. Our data showed that ALA but not LA supplementation alleviated systolic blood pressure elevation and improved ACh-induced, endothelium-dependent vasodilation in both spontaneously hypertensive rats (SHRs) and AngII-induced hypertensive mice. In addition, SHRs displayed reduced vascular Sirtuin 3 (SIRT3) expression, subsequent superoxide dismutase 2 (SOD2) hyperacetylation and mitochondrial ROS overproduction, all of which were ameliorated by ALA but not LA supplementation. In primary cultured endothelial cells, ALA treatment directly inhibited SIRT3 reduction, SOD2 hyperacetylation, mitochondrial ROS overproduction and alleviated autophagic flux impairment induced by AngII plus TNFα treatment. However, these beneficial effects of ALA were completely blocked by silencing SIRT3. Restoration of autophagic flux by rapamycin also inhibited mitochondrial ROS overproduction in endothelial cells exposed to AngII plus TNFα. More interestingly, SIRT3 KO mice developed severe hypertension in response to a low dose of AngII infusion, while ALA supplementation lost its anti-hypertensive and endothelium-protective effects on these mice. Our findings suggest that ALA but not LA supplementation improves endothelial dysfunction and diminishes experimental hypertension by rescuing SIRT3 impairment to restore autophagic flux and mitochondrial redox balance in endothelial cells. Schematic diagram of the mechanisms whereby α-linolenic acid protects against endothelial dysfunction and hypertension. Considering its high prevalence and consequent global disease burden, preventive strategies from a public health perspective are urgently needed, and nutritional interventions have been advanced to fight hypertension epidemic2.As the leading risk factor for cardiovascular disease, hypertension has become the most prevalent chronic disease, affecting more than 30% of adults aged ≥25 years worldwide4. Dietary ALA intake was reported to be epidemiologically associated with a lower prevalence of hypertension, and ALA has been indicated as a promising alternative addition to available lifestyle medications for the prevention of cardiovascular diseases6. In contrast to the recognized benefits of n-3 PUFAs, diets enriched with omega-6 (n-6) PUFAs have traditionally been viewed as detrimental primarily because they are precursors for pro-inflammatory eicosanoids7. However, a recent epidemiological study reported that higher plasma levels of n-6 PUFA linolenic acid (LA) were significantly associated with a lower prevalence of hypertension8. Therefore, comparative data and strong experimental evidence are urged to draw a conclusion about the effects of both dietary n-3 and n-6 PUFAs on hypertension.As a plant-derived omega-3 polyunsaturated fatty acid (n-3 PUFA), α-linolenic acid (ALA) is abundant in nuts, leafy vegetables and plant seed oils, such as rapeseed, soyabean and flaxseed oils+-dependent deacetylases and ADP-ribosyltransferases, among which Sirtuin 3–5 (SIRT3-5) are located in the mitochondria. Mitochondrial Sirtuins regulate numerous aspects of mitochondrial biology, including metabolic homeostasis, redox balance and mitochondrial dynamics9. The associations between mitochondrial Sirtuins impairment and cardiovascular disease have been well established by many preclinical studies. In particular, SIRT3 impairment and the resultant SOD2 hyperacetylation have been demonstrated to induce vascular oxidative stress and contribute to the development of hypertension10. SIRT3 has also been revealed to regulate the autophagy-lysosome pathway and autophagic flux is implicated in the management of nitric oxide (NO) bioavailability and endothelial function12. Although the anti-oxidative property of ALA has been reported previously, whether SIRT3 plays an important role in the benefits of ALA remain unknown.The Sirtuins are a family of NADGiven the increasing evidence suggesting ALA or LA as a nutritional supplement with cardiovascular-protecting potential, the present study was designed to comparatively investigate the effects of ALA and LA supplementation against hypertension and the underlying molecular mechanisms.MitoSOX Red  and MitoTracker Green  probes were supplied by Invitrogen . SIRT3 (ab118334), acetyl-K68-SOD2 (ab137037), SOD2 (ab68155), p62 (207305), LC3B (ab63817) and β-actin (ab6276) antibodies were obtained from Abcam . All other chemicals and reagents were obtained from Sigma .14, were obtained from Research Diets . The animals were randomly divided into four groups with different diets for 8 weeks using a completely randomized design: (1) WKY: the normotensive WKYs fed with the control diet; (2) SHR: the SHRs fed with the control diet; (3) SHR + LA: the SHRs fed with LA-supplemented diet; (4) SHR + ALA: the SHRs fed with ALA-supplemented diet. Food consumption of the SHRs fed with the control diet was firstly recorded by weight, and these data were used to pairfeed animals on the LA-supplemented diet and ALA-supplemented diet to maintain caloric intake. Diet fatty acid composition was confirmed by gas chromatography.All procedures involving animals were performed according to the National Institutes of Health Guidelines for the Use of Laboratory Animals, and were approved by The Fourth Military Medical University Committee on Animal Care. Four-week-old male spontaneously hypertensive rats (SHRs) and age-matched normotensive Wistar-Kyoto (WKY) control rats were purchased from Vital River Laboratory . The control diet , LA-supplemented diet  and ALA-supplemented diet , as reported previously10. SIRT3 knock-out (SIRT3KO) and wild-type (WT) mice were obtained from Jackson Laboratory , and hypertension was also induced by AngII infusion .For angiotensin (Ang) II-induced hypertensive model, male eight-week-old C57BL/6J mice were fed with the ALA-supplemented diet or control diet for 4 weeks, and then randomized to receive a low-suppressor dose of AngII  or vehicle via an osmotic minipump  as described previouslySystolic blood pressure was measured in conscious animals by tail-cuff system  to monitor the progression of hypertension. At the end of the experiments, systolic blood pressure was assessed by inserting a heparinized saline-filled catheter into the left carotid artery after an initial 15-min equilibration period in anaesthetized animals. Blood pressure was measured between 9:00 a.m. and 11:00 a.m.Blood samples were obtained from rats after overnight fasting. Fasting blood glucose and insulin levels were respectively measured by a blood glucose meter (Lifescan) and an ELISA test kit . Plasma NO was evaluated by the NO fluorometric assay  according to the manufacturer’s instructions.15.For fatty acid analysis, 250 μL aliquots of serum from different group animals were analyzed and quantified by GC-MS (QP 2010 ultra) as described previously16. Experiments were performed in a horizontal wire myograph  containing PSS. The contractile force was recorded using a PowerLab Chart v7.2.1 program . After a 60-min equilibration period, one dose of physiological saline solution containing 60 mM KCl (KPSS) was administered to verify vessel viability. Aortic rings were precontracted with phenylephrine . Endothelium-dependent vasorelaxation evoked by acetylcholine  and endothelium-independent vasorelaxation evoked by cumulative sodium nitroprusside  were expressed as percent contraction determined by the percentage of inhibition to the precontracted tension.Mice were sacrificed and the descending aorta was carefully excised and placed in ice-cold physiological saline solution (PSS) as described previously−5 M, Sigma, USA).In another experiment, ACh-induced, endothelium-dependent vasorelaxation and SNP-induced, endothelium-independent vasorelaxation in 2 mm mesenteric artery rings isolated from WKYs and SHRs fed with control diet, LA-supplemented diet or ALA-supplemented diet were measured after precontracted with PE  were purchased from Lonza  and cultured in EGM-2 medium supplemented with 2% FBS without antibiotics. The cell line has been routinely checked for mycoplasma contamination with the MycoProbe detection kit . Only cells negative for mycoplasma contamination were used.2•− was measured as accumulation of mitochondrial 2-hydroxyethidium in MitoSOX supplemented samples as described previously10. Three 2-mm aortic rings from each animal are incubated in 2 μM MitoSOX for 30 min at 37 °C. Next, buffer was aspirated and the tissue is then homogenized in 300 μL of methanol, and 50 μL of homogenate are used for protein determination. The remainder of the sample is passed through a 0.22-μm syringe filter and then used for HPLC analysis. MitoSOX and its oxidation products, 2-hydroxyethidium and ethidium, were separated using a C-18 reverse-phase column (Nucleosil 250 to 4.5 mm) and a mobile phase containing 0.1% trifluoroacetic acid and an acetonitrile gradient (from 37 to 47%) at a flow rate of 0.5 ml/min. Ethidium and 2-hydroxyethidium were detected with a fluorescence detector using an emission wavelength of 580 nm and an excitation of 480 nm. The mitochondrial 2-hydroxyethidium peak reflects the amount of mitochondrial O2•− formed in the tissue during the incubation and is expressed per milligram of protein. H2O2 was measured by Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit (Thermo Fisher A22188) according to the manufacturer’s instructions.Stock solutions of MitoSOX  in DMSO were prepared and were diluted in Kreb-Hepes buffer to a final concentration of 2 μM MitoSOX. Production of mitochondrial OImmunofluorescent staining of mitochondrial ROS was also performed on aortic frozen sections from WKYs and SHRs fed with different diets. Aortic frozen sections were firstly permeabilized with 0.4% Triton X-100 in PBS for 10 min, and then blocked with 10% goat serum in PBST containing 0.1% Tween-20 for 1 h at room temperature. Aortas were incubated with rabbit anti-CD31  primary antibody overnight at 4 °C, followed by incubation of MitoSOX at the concentration of 10 µM for 20 min. Images were acquired using a Zeiss (LSM 800) confocal microscope.2•− was visualized in intact cultured HAECs using the fluorescent probe MitoSOX. HAECs were incubated with 10 μM MitoSOX in Kreb-Hepes buffer for 20 min at 37 °C in a CO2 incubator. The mitochondrial subcellular location of MitoSOX was confirmed by co-labeling with 50 nM MitoTracker Green FM .In vitro, production of mitochondrial OAutophagy was visualized in HAECs by transfection of the lentivirus expressing tandem mCherry-GFP-LC3 . GFP fluorescence is quenched in the acidic pH of the lysosomal compartment, thus limiting the application of GFP-LC3 to the identification of autophagosomes. However, mCherry continues to fluoresce, and mCherry-GFP can be applied to the identification of both autophagosomes and autolysosomes. Quantification of autophagosomes can be achieved by using tf-LC3 and determining the number of red dots that overlay green dots and appear yellow in merged images. The red dots which do not overlay green dots and appear red in merged images indicate autolysosome formation. After infection of tf-LC3 lentivirus for 24 h, endothelial cells were subjected to the corresponding treatment and then imaged for GFP and RFP by using a Zeiss (LSM 800) confocal microscope.The siRNA-mediated SIRT3 knockdown was performed by transfecting synthetic siRNA duplexes  at a concentration of 20 nM with Lipofectamine RNAiMAX regent  according to the protocols provided by the manufacturer. The siRNA sequences are as follows: human SIRT3, 1. sense 5′-CCAGCAUGAAAUACAUUUATT-3′, anti-sense 5′-UAAAUGUAUUUCAUGCUGGTT-3′. 2. sense 5′-CCAGUGGCAUUCCAGACUUTT-3′, anti-sense 5′-AAGUCUGGAAUGCCACUGGTT-3′. Scarmbled siRNA, sense 5′-UUCUCCGAACGUGUCACGUTT-3′, anti-sense 5′-ACGUGACACGUUCGGAGAATT-3′. The transfected cells were cultured for 48 h and used for further experiments.t test (two groups) or ANOVA (three or more groups), followed by Bonferroni’s correction if needed, were performed to determine statistical significance between different treatment groups. The data of blood pressure over the time course were statistically analyzed by repeated measures analysis of variance. P levels of <0.05 were considered significant.Data were presented as mean ± standard error of the mean (n noted in specific figure legends). The sample size was determined based on previous studies with similar experimental design and on the known variability of the assay. Analysis of the immunofluorescence-related data was performed in a blinded way. Normality of data distribution was assessed by Shapiro-Wilk normality test. An unpaired, two-tailed Student In order to clarify the potential beneficial effects of diets enriched with n-3 or n-6 PUFAs on SHRs, four-week-old male SHRs were respectively fed with the control diet, LA-supplemented diet or ALA-supplemented diet for 8 weeks. There was no significant difference in body weight among various groups of SHRs  or vehicle via an osmotic minipump. Infusion of wild-type C57Bl/6J mice with a low dose of AngII led to a mild but significant increase in SBP to approximately 140 mmHg  by western blot in aortas isolated from SHRs fed different diets. Mitochondrial O2•− and H2O2 levels were respectively detected by MitoSOX/HPLC and Amplex red. SHRs exerted reduced vascular SIRT3 expression  bioactivity, due to oxidative inactivation of this radical gas. Indeed, we found that vascular NO level was significantly decreased in SHRs, which was largely rescued by ALA but not LA supplementation Fig. . Taken t19. Previous studies have indicated that AngII and TNFα are commonly encountered in hypertension10. Indeed, AngII plus TNFα administration reduced SIRT3 expression  were exposed to AngII  plus TNFα . ALA dosage  was chosen in accordance with the plasma levels achieved upon dietary ALA intake with reference to a previous studyion Fig. , increasion Fig.  and enhaion Fig.  in endotion Fig. . These d20. The autophagy markers LC3-II and p62 expression  infusion, while AngII  infusion increased SBP of WT mice to 143 mmHg as measured by carotid artery catheterization method Fig. . InteresOur findings suggest that ALA but not LA supplementation rescues SIRT3 reduction, improves endothelial dysfunction and reduces blood pressure elevation in hypertensive animals, thus providing the proof of principle for the benefits of dietary ALA intake against hypertension. SIRT3 plays an important role in the anti-hypertensive effects of ALA by alleviating autophagic flux impairment and inhibiting mitochondrial ROS overproduction in endothelial cells.21. The three most commonly consumed n-3 PUFAs are ALA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Increased consumption of marine-derived EPA and DHA exerts anti-arrhythmic, lipid-lowering, antihypertensive and anti-thrombotic effects24. However, the availability of EPA and DHA remains restricted in many countries due to unfavorable geography, expensive supply and cultural preference. Therefore, ALA, a plant-derived n-3 fatty acid, may provide an ideal cardioprotective alternative to marine-derived n-3 fatty acid. Given that ALA can be catabolized into EPA and DHA by the body after absorption, it is controversial whether the cardiovascular effects upon ALA dietary intake reflect the activity of ALA or the activity of its longer-chain n-3 fatty acid derivatives. Although biochemical pathways exist to convert ALA to EPA and DHA, such endogenous conversion is limited in humans: between 0.2 and 8% of ALA is converted to EPA and 0 to 4% of ALA to DHA27. In the PREDIMED study, dietary intake of ALA still conferred protection against cardiovascular disease and all-cause mortality even in a background of high intake of marine-derived long-chain n-3 PUFAs28. Our in vitro experiments also indicated that ALA exerted direct antioxidant effect and restored autophagic flux in endothelial cells, which might contribute to the improved endothelial dysfunction and decreased blood pressure in hypertensive animals afforded by ALA supplementation. Therefore, in addition to its derivatives (EPA and DHA)-dependent cardioprotective effects, ALA may exert direct benefits against hypertension.Epidemiological studies have revealed an inverse association between dietary intake of long chain n-3 PUFAs and cardiovascular diseases29. Observational studies also yielded conflicting results about the relationship between LA intake and blood pressure in humans31. In the present study, LA supplementation had no effects on blood pressure and endothelial dysfunction in SHRs, which may be partially attributable to the change of prostacyclin from dilator to constrictor in the LA metabolism of SHRs. LA is a precursor of AA, and prostacyclin is the principal metabolite of AA released by ACh in the vessels. Prostacyclin has been reported to be an endothelium-derived vasodilator and is involved in blood pressure regulation32. The anti-hypertensive action of LA is partially mediated through prostacyclin metabolism34. However, prostacyclin cannot evoke vasodilation in aortas from SHRs, and in contrast, it exerts a vasocontracting effect by activating thromboxane A2/endoperoxide thromboxane-prostanoid receptor36. Different from our studies, an anti-hypertensive effect of LA was observed in the hypertensive animal model of Dahl S and deoxycorticosterone acetate-salt hypertensive rats38. Noteworthy, a relatively higher LA diet (20% safflower oil) was used in these studies, while 10% safflower oil was used in our study. Another point we should make it clear is that we only included male rats in the present study. Compelling evidence has demonstrated that sex hormones are involved in the regulation of cardiovascular function and play pathological roles in the development of cardiovascular disease39. In particular, estrogen at a physiological level was demonstrated to confer protection against cardiovascular diseases including hypertension and ischemia/reperfusion-induced myocardial injury41. Premenopausal females have been shown both clinically and experimentally to be less sensitive to hypertension compared to the males. In addition, a clear sex difference in many aspects of cardiovascular function in SHRs has been demonstrated previously, including the degree of hypertension and vascular function42.In contrast to the recognized benefits of n-3 PUFAs, there is still controversy regarding the cardiovascular effects of n-6 PUFAs. Some investigators speculated that a large amount of n-6 PUFA consumption may promote the incidence and development of cardiovascular disease, which may result from its potential pro-inflammatory and thrombogenic properties+-dependent deacetylases and ADP-ribosyltransferases, which have been extensively implicated in modulating a myriad of cellular processes, including energy metabolism, stress responses and cell survival43. SIRT3, located in the mitochondria, has been demonstrated to orchestrate global mitochondrial lysine acetylation, enhance antioxidant defense and preserve mitochondrial function44. SIRT3 regulates cellular redox homeostasis through deacetylation and activation of SOD2, the mitochondrial isoform of major superoxide-scavenging enzyme. Clinical studies have revealed that SIRT3 expression decreases by 40% by 65 years of age45, which is paralleled with the increased incidence of hypertension. SIRT3 deficiency and redox inactivation of SIRT3 result in SOD2 inactivation and contribute to the pathogenesis of endothelial dysfunction and hypertension10. In our study, ALA supplementation rescued SIRT3 reduction and SOD2 hyperacetylation, improved endothelial dysfunction and reduced blood pressure elevation in hypertensive animals. SIRT3 knockout rendered the mice more susceptible to AngII-induced hypertension and abolished the endothelium-beneficial and antihypertensive effects of ALA supplementation, indicating the critical role of SIRT3 in vascular protective effects of ALA.The Sirtuins are a family of NAD47. Aberrant autophagic activity has recently been implicated in the pathogenesis of hypertension-related endothelial dysfunction48. In the present study, the expression of both LC3-II and p62 were upregulated in aortas from SHRs and in endothelial cells exposed to AngII plus TNFα treatment. Given that the autophagosome is an intermediate structure in a dynamic autophagic pathway, the number of autophagosomes observed at any specific time point is a function of the balance between the rate of their generation and the rate of their conversion into autolysosomes49. So, increased LC3-II levels may be indicative of either enhanced autophagy induction or defective autophagosomes clearance. In order to identify the exact point of dysregulation of autophagic flux, we further applied the lysosomal enzyme inhibitors E64d and Pepstatin A to impede autophagosome clearance. Interestingly, blockade of autophagosome clearance did not further increase LC3-II levels in endothelial cells exposed to AngII plus TNFα, indicating a defective autophagosome clearance in these cells. The syntropic change of LC3-II and p62 also happened in other pathological models, such as amyloidogenic light chain-mediated cardiotoxicity, ischemia/reperfusion-induced myocardial injury and ethanol-induced myocardial injury, indicating an impaired autophagic activity52.Emerging evidence indicates that basal autophagy is an important in vivo process regulating proper cardiovascular homeostasis and function; either excessive or insufficient levels of autophagic flux can contribute to the pathogenesis of cardiovascular disease53. In particular, SIRT1 was demonstrated to regulate autophagy directly through deacetylation of the autophagy-related genes ATG5, ATG7 and ATG811. SIRT1 has also been demonstrated to induce the expression of autophagy pathway components via the activation of FoxO transcription factors54. However, less information is available about the association between SIRT3 and endothelial autophagy in hypertension. Our data demonstrate that endothelial SIRT3 reduction is concomitant with dysregulation of autophagic flux in endothelial cells of hypertension, and both could be rescued by ALA administration. More interestingly, silencing SIRT3 blunted the beneficial effect of ALA on autophagic flux in endothelial cells, indicating SIRT3 as an important factor in ALA-regulated autophagy.Similar to autophagy-related genes, the expression and activity of Sirtuins are influenced by stress and nutrient availability. Sirtuins have been shown to be involved in regulating the autophagy-lysosome pathwayIn conclusion, our data have demonstrated that ALA supplementation ameliorates endothelial dysfunction and hypertension by rescuing SIRT3 impairment and thus alleviating SOD2 hyperacetylation and autophagic impairment to inhibit vascular oxidative stress, suggesting a novel cardioprotective effect of ALA. These findings could be of interest in translational medicine, where ALA may be applicable in nutritional interventions for the prevention and treatment of hypertension.Supplementary Figure LegendsSupplementary Figure. 1Supplementary Figure. 2Supplementary Figure. 3


Plasmodium serve as a silent reservoir of disease, critical to sustaining a low level of remanent malaria in the population. These infections must be effectively identified and targeted for elimination. The sensitivity of light microscopy, the traditional method used for diagnosing Plasmodium infections, is frequently insufficient for detecting asymptomatic infections due to the low density of parasitaemia. The objective of this study was to explore the current prevalence of asymptomatic sub-microscopic Plasmodium carriages to evaluate the parasite reservoir amongst residents from 7 hamlets in Tak Province in northwestern Thailand using a highly sensitive molecular method.Asymptomatic infections with sub-microscopic Malaria infection was screened in a real-world setting from 3650 finger-prick blood specimens collected in a mass cross-sectional survey using light microscopy and loop-mediated isothermal amplification (LAMP). LAMP results were later confirmed in a laboratory setting in Bangkok using nested PCR, restriction enzyme digestion and DNA sequencing. The association of malaria infection with demographic factors was explored.Plasmodium falciparum, 52.9% (45/85) with Plasmodium vivax, 2.4% (2/85) with Plasmodium malariae, 4.7% (4/85) with mixed P. falciparum and P. vivax, and 9.4% (8/85) had parasite densities too low for species identification. Asymptomatic carriages (T < 37.5 °C) accounted for 95% (76/80) of all sub-microscopic cases with the highest prevalence occurring in the subjects 31–45 years of age (p ≤ 0.035). Participants working on plantations or as merchants had an increased infection risk. Evaluation by microscopy identified 10.53% (10/95) of all Plasmodium infected participants.Parasite prevalence was 0.27% (10/3650) as determined by microscopy. Sub-microscopic infection prevalence was 2.33% (85/3650) by LAMP. Of these, 30.6% (26/85) were infected with Plasmodium infections with sub-microscopic parasite densities are considerable in this area. These findings provide the true disease burden and risk factors in this region. This information helps to direct policy makers towards better schemes and delivery of targeted interventions. Moreover, this is the first study to use LAMP in mass screening for sub-clinical and sub-microscopic infections in a field setting in Thailand. LAMP proves to be a sensitive and field-deployable assay suitable for national malaria control screening campaigns.Participants carrying asymptomatic  Based on the world malaria report in 2017 from 91 countries and areas with ongoing malaria transmission, the progress of malaria control efforts has stalled after an unprecedented session of successfulness in worldwide malaria control [Plasmodium species, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi, Plasmodium o. wallikeri as well as Plasmodium knowlesi that can infect humans. Of these, two species, P. vivax (76.70%) and P. falciparum (15.23%) are the predominant species found in Thailand [According to malaria statistics for Thailand in 2017, malaria control efforts in Thailand have been highly successful, resulting in an 82.2% decrease in clinical cases over the last 10 years since 2007 from 63,354 to 11,263 cases [Plasmodium infections for improving malaria elimination strategies. In addition to being a highly sensitive diagnostic, the ideal method needs to be readily field-applicable for pre-elimination field screening teams.As the malaria burden still poses serious challenges, the Thai Government has declared a national malaria elimination goal of 2024 . In ThaiPlasmodium infections. This method had to be conducted in Bangkok [The study site in this current study is located in Tak, a Myanmar-border province, previously the location of Thailand’s highest malaria burden. In the past few years, however, this province experienced a substantial decrease in transmission , 9. Howe Bangkok , 11. Up Plasmodium infections with low-density parasites, that otherwise go undetected by microscopy.A newer molecular method, loop-mediated isothermal amplification (LAMP) has been shown to have an estimated detection limit of 1 parasitized red blood cell (RBC)/500 μL of blood . LAMP isPlasmodium infections with sub-microscopic and low-density malaria reservoirs by LAMP in a real-world field setting of communities in Thailand. Accordingly, a cross-sectional survey was conducted in 3650 people living in malaria-endemic communities in Tak Province, Thailand. Here, a malariometric survey performed in the field by LAMP assay to determine the current epidemiology of asymptomatic sub-microscopic carriers was described. This study explored the risk factors associated with residual malaria parasitaemia amongst local residents, for a more comprehensive understanding of the malaria epidemiology.To date, there have been no studies done to determine the prevalence of asymptomatic Plasmodium vivax and P. falciparum are predominant in this region, although all human malaria parasites, as well as the simian malaria species P. knowlesi, can be found [Plasmodium prevalence.The community-based cross-sectional survey was conducted in Tha Song Yang district, Tak Province, northwestern Thailand Fig. . Tha Sonbe found . There aPlasmodium carriages is shown in Fig. A mass cross-sectional study was conducted to determine the prevalence of asymptomatic sub-microscopic malaria carriages from 13 to 27 July, 2015. An ethical consent for this study was provided by an ethical committee, Department of Disease Control, Ministry of Public Health, Nonthaburi, Thailand (Ref No 7/54-479). All individuals and all age groups were invited to participate in the study. Signed informed consents were obtained from all participants before enrolment. Interviews were performed with parents and/or legal guardians for participants 7 years or younger. A selection criteria for participation was the absence of anti-malarial drug treatment within the previous 2 weeks. The participant’s history of previous malaria infection, body temperature, occupation, age and gender, were recorded at the time of enrolment. The study design for determination of Plasmodium genus-specific LAMP assay in the field, 50 μL of blood was used. LAMP genus-positive blood samples were further characterized by a LAMP species-specific assay in the field. The remainder of blood samples was kept at − 80 °C for repeating LAMP analyses in a laboratory setting in Bangkok. Results of the repeated LAMP reactions were later confirmed using a combination of nested PCR and restriction enzyme digestion, followed by DNA sequencing.EDTA microtainer tubes were used to collect finger-prick blood samples from 3660 participants who met the inclusion criteria. However, 10 blood samples were excluded from the study since the blood was not sufficient to perform LAMP analysis. For each of the finger-prick blood sample, a portion of the blood was utilized directly to prepare thick and thin blood smears for microscopic analysis. For screening by a Plasmodium density obtained from thick smear analysis was interpreted as the parasite number detected per 500 white blood cells (WBCs) and was converted into the parasite number/ml of blood presuming a WBC count of 8000 WBCs/μL of blood [Thick and Thin blood smear preparation was done on one slide, air-dried, fixed with methanol (thin blood smears) and stained for 10 min with 10% Giemsa. Thick blood films were examined immediately under a light microscope (1000× magnification) by clinical staff in the field as per routine malaria diagnosis. of blood . If no pg. For each LAMP reaction, 100 µL of the clear supernatant was transferred to a new clean tube and 5.5 µL of the supernatant was used immediately. Based on the observation, the supernatant containing gDNA could be kept at − 80 °C for not more than 7 days without compromising stability.For the LAMP assay, a simple boiling technique was used in this study to extract genomic DNA , 17. Bri® Blood mini kit  according to the manufacturer’s protocol. For nested PCR analysis, 5 µL of the eluted DNA was used directly.For nested PCR, DNA extraction was done from frozen EDTA blood samples using a NucleoSpin18srRNA) as described previously by Han et al. [Plasmodium spp. and 4 human Plasmodium parasites including P. falciparum, P. vivax, P. malariae, and P. ovale [AMA-1) gene, which is found during the late schizont stage of all Plasmodium parasites, was used to identify P. knowlesi as described by Lau et al. [Plasmodium genus, P. falciparum, P. ovale, and P. knowlesi and 2.4 µM of each FIP and BIP for P. vivax and P. malariae, 0.2 µM of each forward outer primer (F3) and backward outer primer (B3), 0.8 µM of each loop primer forward (LPF) and loop primer backward (LPB) 12.5 µL of 2× reaction mix, 1 µL of Bst DNA polymerase and 5.5 µL of DNA template. The LAMP reaction mixture was incubated in a Loopamp real-time turbidimeter  or in a water bath for 90 min at 60 °C and for 5 min at 80 °C for enzyme inactivation. A negative control was included in each run. The LAMP reaction was assessed by the naked eye for endpoint detection or by a Loopamp real-time turbidimeter (LA-320C) for real-time detection. LAMP reactions for Plasmodium genus and species were interpreted as positive when an obvious increase in turbidity was detected by the naked eye or by real-time turbidimeter readings. The results were interpreted as valid when the turbidity was absent in the negative control.LAMP primer sets targeting 18S ribosomal RNA  genes of P. falciparum, P. vivax, P. malariae, and P. ovale were amplified in two successive rounds of PCR as described previously [Plasmodium spp. In the second round of PCR, the primers were specific for 4 human malaria species . The PCR reaction was carried out in a 25-µL reaction volume consisting of 12.5 µL GoTaq® Green Master Mix , 0.4 µM of each forward and reverse primers for Plasmodium genus, and 5 µL of template DNA. In the second round of PCR, the PCR product from the first round was diluted 10-fold in nuclease-free distilled water, and 5 µL was added to a new PCR reaction mixture containing the same concentration of species-specific primers as the genus-specific primers in the first round. For P. knowlesi, 2 sets of primers were used to amplify the small sub-unit ribosomal RNA (ssrRNA) gene. Plasmodium-specific primers (rPLU1 and rPLU5) were utilized in the first-round [Plasmodium knowlesi-specific primers (Pmk8 and Pmkr9) were utilized in the second round [rPLU1 and rPLU5), 12.5 µL of GoTaq® Green Master Mix . In the first PCR, the ssrRNA gene was amplified following the conditions as described previously [The species-specific nucleotide sequences of the 18S ribosomal RNA eviously . For thend round . Amplifieviously . The ampDdeI was used for analysis of Plasmodium genus-specific LAMP products. HpyCH4V was used for P. falciparum, P. vivax, and P. malariae. AluI was used for P. ovale. LAMP products were digested with restriction enzymes at 37 °C for overnight and were incubated at 65 °C for 20 min to inactivate the enzyme. Digested LAMP products were analyzed by electrophoresis in a 3% agarose gel, stained with ethidium bromide and visualized under a UV light.For further confirmation, LAMP amplified products were validated by restriction enzyme digestion, followed by comparison with the restriction map of the target sequences of each of the LAMP products. For restriction enzyme analysis, Plasmodium genus and species of the LAMP-positive blood samples, the LAMP products were used as template DNA for PCR amplification using LF and LB primers. The products were run on an agarose gel, stained with SYBR safe DNA gel stain , and the specific band was cut and purified with a QIAquick Gel Extraction kit . The purified amplicons were cloned into the pGEM®-T Easy Vector  and sequenced using an ABI3730XL DNA Analyzer  with 2 universal primers (T7 and SP6). The sequences were aligned with standard sequences of the Plasmodium 18srRNA gene.In order to confirm the Plasmodium infections was done using binary logistic regression. Chi square (χ2) test with 95% CI was used for p-value calculation. The SPSS 17.0 statistical software package  was used for statistical analysis.Data analysis of factors associated with P. vivax infection. The other 2 participants with body temperature of 38.1 °C (Thai) and 39 °C (foreigner) had P. vivax infection and P. falciparum infection, respectively. The most frequent occupation among participants  was student. Among the 7 study sites, Nong Bua had the highest population number  followed by Tala Oka, Mae Salid Noi, Suan Oi, Ko Ma Nae, Pha Man, and Orphanage, respectively.The mass cross-sectional survey was conducted between 13 and 27 July, 2015. The blood samples were collected with informed consents from 3660 residents living in 7 hamlets in Tha Song Yang district, Tak Province, Thailand. Ten samples had to be excluded from the study since there was insufficient blood to perform a LAMP reaction. Of these 3650 participants, 2035 (55.75%) were females and 1615 (44.25%) were males . Among these LAMP-positive samples, 0.16% (6/3650) were also positive by microscopy. Thus, LAMP identified a substantially higher number of Plasmodium carriages  as shown in Table P. falciparum and 2 samples positive for P. vivax by LAMP. These samples were negative by restriction enzyme digestion. In summary, overall the prevalence of sub-microscopic infections as determined by LAMP was 2.33% (85/3650). Details of the prevalence of sub-microscopic Plasmodium infections detected by LAMP in the 7 hamlets are presented in Table Of the 3650 blood samples screened in the field, LAMP successfully amplified Plasmodium infections , representing an overall prevalence of 0.27% (10/3650) (Table 3). These 10 samples classified as positive by microscopy originated from 8 male and 2 female subjects. Among 10 samples positive by microscopy, 4 samples were negative by LAMP.Thick and thin blood films from the 3650 blood samples were analysed by Giemsa-stained smear examination by local staff in the field and later confirmed by an expert microscopist. After the initial and second round of microscopy, only 10 samples were found positive for Plasmodium carriages was a single P. vivax infection, accounting for 52.9% (45/85) of the cases. A single P. falciparum infection was present in 30.6% (26/85) of the subjects. A single P. malariae infection accounted for 2.35% (2/85) of the subjects. Mixed infections between P. falciparum and P. vivax accounted for 4.75% (4/85) of the cases and 9.41% (8/85) of the cases identified as Plasmodium genus-positive.As shown in Table Plasmodium infections is shown in Table Plasmodium infections were found in adults and were significantly higher in subjects aged 31–45 years  p ≤ 0.035. Among the positive samples, the youngest and oldest subjects were 1 and 78 years of age, respectively. Most of the sub-microscopic malaria infections  were asymptomatic, since indications of malaria-like symptoms, such as headache, chills and fever were not observed. The majority of the sub-microscopic cases of malaria occurred in the participants with no Thai citizenship . Furthermore, a strong association between asymptomatic Plasmodium infections and patient occupations was observed in this study. The risk of acquiring asymptomatic sub-microscopic Plasmodium infections significantly increased in individuals working on the plantation (p ≤ 0.013) or as merchants (p ≤ 0.015). The odds ratios of plantation and merchant occupations were approximately 2 and 4 times greater than other occupations, respectively. Among 7 hamlets, the prevalence of asymptomatic Plasmodium infections was significantly high in Nong Bua  p ≤ 0.004 and Suan Oi  p ≤ 0.05.Detailed analysis of the association of the demographic characteristics of the study population and sub-microscopic In 2016, Thailand had approximately 11,522 malaria cases, or 0.17 cases/people , and wasThe studied area in this current study is one of the most malaria-endemic regions of Thailand. Although there has been a marked reduction in malaria transmission, Tak Province in 2017 was still classified as a high transmission area based on the WHO 2016 index, with an estimated annual incidence of 5.04 cases/1000 people . In 2017Plasmodium infections in Thailand conducted in field clinics using a LAMP method. LAMP results revealed that there were 3 samples positive by LAMP but negative after LAMP product confirmation by restriction enzymes digestion. These false positives could be due to haemolysis or high levels of lipid in the plasma of the blood samples. Moreover, 4 samples were positive by microscopy but negative by LAMP. In these samples, the parasite density ranged between 16 and 1440 parasites/µL, which could be detected by LAMP. Thus, there should be other causes for obtaining LAMP-negative results including a lower efficiency of template DNA preparation or DNA degradation during the preparation process. Overall, the LAMP method performed in the real-world field setting demonstrated reliable results. In this study, LAMP proves to be easy to operate and simple to adapt for use in any environment and field condition, and can be used as a reliable method for discerning the hidden malaria reservoir in various communities.Quantitative polymerase chain reaction (qPCR) has been used to detect a number of asymptomatic sub-microscopic malaria infections in this study area , 11. AltPlasmodium infections was 2.6% (95/3650) and over 85.6% were asymptomatic sub-microscopic infections. This suggests that there is a substantial proportion of Plasmodium-infected individuals with both asymptomatic and sub-microscopic densities in this area. This current finding is in agreement with the results from the previous study by Baum et al. conducted in the same area using qPCR, which found that the prevalence of asymptomatic and sub-microscopic Plasmodium infections in the population was 3.0 and 90.2%, respectively [18srRNA) gene used in this study, this limit of detection may be decreased making a LAMP assay to be more sensitive.This current study found that malaria infection rates estimated by LAMP in the mass blood survey of this community were 8.6 times higher than those estimated by an expert microscopist. At the time of this survey, the prevalence of ectively . In BaumP. vivax. This malaria species distribution is similar to that reported in Imwong’s study conducted in the northwestern border of Thailand [Plasmodium species in western Thailand.Amongst the low-density asymptomatic cases in this study, the most prevalent species was Thailand  and NguiThailand . The simPlasmodium prevalence of asymptomatic malaria infections, with some hamlets being completely free of infections, while in other nearby hamlets separated by short distances, approximately 5.57% of the hamlet population was infected. The heterogeneity in the prevalence of malaria infections in this study is in agreement with a previous finding performed in southern Laos PDR [Plasmodium epidemiology at the community level. The heterogeneity in the Plasmodium infection prevalence may at least in part be described by environmental factors, including abundance of mosquito larval habitats, mosquito densities, house location in relation to mosquito breeding grounds, house construction materials and design, and resident protective measures and exposure to mosquitoes due to occupations. It appears that the hamlet with the infected population is a local transmission hotspot that needs special attention by the malaria control programmes.Moreover, this current study also observed evidence of heterogeneity of Laos PDR  and in wLaos PDR . MalariaPlasmodium infections and acquired immunity [Plasmodium infections was in the age group of 31–45 years. A similar peak prevalence in adults has recently been reported in eastern Myanmar which found that all asymptomatic infections were restricted to participants aged > 17 years [The protective immunity in malaria-endemic areas correlates with age, which is considered one of the most important factors . Generalimmunity , 34. Thi17 years . This inPlasmodium infections. Interestingly, a significantly increased risk was also found in merchants, which is consistent with the correlation with extent of outdoor-related activities. Moreover, many past findings have observed that males are more likely to get infected by malaria than females [Previously, the study by Zaw et al. conducted in eastern Myanmar found that there was a statistically significant difference in infection rates among occupations. This previous study showed that much higher infection rates were observed in men working in the forests . Consist females , 30, 35.In this study, the LAMP malaria assay provides a promising tool for being used in remote field settings, which could render increased efficiency of ongoing malaria control interventions. However, there are some limitations. The false positive could be occurred when using the supernatant, which was not completely clear from the cell pellet especially in blood samples with haemolysis or with high level of lipid in the plasma. The supernatant should be cleared by centrifugation twice before being used. The turbidimetric determination of the LAMP reaction by visual observation is sometimes difficult especially in the weak positive result. The false negative results of LAMP assay could be observed when using the supernatant, which was repeated freezing and thawing or kept on ice for more than 4–5 h. The supernatant should be immediately used to prevent DNA degradation.Plasmodium carriages among asymptomatic individuals in Tak Province in northwestern Thailand. This study indicates that light microscopy, a rountinely used diagnostic technique, excessively underestimates true malaria parasite prevalence. Thus, it is imperative to employ a higher sensitivity method such as LAMP for parasite field detection in the phase of malaria elimination. These findings also provide an understanding of the current status of asymptomatic sub-microscopic Plasmodium infection prevalence, and determine the key demographic characteristics of Plasmodium infected cases, such as age groups and occupations. This knowledge could provide crucial information regarding potential infection sources, and could assist in determining the risk of infection within individual hamlets. These findings have important practical implications for targeting activities to eliminate malaria including mass screening and mass anti-malarial drug administration.These current findings based on the LAMP survey in a real-world fielding setting reveal substantial sub-microscopic


Plasmodium falciparum asexual parasite and gametocyte carriage remains unclear. This study examined association between prevalence of α-thalassaemia and P. falciparum asexual stage parasitaemia and gametocytaemia in children, pregnant women and adults, which was part of a bigger study that investigated some key factors that influence gametocyte carriage.The alpha-thalassaemia trait has been associated with protection against severe malaria but its role in P. falciparum positive individuals, gametocyte prevalence was 38.5% (15/39) in children, 29.7% (11/37) in pregnant women and 17.4% (4/23) in adults. Heterozygous α-thalassaemic children were less likely to harbour asexual parasites, compared with normal and those deficient  under the dominant model. These heterozygous children were also associated with reduced risk of parasitaemia compared to heterozygous adults and pregnant women. Children with heterozygous α-thalassaemia trait had reduced risk of asexual parasite carriage. There was however, no association between α-thalassaemia trait and risk of gametocyte carriage.Overall prevalence of heterozygous α-thalassaemia trait among all the groups was 39.0%, while 8.2% were homozygous alpha thalassaemia. Asexual parasite prevalence was significantly higher in children (P = 0.008) compared to adults and pregnant women. Of the asexual  Certain genetic blood disorders such as the thalassaemia’s  and sickle cell anaemia have been shown to influence malaria pathogenesis –3. HeterThe study was conducted in Asutsuare and its surrounding villages in the Shai Osudoku District of Ghana. Baseline samples from a longitudinal study conducted from November 2013–September 2014 were used. Ethical approval was given by the Institutional Review Board of Noguchi Memorial Institute for Medical Research, University of Ghana, Legon. The study included three different demographic groups; children (aged 2–15 years), male and non-pregnant female adults (aged 16–65 years), and pregnant women (aged 18–45 years). Informed consent was obtained from all participants and/or their parents/legal guardians prior to their recruitment and inclusion in the study. A detailed description of the study area and population has been presented elsewhere [All study participants were screened for their sickle cell status using the sodium metabisulphite method, and this grouped all SS and SC under AS phenotypes. Haemoglobin levels were measured using Hemocue-Hb 201 . The ABO blood type was determined using a commercial anti-sera blood grouping kit .P. falciparum parasites were determined by a previously described nested PCR targeting sequences of the small subunit ribosomal (ssrRNA) 18 s genes of four Plasmodium species  [® plus mini kit . Gametocyte detection was done by real-time (RT) quantitative PCR [Pfs25 transcripts and RNA template for the reaction included 3D7 gametocyte-positive standard RNA generated from parasite culture as previously described [Blood smears were stained with 10% Giemsa for detection of both asexual and sexual stage parasites by microscopy under oil immersion (100× magnification). Submicroscopic . vivax) . RNA wastive PCR  targetinescribed .P. falciparum infection, it was analyzed under three models of inheritance  [Polymorphisms in the human α globin gene resulting in α-thalassaemia, specifically the—α 3.7 deletion, was determined by PCR . After gcessive)  using loP. falciparum infections was significantly  different among the study groups and majority of these infections were submicroscopic (97%). The prevalence was highest among the children (49.1%), compared to adults (28.7%) and pregnant women (22.2%) (Table The study population comprised of 463 subjects consisting of children 184), adults (154) and pregnant women (125), of which a total of 167 (36.1%) of the population were asexual parasite positive. Generally, majority of the parasites detected were submicroscopic. The distribution of asexual 84, adultThe overall prevalence of heterozygous α–thalassaemia was 39.0% (176/451) and 8.2% (37/451) carried the deficient homozygous trait. The distribution of α–thalassaemia alleles in the study population did not deviate from Hardy–Weinberg equilibrium (P = 0.56).Overall, the heterozygous α-thalassaemia trait was not associated with asexual parasitaemia  than observed in other parts of the country [This study investigated associations between the α-thalassaemia trait and  country , 18, 19.y higher % to 30%  country .P. falciparum infections and are therefore able to clear parasites effectively due to repeated exposure [The association of asexual parasite carriage with α-thalassaemia trait varied among the groups. This difference in association may be due to possible confounding factors since protection from malaria has been shown to depend on several factors including the host’s immune status as well as host genetics . In thisexposure . We can P. falciparum asexual parasitaemia or gametocytaemia.Interestingly, some genetic traits such as sickle cell traits with HbS and HbC variants that protects against severe malaria have been suggested to promote gametocyte carriage , 2. HoweThe study findings showed that children with heterozygous α-thalassaemia trait had reduced risk of asexual parasite carriage under the dominant model. However, there was no association between α-thalassaemia trait and risk of gametocyte carriage, perhaps due to limited gametocyte prevalence. Further studies need to be conducted in different transmission areas to ascertain a probable association and mechanisms of α-thalassaemia and gametocyte carriage and possibly the infectiousness of these parasites in malaria transmission.The very low numbers of both asexual parasites and gametocytes is an important limitation of the study and the findings described will need to be confirmed with a larger sample size.


Plasmodium falciparum and 3 P. knowlesi infections. We additionally identified 2 monoinfections with the zoonotic malaria Plasmodium cynomolgi, both in individuals reporting no history of forest activities or contact with macaques. Results highlight the need for improved surveillance strategies to detect these infections and determine public health impacts.To determine the presence and species composition of malaria infections, we screened a subset of samples collected during a cross-sectional survey in Northern Sabah, Malaysia using highly sensitive molecular techniques. Results identified 54 asymptomatic submicroscopic malaria infections, including a large cluster of  Plasmodium knowlesi in 2004, P. knowlesi is now the main cause of human malaria in Malaysia [P. knowlesi globally and reported 2030 clinical P. knowlesi cases in 2017, comprising 98% of all malaria cases reported [P. falciparum and 8 cases of P. vivax reported in Sabah in 2017. Malaysia aims to eliminate malaria by 2020, making it critical to identify remaining infections in the population.Following the initial description of a large cluster of human infections with the zoonotic malaria Malaysia , 2. The P. knowlesi has been associated with deforestation and environmental change, likely bringing people in closer contact with long-tailed and pig-tailed macaque reservoirs (Macaca fascicularis and M. nemestrina) [Plasmodium species, including the zoonotic species P. cynomolgi [P. cynomolgi had been demonstrated in laboratory studies, the first natural human infection with P. cynomolgi was identified in West Malaysia in 2011 [P. cynomolgi and P. knowlesi infections in 5 clinical cases in Sarawak in Malaysian Borneo [P. cynomolgi in Cambodia in 2015; however, no asymptomatic human infections have been described in Malaysia [The increased incidence of estrina) . These mynomolgi . While h in 2011 . A more n Borneo . SubsequMalaysia .P. cynomolgi and coinfections with P. knowlesi and P. cynomolgi have been described in both macaque and mosquito populations. In Northern Sabah, entomological investigations of the main vector of P. knowlesi, Anopheles balabacensis, identified relatively high numbers of mosquitoes biting humans were infected with P. cynomolgi [P. cynomolgi is the most common and widely distributed malaria infection detected in macaque populations in Southeast Asia [P. cynomolgi have been described [Within Sabah, high prevalences of ynomolgi , 10. Simast Asia . Howeverescribed .P. knowlesi, as measured by serology, as 5.1% (4.8%–5.4%); however, of 10 100 people sampled, only 9 Plasmodium-positive individuals were identified by polymerase chain reaction (PCR) and no individuals were microscopy positive. Species included P. knowlesi, P. vivax, and P. malariae, with no P. falciparum detected despite on-going transmission in the area. Although previous studies had identified geographical clustering of asymptomatic infections in individuals residing in villages where clinical cases were reported [This study aimed to detect whether low-density asymptomatic malaria infections were present at a community level by using a subset of samples collected during an environmentally stratified cross-sectional survey in Northern Sabah . Initialreported , all indA population-based cross-sectional survey was conducted in 4 districts in Northern Sabah in 2015, as described by Fornace et al . BrieflyPlasmodia, using primers PlasmoM_N1F (5′-ATGGCCGTTTTTAGTTCGTG-3′) and PlasmoM_N1R (5′-TTGTGTTAGACACACATCGTTCC-3′) for nest 1 and PlasmoM_N2F (5′-GTTAATTCCGATAACGAACGAGA-3′) and PlasmoM_N1R for nest 2 [Whole-blood samples of selected individuals were extracted on a QIAsymphony SP/AS instrument  using a QIAsymphony DNA Midi Kit  and eluted in 200 µL of elution buffer. Extracted DNA was amplified with a genus-specific seminested PCR assay targeting 18S rRNA Plasmodia positive by PCR. These included 2 P. cynomolgi infections, 3 P. knowlesi infections, 3 P. malariae infections, 1 P. vivax infections, 41 P. falciparum infections, and 4 infections that species could not be identified despite repeated attempts. No infected individuals reported a history of fever. Of the original 9 infections detected, sequencing results were consistent for P. knowlesi and P. malariae monoinfections and the mixed P. malariae, P. vivax, and P. knowlesi infection was identified as P. malariae and P. vivax. Both P. cynomolgi infections were detected by the initial PCR analysis; however, both infections were previously misdiagnosed, 1 as P. vivax and 1 as a mixed P. knowlesi and P. vivax infection.Individuals screened included 423 men and 453 women, with ages ranging from 5 months to 90 years (mean age 27). This included participants from 417 households in 87 villages across 4 districts. Of these, 54 individuals  were identified as P. cynomolgi infections were identified in adult men, aged 43 and 63. The individuals resided in different villages, 1 in Kota Marudu district and 1 in Ranau district . Two individuals resided in the same village in Kudat district, with the other individual residing in Ranau. All individuals reported contact with long-tailed macaques around the house and village.Both district . Both inP. falciparum infections were identified despite no P. falciparum being identified by the previous study. Infections included 16 women and 35 men with a mean age of 31 years . All P. falciparum-infected participants resided in Ranau district, including 12 household clusters with 2 to 4 P. falciparum positive individuals. Infections were also clustered at a village level, with 21 infections identified in 1 village out of 48 samples screened . An additional village in Ranau had 12 P. falciparum infections, 1 P. cynomolgi infection, 1 P. vivax infection, and 1 unspeciated Plasmodium infection and a third village in Ranau had 6 P. falciparum infections, 1 P. knowlesi infection, and 2 further Plasmodium positive infections that species could not be identified.A large number of asymptomatic P. cynomolgi in the Malaysian state of Sabah and the first description of asymptomatic P. cynomolgi monoinfections in Malaysia. Within this rapidly changing environment with close contact reported between human and macaque populations, there is a need to understand the burden and health implications of this zoonotic parasite and monitor changes in incidence, which could indicate an emergence similar to P. knowlesi. Although results from this study cannot be used to estimate prevalence or spatial distribution of malaria infections at a population level, the identification of areas with relatively high numbers of infections suggests on-going transmission at the time of the survey of both zoonotic and nonzoonotic malarias within an elimination setting.This is the first description of natural human infections with Plasmodium genus developed for the 4 main nonzoonotic malaria species [P. falciparum infections may be due to the low sensitivity of the initial methods used or very low parasite densities. However, identifying the species composition of these infections remains challenging without sequencing confirmation. In contrast to other human P. cynomolgi infections identified, these cases were all monoinfections, although this may be due to the limited sensitivity of these methods to differentiate multiple species. A previous study in Malaysian Borneo did not detected P. cynomolgi infections within communities, despite specifically screening for this species [Plasmodium in both P. cynomolgi-positive individuals; however, both were misidentified by a routine PCR assay not targeting this species. This need is further emphasized by the relatively large number of P. falciparum infections detected in Ranau, an elimination setting with only 110 P. falciparum cases reported statewide in Sabah during the year of the survey.Results highlight both the utility of molecular diagnostics and the challenges in applying these techniques. The initial survey screened all samples with a widely used seminested PCR assay targeting the  species . Althoug species . As all P. cynomolgi remain unknown, this study illustrates the presence of naturally acquired human infections in individuals with no history of contacts with macaques or forest activities. Following the emergence of P. knowlesi within this region, innovative surveillance strategies are needed to detect zoonotic malaria infections and identify individuals and environments at risk.Further studies are needed to assess the prevalence of asymptomatic infections and assess implications for current malaria elimination programs. While the public health impacts of


This becomes even more important as the debate moves towards elimination and eradication. This study sought to quantify the burden of Plasmodium malaria infection in seven communities in the Eastern Region of Ghana.Asymptomatic Plasmodium 18S rRNA gene using species specific PCR.The cross-sectional study recruited 729 participants aged 85 years old and below from 7 closely linked communities. Finger pricked blood was used to prepare thick and thin blood smears as well as spot filter paper and an histidine rich protein 2 (HRP2) rapid diagnostic test kit (RDT). Genomic DNA was extracted from the filter paper dry blood spot (DBS) and used in PCR to amplify the P. malariae and 7 P. ovale mono-infections accounting for 2.2% and P. falciparum combined with either 36 P. malariae or 25 P. ovale infections, accounting for 13.3%. Parasite prevalence by microscopy (32%) was similar to the RDT positivity rate (33%). False positive RDT results ranged from 64.6% in children aged between 5 and 9 years to 10% in adults aged 20 years and above. No significant differences were observed in falciparum and non-falciparum parasite carriage at the community level, however young adults aged between 15 and 19 years had the highest prevalence (34.8% (16/46)) of P. falciparum and P. malariae parasite carriage whilst children aged between 5 and 9 years had the highest level (11.4% (14/123)) of P. ovale carriage.96.6% of the participants were identified as afebrile, with axillary temperatures below 37.5 °C. PCR identified 66% of the participants to harbor malaria parasites, with 9 falciparum parasites and the total absence of detection of P. ovale by microscopy suggests that more sensitive malaria diagnostic tools including molecular assays are required to accurately determine the prevalence of carriers of non-falciparum parasites and low density P. falciparum infections, especially during national surveillance exercises. Additionally, malaria control interventions targeting the non-falciparum species P. malariae and P. ovale parasites are needed.The high rate of misidentification of non- Plasmodium genus namely P. falciparum, P. malariae, P. ovale, P. vivax and P. knowlesi, remains a devastating public health problem in the tropical and subtropical regions worldwide  or as P. malariae [13.6% (3/22)] by microscopy (Fig. Parasite prevalence estimated by PCR across the five age groupings was significantly different for all the three P. falciparum is the major malaria parasite and represents close to 90% or more of the total malaria parasite population [P. falciparum to represent 100% of the total parasite population in Ghana [Plasmodium species [P. malariae or P. ovale with P. falciparum have been reported to lower the total parasite density of an infection [P. falciparum gametocyte prevalence [falciparum parasites [P. malariae and P. ovale parasites.In most malaria endemic countries in the World Health Organization Africa Region, pulation . The 201 species –30. Co-ievalence . Very fearasites , 32, makPlasmodium carriers was generally high in this study with a PCR estimated average of 66% and a microscopy average of 32%. The increased prevalence determined by PCR is expected as microscopy is known to underestimate parasite prevalence, especially at low parasite densities [P. malariae estimated by microscopy and PCR were similar, which was contrary to the differences between P. falciparum and P. ovale estimates, which were much higher by PCR when compared with microscopy. One main reason for the disparity was the high number of P. falciparum samples that were misclassified as P. malariae by the microscopists. Misclassification of Plasmodium species is particularly common in countries with a low prevalence of non-falciparum malaria primarily because of infrequent encounter by the microscopist and the subsequent low ability to identify morphological differences amongst the various Plasmodium species [P. falciparum and P. malariae are treated with the same dose of artemisinin combination therapy [P. malariae for P. falciparum and vice versa may not result in severe outcomes. However, due to the requirement of a radical cure agent such as Primaquine to clear P. ovale hypnozoites the treatment regimen is artemisinin combination therapy + Primaquine [P. ovale with P. falciparum could lead to severe P. ovale infections.The prevalence of ensities . The higensities –35 is a  species . The Wor therapy  as such,imaquine , thus miPlasmodium carriers, including both P. falciparum asexual and gametocyte as well as P. malariae parasite carriage. High gametocyte carriage in the group with the highest prevalence of both P. falciparum and P. malariae could be due to intra-host competition, which is known to enhance P. falciparum gametocyte production [P. malariae than the older population, which is similar to the findings in this study [Children under the age of 5 years are considered to be the most vulnerable groups , howeveroduction . An earlis study . All thris study .In this study, age influenced the false positive RDT rates, with children having much higher false positive rates than the adult group. One main reason why the three groups of children exhibited high false positive rates could be because children generally have more incidents of clinical malaria as well as have higher density infections , which wPlasmodium parasite carriage was similar in the seven closely linked communities. This suggests that although similar malaria control interventions would be effective in closely linked communities, interventions should be age group specific. There are a number of malaria control interventions that are targeted to protect children below the age of 5 years, one of the most vulnerable groups with respect to malaria from exposure to the malaria parasite [Even though afebrile parasite carriage was significantly affected by age, parasite . The resfalciparum malaria parasites in Ghana vary from less than 1% to more than 12% across different regions of the country [P. vivax in Ghana to date [falciparum malaria in Ghana, it is necessary that nationwide molecular surveys of all human malaria parasites be conducted on a regular basis. This will help determine the geographic distribution and prevalence of non-falciparum malaria parasites, especially P. ovale and decide whether there is a need to implement a revision to the national malaria treatment policy to incorporate a treatment regimen for P. ovale, which is presently absent in Ghana.Previous reports on the distribution and prevalence of non- country , 51, 52,falciparum parasites and the total absence of detection of P. ovale by microscopy suggests that more sensitive malaria diagnostic tools including molecular assays are required to accurately determine the prevalence of carriers of non-falciparum parasites and low-density P. falciparum infections, especially during national surveillance exercises. Additionally, malaria control interventions targeting the non-falciparum species P. malariae and P. ovale parasites are needed.The high rate of misidentification of non-Additional file 1: Table S1. List of primers and their properties


Malaria is still a leading cause of morbidity and mortality in many developing countries including Ethiopia. Its prevalence has been declining among Ethiopian adults, especially in Dembia district. However, it is still at the top of diseases list at the district. Hence, the study aimed to determine the prevalence and the factors that contribute to its being the major public health concern despite different preventive and control measures in place.A community based cross-sectional study was conducted from October 11 to November 16, 2017. The multistage sampling technique was employed to select 832 study participants. A rapid diagnostic test were used to confirm the disease. Data were entered using Epi info version 7 and was analyzed by Statistical Package for Social Science (SPSS) version 20. The logistic regression model was performed to examine the association of factors with malaria parasite.Plasmodium falciparum [46 (82%)]. According to the multivariable logistic regression analysis, male sex , adult age 15–19 years , travel history , and stagnant water around home  increased the probability of malaria infectivity, while Insecticidal Treated Nets (ITN) utilization  decreased it.Out of the 832 adults included in the study, 6.7% ) were confirmed to be malaria parasite carriers. The dominant plasmodium species was Malaria is still an important public health challenge among adults in the study area. Male sex, age 15–19 years, travel history, living around stagnant water, and not using ITN increased the probability of infection. Therefore, the District Health office and Health extension workers should work to increase ITN distribution and focus on reducing malaria breading sites through community participation. Malaria is one of the most severe public health problems and the leading cause of death in many developing countries. Globally, there were an estimated 214 million new cases of malaria and 438,000 deaths in 2015. Of this, 88% of the cases and 90% of the deaths occurred in the African region [Ethiopia is generally considered as a low to moderate malaria transmission intensity country with about 75% of the land and 60% of the people is exposed. Due to the unstable and seasonal transmission of malaria in the country, the protective immunity of the population is generally low causing all age groups to be at risk . StudiesMalaria imposes substantial costs on both individuals and governments. Direct costs  have been estimated to be at least USD 12 billion per year globally . DespiteEthiopia has achieved a remarkable progress in the fight against malaria in the last decade following the launching of the Health Extension Program in 2003 . The majA community based cross-sectional study was conducted on the residents of Dembia district, located in North Gondar zone, northwest Ethiopia, from October 11 to November 16, 2017. The total population of the district was about 321,856 of whom 159,962 (49.7%) were male. The district had 45  malarious kebeles. The altitude of the district ranges from 1800 m–2600 m above sea level. The climate of the district was 100% woina-dega, and annual rain fall ranged from 772 mmHg–1260 mmHg. Annual temperature ranged from 18 to 30 degree Celsius. The district had one hospital, 10 health centers, 40 health posts and 20 private clinics at its services.All adults (age > =15 years) in Dembia district were taken as a source population, while all adult individuals in randomly selected kebeles were considered as a study participants. Any adult individuals who took anti-malarial drugs 2 weeks before the survey, severely ill and unable to respond were not included.A sample of 310 adults was estimated using the single population proportion formula and considering a 95% confidence level, 5% margin of error, and 28.1% prevalence of malaria infection . ConsideA multistage sampling technique was used to select representative households. First a list of 45 kebeles was obtained from the District Health Office, at which 9(20%) were selected using the simple random sampling technique and considering as a primary sampling unit. Then the estimated sample was proportionally distributed to the selected kebeles based on their number of households and considering them as a secondary sampling unit. Finally, the lottery method was used to select any single household member for a rapid diagnostic test., travel history, occupation, toilet facilities, outdoor activities, stagnant water, ITN possession, ITN utilization, and IRS.The dependent variable of this study was the malaria test result . The independent variables were age, sex, educational status, family size, knowledge about malaria , main source of drinking waterBlood samples were drawn and tested for malaria presence using a Rapid Diagnostic Test (RDT). Carestart™ malaria Pf/Pv (HRP2/pLDH) Ag combo RDT was used. The validity of the kit was checked through 2 replicates of carestart™ malaria Pf/Pv (hrp2/pLDH) Ag combo RDT from each box with negative and positive clinical samples confirmed by microscopic examinations. In addition, a semi-structured questionnaire adapted from previous studies was administered to collect data on socio-demographic, economic and environmental factors, prevention methods, and knowledge relating to malaria infection.Observations have been made on the possession and utilization of ITN and the availability of stagnant water around houses. The data were collected by 8 health extension workers and 2 laboratory technicians, supervised by 2 clinical nurses.Before the actual data collection, a pretest was administered on 5% of the sample not included in the main study. Both data collectors and supervisors were trained for 3 days on how to approach study subjects and collect data. The supervisors and the principal investigator closely followed the daily completeness and appropriateness of the data collection. The validity of the kit was checked by testing 2 replicates of carestart™ malaria Pf/Pv (hrp2/pLDH) Ag combo RDT before the actual data collection. Knowledge of respondents in this study was measured considering respondents knowledge on the basic concepts of malaria prevention and control strategies such as causes, symptoms of malaria and means of prevention and control mechanisms. Accordingly, the mean score of respondents was used as to classify knowledge as good and poor.p-value < 0.25 at the bi-variable analysis were entered into the multi-variable analysis to control confounding effects. The associations between dependent and independent variables were measured and tested using Odds Ratio (OR) with a 95% CI. The parsimonious model fitness was checked by Hosmer and Lemeshow goodness of fit test and a P-value of 0.67 was obtained.Data were entered into Epi info version 7, exported and analyzed using SPSS version 20. Descriptive statistics, like the median with an interquartile range, frequencies, and percentages were employed. Cross-tabulations were plotted before both bivariate and multivariable logistic regression analyses were performed. Bi-variable and multi-variable logistic regression analyses were performed to examine the associations of covariates with malaria infection. All risk factors with A total of 832 individuals participated in this study with a response rate of 100%. Four hundred thirty-seven (52.5%) of the participants were female with about 547 (67.5%) unable to read and write. The median age was 34 (Inter Quartile Range (IQR): 24–45) years. About 64.8% of the households had less than 5 members with the median family size of 4 (IQR: 3–6) (Table The majority (92%) of the participants used household ITNs at bed-times. Out of those who had ITNs, 687(89%) always slept under mosquito nets, while only 277 (33%) households sprayed chemicals in the last 6 months. Five hundred thirty (63.7%) of respondents had poor knowledge about malaria signs, symptoms, and its prevention and control methods Table .Plasmodium falciparum (46(82%)) followed by 5(9%) Plasmodium vivax, and (Pf + Pv) 5(9%) mixed infections.Fifty-six  of the participants had RDT confirmed malaria parasites in their blood samples. The dominant plasmodium species was After adjusting for potential confounding variables, being male sex was 4.5 times more likely to have malaria infections . Individuals in the age group of 15–19 years were 4.5 times more likely to have malaria infections  compared to 20 and above age groups. Those who always slept under mosquito nets decreased the risk of malaria positivity by 80% . Individuals who traveled away from residence had 5 times more chance of having malaria infections  compared to those who did not. Individuals who lived close to stagnant water  were also significantly associated with malaria infection than those who did not . However, the studies conducted in Dilla town and its surrounding Gedeo zone , Chuchu Plasmodium falciparum which accounted for 82% of the cases was the dominant plasmodium species. This finding is in line with the malaria parasite distribution in Ethiopia [Plasmodium vivax was the dominant plasmodium species [Plasmodium falciparum is more widely distributed in most parts of Ethiopia. The possible discrepancy might be due to the fact that climate variation among study areas have effect on life cycle of parasite, parasite adaptation among Plasmodium vivax and falciparum.In this study, Ethiopia . It is aEthiopia  and NigeEthiopia . Convers species . This miThis study also revealed that the odds of asymptomatic malaria parasitemia among male adults were more than in females. This result is also supported by previous studies conducted elsewhere , 18. ThiThe odds of malaria infection among respondents in the age group of 15–19 years were higher compared with over 20 years . The finding is consistent with that of a study conducted in East Shewa which stated that being in the age group of 15–24 was more risks for malaria infection [The utilization of ITN is a powerful vector control tool for reducing malaria transmission . This stIn this study, the odds of malaria infection among adults who had history of travel were higher than who did not travel away from their residence. This finding is similar with that of a study conducted in Jimma town, southwest Ethiopia . This miThough the study did its best to determine the prevalence of asymptomatic malaria parasitemia in the study setting, it is not free from some limitations. The cross-sectional nature of the study did not enable it to show temporal relationships. In addition, since the verification of malaria parasitemia was done using rapid diagnostic tests, it might have detected false results that did not indicate the actual status of the respondents. Furthermore, variables that were not considered in this study, such as occupation, marital status and residence were the other limitations of the study. Despite the aforementioned limitations, the study was able to assess malaria prevalence and associated factors in the community. This enables to show the relative precise figure of the problem and associated factors in the community, and thus, the result can be adequately generalized for similar population.Plasmodium falciparum, is an important public health problem among the adult inhabitants of the study area. Males and those in the age group of 15–19 years are highly vulnerable groups for malaria positivity. ITN utilization is a protective factor for malaria infections, and factors like travel history and availability of stagnant water around dwelling are the risk factors. Therefore, during the implementation of malaria prevention and control activities, attention should be given to males who travel and work away from home and to youngsters in the age range of 15–19 years to use the most possible malaria prevention mechanisms. The District Health Office and health extension workers should be directed to work to increase ITN distribution in the community. The District Health Office should also focus on reducing or eradicating malaria breading sites through community participation. Travelers should use ITN, repellents, and protective clothes at places of arrival.Malaria, especially the predominant