Degradation of the endothelial glycocalyx is associated with mortality in adult falciparum malaria. However, its role in the pathogenesis of non-falciparum malaria is unknown. In Malaysian patients with knowlesi (n = 200) and vivax (n = 61) malaria, and in healthy controls (n = 50), we measured glycocalyx breakdown products plasma syndecan-1 and urinary glycosaminoglycans, and evaluated correlations with biomarkers of disease severity. Urinary glycosaminoglycans were increased in patients with knowlesi and vivax malaria compared to healthy controls, and in knowlesi malaria were highest in those with severe disease. In knowlesi malaria, plasma syndecan-1 was also highest in those with severe disease, and correlated with markers of endothelial activation , asymmetric dimethylarginine (ADMA) and impaired microvascular reactivity. Syndecan-1 also correlated with endothelial activation  and ADMA in vivax malaria. In knowlesi malaria increased syndecan-1 was associated with acute kidney injury, after controlling for age and parasitemia. In knowlesi malaria, the difference in median syndecan-1 between severe and non-severe disease was more marked in females than males. Endothelial glycocalyx degradation is increased in knowlesi and vivax malaria, and associated with disease severity and acute kidney injury in knowlesi malaria. Agents that inhibit glycocalyx breakdown may represent adjunctive therapeutics for severe non-falciparum malaria. Plasmodium falciparum is the major cause of malaria deaths globally, outside of Africa the non-falciparum species Plasmodium vivax and Plasmodium knowlesi cause significant disease burden, with both species able to cause severe and fatal disease6. P. knowlesi is a particular health problem in Malaysia, where this species now accounts for > 98% of all malaria cases7, and severe disease occurs in 6–9% of patients9. Complications include respiratory distress and acute kidney injury (AKI), with the latter particularly common in older adults11. Given the long-term consequences of AKI12, adjunctive treatments of severe malaria are warranted to attenuate this complication. Development of adjunctive treatments requires improved understanding of the pathogenic mechanisms of severe disease.Malaria causes major morbidity and mortality worldwide. While 14. Contributing factors include intravascular haemolysis, impaired nitric-oxide (NO) bioavailability, and endothelial activation14. In falciparum malaria, degradation of the endothelial glycocalyx also occurs18, and in adults is associated with disease severity and mortality15. However, the role of the glycocalyx in knowlesi and vivax malaria has not been evaluated.As with falciparum malaria, severe knowlesi and vivax malaria are characterised by microvascular dysfunction, leading to impaired tissue perfusion and organ dysfunction20). It regulates endothelial cell NO release, prevents leakage of intravascular fluid and proteins, and attenuates leukocyte- and platelet-binding to endothelial adhesion receptors19. The glycocalyx is comprised of membrane-bound proteoglycans, glycosaminoglycan chains (GAGs), glycoproteins, and adherent plasma proteins . The major glycosaminoglycans are heparan sulfate, chondroitin sulfate and the non-sulfated hyaluronan, while the proteoglycans include the syndecans and glycipans. When the glycocalyx degrades, a number of these components are shed into the plasma and/or urine and can be measured as biomarkers of glycocalyx breakdown. The best studied of these biomarkers include plasma syndecan-1, plasma heparin sulfate, urinary hyaluronan, and total urinary sulphated GAGs. Glycocalyx degradation has been increasingly recognized to play a key role in the pathogenesis of a number of other conditions, including sepsis20, dengue21 and most recently in severe COVID-1922. In sepsis, breakdown is mediated by glycocalyx “sheddases” such as heparanase, metalloproteinases, and hyaluronidase, activated by reactive oxygen species and proinflammatory cytokines20. Another activator of heparanase is the endothelial NO synthase (eNOS) inhibitor asymmetric dithemethylarginine (ADMA)23. ADMA is known to be elevated in falciparum malaria25 and sepsis26, and hence may contribute to glycocalyx breakdown in these conditions. While ADMA has not been found to be increased in vivax malaria14, the role of ADMA in knowlesi malaria has not been evaluated.The endothelial glycocalyx is a gel-like layer that covers the endothelium, and maintains vascular haemostasis, tone and permeability , ADMA, microvascular function, and other biomarkers of disease severity. Plasma concentrations of the glycocalyx stabiliser sphingosine-1-phosphate (S-1-P) were also evaluated.27. Baseline demographic, clinical and laboratory features are shown in Table A total of 261 patients with malaria were enrolled and had plasma and/or urine available for measurement of glycocalyx breakdown products, including 200 patients with knowlesi malaria (152 non-severe and 48 severe) and 61 with vivax malaria . Plasma was available for 197 patients with knowlesi malaria , and 58 patients with vivax malaria . Urine was available for 126 patients with knowlesi malaria  and 51 with vivax malaria . Fifty healthy controls were enrolled; plasma syndecan-1 and S-1-P were measured in 20, and urinary GAGS were measured in 47. Clinical and pathophysiological data from these patients have been previously reported10, among the 48 patients with severe knowlesi malaria, WHO severity criteria included hyperparasitemia , jaundice , respiratory distress , severe AKI , shock , metabolic acidosis , severe anaemia , and abnormal bleeding . Using Kidney Disease: Improving Global Outcomes (KDIGO) criteria to define AKI, and the Modification of Diet in Renal Disease [MDRD] equation to estimate baseline creatinine, AKI was present on or during admission in 31 (65%) patients with severe malaria, and 14 (9%) patients with non-severe malaria. Of the 9 patients with severe vivax malaria, severity criteria included shock , jaundice , respiratory distress , metabolic acidosis , abnormal bleeding , and convulsions . AKI by KDIGO criteria occurred in 3 (33%) patients with severe vivax malaria, and 3 (6%) with non-severe vivax malaria.As previously reportedp = 0.016), and were higher in both disease groups compared to controls . Surprisingly, concentrations of syndecan-1 were lower in those with non-severe disease compared to healthy controls (median 177 [IQR 130–519 ng/ml]), although this was not statistically significant. Concentrations of the glycocalyx stabilizer S-1-P were lower in patients with severe compared to non-severe knowlesi malaria ; however, again there was no significant difference between those with non-severe disease and healthy controls (median 1.49 [IQR 1.08–2.44] µM).In patients with knowlesi malaria, urinary concentrations of GAGs were higher in those with severe compared to non-severe disease . Concentrations of plasma syndecan-1 were higher in those with severe (median 180 [IQR 154–326] ng/ml) compared to non-severe disease, although numbers were small and this was not significant. In patients with vivax malaria there was no significant difference in concentrations of S-1-P between disease groups, or between either disease group and controls  and non-severe (1.12 [IQR 0.23–3.17] g/mol creatinine) disease compared to healthy controls , although this did not remain significant after controlling for parasitemia. There was no correlation between plasma syndecan-1 and urinary GAGs in vivax malaria. There was also no correlation between S-1-P and either plasma syndecan-1 or urinary GAGs, in either knowlesi or vivax malaria.Plasma concentrations of syndecan-1 were correlated with urinary GAGs in knowlesi malaria  and in vivax malaria ; in both species this remained significant after controlling for parasitemia . No such correlation occurred with urinary GAGs, in either knowlesi or vivax malaria. There was no difference in syndecan-1 or urinary GAGs in malaria patients enrolled before, or after, commencing antimalarial treatment.Plasma concentrations of syndecan-1 were correlated with duration of fever in knowlesi malaria (p < 0.0001), and higher in those with severe disease compared to controls . As with syndecan-1, concentrations of ADMA were unexpectedly lower in those with non-severe disease compared to controls (p = 0.007). The concentrations of ADMA in patients with severe knowlesi malaria were comparable to those of patients with severe falciparum malaria, enrolled in the same study cohort .In patients with knowlesi malaria, the median concentration of ADMA was higher in patients with severe compared to non-severe disease  and adjusting for parasitemia, syndecan-1 was associated with ADMA , creatinine , lactate , angiopoietin-2 , intercellular adhesion molecule 1 , osteoprotegerin , and inversely with microvascular reactivity . Syndecan-1 was also inversely correlated with albumin , sodium , bicarbonate , and chloride .In patients with knowlesi malaria, biomarkers of severity were more closely correlated with syndecan-1 than with urinary GAGs Table . After ap < 0.0001); this remained significant after controlling for the other biomarkers of severity listed above. After controlling for age and parasitemia, log-transformed syndecan-1 was also associated with acute kidney injury , again remaining significant after controlling for the other biomarkers listed above.In patients with knowlesi malaria, in a logistic regression model controlling for age and parasitemia, log-transformed plasma syndecan-1 was associated with severe disease , ADMA , and ICAM-1 , with the correlation with ADMA and ICAM-1 remaining significant after adjusting for parasitemia (p = 0.0001). After adjusting for parasitemia, plasma syndecan-1 also correlated with ADMA , angiopoietin-2 , and ICAM-1 . In vivax malaria, neither urinary GAGs nor plasma syndecan-1 were independently associated with risk for severe disease, or AKI.In vivax malaria Table , urinary29, and therefore anaemia and/or thrombocytopenia could potentially lead to reduced S-1-P with consequent glycocalyx breakdown. In patients with knowlesi malaria, syndecan-1 was inversely correlated with haemoglobin on enrolment . However, there was no correlation between haemoglobin and S-1-P . In knowlesi malaria, syndecan-1 was also inversely correlated with platelet count on enrolment , again remaining significant after controlling for parasitemia . Platelet count was also correlated with S-1-P , remaining significant after controlling for parasitemia .Red blood cells (RBCs) and platelets are both known to produce the glycocalyx stabiliser S-1-PP. knowlesi, in patients with vivax malaria syndecan-1 was inversely correlated with platelet count on enrolment, remaining significant after controlling for parasitemia . Platelet count on enrolment was also correlated with S-1-P , remaining significant after controlling for parasitemia . In vivax malaria there was no correlation between either syndecan-1 or S-1-P with haemoglobin.As with p = 0.014). Interestingly however, this difference occurred only in those with non-severe disease ; in patients with severe knowlesi malaria, median plasma syndecan-1 was as high in females (281 [IQR 234–610] ng/ml) as it was in males (286 [IQR 192–541] ng/ml). Thus, in females, median syndecan-1 was 2.6-fold higher in severe compared to non-severe knowlesi malaria, while in males, the median was only 1.7-fold higher. Median syndecan-1 was also higher in healthy male controls  compared to healthy female controls , although numbers were small and this was not statistically significant. There were no significant sex differences in urinary GAGs in patients with knowlesi malaria. Median plasma S-1-P was lower in males compared to females , with this difference observed in both severity groups.In patients with knowlesi malaria, median plasma syndecan-1 was higher in males compared to females , and lower S-1-P . There were no significant sex differences in urinary GAGs in patients with vivax malaria.In patients with vivax malaria there was a trend towards higher median plasma syndecan-1 in males compared to females ; however, this did not remain significant after controlling for parasitemia. There was no correlation between age and syndecan-1 in patients with vivax malaria. There were also no significant correlations between age and S-1-P, in malaria patients or in controls.There was a weak correlation between age and syndecan-1 in patients with knowlesi malaria  ligand. In addition to the ability of OPG to bind to syndecan-123. Although ADMA is known to be elevated in severe falciparum malaria, an association with glycocalyx breakdown has not previously been evaluated. In this study we found that ADMA was increased in severe knowlesi malaria, and was associated with both syndecan-1 and urinary GAGs. The findings in our study suggest that in addition to the role of angiopoietin-2 in mediating glycocalxy breakdown in malaria, agents that increase heparanase expression such as ADMA and OPG may also contribute. Importantly, ADMA and OPG are both increased in other conditions associated with glycocalyx breakdown, such as sepsis39, and may represent common mechanisms of glycocalyx degradation. It is notable that in vivax malaria, an increase in ADMA is not observed14, and it is possible that this may account for the lack of any observed increase in plasma syndecan-1 in these patients.Heparanase expression may also be induced by the eNOS inhibitor ADMA40. Mouse models have demonstrated that glycocalyx degradation increases glomerular permeability to albumin, leading to albuminuria41. We have found that microalbuminuria is common in AKI complicating knowlesi malaria (unpublished), further suggesting that glycocalyx breakdown may be a key mechanism of malaria-associated AKI. Albumin is also able to stabilise glycocalyx42, and hence hypoalbuminemia may in itself exacerbate glycocalyx degradation.Our finding that glycocalyx breakdown was an independent risk factor for AKI in knowlesi malaria is consistent with the key role that the glycocalyx in known to play in kidney function, with the glomerular glycocalyx regulating exchange of plasma proteins across glomerular capillaries28; however, we did not find a correlation between S-1-P and haemoglobin, and therefore reduced RBC production of S-1-P does not seem a likely explanation for the increased glycocalyx breakdown and elevated levels of syndecan-1. Alternatively, shedding of glycocalyx components from RBCs themselves may lead to increased plasma syndecan-1, given that RBCs, like endothelial cells, are known to contain a glycocalyx43. Shedding of glycocalyx from RBCs could further exacerbate anaemia, due to exposure of phosphatidylserine or other receptors enhancing eryptosis and/or phagocytosis.A notable finding is our study was the inverse correlation in knowlesi malaria between syndecan-1 and haemoglobin, with this correlation remaining significant after controlling for parasitemia and fever duration. RBCs are known to be major producers of the glycocalyx stabiliser S-1-P29, and in our study S-1-P was independently associated with platelet count. Thus, it is possible that reduced platelet-derived S-1-P could potentiate glycocalyx breakdown in malaria. Alternatively, glycocalyx breakdown may contribute directly to thrombocytopenia by mediating platelet adhesion and aggregation45.We also found independent inverse correlations between platelets and both syndecan-1 and GAGs, in both vivax and knowlesi malaria. As with RBCs, platelets have been shown to produce S-1-P46, and another study demonstrating that males have a thicker glycocalyx than females47. Consistent with this, we also found a trend towards higher syndecan-1 in male controls compared to female controls. Interestingly we found that in severe knowlesi malaria, syndecan-1 was as high in females compared to males, suggesting that severe disease in females may be associated with a greater proportionate loss of glycocalyx. In knowlesi malaria female sex is an independent risk factor for death3. While the cause of this increased risk is not well understood, it is possible that a thinner glycocalyx in females and a greater proportionate loss may contribute. Sex differences in disease severity and outcomes are observed in many acute infections49; whether differences in glycocalyx degradation contribute warrants further evaluation.We observed sex differences in glycocalyx degradation products. In patients with knowlesi malaria, syndecan-1 was higher and S-1-P lower, in males compared to females. This difference however was only observed in those with non-severe disease, and is consistent with a previous study in acute coronary syndrome demonstrating that males shed more glycocalyx than females20, dengue21 and COVID-1922. Finally, although we found associations between markers of glycocalyx degradation and biomarkers of disease severity, the nature of our study does not provide definitive evidence of causation.Our study had some limitations. First, the small number of cases of severe vivax malaria limited our ability to examine relationships between markers of glycocalyx degradation and disease severity in vivax malaria. However, with larger numbers of cases of severe knowlesi malaria clear associations with disease severity were found. Second, the median age of the control group was higher than that of the patients with vivax malaria; however, given that we did not find significant correlations between age and markers of glycocalyx breakdown, this is unlikely to have affected our results. Third, our study did not have a non-malaria fever comparison group; however, it is well documented that glycocalyx degradation is not specific to malaria fevers, with degradation also found in a number of other conditions including sepsisIn conclusion, we found that glycocalyx breakdown occurs in patients with knowlesi and vivax malaria, and is associated with disease severity and AKI in knowlesi malaria. Plasma syndecan-1 appeared to be a more clinically relevant biomarker than urinary GAGs, and may reflect more severe glycocalyx damage, occurring later in disease. Several agents have been demonstrated to stabilise endothelial glycocalyx, and may be potential agents for adjunctive treatment of severe non-falciparum malaria.The study was approved by the Ethics Committees of the Malaysian Ministry of Health and Menzies School of Health Research. Informed written consent was provided by all adults, and by the parent or guardian of any participant aged < 18 years.27. For the current study, patients enrolled between September 2010 and December 2012 were included if they had PCR-confirmed P. knowlesi or P. vivax monoinfection, were non-pregnant, ≥ 12 years old, had no major comorbidities or concurrent illness, were within 18 h of commencing antimalarial treatment, and had stored frozen urine and/or plasma available for measurement of glycocalyx breakdown products. Severe malaria was defined according to modified WHO criteria50, as previously described11. Renal function was further assessed using the Kidney Disease: Improving Global Outcomes (KDIGO) criteria for AKI51. Healthy controls were visitors or relatives of malaria patients, with no history of fever in the past 48 h and with blood film negative for malaria parasites.Patients were enrolled as part of a prospective observational study of all malaria patients admitted to Queen Elizabeth Hospital, an adult tertiary-referral hospital in Sabah, Malaysia53. Patients with severe disease were treated with intravenous artesunate, while those with non-severe disease received oral artemisinin combination treatment , as previously described27.Standardized history and physical examination were documented. Haematology, biochemistry, and lactate  were obtained on enrolment. Parasite counts were determined by microscopy, and parasite species identified by PCR54. Total urinary sulfated GAGs were measured on a subset by the dimethylmethylene blue (DMMB) colorimetric assay as previously described55. Urinary creatinine was measured by the alkaline picrate method, and the urine GAG concentrations were normalised to creatinine levels. All samples were diluted as per manufacturers’ recommendations but were adjusted if the resulting OD was outside the standard curve. Microvascular function was assessed using Near Infrared Spectroscopy , as previously described56. These data have been previously reported11, and are included here to assess correlation with glycocalyx degradation.Venous blood collected on enrolment in lithium heparin and citrate tubes was centrifuged within 30 min of collection and plasma stored at – 70 °C. Urine was also collected on enrolment and stored within 30 min at – 70 °C. ELISA was used to measure heparanized plasma concentrations of syndecan-1 (Abcam); sphingosine-1-phosphate ; angiopoietin-2, ICAM-1 and E-selectin (Quantikine R&D Systems) and osteoprotegerin (DuoSet R&D Systems). Cell free haemoglobin was measured on citrated platelet-free plasma by ELISA (Bethyl Laboratories). Heparanized plasma concentrations of the endothelial nitric oxide synthase (eNOS)-inhibitor asymmetric dimethylarginine (ADMA) were measured by HPLCAll methods were carried out in accordance with relevant guidelines and regulations.2 or Fisher’s exact tests. Associations between continuous variables were assessed using Spearman’s correlation coefficient. Partial correlation was used to evaluate associations between continuous variables after adjusting for parasitemia. For this analysis, non-normally distributed variables were log-transformed to a normal distribution. Since correlations between glycocalyx breakdown products (urinary glycosaminoglycans and syndecan-1) and 11 variables were assessed, Bonferroni’s correction was used to adjust for multiple correlations, with correlations considered statistically significant if p < 0.0045 (p = 0.05/11).Statistical analysis was performed with STATA software (v15). Student’s T-test or Wilcoxon-Mann–Whitney tests were used for two-group comparisons, depending on distribution. Categorical variables were compared using χBackward stepwise regression was used to evaluate factors associated with severe malaria and AKI, with variables removed at a significance level of > 0.05; for this analysis, patients with hyperparasitemia as a sole severity criterion were reclassified as having non-severe malaria.


Plasmodium falciparum malaria. We found that the high prevalence of severe and profound thrombocytopenia in patients with severe non-P. falciparum malaria at 47% and 20%, respectively. Moreover, deaths were seen in patients with severe and profound thrombocytopenia at 11%. Therefore, the present systematic review provided the new insight into the clinical relevance of severe and profound thrombocytopenia in diagnosing and managing non-P. falciparum malaria. Severe thrombocytopenia should serve as a warning sign of increased chances of complications. Meanwhile, profound thrombocytopenia should serve as a marker of mortality among patients with non-P. falciparum malaria.Alteration of platelet count is frequently seen in patients with malaria. However, the platelet biology under severe pathological conditions like severe malaria remains unclear. The present systematic review collated literatures and synthesized the evidence regarding the risks of severe and profound thrombocytopenia in patients with severe non-Plasmodium falciparum malaria. Our overall aim was to identify potential indicators of severe non-P. falciparum malaria and the Plasmodium species that cause severe outcomes. This systematic review was registered at the International Prospective Register of Systematic Reviews (PROSPERO) under registration ID CRD42020196541. Studies were identified from previous systematic reviews (n = 5) and the MEDLINE, Scopus, and Web of Science databases from 9 June 2019 to 9 June 2020. Studies were included if they reported the outcome of severe non-Plasmodium species infection, as defined by the World Health Organization (WHO) criteria, in patients with known platelet counts and/or severe and profound thrombocytopenia. The risk of bias was assessed using the Newcastle–Ottawa Scale (NOS). Data were pooled, and pooled prevalence (PP) and pooled odds ratios (ORs) were calculated using random effects models. Of the 118 studies identified from previous meta-nalyses, 21 met the inclusion criteria. Of the 4807 studies identified from the databases, three met the inclusion criteria. Nine studies identified from reference lists and other sources also met the inclusion criteria. The results of 33 studies reporting the outcomes of patients with severe P. vivax and P. knowlesi infection were pooled for meta-analysis. The PP of severe thrombocytopenia (reported in 21 studies) was estimated at 47% : 33–61%, I2: 96.5%), while that of profound thrombocytopenia (reported in 13 studies) was estimated at 20% . The pooled weighted mean difference (WMD) in platelet counts between severe uncomplicated Plasmodium infections (reported in 11 studies) was estimated at −28.51% , while the pooled WMD in platelet counts between severe non-Plasmodium and severe P. falciparum infections (reported in eight studies) was estimated at −3.83% . The pooled OR for severe/profound thrombocytopenia comparing severe to uncomplicated Plasmodium infection was 2.92 . The PP of death from severe and profound thrombocytopenia was estimated at 11% (95% CI: 0–22%). These results suggest that individuals with severe non-P. falciparum infection (particularly P. vivax and P. knowlesi) who exhibit severe or profound thrombocytopenia should be regarded as high risk, and should be treated for severe malaria according to current WHO guidelines. In addition, severe or profound thrombocytopenia coupled with other clinical and microscopic parameters can significantly improve malaria diagnosis, enhance the timely treatment of malaria infections, and reduce the morbidity and mortality of severe non-P. falciparum malaria. The understanding of platelet biology under physiological and pathological conditions like malaria infection is critical importance in the context of the disease outcome or model systems used. The importance of severe thrombocytopenia  and profound thrombocytopenia  in malaria patients remains unclear. This study aimed to synthesize evidence regarding the risks of severe and profound thrombocytopenia in patients with severe non- Plasmodium falciparum and Plasmodium vivax infections, thrombocytopenia is associated with increased mortality [P. falciparum malaria are increased platelet activation and consumption by factors such as the cytoadherence of infected erythrocytes and subsequent endothelial damage [P. falciparum and P. vivax malaria and is associated with bleeding [An understanding of platelet biology under physiological and pathological conditions like malaria infection is of critical importance in the context of the disease outcome or model systems used. Platelets are produced from megakaryocytes and play a crucial role in hemostasis and thrombus formation . Thromboortality ,6. The pl damage , platelel damage , bone mal damage . Severe l damage . Severe bleeding  and dissbleeding .Plasmodium parasitemia and one or more of the following manifestations: impaired consciousness, prostration, acidosis, hypoglycemia, severe malarial anemia, renal impairment, jaundice, pulmonary edema, significant bleeding, shock, or hyperparasitemia [P. falciparum malaria, although it is not included in the current World Health Organization (WHO) criteria for severe P. falciparum malaria [P. falciparum malaria and profound thrombocytopenia were five times more likely to die than patients with higher platelet counts [P. falciparum infection, information on the prevalence of severe or profound thrombocytopenia in patients with non-P. falciparum malaria  is limited. The most recent systematic review on severe thrombocytopenia in P. vivax malaria demonstrated that severe thrombocytopenia was equally common in P. vivax and P. falciparum malaria [P. vivax infection. To clarify this issue, we conducted a meta-analysis of studies reporting severe or profound thrombocytopenia in patients with severe P. vivax, P. malariae, P. ovale, and P. knowlesi malaria. Our goal was to assess whether severe or profound thrombocytopenia could inform clinical decision-making, and whether the degree of thrombocytopenia had predictive utility independent of current clinical and laboratory prognostic indices.Severe malaria is defined as the presence of asitemia . Severe  malaria . A previt counts . Althoug malaria . HoweverThis systematic review was registered at the International Prospective Register of Systematic Reviews (PROSPERO) under registration ID CRD42020196541.Plasmodium falciparum including P. vivax, P. malariae, P. ovale, and P. knowlesi infections; (3) severe thrombocytopenia  or profound thrombocytopenia ; (4) severe and uncomplicated malaria with known platelet counts; or (5) severe P. falciparum and non-P. falciparum infections with known platelet counts. The following types of studies were excluded: (1) studies examining severe non-Plasmodium infection co-morbidity with other diseases, (2) studies conducted among pregnant patients, and (3) studies in which platelet counts could not be extracted. The systematic review was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (see PRISMA Checklist S1).Original studies were included if they were observational, prospective, or retrospective in design, and reported outcomes of patients with: (1) severe malaria as defined by the WHO ; (2) non-n = 5) reporting outcomes of severe P. vivax, P. malariae, P. ovale, and P. knowlesi infections. The second source of studies was a systematic search of relevant databases  between 1 June 2020 and 9 June 2020 using MeSH headings (Severe OR complicated OR complication) AND  AND (thrombocytopenia OR “low platelet”), with no restrictions on language. The third source of studies was the reference lists of the included studies as well as other sources . Two authors (AM and MK) independently screened the titles and abstracts. Any disagreements in full-text article selection were resolved by consensus. Potentially relevant full-text articles were obtained, read, and extracted by two independent authors concerning the inclusion criteria. Data were extracted into a standardized form using an Excel spreadsheet. For all studies, data on authors, year of publication, study location, year of publication, and study design were extracted. In addition, data on mean/median age of participants, male percentage, mean/median platelet count, parasitemia level, number of patients with severe and profound thrombocytopenia, Plasmodium species, number of patients with severe malaria, number of patients with severe or profound thrombocytopenia, and number of deaths were extracted. Data extraction was undertaken by one author and checked by all authors.Potentially relevant studies were identified from three sources. The first source of studies was previous systematic reviews  and not the comparator groups. Therefore, only three domains were used to assess the included studies: case definition, representativeness of cases, and ascertainment of the outcome. The risk of bias was reported as low (1 star), moderate (2 stars), or high (3 stars) based on the total of the three scores retrieved from each domain. Any discrepancies in ratings were addressed in the discussion.The risk of bias was independently assessed by two authors (A.M. and M.K.). As the included studies were observational retrospective, prospective, and case-control studies, the Newcastle–Ottawa Scale (NOS) tool was used . The NOSP. falciparum infection, estimated using the random-effects DerSimonian and Laird models [P. falciparum infection compared with individuals with uncomplicated non-P. falciparum infection, estimated using fixed- or random-effects models depending on the level of heterogeneity; (3) pooled ORs in individuals with severe non-P. falciparum infection compared with individuals with severe P. falciparum infection, estimated using fixed- or random-effects models depending on the level of heterogeneity. The heterogeneity and level of heterogeneity of studies were assessed using Cochran’s Q and I2 (inconsistency) statistics, respectively. Publication bias was evaluated using visual inspection of funnel plots and Egger’s tests. If any small-study effects were observed, a further contour-enhanced funnel plot was generated to assess if asymmetry resulted from publication bias or other factors. The meta-analysis was carried out using Stata software. To explore whether a single study affected the heterogeneity or the conclusions of the meta-analysis, sensitivity analyses omitting each study were performed in turn. Subgroup analyses were conducted to evaluate potential sources of heterogeneity.The following three outcomes were analyzed by meta-analysis: (1) PP and 95% confidence interval (CI) of severe and profound thrombocytopenia in individuals with severe non-d models ; (2) pooP. falciparum malaria were retrieved from previous systematic reviews and meta-analyses [Twenty-one studies reporting the outcomes of severe non-analyses ,20,21,22analyses ,52,53,54P. falciparum malaria. The included studies were prospective or retrospective observational studies, case-control studies, and cohort studies. All studies excluded individuals with P. falciparum single or mixed infections. Most of the included studies reported the outcomes of severe non-P. falciparum malaria in adults, while Gupta et al., 2016 [P. falciparum malaria in children younger than 7 years. Most of the included studies reported outcomes of predominantly male individuals who developed severe non-P. falciparum malaria, while Sharma et al., 2012 and Singh et al., 2011 reported a higher proportion of women who developed severe non-P. falciparum malaria. All non-P. falciparum malaria cases reported in the included studies involved P. vivax and P. knowlesi malaria. Of the included studies, 30 [P. vivax single infections, while four [P. knowlesi infections. Barber et al., 2013 [P. vivax and severe P. knowlesi infections. Other characteristics of the included studies are shown in Thirty-three studies from Asia  from 18 studies [P. vivax malaria was 42% . The prevalence reported in individual studies varied highly (range: 3–86%). Data on severe thrombocytopenia in patients with severe P. knowlesi malaria were available from three studies [P. knowlesi malaria (77 cases) was 80% . The prevalence reported in the three individual studies of severe P. knowlesi malaria was consistent, ranging from 71% to 84%. Overall, the PP of severe thrombocytopenia (440 cases) in patients with severe P. vivax and severe P. knowlesi malaria (885 cases) was 47%  (Data on severe thrombocytopenia 349 cases) in patients with severe 9 cases i studies ,50,51,52 studies ,53,54. T: 96.5%) .P. vivax malaria (954 cases) from 12 studies [P. vivax malaria was 21% . The prevalence reported in individual studies varied highly (range: 2–67%). Data on profound thrombocytopenia (13 cases) in patients with severe P. knowlesi malaria (60 cases) were available from two studies [P. knowlesi malaria (77 cases) was 17% . The prevalence reported in individual studies varied highly (range: 6–26%). Overall, the PP of profound thrombocytopenia (202 cases) in patients with severe P. vivax and severe P. knowlesi malaria (1014 cases) was 20%  (Data on profound thrombocytopenia (189 cases) in patients with severe  studies ,45,48,49 studies ,53. The : 85.2%) .P. vivax malaria (557 cases) and uncomplicated P. vivax malaria (2451 cases) were available from eight studies [P. vivax malaria were significantly lower than those of patients with uncomplicated P. vivax malaria . Data on mean platelet counts in patients with severe P. knowlesi malaria (124 cases) and uncomplicated P. knowlesi malaria (318 cases) were available from four studies [P. knowlesi malaria were significantly lower than those of patients with uncomplicated P. knowlesi malaria . Overall, the pooled WMD showed the platelet counts of patients with severe non-P. falciparum malaria (681 cases) were significantly lower than those of patients with uncomplicated non-P. falciparum malaria (2769 cases)   .P. vivax malaria (252 cases) and severe P. falciparum malaria (576 cases) were available from seven studies [P. vivax malaria and severe P. falciparum malaria were comparable . Data on mean platelet counts in patients with severe P. knowlesi malaria (85 cases) and severe P. falciparum malaria (26 cases) were available from two studies [P. knowlesi and severe P. falciparum malaria were comparable . Overall, the pooled WMD showed that the platelet counts of patients with severe non-P. falciparum (337 cases) and severe P. falciparum malaria (610 cases) were comparable   .P. falciparum malaria was performed using age, male ratio, and parasite counts as co-variates. The results of the univariate meta-regression analysis showed that the difference in platelet counts between the two groups was not confounded by age, male ratio, or parasite counts  and uncomplicated P. vivax malaria (1647 cases) were available from five studies [P. vivax malaria had significantly higher odds of severe or profound thrombocytopenia than those with uncomplicated P. vivax malaria . Data on severe thrombocytopenia in patients with severe P. knowlesi malaria (55 cases) and uncomplicated P. knowlesi malaria (185 cases) were available from two studies [P. knowlesi malaria had significantly higher odds of severe or profound thrombocytopenia than those with uncomplicated P. knowlesi malaria . Overall, the odds of severe or profound thrombocytopenia were significantly higher in patients with severe non-P. falciparum malaria compared with those with uncomplicated non-P. falciparum malaria   .P. vivax malaria (381 cases) and severe P. falciparum malaria (396 cases) were available from seven studies [P. vivax and severe P. falciparum malaria were comparable . Data on severe thrombocytopenia in patients with severe P. knowlesi malaria (32 cases) and severe P. falciparum malaria (10 cases) were available from one study [P. knowlesi and severe P. falciparum malaria . Overall, no significant difference was observed in the odds of severe or profound thrombocytopenia between patients with severe non-P. falciparum and severe P. falciparum malaria   .P. vivax malaria were available for extraction. Deaths resulting from severe P. vivax malaria in patients with severe or profound thrombocytopenia were investigated. Mortality related to severe thrombocytopenia in patients with severe P. vivax malaria  was 24% (95% CI: 0–45%) [Data on deaths from severe  studies ,39,50) w: 0–45%) .p = 0.041, t-statistic: 2020, coefficient: 4.48, standard error: 2.04). A contour-enhanced funnel plot was generated and showed that the asymmetry of the funnel plot resulted from publication bias in most studies  and severe P. knowlesi malaria (80%). In addition to severe thrombocytopenia, we found that the PP of profound thrombocytopenia was low in patients with severe P. vivax malaria (21%) and severe P. knowlesi malaria (17%). The PP of severe and profound thrombocytopenia in patients with severe P. vivax malaria could not be precisely estimated because of the high heterogeneity among the included studies (I2 > 86%). In addition, the PP of severe thrombocytopenia in patients with severe P. knowlesi malaria could be reliably estimated because of the homogeneity of the included studies (I2: 0%). Nevertheless, we caution that the homogeneity among studies reporting the outcomes of patients with severe thrombocytopenia in P. knowlesi malaria could result from the relatively small number of included studies. Severe thrombocytopenia is not currently considered an indicator of severe malaria according to the WHO criteria [P. falciparum malaria, it may be worth investigating individuals with severe malaria and severe or profound thrombocytopenia. Clinicians managing patients with malaria should be aware of the risks, as these individuals are more likely to die than patients with higher platelet counts [Thrombocytopenia frequently occurs in individuals with  malaria ,56. The of cases . In addi malaria . Moreoveytopenia ,58,59. Tcriteria . Interest counts . Furthert counts .P. falciparum, P. vivax, and P. knowlesi infection [Plasmodium species [Plasmodium infections, significant bleeding is often unrelated to the level of thrombocytopenia [P. vivax and severe P. knowlesi malaria were lower than those of patients with uncomplicated P. vivax and P. knowlesi malaria. Although high heterogeneity was present in the pooled analysis, these results demonstrated the significant diagnostic value of lower platelet counts in patients with severe non-P. falciparum malaria compared with patients with uncomplicated malaria. In the two-by-two meta-analysis of proportion, individuals with severe P. vivax malaria had more than three times the odds of developing severe or profound thrombocytopenia than those with uncomplicated P. vivax malaria. Lower odds of severe or profound thrombocytopenia were observed in patients with severe P. knowlesi malaria. The pooled ORs from the meta-analysis were reliable because of the low heterogeneity among the included studies. From the results of meta-analysis, most of the included studies showed lower mean platelet counts in patients with severe P. vivax malaria than in those with uncomplicated malaria [P. vivax malaria. Arévalo-Herrera et al., 2015 reported a wider range of platelet counts in patients with severe P. vivax malaria  than in those with uncomplicated P. vivax malaria, but no difference in the risk of spontaneous bleeding associated with thrombocytopenia [P. knowlesi malaria were similar to those in patients with severe P. vivax malaria: all included studies reported a significantly lower mean platelet count in these patients than in those with uncomplicated P. knowlesi malaria. Thrombocytopenia in patients with P. knowlesi infections is common, and platelet counts were lowest in cases of severe disease [P. knowlesi malaria exhibited higher mean platelet counts than those with severe P. vivax malaria.Thrombocytopenia is a common feature of malaria resulting from nfection ,30,55. T species . Althougytopenia . Therefoytopenia  and not ytopenia . The potytopenia . The pre malaria ,38,43,45 malaria  demonstrytopenia . Trends  disease . HoweverP. vivax/P. knowlesi malaria and severe P. falciparum malaria. The two-by-two meta-analysis of proportion confirmed that neither severe nor profound thrombocytopenia was significantly different in patients with severe P. vivax/P. knowlesi malaria and severe P. falciparum malaria. The meta-analysis results were reliable because of the low heterogeneity in the pooled analysis of proportion. Our results demonstrated that severe and profound thrombocytopenia might affect the risk of death in individuals with severe P. vivax malaria. The data reported by four included studies [P. vivax malaria exhibited severe and profound thrombocytopenia. Infection by other Plasmodium species, including P. malariae and P. ovale malaria, resulted in severe thrombocytopenia less frequently than P. vivax malaria [P. falciparum malaria [P. ovale wallikeri infection compared with those with P. ovale curtisi infection [P. falciparum infection, patients with profound thrombocytopenia were five times more likely to die than patients with higher platelet counts, suggesting that thrombocytopenia might be a marker of disease severity in patients with falciparum malaria [P. falciparum and P. vivax suggested that severe thrombocytopenia had a sensitivity and specificity of 65.6% and 70.6%, respectively, to discriminate against severe malaria. Therefore, it is valuable to further investigate the sensitivity and specificity of thrombocytopenia using large longitudinal studies to conclude the performance of using platelet as an indicator for severe non-P. falciparum malaria. In light of our analyses for non-P. falciparum malaria, the mortality rate in patients with profound thrombocytopenia was higher than those with severe thrombocytopenia, suggesting the possibility of using thrombocytopenia to identify the risk of death in patients with severe non-P. falciparum malaria.We found no significant differences in the mean platelet counts of patients with severe  studies ,39,49,50 malaria ,65 and Pnfection . In P. f malaria ,57. The P. falciparum malaria deficient in a disintegrin and metalloprotease with thrombospondin type I repeats, member 13 (ADAMTS13), had higher frequencies of severe thrombocytopenia and other complications [P. falciparum molecules and release prepackaged inflammatory mediators, leading to decreased signaling and inflammatory responses [The pathogenic mechanisms underlying thrombocytopenia and the mechanisms through which platelet numbers affect severe malaria and death remain unclear. However, previous studies have shown that endothelial activation  results ications . The higesponses ,71. Anotesponses .In addition to identifying new candidate makers for severe malaria, new diagnostic tools for detecting malaria parasites are challenging. Recently, magnetic resonance relaxometry (MRR) ,74,75, oP. falciparum malaria, particularly of P. knowlesi are fragmented, and is a research gap for malaria studies. The present study, P. vivax studies and two P. knowlesi studies. P. vivax studies and one P. knowlesi study, while P. vivax studies. It would be interesting to determine whether such comparability in the severity or profoundness of thrombocytopenia and related outcomes was due to the limitations in the available studies or unique biological traits that exist within non-P. falciparum species that induce host responses. The detection of human cases of P. knowlesi demonstrated an ongoing increase despite effective control programs to disrupt the transmission of human-only Plasmodium species [P. knowlesi malaria can be found in the changing patterns in land use where spillover opportunities for human infection due to increased contact with natural reservoirs and infected vectors have been aggravated. In addition, the range and distribution of primary hosts and vectors including their bionomics are still lacking in data [P. falciparum malaria and host immunological responses to the same.Biological and observational studies of non- species . Some no in data . These aP. vivax and severe P. knowlesi malaria. Therefore, the prevalence of severe or profound thrombocytopenia was not assessed among patients with severe P. malariae, P. ovale, or mixed malaria. Second, the prevalence of severe or profound thrombocytopenia-related death could not be precisely estimated because of the limited number of included studies. Despite these limitations, the presence of severe or profound thrombocytopenia in malaria patients may support the diagnosis of malaria, particularly in patients with severe non-P. falciparum malaria, because low levels of parasitemia are frequently encountered in routine diagnosis. In addition, severe or profound thrombocytopenia could be used as an indicator of increased risk of poor outcome and mortality if there are delays in diagnosis. Our results suggest that severe and/or profound thrombocytopenia should be included in the WHO criteria for severe non-P. falciparum malaria because a high prevalence of severe thrombocytopenia was observed in these patients (47% for severe thrombocytopenia and 20% for profound thrombocytopenia).Our study had several limitations. First, the data on platelet counts and the frequency of severe or profound thrombocytopenia could only be extracted from studies of patients with severe P. falciparum malaria. Severe thrombocytopenia should serve as a warning sign of increased chances of complications. Meanwhile, profound thrombocytopenia should serve as a marker of mortality among patients with non-P. falciparum malaria.The present systematic review provided the new insight into the clinical relevance of severe and profound thrombocytopenia in diagnosing and managing non-P. falciparum malaria and thrombocytopenia. Our results suggest that individuals with severe non-P. falciparum malaria, particularly P. vivax and P. knowlesi malaria, with severe or profound thrombocytopenia should be regarded as high-risk patients and should subsequently be treated for severe malaria according to the current WHO guidelines. In addition, severe or profound thrombocytopenia coupled with other clinical and microscopy parameters, may significantly improve malaria diagnosis, provide timely treatment for malaria infections, and reduce the morbidity and mortality of severe non-P. falciparum malaria. Observational studies and research on the parasite, vector, and host biology and outcomes for non-P. falciparum malaria is still fragmented. Moreover, the changing patterns of land use that increase the exposure of natural reservoirs and infected vectors, range distribution of hosts, and vectors, including the biomics of non-P. falciparum malaria, require further and more in-depth exploration.A meta-analysis provides enhanced statistical power, thereby providing more robust and reliable knowledge of the effects of severe non-


Plasma concentrations of Pentraxine-3, Procalcitonin, Ang-2 and the soluble receptors were significantly higher in children with coma as defined by a Blantyre Coma Score < 3 . Moreover, except for the PCT level, the concentrations of Pentraxine-3, suPAR, sEPCR, sICAM-1, sTie-2 and Ang-2 were higher among children who died from severe malaria compared to those who survived . These findings indicate the ability of these molecules to accurately discriminate among clinical manifestations of malaria, thus, they might be potentially useful for the early prognostic of severe and fatal malaria, and to improve management of severe cases.Malaria-related deaths could be prevented if powerful diagnostic and reliable prognostic biomarkers were available to allow rapid prediction of the clinical severity allowing adequate treatment. Using quantitative ELISA, we assessed the plasma concentrations of Procalcitonin, Pentraxine-3, Ang-2, sTie-2, suPAR, sEPCR and sICAM-1 in a cohort of Beninese children with malaria to investigate their potential association with clinical manifestations of malaria. We found that all molecules showed higher levels in children with severe or cerebral malaria compared to those with uncomplicated malaria ( Plasmodium falciparum is the most widespread malaria agent in Africa, and the most virulent species responsible for severe and cerebral malaria. The pathogenicity of severe malaria is mainly attributable to the cytoadherence of infected erythrocytes (IEs) within microvascular endothelia of vital organs modulating properties resulting in immune and splenic evasion characterized by the different pathophysiology of the disease . Addiiewed in ,6,7,8. Tiewed in .In addition, angiopoietin 2 (Ang-2) and its tyrosine-kinase-receptor 2 (Tie-2) were found to be increased and associated with retinopathies in Malawian children with cerebral malaria. In combination with clinical parameters, Ang-2 improved mortality prediction among these children . The resIn this study, we assessed in a cohort of 339 Beninese children presenting with various clinical manifestations of malaria, the level of four soluble receptors  and two other bioactive molecules including PTX3 and the calcitonin-precursor-hormone procalcitonin (PCT).This cross-sectional study was carried out from December 2017 to July 2019 in the south of Benin where children under 6 years consulting at hospitals including Centre-Hospitalier-Universitaire Mère-enfant, de la Lagune, Centre-Hospitalier-Universitaire of Suruléré, and Ménontin Hospital, in Cotonou, l’Oueme/Plateau Hospital in Porto-Novo, and Ouidah Hospital in Ouidah were enrolled. Porto-Novo is 41.2 km east of Cotonou, while Ouidah is 39 km west of Cotonou, both settings share a subtropical climate, with 2 rainy seasons (April to July and September to October) and 2 dry seasons. This makes the south of Benin a malaria holoendemic area with a mean entomological inoculation rate of 33 infective bites per person per year .P. falciparum  and met the World Health Organization clinical malaria definition criteria. All malaria cases were defined by a trained clinician as a microscopically confirmed P. falciparum mono infection. Children were grouped according to the clinical manifestations of malaria they presented at the time of admission to hospital. The cerebral malaria group (CM) had a Blantyre Coma Score (BCS) < 3, excluding any other cause of coma with or without other signs of severe malaria. The severe non-cerebral malaria group (SNCM) included children presenting with one or more of the following symptoms: pulmonary edema, acute respiratory distress syndrome, acute kidney failure, abnormal liver function, hemoglobinuria or severe anemia with a BCS > 2. The uncomplicated-malaria-group (UM) had P. falciparum parasitemia infection with fever, and either headache or myalgia without signs of life-threatening malaria or evidence of vital organ dysfunction and had a hemoglobin level above ≥5 g/dL. Children presenting other causes of morbidity in addition to malaria were excluded.Children spontaneously presenting at the hospitals were recruited into the study if they presented a positive rapid diagnostic test for ® ZI des Iles-BP4-38801-Le Pont de Claix, France) from all participating children before treatment. Clinical, biological and demographic data of patients were captured in a questionnaire and entered in an ad hoc file for further analysis. All patients were treated according to the guidelines of the Beninese Ministry of Health.Up to 4 mL peripheral blood was collected into citrate-phosphate-dextrose-adenine containing tubes  for PTX3, Ang-2 and soluble receptors. Plasma samples were diluted to 1/4 for Ang-2, and sTie-2, 1/10 for PTX3, suPAR, and sICAM-1 and 1/20 for sEPCR. For PCT quantification, the one-step enzyme-linked fluorescent immunoassay  was performed using 200 µL plasma. In all experiments, analyte concentrations were calculated according to standard curves obtained by the assessment of specific recombinant human proteins which systematically included negative control sera. The final results were expressed as ng/mL.Plasma samples were assayed in duplicate blindly. Standard ELISA experiments were performed according to the manufacturer’s instructions  using the Kruskal–Wallis test in conjunction with Dunn’s multiple comparison post-hoc tests and chi-square test, respectively. The median and IC 95 levels of analytes were compared between the groups, UM versus CM-SNCM, patients who survived versus those who died and BCS < 3 versus BCS > 2 among severe cases using the Mann–Whitney U-test and binary logistic regression for which the effect described was independent of covariates such as ethnicity, age, parasitemia and sex. The validity of each molecule as a biomarker for malaria-related severity, coma and fatality was assessed by a receiver operating characteristic (ROC) curve analysis, a two-dimensional measure of classification performance where the area under the ROC curve (AUC) accurately measures discrimination, i.e., reflects the power of a quantified parameter to distinguish between two clinical groups. The greater the AUC, the better the test .Blood samples were collected from 339 children aged between 4 and 66 months in a prospective cohort study of children who came to the hospitals for consultation and who were subsequently admitted to the pediatric ward for intensive care or outpatient consulting for uncomplicated malaria. Among them, 234 had severe malaria, including 101 with cerebral malaria (CM) having coma with BCS < 3. Twenty-nine children also had impaired consciousness with BCS = 3 accompanied by other symptoms of severe malaria, while 104 children had severe malaria without impaired consciousness and were all ranked in the group of severe non-cerebral malaria (SNCM). Severe anemia was very common among children and was detected in 45.71% of children with SNCM and 42.57% of those with CM. Forty children (11.79%) died including 29 (28.71%) among the CM group and 11 (8.27%) among the SNCM group, with six of them having consciousness alteration with a BCS of 3, and five suffering from SNCM without neurological impairment with a BCS of 5. Most children died within 10–24 h after hospitalization. One hundred and five children suffered from UM: all were treated and recovered .p < 0.005. However, with the exception of the sEPCR level, the concentrations of the other molecules were also significantly increased in children with CM defined as coma with a BCS < 3 compared to those among children with SNCM as defined by a BCS > 2. Furthermore, apart from the level of PCT which did not differ between children who died and those who survived, the levels of suPAR, sEPCR, sICAM-1, sTie-2, Ang-2 and PTX3 were higher in children who died from SNCM or CM than those who survived these pathologies , presence versus absence of coma (defined by BCS < 3 versus BCS > 2) and mortality versus survival among severe cases, as outcome variables while the effect described was independent of covariates such as ethnicity, age, parasitemia and sex. The odds ratios show an increase of 51% for Ang-2 and 21% for sTie-2 in children with CM or SNCM compared to UM, indicating that SNCM was 1.51 and 1.21 times more likely to occur with a unit increase in Ang-2 and sTie-2, respectively. Only a slight increase in the odds (between 1.03 and 1.06) for suPAR, PCT and PTX3 was noticed with a statistically significant difference between the two groups of patients. Except for sICAM-1, for which the increase in the odds was close to zero (but still significant), the odds for sTie-2, PTX3 and suPAR levels were 1.09, 1.04, and 1.04 times higher in patients with CM compared to those with SNCM. There was also a 1.03, 1.0, and 1.03 times increase in the odds of suPAR, PTX3 and sEPCR, respectively, in fatal cases compared to survivors of CM or SNCM pathologies which were statistically significant.p < 0.001) and 0.71  for PTX3 and sICAM-1, respectively (p < 0.001) and , respectively  for PCT and 0.78  for sTie-2 . For comectively , while tectively . The thrBoth the innate and adaptive immune responses depend critically on leucoytes migration across the vascular endothelium; therefore, factors associated with endothelial cell response including inflammation, tissue damage and injury aroused a high interest in studies of malaria patients. In this study, we assessed the level of seven markers of vascular and tissue integrity, angiogenesis and inflammation in the context of malaria. The interactions between these molecules are illustrated in Our findings showed the potential of four soluble receptors including sEPCR, sICAM-1, suPAR and sTie-2 with its ligand Ang-2, and two other molecules PCT, and PTX3, as informative biomarkers of malaria disease severity, coma and mortality.On admission, the levels of all the analytes considered were higher in children with CM or SNCM compared to children with UM as shown by a logistical regression analysis  and non-Interestingly, we found that the levels of suPAR, sICAM-1, sTie-2, Ang-2, PTX3 and PCT were associated with coma and that sEPCR, sICAM-1, suPAR, sTie-2, Ang-2 and PTX3 were associated with fatality. These molecules have also been found to be increased in patients with other infectious or chronical diseases such as sepsis, HIV-1-AIDS, tuberculosis, rheumatoid-arthritis and various cancers with suPAR, sEPCR and PTX3, often predicting poor clinical outcomes as in our present study ,14,15,16sEPCR, sTie-2/Ang-2 and sICAM-1 are involved in cytoprotective activity to maintain endothelial barrier functions of microvessels. The soluble as well as the membranous form of EPCR bind with similar affinity to activated C-reactive-protein (APC) to maintain endothelium integrity through the activation of protease-activator-1 ,18 promoSimilarly, the level of Ang-2 and its receptor sTie-2 was higher in SNCM or CM groups as well as in coma and in fatal cases suggesting a pathophysiological association with CM. Interestingly, Ang-2 and the Ang-2/Ang-1 ratio were previously shown as independent predictors of metabolic acidosis, coma and mortality ,20,21. TMoreover, the enhanced level of Ang-2 and sTie-2 was concomitant with the expression of sICAM-1 which was significantly higher in young Beninese patients with CM or SNCM as well as in children with coma and those who died  C–E.We also found that the suPAR level was significantly higher in children with SNCM or CM compared to those with UM, in children with BCS < 3 compared to those with BCS > 2 and in children who died from SNCM or CM making this molecule a potential marker of severity, coma and fatality  A. Produp = 0.004) and showed moderate predictive value for coma with an AUC of [0.65], which is to our knowledge a novel finding. This molecule is able to discriminate between viral, bacterial, fungi or parasitic infections and is also used in guiding antibiotic treatments in patients  and [0.78], respectively. Except for the sEPCR level, the levels of all the molecules tested were higher in children with BCS < 3 compared to children with BCS > 2, with PTX3 and sICAM-1 presenting the best diagnosis performance for coma with an AUC of [0.78] and [0.71], respectively. In addition, the sEPCR, sICAM-1, sTie-2, Ang-2, PTX3 and suPAR levels discriminated between children who died from SNCM and CM and those who survived these pathologies with the highest predictive value for sEPCR, and sICAM-1 with an AUC of [0.77]. Overall, the expression of these molecules during malaria, either at the onset of infection when triggering the inflammatory response such as suPAR and PTX3, or resulting later from an acute inflammatory response such as sICAM-1 and sEPCR, is associated with the pathogenesis of severe malaria in which the site of initiation of this response and its intensity may play a major role. Therefore, further research on the mechanisms of action of these molecules is needed to better understand their role in the pathogenesis of SNCM and CM and explore their potential as host-targeted therapeutics.


Plasmodium falciparum and can therefore affect the risk of clinical malaria. We assessed here the effect of helminth infections on both the immunogenicity and efficacy of the GMZ2 malaria vaccine candidate, a recombinant protein consisting of conserved domains of GLURP and MSP3, two asexual blood-stage antigens of P. falciparum. Controlled human malaria infection (CHMI) was used to assess the efficacy of the vaccine.Helminths can modulate the host immune response to P. falciparum sporozoites  approximately 13 weeks after last vaccination to assess vaccine efficacy. Participants were followed-up on a daily basis with clinical examinations and thick blood smears to monitor P. falciparum parasitemia for 35 days. Malaria was defined as the presence of P. falciparum parasites in the blood associated with at least one symptom that can be associated to malaria over 35 days following DVI of PfSPZ Challenge. Soil-transmitted helminth (STH) infection was assessed by microscopy and by polymerase chain reaction (PCR) on stool, and Schistosoma infection was assessed by microscopy on urine. Participants were considered as infected if positive for any helminth either by PCR and/or microscopy at D0 and/or at D84 (Helm+) and were classified as mono-infection or co-infection. Total vaccine-specific IgG concentrations assessed on D84 were analysed as immunogenicity outcome.In a randomized, double-blind Phase I clinical trial, fifty, healthy, lifelong malaria-exposed adult volunteers received three doses of GMZ2 adjuvanted with either Cationic Adjuvant Formulation (CAF) 01 or Alhydrogel, or a control vaccine (Rabies) on days (D) 0, D28 and D56, followed by direct venous inoculation (DVI) of 3,200 Schistosoma haematobium and STH were significantly associated with earlier malaria episodes following CHMI, while no association was found in case of coinfection. In further analyses, the anti-GMZ2 IgG concentration on D84 was significantly higher in the S. haematobium-infected and significantly lower in the Strongyloides stercoralis-infected groups, compared to helminth-negative volunteers. Interesting, in the absence of helminth infection, a high anti-GMZ2 IgG concentration on D84 was significantly associated with protection against malaria.The helminth in mono-infection, particularly Our results suggest that helminth infection may reduce naturally acquired and vaccine-induced protection against malaria. Vaccine-specific antibody concentrations on D84 may be associated with protection in participants with no helminth infection. These results suggest that helminth infection affect malaria vaccine immunogenicity and efficacy in helminth endemic countries. Helminths, mainly because of their immune regulatory effects, are able to impact the response induced by vaccines. In the context of clinical trial designs that measure accrual of natural infections during follow up or outcome of controlled human malaria infection (CHMI), their effect on vaccine efficacy can be measured. Indeed, most of such clinical trials on malaria vaccine candidates conducted in Africa, especially where the prevalence of helminths is high, have shown a certain limit in their efficacy and immunogenicity, as compared to results observed in European and U.S volunteers. The present analysis assessed the effect of helminths on GMZ2, a malaria vaccine candidate. We found a high level of anti-GMZ2 antibodies among volunteers not infected with helminths and protected against CHMI, indicating efficacy of the candidate vaccine in this population. We found a species-dependent effect of helminths on the level of post-immunization GMZ2-specific IgG concentration, and an association of helminths with an early onset of malaria in CHMI. Our findings reveal that helminths are associated with immunogenicity and may decrease the protective effect of antibodies induced by vaccination. Helminth infection status shall be determined when measuring the immunogenicity and efficacy of malaria vaccine candidates in helminth endemic countries. Schistosoma haematobium and the group of soil-transmitted helminths (STH) including Ascaris lumbricoides, hookworm, Trichuris trichiura and Strongyloides stercoralis are most prevalent in developing countries, particularly in sub-Saharan Africa [Plasmodium falciparum antigens or against vaccine antigens, although with contradictory findings. Some studies reported a down-modulating effect of S. haematobium infection on anti-P. falciparum immune responses such as negative association between the intensity of S. haematobium infection and IgG1, IgG3 and IgG4 antibody subclass levels directed to malarial total schizont extract [S. haematobium infection has also been shown to affect specific IgG1 directed to P. falciparum (Pf) MSP1 and GLURP [S. haematobium infection [S. haematobium-P. falciparum coinfection [Plasmodium infection [P. falciparum infection due to STH in schistosomiasis-positive children [Helminth infections remain widespread and cause important neglected tropical diseases. The clinical presentations include anemia, malnutrition, developmental deficiencies causing significant morbidity and mortality . Blood fn Africa , where mnd GLURP . By contnfection , or antinfection . With renfection  while otnfection ,10. A rechildren . Many stchildren –18, but children –22.P. falciparum GLURP and MSP3 [T. trichiura had low vaccine-specific IgG responses compared to their non-infected counterparts or those infected with A. lumbricoides [S. haematobium mainly. Indeed, the study area is endemic for S. haematobium [S. mansoni infections may also rarely occur [S. haematobium and STH on the time of the occurrence of malaria episodes after CHMI. In addition to that, we also assessed the effect of that helminth on the immunogenicity and the efficacy of GMZ2 when adjuvanted with either CAF01 or alum in Gabonese adults.GMZ2, a malaria vaccine candidate, is a recombinant fusion protein with fragments of and MSP3  that shoand MSP3 –26 or Caand MSP3 . Importaricoides . That stmatobium ,11,28,29ly occur . In the The original study was approved by the Comité National d’Ethique de la Recherche of Gabon, under the reference N°004/2015/SG/P. The trial was performed according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use Good Clinical Practice guidelines and the Declaration of Helsinki. Written informed consent was obtained from all participants included in the study.S. haematobium and STH [The original study was conducted at the Centre de Recherches Médicales de Lambaréné (CERMEL), Gabon (April 2015 –November 2015). Lambaréné is a semi-urban town, an area in which malaria  and othe and STH  are endeP. falciparum sporozoites  approximately 13 weeks after the last immunization (on Day 56), and participants were followed-up on a daily basis with clinical and parasitological  examinations for 35 days , fourteen controlled parasitaemia (low oscillating parasitemia and no symptoms) and fifteen had developed malaria (monotone increase of parasitemia with symptoms). The proportion of participants who developed malaria and the time to develop malaria were similar in all vaccinated groups [P. falciparum infection was treated with artemether-lumefantrine as the first line treatment. Urine samples were collected at D0 and at D84 on three consecutive days whereas stool samples were collected once at D0 and once at D84 , as described elsewhere . BrieflyA. lumbricoides, and T. trichiura in stool samples, and coproculture was performed to detect larvae of hookworms and S. stercoralis as described elsewhere [S. haematobium eggs in urine [Loa loa and Mansonella perstans in blood was assessed using the leuco-concentration technique [The Kato Katz enrichment and microscopy was used to detect eggs of hookworms, lsewhere ,32,33. Uin urine . Presencechnique .3 PFU/ml phocine herpes virus 1 (PhHV-1) was added within the isolation lysis buffer in each sample [A. lumbricoides, S. stercoralis, Necator americanus and T. trichiura. Those species were the most detected by microscopy in our study and are known to be most prevalent in the study area [DNA extraction, amplification, and detection Parasite DNA from stool was isolated and amplified following procedures previously described with minor modifications –37. For h sample ,39 were udy area . Primer S. haematobium (Sh+) and S. stercoralis (Str+). Due to the very low number of cases, and as no considerable difference in concentration of anti-GMZ2 antibodies was found between these both STH, T. trichiura and hookworm infections were merged into the third Tt+/Hw+ sub-study group. The remaining study participants constituted the coinfection (CoInf+) sub-study group as they were infected with more than one parasite. The Mann-Whitney non-parametric test was used to compare characteristics and the haematological profiles of the study population, while linear regression was used for univariable and multivariable analyses to compare specific IgG levels between study groups, using baseline vaccine-specific total IgG concentration as variable of adjustment. The unpaired t test was performed to compare the specific IgG concentration between each combination of two groups after stratification of the helminth infected group, following by ANOVA test with Holm-Sidak’s multiple comparisons. A Kaplan-Meier curve and Log-rank tests were used to assess the time to malaria episodes between study groups and subgroups. All analyses were considered exploratory. The tests were considered statistically significant for a p-value less than 0.05.Data were exported in R version 3.4.2 for statistical analysis. Graphs were made using GraphPad Prism Version 6 and R. Study participants were divided in two main groups; those found to be uninfected with helminths by both qPCR and microscopy formed the helminth negative (Helm-) group, and those shown to be infected with helminths either by qPCR and/or by microscopy formed the helminth positive (Helm+) group. In order to assess the effect of individual helminth species, subgroups were formed based on the respective mono-infections i.e. A. lumbricoides, T. trichiura, S. stercoralis) as determined by the presence of eggs or larvae in stool using microscopy, while twenty-six (26) were considered uninfected. Using the PCR method, a total of nineteen (19) participants were found to be infected with at least one species of STH, including 6 of those for whom microscopy was negative, while the twenty (20) other participants were uninfected (Of the forty-five (45) participants from the fifty (50) included in the clinical trial and for whom all data are available, five (5) were excluded from analysis as they received the control vaccine (Verorab) and one further patient was excluded due to missing data for the PCR on stool samples for STH detection. Data on thirty-nine participants are therefore considered for analysis . Of theminfected .Schistosoma eggs in urine, including ten (10) participants also infected with STH using microscopy and/or PCR, and twenty-five (25) participants had no Schistosoma eggs, including nine (9) participants infected with STH using microscopy and/or PCR. No Schistosoma eggs were found in stool samples, and no filarial infection was detected in the study population. In summary, 16 (41%) participants were classified as uninfected (Helm-), and 23 (59%) were classified as infected (Helm+)  participants had  (Helm+) .S. haematobium alone, 4 had S. stercoralis, 2 had T. trichiura and 1 had hookworm. As detailed in S. haematobium being the most prevalent species (34%), followed by T. trichiura (28%), Hookworm (21%), S. stercoralis (10%) and A. lumbricoides (7%), respectively. All 39 participants included in this analysis were men with a median age (interquartile range) of 23 (5.5) years. Compared to the Helm- group, the hemoglobin level was lower in the Helm+ group  while the levels of white blood cells , lymphocytes , eosinophils  and basophils  were increased , anti-MSP3  and anti-GLURP IgG  concentration , S. stercoralis  status and vaccine-specific IgG concentration on D84. Compared to anti-GMZ2 IgG concentration on D84 in those uninfected (mean log concentration ±SD: 3.49 ±0.14), participants infected with S. haematobium alone presented a higher level of anti-GMZ2 IgG (3.69 ±0.10), while those infected with S. stercoralis alone had lower anti-GMZ2 IgG levels (3.32 ±0.19) , Tt+/Hw+ individuals  and CoInf+ individuals   . Additio = 0.03) . No sign = 0.03) .p-value = 0.039), as depicted in S. haematobium  and STH  infections were associated with faster times to malaria  and Helm+  groups (A), for Helm- and Sh+  (B), for Helm- and Sth+  (C), and in Helm- and CoInf+  (D).In additional analyses in the helminth coinfected group assessing the distribution of helminths species between malaria outcome groups following CHMI, the results showed that the distribution of helminth species was different between participants who did and those who did not develop malaria .p-value = 0.048) on D84 in the Helm- group. The anti-GMZ2 IgG concentration was higher among those who did not experience malaria (3.51 ±0.13) compared to those who experienced malaria Click here for additional data file.S2 TableDifferent primers and probes concentrations were tested to evaluate the best one for our assay. We used the following primers and probes concentration for each species: 0.1 μM, 0.2 μM, 0.3 μM and 0.4 μM. After checking and analyzing the PCR amplification curves, we decided to use the 0.2 μM concentration. (DOCX)Click here for additional data file.S3 TableInfection (+) either by PCR and/or microscopy at D0 and/or at D84 No infection (-) either by PCR and/or microscopy at D0 and/or at D84.(DOCX)Click here for additional data file.S4 TableThe percentage of each helminth specie in the helminth coinfected group in those who did and did not develop malaria was calculated following the formula: (Number of volunteers infected at D0 and/or D84 x 100) / Total number of volunteers who did  or did not develop malaria  following CHMI.(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 FigBlood collection was done before each immunization and at seven, fourteen days after the first and second immunization, and seven, fourteen and twenty-eight days after the third immunization, as well as one day before the CHMI and thirty-five day after the CHMI. The asterisk represents the time of urine and stool collection.(TIF)Click here for additional data file.S2 FigAnti-GMZ2, anti-MSP3 and anti-GLURP IgG concentrations between D0 and D84 in vaccination groups Figures show the log of GMZ2-specific (A), MSP3-specific (B), GLURP-specific (C) IgG concentrations at D0 (filled dot) and at D84 (unfilled dot) in participants vaccinated with Rabies vaccine, 100 μg GMZ2-Alhydrogel, 30 μg GMZ2-CAF01; and 100 μg GMZ2-CAF01. The comparison was performed using a paired t-test. The graphs show mean ± standard deviation. Statistical significance was set for p value below 0.05 *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. NS = Non-significant.(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Plasmodium falciparum infections was measured in cohort participants from randomly selected households in the Kihihi sub-county of Uganda .Evaluation of genetic relatedness of malaria parasites is a useful tool for understanding transmission patterns, but patterns are not easily detectable in areas with moderate to high malaria transmission. To evaluate the feasibility of detecting genetic relatedness in a moderate malaria transmission setting, relatedness of Plasmodium-specific loop mediated isothermal amplification from passive and active case detection during August 2011-March 2012 were genotyped at 26 microsatellite loci, providing data for 349 samples from 230 participants living in 80 households. Pairwise genetic relatedness was calculated using identity by state (IBS).All infections detected via microscopy or e] = 0.73), and the majority (76.5 %) of samples were polyclonal. Despite the high genetic diversity, fine-scale population structure was detectable, with significant spatiotemporal clustering of highly related infections. Although the difference in malaria incidence between households at higher (mean 1127 metres) versus lower elevation (mean 1015 metres) was modest , there was a significant difference in multiplicity of infection  and, more strikingly, a higher proportion of highly related infections within households  at higher elevation compared to lower elevation.As expected, genetic diversity was high (mean heterozygosity [HGenetic data from a relatively small number of diverse, multiallelic loci reflected fine scale patterns of malaria transmission. Given the increasing interest in applying genetic data to augment malaria surveillance, this study provides evidence that genetic data can be used to inform transmission patterns at local spatial scales even in moderate transmission areas. Falciparum malaria remains one of the most important infectious diseases in the world, causing significant morbidity and mortality. More than 90 % of malaria deaths occur in sub-Saharan Africa, with the majority of these in children less than 5 years old . ConsidePlasmodium falciparum infections identified in participants living in 80 households in Kihihi, Uganda.As with most infectious diseases, malaria transmission within a geographical area is heterogeneous and can vary greatly between villages and between households in a village –10. Vari2 in southwestern Uganda  [(1 – ∑Pi2 )], where n = sample size and Pi = allele frequency. The mean multiplicity of infection (MOI) was calculated by dividing the total number of clones by the number of PCR-positive samples for each marker gene. Pairwise time was calculated as the difference in days between obtaining the two samples. Pairwise spatial distance was calculated as the Euclidian distance in metres between the households from which samples were obtained. Pairwise identity by state (IBS) was calculated from allele similarity between isolates using a modified IBS metric to measure parasite relatedness [n = number of genotyped loci, Si = total number of shared alleles at locus i between samples X and Y; Xi = number of alleles in sample X at locus i and Yi = number of alleles in sample Y at locus i. An IBS of ≥ 0.6 was used as a cut off for the most highly related samples, as per Fig. All data were analysed using R (version 3.5.0) with R studio . Expected heterozygosity . Comparisons of proportions were performed using generalized estimating equations to account for repeated measures. Pairwise distance and time means were compared using the Wilcoxon test for independent samples.versus below the median (lower elevation). Using data from all children in the cohort through one year, there was lower malaria incidence in children living at higher compared to lower elevation (1.4 versus 1.9 episodes per person-year (ppy) . In addition, the daily human biting rate for anopheline mosquitoes (dHBR) was considerably lower in households at higher versus lower elevations , adding evidence to support higher transmission at lower elevation. Thus, the data suggest modest but meaningful differences in transmission within the study area by both clinical and entomologic measures, with transmission higher in houses at lower elevation.Despite the small size of the study area, there were noticeable differences in transmission intensity comparing cohort households above the median elevation (higher elevation) A total of 408 samples with ≥ 5 parasites/µL of blood from 80 households were genotyped using 26 microsatellite markers. Of these, 349 (85.5 %) samples were successfully genotyped at 15 or more loci and included in subsequent analysis; these samples were collected from 230 unique individuals (142 children and 88 adults) (Table e) of 0.73 (range: 0.37–0.91) with no difference between elevations or between adults and children.The majority of genotyped infections were polyclonal (76.5 %) with a mean MOI of 2.4 , suggesting that transmission-related infections cluster within households. To test the hypothesis that highly related infections were more likely to be observed within the same household where transmission was lower, the proportion of highly related infections within households was compared by elevation. As hypothesized, a higher proportion of highly related pairs of infections was found within households at higher elevation compared to lower elevation   to evaluate within-household transmission events. However, the proportions of highly related infections in child-child, child-adult, and adult-adult paired samples both within and between households were calculated. In child-child pairwise comparisons, the proportion of highly related infections was higher than in comparisons for child-adult or adult-adult pairs Fig. , althougTo evaluate the potential to detect genetic relatedness of malaria parasites in a moderate malaria transmission setting, relatedness of infections was measured using 26 microsatellites in Kihihi sub-county, southwestern Uganda. As expected, genetic diversity was high and the majority of samples were polyclonal. Despite the high diversity, spatiotemporal structure in genotypes was detectable, with significant spatial and temporal clustering of highly related infections identified in both higher and lower transmission areas of Kihihi, including marked clustering of related infections within households. Despite modest differences in malaria incidence found at higher vs. lower elevations within the study site, there were differences in MOI and spatial clustering of infections, reflecting underlying differences in transmission identified using detailed entomologic measures. Of note, statistical power for both of these molecular metrics was higher than that observed for malaria incidence. Thus, these data demonstrate the potential utility for genetic measures of parasite diversity in stratifying malaria transmission, even in moderate to high transmission settings where the majority of infections are polyclonal.versus local transmission in Namibia, Tessema et al.. showed that microsatellite data could capture strong spatial signals over both local and regional scales in a study with limited travel history and without cross-border mobile phone data. In a study conducted along the Thai-Myanmar border, single nucleotide polymorphism (SNP) barcode data were used to show that identity by descent (IBD), a measure of parasite relatedness, declined with increasing inter-clinic distance [While it is clear that there is heterogeneity in malaria transmission at all levels of endemicity, this phenomenon is most obvious in areas of low transmission , 22. Thedistance . In Zambdistance . Two othdistance  or at thdistance , using Sdistance –28. NotaAdditionally, this study showed that relatedness of infections between children was greater than that of infections between children and adults or infections between adults. This pattern was evident both between and within households, but was much more pronounced within households. This pattern may be due to age-related immunity, since adults may more readily suppress or clear a proportion of parasites to which they had been previously exposed. Alternatively, because children harbour higher parasite densities than adults, they may be more likely to transmit to other children, including those in the same household . A recenThe study had some limitations. For example, IBS is a biased measure of parasite relatedness if one is attempting to compare IBS measures across different studies or populations, because it is sensitive to allele frequencies . An advaIn this study, spatiotemporal clustering of malaria infections was identified in a moderate transmission setting by analysing all infections and focusing on those which were highly related. There was also evidence of clustering of related malaria infections within households, which was more evident in areas of lower transmission. Importantly, genetic data were able to clearly discriminate transmission intensity in this small study area where only modest differences in malaria incidence were evident. Given the increasing interest in applying genetic data to augment malaria surveillance, this study provides evidence that genetic data can be used to inform transmission patterns at local spatial scales even in moderate transmission areas.Additional file 1: Table S1. Diversity at each microsatellite marker. Avg He 0.73. Figure S1. MOI distribution. A) Overall distribution of MOI B) Distribution of MOI at lower elevation C) Distribution of MOI at higher elevation.


In recent years, the incidence rate of vivax malaria recurrence still had 3.1% in Yunnan Province population after eradication therapy using primaquine (PQ). In order to understand the specific failure reasons for preventing vivax malaria relapses, a preliminary exploration on the CYP2D6 enzyme activity was carried out in the vivax malaria patients in Yunnan Province population by analysing mutational polymorphism in the coding region of CYP2D6 gene.Blood samples were collected from vivax malaria patients with suspected relapse (SR) and non-relapsed (NR) malaria in Yunnan Province. The DNA fragments containing 9 exons regions of human CYP2D6 gene were amplified by performing PCR and sequenced. The sequencing results were aligned by using DNAStar 11.0 to obtain the coding DNA sequence (CDS) of CYP2D6 gene. DnaSP 6.11.01 software was used to identify mutant polymorphisms and haplotypes of the CDS chain. The waterfall function of GenVisR package in R was utilized to visualize the mutational landscape. The alleles of CYP2D6 gene were identified according to the criteria prescribed by Human Cytochrome P450 (CYP) Allele Nomenclature Committee Database and the CYP2D6 enzyme activity was predicted based on diploid genotype.P < 0.05), as opposed to the NR group. Of the 23 haplotypes (from Hap_1 to Hap_23), the nomenclatures of 11 allelic forms could be found: Hap_3 was non-mutant, Hap_2 accounted for the highest frequency , and Hap_9 had the most complex sequence structure, containing 7 loci mutations. Allele *10 was the most frequent among these genotypes (0.423). Among the allele *10 standard named genotypes, *1/*10, *1/*1 and *2/*10 were significantly more frequent in the NR group (P < 0.05) and all showed uncompromised enzyme activity; the impaired genotype *10/*39 was more frequent in the SR group (P < 0.05), and genotype *4/*4was detected only in the SR group.A total of 320 maternal CDS chains, including 63 from SR group and 257 from NR group, were obtained. Twelve mutant loci, including c.31 (rs769259), c.100 (rs1065852), c.271 (rs28371703), c.281 (rs28371704), c.294 (rs28371705), c.297 (rs200269944), c.336 (rs1081003), c.408 (rs1058164), c.505 (rs5030865), c.801 (rs28371718), c.886 (rs16947), and c.1,457 (rs1135840) were observed on the 640 CDS chains . The high-frequency mutation at rs1135840 (0.703) and low-frequency mutation, such as rs28371703, were detected only in the SR group. The frequency of mutant rs1058164 and rs1135840 were significantly increased in the SR group (In the patients receiving PQ dosage in Yunnan Province population, both rs1135840 single nucleotide polymorphism and *10 allele form was common in the CYP2D6 gene. Low-frequency mutation sites, such as rs28371703, were only presented in patients with vivax malaria relapse.The online version contains supplementary material available at 10.1186/s12936-021-03988-5. Plasmodium vivax [PQ currently remains the only reliable drug to eliminate the hypnozoites of um vivax . Howeverum vivax –4. CYP2Dum vivax . Previouum vivax , 8. Evidum vivax , 9.In relapsed vivax malaria cases in Papua New Guinea, CYP2D6 enzyme activity was mostly deficient star alleles *3, *4, *5, *6, *7, and *8, which indicates the phenotype of poor metabolizer (PM) , 11. HowRecently, the World Health Organization (WHO) classified the impaired CYP2D6 function as one of the four risk factors for PQ therapy, in addition to G6PD deficiency, pregnancy and/or lactation and infancy , 13, 14.Molecular epidemiological investigation on G6PD deficiency in vivax malaria patients has been initiated in Yunnan Province , 17 to cPlasmodium in peripheral blood [All vivax malaria cases included in the current study had been diagnosed and reported by county-level laboratories in Yunnan Province since 2014. The initial diagnosis from county-level laboratories were re-tested by Yunnan Provincial Malaria Diagnostic Reference Laboratory (YPMDRL) using both microscopic examination and genetic testing  within 3 days, followed by the subsequent 8-day course of primaquine therapy (22.5 mg/day) (commonly known as 8-day chloroquine/primaquine therapy). The vivax malaria recurrence cases were found by the county-level laboratories, too. Everyone received 8-day chloroquine/primaquine therapy was able be identified as recurrence case when his peripheral blood showed  malaria .pvcsp gene and pvmsp-1 gene [pvcsp genes in different Plasmodium vivax stains), the case with recurrent experience can be confirmed as vivax malaria suspected-relapse.Then, it is necessary that the paired infection strains between previous and recurrence episodes were molecularly identified by YPMDRL after that. The paired strains, between the previous and recurrent infections, were molecularly identified by YPMDRL, and verified as the single clone with homologous or the sibling clones with weak heterologous DNA sequences including both p-1 gene , 21  and non-relapse group (NR group). The NR group, which included the cluster patients from 2018 to 2020, had not experienced recurrent symptoms after being clinically cured by 8-day chloroquine/primaquine therapy. In the SR group, all patients from 2014 to 2020 were included.The DBS method was used on filter paper dried blood stain (diameter = 5 mm). QIAgen Mini Kit  was used to extract human genomic DNA from the blood stain, according to manufacturer’s instructions. DNA samples were stored at -20 °C for subsequent assay.Reference sequence (ID: NC_000022.11) was used as the template to design the PCR primers and to determine the reaction conditions for CYP2D6 gene, according to methods described in previous studies , 22, 23.https://www.ncbi.nlm.nih.gov/snp/). GenVisR package in R (version: 4.0.2) was used to visualize the mutational waterfall plots of the haplotypes of the loci in the allelic form.The sequencing results were collated by using DNAStar 11.0 and BioEdit 7.2.5, and the first coding DNA sequence (CDS) chain of CYP2D6 gene of each sample, known as maternal CDS chain, was obtained by the splicing method . The matThe haplotypes  of the CDS chain of CYP2D6 gene were determined in accordance with the criteria of the Human Cytochrome P450 Allele Nomenclature Committee Database (NM_000106.5)  and publThe genotype of each sample was presented in the diploid form consisting of maternal and paternal CDS chains, such as *10/*39, *1/*10 and so forth. The diploid allelic form of the genotype was no longer refined to the sub-allelic level, as the sub-allelic forms should be merged in the first place. For example, *10.001 and *10.002 could be combined into the allelic form *10 to count the frequency.http://provean.jcvi.org/index.php) was used to predict the function of CYP2D6 enzyme protein of different genotypes as deleterious (DT) or neutral (NT).For the genotypes with standard nomenclature, the CYP2D6 enzyme activity was predicted by calculating the genotype score based on diploid genotypes, following the previously described method , 27, 28.P < 0.05 was regarded statistically significant.The frequency of mutation site was presented by the ratio of the number of base substitutions to the total number of CDS chains. Each study subject had two CDS chains, including maternal chain and paternal chain. Chi-square test A total of 345 blood samples were collected from vivax malaria patients for subsequent PCR amplification of human genomic DNA, which covers exon1-4 and exon5-9 regions of CYP2D6 gene. PCR amplification products showed the target fragment more than 2000 bp bands at gel electrophoresis. After collating the sequencing results of PCR amplification products, all 320 maternal CDS chains of CYP2D6 gene (1491 bp in length) were obtained in 345 subject blood samples , including 63 SR cases and 257 NR cases.The blood samples of obtained 320 maternal CDS chains were collected from diagnosed vivax malaria cases in the following prefectures of Yunnan Province: Yingjiang (182), Mangshi (15), Longchuan (8), Ruili (16), Tengchong (42), Baoshan (20), Lianghe (3), Mengla (1), Menglian (1), Diqing (1), Nujiang (1), Jinping (4), Yuanyang (2), Lincang (20), Dali (2) and Kunming (2).The demographic and clinical characteristics of 320 cases are listed in Table The polymorphism of 640 CDS chains, 320 maternal and 320 paternal, of CYP2D6 genes revealed base substitutions  at 12 loci, includingc.31, c.100, c.271, c.281, c.294, c.297, c.336, c.408, c.505, c.801, c.886, and c.1,457. Most of these mutation sites have been registered as single nucleotide polymorphism (SNP) loci , and the minor allele frequency (MAF) was 0.698 for c.408 (rs1058164) Table . Loci muThese 640 CDS chains could be subdivided into 23 haplotypes  , Hap_9 showed the most complicated sequence composition, exhibiting mutations sites at c.100, c.271, c.281, c.294, c.297, c.408, c.1,457 and so forth. Hap_14 showed the simplest sequence composition, with mutation detected at c.408  (Table P < 0.05) than in the SR group (Table P < 0.05), compared with the NR group , c.281 (rs28371704), c.294 (rs28371705), and c.505 (rs5030865), were present only in samples of SR group  of the samples , 37, andThe radical cure of vivax malaria is a critically important intervention to prevent the re-transmission and repeated attack of malaria . As we kPlasmodium reservoir in bone marrow into peripheral blood [Plasmodium to PQ [This study has certain shortcomings. First, the determination of the recurrence of vivax malaria was somewhat flawed, as the al blood , 41 or tum to PQ , despiteum to PQ , 44. ThiIn this study, only 12 mutation loci, including c.31, c.100, c.271, c.281, c.294, c.297, c.336, c.408, c.505, c.801, c.886, and c.1,457 were detected in Yunnan Province vivax malaria patients, which is consistent with the findings of a previous small sample. In those patients receiving PQ dosage in Yunnan Province, both rs1135840 SNP and allele form *10 was common in the CYP2D6 gene coding region. Low-frequency mutation sites, such as rs28371703, were presented only in patients with vivax malaria relapse. Genotype *10/*39 could be suitable for indicating impaired CYP2D6 enzyme activity in this population to be treated with PQ.Additional file 1. The details of nested PCR testing for differentiating between various Plasmodium species.Additional file 2. Records of basic personal information of subjects