Plasmodiumfalciparum and non–P. falciparum infections. We determined prevalence and geographic distribution of 4 Plasmodium spp. by performing PCR on dried blood spots collected within 8 regions of Tanzania during 2017. Among 3,456 schoolchildren, 22% had P. falciparum, 24% had P. ovale spp., 4% had P. malariae, and 0.3% had P. vivax infections. Most (91%) schoolchildren with P. ovale infections had low parasite densities; 64% of P. ovale infections were single-species infections, and 35% of those were detected in low malaria endemic regions. P. malariae infections were predominantly (73%) co-infections with P. falciparum.P. vivax was detected mostly in northern and eastern regions. Co-infections with >1 non–P. falciparum species occurred in 43% of P. falciparum infections. A high prevalence of P. ovale infections exists among schoolchildren in Tanzania, underscoring the need for detection and treatment strategies that target non–P. falciparum species.Achieving malaria elimination requires considering both  Plasmodiumfalciparum prevalence  malaria species, such as P. malariae, P. vivax, and P. ovale curtisi or P. ovale wallikeri   or histidine-rich protein 2 (HRP2)–based rapid diagnostic tests (RDTs), lack sensitivity to detect nonfalciparum species  was a cross-sectional study of children who were 5–16 years of age and enrolled in public primary schools in mainland Tanzania. Methods for site selection and survey design mirrored the 2015 SMPS and have been previously described .https://www.accessbio.net) that were specific for P. falciparum HRP2 and pan-pLDH antigens. RDTs were considered positive if they were positive for either antigen. Schools and councils were grouped into epidemiologic malaria transmission risk strata on the basis of P. falciparum prevalences in children estimated from the 2014–15 Tanzania SMPS  were shipped to the University of North Carolina  for molecular testing.During the survey, malaria detection was conducted by using CareStart Malaria Pf/PAN (HRP2/pLDH) Ag Combo RDTs  to create mock DBS samples and for nonfalciparum species detection by using plasmid DNA (BEI Resources). We serially diluted the control samples and extracted DNA as described. We estimated semiquantitative parasitemias for nonfalciparum species by assuming 6 18S rRNA gene copies/parasite  and used the eulerr (https://cran-r-project.org/package=eulerr) and sf version 0.9–7 (https://gadm.org) and collected elevation measurements from the US National Aeronautics and Space Administration, Shuttle Radar Topography Mission (https://www.nasa.gov). We performed analyses by using R version 4.0.2 , P. ovale in 24% , P. malariae in 4% , and P. vivax in 0.3%  of students, including single- and mixed-species infections  were co-infections with P. falciparum only  were co-infections with P. falciparum only. We determined 36% (n = 4) of P. vivax infections were single-species infections, and 43% (n = 326) of P. falciparum infections were co-infections with >1 nonfalciparum malaria species.We identified fections  Table 5.rum only . ConversP. falciparum and P. malariae by using PCR cycle thresholds of <45 to evaluate different PCR cycles between assays. We observed 25% (95% CI 21%–29%) P. falciparum and 3% (95% CI 2%–5%) P. malariae prevalences, weighted according to student distribution within the total nonfalciparum population by transmission risk strata  of P. malariae infections were detected at cycle thresholds of 40–45. Thus, >97% of P. falciparum and 90% of P. malariae infections were detectable by the primary 40-cycle assay in our study.We conducted a sensitivity analysis, detecting k strata  Table 6.Plasmodium spp. infection  and female  students. Comparing RDT sensitivity to PCR, we observed 8% (n = 40) of students with P. ovale single-species infections were RDT-positive for any band, whereas 33% (n = 8) of those with P. malariae and 69% (n = 295) with P. falciparum single-species infections were RDT-positive. Co-infections with P. falciparum and nonfalciparum were RDT-positive in 78% (n = 253/325) of cases detected by PCR. Although only 3% (n = 13) of P. falciparum single-species infections and no P. malariae or P. vivax single-species infections were detected in low transmission risk strata, 35% (n = 181) of P. ovale single-species infections occurred in regions classified as low or very low malaria transmission risk. High epidemiologic risk strata harbored most single-species infections across all 4 Plasmodium spp. and also mixed infections with P. falciparum.We evaluated differences in student characteristics according to nfection  Table 7.P. ovale density was 7.2  parasites/µL, comparable to P. malariae density at 11.7  parasites/µL. P. vivax density was ≈0.6  parasites/µL. Although 18% (n = 25) of P. malariae infections had a parasite density >100 parasites/µL, we rarely observed that level for P. ovale  and never for P. vivax. P. falciparum density was also low at 13.1  parasites/µL; however, 17% (n = 132) of P. falciparum cases had a parasite density >100 parasites/µL, and 3% (n = 24) had >500 parasites/µL. Median (IQR) density among P. ovale mixed infections was 3.1 (1.2–11.4) parasites/µL and 13.5 (1.3–30.1) parasites/µL for P. ovale single-species infections (p<0.001), whereas densities were similar between single- and mixed-species infections among the other malaria species  samples with the highest parasitemia were selected for PCR to distinguish between P. ovale wallikeri and P. ovale curtisi. Species determination by PCR was successful in 35% (n = 21) of samples; P. ovale curtisi was detected in 17 samples and P. ovale wallikeri in 9 samples. We identified P. ovale curtisi and P. ovale wallikeri co-infections in 5 students. We did not perform further characterization because of limited sample sizes.Among 814 samples positive for P. ovale across all 8 regions sampled in Tanzania, indicating widespread distribution  and central Tabora  regions. We detected P. ovale curtisi infections in 6 of 8 regions  and P. ovale wallikeri in 5 of 8  regions. We observed high prevalence of P. malariae in Kagera  and in southernmost Mtwara , and P. vivax was predominantly distributed along the northwestern borders of Tanzania in Kagera ; select, isolated cases of P. vivax were also detected in southern and eastern regions. Arusha and Iringa did not have any cases of P. malariae or P. vivax infections and had the lowest frequencies of P. ovale  and P. falciparum  infections.We detected ribution , 4. P. oP. ovale, 94% (n = 707) by P. falciparum, 95% (n = 129) by P. malariae, and 100% (n = 11) by P. vivax  of students compared with 6% (n = 48) infected with P. falciparum, 1% (n = 7) infected with P. malariae, and 0% infected with P. vivax.We detected malaria infections in students who were predominantly located at elevations <1,500 m, including 85% (n = 693) infected by P. vivax . Most , scaled P. ovale prevalences were higher than P. falciparum prevalences. Scaled prevalence of P. malariae was notably higher than that of P. falciparum in the Karagwe council in Kagera and Mtwara municipal council in Mtwara. In most other areas, scaled prevalence of P. malariae was similar to or lower than P. falciparum prevalence.We compared scaled prevalence estimates for nonfalciparum species with requency ; P. vivaP. ovale parasites, comparable to the 22% P. falciparum prevalence in the population, and 64% of P. ovale infections were single-species infections. P. malariae was observed in 4% of students, of which most were co-infected with other malaria species. P. vivax infections were rare .Our study describes a large nationally representative molecular survey of nonfalciparum malaria epidemiology across Tanzania. We used real-time PCR to estimate nonfalciparum infection prevalences in school-aged children in 8 regions of the country selected to maintain geographic diversity and malaria transmission risk heterogeneity. One quarter (24%) of schoolchildren harbored P. ovale prevalence could be attributed to several factors. First, we increased the number of PCR cycles for P. ovale detection to 45 to enable detection of low-density infections, which comprised 91% of all P. ovale infections identified , confirming most infections occurred at very low parasite densities. Second, many large-scale molecular surveys of nonfalciparum malaria have focused on adults or all-age cohorts, whereas school-aged children are increasingly recognized as the main contributors to asymptomatic and infectious malaria reservoirs  , likely because of the predominantly asymptomatic population. In addition, mapping confirmed low or nonexistent prevalence of nonfalciparum malaria within the northern highlands of Arusha and southern highlands and midlands of Iringa.Detection of P. malariae and P. falciparum assays were run at 40 rather than 45 cycles, their relative prevalences compared with prevalence for P. ovale might be underestimated. However, we performed a sensitivity analysis to quantify this bias, which indicated that only an additional 2.5% of P. falciparum and 10% of P. malariae infections would be detected by using 45 cycles, suggesting minimal underestimation of reported P. falciparum and P. malariae prevalences and no meaningful effect on overall conclusions. Weighting sensitivity analysis results to the total study population yielded a P. falciparum prevalence of 25% if 45 cycles were used compared with the observed prevalence of 22%. Despite this result, prevalences could still be underestimated given lower probabilities of detecting very low density infections because of PCR limits of detection in concert with small volumes of template DNA used in the assays . Second, our study did not sample all geographic regions in Tanzania, and findings cannot be extrapolated to other age groups with differing malaria risk profiles. School-based sampling likely underestimated prevalence of symptomatic or severe malaria infection in school-aged children because children might have been absent because of illness. Finally, the cross-sectional survey design revealed little about clinical implications of prevalent nonfalciparum infections, especially given substantial nonrandom missingness in fever data, or the extent to which infections represented chronic infection carriage versus transient parasitemia.The first limitation of our study is that using different PCR cycling times for different species introduces ascertainment bias. Because P. ovale and, to a lesser extent, P. malariae and the more focal distribution of P. vivax in this study underscore an urgent need to elucidate clinical prevalence and transmission patterns of those species to inform malaria control programs in Tanzania. Current treatment protocols in Tanzania do not regularly address hypnozoite liver-stage P. ovale infection, and relapses are expected after blood-stage clearance by artemisinin-based combination therapy (In conclusion, the overall high prevalence and broad geographic distribution of Additional information for similar prevalence of Plasmodium falciparum and non–P. falciparum malaria infections among schoolchildren, Tanzania.


When compared with the levels of immune complexes and of autoimmune antibodies to phosphatidylserine, factors previously associated with severe anemia in malaria patients, we observed that XO is not associated with them, suggesting that XO is associated with severe anemia through an independent mechanism. XO was associated with prostration, acidosis, jaundice, respiratory distress, and kidney injury, which may reflect a broader relation of this enzyme with severe malaria pathology. Since inhibitors of XO are inexpensive and well-tolerated drugs already approved for use in humans, the validation of XO as a contributor to severe malarial anemia and other malaria complications may open new possibilities for much needed adjunctive therapy in malaria.Severe anemia is an important contributor to mortality in children with severe malaria. Anemia in malaria is a multi-factorial complication, since dyserythropoiesis, hemolysis and phagocytic clearance of uninfected red blood cells (RBCs) can contribute to this syndrome. High levels of oxidative stress and immune dysregulation have been proposed to contribute to severe malarial anemia, facilitating the clearance of uninfected RBCs. In a cohort of 552 Ugandan children with severe malaria, we measured the levels of xanthine oxidase (XO), an oxidative enzyme that is elevated in the plasma of malaria patients. The levels of XO in children with severe anemia were significantly higher compared to children with severe malaria not suffering from severe anemia. Levels of XO were inversely associated with RBC hemoglobin (ρ =  − 0.25,  Severe anemia is a common complication of malaria, which occurs most frequently in children under the age of five and is a major contributor of death by malaria1. Although mortality associated with severe anemia in children with malaria is low with appropriate transfusion, unreliable blood availability in hospitals frequently results in avoidable deaths2. Additionally, children with severe malarial anemia (SMA) are at risk of repeated malarial episodes and increased post-discharge mortality3.Malaria is a major cause of anemia in the areas of the world where this disease is endemic4. While Plasmodium infection of RBCs can lead to destruction and clearance of these cells, the majority of RBCs lost during Plasmodium infection are uninfected RBCs. For each RBC lysed directly due to Plasmodium infection, about eight uninfected erythrocytes are lost in P. falciparum6 infections. Although the mechanisms underlying the loss of uninfected RBCs during malaria are not completely understood, two mechanisms have been proposed: (1) increased RBCs rigidity from oxidative stress leads to subsequent splenic clearance of these cells8, and (2) immune dysregulation resulting in binding of antibodies and immune complexes (IC) to uninfected RBCs10 together with complement dysfunction12 would contribute to increased uptake by macrophages.Malaria-induced anemia involves both dyserythropoiesis and increased loss of infected and uninfected red blood cells (RBCs)13, including anemia14, but also respiratory distress and acute kidney injury, as well as mortality after 6 months16. Here we have determined the levels of xanthine oxidase (XO), an oxidative enzyme not found in RBC17 that is upregulated during malaria18, in a cohort of Ugandan children with severe malaria. We have observed a highly significant correlation between the levels of this enzyme and severe anemia in these patients. We also measured the levels of other immune-related parameters previously described to correlate with SMA: anti-phosphatidylserine (anti-PS) antibodies23, anti-DNA antibodies23 and immune complexes (IC)10, finding that they are only weakly associated with the levels of XO. Taken together, these results indicate that XO is associated with SMA independently of autoimmune-related mechanisms. A significant association of XO levels and other common complications of severe malaria suggests a possible broad relation of XO with malaria-induced pathology.Oxidative stress is elevated during malaria and has been associated with different complicationsEthics and consent to participate: Written informed consent was obtained from parents or guardians of study participants. Ethical approval was granted by the Institutional Review Boards for human studies at Makerere University School of Medicine and Indiana University School of Medicine. All methods were performed in accordance with the relevant guidelines and regulations.24, between 2014 and 2017, 600 children with severe malaria were enrolled in the study designed to assess cognition in children < 4 years of age at 12 months’ follow-up25. Children were eligible if they: (1) were between 6 months and 4 years of age; (2) had diagnostic evidence of malaria with either a positive rapid diagnostic test for P. falciparum histidine-rich protein-2 (HRP2) or direct visualization of parasites by Giemsa microscopy; (3) required hospitalization; and (4) had 1 or more of the following severe malaria criteria: coma (Blantyre coma score < 3), respiratory distress , multiple seizures , severe anemia (hemoglobin ≤ 5 g/dL), or prostration . Children were recruited at 2 referral hospitals in Central and Eastern Uganda: Mulago National Referral Hospital in Kampala and Jinja Regional Referral Hospital in Jinja. Exclusion criteria included the following: known chronic illness requiring medical care, history of coma, head trauma, known developmental delay, cerebral palsy, or prior hospitalization for malnutrition. Delayed exclusion criteria included an elevated cerebrospinal fluid white blood cell count in a child with coma. Children with severe malaria returned for an interim health assessment and blood draw at one-month follow-up.As described beforeFor controls, 120 community children from the nuclear family, extended family, or household area of the children with severe malaria were enrolled. Additional exclusion criteria for the community children included an illness requiring medical care within the previous four weeks, a major medical or neurologic abnormality at screening physical examination, or active illness. Samples from 98 children in this group were used for this study. Samples from 11 children in this group presented positive levels of HRP2 (> 100 ng/mL), of which five were confirmed by Giemsa microscopy.26; Acidosis: base deficit of > 8 mmol/L or, if unavailable, a plasma bicarbonate of < 15 mmol/L or venous lactate of > 5 mmol/L27.Other clinical complication definitions: Acute kidney injury (1.5-fold increase in creatinine over estimated baseline or a 0.3-mg/dL change in creatinine within 48 h)At enrollment, study participants had serum collected for long-term storage at − 80 °C. All children had a complete blood count, blood smear to quantify malaria, stool testing for helminth infection, and were tested for HIV. At one month follow up the children with severe malaria returned for a health assessment and had serum collected and stored at − 80 °C.. ROS production in plasma from five healthy individuals were averaged and used as a reference to calculate relative units (RU) for each determination. RU at the different time points was averaged for each plasma sample and reported as the final XO activity. To determine XO levels at the one-month follow-up, we first selected the 100 patients with the highest levels of XO activity in plasma upon admission and determined XO levels from the 67 that had follow-up samples, all from children with severe malaria.XO activity in plasma samples from this cohort was measured using the Amplex Red Xanthine/Xanthine Oxidase Assay kit (Invitrogen A22182) according to the manufacturer’s instructions. Briefly, plasma samples (10 µl) taken from children upon hospital admission were incubated with hypoxanthine, the substrate of XO, Amplex Red reagent, and horseradish peroxidase (HRP). XO breaks down hypoxanthine into xanthine, which is then broken down into uric acid, and generates reactive oxygen species (ROS) along the way. In the presence of HRP, Amplex Red reacts with ROS to generate a fluorescent product (Excitation-530 nm/Emission-590 nm) that was quantified using a plate reader (Victor X3 from Perkin Elmer). ROS production was measured at different time points  after the start of the reactionCostar 3590 96-well ELISA plates were coated for16h at 4 °C with 100 µL of PS (Sigma) at 20 μg/ml in 200-proof Molecular Biology ethanol or calf thymus DNA (Sigma) at 10 μg/ml in PBS. DNA plates were allowed to evaporate at RT after coating. Plates were washed 5 times with 200 µL of PBS and then blocked for 1 h with 200 µL of PBS 3% BSA. Plasma from patients was diluted at 1:100 in PBS 1% BSA and incubated for 2 h at 37 °C. Plates were washed again five times and incubated with anti-human IgG-HRP  for 1 h at 37 °C. Plates were washed five more times and TMB substrate (BD Biosciences) was added until the desired color was obtained. The reaction was stopped by with Stop buffer (Biolegend) and absorbance was read at 450 nm. Positive and negative controls were the same used for XO determinations. The mean OD at 450 nm from triplicate wells was compared with the same dilution of the reference positive serum to calculate relative units (RU). Anti-PS and anti-DNA levels were determined in all available samples at one-month follow-up (n = 443).Costar 3590 96-well ELISA plates were coated with 100 µL of C1q capture antibody (1 µg/mL) (Thermofisher PA5-29586) in sodium bicarbonate-sodium carbonate buffer  overnight at 4 °C. Plates were washed 2 times with PBS 0.05% Tween 20 washing buffer and incubated with 100 µL 0.1% BSA in PBS blocking buffer for 1.5 h at RT. Plasma samples were diluted 1:80 in 0.1% blocking buffer. 100µL were added to the wells and incubated for 2 h at RT. Plates were washed 2 times and incubated with 100uL polyclonal goat anti-human IgG-HRP diluted 1:5000 (Invitrogen) for 1 h at RT. Plates were washed 3 times before developing with TMB substrate as described for anti-PS. Positive and negative controls were the same used for XO determinations. The mean OD at 450 nm from triplicate wells was compared with the same dilution of the reference positive serum to calculate relative units (RU).Data were analyzed using Microsoft Excel, Graph Pad Prism and the StatsModels Python library. Differences in continuous variables between SMA group were analyzed using Wilcoxon rank sum analysis with Bonferonni correction to adjust for multiple testing. Differences in categorical variables were assessed using Fisher’s exact test or Pearson’s Chi squared test, as appropriate. Spearman’s rho was used to evaluate the correlation between the measured parameters and different clinical parameters. To evaluate differences in biomarker levels at admission and one-month follow-up, Wilcoxon signed rank test was used. To assess the interaction between XO levels and IC and anti-PS, we fitted two logistic regression models including the XO:IC and XO:anti-PS interaction terms, respectively. The p-value of the interaction term coefficient was considered as a measure of significant interaction. In addition, the log-likelihood of the fitted models was compared, correspondingly, to that of an additive model that did not include the interaction term. The log-likelihood ratio test was to evaluate model improvement upon inclusion of the interaction term.28, of which 251 presented with severe anemia (hemoglobin ≤ 5 g/dL), and 98 community children , and heme-oxygenase 1 (HO-1), measures of RBC lysis and oxidative response, than community children or children with severe malaria but without SMA , we still observed increased levels of XO and anti-PS in this group compared to controls and children with severe malaria without SMA . Moreover, this model was not significantly better than an additive model where the interaction was not present . Similar results were observed with a model based on XO and IC . Overall, these findings indicate that XO, anti-PS and IC levels are independently associated with severe anemia during malaria.To further investigate the relation of the measured parameters with severe anemia, a logit model based on XO levels, anti-PS levels, and an XO:anti-PS interaction term showed that the latter did not significantly contribute to the SMA outcome  and creatinine Fig. , suggestWe also determined whether the measured parameters were related to specific malaria complications and clinical definitions of disease severity on admission Table . In thisWe observed that XO levels were significantly higher in severe malaria patients with acute kidney injury, prostration, acidosis or jaundice, indicating a relation of this enzyme to these severe manifestations of malaria. However, XO levels were not associated with multiple seizures or with the most severe neurological complication, coma . IC were also significantly elevated in children with jaundice, indicating a relation of IC to this complication.p < 0.0001 for both parameters), but still showed significant correlations with the levels at the time of admission , as well as with related complications such as jaundice.Since the oxidative enzyme XO is frequently elevated in malaria patients and correlates with disease severity8, and ultimately leading to their clearance in the spleen7. This hypothesis is supported by previous patient-based studies showing that, in the context of P. falciparum infections, RBCs rigidity was mostly observed in uninfected RBCs and correlated strongly with severe anemia34 and death35. Some potential factors that contribute to the oxidative burden during malaria include elevated levels of oxidative enzymes, free heme released after the lysis of infected and uninfected RBCs, and the phagocytic oxidative burst that is induced after phagocytosis of infected RBCs by innate immune cells13. It is still unclear what is the relative contribution of these factors to the high levels of oxidative stress that are observed in malaria patients, but XO is probably an important contributor, since this enzyme degrades hypoxanthine and xanthine in plasma, releasing high levels of reactive oxygen species. XO is elevated in the blood of malaria patients, correlating with disease severity18 and with inflammatory cytokines36. Our results further support that XO may be an important source of oxidative stress in malaria given its association with severe anemia, hemolysis and acidosis parameters.Oxidative stress has been proposed as a contributor to SMA, since it causes peroxidation of lipids on the surface of erythrocytes, reducing the deformability of these cells17. It is also unlikely that the observed increase in XO levels in patients may be caused by high levels of its substrates hypoxanthine/xanthine in plasma, since the concentration of hypoxanthine in uninfected RBCs that would be released upon lysis is very low (9.3 nM)37 and there is a marked decrease in the levels of hypoxanthine in plasma during P. falciparum infections38, probably caused by the robust transport of hypoxanthine and its precursors into infected RBC39. However, mice studies showed that the enzyme precursor of XO, xanthine dehydrogenase, is upregulated early during Plasmodium infection by the activation of type I IFN receptor40, proposing an immune-related mechanism for the upregulation of XO during malaria. Since hemolysis can induce secretion of type I IFN in liver macrophages41, it is also possible that the correlation between hemolysis and XO levels may be driven by the levels of this cytokine. However, if this was the case, a strong correlation between XO levels and other parameters associated with hemolysis (anti-PS and IC) would have been expected.Since the relation between XO and anemia is associative, it is possible that XO directly contributes to the loss of RBC, but also that elevated levels of XO are a consequence of anemia or other associated factors. A direct relation involving the release of XO by lysed RBC is not likely since XO activity is not found in RBC8. It is possible that XO contributes to this mechanism, but in this study we did not quantify any parameter specifically associated to RBC phagocytosis in patients. However, we observed a significant association between XO levels and hemolysis parameters  that are compatible with the hypothesis that lysis of uninfected RBCs is directly induced by XO, as was observed in mice studies in the context of sickle cell disease42.Oxidative stress has been proposed to induce SMA by causing increased rigidity of these cells that are subsequently recognized by phagocytes and cleared from the circulation1, which increase their susceptibility to phagocytosis9 and their levels are associated with anemia in patients43. Mechanistic studies in a mouse model showed that binding of antibodies to the phospholipid PS, that is exposed in uninfected RBCs during malaria, increases RBCs phagocytosis and delays recovery from anemia20. In patients with P. falciparum and P. vivax infections, levels of anti-PS are associated with anemia23. However, we observed that they also correlate with parameters of hemolysis in patients, as described before22, suggesting they may contribute to SMA through both enhancing phagocytic clearance and hemolysis.Other proposed causes of SMA include the binding of autoimmune antibodies to uninfected RBCs11 and correlate with SMA10. Here we observed that they correlate with parameters of hemolysis and also with anti-PS and anti-DNA, which probably indicates that part of the circulating IC are formed by these autoantibodies. It has been proposed that IC could contribute to anemia by increasing complement susceptibility of RBCs and increasing phagocytosis44. In this cohort, we also observed a correlation between IC and severe anemia, as well as hemolysis associated parameters.IC are also thought to contribute to phagocytosis of uninfected RBCs46. It is also possible that since XO is a highly oxidative and inflammatory enzyme36, the association with complications such as acute kidney injury may be driven by increased inflammation, which is thought to contribute to this complication47.The observed association of XO with different malaria complications points to a broader detrimental effect of this enzyme in severe malaria. It is possible that XO may contribute to complications such as jaundice and acute kidney injury as a consequence of increased hemolysis, since these complications are associated with it48. In mice studies, treatment with the XO inhibitor febuxostat reduced hemolysis induced in the context of sickle cell disease42. Given that elevated levels of XO may contribute to the development of severe anemia, treatment with the XO inhibitors allopurinol or febuxostat49, could contribute to alleviate this complication in malaria. Since febuxostat is not approved for use in children by the US Food and Drug Administration, and has a black box warning for adults, allopurinol is likely to be the preferred drug for studies in animals to evaluate prior to human use for malaria.The novel finding of an association between XO and different severe malaria complications may open a possibility for adjunctive treatment, provided that a causative effect of XO in malarial anemia could be inequivocally demonstrated. Inexpensive and well-tolerated inhibitors of XO have been developed for the treatment of gout, since this condition is caused by accumulation of urate, the product of XOSupplementary Information.


Plasmodium infection and the associated factors among communities of the selected districts in the Kaffa zone.  Malaria remains a serious public health problem, particularly in resource scarce areas of the world. The number of malaria cases has dropped remarkably in Ethiopia over the last decade, and efforts to eliminate the disease are underway. Asymptomatic infections may pose significant challenges to the elimination program. The essence of this study was to assess the prevalence of asymptomatic  April to May and September to October 2021, were the two seasons in which the community-based cross-sectional survey was conducted. Capillary blood from a finger prick was examined by light microscopy (LM) and screened using rapid diagnostic tests (RDTs). The participants' sociodemographic characteristics and malaria prevention measures were collected using a pretested semistructured questionnaire. Data entry and analyses were carried out using EpiData and SPSS version 25.0. Logistic regression (bivariate and multivariable) analyses were carried out to assess the possible associations between the dependent variable and the associated factors. Plasmodium species tested by both LM and RDT. Last night's use of long-lasting insecticidal net (LLIN) , presence of eave , and house sprayed in the last year  were among factors that showed significant association with asymptomatic Plasmodium infection.  566 study participants were involved in the two cross-sectional surveys, including 234 male and 332 female subjects with a mean age of 18.486 (SD ± 15.167). Thirty-eight blood samples (6.7%) were found to be positive for Plasmodium infection prevalence recorded in the study area was low. Last night's LLIN usage, the presence of an eave, a house sprayed in the last year, and the presence of stagnant water near the home of the study participants were among the factors associated with an increased risk of catching the disease. The asymptomatic  Many countries in the world are still suffering from malaria, the deadliest and most common protozoan infection in humans. 627,000 malaria deaths were reported in 2021, which is also evidenced by an increase of the cases from the previous two years, 2019 and 2020, which accounts for approximately 95% of cases, with WHO African region countries accounting for most of this increase .P. falciparum and P. vivax [Plasmodium parasites leads to partial immunity and, consequently, creates asymptomatic carriers [Malaria transmission in Ethiopia has a dynamic pattern, with unstable and seasonal transmission occurring in the majority of the country's transmission areas. Perennial transmission occurs in the country's western lowlands, where the climate is conducive to malaria transmission all year . AltitudP. vivax . In highcarriers . When trcarriers –9. The fcarriers . Asymptocarriers . Light mcarriers . Individcarriers .As vector control interventions, including IRS and LLINs, have been widely used in the last decade for the control of malaria, the burden of malaria has significantly declined in many parts of Ethiopia. These vector control interventions have likely played a vital role in the control of malaria , 13. DruPlasmodium infection is impeding Ethiopia's efforts to eradicate malaria by 2030. As a result, it is crucial to ascertain the disease prevalence in the study area. That could aid in lowering the local parasite reservoir and in developing methods for effective control and, eventually, elimination.Asymptomatic This study was conducted from April to May and September to October 2021 in selected districts of the Kaffa zone (Decha and Gimbo), which are among the 13 districts in the Kaffa zone. These districts were known for their history of having high malaria transmission. The research was carried out 451 kilometers from Addis Ababa, the country's capital city. According to the CSA's 2007 Census, these districts have a total population of 128,887, of which 64,438 are men and 64,449 are women (Decha), and 89,892, of which 44,774 are men and 45,118 are women (Gimbo).A community-based cross-sectional study was employed.All residents in the selected districts  of the Kaffa zone.All individuals within the selected households who took part in the study.All residents aged 6 months and older, who did not present any malaria-related symptoms/Apparently healthy individuals  were taken as eligible to participate in the study.Participants who were not able to respond to the questionnaire and were unable to provide a blood sample for different reasons. Individuals who did not consent to participation, as well as those who were taking antimalarial medication two weeks before data collection, were excluded.n = the minimum sample size required for the study. Zα/2 = is the standard normal variable at a 95% confidence level (1.96).p = proportion of the population that has asymptomatic malaria infection was taken from a study in Northwest Ethiopia (6.7%) [d = margin of error (0.03).The sample size was calculated using the formula for a single population proportion.a (6.7%)  d = margAdding a 10% nonresponse rate, the final sample size was 293.7 ≈ 294.588.In the two-round surveys, the total sample size was taken to be K” of 3, but the first household was selected randomly. Finally, all individuals in a household that was available during the survey and fulfilled the inclusion criteria were included. After obtaining written informed consent from parents/guardians, all members of the randomly selected households were asked to give a blood sample.Of the two selected districts, Decha with 43 villages and Gimbo with 35 villages, six villages, namely  villages from Decha district and  villages from Gimbo district, were selected purposely due to their previous history of being severely affected by malaria. The sampling frame of households was obtained from the health posts of the selected villages. To recruit the desired sample size, a systematic random sampling method was used with an interval of “Sociodemographic information and data on risk factors for malaria were collected using a pretested semistructured questionnaire. The questionnaire contained information about the study participants' sociodemographic characteristics (age and gender), housing conditions, indoor residual spray, the presence of stagnant water near the study participants' homes, bed net usage, and bed net availability for sharing the living house with domestic animals. The questionnaire was first prepared in English and then translated into the local language, which is spoken by the residents of the villages or “kebeles.” The data were collected by laboratory professionals and health extension workers.μl) of blood sample and 2 drops (60 μl) of buffer solution were used for the RDT malaria screening following the manufacturer's instruction, whereas, 6 μl and 2 μl of blood sample were taken for thick and thin blood film preparation.Finger prick samples were collected from consenting individuals for blood film preparation for microscopic examination and multispecies malaria rapid diagnostic tests. Five microliters  kit to confirm the presence of pregnancy.P.f/P.v POCT test kits  were used and was based on lateral flow immunochromatography in cassette format, which can detect P. falciparum histidine-rich protein II (PfHRP-II) and other Plasmodium species  . The test was carried out on the field, and the result was read within 20 minutes, according to the manufacturer's instructions.In the study, the RDT CareStart™ combo test , and “not sufficient” when the ratio is less than 0.5 .By discussing the tools before data collection, the data were checked for completeness and consistency. Clean and grease-free slides were used for blood film preparation to avoid scratches on the slides. To reduce the effect of tissue fluids, the first drop of blood was swiped away. All CareStart malaria test kits were labeled with participant identification numbers specifically given for this purpose, and the test was carried out according to the manufacturer's instructions. In all cases, the RDTs test results were determined before the microscopic results, with the microscopic examination of the thick and thin blood smears strictly blinded. Discordant slides between the microscopic readings were reanalyzed for the third time by the most experienced laboratory technician from the MTU laboratory. All laboratory procedures were conducted based on the standard operating procedures. The laboratory technicians who participated in the blood film examination were blinded to the RDTs results. At least 5% of the negative slides and all the positive slides were randomly selected for internal quality control to be checked by another laboratory technician.P value less than 0.05 was taken as statistical significance.Data were entered into EpiData version 3.1 software  and exported to SPSS version 25  for analysis. Descriptive statistics were utilized to summarize the demographic profile of the study participants. To determine the relationship between independent variables and the outcome/dependent variable, both bivariate and multivariable logistic regression analyses were employed. A More than half of the participants were females, with seven of them pregnant, and nearly two-thirds were illiterate, with nearly one-fourth being farmers. Only one-fourth of them had traveled before the start of the survey, and more than two-thirds had at least five family members .Plasmodium falciparum was the cause of seven of these cases. In the second survey, which was conducted during a major transmission season involving 281 study subjects, 27 study participants were found to be positive for Plasmodium species. In this survey, thirteen positive cases were identified to be Plasmodium falciparum, while eleven cases were accounted for Plasmodium vivax, and the remaining three cases were mixed infections. Sixty-four (11.3%) of the study participants reported that they had experienced malaria in the last year. Nearly one-third of the households that were included in the surveys had at least one long-lasting insecticidal net (LLIN), and 36.6% of them used it the previous night before the data collection. Multivariable analysis revealed that last night's LLIN usage , presence of eave , house sprayed in the last one year , and presence of stagnant water nearby the home of the study participants  showed significant association with asymptomatic Plasmodium infections  because it was low in the investigation.Of the total blood samples collected from the study participants, Plasmodium-positive cases were found in combined tests performed by both light microscopy and rapid diagnostic tests., of which seven (1.2%) were found to be gametocyte stages of Plasmodium falciparum, while three more malaria cases  were found upon screening by RDTs but not by LM  ot by LM .Plasmodium infection was observed in the first survey (eleven cases), whereas higher number of cases (twenty-seven) were recorded in the second survey, which was conducted during the high malaria transmission season in the study areas. Malaria prevalence was higher among female participants and those whose ages are above 15 years. It is revealed that the prevalence of asymptomatic Plasmodium infection found in the study was low. However, the prevalence and burden of the disease could even worsen given that ongoing malaria elimination efforts were not in place in the country, as those cases could be reservoirs of infection for the communities of the study areas. It should also be noted that the Plasmodium species were detected using microscopy and RDTs, both of which are less sensitive than molecular methods [Plasmodium infection could be higher than the recorded one in the study area. A study carried out in Jimma town also reported a low prevalence of asymptomatic malaria infection [This study was conducted in districts of the Kaffa zone from April to May and September to October 2021, the two seasons which are known to favor malaria transmission across the country. Five hundred sixty-six study participants were involved in the study with all of them living in houses made of iron sheets, of which more than half were sprayed with insecticide chemicals in the last year before the survey. A high number of the study participants reported that they share a common house with their domestic animals, and nearly two-thirds of them were illiterate. Regarding LLIN coverage, nearly one-third of the study participants reported that they have sufficient bed net coverage in their home, and the level of its usage was also below half. A low prevalence of asymptomatic  methods . TherefoPlasmodium infection prevalence discovered in this study by microscopy and RDTs compared to other similar studies could be due to factors such as storage temperature, particularly during transportation from the point of manufacture or supply to the final point of use, low sensitivity at low parasite densities, as well as exposure to adverse environmental conditions during distribution [Plasmodium infection, while educational status (being literate) seems to play a protective role against malaria infection. The study revealed almost one-third of households owned at least one-bed net, and only one-third of them used the LLIN the previous night before the survey; this calls for interventions on human behavior to effectively utilize the LLIN for malaria prevention. Much higher use of LLIN (68.3%) was reported in a study from the Limmu Seka District of Jimma Zone, Ethiopia [The low asymptomatic ribution . When paribution , both miribution . Anotherribution . Moreoveribution . The perribution . But in Ethiopia .Plasmodium infection was 6.7% (38/566), of which sixteen malaria cases were detected by light microscopy (LM) and twenty-two cases were identified by rapid diagnostic tests (RDTs), which is lower compared to studies from other parts of the worlds: Ghana  [Plasmodium infection prevalence might be due to the differences in geographical location, i.e., the climate conditions , which contribute a lot to vector abundance in the study areas. Improving the use of LLINs is critically required as high coverage and use has a herd effect whereby a larger proportion of individuals utilizing the LLINs could be protected from malaria infections [The overall prevalence of asymptomatic , 31.2%) , Ethiopi, 31.2%)  by LM an, 31.2%) , and  , Southwe, 31.2%) , and a t, 31.2%)  by both fections .This could have far-reaching implications for efforts to control and eliminate malaria. Therefore, it is essential to put in place effective treatment protocols that can completely remove the gametocyte stages from the bloodstream of asymptomatic carriers of the malarial parasite, which is mainly taken by mosquitoes and responsible for the sustained transmission of the disease.Plasmodium infection by both diagnostic methods (light microscopy and RDTs). In both surveys, the study participants' LLINs coverage and LLINs usage were found to be low. Last night's LLIN usage, the presence of an eave, house sprayed in the last year, and presence of stagnant water nearby the home was among the associated factors that showed significant association with asymptomatic Plasmodium infection.This study showed a low prevalence of asymptomatic Although polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) offers a more accurate assessment of the magnitude of asymptomatic infections; quality-assured microscopy is still officially considered the gold standard by the WHO, despite its limited performance compared to the above-advanced techniques. Moreover, surveillance that covers wide areas of the country is needed to detect all the hidden malaria cases, and on top of that mass treatment of the community (those reservoirs) in the study area is crucial.


Schoolchildren with asymptomatic malaria infections often go undiagnosed and untreated, serving as reservoirs for infection that hamper malaria control and elimination efforts. In this context, little is known about the magnitude of asymptomatic malaria infections in apparently healthy schoolchildren in Ethiopia. This study was aimed at determining the prevalence of asymptomatic malaria infection and its associated factors in apparently healthy schoolchildren in Ethiopia.From September 2021 to January 2022, a school-based cross-sectional study was conducted on 994 apparently healthy schoolchildren (aged 6–15 years) selected from 21 primary schools in the Gomma district, of Jimma zone, southwestern Oromia, Ethiopia. A multi-stage sampling technique was used to select schools and participants. After allocating the total sample proportionally to each school and then to each grade, participants were selected using the lottery method from a list of student records (rosters). Finger-pricked blood samples were collected for microscopy blood film preparation and malaria rapid diagnostic test (RDT) . Moreover, dry blood spots (DBSs) were prepared onto filter papers for quantitative real time polymerase chain reaction (qPCR) analysis.Plasmodium falciparum, Plasmodium vivax and mixed infections accounted for 90%, 6% and 4%, respectively. Submicroscopic asymptomatic malaria infection was also accounted for 70% (35/50) of the overall prevalence. Household head age, nighttime outdoor activities of household heads, family history of malaria, absence of insecticide-treated nets (ITN), and presence of stagnant water around the houses are all significantly associated with asymptomatic malaria infections among schoolchildren.As determined by RDT and microscopy, the prevalence of asymptomatic malaria was 2.20% and 1.51%, respectively. Using qPCR, the overall prevalence was 5.03% (50/994). Of this, This study found that both RDT and microscopy underestimated the prevalence of asymptomatic malaria in schoolchildren. However, qPCR was able to detect even low levels of parasitaemia and revealed a higher prevalence of asymptomatic submicroscopic malaria infections. The findings imply that schoolchildren with asymptomatic malaria infection are potential hotspot for malaria reservoir that fuels ongoing transmission. Therefore, it is imperative to include schoolchildren and schools in malaria intervention package and equally important is the adoption of more advanced and sensitive diagnostic tools, which would be crucial for successful malaria control and elimination efforts. Targeted interventions for asymptomatic malaria-infected schoolchildren can provide invaluable support to the National Malaria Control Programme in controlling and eventually eliminating the disease.The online version contains supplementary material available at 10.1186/s12936-023-04736-7. Plasmodium parasites , which are transmitted into the human body through the bite of female Anopheles mosquitoes, which seek a blood meal to sustain themselves  and mixed infections [5.70% (2/35)]. Although the RDT detected seven-fold more cases than microscopy, it also missed 56% (28/50) of infection reservoirs detected by qPCR. Generally, both the conventional malaria diagnostic tools missed a high number of infection reservoirs, which might jeopardize malaria elimination efforts.In this study, the proportion of cases missed by microscopy is translated to asymptomatic submicroscopic malaria infection, which is obtained by subtracting the microscopic prevalence from the qPCR prevalence 50–15 = 35). This means that 35 children went un diagnosis and left un treatment. ]. Also, children whose HHHs were 55 years or older were five times more likely to be infected with malaria than children whose HHHs were younger than 35 years [AOR = 4.98 ]. In the same vein, children whose HHHs engaged in nighttime outdoor activities had a 3.27 [AOR = 3.27 ] times greater risk of developing asymptomatic malaria compared to whose heads of households did not engage in such activities. What stands out in the Table Asymptomatic malaria infections pose a hidden threat to malaria elimination efforts. This is because large segments of the population, asymptomatic schoolchildren with malaria, are sources of persistent infection and contribute to continued transmission , 34, 41.P. falciparum (90%) followed by P. vivax (6%), and mixed infections (4%). The prevalence of falciparum malaria infection was 1.51% using RDT and 2.20% using microscopy. This is consistent with other reports [P. falciparum infections account for the vast majority of infections in sub-Saharan countries. Importantly, 70% of the schoolchildren in this study were asymptomatic submicroscopic malaria reservoirs. The majority of infections (85.70%) were caused by P. falciparum, followed by P. vivax (8.60%) and mixed infections (5.70%), highlighting that microscopic asymptomatic infection is only the tip of the iceberg, consistent with previous findings in Ethiopia [. [In this study, the overall prevalence of asymptomatic malaria infection was 5.03%, as determined by qPCR. The majority of infections were caused by  reports , 25, accEthiopia . The resiopia [. , that loiopia [. , the appWhitttaker et al.  also conP. falciparum infection demonstrates that it can remain in the circulation for months, up to 200 days [Plasmodium parasite life cycle, particularly P. vivax, in the human spleen [P. vivax and 93.1% of P. falciparum hidden biomasses were found in the human spleen rather than in the circulation. It is therefore safe to conclude that malaria parasites escape detection by conventional methods by circumventing circulation in chronically asymptomatic carriers and evolving a novel survival strategy. Additionally, Andrade et al. [P. falciparum circulating time is linked to increased splenic clearance in asymptomatic carriers during the dry season, and maintains parasitaemia below clinical and immunological thresholds. A similar study in Uganda reported that in areas where malaria is effectively controlled, asymptomatic school-age children were sources of persistent infections [In broad sense, the current study showed that males had a higher prevalence of asymptomatic malaria than females, which is consistent with previous research from Ghana . Despite200 days . The fin200 days . Because200 days –53. Anot200 days , 55 dealn spleen , 57. Thee et al.  showed tfections ..[The microscopic result of this study is lower than reports from northern Ethiopia , 22, 58,. on schooFurthermore, the present study identified several risk factors for asymptomatic malaria infection in schoolchildren, which may help malaria control programmes focus on targeted interventions. Proceeding sequentially, anemic schoolchildren were at a significantly higher risk of having asymptomatic malaria infection, which is consistent with reports from previous studies , 70, 71.The current study revealed that apparently healthy schoolchildren were potential hotspot malaria infection reservoirs. The study also showed that infection was more common in male children than females and that both RDT and microscopy missed a significant portion of infection which tested positive with qPCR. The study also uncovers a multitude of factors associated with increased risks of apparently healthy schoolchildren to asymptomatic malaria. Hence, it was revealed that anaemia in children, the age of household heads, their nighttime outdoor activities, and the presence of stagnant water around housed all play pivotal roles in this phenomenon. Moreover, the study highlights the significance of a family history of malaria and the absence of ITNs as crucial contributors to this alarming issue. By shedding light on these interconnected factors, this study provides invaluable insights into the intricate web of connections associated with the increased risks of asymptomatic malaria in schoolchildren.Inclusion of schoolchildren and schools in the malaria intervention package would be the most effective strategy for the success of malaria control and elimination programme. Given that asymptomatic malaria infected schoolchildren are the source of persistent infections, targeted intervention in conjunction with identified risk factors provides an additional opportunity to strengthen the National Malaria Control Programme. Hence, to ensure the elimination goal, such new interventions are strongly recommended to support and improve existing measures and it has the potential to provide both immediate and long-term benefits to schoolchildren. The authors also strongly recommend using molecular tools to screen for low-density parasitaemias in the malaria elimination programme.Additional file 1: Table A. Schools with number of asymptomatic malaria positive students in Gomma district, Jimma zone, Southwestern Oromia; from September 2021 to January 2022, Ethiopia. Table B. Study schools with their respective numbers of students participated in the study in Gomma district, Jimma zone, Southwestern Oromia; from September 2021 to January 2022, Ethiopia.


Anopheles mosquito injects MPs into its hosts during a blood meal, and MPs invade the host skin and the hepatocytes without causing any serious symptoms. Symptomatic infections occur only during the erythrocytic stage. In most cases, the host’s innate immunity  and adaptive immunity  mount severe attacks and destroy most MPs. It is increasingly understood that MPs have developed several mechanisms to escape from the host’s immune destruction. This review presents recent knowledge on how the host’s immune system destroys invading MPs as well as MPs survival or host immune evasion mechanisms. On the invasion of host cells, MPs release molecules that bind to cell surface receptors to reprogram the host in a way to lose the capacity to destroy them. MPs also hide from the host immune cells by inducing the clustering of both infected and uninfected erythrocytes (rosettes), as well as inducing endothelial activation. We hope this review will inspire more research to provide a complete understanding of malaria biology and promote interventions to eradicate the notorious disease.Malaria continues to cause untold hardship to inhabitants of malaria-endemic regions, causing significant morbidity and mortality that severely impact global health and the economy. Considering the complex life cycle of malaria parasites (MPs) and malaria biology, continued research efforts are ongoing to improve our understanding of the pathogenesis of the diseases. Female  Anopheles mosquito, leading to the injection of malaria parasites . Among the species of Plasmodium that infect humans, P. falciparum is the most notorious, followed by P. vivax, with weaker contribution by P. ovale, P. malarae, and P. knowlesi  migrate into the human host’s liver and infect the hepatocytes. They develop into schizonts and release many merozoites into the bloodstream to infect erythrocytes. Most of the pathological effects of malaria infection are accrued to the erythrocytic life cycle stage , leading to the clearance of the invading MPs in most cases  molecules  produce pro‐inflammatory responses such as upregulated expression of interleukin (IL)-12, interferon-gamma (IFN-γ), and inducible nitric oxide synthase (iNOS)  , on the other hand, attack pathogens and/or pathogen-infected cells through secretion of cytokines   , skin damage inflicted by a probing bite of a mosquito (even a sterile one) stimulates the degranulation of mast cells, leading to recruitments of immune cells to the dermis and epidermis  or vaccinated with sporozoite-based vaccines, IgG1 and IgG3  dominate the antibody-mediated responses  through its melanoma differentiation-associated gene 5 (MDA5) [an example of cytoplasmic pattern recognition receptor (PRR)], which triggers mitochondrial antiviral signaling protein (MAVS) [another PRR]. MAVS then triggers the indispensable interferon regulatory factors, IRF3 and IRF7, to activate transcription and expression of IFN-α and IFN-β (type I IFNs). These two IFNs then bind to IFNAR (IFN-α/β receptors) on hepatocytes and on leukocytes to activate interferon-stimulated genes (ISGs) , and IRF3/7 , and hepatocytes .Many cells in the liver express the INFAR. Still, only hepatocytes seem indispensable for recognizing pathogens as the specific deletion of INFAR on hepatocytes, but not the specific deletion on macrophages and neutrophils, hampered the induction of ISGs  mice , especially in unvaccinated malaria-naïve individuals, the sporozoites can still escape recognition and destruction by the liver-stage immune response, grow, and reproduce to form merozoites-containing schizonts Fig. . The rupPlasmodium parasites as well as their transmission vector . Mosquito infections with P. falciparum involve cascades of events immediately after a blood meal (via mosquito bites) containing the Plasmodium gametocytes  is becoming popular in recent studies and is known for its activity in promoting the evasion of mosquito complement-like response. This protein was reported to be expressed on the surface of both ookinete and sporozoites  , which then fosters the melanization of matured oocysts, upregulated expression of hemocyte TEP1 and a corresponding distorted release of sporozoites  was reported as another important protein that fosters the evasion of mosquito immunity by P. falciparum or P. vivax has myriad symptoms as the parasite gains entries and infects the erythrocytes   RBCs. Studies have identified the merozoite surface proteins (MSP-1) and other erythrocyte binding-like (EBL) proteins to facilitate the parasite attachment to the surface membrane of erythrocytes through a protruded GPI anchor , Var, Rifin, and Stevor multigene families, also facilitate the formation of rosette clusters and have been implicated in other pathological challenges such as malaria anemia, placental malaria in pregnant women, and cerebral anemia  secrete extracellular vesicles (EVs) to foster their survival and infectivity , having a band at 175 kDa on the SDS-PAGE, to bind to the glycophorin A (a glycoprotein found on human erythrocytes) , resulting in immunosuppression, thereby enhancing the infection , among which miR-16-5p, miR-15a-5p, and miR-181c-5p foster lymphocyte apoptosis. This process, therefore, promotes a survival advantage for the parasites.It was also recently discovered that microRNA (miRNA) plays a crucial role in fostering immunological evasion by malaria parasites  have been reported to be present on the surface of parasites (predominantly from Africa) and responsible for fostering the transmission of the parasite from mosquito to human after a blood meal (mosquito bites). Inhibition of PIMMS43 by complete gene knockdown and blocking by antibodies inhibited malaria parasites –A and its receptor vascular endothelial growth factor receptor 2 (VEGFR2), c-reactive protein (CRP), platelet factor–4, intercellular adhesion molecule (ICAM)–1, and von Willebrand factor (vWF), endothelial protein C receptor (EPCR), leading to their sequestration into tissues to cause tissue damage while escaping from splenic clearance (Furuta et al. This review provided an exciting discussion of current knowledge on how the immune system mounts an attack on MPs upon invasion of the skin, live, and erythrocytes, as well as the cellular and molecular mechanisms via which MPs escape from the wrath of the host immune system. Despite the array of publications on malaria biology, curbing the burden of the disease has remained a big challenge, warranting more research efforts towards improving our understanding of other pathways yet to be unraveled through which parasites evade the host immune system. A competent and effective malaria vaccine remains the major hope for eradicating malaria or at least reducing the burden to the barest minimum. However, discovering a clinically effective malaria vaccine is highly dependent on a comprehensive understanding of malaria biology. However, boosting individuals’ immune systems in malaria-endemic regions, as suggested previously (Okagu et al.


Here, we demonstrate the challenges of malaria diagnosis, disease course, and treatment, including post-artesunate hemolysis in a Syrian refugee with severe  Plasmodium falciparum, the pathogen causing malaria tropica, remains one of the most potent and deadliest of parasites known to humankind, causing an estimated 619,000 in 2021 [P. falciparum predominantly occurs in the (sub)tropical zones of sub-Saharan Africa, Asia, and Latin America.plasmodia infecting humans, need to develop within blood-feeding insects from gametes to sporozoites over ten to 18 days before becoming infectious and being injected into the human blood stream. Plasmodia subsequently infect human liver cells (schizonts), followed by erythrocytes (trophozoites), causing both hepatocellular damage and sequestration of mature-staged red blood cells. Severe malaria is thus facilitated by microcirculatory congestion and organ failure [plasmodia, from the initial presentation with (mild) periodic fever to signs of cerebral malaria and full-scale multi-organ failure.To complete their life cycle, any of the known  failure . The incplasmodia are only transmitted through the bite of an infected endemic female Anopheles mosquito [P. falciparum malaria are thus exceedingly rare in both continental Europe and North America. Cases with symptomatic malaria are mainly imported through travelers and immigration. Autochthonous P. falciparum transmission has sporadically been witnessed through ‘backpack mosquitoes’, in the vicinity of airports, or even in hospitals [P. vivax was considered eradicated in Europe, but has since been re-introduced to Greece via imported cases [In humans, mosquito . Patientospitals , 5. Idened cases .plasmodium infections is dangerously limited within colder regions and hospital personnel without access to infectious disease specialists.As transmissions seldomly occur outside of the tropical zones, daily experience with diagnostics, disease course, and treatment options in Fueled by rising temperatures and large-scale refugee crises, incidence rates of infectious diseases will increase. It is thus highly necessary to report on and reshape awareness for  infectious disease, preventing misdiagnosis -and treatment.Here, we report on the case of a 38-year-old female, who was transferred from an outside hospital to our intensive care unit (ICU) with severe malaria. Four days prior she had developed fatigue, jaundice, headaches, and exertional dyspnea. Before admission, the patient reported subjective fever, but the temperature was not elevated in the emergency department. Chest X-rays did not demonstrate pathologies and PCRs for respiratory viruses, such as influenza or SARS-CoV-2 remained negative.Upon transfer to our service, laboratory parameters demonstrated hypoglycemia, severe hemolysis and thrombopenia as well as acute hepatopathy with a total serum bilirubin of 8.2 mg/dL , conjugated bilirubin of 6.7 mg/dL , and an international normalized ratio of 1.32. Serum alanine aminotransferase (ALT) was 102 U/L (reference < 35 U/L) and aspartate aminotransferase (AST) was 76 U/L (reference < 35 U/L). Additionally, the patient developed elevated inflammatory markers with a C-reactive protein (CRP) of 154 mg/L (reference < 5 mg/dL) and procalcitonin (PCT) of 4.7 ng/mL (reference < 0.5 ng/mL). No leukocytosis was present, and hemoglobin at presentation was 16 g/dl (reference 11.2–15.7 g/dL) and decreased to 13 g/dl within 16 h, with lactate dehydrogenase (LDH) initially at 471 U/L (reference < 250 U/L). Renal function remained within the normal range.P. falciparum being highly positive [During a microscopic examination of a routine blood smear, performed to rule out hematologic malignancies as the cause, the red blood cells (RBCs) showed a significant number of ring-shaped early trophozoites Fig. . The thipositive . ParasitThe patient was transferred to our university hospital with early signs of cerebral malaria, such as headaches, somnolence, and a high fever and thus directly admitted to our intensive care unit. Within hours she developed severe hypotension requiring vasopressor therapy. The initial regimen of artemether/lumefantrine was escalated to intravenous artesunate (IVAS) when we received further results of the thin smear with a parasitemia of 13% (130 per 1000 RBC) , an uncommon but in many cases self-limiting adverse event in IVAS treatment that mainly occurs within 14 days of intravenous administration [Ten days after receiving artesunate treatment, the patient experienced significant hemolysis, which became increasingly severe. The level of LDH peaked at 2013 U/L on the 15th day post-treatment. Hemoglobin decreased to 5.9 g/dL and the patient needed transfusions of erythrocyte concentrates. Blood smears remained negative for In this case, hemolysis was self-limiting; the patient showed significant improvement and was discharged 25 days after the onset of symptoms.The Syrian patient arrived in Germany only ten days prior to the presentation. She had fled from northern Syria on foot to a rural village in Türkiye where she remained for several days. From there she went to Istanbul where she was picked up by traffickers. The patient and other migrants, possibly from malaria-endemic countries, were loaded into the back of a van without windows and driven through Europe. The trip took four days and involved one transfer into another van. Upon arrival in Germany, all migrants were instructed to delete their phone records and were provided with train tickets to a destination of their choice within Germany.P. falciparum is not endemic to Northern Syria, Türkiye, or the Balkans. Due to the short time span of incubation for P. falciparum, a transmission prior to departure from Syria seems highly unlikely [P. vivax [P. falciparum in this patient might have occurred during the transportation to Europe, possibly through an infected mosquito hidden in the bags of another migrant from a P. falciparum-endemic country, travelling in the same van. As all migrants were instructed to delete their phone records and were heading to different locations within Germany, tracking and treating other infected individuals proved impossible, thus highlighting that especially refugees are vulnerable to a delayed diagnosis. A global movement of people and goods in combination with global warming poses a risk for (re)introduction of new or locally extinct diseases, as demonstrated by the autochthonous malaria transmission in Greece, starting in 2009, as well as Italy in 2017 [unlikely . Until tP. vivax . The pat in 2017 , 6, 12.  in 2017 .Apart from highlighting the unexpected circumstances of transmission within the challenges of the modern world, this case stresses several notable aspects of clinical management of (a)typical falciparum malaria: The necessity of quick differential diagnoses to different and unexpected forms of hemolysis and hepatopathy, the management of high parasitemia with ACT and IVAS, as well as the severity of post-artesunate delayed hemolysis.Risk factors for PADH include non-immune travel status, high initial parasitemia, and cumulative IVAS dose of > 9.5 mg/kg, of which only the parasitemia applied to our patient . SomewhaPatients treated with IVAS require routine laboratory checks for four to six weeks following treatment. Optimally, monitoring should include hemoglobin, reticulocyte count and production index (RPI), lactate dehydrogenase, haptoglobin, and total bilirubin.Globally, the debate on IVAS treatment and its clinical consequences is of high relevance. After isolation in China in the mid-1970s, intravenous artesunate has only been approved for use by the U.S. Food and Drug Administration (FDA) in May 2020, ending a period without FDA-approved intravenous malaria medication since the discontinuation of quinine in 2019. In Europe, the European Medicines Agency (EMA) approved artesunate in November 2021 for marketing with additional monitoring . KnowledP. falciparum malaria, which could have been easily missed, and could have been fatal, had the parasitemia not been this high by the time a manual blood smear was done, or no manual blood smear had been ordered.This case impressively demonstrates the necessity for increased awareness towards tropical diseases as a challenge for physicians in an ever more globalized world. Despite the lack of a clear exposure, this patient suffered from