Cerebral malaria (CM), is a life-threatening childhood malaria syndrome with high mortality. CM is associated with impaired consciousness and neurological damage. It is not fully understood, as yet, why some children develop CM. Presented here is an observation from longitudinal studies on CM in a paediatric cohort of children from a large, densely-populated and malaria holoendemic, sub-Saharan, West African metropolis.Plasma samples were collected from a cohort of children with CM, severe malarial anaemia (SMA), uncomplicated malaria (UM), non-malaria positive healthy community controls (CC), and coma and anemic patients without malaria, as disease controls (DC). Proteomic two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry were used in a discovery cohort to identify plasma proteins that might be discriminatory among these clinical groups. The circulatory levels of identified proteins of interest were quantified by ELISA in a prospective validation cohort.The proteome analysis revealed differential abundance of circulatory complement-lysis inhibitor (CLI), also known as Clusterin (CLU). CLI circulatory level was low at hospital admission in all children presenting with CM and recovered to normal level during convalescence (p < 0.0001). At acute onset, circulatory level of CLI in the CM group significantly discriminates CM from the UM, SMA, DC and CC groups.The CLI circulatory level is low in all patients in the CM group at admission, but recovers through convalescence. The level of CLI at acute onset may be a specific discriminatory marker of CM. This work suggests that CLI may play a role in the pathophysiology of CM and may be useful in the diagnosis and follow-up of children presenting with CM. Plasmodium falciparum severe malaria syndromes [Malaria is one of the major infectious disease challenges in sub-Saharan Africa, particularly in Nigeria. The global burden of malaria in 2017 was estimated to be 219 million cases with 435,000 deaths [It is still unknown why some children develop severe malaria complications, including cerebral malaria (CM) and severe malaria anaemia (SMA), whilst in others the infection may result in mild or uncomplicated malaria (UM). Childhood CM is a poorly-understood life-threatening syndrome where several complex pathophysiological processes such as mechanical obstruction of cerebral microvasculature by infected red blood cells , 5, neurP. falciparum, normal cerebrospinal fluid, and no other cause of encephalopathy [P. falciparum malaria setting of the city of Ibadan in sub-Saharan West Africa.Cerebral malaria is clinically defined by an unarousable coma lasting for at least 1 h with or without generalized convulsions, circulatory asexual stages of alopathy , 5. Howealopathy . In thisThe report shows that circulatory complement-lysis inhibitor (CLI), also known as Clusterin (CLU), is highly depleted in severe malaria. A very low plasma CLI level is associated with children presenting with CM, which recovers with convalescence in the entire CM cohort. More importantly, the circulatory CLI differentiates the CM group from other clinical manifestations of malaria, as well as from malaria-negative encephalopathy-like syndromes with CM-like pathology, including convulsions and meningitis.The Ethics Committee at the Institute for Advanced Medical Research and Training of the College of Medicine, University of Ibadan, Nigeria, reviewed the study and gave ethical approval for the sample collection from the hospital platform, primary care centres and schools in the city of Ibadan, Nigeria. Parents and/or guardians of study participants gave informed written consent in accordance with the World Medical Association’s ethical principles for research involving human subjects.All study participants were recruited from 2008 to 2013 at the University College Hospital (UCH) in the city of Ibadan, Nigeria, a densely populated malaria-hyperendemic city . It has P. falciparum parasitaemia who did not require hospital admission and were recruited as part of a daily malaria parasite screening routine at children outpatient clinics. The SMA group was comprised of conscious children with a packed cell volume (PCV) of less than 16% or a hemoglobin concentration of less than 5 g/dl, with asexual forms of P. falciparum detected in blood films and no other evident cause of the anaemia. The CM group consisted of children with an unarousable coma that persisted for more than an hour, with generalized convulsions and the presence of asexual forms of P. falciparum and normal cerebrospinal fluid. Coma status was defined by a Blantyre coma score < 2 [This study was part of the larger case–control study on severe malaria as reported previously , 10–12. core < 2 . Two addAll children diagnosed with severe malaria syndromes were admitted to the emergency ward and those with severe anaemia were transfused within 2 h of admission and confirmation of malaria diagnosis. However, samples were collected prior to the blood transfusion. All children diagnosed with malaria were treated and followed through convalescence to recovery following Nigerian anti-malarial treatment guidelines, which are based on the WHO guidelines . Uncomplg for 5 min. The cell volume was calculated as a percentage of the whole tube volume [Clinical data were collected using a malaria-focused questionnaire designed by the Childhood Malaria Research Group (CMRG) at UCH Ibadan. Malaria parasites (MPs) were detected and counted by microscopy following Giemsa-staining of thick and thin blood films. The criterion for declaring a participant to be malaria parasite-free was no detectable parasites in 100 high-power (1000×) fields in both thick and thin films , 10. Thie volume .g, 10 min) and frozen in aliquots at − 80 °C.Plasma samples were collected at recruitment and following recovery for the malaria-positive patients. The first samples collected on the day of recruitment or hospital admission, prior to drug treatment or blood transfusion, were labeled as onset. Samples following recovery were collected between 28 and 35 days after recruitment and were designated as ‘recovery’. A 2.5 ml blood sample was obtained from each participant in an EDTA blood tube and transferred on ice to the central malaria laboratory at UCH. Plasma was harvested immediately following centrifugation , and the combined flow-through and first wash were retained for analysis by 2D-DIGE.Plasma samples collected from children aged between 6 and 165 months were pooled based on the clinical groups . For crude plasma analysis, pools were directly processed by 2D-DIGE. For immunodepleted plasma, pooled samples were passed through Seppro IgY14Typically, 120 µg protein was labelled with CyDye DIGE Fluor Labeling Kit, following the manufacturer’s instructions . The Cy5 and Cy3 dyes were used to label pooled plasma proteins from two different disease groups, whilst Cy2 was used to label proteins from a pool of plasma samples combining CC, UM, SMA and CM groups to allow comparison between gels. For each gel, all three labeled samples were combined and buffered in DIGE Isoelectric focusing (IEF) buffer  to a total of 200 µL. These mixed samples were applied onto ReadyStrip IPG strips  and actively rehydrated for 13 h 40 min at 50 V followed by IEF with the voltage ramping option , using a PROTEAN IEF cell . The IPG strips were then immediately prepared for SDS-PAGE by transfer into equilibration buffer  for 15 min followed by alkylation by the addition of 2.5% (w/v) iodoacetamide for 15 min.Following SDS-PAGE, the gels were digitally imaged prior to staining with CooBlue following the manufacturer’s instructions . The 2D-DIGE pattern was analysed using Bio-Rad PDQuest software . Protein spots were defined as being differentially abundant between the pooled samples if they were statistically different using a two-tailed unpaired t-test between groups (n = 4), and a difference of greater than 1.5-fold abundance was detected Table . The Coo4HCO3), 50% acetonitrile (ACN) for 30 min at 37 °C until all the stain had been removed. Spots were then incubated in 20 mM DTT  for 1 h at 37 °C and washed in the same solution without DTT, follow by an incubation in 5 mM iodoacetamide  for 20 min at 37 °C. Gel pieces were dehydrated in 100% ACN for 15 min and the supernatant removed. In-gel digestion was performed by adding 2 µg/ml Trypsin Gold (Promega) at 32 °C for 16 h. Soluble tryptic peptides were analysed by LC–MS/MS using an OrbiTrap LC–MS (Thermo-Scientific). The peptide and fragment ion profiles were extracted and compared to the Mascot database for protein identification.Excised gel spots containing proteins for analysis  ELISA plate reader. Computer software  capable of generating four and five parameters logistic (4/5-PL) curve-fit was used to generate standard curves and the absorbance readings for each sample were used to calculate the CLI concentration from the standard curve.Complement Lysis Inhibitor (CLI) levels in plasma were measured using a human CLI ELISA kit  and the manufacturer’s procedure. Briefly, the plasma samples stored at − 80 °C prior to the assay were thawed on ice and diluted serially in 1× assay diluent to a final dilution of 1/50,000. CLI standard and diluted samples were added to 96-well plates coated with anti-CLI antibodies and incubated at 4 °C overnight. Unbound proteins were removed by washing and then biotinylated antibody (1×) was added to the plate and incubated for 1 h at room temperature. After another washing step, HRP-streptavidin solution was added for 45 min followed by subsequent washing steps to remove any unbound proteins. The amount of bound HRP was assayed by adding 3,3,5,5, tetramethylbenzidine (TMB) solution for 15 to 20 min, and the reaction was stopped by addition of 50 µl 1 M HPlasma cytokines levels were measured to ascertain whether the observed levels of CLI correlated with and/or were regulated by pro-/anti-inflammatory cytokines. The cytokines levels were measured simultaneously using the Bio-Rad seventeen human cytokine Bio-plex bead immunoassay kit, the Bio-Plex 200 System and the manufacturer’s instructions. The amounts of the individual analytes were assayed for each sample using the Bio-Plex Manager 6.0 software (Bio-Rad Laboratories).2-fold change was used to compute differences in protein levels in the disease groups relative to the CC group  and CC. To achieve this, 30 patient samples were pooled together for each disease group Table .Proteomic profiling of crude pooled plasma samples Fig. a detecteCLI changes observed in the discovery cohort were re-examined in a separate validation cohort of individual (non-pooled) samples at disease onset, using an ELISA-based assay Fig. a with obThe low levels of CLI at acute onset observed in the CM group had returned to normal levels in all the patients following convalescence Fig. b within At hospital admission (designated as onset), the CM group showed a significant negative correlation in the level of CLI with the pro-inflammatory cytokines IL-6 and IL-8, and the anti-inflammatory cytokine IL-10 Table . At recoIn the context of a large urban, densely-populated, high-transmission, malaria holoendemic setting, the specific diagnosis of CM is extremely challenging, because of the occurrence of other confounding causes of coma and convulsions, such as meningitis and viral encephalitis in the presence of circulatory malaria parasites. To provide solutions to this challenge, well-defined childhood malaria cohorts from the city of Ibadan, Nigeria were studied using well-established plasma proteomic methods.2D-gel electrophoresis analysis allowed the discrimination of 13 polypeptides corresponding to 9 unique proteins. The pathways involving these proteins are increased coagulation products (fibrinogen subunits), increased acute phase reaction , dysregulation of cholesterol transport (Apolipoprotein IV) and heme-induced oxidative stress , all previously established as hallmarks of malaria syndromes , 17. In This study reveals that level of CLI is depleted in childhood severe malaria, a finding that is consistent with a previous report of low CLI levels in malaria . The dynAnalysis of the discovery cohort used a proteomic approach with pools of plasma to identify proteins with significant differences in abundance. Whilst the approach successfully identified CLI as a protein of interest, it does not allow a detailed analysis of the protein in individual plasma samples or the resolution provided by a specific ELISA-based assay to quantify the protein. Therefore, to analyse the validation cohort, a specific antibody-based assay was used to provide greater specificity and sensitivity and to show that a very low plasma CLI level is uniquely associated with children presenting with CM. The results also show that circulatory CLI levels at acute onset differentiate the CM patient group from patients with other clinical manifestations of malaria as well as malaria-negative encephalopathy-like syndromes with CM-like pathology including convulsions and meningitis with concurrent anaemia. Furthermore, the CLI level in the entire CM patient cohort returned to normal upon recovery, an observation that strengthens the idea of a possible mechanistic role of this multi-faceted protein in the pathogenesis of CM. Moreover, measuring the level of CLI may provide a novel tool to monitor CM disease progression towards convalescence.Complement-lysis inhibitor is highly conserved between species with 70–80% sequence homology. It binds to IgG, lipids, heparin, bacteria, complement, beta amyloid and leptin , 20. CLIComplement-lysis inhibitor expression is induced by TGF-β and TNF , 35. WhiComplement-lysis inhibitor depletion may indicate an enhanced demand for the protein or an accelerated scavenging associated with CM pathology. In addition to the potential value of using depleted CLI as a specific CM biomarker at onset of disease and its reconstitution on convalescence, the study suggests that it will be of interest to evaluate further its significance in CM pathophysiology, for which further studies are needed.In densely-populated urban cities of sub-Saharan West Africa, with high-transmission all-year-round malaria, childhood populations are under a large burden of severe malaria. In this context, where other causes of coma such as meningitis and viral encephalitis may also occur in the presence of circulatory malaria parasites, there is an urgent need for specific cerebral malaria onset and recovery biomarkers. This study shows that circulatory levels of complement-lysis inhibitor (CLI) can play a role as a specific discriminatory biomarker of CM both at admission as well as during convalescence. These findings also suggest that CLI may play a role in the pathophysiology of CM. Further studies are needed to fully evaluate CLI potential across other regions with varying degrees of malaria disease burden.Additional file 1. Supplementary material.


Plasmodium falciparum infections. Thus, to identify key prospective malaria antigens for vaccine development, there is the need to evaluate the associations between malaria antibodies and antibody dependent host factors with more rigorous statistical methods. In this study, different statistical models were used to evaluate the predictive performance of malaria-specific antibodies and host gene polymorphisms on P. falciparum infection in a longitudinal cohort study involving Ghanaian children.Malaria antigen-specific antibodies and polymorphisms in host receptors involved in antibody functionality have been associated with different outcomes of FCGR3B polymorphisms shown to mediate antibody-dependent cellular functions. Malaria antigens studied were Merozoite surface proteins (MSP-1 and MSP-3), Glutamate Rich Protein (GLURP)-R0, R2, and the Apical Membrane Antigen (AMA-1). The models were evaluated through visualization and assessment of differences between the Area Under the Receiver Operating Characteristic Curve and Brier Score estimated by suitable internal cross-validation designs.Models with different functional forms were built using known predictors  and malaria antigen-specific immunoglobulin (Ig) G and IgG subclasses and FCGR3B-c.233C>A genotype and IgG against AMA1 were relatively better compared to the other antibodies and FCGR3B genotypes studied in classifying or predicting malaria risk among children.This study found that the P. falciparum, AMA1 as an important malaria vaccine antigen, while FCGR3B-c.233C>A under the additive and dominant models of inheritance could be an important modifier of the effect of malaria protective antibodies.The data supports the  Plasmodium falciparum and polymorphisms in host FCGR3B gene, encoding the Fc Gamma Receptor IIIB (FcγRIIIB) plays a key role in immunity against malaria [P. falciparum [FCGR3B gene polymorphisms on P. falciparum infection outcome. To assess the performance of the models, this study used Brier scores and area under the receiver operating characteristic curve (AUROC) through appropriate bootstrap cross-validation design.Malaria remains a major public health concern globally and is considered as one of the most prevalent and lethal human infectious diseases among children in sub-Saharan Africa . Despite malaria . The Fcγ malaria , which klciparum . It is clciparum –10. Tradlciparum  developelciparum . Severallciparum –21 have lciparum –10, 22, The identified model was obtained by comparing Brier score estimates and the AUROC curve of several models that integrate malaria antibodies and host gene polymorphisms.P. falciparum parasitaemia plus at least one clinical symptom of malaria, such as vomiting, joint pains, diarrhoea. In this study, the term “Susceptible” and “Protected” are used to represent clinical malaria and no malaria case detection respectively over the study period. The proportion of children that developed malaria in the 1 year follow up was 15.0% (incidence proportion) and there were approximately 1.7 malaria cases per 100 children per month (incidence rate) [FCGR3B polymorphisms . Malaria antigens studied were Merozoite surface proteins (MSP-1 and MSP-3), Glutamate Rich Protein (GLURP)-R0, R2, and the Apical Membrane Antigen (AMA-1). Antibody levels were measured by sandwich ELISA and optical density values converted to antibody units using a four parameter curve fitting program by means of a reference curve on each plate generated by serial dilution of malaria hyperimmune sera [Data used for modelling the risk of malaria was secondary data obtained from a prospective longitudinal malaria cohort study which was conducted from May 2008 to January 2009 among children under 13 years of age in five different communities in the Shai Osudoku (formerly Dangme West) district of Ghana  . These cce rate) . The preThis study includes a summary of the climatic variables at the time of the study to serve as a guide for future studies that may wish to compare their findings to this study. Changes in relative humidity (RH), which is the ratio of the partial pressure of water vapor relative to saturated vapour at the specified temperature was assessed over the study period. The minimum and maximum relative humidity over the study period were 71.0% and 98.0% respectively. The total monthly rainfall ranged between 0 and 273.5 mm with the month of May 2008 recording the highest total rainfall. The total rainfall between June and December 2008 ranged from 19.7 to 125.4 mm. The maximum daily rainfall was recorded in the month of June 2008 (60 mm). There was no rainfall in the month of January 2009. The minimum and maximum air temperature ranged from 19.3 to 36.8 °C.n number of children aged less than 13 years. P predictors of malaria  andLet ith child with baseline characteristics Let R2) using bootstrap cross-validation from 200 bootstrap samples with replacement. In selecting predictor variables to be included in the final model, candidate predictor variables were screened using tools for discriminative and calibration abilities such as the area under the receiver operating characteristics curve, the Brier score and explained variation in predicted risk. The model’s ability to predict accurately was further assessed by means of the calibration plot. According to Gerds et al. [p is the number of predictors (regression parameters including both linear and non-linear and possible interaction terms), p parameters to determine whether any of the predictor(s) is/are associated with log-odds of developing clinical malaria. For the model to be calibrated well for future data, e in subsequent analysis. All models were fitted with R programming software version 3.2.4 with the following specialized packages: Design [p-value of < 0.05 was considered statistically significant.The modelling strategy was to first identify a baseline model out of several other competing models relating to the prevalence of malaria to baseline covariates. Comprehensive discrimination and calibration assessments of the fitted models were explored based on the AUROC curve, Brier score, root mean squared error and the total explained variations in predicted probabilities . Distribution of other predictors and clinical malaria status can be found in Table The study recruited 799 children of which 393 (49.2%) were males and 406 (50.8%) were females. Complete information on candidate predictors was available for 395 children. The missing data were due to children for whom either no or insufficient plasma was available for all antibody measurement. Additionally, children for whom there was no DNA or poor quality DNA that resulted in poor genotyping data were excluded. The overall median age for this study sample was 5.0 years (Interquartile range = 3.0–8.0). The cumulative incidence of malaria was 13% (53 out of 395 children). The bed net use among the children was 40.5%. The analysis of sociodemographic characteristics and baseline biomarkers on the risk of clinical malaria indicated that the cumulative incidence of malaria did not differ significantly among the baseline predictors that were studied  as shown in Table FCGR3B could significantly improve the performance of the PMLE.model after introduction but it was observed that IgGAMA1, IgG1AMA1, and FCGR3Bc.233C>A were better than all the other predictors in relation to their bootstrap cross-validated estimates of relatively higher AUROC, lower Brier score, and and relatively higher R2.The predictive effect of each antibody (IgG and subclasses) and 2, and a smaller Brier Score as previously shown in Table c.233C>A) but with no shrinkage adjustment to the regression coefficients. This model was denoted as the final model with no shrinkage (FMNS). The second model was a baseline model (PMLE.model) together with the three predictors , but with shrinkage of the regression coefficient using van Houwelingen-Le Cessie heuristic estimate. This was done to improve the calibration ability of the model. This was denoted as slope corrected optimism with the van Houwelingen-Le Cessie heuristic (SCOV) model. Finally, a model was fitted and evaluated with the only IgGAMA1, IgG1AMA1 and dominant gene c.233C>A. This was denoted as the no baseline line covariate (NBC) model. The predictive performance indices of these three final models were evaluated and the results showed that the NBC model with only IgGAMA1, IgG1AMA1, and dominant gene c.233C>A predicted malaria incidence better as this model had the highest bootstrap cross-validated AUROC and R2 with a smaller Brier score as shown in Table The selection of IgGAMA1, IgG1AMA1, and dominant gene c.233C>A in the subsequent model building were based on the fact that they had a higher AUROC and RThe prediction performance of these models was explored by examining calibration plots, that is the plots of sensitivity versus (1-specificity) (AUROC). There was no significant difference in their respective areas under the curve before internal validation, although the model with penalty corrected parameter estimates (SCOV model) had a higher AUROC (75.3%). After internally validating these models, it was observed that the AUROC of the model with only IgGAMA1, IgG1AMA1, and dominant gene c.233C>A (NBC model) performed better than the other two models (AUROC = 61.5%)  and  studies , 28–34.  studies , 34–38 wP. falciparum [Traditional statistical methods of estimating odds ratios, relative risk and hazard ratio’s in epidemiological studies to assess associations between antibodies, genetic polymorphisms, and clinical malaria may not adequately determine the performance of each biomarker for predicting the risk of malaria for a child . An antilciparum . In situlciparum . Other ilciparum . The inclciparum  because lciparum –43 but tlciparum . If for Other factors that contribute to the incidence of malaria but were not measured to assess their predictive effect may also be of interest. These include drug resistance in parasites, vector species, parasite strain and insecticide resistance in mosquitoes , 45–48. FCGR3B-c.233C>A genotype and IgG against AMA1 were relatively better compared to the other antibodies and FCGR3B genotypes in classifying or predicting malaria risk among children. The findings support Apical Membrane Antigen 1 as an important malaria vaccine antigen while FCGR3B-c.233C>A under the additive and dominant models of inheritance were also identified as an important modifier of the effect of malaria protective antibodies. Furthermore, the goal of malaria etiological risk factor studies may be quite different from studies where antibodies and host genes are used in classifying a child into high or low-risk groups. Hence the statistical methods between such studies differ to a large extent. If the latter is required, then it would be appropriate to e use model discrimination and calibration indices, such as Brier score, RMSE, and AUROC.This study has found that the


Plasmodium infections in Malaysia over the last four decades.Malaysia has already achieved remarkable accomplishments in reaching zero indigenous human malaria cases in 2018. Prompt malaria diagnosis, surveillance and treatment played a key role in the country’s elimination success. Looking at the dynamics of malaria distribution during the last decades might provide important information regarding the potential challenges of such an elimination strategy. This study was performed to gather all data available in term of prevalence or incidence on A systematic review of the published English literature was conducted to identify malaria distribution from 1980 to June 2019 in Malaysia. Two investigators independently extracted data from PubMed, Scopus, Web of Science and Elsevier databases for original papers.Plasmodium knowlesi incidence rate throughout the year of sampling period. The summary estimates of sensitivity were higher for P. knowlesi than other Plasmodium species for both microscopy and PCR. Nevertheless, the specificities of summary estimates were similar for microscopy (40–43%), but varied for PCR (2–34%).The review identified 46 epidemiological studies in Malaysia over the 39-year study period, on which sufficient information was available. The majority of studies were conducted in Malaysia Borneo , followed by Peninsular Malaysia  and in both areas . More than half of all studies  were assessed by both microscopy and PCR. Furthermore, there was a clear trend of decreases of all human malaria species with increasing Plasmodium species distribution in Malaysia. Malaria cases shifted from predominantly caused by human malaria parasites to simian malaria parasites, which accounted for the majority of indigenous cases particularly in Malaysia Borneo. Therefore, malaria case notification and prompt malaria diagnosis in regions where health services are limited in Malaysia should be strengthened and reinforced to achieving the final goal of malaria elimination in the country.This study outlined the epidemiological changes in  Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Plasmodium falciparum is the most prevalent and causes the highest mortality particularly in the African region  of combination to obtain relevant articles. This systematic review was accorded to the protocol and followed the PRISMA  guidelines ), followed by P. malariae (25% [95% CI 24–26]) and P. vivax (14% [95% CI 14–15]), and lowest for P. falciparum (11% [95% CI 10–11]). Nevertheless, the summary estimate of specificity by microscopy was similar in all species ranged 40–43%.The performance of microscopy and PCR in detecting P. knowlesi with 56% (95% CI 55–57). Whereas, the summary estimate of sensitivity by PCR for P. vivax, P. falciparum and P. malariae were 14% (95% CI 13–15), 11% (95% CI 10–11) and 1.6% (95% CI 1.4–1.9), respectively. On the other hand, the specificities of summary estimate for species-specific were less than 35% with P. knowlesi, P. vivax, P. falciparum and P. malariae were 34.5% (95% CI 34–35), 34% (95% CI 33–35), 16.5% (95% CI 16–17) and 2.3% (95% CI 2–3), respectively.Similar to microscopy, the summary estimate of sensitivity by PCR was highest for P. knowlesi continues to infect a large number of people in remote parts of Malaysia [Malaysia aims to eliminate malaria nationwide by 2020. Although the country has successfully eliminated indigenous transmission of all human malaria species , the incMalaysia , 11, 69.P. knowlesi) in the Peninsular region by 2015 and in the Malaysia Borneo region by 2020 [In this study, malaria incidence caused by human malaria species indicates a decreasing trend from 2006 onwards. This finding is consistent with a recent study by Hussin et al. that showed the decreasing incidence of human malaria notified to the Ministry of Health, Malaysia between 2013 to 2017 [ by 2020 , 17. TheP. knowlesi was the most dominant species, particularly in Malaysia Borneo i.e. East Malaysia. Plasmodium knowlesi cases rose steadily year by year and the incidence rate was highest at 5.909 per 100,000 in 2017. Other than the wide utilization of molecular diagnosis in health facilities, it has been hypothesized that the rise of knowlesi malaria cases in the country was associated with ecological changes, particularly by deforestation [Anopheles balabacensis seems to have been replaced by Anopheles donaldi [Over the past years, malaria species-specific analysis showed that estation , 36, 70. donaldi , 71. Thi donaldi . Besides donaldi .P. vivax and P. falciparum in Malaysia. Plasmodium vivax has been the main cause of human malaria in the country for the past 10 years and remains a health concern today [P. vivax [P. vivax observed in the present study also follows a steady increase in notification rate of P. malariae/P. knowlesi. In fact, over the past decade, a strong inverse correlation was observed between notification rates of P. malariae/P. knowlesi and P. vivax or P. falciparum [P. falciparum or P. vivax infections as P. malariae/P. knowlesi [P. knowlesi as P. falciparum or P. vivax [P. knowlesi and reducing incidence of P. vivax and P. falciparum.It was interesting to note that there was a dramatic reduction in both parasite and incidence rates of rn today . In 2010P. vivax . MoreoveP. vivax . The dralciparum , 72. Thiknowlesi . MoreoveP. vivax . The effPlasmodium detection and species differentiation. The use of microscopy as the sole diagnostic method likely leads to an underestimation of the malaria burden in a specific population [P. knowlesi and P. malariae infections that usually present at densities below the limit for microscopic detection [P. knowlesi infection is commonly misdiagnosed as P. malariae infection and other Plasmodium infections  due to their morphological similarities [Plasmodium species is known to be problematic, this study demonstrates that the increase in notifications is likely to represent a real increase in the incidence of P. knowlesi. In this regard, PCR tests play an important role in order to confirm that the infection is due to knowlesi malaria.Widely, the detection of malaria parasites by light microscopy of Giemsa-stained blood films continues to be the gold standard for malaria diagnosis . It is hpulation , particuetection . This relarities , 10. AltPlasmodium based on clinical samples and not in the population. This is possibly due to logistical difficulties and financial costs to carry the surveys, with most studies conducted in the Malaysia Borneo, where access to household is limited by geographic barriers. Second, our search strategy could have missed potentially eligible studies, because identification of Plasmodium infections were often not the primary target of many epidemiological studies in Malaysia. Third, the small sample size in some studies did not allow the evaluation of possible source of a high variation between studies.A number of caveats should be considered in this study. First, the data used in the included studies were not uniform. The data gathered was solely based on published articles readily available on the internet and did not include data from the ministry of health. Thus, the findings of this study might not reflect the totality of malaria situation in Malaysia. In addition, most studies focused on the distribution of Plasmodium species distribution in Malaysia. Malaria cases shifted from predominantly human malaria especially P. falciparum and P. vivax to P. knowlesi in the early 2000s. Plasmodium knowlesi is now responsible for the majority of malaria cases in the country. Therefore, malaria case notification and interventions in Malaysia should be strengthened and reinforced to achieving the final goal of malaria elimination in the country.This study outlined the epidemiological changes in


Glucose-6-phosphate dehydrogenase (G6PD) deficiency is currently a threat to malaria elimination due to risk of primaquine-induced haemolysis in G6PD deficient individuals. The World Health Organization (WHO) recommends G6PD screening before providing primaquine as a radical treatment against vivax malaria. However, evidence regarding the prevalence and causing mutations of G6PD deficiency in Nepal is scarce.A cross-sectional, population-based, prevalence study was carried out from May to October 2016 in 12 malaria-endemic districts of Nepal. The screening survey included 4067 participants whose G6PD status was determined by G6PD Care Start™ rapid diagnostic test and genotyping.The prevalence of G6PD deficiency at the national level was 3.5% . When analysed according to ethnic groups, G6PD deficiency was highest among the Janajati  and low among Brahman and Chhetri (1.3%). District-wise, prevalence was highest in Banke (7.6%) and Chitwan (6.6%). Coimbra mutation (592 C>T) was found among 75.5% of the G6PD-deficient samples analysed and Mahidol (487 G>A) and Mediterranean (563 C>T) mutations were found in equal proportions in the remaining 24.5%. There was no specific geographic or ethnic distribution for the three mutations.Plasmodium vivax.This study has identified populations with moderate to high prevalence of G6PD deficiency which provides strong evidence supporting the WHO recommendations to screen G6PD deficiency at health facility level before the use of primaquine-based radical curative regimen for  Malaria remains a global problem with an estimated 228 million cases occurring globally and 405,000 deaths in 2018. It is still endemic in 91 countries with about half of the world’s population at risk, particularly those living in lower-income countries , 4.The presence of G6PD deficiency among the population is a huge challenge for malaria treatment due to the associated haemolytic risk with radical treatment using 8-aminoquinolines. G6PD deficiency is the most common human enzyme defect and is mostly asymptomatic. However, G6PD-deficient individuals may suffer from acute haemolytic anaemia (AHA) when exposed to several oxidative triggers, such as fava beans, infections or anti-malarial drugs such as primaquine. AHA is the most common manifestation of this deficiency associated with intra and extra-vascular haemolysis, jaundice and haemoglobinuria with acute renal failure as the most severe outcome –11. FactAbout 400 million people are estimated to be G6PD deficient worldwide , 15–18, Studies on G6PD deficiency among a small number of individuals of Nepalese origin were conducted as early as 1960s in Calcutta . StudiesPlasmodium vivax [P. vivax cases have been recorded in non-endemic areas representing a challenge for malaria elimination efforts. The Epidemiology and Disease Control Division (EDCD) has adopted the World Health Organization (WHO) policy to screen malaria patients for G6PD deficiency in its National Malaria treatment protocol published in 2015 [Nepal has a long history of malaria control programmes starting from 1954  and its um vivax , 29. How in 2015 .Understanding the burden of G6PD deficiency and identifying vulnerable populations is crucial for the success of the malaria control programme in Nepal. In this regard, this study aims to estimate the prevalence of G6PD deficiency and to describe the underlying mutations in areas where malaria transmission is endemic. It is anticipated that the study findings will inform strategic plans for the malaria elimination programme in Nepal.A cross-sectional prevalence survey was conducted among 4067 study participants of 12 malaria-endemic districts of Nepal. Data were collected from May 2016 to October 2016. The study population comprised both male and female participants above 6 years of age and residing in the study districts.http://edcd.gov.np/resource-detail/report-on-malaria-microstratification-2013).The study districts were Banke, Bardiya, Chitwan, Dadeldhura, Kailali, Kanchanpur, Kapilvastu, Makwanpur, Nawalparasi, Rautahat, Sindhuli, and Surkhet. These districts were selected according to malaria endemicity data based on the malaria micro-stratification report 2013 , 54 were identified to be at high risk of malaria based on the malaria micro-stratification report 2013. These VDCs were further divided into clusters and 30 of them were selected using systematic random sampling with probability inclusion proportional to size (PPS) method from the VDC.The field teams visited each selected cluster to prepare a list of households for the study and 132 households were selected by systematic random sampling method from each cluster. From this list, a male participant was selected from the first and second household and a female participant was selected from the third household; this was repeated for every block of three successive households resulting in the selection of a total of (30 × 132) = 3960 participants with a male to female ratio of 2:1. The total final sample size of the study was 4067 due to additional samples taken in some sites.A structured questionnaire was used for data collection, including socio-demographic characteristics. Interviews were conducted in Nepali language by researchers of the same gender of interviewed participants. Screening of G6PD status was done on capillary blood using CareStart™ . CareStart™ retraining was supported by investigators of the Shoklo Malaria Research Unit (SMRU) prior to survey activities. The test was run following manufacturer’s instructions. Test interpretation was based on colour change in the reading window, with a pink purple colour interpreted as ‘G6PD normal’ result and a faint or no colour development interpreted as ‘G6PD deficient’ result. Blood for human genotyping was spotted on filter papers, dried, and stored at − 20 °C.The study variables were socio-demographic variables , previous history of malaria and G6PD screening results. Since the ethnicity and caste classification of Nepal is extremely complex , both thData were collected in the field by district level laboratory staff, peripheral level laboratory and health workers supervised by District Public Health Office (DPHO) team and EDCD team. Data were quality controlled to assure uniformity across all sites.Dried blood spots of subjects who tested deficient were shipped on ice packs to SMRU in Mae Sot (Thailand). DNA was extracted by mini-column  or saponin-chelex method for PCR–RFLP and gene sequencing from exon 2 to exon 13 of G6PD gene. Primer list and PCR conditions are reported in Additional file Survey data were entered in Microsoft Excel and then transported into IBM SPSS version 20. Double entry was done to ensure accuracy. Prevalence of G6PD deficiency was calculated from the pooled number of subjects with deficient or suspected deficient phenotype over the total of subjects included. Allelic frequencies were calculated as the number of mutated alleles  over the total number of alleles . Allelic frequencies among groups were compared by Chi square test. A P < 0.05 was considered significant.The screening of G6PD deficiency was done among 4067 participants. Ten participants whose data were missing were removed from further analysis, thus 4057 participants were included in the final analysis.The demographic characteristics of the participants are summarized in Table The G6PD phenotypic status among the participants is presented in Tables Prevalence of G6PD deficiency was highest in Banke district (7.6%) followed by Chitwan (6.6%), Nawalparasi and Bardiya 4.3%), and Kailali (4.0%)  are shown in Table In the current study, the overall prevalence of G6PD deficiency was 3.5% at the population level, with a wide geographical variation (from nil to 7.6%) and across ethnicities (from nil to 17.6%). This prevalence is consistent with the worldwide prevalence (4.9%), and the prevalence in Asia (4.7%) and across much of the Indian Sub-continent (2–5%) . In a siThe sensitivity and specificity of the G6PD CareStart™ RDT could not be formally assessed in this study; the inconsistencies observed in this context hint to a more general consideration on the necessity to use fully validated and quality-controlled G6PD tests for malaria elimination strategies to be effective.The three G6PD variants found had a broad distribution geographically and among ethnicities in the studied districts. Interestingly, Mahidol is a mutation found mainly in the most western countries of Southeast Asia  andP. vivax [P. vivax radical cure treatment regimens be based on the level of G6PD activity [Primaquine and tafenoquine are the only currently available drugs effective against hypnozoites of P. vivax . The advP. vivax , 11, 42 P. vivax , 43. Theactivity , 45.The findings of the current study have therefore very clear programmatic and policy-making implications. They show that G6PD deficiency is present broadly in the Terai regions of Nepal and is caused by mutations associated to described primaquine-induced haemolysis. Some ethnic groups are more affected but the prevalence of deficiency within the same ethnicity varies widely in different districts. Since the risk of haemolysis due to G6PD deficiency cannot be excluded on the basis of the geographic region or ethnicity alone, G6PD testing will be always necessary before deployment of primaquine. The EDCD in Nepal has provided directives for G6PD screening among the population at health facility level . HoweverAdditional file 1. Tables S1–S5 and Figure S1.


Malaria infection during pregnancy has negative health consequences for both mothers and offspring. Sub-microscopic malaria infection during pregnancy is common in most African countries. We sought to identify factors associated with sub-microscopic placental malaria, and its association with adverse pregnancy outcomes among HIV-negative pregnant women in Dar es Salaam, Tanzania.Plasmodium falciparum DNA by polymerase chain reaction (DNA PCR) amplification in a negative histopathology test. Adverse pregnancy outcomes investigated included low birth weight (birth weight below 2.5 kg), prematurity (live birth below 37 weeks), and small-for-gestational-age (SGA) . Weighted baseline category logit, log-binomial, and log-Poisson models were used to assess factors associated with placental malaria, and its association with adverse pregnancy outcomes.We recruited a cohort of pregnant women during their first trimester and assessed for the occurrence of placental malaria and pregnancy outcomes. The follow-up was done monthly from recruitment until delivery. Histopathology placental malaria positive results were defined as the presence of malaria pigment or parasitized erythrocytes on the slide (histology-positive (HP)), and the sub-microscopic placental infection was defined as positive P = 0.03) and in women who were anemic . Women who were underweight had reduced odds of sub-microscopic placental malaria infection . Women who were overweight/obese had 1.48 times higher the odds of HP placental malaria compared to normal weight . HP placental malaria infection was associated with an increased risk of SGA births . In contrast, the sub-microscopic infection was associated with a reduced risk of SGA births . Placental malaria was not associated with low birth weight or prematurity.Among 1115 women who had histopathology and DNA PCR performed, 93 (8%) had HP placental infection, and 136 (12%) had the sub-microscopic placental infection. The risk of sub-microscopic placental malaria was greater in women who did not use mosquito prevention methods such as bed nets, fumigation, or mosquito coils (odds ratio (OR) = 1.75; 95% confidence interval (CI): 1.05–2.92; Malaria prevention methods and maternal nutrition status during early pregnancy were important predictors of sub-microscopic placental malaria. More research is needed to understand sub-microscopic placental malaria and the possible mechanisms mediating the association between placental malaria and SGA. Plasmodium falciparum  infections each year in sub-Saharan Africa [Worldwide, 75,000 to 200,000 infant deaths every year are attributable to malaria infection during pregnancy (MiP) . Approxin Africa , 2. MiP n Africa –3. Furthn Africa , 5.Histopathology examination of stained placental biopsies is considered as the gold standard for diagnosis of placental malaria (hereafter referred to as histology-positive (HP) placental malaria), but this test is impractical or not available in some settings , 7. PrevPeripheral sub-microscopic malaria is strongly associated with placental malaria and low maternal hemoglobin levels , 15, butLimited evidence is available on the association between adverse pregnancy outcomes and placental malaria infections (particularly sub-microscopic variant) in tropical African cities such as Dar-es-Salaam, Tanzania, where the malaria burden has been significantly reduced, but low-level transmission remains. We, therefore designed a study with the objectives of identifying factors associated with sub-microscopic placental malaria and examined its association with LBW, prematurity, and small-for-gestational-age (SGA) births among HIV-negative pregnant women in their first and second pregnancy in Dar Es Salaam, Tanzania.We used data from a trial that examined the effect of prenatal vitamin A and zinc supplements on malaria in pregnancy among women in their first or second pregnancy . In brie2 (overweight/obese) [Socio-demographic and clinical variables were assessed at recruitment (baseline); these include age, parity, twin pregnancy status, years of education, marital status, gestational age, employment status, and use of fumigation, mosquito coil or bed nets at night. Selected possessions were asked about, from which a Filmer-Pritchet wealth index was computed . Body mat/obese) , and hemt/obese) .P. falciparum DNA by PCR amplification in samples negative for histopathology test. Details on the two methods have been previously described [Placental malaria was evaluated using both histopathology and DNA PCR. HP placental malaria was defined as the presence of malaria pigment or parasitized erythrocytes on the slide, and sub-microscopic placental malaria infection was defined as positive escribed . BrieflyP. falciparum-specific [Plasmodium 18S rRNA genes [For the nucleic acid studies, tissue was stabilized in RNA later  and homogenized. Genomic DNA was extracted using DNeasy (Qiagen). Taqman® qRT-PCR  was used for amplification using published primer and probe sets (specific  and geneNA genes ). The tiNA genes . At recrth percentile for the exact day of gestation and sex based on INTERGROWTH-21st standards [Adverse pregnancy outcomes investigated in this study were LBW (birth weight below 2.5 kg), SGA births  as p-values less than 0.2 were considered potential variables for the multivariable models . Other covariates were included based on prior knowledge of the topic . Due to the positive correlation between maternal age and parity in this population (ρ = 0.3 with P < 0.0001) maternal age was not included in any multivariable models. The trial regimens were forced into the multivariable models. Lastly, results were presented as odds ratio (OR) and their 95% confidence intervals.Baseline continuous variables were presented as medians and their interquartile ranges (IQR) and categorical variables as numbers and their percentage. To assess factors associated with placental malaria, baseline category logit regression models were used. Since we were unable to obtain placental samples for over 40% of women recruited for the original study due to changes in delivery plans, we weighted observations to account for non-response bias by the inverse of the probability of women having placental samples (for both tests) as determined by a multivariable logistic regression model . In builIn each outcome, the distributions of outcomes by exposure were presented in numbers and their percentage. To investigate the relationship of placental malaria with LBW, premature birth, and SGA birth, weighted log-binomial models, and log-Poisson models (with weights obtained from logistic regression models as described above) were used, , 28. ForAnalyses were performed among women with both tests done. In this case, placental diagnoses were classified as HP placental malaria, sub-microscopic positive placental malaria when histopathology was negative, and negative for both diagnoses. The level of significance was 0.05, and all data analyses were conducted in SAS version 9.4 .The original trial recruited 2500 pregnant women in their first trimester Fig. . Of thesAmong 1404 women, there were 1115 (79%) pregnant women with sufficient placenta samples for both diagnostic methods, 886 (80%) tested negative by both methods, 93 (8%) had HP placental malaria, and 136 (12%) had sub-microscopic placental malaria.The distribution of baseline characteristics was presented in Table P = 0.05 and OR = 0.49; 95% CI: 0.28–0.86; P = 0.01 respectively). Compared to those with a higher education level, pregnant women with the least education (0–7 years of education) had an increased odd of sub-microscopic placental malaria . Similar findings for HP placental malaria infection were observed but estimates were not stable due to the low number of outcomes. Pregnant women who did not use mosquito prevention methods such as fumigation, mosquito coil, or a bed net at night had a 75% increased risk of sub-microscopic placental malaria .Results of the multinomial model for factors associated with placental malaria are summarized in Table P = 0.001). In contrast, being overweight/obese was associated with increased odds of HP placental malaria . Compared to pregnant women who were not anemic in the first trimester, anemic pregnant women had increased odds of sub-microscopic  and HP placental malaria .Being underweight was associated with reduced odds of sub-microscopic placental malaria compared to normal BMI .The prevalence of LBW was 8% among HP positive women and 5% among sub-microscopic positive women Table . LBW wasP = 0.07), while sub-microscopic infection was associated with reduced risk of SGA births .A high prevalence of SGA births was found in women with HP placental malaria (23%), while women with sub-microscopic placental malaria had a low prevalence of SGA births (12%). Compared to women with negative results for both tests, HP placental malaria infection was associated with increased risk of SGA births  [P. falciparum immune responses among primiparous women showed low-to-medium pregnancy-associated malaria IgG concentration among women with acute or chronic MiP, which was an indication of the development of the immune response to infection [Our findings that sub-microscopic placental malaria was associated with a reduced risk of SGA birth was similar to results from a study done in Benin  despite  A (CSA) . High le A (CSA) . Further A (CSA)  found th A (CSA) , the relnfection . On othenfection . Sub-micnfection , we alsoBeing underweight weakens antibody-mediated anti-malarial immunity . This stWe also noted the increased odds of HP placental malaria in women classified as overweight/obese. This finding was similar to that of a previous study done in Sweden , which fBeing anemic during the first trimester was associated with an increased risk of sub-microscopic placental malaria, consistent with a study done in Malawi , but incIntermittent preventive treatment in pregnancy (IPTp) with SP is among the strategies used for preventing adverse pregnancy outcomes associated with MiP in Tanzania , and it n = 41). DNA PCR diagnosis for placental malaria, though highly sensitive compared to histopathology, may detect both viable, and non-viable or gametocytes [To our knowledge, our study is one of the first large studies to investigate the associations of placental malaria and adverse pregnancy outcomes among women in their first and second pregnancy in urban Tanzania. However, the study has some limitations first, placental malaria was diagnosed at delivery. Establishing the temporal relationship between malaria and baseline characteristics such as maternal anemia and underweight is therefore challenging, especially if malaria infection may have happened before enrolment. However, past infections diagnosed at delivery are informative with respect to the presence of MiP . We alsoetocytes , which metocytes . It has etocytes . Therefoetocytes , 57. HowIn summary, social determinants such as low educational level may influence the understanding and interpretation of anti-malaria messages delivered at antenatal clinics. It is crucial that these programs tailor their messages and campaigns such that even those with low or no education are well informed. Further, even though over 90% reported the use of at least one malaria preventive measure, more effort should be directed to pregnant women who do not use recommended preventive measures. The integration of nutrition services that target both extremes of the malnutrition spectrum is warranted. Lastly, further research is also needed to understand sub-microscopic placental malaria and the underlying malaria immune response mechanisms that might be linked with the protective effect of SGA births among those infected.


Plasmodium niches outside the liver possibly represent a major source of hypnozoite-unrelated recrudescences in malaria. Maurizio Ascoli, an Italian physician and scientist, suggested that infection was maintained as a result of the persistence of endoerythrocytic parasites in the circulatory bed of some internal organs, mainly the spleen. This would explain a proportion of the recurrences in patients, regardless of the Plasmodium species. Ascoli proposed a method that included the co-administration of adrenaline, in order to induce splenic contraction, and quinine to clear expelled forms in major vessels. Driven by controversy regarding safety and effectiveness, along with the introduction of new drugs, the Ascoli method was abandoned and mostly forgotten by the malaria research community. To date, however, the existence of cryptic parasites outside the liver is gaining supportive data. This work is a historical retrospective of cryptic malaria infections and the Ascoli method, highlighting key knowledge gaps regarding these possible parasite reservoirs.Cryptic  Plasmodium vivax. In fact, the discovery of a dormant exoerythrocytic stage, the hypnozoite , a term coined in relation to studies on other non-plasmodial apicomplexan parasites [P. vivax parasites in the bloodstream to an activation of quiescent hepatic hypnozoites [The possible existence of malaria parasites outside red blood cells (RBCs) has been considered since the very beginning of malariology –3. It isarasites , was madnozoites .P. vivax and Plasmodium ovale. This biological characteristic makes P. vivax a major cause of ongoing febrile illness in endemic areas of Asia, Central and South America, and the horn of Africa [P. vivax from different geographical areas are thought to exhibit varied relapse timing. In tropical regions, strains relapse quickly (3–6 weeks), whereas those in temperate regions do so more slowly (6–12 months) [Malaria relapses occur at variable times after infection and represent a major hurdle in malaria epidemiology . A relapf Africa . Strains months) , 9. Acti months) . HoweverP. vivax and P. ovale malaria and has played a prominent role in several successful elimination campaigns. The long-acting tafenoquine is a recently FDA-approved 8-aminoquinoline, which opens venues to an easier radical cure regimen with a single dose [ Plasmodium liver-stage models and the possibility of using humanized mouse models engrafted with functional human cells and tissues could facilitate understanding of Plasmodium liver-stage biology, further enabling the search for compounds that kill hypnozoites or disrupt their quiescence [The achievement of malaria eradication is thought to be unlikely without the development of effective hypnozoiticides. Primaquine is routinely used to prevent relapses of gle dose . The avaiescence –14.Plasmodium, including non-relapsing species, in the peripheral blood of persons who remain asymptomatic for long periods of time. For instance, although Plasmodium malariae is thought to lack the ability to relapse from dormant liver stages, it is well known for possessing a capacity for “dormancy”, lasting for decades in a well-documented clinical case [Plasmodium falciparum malaria up to at least a decade after people have left endemic regions [P. falciparum infection is also related to transfusion-transmitted malaria in non-endemic areas, transferred from asymptomatic individuals years after they were in a malarious area [P. falciparum maintains itself over extended periods of time within human blood and bone marrow in order to bridge periods between transmission times. Moreover, a hypothesis compatible with the continuity concept of biological evolution has been proposed, in which relapse may potentially be caused by all Plasmodium spp. [Plasmodium spp. are quantitative rather than qualitative. Plasmodium vivax causes relapses most frequently, whereas other species, such as P. ovale and sometimes P. malariae do so less often, and P. falciparum rarely causes relapses [The existence of non-hypnozoite stages lasting longer than the acute malarial disease has been suggested in several studies. In fact, in endemic areas it is not unusual to find cal case . Moreove regions –19. Crypous area . Shanks ous area  suggestsium spp. . Accordirelapses .P. falciparum can persist as a dormant blood-stage form [P. falciparum occur in the bone marrow [Some evidence of merozoite sequestration and persistence as cryptic infections in extrahepatic and non-bloodstream sites can also be found in the literature. Although prompt treatment of women with peripheral parasitaemia during pregnancy limits placental pathology , a studyage form . In addiage form , 27. Fure marrow . Interese marrow . Histoloe marrow .P. vivax, even though the presence of parasites in the bone marrow was first acknowledged in 1894 [P. vivax in the bone marrow was reported in a clinical case with both gametocytes and schizonts readily detected [P. vivax infections in splenectomized monkeys also revealed that the bone marrow is a niche for parasites during active infections [In the case of  in 1894 , studies in 1894 , 33. Yet in 1894 . More redetected . In addifections . In splefections .Plasmodium berghei and Plasmodium yoelii infections in mice, where a small fraction of the sporozoite inoculum remains in the skin and begins to develop into exoerythrocytic forms not eliminated by primaquine and which can persist for days [Plasmodium chabaudi experimental infections, dendritic cells were shown to hold infective parasites capable of inducing new infections upon injection into naïve mice [P. berghei within the spleen has also been demonstrated [Non-bloodstream sites of parasite occurrence have also been described in for days . Therefofor days , 40. Morïve mice . Adherennstrated , 43. AltPlasmodium-parasitized RBCs during malaria attacks [Plasmodium is the most common cause of splenomegaly, albeit variably. Splenic enlargement is a common clinical characteristic of malaria, estimated to occur in 70–80% of acute cases with the size of the spleen generally normalizing after anti-malarial treatment [The spleen is a complex organ that is perfectly adapted to selectively filtering and destroying senescent RBCs, infectious microorganisms and  attacks , 45. In reatment . In areareatment .Plasmodium was first seen in avian malaria. MacCallum, in 1898, observed developmental stages of Plasmodium relictum in the spleen of infected birds [Evidence of splenic exoerythrocytic reproduction of ed birds . By the ed birds .Plasmodium strains [In the early twentieth century, experiments suggested a role for the human spleen as a reservoir of RBCs (and other ‘figured elements’ of the blood), establishing the known theory of erythrocyte reservoirs in the spleen , 51. Acc strains , 53, theP. vivax patient [P. vivax infection was reported. Histological analyses revealed the presence of large numbers of intact P. vivax–infected RBCs in the splenic cords [Indirect evidence of human malaria infections in the spleen has been suggested by clinical observations of the Hackett’s index, the hyper-reactive malarial splenomegaly syndrome (HMS) and clinical cases of untreated spontaneous spleen ruptures. Firstly, for epidemiological studies, the average size of enlarged spleens was a very helpful malariometric index (Hackett’s index), which was calculated from the frequency distribution of five classes of enlarged spleen and ranged from non-palpable on deep inspiration to lowest point palpable in the right iliac fossa . Since s patient . More reic cords .Fig. 1ThWhat happens in spleens of humans seemed clarified by the clinical observations of the Italian physician Maurizio Ascoli   [P. vivax prevails amongst Duffy-positive population, an impressive reduction in mortality was attributed to quinine-adrenaline treatment, with a further decrease in the incidence in malaria severity [P. falciparum and P. vivax endemic areas, spleen enlargement seems to be more pronounced in the latter infection [Unfortunately, the effectiveness of the method in patients infected with different resolved . In the fection) . It is oseverity . In Vietseverity , an expeseverity , 85. Altnfection , thus suPlasmodium.Safety concerns led to strong debates among clinicians early in the adoption of the treatment worldwide . The actPlamodium niches during asymptomatic chronic infection is warranted. Whether Ascoli’s theory was right is difficult to determine because it was never tested in experimental models, and clinical trials nowadays would be considered unethical due to the severe side effects of adrenaline. Furthermore, there might have been a bias toward publication of only positive experiences. However, all data generated by this worldwide used technique could provide useful insight into cryptic Plasmodium stages, particularly P. vivax, which might have therapeutical implications when pursuing elimination in the field. As stated by Aluízio Falcão Filho, the Brazilian writer, in his novel ‘The journalist, the writer and the aviator’: ‘The past is unpredictable’.Asymptomatic carriers of malaria parasites are a major challenge for malaria elimination. The old and new evidence reviewed here indicates that cryptic niches outside the liver, in particular in the spleen, can represent a major source of hypnozoite-unrelated recrudescences. It is noteworthy, however, that there is no direct evidence that adrenaline disrupts the spleen in a manner that redistributes the anatomic locations of any parasites residing in them. Furthermore, no information on the impossibility of quinine to reach spleen niches as much as the peripheral blood is available. Thus, the results of the Ascoli method should be interpreted with caution. Nevertheless, they indicated the spleen as a major parasite reservoir and new evidence is indicating that parasites are able to create cryptic infections in this organ as well as in the bone marrow. Moreover, in cases of non-relapsing malaria, evidence from human infections and animal models indicates that these parasites have also evolved cryptic niches that are associated with chronic infections. The source of such recrudescent parasites is controversial and many key knowledge gaps concerning cryptic niches remain to be addressed Box . In addi


Plasmodium vivax, once considered benign species, is recently being recognised to be causing severe malaria like Plasmodium falciparum. In the present study, the authors report the trends in malaria severity in P. vivax among patients from a Delhi government hospital. The aim of the study was to understand the disease severity and the burden of severe vivax malaria.A hospital based study was carried out from June 2017 to December 2018 at a tertiary care centre from Delhi, India. Patients were tested for malaria using peripheral blood smear (PBS) and/or rapid malaria antigen test (RMAT). The severe and non-severe vivax malaria categorization was done as per the WHO guidelines. Sociodemographic, clinic and paraclinical data were collected from patients and their medical records.P. vivax infection, 22 (10.7%) had P. falciparum infection and six (2.9%) had mixed infection with both the species. Out of 177 P. vivax cases included in this study one or more manifestations of severe malaria was found in 58 cases (32.7%). Severe anaemia (56.9%), jaundice (15%) and significant bleeding (15%) were the most common complications reported in most of patients, along with thrombocytopenia.Of the 205 patients, 177 (86.3%) had P. vivax pathogenesis. The spectrum of complications and alterations in the laboratory parameters in P. vivax clinical cases also indicate the recent shift in the disease severity.In this study, it is evident that vivax malaria is emerging as the new severe disease in malaria patients, a significant shift in the paradigm of  Plasmodium vivax is solely responsible for about 50% of the reported malaria cases . A written informed consent was obtained from each study participant or guardian of the wards, and confidentiality of the participant’s test results was ensured throughout the research. Each study participant was given a unique numerical code in order to streamline the data collection. The inclusion and exclusion criteria of the study were as following:Patients presenting with undifferentiated fever for minimum five days with clinical suspicion of malaria.Patients whose blood sample was positive for malaria by peripheral blood smear (PBS) and Rapid malarial antigen test (RMAT), with or without clinical suspicion of malaria.Availability of informed written consent and completed performa.Non- availability of informed written consent.Patients positive for any other illness other than malaria.2/d2 where N is the minimal sample size; p is the prevalence of severe vivax malaria (62.9%) reported previously [The minimal sample size required for the study was computed using the following formula of Lorentz: N = p (1-p) zeviously ; d the aA detailed clinical history of the consenting patients was taken and a thorough physical examination was performed. Laboratory investigations were performed to establish the diagnosis of malaria and assess the severity of the disease in these patients.P. falciparum, and (2) Pan malarial lactate dehydrogenase (p-LDH), produced by all malarial species. Giemsa stained thick and thin PBS were examined for the identification of species and counting of malarial parasites, respectively. PBS and RMAT were performed by skilled operators to ascertain the quality of sample. RMAT results were classified as valid (positive or negative) and invalid [P. vivax infections, 2) P. falciparum infections and 3) mixed infections of P. falciparum and P. vivax. The classification as severe and non-severe vivax malaria was done following WHO guidelines [P. vivax and P. falciparum [PBS and RMAT were the diagnostic methods used to detect malaria species in blood samples of patients. The RMAT used in the study is designed to detect two malarial antigens: (1) Histidine rich protein-2 (HRP-2), a protein specifically produced by  invalid . The par invalid . On the idelines . The patlciparum . Patientlciparum .Data regarding age, gender and pregnancy status were collected using an ad hoc investigation form conceived for the need of the study.P. vivax. Appropriate serological tests were also performed to exclude enteric fever, dengue, chikungunya, scrub typhus and leptospirosis.The venous blood samples were collected in ethylenediaminetetraacetic acid (EDTA) anticoagulant vacutainer tubes for serological, haematological and biochemical investigations. Complete blood counts, coagulation profile tests, blood glucose, blood urea, serum creatinine, serum electrolytes, serum bilirubin, serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT) were measured for patients positive for 10-transformed before statistical analysis. Pearson’s chi square and Fisher’s exact tests were used to compare percentages, while unpaired sample t test was used to compare mean values. Significance was set at p-value < 0.05.Data were keyed into an Excel spreadsheet  and analysed using the statistical package for social sciences v16 for Windows . Qualitative and quantitative variables were expressed as frequency (percentages) and mean ± standard deviation (SD), respectively. Confidence interval at 95% (95% CI) of percentages was computed. Parasitemia values were logP. vivax infection, 22 (10.7%) had P. falciparum infection and six (2.9%) showed mixed infections as diagnosed by PBS and RMAT  and 77 adults. The age of the participants ranged from 2 to 58 years with a median age of 13 years. The number of male patients was higher in both age groups i.e., below 14 years and above 14 years  in Delhi and other neighbouring regions. Maximum number of cases were recorded in October 2018 (65 cases) followed by September 2018 (51 cases). A total of 205 confirmed cases of malaria were enrolled in the study, after inclusion criteria were satisfied. Of the 205 patients, 177 (86.3%) were positive for P. vivax cases, fever was present in all the patients. Of these, 119 patients (67.2%) had uncomplicated malaria while the remaining 58 (32.7%) were suffering from complicated malaria. In addition, 41 of these 58 patients, had only a single complication, while 17 cases presented > 1 or multiple complications  CNS infections. Abnormal bleeding was observed in 11 patients (6.2%) in whom epistaxis, hematemesis, haematuria and melena were also observed. On physical examination 50 patients (30.5%) had hepato-splenomegaly, 4 individuals (23.2%) showed pallor, while 15 (47%) were icteric. Apart from these observations, the other clinical findings were oedema, rashes and hypovolemic shock. Clinical details of the patients are shown in Table All the patients who presented with either seizures or altered sensorium were suspected to be having cerebral malaria attributed to tients 6.% in whomThe comparative analysis of the prevalence of clinical features in patients diagnosed with severe and uncomplicated malaria revealed statistically significant variations between both groups (P < 0.05). Prevalence of these features were significantly higher in patients with severe complications in comparison to their counterparts diagnosed with uncomplicated malaria Table .In patients with severe manifestations hepatosplenomegaly was observed to be significantly higher  in comparison to patients presenting uncomplicated malaria 4.2%). The same pattern was observed for altered sensorium , jaundice , hypovolemic shock , oedema   were also included in the study group. Later, during the study intra-uterine deaths were reported in these pregnant patients as neither foetal movements were felt, nor foetal heart sounds were heard and with no other co-morbid conditions. There were 3 cases with dengue co-infections with 1 patient had enteric fever co-infection. No mortality was observed during the course of this study.Pancytopenia was seen in 15 cases (8.5%) and packed red cells infusion was given in 2 patients. Deranged liver enzymes and high concentration of serum bilirubin was observed in 24.3% of All laboratory parameter levels significantly varied according to the severity of vivax malaria Table . Levels The evolution of different haematological features according to vivax parasitemia levels is depicted in Table P. falciparum infection, in which increased sequestration of RBC in the microvasculature and massive haemolysis lead to complications like cerebral malaria (CM), renal dysfunction, hepatic dysfunction and ARDS [P. falciparum through the cytoadherence phenomenon, whereas little is known about molecular basis of CM due to P. vivax [P. vivax to also elicit cytoadherence [P. vivax infections include CM, hepatic dysfunction, acute renal injury, severe anaemia, ARDS, splenic rupture and multiple organ failure [P. vivax to elicit severe complications [The clinical course of malaria depends on several host and parasite-related factors and it manifests with a multitude of signs and symptoms. The disease spectrum progresses from stage of asymptomatic parasitemia to uncomplicated malaria, severe malaria and leading to death in some cases. Severe or complicated malaria is more often associated with and ARDS . The bioP. vivax . There adherence , 22. Beydherence . Recentl failure . Variousications .P. vivax accounts for one third of all malaria cases detected in India, with high prevalence in the urban areas [P. vivax immunity observed frequently in children may explain a higher burden of malaria infection in them as found in P. falciparum [an areas . In the lciparum . A totallciparum , 25–32. lciparum –36.TableP. vivax malaria  in severe malaria group, and 53.8% 64/119) in uncomplicated malaria group as reported also from other regions in the country (Table 2/58 in s9 in uncoAnaemia was observed in all three categories of mild, moderate and high parasitemia patients, but a significant relationship between haematological changes and parasitemia levels could not be established, unlike previous studies . This coP. vivax malaria in several areas, our observations provide an insight into increased number of clinical issues related to the severity of P. vivax malaria (Table P. vivax infections is poorly understood, in addition; there is paucity of sufficient data on this aspect. A further large-scale study is required to determine the underlying pathogenesis of the severity of the disease, and the degree to which it is related to the emerging multidrug resistance in P. vivax malaria. Thus, there is an urgent need to re-examine the clinical spectrum and burden of P. vivax malaria in our settings, so that adequate control measures can be implemented against this disease. Despite effective reduction in the transmission, there is an increase in the number of complicated P. vivax cases which significantly contributes to the severity and morbidity in malaria and every effort should be made to reduce or eliminate the malaria burden. We should also target towards reducing infections due to P. vivax along with P. falciparum in regions where both these species coexist.Considering the re-emergence of ia Table . BiologiP. falciparum and P. vivax due to low sample size of P. falciparum cases in the present study. Second, comorbidities such as malnutrition eliciting haematological disorders along with the aetiology of anaemia cases were not investigated. Finally, study findings drawn from low sample size of vivax patients attending this heath facility cannot reflect the whole malaria situation in the Delhi State.This study has few limitations. First, we were unable to make comparison of severity signs between P. vivax is emerging as an important cause of malaria and morbidity in several endemic and non-endemic regions in India. In addition, the present study demonstrates the important role of P. vivax in complications observed in malaria disease pathogenesis. Considering the observed trend in the disease severity from the reported study there is need to strengthen the control programs targeted towards disease severity due to P. vivax.It is notable that


Plasmodium knowlesi is a simian malaria parasite currently recognized as the fifth causative agent of human malaria. Recently, naturally acquired P. cynomolgi infection in humans was also detected in Southeast Asia. The main reservoir of both parasites is the long-tailed and pig-tailed macaques, which are indigenous in this region. Due to increased urbanization and changes in land use, there has been greater proximity and interaction between the long-tailed macaques and the general population in Singapore. As such, this study aims to determine the prevalence of simian malaria parasites in local macaques to assess the risk of zoonosis to the general human population. Screening for the presence of malaria parasites was conducted on blood samples from 660 peridomestic macaques collected between Jan 2008 and Mar 2017, and 379 wild macaques collected between Mar 2009 and Mar 2017, using a Pan-Plasmodium-genus specific PCR. Positive samples were then screened using a simian Plasmodium species-specific nested PCR assay to identify the species of parasites  present. All the peridomestic macaques sampled were tested negative for malaria, while 80.5% of the 379 wild macaques were infected. All five simian Plasmodium species were detected; P. cynomolgi being the most prevalent (71.5%), followed by P. knowlesi (47.5%), P. inui (42.0%), P. fieldi (32.5%), and P. coatneyi (28.5%). Co-infection with multiple species of Plasmodium parasites was also observed. The study revealed that Singapore’s wild long-tailed macaques are natural hosts of the five simian malaria parasite species, while no malaria was detected in all peridomestic macaques tested. Therefore, the risk of simian malaria transmission to the general human population is concluded to be low. However, this can be better demonstrated with the incrimination of the vectors of simian malaria parasites in Singapore. Malaria is one of the most important vector-borne diseases in the world. In Southeast Asia, the incidence of human malaria infections caused by simian malaria parasites that originate from macaques has been increasing in the last decade, possibly due to closer proximity between the macaque and human population as a result of deforestation. In Singapore, urbanization and change in land use have resulted in closer proximity and interactions between macaques and the general human population. In order to assess the risk of potential simian malaria zoonosis, 1039 macaques’ blood samples were screened in this study for simian malaria parasites. Our results showed that simian malaria parasites were only detected in macaques living deep in the forest within the military protected zone, while macaques living close to human habitations are malaria-free. This study illustrated a low risk of zoonotic transmission of simian malaria parasites to the general population in Singapore. Plasmodium knowlesi being an emerging zoonotic pathogen in many Southeast Asian countries [Macaca fascicularis) and pig-tailed macaques (Macaca nemestrina) [P. knowlesi infections in humans were initially thought to be rare. However, a paradigm shift occurred in 2004 when a large cluster of human infections was detected in the Kapit division of Sarawak, East Malaysia [P. knowlesi is morphologically similar to Plasmodium falciparum and/or Plasmodium malariae species during the early ring and late trophozoite stages, respectively [P. knowlesi infection using molecular techniques. With an increased awareness of P. knowlesi infections in humans and the availability of determinative molecular assays, naturally acquired human knowlesi cases were subsequently reported in many parts of Southeast Asia: Myanmar [Plasmodium knowlesi has since been proposed as the fifth human malaria [Simian malaria parasites have been playing an increasing role in the human malaria burden , with Plountries –13. The estrina) . As mostectively . These c Myanmar ,16, Laos Myanmar , Vietnam Myanmar ,17, Camb Myanmar ,18,19, P Myanmar , Indones Myanmar ,22, Nort Myanmar , and Phi malaria ,25.P. knowlesi infection after a period of training in a forested area in Singapore. This prompted a fever monitoring and surveillance programme for all military personnel who had spent periods of time in this area, and led to the detection of five additional cases of P. knowlesi infections; four cases in 2007 and one in 2008 [et al [Macaca fascicularis) caught in the forest where transmission was thought to occur, were infected with P. knowlesi. Phylogenetic analysis inferred from the Plasmodium circumsporozoite protein (csp) gene of the parasite revealed identical genotypes between the human cases and the infected macaques, suggesting that local human knowlesi cases had acquired the infection in the vicinity where these monkeys were found.Singapore reported its first naturally acquired human zoonotic malaria in 2007 . The cas in 2008 . All theP. knowlesi in Singapore is of no surprise as this parasite was first discovered in a long-tailed macaque exported from Singapore to India in 1931 [P. knowlesi from long-tailed macaques, 80 years since its discovery, suggests the continuous and ongoing transmission of P. knowlesi in the local macaque population. The report of the locally acquired human knowlesi cases and the subsequent detection of P. knowlesi in wild macaques demonstrated the risk of zoonotic transmission of P. knowlesi in Singapore. Apart from P. knowlesi, long-tailed macaques in many Southeast Asian countries have shown to harbour P. cynomolgi, P. inui, P. fieldi and P. coatneyi [Plasmodium infections are on a decline, the bulk of malaria infections were attributed to P. knowlesi [Plasmodium parasites, of which 60% were P. knowlesi [The finding of  in 1931 . The reccoatneyi ,28–32, aknowlesi . Simian knowlesi ,35.2. Due to land scarcity, the majority of the forest cover had to make way for urbanization and industrialization. Currently, only the forests within the Bukit Timah Nature Reserve, Central Catchment Nature Reserve and the military training areas are the major forest reserves left. There are little buffer zones between the forest patches of the Nature reserve and human habitations, except for roads or highways separating the two. On the other hand, forest patches within the military training zone are generally away from the human settlement as it is a state-declared protected place with restricted access to the public.Singapore is a small island state of area approximately 721.5 kmMacaca fascicularis, the most predominant macaque species in Singapore, inhabits in the remaining patches of forests locally. In a population census report, it was noted that 70% of the macaque population resides in the Bukit Timah and Central Catchment Nature reserve [ reserve ,37. Due  reserve ,39, thisSample collection in this study was approved by the Singapore Armed Forces Joint Medical Committee and the DSO National Laboratory’s Institutional Animal Care and Use Committee.Macaques in this surveillance study were categorized into two groups–“wild” and “peridomestic”. “Wild” macaques were caught in forested areas within the military protected zone in the Western Catchment area see , where lOn the other hand, “peridomestic” macaques were found in close association with human habitations and were habituated to human presence and their food provision. Due to the closer macaque-human interactions and conflicts which arose, they were trapped and removed from various parts of Singapore see . Six hunAll blood samples in this study were not collected systematically; the collection was based on convenience sampling from the two operational programmes. All macaques trapped were sent to Agri-Food and Veterinary Authority of Singapore (AVA) for age, sex, and blood sampling. The age of the macaques was estimated based on dentition ; those aDNA was extracted from 200 μL of whole EDTA blood using DNeasy Blood and Tissue kit  according to the manufacturer’s instructions. Purified DNA was stored at −20°C until further use.Plasmodium PCR assay primers which target the 18S ribosomal RNA genes  [2 , 200 mM of each dNTP , 0.25 μM of each primer and 1.25U of GoTaq DNA polymerase . The PCR was carried out using Veriti Thermal Cycler  with cycling conditions of 95°C for 4 min, 44 cycles of amplification at 95°C for 30 sec, 57°C for 30 sec, 72°C for 30 sec, followed by a final extension step of 2 min. Samples with Plasmodium parasites’ DNA will have an amplification product size of 188 bp. Summary of positive controls used in this study can be found in Initial screening for the presence of malaria parasites was conducted using a Pan-AGA -3’) . PCR ampPlasmodium parasites present were determined using published primers designed to target the 18S small sub-unit ribosomal RNA genes [For samples tested positive for malaria parasites, the species of NA genes ,41–43,44All PCR reactions were carried out in Veriti Thermal Cycler  and PCR products were analyzed by 2% agarose gel electrophoresis.The linear trend of malaria parasites prevalence over the years was tested by regress function using STATA 14.2 .Plasmodium screening conducted on 379 wild and 660 peridomestic long-tailed macaques revealed that 80.5% (305/379) of the wild macaques were infected with malaria parasites, while none of the 660 peridomestic macaques were infected , followed by P. knowlesi (47.5%), P. inui (42.0%), P. fieldi (32.5%), and P. coatneyi (28.5%). Co-infection with multiple species of Plasmodium parasites was also observed; double infection was detected in 85 macaques, triple infection detected in 47 macaques, quadruple infection detected in 37, and quintuple infection detected in 23 macaques. Using the published assay, all five simian malaria parasites were detected, with p-value for linear trend p<0.001 for Pan-Plasmodium, p<0.001 for species-specific PCR). After stratifying by species of malaria, all species had a significant linear trend over the years  except for P. cynomolgi.The observed increasing trend of the proportion of infected macaques sampled across the years from 2009 to 2017 was found to be statistically significant.  were tested positive using the Pan-Malaysia , wherebyMalaysia .P. knowlesi, P. cynomolgi and P. inui are also common malaria parasites found in the local macaque population, with 71.5% and 42.0% respectively. Naturally acquired P. cynomolgi infections have been reported in Peninsular Malaysia [P. inui is infectious to humans under laboratory studies [P. cynomolgi is the highest, followed by P. knowlesi, P. inui, P. fieldi and then P. coatneyi. The prevalence obtained in a simian malaria study conducted in wild macaques from Selangor, Peninsular Malaysia was slightly different [P. inui was the predominant (65.7%), followed by P. knowlesi (60%), P. cynomolgi (51.4%) P. coatneyi (45.7%) and P. fieldi (2.9%). Nevertheless, except for P. fieldi the other species are in the similar range with what we have observed in this study.Apart from Malaysia , CambodiMalaysia , and MalMalaysia ,46, whil studies . In our ifferent . In the Plasmodium and simian malaria species-specific PCRs. This demonstrated an ongoing transmission of simian malaria parasites among the wild macaque population in the military protected forest within the Western Catchment area. The increasing trend was contributed by all species except for P. cynomolgi, which prevalence remained stable over the years. Apart from these five simian malaria species, there have been reports of P. simiovale and P. inui-like species detected in long-tailed macaques in Sarawak, Malaysian Borneo [There was an increasing trend of the proportion of infected macaques sampled across the years from 2009 to 2017, based on both Pan-n Borneo . Hence, Although macaques caught near human habitations were tested to be free from malaria infection, the possibility of wild macaque populations emigrating from the military protected forest within the Western Catchment area, alongside with the simiophagic mosquito vectors, cannot be ignored. This is particularly true in the land-scarce Singapore where residential areas may be built close to forest fringes and vegetation is cleared to meet increased demands for land use. As such, a thorough entomological surveillance targeting potential vectors of simian malaria is needed, particularly at the boundary of forests where housing estates are being built.Anopheles leucosphyrus group have been incriminated in the transmission of P. knowlesi and other simian malaria parasites. These include Anopheles hackeri [An. cracens [An. latens, An. donaldi and An. balabacensis in Sarawak and Sabah of East Malaysia [An. dirus in Vietnam [Anopheles leucosphyrus group [Despite the on-going transmission of malaria parasites among the wild macaques, the vectors involved in the transmission of zoonotic knowlesi in Singapore are yet to be identified. To date, only mosquitoes from the  hackeri , An. cra cracens ,50 in PeMalaysia –54, and  Vietnam ,56. Althus group , there hus group ,57.Bacillus thuringiensis var. israelensis and environmental control works were undertaken to eliminate mosquito breeding grounds within the forested grounds. Enhanced personal protection measures were enforced for all personnel training within these sites and these measures include the routine application of mosquito repellent on areas of exposed skin, the use of permethrin insecticide-treated uniforms and rolling down of long sleeves to prevent further transmission and infections [The major elements in determining the suitability of control measures to be initiated highly depend on the identity and bionomics of the vectors found in the area . Since tfections . These mDespite the reduced mosquito population, the infection rate of wild macaques across the years remained high with an increased prevalence in recent years. There are a few plausible explanations for this. The trapping and removal of the macaques could have resulted in the reduction of their population in the Western Catchment area. The remaining macaque population would therefore been exposed to higher biting pressure, resulting in increased simian malaria transmission. In addition, the control measures taken to prevent human cases might not be effective against simiophagic vectors that are transmitting the infection among the macaques. As such, identifying the principal vector responsible for zoonotic knowlesi transmission is necessary for an appropriate vector control strategy to be developed. Another possibility for the observed increase in simian malaria prevalence over the years could be the degradation of DNA in samples collected in the earlier part of the study, resulting in the apparent lower prevalence rate detected in these samples.The high malaria prevalence among the macaques suggests an active transmission among the macaque population in Western Catchment area. Therefore, to incriminate the vectors, mosquito surveillance should preferably include the use of monkey-baited traps ,51 in adThe main limitation of this study is the obtainment of samples through convenience sampling, which may result in sampling bias and inaccurate estimates. Therefore, the prevalence values for each species should be analysed with caution. Nonetheless, this study provided evidence that simian malaria infection found mainly in the macaque population from the Western Catchment forest, while no infection was detected among the peridomestic macaques sampled from other regions of Singapore which are accessible to the general public. As such, the risk of the general human population acquiring zoonotic malaria from the macaque is low.P. knowlesi, wild long-tailed macaques also harbour P. inui, P. cynomolgi, P. fieldi, and P. coatneyi. Despite a high infection rate among the wild macaques, the risk of the general human population acquiring zoonotic malaria is low. However, the overall risk can be better demonstrated with information on the spatial distribution of macaques and the identification of vectors involved in the transmission among macaques and between macaques and humans.This study constitutes the first comprehensive report for surveillance of simian malaria parasites in long-tailed macaques in Singapore. In this study, we found that apart from S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.