Plasmodium falciparum characterized by a highly inflammatory response. This systematic review aimed to investigate the association between TNF-α levels and cerebral malaria.Cerebral malaria is the most severe form of infection with P. falciparum-infected humans with or without cerebral malaria and TNF-α dosage level. All studies were evaluated using a risk of bias tool and the GRADE approach.This review followed the Preferred Reporting of Systematic Review and Meta-analyses (PRISMA) guidelines. The search was performed at PubMed, LILACS, Scopus, Web of Science, The Cochrane Library, OpenGrey and Google Scholar. We have included studies of Our results have identified 2338 studies, and 8 articles were eligible according to this systematic review inclusion criteria. Among the eight articles, five have evaluated TNF- α plasma dosage, while two have evaluated at the blood and one at the brain (post-Morten). Among them, only five studies showed higher TNF-α levels in the cerebral malaria group compared to the severe malaria group. Methodological problems were identified regarding sample size, randomization and blindness, but no risk of bias was detected.Although the results suggested that that TNF-α level is associated with cerebral malaria, the evidence is inconsistent and imprecise. More observational studies evaluating the average TNF-alpha are needed. Plasmodium. Over 100 species of Plasmodium can infect several groups of vertebrates like reptiles, birds, and mammals. However, only six of them are capable of infecting humans: Plasmodium vivax, Plasmodium over, Plasmodium malariae, Plasmodium knowolesi, Plasmodium simiu and Plasmodium falciparum [Malaria is an acute infection caused by the protozoan parasite lciparum .P. ovale is the least common. It is primarily concentrated in Western Africa, while P. malariae is widely distributed with low incidence. Infection by P. vivax and P. falciparum is the most common and can cause severe anemia. However, only P. falciparum may lead to the most severe form of the disease, cerebral malaria (CM) [Among these species, the ria (CM) , 7–9. Thria (CM) .The main obstacle to eradicating malaria are the decrease of international and domestic funding, the presence of endemic malaria regions, anomalous weather patterns, parasite resistance of treatment, and mosquito resistance of insecticides . CountriIn 2016, 216 million cases of malaria were registered in 91 countries, with 445.000 deaths. Among the cases, 80% came from countries are in sub-Saharian Africa . The firAccording to the World Health Organization , CM is aA study in 2006 proposed the unification of theories, suggesting that cerebral malaria is a consequence of hypoxia since parasitized erythrocytes adhere to the endothelium wall and induce the blockage of blood flow , 7, 21. + has gotten attention. TNF-α production on the initial CM phase seems to be related to a reduction of parasitic load. However, the overproduction of TNF-α in the late phase is associated with disease severity [Among them, the TNF-α secretion by macrophages and T CD4severity . This duseverity . On endeseverity .Plasmodium falciparum and TNF-α level increasing in humans.In this way, this systematic review aimed to gather clinical evidence of the association of cerebral malaria by Preferred Reporting of Systematic Review and Meta-analyses (PRISMA) were followed [This systematic review was registered on the International prospective register of systematic reviews (PROSPERO), under the number CRD42016042745. The followed .From July to September 2018, searches were performed on the following literature bases: PubMed, Latin American and Caribbean Health Sciences Literature database (LILACS), Scopus, Web of Science, The Cochrane Library, OpenGrey and Google Scholar. There was no restriction regarding language or year of publication. In each database, a specific search strategy, combining MESH and free terms was devolved following the rules of each database (Table P- population) Humans; (E- exposition) presence of cerebral malaria induced by Plasmodium falciparum; (C- comparison) presence of severe malaria but no cerebral commitment; (O- outcome) Association between TNF-α immunologic response on a blood sample from patients that have been infected by Plasmodium falciparum and have developed cerebral malaria.After searches, duplicated studies were identified and excluded in a reference manager software . The selection of studies was performed independently by two reviewers (LKRL and BP) according to the following PECO:  and reference group . We also included studies with TNF-α level dosage, at least in one sample. We excluded reviews, animals, and in vitro studies, clinical cases, case series, guidelines and editorials.We first selected studies by title and abstract and then by full reading. A third reviewer (RRL) was consulted in case of disagreement. To the final selection of eligible studies, we took into consideration studies that have infected groups , quality of methodology and results, withdrawals or loss of sample, experimental bias and confounding factors. Each question was answered according to the following code: (0) no problem; (+) minor problem; (++) major problem; (NA) not applicable  tool . Includen = 1046), followed by Web of Science (n = 635), Pubmed (n = 248), Google Schoolar (n = 237), Cohcrane (n = 109) and Open grey (n = 01). Alerts did not informed non studies that satisfied the inclusion criterion of this present review.After search we identified 2338 studies. Most of which were at Scopus  and reference group (without CM), five showed an increase on TNF-α at CM group when compared to non-CM group , 52, 53.The levels of TNF-alpha were most increased in cerebral malaria than severe malaria groups for adults and children. The overall level of certainty was classified as low for studies involving adults and children. This result can be associated with the observational nature of the included studies Table .Table 5In this systematic review, we gathered evidence of the association between Cerebral Malaria and TNF-alpha levels. Among the eight elected studies, five reported an increase in TNF-alpha in patients with cerebral malaria compared to patients with malaria who did not develop Cerebral Malaria; another three articles did not show significant differences between the groups. However, even in the face of the association results presented by most of the selected articles, the low level of evidence presented by them does not offer certainty of the results regarding the association of TNF-alpha with cerebral malaria, according to GRADE criteria.To gather the evidence presented in this article, we opted for a type of bibliographic research recognized today as the top of the evidence pyramid: the systematic review. A systematic review is a type of study that seeks to gather and analyze articles with similar designs, evaluate them methodologically and, if possible, bring them together in statistical analysis. This type of review seeks to synthesize similar primary studies. It is considered the best level of evidence for decision making in questions about therapy and health conduct –67. In tP.falciparum, the most lethal of the six human malaria parasites [Anopheles gambiae mosquito, the most efficient and most difficult to control malaria vector. It is estimated that 1 million people in Africa die each year from malaria 308, with a prevalence of children under 5 years of age [Of the eight eligible studies, only one has not clarified the source of 298 samples. Two were conducted in India, two in Ghana, one in Mali, one in the Republic of 299 Cameroon and one in the Republic of the Gambia (Table arasites . Anothers of age .In areas of stable malaria transmission, incredibly Young children are the main group with a major risk of malaria morbidity and mortality. The highest incidence of infection on children is in the first or second year of life when they have not yet acquired proper clinical immunity. This makes the early years particularly dangerous, accounting for 90% of all deaths in Africa. Three reasons for this high mortality rate are the development of cerebral malaria, the frequency of malaria infections that leads to severe anemia and the low weight at birth that is frequently associated with malaria infection during pregnancy . Also, rTNF-α is closely associated with the reduced parasitic load. However, it is shown to be associated with the severity of cerebral malaria if production occurs in its later phase, which may suggest an attempt by TNF-α to control infection, and possibly contribute to the reduction of deaths, especially in children under five, who are the most susceptible to this in endemic areas , 24, 33.After we have analyzed the eight selected studies in this systematic review, we identified that the TNF-α level was found to be increased on the cerebral malaria group, suggesting TNF-α level could be closely linked to malaria. In the quality assessment, all studies showed major problems regarding sample size and blindness. The study performed by Kinra and Dutta  was the All studies that have been selected showed their criterion to determine if patients had or not CM. To characterize CM’s development, patients should present a coma score lower than 2 . On the Regarding sample representativeness, one of the major problems identified in all selected studies is related to the absence of sample size calculation fewer than 50. This problem was identified in seven out of eight selected studies. Malaria studies performed in humans are fundamental to have a high sample size to ensure the population’s representativeness . In addiThe antigen production induced by CM leads to the activation of the immunologic system, such as monocytes activation that is responsible for pro-inflammatory cytokines such as IL-1, IL-6 and TNF-α . HoweverP. falciparum on children. However, the circulating concentrations are unusually high in the minority of CM patients with CM when compared to the majority that develops fever without severe complication. However, high concentrations of TNF are associated with death even after the correction of parasitemia and glucose concentrations by multivariate analysis. This observation is following the hypothesis that excessive TNF production could contribute to the pathogenesis of malaria.Kwiatkowski et al.  have shoP.falciparum. However, it is still necessary for the development of studies that make use of a greater sample size to have a more reliable and representative analysis. Even malaria is no longer considered a neglected disease; it is still necessary to investigate this association’s temporal course, especially on children under 5 years old.We may conclude that the TNF level seems to be associated with cerebral malaria by


Plasmodium vivax malaria is unclear. As platelets have been reported to be activated and to play a role in inflammatory response during malaria, we hypothesized that this would correlate with endothelial alterations during acute illness. We performed platelet flow cytometry of PAC-1 and P-selectin. We measured platelet markers  and endothelial activation markers  in plasma with a multiplex-based assay. The values of each mediator were used to generate heatmaps, K-means clustering and Principal Component analysis. In addition, we determined pair-wise Pearson’s correlation coefficients to generate correlation networks. Platelet counts were reduced, and mean platelet volume increased in malaria patients. The activation of circulating platelets in flow cytometry did not differ between patients and controls. CD40L levels  were significantly higher in patients, while P-selectin and CXCL4 showed a nonsignificant trend towards higher levels in patients. The network correlation approach demonstrated the correlation between markers of platelet and endothelial activation, and the heatmaps revealed a distinct pattern of activation in two subsets of P. vivax patients when compared to controls. Although absolute platelet activation was not strong in uncomplicated vivax malaria, markers of platelet activity and production were correlated with higher endothelial cell activation, especially in a specific subset of patients.Platelets drive endothelial cell activation in many diseases. However, if this occurs in  Plasmodium vivax malaria. Platelets are classically involved in endothelial cell activation in several diseases, but their role in the context of vivax malaria remains unclear. Thrombocytopenia is the most common hematological disturbance in P. vivax-infected patients, and platelets have been implicated in parasitemia control. In this work, we studied the activation of platelets in association with endothelial cell activation in vivax malaria. Platelets retrieved from infected peripheral blood were non-activated when analyzed by flow cytometry; however, they displayed higher mean volume and significantly reduced counts. We also found higher levels of circulating factors associated with platelet activation , thrombopoiesis and endothelial cell activation in infected patients. Further, through pair-wise correlation and clustering analysis, we found a subgroup of patients showing significant associations between markers of platelet and endothelial activation in a pattern different from that of endemic controls. Collectively, our findings indicate a role of platelets in endothelial cell activation in vivax malaria and indicate a heterogeneous host response in the setting of uncomplicated disease, a finding to be further explored in future studies.Endothelial cell activation is a key process in the pathogenesis of  Plasmodium parasites of all species, effectively controlling parasitemia and, possibly, disease progression [Platelets play a significant role in malaria, being involved both in the immune response and the pathogenesis of the disease. Recently, platelets have been shown to kill gression , 2. Howegression , 4. Givegression , 5, whilgression , 5–8.Plasmodium falciparum malaria had lower platelet counts in comparison to those with less severe disease [P. vivax malaria, platelets counts were significantly lower in those with complications [Besides their role in immunity, thrombocytopenia  is the most common hematological alteration in malaria, and there is no definitive mechanistic explanation to its occurrence , 10. Bes disease . In patiications  or severications . In addiications . This asications , and theP. falciparum-infected erythrocytes adhesion to microvasculature, avoiding immunological clearance and leading to severe disease, while inducing more EC damage [P. vivax-infected erythrocytes also adhere to EC [EC activation is present in all malaria species, occurring in both mild and severe cases –20. In tC damage , 22. P. re to EC , 24, butre to EC . Nonethere to EC , 25, 26,in vitro studies. Importantly, in pediatric cerebral malaria, the most severe presentation of the disease, platelets have been shown to accumulate in the brain microvessels of affected children [P. falciparum adhesion to and activation of cultured EC [There is limited information regarding the interplay of platelets and EC in human malaria, with most insights coming from animal models and children . Experimchildren , and thatured EC , 29.Plasmodium species, whether they play a role in EC activation during P. vivax malaria remains to be investigated. In this study, we show that platelet counts were reduced in P. vivax malaria patients, while circulating markers of platelet activation showed a trend towards elevation. Importantly, platelet activation markers correlated with those related to endothelial activation, indicating a role for platelets in EC pathology in this disease.Therefore, while platelets are implicated in endothelial pathology in diverse disease settings, including malaria due to other P. vivax malaria, as diagnosed by light microscopy, seen at FMT-HVD and 34 healthy controls were enrolled. All patients included were outpatients that did not meet World Health Organization (WHO) criteria for severe malaria. We further confirmed diagnosis through qPCR assays for both P. vivax and P. falciparum. We used previously published nucleotides sequences [All subjects enrolled in the study were adults, and samples were taken only after signing of informed consent. The study was approved by the local Research Ethics Committee at Fundação de Medicina Tropical Dr. Heitor Vieira Dourado , under #CAAE: 54234216.1.0000.0005. Seventy-nine patients with equences  . The PRP was centrifuged at 100 g for 10 minutes for removal of residual leukocytes, and subsequently centrifuged at 800g for 20 minutes to obtain the platelet pellet. Prostaglandin E1 300 nM was used to minimize platelet aggregation. The supernatant of this centrifugation was centrifuged at 1000 g for 10 minutes to obtain platelet poor plasma (PPP).Within 15 minutes of sampling, complete blood counts were performed. Platelet activation was assessed in PRP using anti-CD61 antibody (PerCp-Cy5.5), PAC-1 (FITC) and anti-P-selectin (PE) antibodies, by flow cytometry  and analysis with FlowJo software (Free Star). The same panel was used to assess whether the incubation of PPP (50% v/v for 10 min at 37°C) from malaria patients was capable to activate platelets from a healthy donor. For this assay specifically, we used mean fluorescence intensity to compare the groups.Pv-IE/μL). This subgroup did not differ from the overall population of patients regarding severity of disease, sex proportion, platelet counts and mean parasitemia : sCD40L, P-selectin, CXCL4 and thrombopoietin (TPO), IL-11, as well as circulating markers of EC activation ). We selected 31 patients for the multiplex assay to represent a wide range of parasitemias  to generate heatmaps and to perform K-means clustering. After running the algorithms, individuals were clustered according to the levels of expression of the mediators in 3 groups, which were named Control, Vivaxlow and Vivaxhigh. The heatmap represents z-scores obtained by centering variables  with the Scale Function in R, followed by hierarchical and k-means clustering analysis. In addition, the same software was used to determine pair-wise Pearson’s correlation coefficients to generate correlation networks and the p value to test for non-correlation was evaluated using p ≤ 0.05 as a cut-off. In order to analyze the structure of the networks, the graphics for the network analysis were customized in the Cytoscape software (v 3.5.1) using the prefuse force-directed layout, which in the equilibrium state for the system of forces, determined by the correlation strength, the edges tend to have uniform length, and nodes that are not connected by an edge tend to be drawn further apart.The values of each circulating factor measured in the plasma samples, hematological parameters and parasitemia from endemic controls and Fisher’s exact test was used for categorical data. Student’s t-test was used to compare means between groups with normally distributed data, and data sets with non-normal distributions were compared using the Mann–Whitney test, with p<0.05 considered significant. Data are presented as means and SD unless otherwise stated. Analysis were performed, and the graphs generated in GraphPad Prism5 and R software.9/L vs. 236.0±54.2 x109/L, P<0.0001), yielding an 87.3% frequency of thrombocytopenia. Mean Platelet Volume (MPV) was increased in patients (9.1±1.1 fL) compared to controls , and was inversely correlated with platelet counts in both patients and control groups  (Platelet counts were significantly reduced in patients (Mean ± SD: 91.6±43.3 x10<0.0001) . PlatelePlatelet activation is a feature of some thrombocytopenic infections as well of diseases associated with endothelial cell dysfunction , 16, 31.P = 0.0001; P = 0.0621; P = 0.1236) were not different between the groups  in vitro [in vivo, through measurement of circulating factors, have been shown by other groups [Whether a systemic activation of circulating platelets occurs in malaria is still unclear. While a study has reported altered platelet responses after exposure to in vitro , direct in vitro , 5. Hower groups , 7. Therr groups . As CD40r groups .P. vivax during vertebrate infection [Endothelial cell activation is a major component of malaria pathogenesis , 25, 26,nfection , especianfection –49 and Anfection , are assnfection , 52 and As platelets are classically associated with endothelial pathology, we searched for patterns of association between platelet factors and markers of endothelial cell activation. Interestingly, our networks revealed the association of soluble CD40L and P-selectin with ICAM-1 and E-selectin, indicating the interplay between these two cell populations in vivax malaria. Indeed, these platelet markers have been implicated in EC activation in different diseases. Incubation of activated platelets with cultured endothelium leads to its activation through CD40L-CD40 interaction , and CD4P. vivax malaria [Notably, the heatmaps further confirmed a distinct pattern of platelet and endothelial cell activation in two subsets of patients , with on malaria .This study has several limitations, with the most important being the small number of patients included in the multiplex-based assay, limiting the generalizability of the findings and the statistical analyses. Moreover, while a role for platelets in endothelial activation in malaria is indicated by our results and has biological foundation, the mechanisms behind this association were not elucidated. Finally, although all patients included in the study presented with uncomplicated malaria, we did not follow patients throughout their illness to assess potential clinical implications of our findings.In this study, we found evidence that markers of platelet activation and production correlate with endothelial cell activation during vivax malaria. Importantly, these markers categorize patients in clearly distinct groups, further indicating that they are indeed intertwined. Collectively, our findings indicate a role of platelets in endothelial cell activation in vivax malaria and indicate a heterogeneous host response in the setting of uncomplicated disease. Therefore, we believe that future studies to identify the mechanisms of how platelets induce EC pathology in relevant models of vivax malaria are warranted.S1 Table(PPTX)Click here for additional data file.S2 Table(PPTX)Click here for additional data file.S3 Table(PPTX)Click here for additional data file.


Plasmodium vivax malaria and 1408 population controls. G6PD Med was associated with reductions in symptomatic P. vivax malaria incidence of 76%  in hemizygous males and homozygous females combined and 55%  in heterozygous females. Unless there is very large population stratification within the Pashtun (confounding these results), the G6PD Med genotype confers a very large and gene-dose proportional protective effect against acute vivax malaria. The proportion of patients with vivax malaria at risk of haemolysis following 8-aminoquinoline radical cure is substantially overestimated by studies measuring G6PD deficiency prevalence in healthy subjects.X-linked glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. The severe Mediterranean variant (G6PD Med) found across Europe and Asia is thought to confer protection against malaria, but its effect is unclear. We fitted a Bayesian statistical model to observed G6PD Med allele frequencies in 999 Pashtun patients presenting with acute  In red blood cells, glucose-6-phosphate dehydrogenase  is the only source of reduced nicotinamide adenine dinucleotide phosphate (NADPH) . G6PD dePlasmodium falciparum or Plasmodium vivax  variant prevalent in the sub-Saharan African populations , and in people with their genetic origin there. The most severe of the commonly found G6PD variants is the ‘Mediterranean’ variant (‘G6PD Med’). This results from a single C-T transition at nucleotide position 563, causing a serine phenylalanine replacement at amino acid position 188. G6PD Med is the predominant genotype in the Pashtun who live in Afghanistan, Pakistan, and India .In total, 764 Pashtun patients presenting with acute vivax malaria  and 699 Pashtun controls  were studied; 236 healthy males came from the epidemiology study reported previously  and the P. vivax parasite densities were very similar in patients with wild-type G6PD  and female G6PD Med heterozygotes  and were slightly lower in the six patients with quantitative parasite counts who were G6PD Med hemizygotes or homozygotes .Geometric mean (range) In our earlier studies, patients with moderate or severe anaemia (haemoglobin <8 g/dL) were excluded, which could have biased our results. We therefore compared distributions of haemoglobin concentrations in the earlier clinical trials, in which this exclusion was applied, with those in the more recent studies in which there was no anaemia exclusion criterion . This indicated that 9.6% (29 of 302) patients would have been excluded if the earlier study threshold had been applied. This is predicted to have resulted in exclusion of a single female heterozygote. There was no significant difference in the haemoglobin concentrations at presentation comparing G6PD Med hemizygotes or homozygotes, heterozygotes, or wild type . This shows that exclusion of anaemic patients in the first part of the study did not affect the interpretation of the protective effect of G6PD deficiency on vivax malaria.These data were then combined with data from the two previously reported studies on the prevalence of G6PD deficiency in the Pashtun ethnic group from Afghanistan. One was in healthy subjects only , and thePlasmodium vivax malaria. This is a much greater protective effect than observed against P. falciparum malaria elsewhere   (P. vivax is now the predominant (90%) cause of malaria, there was also a very strong protective effect of G6PD Med against self-reported previous malaria (AOR: 0.010) , but they are underused because G6PD deficiency testing is usually not available and prescribers are naturally concerned about precipitating dangerous haemolysis. The proportion of patients with vivax malaria at risk of serious haemolysis with G6PD Med in these studies is nearly four times lower than would be predicted from gene frequencies in the healthy population.Mediterranean-type glucose-6-phosphate dehydrogenase deficiency (G6PD Med) prevalent in the Pashtun provided a strong and gene-dose-related protective effect against lsewhere . It is pnolines) . P. viva16–1.02) . These e16–1.02) , are all: 0.010) . In compP. vivax densities were lower in hemizygote males and also in heterozygote females presenting to clinics with vivax malaria, but there was no corresponding effect in falciparum malaria, and there was no apparent protective effect against malaria illness   . ConversP. vivax .P. vivax malaria, although there were limited data to assess the protective effects in P. vivax infections.A recent meta-analysis of 28 studies  addressiP. vivax illness estimated in this study is large; it is similar in magnitude to the well-described protection conferred by Hb AS (sickle cell heterozygotes) against falciparum malaria  was not possible. We do not have additional genetic marker data that could be used to adjust for population stratification. However, in the earlier study by Overall, this study shows that the G6PD Mediterranean genotype confers a very large and gene-dose proportional protective effect against vivax malaria. If G6PD Med is approximately at an equilibrium allele frequency in the Pashtun population , this implies that it must be harmful, i.e. malaria alone cannot create balancing selection. The main risks associated with G6PD deficiency are neonatal hyperbilirubinaemia and haemolysis following oxidant food and drugs, while the benefit is protection from malaria. Importantly for malaria treatment, the population risks associated with 8-aminoquinoline radical cure are greatly overestimated from G6PD prevalences in the healthy population.In Afghanistan, malaria is confined to the northern plains, the Jalalabad basin and river valleys, which fringe the central mountains to the west and south . MalariaP. vivax transmission areas for two main reasons: the Pashtun comprises the majority of people in the Eastern provinces where malaria incidence is highest, and G6PD variants other than G6PD Med are rare in this ethnic group , Mehtherlam (Laghman), Asadabad (Kunar), Maimana (Faryab), Taloqan (Takhar), Imamsahib (Kunduz), Pulikhumri (Baghlan), Pulialam (Lowgar), and the capital, Kabul (Kabul province) . Provincic group .In the initial prevalence study in healthy subjects conducted in 2009, only males were studied . A conveP. vivax, axillary temperature ≥ 37.5°C or oral/rectal temperature ≥ 38°C, or history of fever in preceding 24 hr, and able to swallow oral medication and comply with study requirements. Exclusion criteria were any clinical or laboratory feature of severe malaria, significant comorbidity, known hypersensitivity to any study drugs, mixed species Plasmodium infection, and pregnancy or lactation. In the initial studies, patients with haemoglobin concentrations ≤ 8 g/dl were excluded, but in the later observational studies in 2019, anaemic patients were not excluded.Patients with symptomatic malaria presented to provincial malaria control centres adjacent to the Pakistan border. Jalalabad, the capital city of Nangarhar province, is a referral centre for the whole eastern region. Population movement takes place in both directions across this largely mountainous area bordering Pakistan. The malaria treatment protocols have been reported previously . Patient2, 125 µM 4-deoxynucleotide triphosphate, and 0.05 units PlatinumTaq DNA polymerase . After pre-denaturation at 95°C for 5 min, 45 PCR cycles were performed involving denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, with post-extension at 72°C for 7 min. Ten microlitres of each PCR product was digested with 10 units of the restriction enzyme MboII (New England Biolabs Inc) at 37°C for 3 hr and visualised by 2% agarose gel electrophoresis. A product pattern of 104+98+28 bp indicates the G6PD Med mutation, whereas 202+28 bp indicates wild type. All PCR products of samples with mutant type and 10% of the wild-type samples were purified subsequently and the DNA was sequenced . Gene sequences of the PCR amplification products of g6pd (accession number: NM_001042351.2) were confirmed using the NCBI’s Blastn and Blastx programmes. Gene polymorphisms were assessed by comparison with the reference sequences using BioEdit v7.2.5.Capillary blood was collected for haemoglobin measurement (HemoCue), Giemsa staining of thick and thin blood films for parasite speciation and counts and G6PD testing. Before 2018 G6PD deficiency screening used the G-6-PD OSMMR 2000 (R and D Diagnostics) and NADPH fluorescent spot test kit (10 min incubation). From 2018, the CareStart rapid G6PD test was substituted. Dried blood spots were stored for later genotyping . GenomicP. vivax case–control study in which patients who had been enrolled in earlier clinical trials assessing vivax malaria treatments were matched to community controls. This was also an Afghan refugee population. The refugee villages were located close to Peshawar in the North-West frontier province of Pakistan  because we can assume that the protective effect in G6PD-deficient hemizygous males and homozygous females is the same . Log-odds estimated from a logistic regression model are not collapsible; therefore, this assumption cannot be input into a logistic regression model. Our formulation, in terms of relative reduction, allows for this assumption to be hard-wired into the model. It should be noted that this assumption applies to the marginal effect of G6PD deficiency, that is females could be more or less likely to have clinical vivax malaria relative to males, but the protective effect of hemizygote and homozygote G6PD deficiency is assumed identical, conditional on having clinical vivax malaria.p. The total number of healthy males (controls) sampled is denoted P. vivax infected males (cases) is denoted HetD refers to heterozygous deficient, HomoD refers to homozygous deficient, and WT refers to G6PD normal.The model assumed that Hardy–Weinberg equilibrium held in the studied population(s). We tested this assumption by applying an exact test appropriate for X-linked biallelic markers in the control populations . Convergence of chains was checked visually with trace plots.This model allows for an additive effect  or an increased effect in heterozygotes . The Bayesian model was written in Stan , and a rhttp://www.clinicaltrials.gov/ct under the identifier NCT01178021.The clinical studies were approved by the Institutional Review Board of the Afghan Public Health Institute, Ministry of Public Health, Afghanistan, the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Thailand, and the Oxford Tropical Research Ethics Committee, Oxford University, UK. The clinical trial was registered with the clinical trials website  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Plasmodium vivax disease. It uses a Bayesian statistical approach which specifically elucidates the particular protection which female heterozygotes versus male hemizygotes  for the Med mutation may experience. This is an important contribution to our understanding of the relationship between G6PD deficiency and P. vivax.The exact relationship between G6PD deficiency and malaria protection remains uncertain. This study provides evidence that the G6PD Med mutation (563 C>T) protects against clinical Decision letter after peer review:Plasmodium vivax malaria" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Bridget S Penman (Reviewer #1); Anirban Mondal (Reviewer #2).Thank you for submitting your article "Protective effect of Mediterranean type glucose-6-phosphate dehydrogenase deficiency against The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife, either of which would be linked to the original paper.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in SummaryPlasmodium vivax disease. It uses a Bayesian statistical approach which specifically elucidates the particular protection which female heterozygotes versus male hemizygotes  for the Med mutation may experience. This is an important contribution to our understanding of the relationship between G6PD deficiency and P. vivax.The exact relationship between G6PD deficiency and malaria protection remains uncertain. This study provides evidence that the G6PD Med mutation (563 C>T) protects against clinical Overall, the reviewers were positive about the work and its potential, but have some clear concerns that will require additional data, analyses, and interpretation. Below are the main points raised by the reviewers that would need to be addressed to for a revised manuscript.P. vivax, the majority (95%) of malaria in Afghanistan is caused by P. falciparum that means mixed species infections are likely high and P. falciparum infections may be obscuring P. vivax infections. It is not clear to what extent G6PD deficiency may impact the chance of being coinfected with both falciparum and vivax. Ideally, PCR verification of these samples would be performed to confirm the species for samples included in the analysis. Without this molecular data, the overall assessments of susceptibility to vivax malaria in association with G6PD Med is incomplete.1) The presence of mixed infections: although the work is focused on 2) The analysis relies on a number of assumptions made about Pashtun population genetics  and it is not clear to what extent these assumptions are justified since little evidence/support is provided. In particular, the assumptions about Hardy Weinberg equilibrium of G6PD Med within the Pashtun population need to be justified and supported since the analysis is highly reliant on this assumption.3) The exclusion criteria does not appear to have been uniformly applied – in particular anemia was an exclusion criteria for only part of the data. This was not clear and may impact the overall significance of statistical results.4) While the manuscript makes a number of conclusions about female homozygotes, these are not strongly supported by the evidence. In particular, the study is likely under-powered with regard to clinical associations among female homozygotes with G6PD Med, but this is not addressed and the stated conclusions are likely stronger than what can be supported by the data/analyses provided.Reviewer #1:There are aspects of the study which I would like to see explained further before I can be certain of the strength of the evidence.1) The key result rests on the fact that the proportion of G6PD deficient individuals is lower among patients with vivax malaria than in controls without malaria. However, as the authors note: "In the initial clinical studies reported here severe anaemia was an exclusion criterion and this could have reduced the proportion of G6PD deficient patients [among malaria patients – my insertion]". To enable readers to judge for themselves how big an effect this could have had, it would be useful to indicate the numbers of patients affected -e.g. by providing a supplementary table which splits up the data in table 1 according to before and after the anaemia criterion was changed. It may also be useful to conduct a supplementary analysis among just the data where severe anaemia was not an exclusion criterion – but I do not want to request that without knowing the numbers involved.2) The Leslie study [14], from which data was included in the meta-analysis had "372 cases and 743 controls", but from the numbers in Table 1 it seems that not all of these have been included in the present meta-analysis. The Materials and methods need to make clear which of the samples from the Leslie study were included in the meta-analysis, and why. I can imagine this may be something to do with the geographical origins of the refugee populations in the Leslie study, since this study focused on high malaria transmission regions only, but this needs to be explained.3) The method used here implicitly assumes that there is little heterogeneity in G6PD Med frequencies between different Pashtun tribal groups currently living in malaria endemic regions. It is hard to know whether or not this is a reasonable assumption, because inevitably the evidence for G6PD Med frequencies in different tribal groups is based on small numbers (e.g. the Leslie study [14]). The authors do acknowledge this with the statement in the Discussion that "genetic heterogeneity within the Pashtun group" may be a confounder. However, I would like to see this potential limitation of the study discussed in more detail in a specific section of the Discussion or Introduction, including reasons why it is reasonable to assume a consistent Med frequency. Are there any other sources of evidence about genetic diversity within the Pashtun group, e.g. from studies at other loci?P. vivax infection for the majority of the data (that which was obtained by the authors). However, it is not stated that the case definition for P. vivax infection was equivalent in the Leslie study, from which data was included in the meta-analysis.4) The Materials and methods explain the case definition for 5) The number of female homozygotes in this study is (inevitably) very small. I do not object to the authors including female homozygotes and male hemizygotes in the same category, I can see the biological logic behind it, and I like their entire approach to estimate the gene dose effect. However, on the other hand, surely it remains possible that, despite their similar red blood cells, a female homozygote for G6PD Med may not be exactly the same as a male hemizygote for G6PD Med with respect to their likelihood of displaying clinical vivax malaria. If the authors had fitted α, β and γ , it seems likely to me that α and β would have been similar to the current estimates and γ would have had a huge credible interval because there is little data. If the authors concur, it would be worth acknowledging this explicitly in the Discussion – because to me this means that this study tells us quite a lot about male hemizygotes and female heterozygotes, but doesn't tell us much about female homozygotes.Reviewer #2:This review is mainly focussed on the statistical analysis part of the article.The authors have used a well-established and standard Bayesian methodology for inference on the role of G6PD Med as a protective effect against vivax malaria. The Bayesian model is appropriate assuming that the Hardy-Weinberg equilibrium is held in the studied population(s). A Markov Chain Monte Carlo sampling based inference is used for the Bayesian inference.I have the following concerns and/or comments which should be addressed.1) The author states that weakly informative Bayesian priors were used in the model. But this is not true for the allele frequency parameter p. I believe Β is a strongly informative prior for p. Some explanation for choosing such a strong prior for p should be included in the article. How are the values for hyperparameters (2 and 18) chosen? How sensitive are the results with respect to these hyperparameter values?hat), in the R-markdown document.2) It is desired that the authors include some plots and statistics assessing the convergence of the MCMC method, e.g. trace plots of the posterior samples, the Gelman-Rubin statistics (R3) The Bayesian model assumed that the Hardy-Weinberg equilibrium is held in the studied population(s). Can the authors discuss why this assumption is reasonable in the studied population(s) and how the model can be modified if the equilibrium is disturbed by mutations, natural selection and non-random mating?Reviewer #3:Plasmodium vivax malaria in the Pashtun ethnic group in Afghanistan. Results suggesting that G6PDd Med hemizygous males and homozygous females have 67% relative reduction against acute P. vivax malaria and G6PDd Med heterozygous females have 51% relative reduction is of particular interest for malaria and human genetics researchers. The authors present a very thoughtful discussion of P. vivax sensitivity to G6PD deficiency oxidant effects on parasite viability.The Short Report article suggests that the Mediterranean variant of G6PD deficiency confers a strong gene-dose proportional protective effect against symptomatic 1) The authors assume that Hardy-Weinberg equilibrium exists in the populations studied but they provided no evidence that it exists. Please provide the evidence that Hardy-Weinberg equilibrium exists for the alleles/genotypes for G6PD. Please also discuss how a deviation from Hardy-Weinberg equilibrium would affect the results of this study.2) Previous studies have shown that the G6PD Mediterranean allele is the predominant G6PD deficiency allele in the region. The G6PD gene is, however quite polymorphic, with over 180 mutations and at least two different G6PD Mediterranean allelic haplotypes. Thus the possibility that further genetic polymorphism is associated with G6PD deficiency and significant potential for population mixing (particularly in large cosmopolitan urban settings) may introduce genetic diversity in the G6PD gene. Please discuss how additional G6PD deficiency alleles could affect the results of this study.P. falciparum accounts for 95% of malaria cases in Afghanistan; P. vivax 5% . Diagnosis of P. vivax and accurate assessment of mixed species infections in areas where these two species are co-endemic is known to confound malaria microscopy. Additionally, submicroscopic Plasmodium species infections (involving P. vivax in particular), revealed by parallel PCR methods, is now very well established. Please provide further details as to how Plasmodium species diagnosis was performed, how monoinfection by P. vivax was confirmed and how exclusion of other Plasmodium species and/or mixed species infections were ruled out. If the complexity of Plasmodium species infections has been confirmed by PCR, the authors should revise their Laboratory Procedures section and present their data in these Result section. If Plasmodium species infections have not been confirmed by PCR, the authors should perform this work.3) Brief details were provided on malaria diagnosis by Giemsa-stained thick and thin blood films. Plasmodium vivax malaria" for further consideration by eLife. Your revised article has been evaluated by a Senior Editor and a Reviewing Editor.Thank you for resubmitting your work entitled "Protective effect of Mediterranean type glucose-6-phosphate dehydrogenase deficiency against The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:1) Include information in the main text about co-infection. The Who data seems to suggest that co-infection may be possible, although unlikely, and this is not likely to impact the study's outcome. However, the authors should address this point with a few sentences in the manuscript since readers may be concerned by the absence of PCR confirmation.2) Include a figure to highlight/provide more context/information about the data on the haemoglobin levels in the different genotypes. While the code provide is helpful, it would be much easier for the reader to interpret if it was summarized with a figure/legend. Also, all figures/etc. should be labeled and well references in the supplement.3) We propose the following smaller edits :In the Abstract: "Thus, in THE absence of very large population stratification within the Pashtun ethnic group (i.e. confounding these results), the G6PD Med35 genotype confers a very large and gene dose proportional protective effect against acute vivax malaria. "P. falciparum, but protects only against life threatening illness." could be improved by replacing "infection by P. falciparum" with something like "mild falciparum disease" or "uncomplicated clinical falciparum malaria".… just because protecting against "infection" could also mean protecting against completely asymptomatic infection, whereas the bulk of the evidence that has been discussed relates to disease with clinical symptoms.In the Discussion, when the protective effects of G6PD deficiency against falciparum are discussed, this sentence "It is possible that G6PD deficiency confers no significant protection against infection by  Overall, the reviewers were positive about the work and its potential, but have some clear concerns that will require additional data, analyses, and interpretation. Below are the main points raised by the reviewers that would need to be addressed to for a revised manuscript.P. falciparum that means mixed species infections are likely high and P. falciparum infections may be obscuring P. vivax infections. It is not clear to what extent G6PD deficiency may impact the chance of being coinfected with both falciparum and vivax. Ideally, PCR verification of these samples would be performed to confirm the species for samples included in the analysis. Without this molecular data, the overall assessments of susceptibility to vivax malaria in association with G6PD Med is incomplete.1) The presence of mixed infections: although the work is focused on P. vivax, the majority (95%) of malaria in Afghanistan is caused by P. falciparum and P. vivax mixed up? The World Malaria report (2020) gives tells the true story. In fact, P. falciparum has now almost gone from Afghanistan, and was rare during the course of this study. It is certainly not a confounder.We understand this reasonable concern but this description of the epidemiology in Afghanistan is wrong. It is based upon incorrect information provided (unfortunately) on the WHO EMRO site. The author of the WHO situation report may have got 2) The analysis relies on a number of assumptions made about Pashtun population genetics  and it is not clear to what extent these assumptions are justified since little evidence/support is provided. In particular, the assumptions about Hardy Weinberg equilibrium of G6PD Med within the Pashtun population need to be justified and supported since the analysis is highly reliant on this assumption.We agree that this is an important point and population stratification between cases and controls could bias the effect estimates. We cannot adjust for population stratification as we do not have other genetic marker data. We have added to the section in the Discussion outlining this limitation. However, the population stratification would have to be very large to account for such a large protective effect. The data from Leslie et al.  do show that there are different allele frequencies between the different tribes comprising the Pasthtun ethnic group. We do not have data on case-control allele counts broken down by tribe, so we cannot adjust for this. However, we note that this does not make the model mis-specified per se . The data from Leslie et al. are carefully matched so are no large differences between cases and controls. It is reassuring that the effect sizes between our data and the data from Leslie are very similar.We have added a statistical test for deviations from Hardy –Weiberg equilibrium (H-W). This shows no evidence for a departure from H-W in the controls (i.e. data from the controls are entirely consistent with the H-W assumption). This is referred to in the Materials and methods and given in the Source code 1 file.3) The exclusion criteria does not appear to have been uniformly applied – in particular anemia was an exclusion criteria for only part of the data. This was not clear and may impact the overall significance of statistical results.We fully accept this criticism of our retrospective study, which we did acknowledge in the paper. We now present a sub-analysis to see if there are significant differences between the two groups (i.e. before versus after: when the exclusion criterion was applied versus afterwards when it was lifted) which might have affected our overall estimates. This shows that the 8g/dL criterion excludes about 10% of patients  if applied to the later series. There is no evidence that the exclusion criteria would have affected the effect estimates as there was no difference in the distribution of haemoglobin concentrations between G6PD deficient and G6PD normal patients .4) While the manuscript makes a number of conclusions about female homozygotes, these are not strongly supported by the evidence. In particular, the study is likely under-powered with regard to clinical associations among female homozygotes with G6PD Med, but this is not addressed and the stated conclusions are likely stronger than what can be supported by the data/analyses provided.https://ashpublications.org/bloodadvances/article/4/23/5942/474381/Glucose-6-phosphate-dehydrogenase-deficiency-and ). It also the norm for general tests of association on the X-chromosome, see https://pubmed.ncbi.nlm.nih.gov/18441336/. Given the strong background biological knowledge, the rationale for not pooling hemizygotes and homozygotes is unclear.This is correct-the number of female homozygotes is very small, as expected. We are unaware of any evidence that the red cells of hemizygotes and homozygotes are phenotypically different. The assumption that red cells of female homozygotes and male hemizygotes are identical is fully supported by the underlying biology (due to Lyonisation). The model only makes an assumption about relative effects (hemi or homo-zygous deficiency versus WT), so males and females could differ in their absolute risk but this would not affect the model estimates unless there was some other genetic variation that had a direct interaction with G6PD deficiency. Pooling of male hemizygotes and female homozygotes is now the norm for the G6PD deficiency  The key result rests on the fact that the proportion of G6PD deficient individuals is lower among patients with vivax malaria than in controls without malaria. However, as the authors note: "In the initial clinical studies reported here severe anaemia was an exclusion criterion and this could have reduced the proportion of G6PD deficient patients [among malaria patients – my insertion]". To enable readers to judge for themselves how big an effect this could have had, it would be useful to indicate the numbers of patients affected -e.g. by providing a supplementary table which splits up the data in Table 1 according to before and after the anaemia criterion was changed. It may also be useful to conduct a supplementary analysis among just the data where severe anaemia was not an exclusion criterion – but I do not want to request that without knowing the numbers involved.We present a supplementary analysis split as requested.2) The Leslie study [14], from which data was included in the meta-analysis had "372 cases and 743 controls", but from the numbers in Table 1 it seems that not all of these have been included in the present meta-analysis. The Materials and methods need to make clear which of the samples from the Leslie study were included in the meta-analysis, and why. I can imagine this may be something to do with the geographical origins of the refugee populations in the Leslie study, since this study focused on high malaria transmission regions only, but this needs to be explained.This is partly a mistake on our part and partly a lack of clarity. We had only included data from Leslie et al. for the individuals who had been genotyped (418 samples). However, genotyping data are only necessary for females (where the phenotypic test cannot determine heterozygosity). Therefore, we can augment the total number of samples in the meta-analysis to 316 male controls and 157 male cases . We can only use data from the genotyped females reported in Leslie for the meta-analysis (124 controls and 78 cases). We have added one line to the Materials and methods to clarify this.3) The method used here implicitly assumes that there is little heterogeneity in G6PD Med frequencies between different Pashtun tribal groups currently living in malaria endemic regions. It is hard to know whether or not this is a reasonable assumption, because inevitably the evidence for G6PD Med frequencies in different tribal groups is based on small numbers (e.g. the Leslie study [14]). The authors do acknowledge this with the statement in the Discussion that "genetic heterogeneity within the Pashtun group" may be a confounder. However, I would like to see this potential limitation of the study discussed in more detail in a specific section of the Discussion or Introduction, including reasons why it is reasonable to assume a consistent Med frequency. Are there any other sources of evidence about genetic diversity within the Pashtun group, e.g. from studies at other loci?https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076748). Having additional genetic marker data would allow for adjustment for population stratification but this is not available.We accept that this is a limitation. As pointed out above the populations studied in this investigation, and those reported earlier by Leslie et al. were located in a similar geographic area divided by the Afghanistan-Pakistan border. There are few population genetic studies of the Pashtun ethnic group, but there is evidence that it is a diverse group (4) The Materials and methods explain the case definition for P. vivax infection for the majority of the data (that which was obtained by the authors). However, it is not stated that the case definition for P. vivax infection was equivalent in the Leslie study, from which data was included in the meta-analysis.P vivax malaria, age 3 years or older, written or witnessed verbal consent (by parents in the case of minors), available for the duration of follow-up, and willingness to be tested for G6PD deficiency at admission. Exclusion criteria were general condition requiring hospital admission, evidence of any concomitant infection or disease likely to mask treatment response, known allergy to any classes of the study drugs, known methemoglobin reductase deficiency, treatment within the past 7 days with any drug with known antimalarial properties or with an investigational drug within 30 days, severe anaemia (haemoglobin <7 g/dL), mixed infection , and pregnancy (confirmed by testing), lactation, or both. Thus, the criteria, case definition and the patient populations were all very similar to the current studies by Awab et al. with which they were pooled.Inclusion criteria in the Leslie et al. clinical trial  were microscopically confirmed 5) The number of female homozygotes in this study is (inevitably) very small. I do not object to the authors including female homozygotes and male hemizygotes in the same category, I can see the biological logic behind it, and I like their entire approach to estimate the gene dose effect. However, on the other hand, surely it remains possible that, despite their similar red blood cells, a female homozygote for G6PD Med may not be exactly the same as a male hemizygote for G6PD Med with respect to their likelihood of displaying clinical vivax malaria. If the authors had fitted α, β and γ , it seems likely to me that α and β would have been similar to the current estimates and γ would have had a huge credible interval because there is little data. If the authors concur, it would be worth acknowledging this explicitly in the Discussion – because to me this means that this study tells us quite a lot about male hemizygotes and female heterozygotes, but doesn't tell us much about female homozygotes.We agree that the study conclusions regarding the fully deficient state are almost entirely based on males hemizygotes. However, the model assumption is about a marginal relative effect: it could be the case that female homozygotes and male hemizygotes do not have the same absolute probability of having clinical vivax (for reasons unrelated to G6PD deficiency). However, the model assumption is that, conditional on having clinical malaria, the protective effect of the hemizygote and homozygotes states are equal. There is no evidence that this would not be the case but, as mentioned, the data are too sparse to support this claim . We have one sentence in Discussion making this assumption explicit.Reviewer #2:This review is mainly focussed on the statistical analysis part of the article.The authors have used a well-established and standard Bayesian methodology for inference on the role of G6PD Med as a protective effect against vivax malaria. The Bayesian model is appropriate assuming that the Hardy-Weinberg equilibrium is held in the studied population(s). A Markov Chain Monte Carlo sampling based inference is used for the Bayesian inference.I have the following concerns and/or comments which should be addressed.1) The author states that weakly informative Bayesian priors were used in the model. But this is not true for the allele frequency parameter p. I believe Β is a strongly informative prior for p. Some explanation for choosing such a strong prior for p should be included in the article. How are the values for hyperparameters (2 and 18) chosen? How sensitive are the results with respect to these hyperparameter values?We disagree that this is a strong prior. The Β distribution has 80% of its mass between 2.8% and 19%  Given our prior knowledge , this fits the standard usage of the “weakly informative”. A plot is shown for illustration in hat), in the R-markdown document.2) It is desired that the authors include some plots and statistics assessing the convergence of the MCMC method, e.g. trace plots of the posterior samples, the Gelman-Rubin statistics . Traceplots are now included in the RMarkdown and referred to in the Materials and methods.3) The Bayesian model assumed that the Hardy-Weinberg equilibrium is held in the studied population(s). Can the authors discuss why this assumption is reasonable in the studied population(s) and how the model can be modified if the equilibrium is disturbed by mutations, natural selection and non-random mating?We now include standard tests for H-W equilibrium in the RMarkdown (reported in the main text as well). There is no evidence in the controls for departure from H-W.Reviewer #3:The Short Report article suggests that the Mediterranean variant of G6PD deficiency confers a strong gene-dose proportional protective effect against symptomatic Plasmodium vivax malaria in the Pashtun ethnic group in Afghanistan. Results suggesting that G6PDd Med hemizygous males and homozygous females have 67% relative reduction against acute P. vivax malaria and G6PDd Med heterozygous females have 51% relative reduction is of particular interest for malaria and human genetics researchers. The authors present a very thoughtful discussion of P. vivax sensitivity to G6PD deficiency oxidant effects on parasite viability.1) The authors assume that Hardy-Weinberg equilibrium exists in the populations studied but they provided no evidence that it exists. Please provide the evidence that Hardy-Weinberg equilibrium exists for the alleles/genotypes for G6PD. Please also discuss how a deviation from Hardy-Weinberg equilibrium would affect the results of this study.As above. There is no evidence for large departures from H-W.2) Previous studies have shown that the G6PD Mediterranean allele is the predominant G6PD deficiency allele in the region. The G6PD gene is, however quite polymorphic, with over 180 mutations and at least two different G6PD Mediterranean allelic haplotypes. Thus the possibility that further genetic polymorphism is associated with G6PD deficiency and significant potential for population mixing (particularly in large cosmopolitan urban settings) may introduce genetic diversity in the G6PD gene. Please discuss how additional G6PD deficiency alleles could affect the results of this study.Previous studies have only found the G6PD Mediterranean variant in the Pashtun, but these have not been exhaustive. If additional G6PD deficiency variants did occur in this study they would have diluted the protective effect estimate. We have added a sentence in the Discussion.P. falciparum accounts for 95% of malaria cases in Afghanistan; P. vivax 5% . Diagnosis of P. vivax and accurate assessment of mixed species infections in areas where these two species are co-endemic is known to confound malaria microscopy. Additionally, submicroscopic Plasmodium species infections (involving P. vivax in particular), revealed by parallel PCR methods, is now very well established. Please provide further details as to how Plasmodium species diagnosis was performed, how monoinfection by P. vivax was confirmed and how exclusion of other Plasmodium species and/or mixed species infections were ruled out. If the complexity of Plasmodium species infections has been confirmed by PCR, the authors should revise their Laboratory Procedures section and present their data in these Result section. If Plasmodium species infections have not been confirmed by PCR, the authors should perform this work.3) Brief details were provided on malaria diagnosis by Giemsa-stained thick and thin blood films. This is an unfortunate mistake by WHO EMRO.


Plasmodium falciparum malaria has been challenging, primarily due to high levels of antigen polymorphism and a complex parasite lifecycle. Immunization with the P. falciparum merozoite antigens PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5 has been shown to give rise to growth inhibitory and synergistic antisera. Therefore, these five merozoite proteins are considered to be promising candidates for a second-generation multivalent malaria vaccine. Nevertheless, little is known about IgG and IgM responses to these antigens in populations that are naturally exposed to P. falciparum. In this study, serum samples from clinically immune adults and malaria exposed children from Ghana were studied to compare levels of IgG and IgM specific for PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5. All five antigens were found to be specifically recognized by both IgM and IgG in serum from clinically immune adults and from children with malaria. Longitudinal analysis of the latter group showed an early, transient IgM response that was followed by IgG, which peaked 14 days after the initial diagnosis. IgG levels and parasitemia did not correlate, whereas parasitemia was weakly positively correlated with IgM levels. These findings show that IgG and IgM specific for merozoite antigens PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5 are high in children during P. falciparum malaria, but that the IgM induction and decline occurs earlier in infection than that of IgG.Developing a vaccine against  Plasmodium falciparum malaria is estimated to cause more than 400,000 deaths annually [P. falciparum is rampant, young children are particularly vulnerable to the disease. This is primarily because they lack protective immunity [annually . In sub-immunity  which isimmunity –6.P. falciparum malaria has proved difficult primarily due to high levels of antigen polymorphism and a complex parasite lifecycle [Parasite resistance to antimalarial drugs and insecticides poses a major threat –9 and maifecycle –12. To difecycle . Neverthifecycle . Howeverifecycle , 16 meanifecycle , 18.P. falciparum-specific IgM in protective immunity [P. falciparum-infected children and adults, and is long-lived in the absence of re-infection [Plasmodium-specific IgM memory B cells have been found in naturally exposed human subjects and in mouse models of malaria infection [Immunoglobulin G (IgG) is thought to play a major role in NAI , 20, howimmunity –24. The nfection . Furthernfection .Understanding IgM and IgG acquisition and maintenance is important for the development of antibody-based vaccines. IgM has been shown to be critical for protection against multiple human pathogens including West Nile virus, HIV, Hepatitis C virus, chikungunya and influenza viruses –31. FurtP. falciparum merozoite antigens PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5 have recently been identified as promising vaccine candidates [in vitro when combined [The ndidates . This isndidates , 34. Thecombined . Furthercombined –38 - is combined –41. Nevecombined , 41–47.P. falciparum malaria [P. falciparum malaria. Furthermore, we show, that in children IgM induction and decline towards PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5 occurs earlier in infection than IgG.Previously, we and others have shown that IgG responses are induced against PfRH5 and PfCyRPA during  malaria , 47. In P. falciparum malaria transmission intensity, from May – August 2015, as described previously [Samples from adults were collected from Asutsuare, a town within the Shai-Osudoku district, Greater Accra Region, from April – June 2014, as previously described . Sampleseviously , 48.The cohorts collected here are representative of the area in which they were collected. Study participants were enrolled on admission at Hohoe municipal hospital (Febrile children) or from within the Hohoe and Asutsauare communities after engagement with community/opinion leaders. This was followed by a durbar in the community to explain the study in the local language to the residents . SubjectP. falciparum but were malaria negative by rapid diagnostic test (RDT) and microscopy were recruited from within the Asutsuare community and included in the study  [P. falciparum malaria was confirmed by a positive rapid RDT and by light microscopy with a parasite density > 2,500 parasites/μL. These malaria patients were divided into those with severe malaria  or uncomplicated malaria , respectively, as previously described [. All children were treated according to Ghanaian medical treatment guidelines. Children with SM and UM were seen again at 14 days (Day 14) and 42 days (Day 42) after the initial day of diagnosis (Day 0). In addition, 85 clinically healthy, age-matched children from within the Hohoe community were recruited in a similar manner, and grouped as either asymptomatic  ) or uninfected  controls (where children were negative by microscopy and RDT) .In brief, 78 healthy adult (18 – 69 years) male and female volunteers who were likely to have been exposed on numerous occasions to e study  . Febrileescribed . FebrileHuman research ethics approvals were obtained from the Ethics Committee of the Noguchi Memorial Institute for Medical Research, University of Ghana (NMIMR-IRB: CPN: 010/12-13) and from the Ghana Health Service (GHS-ERC 08/05/14). Plasma samples for the 10 non-exposed, anonymous Danish adults used as negative controls in the ELISAs were approved by the Regional Research Ethics Committees for the Capital Region of Denmark (Protocol H-4-2013-083). All methods were performed in accordance with the relevant guidelines and regulations.Drosophila melanogaster Schneider 2 stable cell line system as previously described [Recombinant PfRH5, sourced from Prof. Simon J Draper , was expressed using a escribed .TM Expression System Kit (ThermoFisher) as per manufacturer’s instructions. Media was harvested four days post transfection for purification. This supernatant was then passed through a 5 mL HisTrap HP chromatography column  charged with 0.1M NiSO4.6H2O and washed with sodium phosphate buffer with 25 mM Imidazole. Proteins were eluted from the column by passing through 500 mM imidazole in sodium phosphate buffer. Elution fractions were pooled and buffer exchanged into PBS. Quality of antigens were assessed using SDS-PAGE and western blotting performed with Anti-his-HRP (C-term) antibody (Miltenyi Biotec).Recombinant extracellular domains of merozoite antigens PfMSRP5-bio-his (Addgene plasmid #50805), PfSERA9-bio-his (Addgene plasmid #50820), PfRAMA-bio-his (Addgene plasmid #50737), and PfCyRPA-bio-his  were gifts from Dr. Gavin Wright  . TransieMicrotiter plates, 96 well  were prepared with 2 μg/mL of either PfMSRP5, PfSERA9, PfRAMA, PfCyRPA or PfRH5 and incubated overnight. Plates were washed three times with PBS-T and then blocked for one hour with blocking buffer (5% non-fat dry milk in PBS-T). The bound antigen was then incubated with plasma at a final dilution of 1:100 for 1 hour, followed by a 1 hour incubation with anti-human IgG-AP  or anti-human IgM-AP . Next, an incubation with 4-Nitrophenyl phosphate disodium salt hexahydrate tablets (Sigma Aldrich) dissolved in 1 × Diethanolamine Substrate Buffer (Sigma Aldrich) was performed. Color development and absorbance were measured at 405 nm.sample-ODblank)/ (ODpositive control -ODblank). In order to define a true positive result, a cut-off value was calculated for each assay as the mean + 3SD of OD values in plasma from 10 healthy Danish donors.Plasma pools of semi-immune adults against each antigen were used as positive controls and to normalize plate to plate variability. Antibody levels were presented as arbitrary units (AU) calculated as . A Kruskal-Wallace and Dunn’s post-hoc test were used to evaluate antibody levels in the different donor groups, and a Friedman and Dunn’s post-hoc test for longitudinal samples. Fisher’s exact tests were performed to compare proportions. A Wilcoxon matched-pairs signed rank test was performed to compare IgM and IgG levels. Spearman’s correlation co-efficient were performed to compare; IgM and IgG levels to merozoite antigens, antibody levels to merozoite antigens, antibody levels to age, and antibody levels and log-transformed parasitemia. Principle component analysis was performed with the ggbiplot package in R  using default parameters. For each IgG and IgM reactivity combination, the parasitemia (log10) were compared for positive and negative responders using Wilcoxon test and Benjamini–Hochberg adjusted P values . The analysis was limited to antigen combinations for which there were at least six positive and negative responders. Analysis was performed using R and GraphPad Prism Software. Temporal changes in antibody levels were evaluated using cohort running means as previously described . Linear P. falciparum malaria. At baseline (Day 0), more than two thirds of the children had IgM specific for PfMSRP5 (83.2%), PfSERA9 (73%) and PfRAMA (76.8%) . The prevalence of IgM specific for PfCyRPA and PfRH5 was slightly lower, with 63.2% having PfCyRPA-specific IgM, and 56% having PfRH5-specific IgM . IgM responses for all antigen combinations tested were all significantly positively correlated , suggesting that these antigens are immunologically regulated in a similar way following P. falciparum malaria.Recently, merozoite-specific IgM has been found to be important in NAI against malaria . However. When IgG responses were assessed for correlation between antigens, significant positive correlation was observed for PfMSRP5, PfSERA9 and PfRAMA .We next investigated whether IgG is induced against PfMSRP5, PfSERA9, and PfRAMA. PfCyRPA and PfRH5 IgG responses were not examined as these have been previously reported . Half ofThe analyses above confirm that both IgG and IgM to each of the merozoite antigens tested are induced following natural exposure to malaria. These results support the previously published data on PfMSRP5, PfSERA9 and PfRAMA -specific IgM and IgG , 33.P. falciparum malaria into two groups: uncomplicated (UM) and severe malaria (SM). Furthermore, we tested IgG and IgM responses against the five merozoite antigens in clinically immune adults (CI), and in children from three additional distinct clinical groups: healthy control children (HC), febrile control children (FC), and asymptomatic control children (AC)  see . Clinica. Overall, IgM prevalence and IgM levels in children without P. falciparum malaria (FC and HC) were significantly lower than those in children with P. falciparum malaria  . Furthermore, for all antigens, IgM prevalence and IgM levels (with the exception of PfRH5 levels) in clinically immune adults were significantly lower than in children with malaria (UM and SM) . Together, these results indicate that levels of IgM specific for merozoite antigens PfMSRP5, PfSERA9, PfRAMA and PfCyRPA are higher in children with P. falciparum malaria than in those without.All merozoite antigens tested were recognized by IgM from all the clinical categories . Breadth analysis of IgM showed that children with acute P. falciparum malaria (SM and UM) had a significantly broader response than clinically immune adults (CI) and uninfected children (HC and FC). These results further indicate that children with acute P. falciparum malaria have higher antibodies specific for selected merozoite antigens than children without P. falciparum malaria.Immunity to malaria is thought to result from high antibody titers (magnitude) to multiple antigens (breadth) . Therefo. IgG levels towards PfCyRPA and PfRH5 have been previously reported in all children used in this study [. Overall, IgG prevalence and levels in uninfected healthy children (HC) were lower than those in clinically immune (CI) adults and in children with P. falciparum malaria  . Specifically, for PfSERA9, levels of IgG were significantly higher in asymptomatic (AC) children than in children symptomatic uncomplicated malaria (UM) children .Next, we assessed PfMSRP5, PfSERA9 and PfRAMA-specific IgG levels in the six distinct groups is study , thus on. These results suggest that children with P. falciparum malaria have higher merozoite antigen- specific IgG than children without P. falciparum malaria regardless of symptoms. Overall, the IgM and IgG responses to each of the antigens tested, mostly indicate parasite exposure.Breadth analysis of IgG showed that clinically immune adults (CI) and children with severe malaria (SM) and asymptomatic malaria (AC) had a broader response than healthy children (HC) To better understand the contribution of IgG and IgM to protection, IgG and IgM responses within clinical categories for each antigen were compared. However, because of possible sensitivity differences of secondary detection antibodies and different affinities of the primary antibodies, caution must be taken when comparing IgM to IgG. To allow for direct comparisons the data was standardized and compared as level of antibody over background cut-off. IgG responses specific for PfCyRPA and PfRH5 in cohorts of children previously published  were inc(S1 File). For the remaining antigens, IgM prevalence and levels were higher than IgG prevalence and levels in children with acute P. falciparum malaria (SM and UM) . For CI adults, IgM prevalence and levels were higher than IgG prevalence and levels for PfCyRPA and PfRH5 .PfRAMA-specific IgG prevalence and levels were significantly higher than PfRAMA-specific IgM for all groups, indicating that regardless of clinical category PfRAMA induces a strong IgG responses (S1 File). For subjects with symptomatic P. falciparum malaria (SM and UM), IgM responses were significantly broader than IgG responses (S1 File) indicating that high levels of IgM are induced against multiple antigens in non-immune individuals.Breadth analysis further revealed that IgG responses were broader than IgM responses for subjects who were either aparasitemic or asymptomatic  . Correlation was observed for all antigens for clinically immune adults (CI), children with UM (with the exception of PfRH5) and children with asymptomatic malaria (AC) (with the exception of PfSERA9). For subjects with severe malaria (SM), correlation was observed for PfRAMA and PfCyRPA only. These results suggest co-occurrence of IgM and IgG in subjects with less severe disease. The co-occurrence of IgM and IgG in subjects with less severe disease observed here is consistent with the limited published data on IgM [To investigate the potential importance of IgM alongside IgG, IgM and IgG responses were assessed for correlation between antigens a on IgM .P. falciparum malaria (SM and UM group).Higher merozoite antigen-specific IgG and IgM responses have been shown to be associated with both age and lower parasitemia , 33. To S4 Table in S1 File). Additionally, levels of IgG specific for PfMSRP5, PfSERA9 and PfRAMA did not increase significantly with age (S4 Table in S1 File). To assess age and antibody responses globally, a PCA analysis was performed. This analysis included previously published IgG responses to PfCyRPA and PfRH5 [Fig 5A).First, the relationship between age and IgM and IgG responses were examined. IgM levels did not correlate with age for any of the merozoite antigens tested  or antigens tested  and parasitemia.Next, the relationship between parasitemia and Ig-specific merozoite antigen responses were examined  showed that subjects with lower parasitemia (<100000 parasites/ul) clustered together and were partially segregated by IgG specific for PfMSRP5, PfSERA9, PfRAMA and PfRH5, although there was some overlap. This suggests that IgG reactivity to multiple antigens is associated with lower parasitemia.To assess the relationship between parasitemia and antibody responses to the merozoite antigens studied here, a PCA analysis was performed. This analysis included previously published IgG responses to PfCyRPA and PfRH5 . AnalysiP. falciparum malaria using Wilcoxon test and Benjamini–Hochberg adjusted P values . Although IgM responses to MSRP5, RAMA, CyRPA, MSRP5+RAMA, MSRP5+CyRPA, SERA9+CyRPA, RAMA+CyRPA, MSRP5+RAMA+CyRPA and SERA9+RAMA+CyRPA (S5 Table in S1 File) were associated with higher parasitemia, and IgG responses to MSRP5+SERA9+RH5 and MSRP5+SERA9+RAMA+RH5 (S6 Table in S1 File) were associated with lower parasitemia, these associations were not significant after adjusting for false discovery rate.To determine whether certain combinations of antigen-specific IgG or IgM responses affected parasitemia, we compared parasitemia_(log10) between negative and positive responders for all antigen combinations possible. This was performed on subjects with acute P. falciparum malaria (SM and UM). Responses were measured at 2 weeks (Day 14) and 6 weeks (Day 42) after initial diagnosis and sampling (Day 0) .We previously reported that IgG levels towards PfRH5 and PfCyRPA increased in a minority of children at 2 weeks post-diagnosis, but quickly declined, leading to the conclusion that IgG responses to these antigens were short-lived. To determine whether IgM responses specific for the merozoite antigens PfMSRP5, PfSERA9, PfRAMA, PfCyRPA, PfRH5, and IgG responses specific for the merozoite antigens PfMSRP5, PfSERA9, PfRAMA were sustained or transient, IgM and IgG levels to each antigen were measured longitudinally in children with acute . Additionally, when slopes were compared, responses to PfMSRP5 (p = 0.1732), PfSERA9 (p = 0.2940) and PfCyRPA (p = 0.2141) followed that of the predicted half-life of IgM whereas responses to PfRAMA (p = 0.0330) and PfRH5 (p = 0.0151) declined more slowly than predicted half-life. Nevertheless, by Day 42, mean responses for PfRH5 and PfRAMA were below cut-offs.IgM declined significantly throughout follow-up with responses to all antigens significantly lower on Day 42 compared to those on Day 0 and Day 14 . An increase was also observed for PfRAMA, however, this finding was not significant. The increase of IgG levels at Day 14 was followed by a significant decline in IgG levels to all antigens at 42 days post-diagnosis . When slopes were compared, responses to PfMSRP5 were not significantly different to that of predicted IgG half-life (p = 0.5887), whereas IgG responses towards PfSERA9 and PfRAMA declined more slowly than predicted by the IgG metabolic half-life . Furthermore, PfSERA9 responses at Day 42 were significantly higher than those at time of diagnoses . These results show that in children IgG responses to PfMSRP5 are transient, whereas IgG responses specific for PfSERA9 and PfRAMA do not rapidly decline following acute P. falciparum malaria. This suggests that long-lived plasma cells are induced only in response to the latter antigens. Overall, we observed that IgM responses in children to the antigens tested here were transient, supporting the general view that IgM induction and decline occur earlier in infection than that of IgG [IgG levels increased significantly from diagnosis (Day 0) to 14 days post-diagnosis for PfMSRP5 and PfSERA9  antibodies were already induced and so it would be beneficial to include a pre-infection time-point to correctly evaluate induction and waning. Additional longitudinal sampling would also help to strengthen this data as re-infection and further protection of antibody responses could be evaluated. Finally, as mentioned above, caution must be taken when comparing IgM and IgG responses due to possible differences in the sensitivity of detection antibodies.Although the results reported here are informative for P. falciparum malaria exposure we observed high levels of IgM and IgG where IgM induction and decline occur earlier in infection than that of IgG. Although both IgM and IgG responses in children reported here are transient, vaccine induced antibodies against the five antigens analyzed, especially PfRH5, may prove more effective and reach protective levels [in vitro [Overall, after e levels . Howeverin vitro . This stS1 File(PDF)Click here for additional data file.


Thrombocytopenia in malaria involves platelet destruction and consumption; however, the cellular response underlying this phenomenon has still not been elucidated.Plasmodium vivax infected (Pv-MAL) patients. To find associations between platelet indices and unbalanced Th1/Th2/Th17 cytokines as a response to thrombocytopenia in P. vivax malaria and 37 healthy donors from the same area (endemic control group - ENCG). Platelet counts and quantification of Th1/Th2/Th17 cytokine levels were compared in 77 patients with uncomplicated  Thrombocytopenia was the main manifestation in 55 patients, but was not associated with parasitaemia. The Pv-MAL patients showed increases in the mean platelet volume (MPV), which may be consistent with larger or megaplatelets. Contrary to the findings regarding the endemic control group, MPV and platelet distribution width (PDW) did not show an inverse correlation, due the increase in the heterogeneity of platelet width. In addition, the Pv-MAL patients presented increased IL-1β and reduced IL-12p70 and IL-2 serum concentrations. Furthermore, the reduction of these cytokines was associated with PDW values.P. vivax malaria. Our data demonstrate that an increase in MPV and the association between reductions of IL-2 and IL-12 and PDW values may be an immune response to thrombocytopenia in uncomplicated  Plasmodium falciparum and Plasmodium vivax malaria (Pv-MAL).,,,,,,Thrombocytopenia is one of the most frequent haematological alterations in acute malaria infections involving ,,,The pathogenesis of thrombocytopenia caused by malaria involves multifactorial phenomenon and leads to destruction and consumption of platelets.9 platelets per day and lifespan is 7-10 days. Under inflammatory and thrombocytopenia conditions, such as malarial infection, platelet lifespan is reduced to 2-3 days and MKs differentiate in order to replenish platelets.,Platelets are small disc-shaped cellular fragments with no nucleus, and which have regenerated continuously from megakaryocyte (MK). The MKs are large, polyploid cells that have lost their proliferative ability and have become progressively differentiated, creating an invaginated membrane system for platelet formation.,,,,,,,,,,P. vivax infections.In general, thrombocytopenia in malaria is accompanied by both higher mean platelet volume (MPV) and larger platelet distribution width (PDW), which arise concomitantly with reduced platelet counts.Study area and population - 77 Pv-MAL patients (diagnosed as confirmed cases via examination of the thick blood smear) were enrolled at the Tropical Medicine Institute in the municipality of Coari, Amazonas State, Brazil. Coari is located at latitude: -4.08488, Longitude: -63.1417; 4º5’6” South, 63º8’30” West, in an area with a high incidence of malaria. It is estimated that approximately 30% of its inhabitants are at risk of malaria . P. vivax is responsible for 99.6% of cases of malaria.3) or mild (> 500 parasites/mm3). Blood cells were examined using an ABX Micros 60 haematology analyser (Horiba). 37 healthy donors were included in the study to form an endemic control group (ENCG). These were health professionals who are residents of this area, who had no previous history of malaria and tested negative in two thick blood smear examinations which were performed at intervals of 15 days. When detected, thrombocytopenia was classified as being either mild  or severe .Parasitaemia levels were defined as low parasitaemia , and supervised by the Blood Bank (HEMOAM). In addition, participants were also screened for dengue Virus by polymerase chain reaction (PCR). After malaria diagnosis, blood samples were collected in EDTA tubes , and sent in thermal boxes (maintained at 4ºC) to the Molecular Biology Laboratory of the Federal University of Amazonas in Coari.Serum cytokine measurements - Frozen plasma samples were sent on dry ice to the Core Flow Cytometry Facility at the HEMOAM blood bank for cytokine measurement with the cocktail kit . The cytokines IL-2, IL-4, IL-5, IL-6, IL-10, TNF, IFN-γ and IL-17A were measured according to manufacturer’s instructions, and IL-1β, IL-8 (CXCL-8) and IL-12 (IL-12p70) were measured by ELISA .Statistical analysis and data presentation - All data were analysed using GraphPad prism software version 5. The Th1/Th2/Th17 cytokines levels were separated into non-thrombocytopenic, mild and severe thrombocytopenia according to platelet counts. Reference intervals for the platelet parameters according to the recommended European Federation of Clinical Chemistry and Laboratory Medicine were used.Ethics - All protocols and consent forms were approved by the Research Ethics Committee at HEMOAM, under approval No. #449.864/2011. Participants read and signed the written informed consent form prior to enrollment, according to Declaration of Helsinki and Resolution 466/12 of the National Health Council for research involving human subjects. All patients were treated according to recommendations of Brazilian Ministry of Health.All patients included in the study had uncomplicated paroxysms at the time of seeking medical care. None of the patients reported diarrhea or vomiting. 53 (68.8%) patients were male and 24 (31.2%) were female. 15 of the patients (19.5%) reported primary malaria and 62 reported at least one previous malaria infection. None of the patients showed signs of anaemia or severe malaria. 30 patients had leukopenia (38.9%) and lymphopenia was evident in 32 patients (41.5%).3 platelets/μL) or severe (below 50 x 103 platelets/μL). Among the patients, 44 (80%) had moderate thrombocytopenia and 11 displayed severe thrombocytopenia (20%); none of the patients needed platelet transfusion. In Thrombocytopenia was observed in 55 of 77 patients (71.4%), also in five of the 37 individuals in the ENCG. Thrombocytopenia was classified as being either moderate (51-149 x 103), while 39 Pv-MAL patients (50.6%) showed moderate parasitaemia . However, not all platelet indices differed in relation to parasitaemia  and platelet counts A-D, thouThe serum concentrations of Th1/Th2/Th17 cytokines were compared in the Pv-MAL patients and the endemic control group . Pv-MAL To assess predictors of thrombocytopenia using cytokine levels of Th1/Th2/Th17 and platelet indices, a multinomial model was used to exclude parasitaemia as a confounding factor . For thiFinally, a linear regression was performed to assess predictors of thrombocytopenia in Pv-MAL patients in relation to the ENCG. Since the PDW index showed negative correlation with platelet counts, we used it as a dependent variable, while the other platelet indices and cytokine levels were considered independent variables . The lin,,,Thrombocytopenia is the most common haematological disorder in patients with malaria and one of the major concerns when accompanied by multiple organ failures.,,,,The total platelet mass is considered as a product of platelet count X MPV, and this inverse correlation may be closely regulated.,Furthermore, parasitaemia was associated with higher levels of IL-10 and a slight increase in the serum concentration of IL17A. Increased levels of IL-10 have already been seen in patients with higher parasitaemia and patients with recurrent malaria.,,P. falciparum malaria.,,,P. vivax infections.This study had several limitations. The sample size of patients was small and PCR for malaria diagnoses was not performed in all of those enrolled in this study. Nor did we evaluate antimicrobial failure. The population of the municipality of Coari is very well-informed about malaria and knows about the need to diagnose malaria at the onset of the disease, hence, this may explain why all the patients had uncomplicated malaria. Another limitation to the study was the semi-quantification of parasites which is carried out in the Brazilian Amazon, and which made it impossible to assess further correlations, as also observed by a few other studies.In conclusion, the increase in MPV indirectly indicated the presence of mega platelets. The loss of its correlation with PDW in Pv-MAL patients may also be a consequence of the release of mega platelets. The reduction of IL-2 and IL-12 associated with PDW may be consistent with regulation of immune response which is associated with appearance of mega platelets in thrombocytopenia caused by vivax malaria.