In African children the proportion of pigment containing PMNs helps distinguish severe malaria from other life-threatening febrile illnesses, and it adds to the prognostic assessment from simple bedside examination, and to the conventional malaria parasite count. Microscopy assessment of pigment containing PMNs is simple and rapid, and should be performed in all patients hospitalised with suspected severe malaria.Severe falciparum malaria is a major cause of death in tropical countries, particularly in African children. Rapid and accurate diagnosis and prognostic assessment are critical to clinical management. In 6027 prospectively studied patients diagnosed with severe malaria we assess the prognostic value of peripheral blood film counts of malaria pigment containing polymorphonuclear leukocytes (PMNs) and monocytes. We combine these results with previously published data and show, in an individual patient data meta-analysis ( Malaria diagnosis by microscopy provides valuable rapid diagnostic and prognostic information, which is critical to clinical management. In this work, authors carry out a meta-analysis on the prognostic and diagnostic value of polymorphonuclear leukocytes and monocytes containing malaria pigment in peripheral blood film counts taken from patients with severe malaria. Many factors affecting the prognosis of falciparum malaria have been identified in the past from detailed prospective clinical studies6. In routine clinical practice in malaria-endemic areas the initial assessment of disease severity is made in the clinic or at the bedside from the level of consciousness, vital signs, respiratory pattern and degree of anaemia. If the patient is admitted to the hospital then additional laboratory information of prognostic value may become available, notably acid-base status from plasma bicarbonate, lactate or blood gas, plasma creatinine or blood urea, and blood glucose measurements7. Rapid tests for blood glucose measurement are widely available and point-of-care blood lactate measurement is increasingly available in intensive care units.Severe malaria is a major cause of preventable childhood death in tropical countries, particularly in sub-Saharan Africa. The continuing large death toll justifies the substantial global investment in malaria control, although relatively little attention has been paid to severe malaria in recent years1. Microscopy malaria parasite counts are still often semi-quantitative in reports , and other potentially valuable information is not assessed or reported. In assessing a patient suspected of having severe malaria, microscopy provides important diagnostic and prognostic information obtained from counting the malaria parasites, assessing their stage of development8, and counting the proportions of leukocytes that have ingested malaria pigment11. In many locations only a thick blood film is taken, whereas for counting high-density parasitaemias, staging parasite development and assessing intraleukocytic pigment, the thin blood film is more rapidly obtained, easier to read, and more accurate. Haemozoin, or malaria pigment, is released at schizont rupture and some of this material is phagocytosed immediately by circulating phagocytic cells. As a result the amount of this particulate material in the circulation reflects the extent of earlier schizogony. Both monocytes and polymorphonuclear leukocytes (PMNs) ingest malaria pigment. Monocytes have slower turnover in the circulation than PMNs so in blood films  malaria pigment-containing monocytes persist for longer12. Although there is general agreement that the proportions of PMNs or monocytes containing intraleukocytic malaria pigment are higher in more severe disease, there is disagreement about its prognostic value14, notably whether this rapid microscopy assessment adds to simple clinical variables. A prospective study in 26,296 African children with a suspected diagnosis of severe falciparum malaria from the SMAC network reported that, after adjustment for the main indicators of severity , quantitation of intraleukocytic pigment-containing cell counts did not provide additional prognostic value13. As a result of this continuing uncertainty, intraleukocytic pigment-containing cell counts are generally not performed in routine practice, despite their speed and simplicity.The diagnosis of malaria is made either from microscopy of stained blood slides or, increasingly, from rapid diagnostic blood tests (RDT). Unfortunately RDTs do not quantify parasitaemia and they also do not provide other information of prognostic value. However, even small, poorly equipped hospitals usually have a microscope, although the support for, and thus the quality of microscopy vary substantially16, and the largest randomised trial in African children (AQUAMAT) hospitalised with severe falciparum malaria17. Following a systematic review of the literature, we pooled individual patient data from over 32,000 patients clinically diagnosed with severe falciparum malaria and we assessed the prognostic value for in-hospital mortality of the proportion of pigment-containing PMNs and monocytes. This analysis of data from the majority of all patients with severe falciparum malaria studied prospectively in recent years provides conclusive evidence that assessment of intraleukocytic pigment in PMNs has important prognostic and diagnostic value in addition to that provided by a simple bedside assessment.In this study we present new data on the prognostic and diagnostic value of intraleukocytic pigment in severe malaria from three large randomised clinical trials carried out or coordinated by the Oxford University Clinical Research Unit in Ho Chi Minh City, Vietnam and the Mahidol Oxford Research Unit based in Bangkok, Thailand. These are the largest prospective randomised controlled trials of Asian adults and children with severe falciparum malaria (AQ Vietnam and SEAQUAMAT)μL versus 89% of slides (4283 out of 4823) in patients with parasite densities above 10,000 per μL.Quantitative pigment-containing PMN counts were available for 483 patients from the AQ Vietnam trial; 1333 patients from the SEAQUAMAT trial; and 4211 patients from the AQUAMAT trial. For the pigment-containing monocyte counts the corresponding numbers were 301, 1332, and 4186, respectively, and for parasite counts were 560, 1457, and 4786, respectively  were associated with slightly higher than average mortality in African children . This is most likely explained by mis-classification of severe disease, i.e. misdiagnosing severe  illness and incidental parasitaemia as severe falciparum malaria18. It has been estimated from clinical studies that approximately one third of children in sub-Saharan Africa with an initial diagnosis of severe malaria are mis-classified20. In Asian adults and children, in whom the diagnosis of severe malaria is more specific, mortality rose steeply for parasite counts > 10,000 per μL (p = 10−11 for the spline fit) and there was no apparent increase in mortality at low parasite densities.We characterised the prognostic value of pigment-containing PMNs, pigment-containing monocytes, and standard malaria parasite counts in Asian adults and children and African children by fitting flexible generalised additive logistic models to in-hospital mortality Fig. . The prop = 10−8 and p = 10−16 for the spline fits, respectively). The effect was greatest in the Asian adults and children, in whom the mortality in patients with 20% or more of pigment-containing PMNs was at least three times higher than in those with no pigment-containing PMNs . By contrast pigment containing monocytes had a lower prognostic value, although the quantitative counts were still correlated significantly with in-hospital mortality .The prognostic value of pigment-containing neutrophils (PMNs) was consistent across Asian adults and children, and African children. Higher pigment-containing PMN counts were associated with higher mortality , greater than 5% pigment-containing PMNs was associated with an odds-ratio for death of 2.39 (95% CI 2.03–2.82). Greater than 5% pigment-containing monocytes (n = 5819) was associated with an odds-ratio for death of 1.31 (95% CI 1.10–1.55).In the initial report demonstrating the prognostic value of pigment-containing PMN counts a 5% threshold was proposed for clinical practice23. We were able to obtain individual patient data from two of these studies13. We conducted an individual patient data meta-analysis using the pooled data set . There were no identified issues with data integrity in the pooled studies. A flow diagram for the inclusion and exclusion of the screened studies is given in Supplementary Fig. 20 . The proportions of pigment-containing PMNs and pigment-containing monocytes were both correlated positively with in-hospital mortality, with the greatest mortality increase observed for proportions of pigment-containing PMNs greater than 5% .In our pooled data set, 30,868 African children had recorded parasite densities; 30,222 had recorded proportions of pigment-containing PMNs, and 29,377 had recorded proportions of pigment-containing monocytes. Figure n = 32,035), relative to patients with no pigment-containing PMNs, the greatest increase in mortality was seen in the subgroup of patients with >5% pigment-containing PMNs .There was heterogeneity in the prognostic value of >5% pigment-containing PMNs when analysing adults versus children (>15 years vs ≤15 years). As apparent from Fig. The clinical utility of a prognostic test in severe malaria depends on whether it adds information to simple bedside assessment. With the exception of blood glucose, laboratory tests are not usually available immediately at point of care in the rural tropics -whereas most facilities do have a microscope . We assessed the prognostic value of >5% pigment-containing neutrophils (PMNs) and >5% pigmented monocytes before and after adjustment for readily measured known prognostic clinical and laboratory variables 18. The diagnosis of severe malaria is highly specific for patients with a plasma PfHRP2 concentration ≥1000 ng/ml20. We assessed the diagnostic value of the proportion of pigment-containing PMNs and monocytes by comparing their values with the plasma PfHRP2 concentrations in patients from the AQUAMAT trial for whom both were measured. We compared this with the peripheral parasite density. Plasma PfHRP2 concentrations and proportions of pigment-containing PMNs and monocytes were jointly available for a total of 2933 and 2913 patients from the AQUAMAT trial, respectively. In total 1890 of these patients (64%) had a plasma PfHRP2 concentration above 1000 ng/ml. The proportions of pigment-containing PMNs and pigment containing monocytes were both highly predictive of having a plasma PfHRP2 concentration ≥1000 ng/mL . In patients with ≤5% pigment-containing PMNs, 1306 out of 2178 (60%) had a plasma PfHRP2 ≥1000 ng/mL. In comparison, in patients with >5% pigment-containing PMNs, 583 out of 755 (77%) had a plasma PfHRP2 ≥1000 ng/mL. The corresponding proportions with less than and greater than 5% pigment-containing monocytes were 672 out of 1208 (56%) and 1204 out of 1705 (71%), respectively. In comparison, only parasite counts above 100,000 per μL were predictive of having a plasma PfHRP2 ≥1000 ng/mL  can be done immediately but, importantly, this does not distinguish malaria as the cause of severe illness, from other causes of severe febrile illness (e.g. sepsis) and incidental malaria. The diagnosis of malaria has traditionally been made by microscopy of stained blood films. However today, rapid diagnostic tests (RDTs) are increasingly used to diagnose malaria, even in hospitals, and there is less use of microscopy than in previous years. There are concerns that microscopy proficiency and support are declining in malaria endemic areas1, yet microscopy of thin and thick blood films provides important diagnostic and prognostic information which the current rapid tests do not. Blood slide microscopy gives quantitative information which is not vulnerable to technical errors or to mutations in the parasite target genes . Thin blood films have the advantage over thick films of speed and ease of assessment (see29) and they provide valuable prognostic information. In addition to the malaria parasite count (the proportion of red cells containing one or more parasites), the stage of asexual parasite development can be assessed8 and the proportion of leukocytes containing ingested malaria pigment in the blood slide can be counted9. Polymorphonuclear leukocytes  are more abundant than monocytes, are readily identified, and an assessment of 100 PMNs in the tail of the thin blood film takes only a few minutes (see29). To evaluate the diagnostic and prognostic value of counting the proportion of pigment-containing PMNs this study used prospective assessments from three of the largest randomized controlled trials in severe falciparum malaria, and then combined these within an individual patient data meta-analysis of all the large studies which recorded intraleukocytic pigment data. In total this combined data set comprises the majority of all patients with severe malaria who have been studied prospectively in the past half century. The very large data set shows clearly the prognostic and diagnostic value of counting the proportion of PMNs with intraleukocytic malaria pigment. It is a simple and rapid assessment which provides additional prognostic information above that provided by a bedside examination, even when it is supplemented by other point-of-care tests.Severe falciparum malaria is estimated to kill over 1000 people each day. Most of these preventable deaths are in young children in Africa. Rapid recognition of the disease and early administration of effective treatment are critical determinants of outcome24 with incidental parasitaemia)32. Finding a high proportion of pigment-containing PMNs supports the diagnosis of severe malaria and it identifies those patients in need of intensive care or, if this is unavailable, close monitoring. Prognosis and diagnosis are linked: if malaria is not the cause of severe illness, then potential sources of sepsis need to be investigated and parenteral antibiotics need administering rapidly32. This dilution by a different cause of fatal illness explains why the prognostic value of the intraleukocytic malaria pigment counts in African children is less than in Asian adults and children . But they are much easier to read, they provide more accurate counts in high-density infections, and they allow more accurate staging of parasite development, and counting of pigment-containing leukocytes. Thick films take time to dry, and are more difficult to evaluate accurately because cellular morphology is contracted, and artefacts and stain precipitates may be mistaken for malaria pigment. The speed and simplicity of counting pigment-containing PMNs is particularly valuable in rural settings with limited access to laboratory support, and thus limited capability of providing other prognostic information. Health systems should support maintenance of quality microscopy. New inexpensive cell phone-adapted automated methods of microscopy cell counting are now being developed and evaluated. These may be adaptable to parasite counting, staging and neutrophil pigment assessment but they are still years from being implementable in remote areas34. Where there is a microscope, thin film assessment should be a routine part of the assessment of any patient suspected of having severe falciparum malaria.The utility of counting the proportion of pigment containing PMNs in patients with suspected severe malaria has to be understood in the context of delivering critical care in busy resource-limited hospitals and health centres. In higher transmission settings differentiating severe malaria from other causes of severe childhood febrile illness is difficult, even for experienced clinicians, because the key clinical signs are not specific, and asymptomatic malaria parasitaemia is so common. Thus, many children are diagnosed as having severe malaria, whereas in fact they have another severe illness , and thick blood films rather than thin films were evaluated, reanalysis of the SMAC data shows clear prognostic value of intraleukocytic pigment, consistent with the four other malaria studies , and also by the Oxford Tropical Research Ethics committee (OXTREC). Re-use of existing, appropriately anonymized, human data does not require ethical approval under the Oxford Tropical Research Ethics Committee regulations (OxTREC).15. Patients were included in the study if they (or an accompanying relative) gave informed consent, if the patient had asexual forms of P. falciparum on a peripheral-blood smear, was older than 14 years, was not in the first trimester of pregnancy, was not an intravenous drug user, and had received less than 3g of quinine or two doses of artemisinin or a derivative in the previous 48 hours. The severe malaria entry criteria were modified from the WHO criteria as follows: patients could be included if they had one or more of the following: a Glasgow Coma Scale score of less than 11 ; anaemia (haematocrit <20%) together with a parasite count >100,000/μL; jaundice (serum bilirubin >2.5 mg/dL [50 μmol/L]) together with a parasite count >100,000/μL; renal impairment ; hypoglycaemia (blood glucose < 40 mg/dL [2.2 mmol/L]); hyperparasitaemia (>10% parasitaemia); and systolic blood pressure <80 mm Hg with cool extremities (indicating shock). Intensive clinical and laboratory monitoring was performed by a specialist team in a dedicated ward. This trial provided the basis for the design and conduct of the multi-centre trials conducted in adults and children with severe falciparum malaria in South-East Asia (SEAQUAMAT: mainly adults16), and then in Africa (AQUAMAT: mainly children17). The AQ Vietnam and SEAQUAMAT trials were conducted before pre-registration was standard practice; the AQUAMAT trial was registered on ISRCTN (ISRCTN50258054).This work presents new data from three previously published randomised clinical trials in severe malaria. The three clinical trials were conducted by associated research teams. The AQ Vietnam trial was a very detailed single-centre study in Vietnamese adults with severe falciparum malaria15, and data on the prognostic value of pigment-containing PMN and monocyte counts were published for the first 300 patients9.The AQ Vietnam trial was a double-blind randomised controlled comparison of intramuscular artemether and intramuscular quinine in 560 Vietnamese adults with strictly defined severe falciparum malaria. It was conducted between May 1991 and January 1996 in a specialist ward of the Hospital for Tropical Diseases, Ho Chi Minh City. Malaria blood film microscopy was performed on admission before enrolment. Full details have been published previously16. The participating centres were located in Bangladesh, Myanmar , India, and Indonesia. Patients over two years of age were included in the study if they had a positive RDT for P. falciparum histidine rich protein 2  and, in the admitting physician’s clinical opinion, they had severe malaria, and they or their attendant relative of guardian, gave fully informed written consent. Patients were excluded if there was a convincing history of full treatment with quinine or an artemisinin derivative for more than 24 hours before admission. Full details of the trial have been published previously16.The SEAQUAMAT trial was a multicentre open label randomised comparison of parenteral artesunate and parenteral quinine in 1461 Asian adults and children admitted to hospital with severe falciparum malaria17 (ISRCTN: 50258054). The eleven participating centres in nine countries across Africa were: Hospital Central da Beira, Beira, Mozambique; Royal Victoria Teaching Hospital, Banjul, The Gambia; Komfo Anokye Hospital, Kumasi, Ghana; Kilifi District General Hospital, Kilifi, Kenya; Magunga District Hospital, Korogwe, Tanzania; Teule District Hospital, Muheza, Tanzania; University of Ilorin Teaching Hospital, Ilorin, Nigeria; Mbarara Teaching Hospital, Mbarara, Uganda; Kingasani Health Centre, Kinshasa, DRC; Rwamagana Hospital, Rwamagana, Rwanda and Nyanza Hospital, Nyanza, Rwanda. Children less than 15 years were included in the study if they had a positive RDT for PfLDH  and, in the admitting physician’s clinical opinion, they had severe malaria, and they or their attendant relative or guardian gave fully informed written consent. Thus the inclusion criteria of a clinical diagnosis of severe malaria (guided by the WHO criteria) by the admitting clinician were similar in AQUAMAT and SEAQUAMAT trials. Patients were excluded if there was a convincing history of full treatment with quinine or an artemisinin derivative for more than 24 hours before admission. Overall a third of enrolled patients had cerebral malaria. Full details of the trial have been published previously17.The AQUAMAT trial was a multicentre open-label randomised comparison of parenteral artesunate and parenteral quinine in 5425 African children admitted to hospital with severe falciparum malariaIn the three trials, an initial clinical assessment was recorded and a peripheral thick and thin blood smear was made for quantitative malaria parasite counting. In the AQ Vietnam study, blood was also taken for immediate haematocrit, blood glucose, plasma lactate and blood gas measurements, blood culture, cross match if necessary, and same-day laboratory haematology and biochemistry. Pigment containing leukocytes on the admission blood films were counted later by expert microscopists blinded to the patient outcomes. In the SEAQUAMAT and AQUAMAT trials, blood was taken for immediate haematocrit and biochemical analysis using the EC8+ card in a hand held battery operated internally calibrated biochemical analyser . This provided an immediate hard copy readout with time and date. Parasitised erythrocytes were counted on the Giemsa or reverse Field’s stained thin film and reported per 1000 red cells, or if the count was low or the thin film unavailable, was counted in the thick film (and reported as parasites/200WBC). Parasite counts were checked, and parasite staging and leukocyte-associated pigment was assessed at the reference laboratory in Bangkok. In each study the numbers of leukocytes containing visible malaria pigment were counted per 100 PMNs and per 30 monocytes under oil immersion microscopy at X1000 magnification on thin blood films (or thick films if the thin film was unavailable).29.A video showing how to perform a blood smear (for thick and thin blood films), as well as how to assess slide quality, and count intraleukocytic malaria pigment is provided at 10.6084/m9.figshare.20783683.v16, and searched Google Scholar for publications that cited the original report on the prognostic value of intraleukocytic pigment in severe malaria 9. Studies were screened for eligibility by two reviewers (JAW and KS) and any disagreements were adjudicated by a third (NJW).We searched PubMed and EMBASE for the terms  AND  to find all studies that measured the proportions of either pigment-containing PMNs or pigment-containing monocytes in patients diagnosed with severe falciparum malaria. In addition we reviewed all studies included in a recent systematic review of prognostic factors in African children with severe malariaP. falciparum malaria, we identified 19 relevant studies. The median sample size was 67 with a range of 22 to 26,296. Summary details and findings for each study is provided in the Out of a total of 202 publications identified, after removing duplicates, reviews, case-reports, animal studies, studies in nonsevere disease and non-23. For three of the studies we were unable to obtain the individual patient data: for Boeuf et al.22 the P.I was deceased; for Luty et al.21 the original data could not be found by the authors; for Birhanu et al.23 the contact details were out of date and we failed to contact the corresponding author. The systematic review was registered prospectively on PROSPERO, number CRD42021284527 (https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=284527).We contacted investigators of the 5 studies which had data on at least 100 patients diagnosed with severe malaria11. The proportions of pigment containing PMNs and monocytes were quantitated on thin films by counting 100 PMNs and 30 monocytes, respectively. Microscopists were blinded to clinical presentation and outcome. 20% of patients had coma and 15 patients died (9%).This was a prospective study of severe malaria, designed as a pilot for the larger SMAC network study. 172 patients diagnosed with severe malaria were enrolled in Bandiagara, Mali from July 2000 to December 200133. The evaluated population comprised all parasitaemic children suspected of having severe malaria who were admitted to each of the participating hospitals of the SMAC network. The SMAC network included sites in five African countries: Banjul, The Gambia ; Blantyre, Malawi ; Kumasi, Ghana ; Kilifi, Kenya ; and two sites in Gabon, Lambarene and Libreville, both run by the Medical Research Unit of Albert Schweitzer Hospital. All children aged between 1 month and 15 years of age suspected of having malaria and who were sick enough to be hospitalized were screened with a thick blood smear for P. falciparum parasites. Only a minority of the children in the SMAC study had strictly defined severe malaria2. Clinical and laboratory measures were documented and children were followed during their hospital admission. Pigment-containing mononuclear leukocytes, and pigment-containing granulocytes (PMNs) were counted as described by Lell et al. in stained thick blood smears40. The numbers of pigment-containing monocytes and pigment-containing granulocytes were reported per 200 cells. Between December 2000 and May 2005 26,389 patients were enrolled. Overall 9.3% of enrolled patients had coma (BCS ≤ 2) and 4.4% died. Full details have been published previously13.SMAC was a prospective multicentre network observational study41. We adjusted QUADAS-2 so that it was relevant to the key questions of interest using 5 domain questions . Quality assessment was performed by two reviewers . Any disagreements were resolved by a third reviewer (NJW).The risk of bias and the applicability of the 5 included studies was assessed by applying the 2011 revised version of the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) toolPfHRP2, and the peripheral parasite density are proxy measurements for the parasite biomass of varying accuracy, and thus are expected to have prognostic utility20. Clinical measurements such as coma are best defined as mediators, i.e. the effect of parasite biomass on mortality is due to organ dysfunction (which can be measured to varying degrees of accuracy). Thus the prognostic value of indicators of parasite biomass such as intraleukocytic pigment after adjustment for clinical variables can be thought of as the direct effect (not going via the mediators).The analyses were guided by the simplified causal diagram shown in Supplementary Fig. In all analyses we used in-hospital mortality as the primary endpoint . We used the log-odds scale to estimate effects. Only patients with available and readable slides were included in these analyses.9. In addition, it is an easy to remember threshold and thus appropriate for use in clinical practice.The primary exposure definition was > 5% pigment containing PMNs or > 5% pigment containing monocytes. The 5% threshold was proposed in the first paper noting the prognostic value of PMN and monocyte countsx is expressed as a percentage and λ = 0.5; and for the parasite counts x is the per μL density and λ = 50 . Sensitivity analyses varied these λ parameters. For the proportions of pigment containing PMNs and monocytes, estimated odds-ratios for death are sensitive to the choice of the additive parameter in this Box-Cox transformation. The value of λ = 0.5 was chosen as this resulted in approximately the same odds-ratio when analysing all data under the Dichotomising continuous variables loses key information. We also performed analyses using the continuous count data. For all the microscopy cell counts, there were a substantial number of zero counts, and the distributions were highly right skewed as a consequence. We used a Box-Cox For each included study, the analysis population was all patients who had available and readable slides (no imputation of missing count data).To estimate the relationship between baseline microscopy quantitative cell counts and in-hospital mortality, we fitted univariable generalised additive logistic regression models with mortality as the dependent (outcome) variable, and the baseline counts on the mgcv which implements penalised regression using smooth splines. We chose a spline based regression approach as the association between mortality and baseline quantitative microscopy cell counts could plausibly be non-linear on the logit scale. To avoid over-fitting we set the maximum basis for the smooth spline component to be 4. Random effect terms were included for country of enrolment and study. Independent models were fitted to the data from Asian adults and children (SEAQUAMAT and AQ Vietnam), and African children (AQUAMAT). In addition we fitted models to all available data from African children .The generalised additive logistic regression models were fit using the R package 5.To characterise the prognostic utility of malaria pigment-containing leukocyte count data after adjustment for key clinical variables, we fitted additional prognostic models of death adjusted for known prognostic clinical variables  or known clinical and readily measured laboratory variables lme4.Coma was defined in children as a Blantyre coma score (BCS) ≤2 and in adults as a Glasgow coma score (GCS) ≤10. Suspected acidosis was defined clinically in the SEAQUAMAT and AQUAMAT trials as acidotic breathing and in SMAC as deep breathing (Kussmaul respirations). In the AQ Vietnam study this was not recorded explicitly so we used the proxy variable of respiratory rate. Hypoglycaemia was defined as a blood glucose ≤2.2mmol/L. Lactate was measured in venous plasma for the AQ Vietnam trial and in venous whole blood in the SMAC study. Blood urea nitrogen in SEAQUAMAT and AQUAMAT was measured with the i-stat handheld analyser. The prognostic models were logistic regression models with the binary predictor of >5% pigment-containing PMNs or monocytes and with nested random intercept terms for hospital, country and study, fitted using the R package mgcv) to the transformed count data (see above section on Box-Cox transformations used). Estimation and forest plot visualisation of the meta-analysis was done with the R package meta.To estimate the meta-analytic prognostic value of a pigment containing PMN count or a pigment containing monocyte count ≥5% with respect to in-hospital mortality, we fitted a logistic regression model with nested random effects by hospital, country and study (for SMAC we had data only at the country level except for Gabon). We looked at heterogeneity in effect between adults versus children (<15 years versus >15 years) by adding an interaction term in the random effects logistic regression model. For children we also fitted generalised additive logistic regression model , and thus latent class model fits are highly sensitive to model assumptions because of the within-class correlation. For this reason, to assess diagnostic utility we simply assessed the predictive value of intraleukocytic pigment for the plasma PfHPR2  with random effects for each study site. We used a bootstrap approach to calculate confidence intervals around the fixed-effect predictions in mixed effects models (R package bootpredictlme4).In previous work we assessed the accuracy of the clinical diagnosis of severe malaria in large patient cohorts from Africa and AsiaThis research has included local researchers throughout: in the study design, study implementation, data ownership, and authorship of publications. This research has practical and implementable consequences for the treatment of severe disease in the included areas. All clinical studies were approved by local ethics review committees.Further information on research design is available in the Supplementary MaterialsPeer review fileReporting Summary


Plasmodium zoonotic cycles that involve non-human primates (NHPs) in sylvatic environments. Since vivax malaria is less responsive to control measures, finding Plasmodium vivax infected NHPs adds even more concern.The groundwork for malaria elimination does not currently consider the potential of Plasmodium species.Both Free-living monkeys in forest fragments inside the urban area and captive monkeys from a local zoo had blood samples tested for P. vivax.In this study, among the Neotropical monkeys tested, three (4.4%), one captive and two free-living, were found to be naturally infected by P. vivax NHP infection contributes to the maintenance of malaria transmission to humans. Therefore, the discussion on wildlife conservation and management must be incorporated into the malaria elimination agenda.This important finding indicates that it is necessary to estimate the extent to which  The world is joining forces, though not just to control malaria cases, but also to try to eliminate this disease, which is an important cause of death in economically disadvantaged countries. The World Malaria Report 2020  and the Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax are the main parasites transmitted by mosquito vectors of the genus Anopheles and cause malaria in humans. This transmission cycle of malaria theoretically makes it easier to fight this disease since it does not involve a wild animal reservoir, compared to other pathogens that have a complex cycle because they can infect more than one vertebrate host. The evolution of malaria parasites highlights their propensity to switch hosts; both P. vivax and P. falciparum first arose as human pathogens after a host switch from great apes in Africa [P. vivax and P. falciparum occurs between infected mosquitoes and humans; however, some research groups have identified the possibility of a malaria reservoir in non-human primates [Plasmodium-infected non-human primates (NHPs), and this is increasing due to habitat destruction and human encroachment into NHP habitats [n Africa , 4. It iprimates , 6. Zoonhabitats .Plasmodium cynomolgi, and mainly Plasmodium knowlesi, are parasites that typically infect forest monkeys in Asia, but they can also cause zoonotic malaria when humans enter this parasite cycle. Another two Plasmodium species that can cause zoonotic malaria in Latin America are Plasmodium brasilianum and Plasmodium simium [P. malariae and P. vivax, respectively [P. brasilianum/P. malariae and P. simium/P.vivax, also have a very close genetic relationship. Plasmodium vivax came from Africa and, based on genomic data, there is a hypothesis that P. simium originated from humans infected with P. vivax that was transferred to New World monkeys [P. brasilianum in platyrrhines is a result of the cross-species transfer of P. malariae that was brought to the New World [m simium –10. Thesectively , 11. The monkeys , 12. Theew World , 13 or wew World .P. vivax [P. simium has only been reported in NHPs of the Atlantic Forest on the southern and southeastern states of the Brazilian coast [Plasmodium infection, including the differential diagnosis of P. simium.In 2019, Brazil was responsible for 19% of all reported malaria cases in the Americas, and 99.9% of all reported Brazilian malaria cases are from the endemic Amazon region. Furthermore, more than 80% of the cases in Brazil were caused by P. vivax . It is ian coast , 16, thoan coast . Thus, tIn a study in Manaus, Amazonas state, Brazil, free-living monkeys were captured in forest fragments inside the urban area using Tomahawk Live  traps baited with fruit. Blood samples were collected via femoral vein puncture (1–1.5 mL), and samples were then transported to the laboratory. The animals were maintained in cages and were held overnight and then released in the early morning of the next day at the site of capture. It is important highlight that all animals seemed healthy and none appeared to be in need of veterinary medical assistance. Captive monkeys from a local zoo also had blood samples taken.coxI gene [Plasmodium species infecting humans [P. malariae were employed to identify P. brasilianum infections in NHPs and the primers for P. vivax were used to identify P. simium infections in NHPs, since these primers do not discriminate among these two pairs of Plasmodium species. The differential diagnosis of P. simium in relation to P. vivax was based on the nested PCR of the coxI gene fragment and subsequent enzymatic digestion, using primers and the protocol described by Alvarenga et al. [It was possible to carry out blood smears for 40% of the samples, but all samples had DNA extraction from blood using the QIAamp DNA Blood Mini Kit , according to the manufacturer’s instructions. The molecular diagnosis was performed using nested PCR targeting the 18 S small subunit (SSU) rRNA and the mitochondrial oxI gene , 17. Thea et al. .P. falciparum DNA from 3D7 strain in vitro culture (IRR-FIOCRUZ MINAS) [P. vivax DNA, from a patient previously diagnosed by microscopy and nested PCR [P. simium DNA from an acute infection in an NHP with parasitaemia confirmed by optical microscopy (BL10) [P. brasilianum DNA from the Malaria Research and Reference Reagent Resource Center . The negative control was from non-human primates from areas without malaria transmission.In the PCR assays, the following were used as positive controls: (i) Z MINAS) ; (ii) P.sted PCR ; (iii) Py (BL10) ; (iv) P.coxI gene PCR products, which resulted in sequences that covered both single nucleotide polymorphisms (SNPs) that are considered specific to P. simium [Plasmodium simium differs from the closely related P. vivax in two unique single nucleotide polymorphisms (SNPs) in the mitochondrial genome, at positions 3535 (T > C) and 3869 (A > G) [P. simium in relation to P. vivax was based on the nested PCR of a coxI gene fragment and subsequent enzymatic digestion, using the primers and the protocol described by Alvarenga et al. [DNA sequencing was performed as described by Alvarenga et al. , using m. simium . Plasmod (A > G) . The difa et al. .P. vivax, as well in all other Plasmodium species tested, is substituted by a “C” in P. simium [The polymorphism at position 3535 generated a new restriction enzyme site for HpyCH4III, and the digestion of the amplified fragment resulted in two fragments of similar lengths (118 and 126 bp), where “T” in . simium .The fragments were electrophoretically separated in an automatic DNA sequencer . Sequences were aligned using the ClustalW software in the Bioedit package  and ChroEvolutionary history was inferred by using the maximum-likelihood method and the Tamura-Nei model . The proPlasmodium spp. using two nested PCR methods. There was no amplification for the 18 S gene; however, three samples (4.4%) were positive in the nested PCR for the coxI mitochondrial gene of Plasmodium spp, of them only one had blood smears carry out and analysed but was negative. After restriction enzyme digestion, the profile of the fragments suggested non-P. simium samples  gene sequences of P. vivax isolated from one captive (H47) and two free-living NHPs had 100% identification with GeneBank sequences from P. vivax  —one male (H52) and one female (H73), and one captive Saimiri sciureus female (H47) from the army zoo—Centro de Instrução de Guerra na Selva (CIGS)  Table  was screcoxI gene fragments were sequenced in both strands, and the consensus sequences were compatible with P. vivax species. This is an important finding when trying to evaluate zoonotic vivax infection, and may help us to begin to understand the real role of NHPs in malaria transmission in the Americas. In 2019, in the western Brazilian Amazon, Silva et al. detected P. vivax and P. falciparum DNA in 2.04% (2/98) and 4.08% (4/98), respectively, of Neotropical primates in captivity, using another protocol [Saguinus bicolor were tested, and one of them was positive for P. falciparum, however 12 Saimiri sciureus were screened for Plasmodium, but none were positive [Saimiri sciureus were experimentally infected with the pooled sporozoites from three specimens of Anopheles cruzi, though all of them were negative on daily examination of thick blood smears [Three samples amplified from Neotropical NHPs using nested PCR of protocol . These apositive . In a std smears .Plasmodium spp.; P. falciparum was detected in two fecal samples of Alouatta seniculus, while Cebus versicolor, Ateles hybridus and Alouatta seniculus were infected with P. vivax/simium, and these last two species and Aotus griseimembra had fecal samples that were positive for P. malariae/brasilianum. Blood samples were also tested in this study, and one Ateles hybridus and one Alouatta seniculus were positive for P. vivax/simium, while these same two species plus Aotus griseimembra and Cebus versicolor were positive for P. malariae/brasilianum [Saguinus bicolor. These are important since, in the majority of previous studies, positive results for P. vivax in NHPs were only serological finds and indistinguishable positive PCR results for P. vivax/P. simium [In Colombia, molecular analysis found Neotropical NHPs that were positive for silianum . The pre. simium , 26.Plasmodium vivax and P. simium can be misdiagnosed due to their morphological and molecular similarity. Malaria caused by P. simium is apparently restricted to regions of the Atlantic Forest on the coast of southeastern and southern Brazil [P. vivax using differential diagnosis. Identification of human malaria parasites in NHPs in the north of this country gets deserves attention since a zoonotic cycle for P. vivax in the Americas has not yet been considered due to a lack of scientific evidence. Sampling was performed in Manaus, capital of the Amazonas state and located in the middle of the tropical rainforest , where more than 2 million people coexist with malaria. Besides socioeconomic difficulties, there are nuances in vivax malaria that complicate all efforts towards malaria elimination in this region, such as its hypnozoite form that causes late relapses, the existence of drug resistant parasites [n Brazil ; despitearasites  and the arasites . It may Plasmodium species, for how long and their importance in the parasite cycle. In addition, the presence of parasites in the blood of NHPs demands attention and discussion, since NHPs represent an animal reservoir in the vivax malaria cycle and, as such, the risk this presents should return to the discussion agenda.The richness of Neotropical primate species (Platyrrhini) is evidenced by the 171 species in 20 genera and five families ; howeverP. vivax positive. In another study, the presence of two Anopheles species was identified at the CIGS zoo, namely Anopheles matogrossensis and Anopheles nimbus, although they are not species associated with human malaria transmission, which reinforces the results present here and strengthens the hypothesis of a zoonotic cycle. In addition, An. nimbus and Anopheles triannulatus were identified at UFAM [An. (Nysorhynchus) triannulatus, is considered to be a secondary human malaria vector in some areas in Brazil [P. vivax between 2018 and 2019.Three primates (two free living and one captive) that were captured in small forest fragments in the urban area were  at UFAM . The latn Brazil , 32 and n Brazil . These aSaguinus bicolor is a critically endangered species [Plasmodium infection can cause on animal health and the conservation of this primate species, which is endemic to Manaus [Since  species , it is no Manaus .P. vivax in the Amazon, which is worrisome since P. vivax is the agent of the greatest number of cases of malaria in the Americas. Knowledge regarding the disease cycle is essential in order to plan measures for mitigation, as well as for defining targets that help us to achieve the ultimate goal, which is, of course, malaria elimination. The assessment of the frequency of P. vivax infection in NHPs and the evaluation of the ecological importance of these primates as the parasite’s reservoir are urgent measures, and the questions presented herein need to be included in the agenda of further studies. As such, it remains to be determined whether wildlife management as a component of malaria elimination programmes is necessary.The data shown here reinforce and bring into question again the possibility of a non-human reservoir of


In summary, patients with severe malaria exhibited higher IFN-γ levels than those with uncomplicated malaria, although the heterogeneity of the outcomes is yet to be elucidated. To confirm whether alteration in IFN-γ levels of patients with malaria may indicate disease severity and/or poor prognosis, further studies are warranted.Interferon (IFN)-γ contributes to the pathogenesis of severe malaria; however, its mechanism remains unclear. Herein, differences in IFN-γ levels between patients with severe and uncomplicated malaria were evaluated using qualitative and quantitative  approaches. The systematic review protocol was registered at PROSPERO (ID: CRD42022315213). The searches for relevant studies were performed in five databases, including PubMed, Scopus, Embase, MEDLINE and Web of Science, between 1 January and 10 July 2022. A meta-analysis was conducted to pool the mean difference (MD) of IFN-γ levels between patients with severe malaria and those with uncomplicated malaria using a random-effects model (DerSimonian and Laird method). Overall, qualitative synthesis indicated that most studies  reported no statistically significant difference in IFN-γ levels between patients with severe malaria and those with uncomplicated malaria. Meanwhile, remaining studies  reported that IFN-γ levels were significantly higher in patients with severe malaria than those in patients with uncomplicated malaria. Only one study (4.17%) reported that IFN-γ levels were significantly lower in patients with severe malaria than those in patients with uncomplicated malaria. The meta-analysis results indicated that patients with severe malaria had higher mean IFN-γ levels than those with uncomplicated malaria ( The majority of these deaths were reported in children under 5 years of age in six countries of Africa, including Nigeria (27%), the Democratic Republic of the Congo (12%), Uganda (5%), Mozambique (4%), Angola (3%) and Burkina Faso (3%)1. Most malaria deaths were caused by Plasmodium falciparum infection; whereas, a minority were caused by other Plasmodium spp.5.Malaria is one of the leading causes of death worldwide, with an estimated 627,000 malaria deaths reported in 20208. During malaria infection, pro- and anti-inflammatory cytokines play a role in protection against infection or disease pathogenesis9. Pro-inflammatory cytokines are produced by leukocytes such as neutrophils, lymphocytes and monocytes or other cells such as macrophages, endothelial cells, fibroblasts and mast cells14. These pro-inflammatory cytokines are interleukin (IL)-1, tumour necrosis factor (TNF)-α, interferon (IFN)-γ, IL-12 and IL-1816. IFN-γ is one of the pro-inflammatory T helper 1 (Th1) cytokines involved in protection against malaria and parasite clearance9. In combination with TNF-α and IL-12, IFN-γ inhibits parasite growth, stimulates phagocytosis and enhances the clearance of parasitised erythrocytes17.Immune responses to malaria have been described previously19 and severe anaemia20, while also being associated with an increased likelihood of death22. Nevertheless, the role of IFN-γ in malaria severity remains controversial, and previous studies have included only a small number of participants with severe malaria26. To date, no meta-analysis has been used to unveil differences in IFN-γ levels among patients with severe and uncomplicated malaria. Therefore, in this study, the differences in IFN-γ levels between patients with severe malaria and uncomplicated malaria were estimated using a meta-analysis. These findings are essential to understanding the pathogenesis of malaria and guiding further studies.Previous studies have indicated that elevated levels of pro-inflammatory cytokines, including IFN-γ, contribute to the severity of cerebral malaria27. The systematic review protocol was registered at PROSPERO (ID: CRD42022315213).A systematic review and meta-analysis were conducted according to the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) (S1 PRISMA Checklist)Plasmodium parasitemia with one or more of the following complications: impaired consciousness, severe malarial anaemia, renal impairment, significant bleeding, acidosis, jaundice, prostration, multiple convulsions, shock, hypoglycaemia and hyperparasitemia. Severe vivax malaria is defined similarly to severe falciparum malaria, but there are no thresholds for parasite density28. Uncomplicated or mild malaria is the presence of Plasmodium parasitemia without the characteristics of severe malaria.Severe falciparum malaria is defined as the presence of Studies reporting IFN-γ levels in patients with severe and uncomplicated malaria that met the PICO question criteria were included in this systematic review. The exclusion criteria were animal studies, in vitro studies, case reports or case series, review articles, studies with missing article information online, studies for which full-texts were unavailable, conference abstracts without full data, studies for which data of IFN-γ in both groups of patients could not be extracted and studies that reported IFN-γ levels after patients were treated.The search terms were chosen based on the Medical Subject Headings. A combination of search terms with Boolean operators was used as follows: ‘(interferon OR IFN OR interferon-gamma OR interferon-g OR IFN-g OR interferon-γ OR IFN-γ) AND (severe OR complicated) AND ’. The searches were conducted in PubMed, Scopus, Embase, MEDLINE and Web of Science between 1 January and 10 July 2022 without a restriction on the publication date Table . The seaTwo authors (MK and KUK) independently performed study selection. First, the titles and abstracts were screened for potentially relevant studies. In cases for which information from titles and abstracts was not sufficient for inclusion, the article was retained for full-text examination. Second, the full-texts of relevant studies were examined to find studies that met the eligibility criteria. Finally, disagreement between the two authors in study selection was resolved by discussion to form a consensus.Two authors (KUK and AM) carried out the data extraction, and data from the included studies were cross-checked by another author (MK). As a result, the following data were extracted to the spreadsheet: the name of the first author, publication year, study location, period of data collection, number of patients, age group, percentage of male participants, data on IFN-γ levels , parasite density, the technique used for detecting malaria parasites and technique used for measuring IFN-γ.29.The methodological quality of the included studies was evaluated using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist for cross-sectional, prospective observational, and case–control studies30. The mean and standard deviation were estimated from the median and range as described previously31. Comparing the mean and standard deviation between groups of participants was carried out using a protocol described previously32. If the standard deviation was unavailable, a value was borrowed from other studies (with the lowest standard deviation) in the same meta-analysis33. The heterogeneity of the estimated effects between studies was assessed using Cochrane Q and I2 tests. A Cochrane Q test result with a significant value (p) less than 0.1 or an I2 result of more than 50% indicated heterogeneity in the estimates of effect between studies. When heterogeneity was revealed, a meta-regression analysis was performed to identify the source(s) of heterogeneity in the effect estimates. Then, a subgroup analysis was performed to determine the differences in the effect estimates between the subgroups of interest. Finally, a sensitivity analysis using the leave-one-out method34 was performed to test whether the meta-analysis was robust. Sensitivity analysis between studies that reported mean/standard deviation and median/range and studies that reported mean without standard deviation was conducted to assess the effect of changing the assumptions made. The sensitivity analysis between studies that reported mean/standard deviation and those with median/range of IFN-γ levels in patients with severe and uncomplicated malaria was conducted using a subgroup analysis. Publication bias was assessed by visually assessing funnel plot asymmetry and Egger’s test for small-study effects. If publication bias was found, the trim-and-fill method35 was applied to adjust the pooled effect estimates. The meta-analysis was performed using Stata version 17.0 .Data syntheses included qualitative and quantitative syntheses. A qualitative synthesis was the narrative synthesis of the difference in IFN-γ levels between patients with severe and uncomplicated malaria. A quantitative synthesis  was conducted to pool the mean difference (MD) of IFN-γ levels between patients with severe and those with uncomplicated malaria. The random-effects model was employed to pool the MDs using the DerSimonian and Laird methodA total of 2668 articles were identified from a database search, including 524 articles from PubMed, 558 from Scopus, 792 from Embase, 325 from MEDLINE and 469 from Web of Science. After study selection, 24 studies that met the eligibility criteria were included in the review Fig. .Figure 1The included studies were published between 1990 and 2000 , 2000 and 2010  and 2011 and 2017 . The study designs were as follows: case–control , prospective observational , cross-sectional  and prospective cohort  studies. The studies were located in Africa , Asia , South America , North America , Europe  and both African and Asian countries . Most of the included studies used only microscopy for identification of malaria parasites . Most of the included studies used enzyme-linked immunosorbent assay (ELISA) to measure IFN-γ in the blood of participants . Other characteristics of the included studies are shown in Table The quality of the included studies was determined using the STROBE checklist. The assessment results showed that seven of nine prospective observational studies (77.8%) were of high quality, while three were of moderate quality (22.2%). Eight of the ten case–control studies (70%) were of high quality, while two were of moderate quality (30%). Two prospective cohort studies were of high quality, and one cross-sectional study was of moderate quality Table .39. Meanwhile, 14 studies (58.3%) reported no statistically significant differences in IFN-γ levels between patients with severe malaria and those with uncomplicated malaria51. Only one study (4.17%) reported that IFN-γ levels were significantly lower in patients with severe malaria than those in patients with uncomplicated malaria  reported severe P. falciparum infections39 and three studies (33.3%) reported severe P. vivax infections37. Meanwhile, 14 studies reported no difference in IFN-γ levels between the two groups51, and one study that reported significantly lower IFN-γ levels in patients with severe malaria than in those with uncomplicated malaria17 reported only P. falciparum infection.The difference in IFN-γ levels between patients with severe and uncomplicated malaria was qualitatively described using the results from individual studies. Overall, nine studies (37.5%) reported that IFN-γ levels were significantly higher in patients with severe malaria than those in patients with uncomplicated malariaia Table . Among s51. The lowest MD was identified in the study conducted in Gabonese children 17. The highest MD was identified in the study conducted in India 24. Four studies demonstrated that patients with severe malaria had lower mean IFN-γ levels than those with uncomplicated malaria51. Two studies demonstrated no difference in mean IFN-γ levels between patients with severe malaria and those with uncomplicated malaria45. Meanwhile, six studies showed that patients with severe malaria had higher mean IFN-γ levels than those with uncomplicated malaria47. Overall, the results demonstrated that patients with severe malaria had higher mean IFN-γ levels than those with uncomplicated malaria , age group (p = 0.001), location  and technique used to measure the IFN-γ levels (p < 0.009) were sources of heterogeneity in the effect estimates among the included studies. Therefore, subgroup analyses of study design, age group, location (continent) and technique used to measure the IFN-γ levels were performed.Because of the high heterogeneity of the effect estimates among the included studies, a meta-regression analysis incorporating study design, location (continent), age group and technique used to measure the IFN-γ levels was conducted to identify whether these covariates were the source(s) of heterogeneity. The findings showed that study design  and case–control studies . Meanwhile, no difference in mean IFN-γ levels between patients with severe and those with uncomplicated malaria was identified in prospective cohort studies  and all age groups . Meanwhile, higher mean IFN-γ levels were found in adults with severe malaria than in those with uncomplicated malaria . However, no differences in mean IFN-γ levels between patients with severe and those with uncomplicated malaria were found among studies conducted in Africa  and all age groups  showed higher mean IFN-γ levels in patients with severe malaria than in those with uncomplicated malaria among studies that were conducted in Asia . However, no differences in mean IFN-γ levels between patients with severe and those with uncomplicated malaria were found among studies using bead-based assays for IFN-γ measurement . Meanwhile, no differences in mean IFN-γ levels between patients with severe and those with uncomplicated malaria were found among studies that reported median/range of IFN-γ levels , showing that publication bias was discovered. The trim-and-fill method was applied to adjust the pooled effect estimate, and the rresults showed that the pooled MD of IFN-γ levels between patients with severe and uncomplicated malaria after adjusting for publication bias was 13.634 pg/mL (95% CI 6.979–20.29 pg/mL).The publication bias of the effect estimates among the included studies was assessed by visualisation of funnel plot symmetry and Egger’s test for small-study effect. In the meta-analysis between IFN-γ levels between patients with severe and those with uncomplicated malaria, asymmetry of the funnel plot was suspected Fig. , and EggThe main feature of this study was the comparison of IFN-γ levels between patients with severe and those with uncomplicated malaria. The results of the qualitative synthesis demonstrated that most studies that investigated IFN-γ levels in both patients with severe and uncomplicated malaria revealed no statistically significant IFN-γ levels between the two clinical outcomes. Meanwhile, few studies have reported that IFN-γ levels were significantly higher in patients with severe malaria than those in patients with uncomplicated malaria. For quantitative synthesis by meta-analysis, the higher IFN-γ levels were observed in patients with severe malaria than those in patients with uncomplicated malaria. Although a high degree of heterogeneity of the outcome existed, the meta-analysis results implied that IFN-γ levels were associated with malaria severity, i.e. increased levels positively correlated with increased severity.54. Nevertheless, a study on children with malaria argued that reduced IFN-γ levels were associated with malaria severity55. A discrepancy in IFN-γ levels and malaria severity between studies might be because of differences in the different participants enrolled in each as suggested previously20. Various severe complications among patients, such as anaemia, parasitemia levels or cerebral malaria, might cause the differences in the MD in IFN-γ levels between severe and uncomplicated malaria. A previous study indicated that IFN-γ production was associated with the reduced prevalence of anaemia caused by P. falciparum; hence, IFN-γ was suggested to be an immunity-based protection against severe malarial anaemia17. A previous study found that IFN-γ levels were negatively associated with parasitemia, suggesting that this cytokine has antiparasitic effects46. Increased IFN-γ levels were also related to high malarial parasitemia52. This association may be due to malarial parasitemia caused by the production of IFN-γ by immune cells56. In comparison to severe malarial anaemia, patients with cerebral malaria showed higher levels of pro-inflammatory/Th1 cytokines19, indicating that the production of pro-inflammatory cytokines in severe malaria is poorly regulated21.IFN-γ levels related to malaria severity were observed by in vitro studies60. Therefore, it is possible that both patients with severe and uncomplicated malaria showed comparable cytokine responses, which causes non-statistical significance between groups in this meta-analysis. In Asia, where falciparum malaria is less endemic, most populations are less exposed to infections; hence, they are non-immune or semi-immune responses to malaria infections61. Therefore, patients with severe malaria in Asian countries might develop a stronger immune response to the infections than those with uncomplicated malaria. For example, Singotamu et al.24 in India indicated that P. vivax infections demonstrated very high mean IFN-γ levels (619.7 pg/mL) in patients with severe malaria compared with uncomplicated malaria (177.3 pg/mL). Additional background histories or co-occurrence with pathogens other than malarial ones in various areas might manifest different immune responses in patients with malaria. The histories of the individuals studied may add a layer of complexity to the findings on the immune response to malaria6.Although the meta-analysis results exhibited higher mean IFN-γ levels in severe malaria than in uncomplicated malaria, the degree of heterogeneity among studies included in the meta-analysis was extremely high. The meta-regression and subgroup analyses of study design, continents, age groups, and techniques for IFN-γ measurement proposed that these parameters were sources of heterogeneity in the outcome. Considering study design as a source of heterogeneity, prospective observational and case–control studies showed higher mean IFN-γ levels were found in patients with severe malaria compared to those with uncomplicated malaria. Meanwhile, the prospective cohort studies indicated no difference. These results might be because only two prospective cohort studies were included in the subgroup analysis, which might bias the results of the subgroup analysis. Considering the continent as another source of heterogeneity, studies conducted in Asia showed higher mean IFN-γ levels in patients with severe malaria than those with uncomplicated malaria. However, there was no difference in mean IFN-γ levels among studies performed in Africa, indicating that the different populations investigated may have had various immune responses to malaria. In Africa, where falciparum malaria is endemic, the populations are more exposed to infections; hence, they may have acquired immune responses against malaria infections or severityP. falciparum. Most infected adults rarely experience severe disease because of the acquired immunity against the infection. In areas where malaria is less endemic, such as Asia, a higher risk of severe disease is frequently observed among adults than children as adults develop a stronger immune response to the infection, but infants and children occasionally do not. This reason explained the possible cause of high cytokine response, including IFN-γ response to the infection.Considering age groups as another source of heterogeneity in the outcome, no differences in mean IFN-γ levels in children with severe malaria and those with uncomplicated malaria, but adults with severe malaria showed higher mean IFN-γ levels than those with uncomplicated malaria. This result might be explained by the fact that studies enrolling children with severe malaria were conducted in Africa, where malaria is endemic. Meanwhile, studies enrolling adults with severe malaria were conducted in Asia, where malaria is less endemic. Across sub-Saharan Africa, where the disease is hyper-endemic, most people are almost continuously infected with 62. A study comparing the overall performance of the two methods for cytokine profiles demonstrated that the ELISA and bead-based assays yielded similar results63. Notably, ELISA was more sensitive in the low concentration range of the standard curve, whereas bead-based assays could detect higher protein concentrations63. Another study that measured IFN-γ levels using both techniques indicated comparable detection of plasma IFN-γ, IL-4, IL-10, and TNF-α, but the ELISA missed a cytokine such as IL-564. Therefore, no study yet warrants the difference in performance between the two techniques for detecting IFN-γ in the blood. However, the results of the subgroup meta-analysis might guide further studies to examine the difference in the real performance of these techniques.Considering the techniques for measuring IFN-γ as another source of heterogeneity of the outcome, studies using ELISA exhibited higher mean IFN-γ levels in patients with severe malaria than in those with uncomplicated malaria. However, studies using bead-based assays indicated no differences in mean IFN-γ levels between patients with severe and those with uncomplicated malaria. Multiplex bead-based assays provide the means to simultaneously measure multiple proteins in a single reaction compared to ELISA, which measures a single protein in a cone reaction67. The IFN-gamma receptor (R) locations were in lymphoid organs such as the B-cell areas of lymph nodes, spleen, tonsils, and in epithelial tissues of the intestinal system, lung, and endometrial mucosa cells68. Studies in mice models indicated that treatment of mice infected with blood-stage P. berghei by anti-IFN-γ antibody failed to control the infection parasites70. Additionally, delayed parasite elimination was found among IFN-γ-deficient or IFN-γ receptor (IFN-γR)-deficient mice or anti-IFN-γ antibody-treated mice73. During Plasmodium infection, γδ T-cells that express CD40 ligand produce IFN-γ in response to infection by enhancement of dendritic cell activation to remove malaria parasites74. In the pathogenesis of severe malaria, many studies have indicated that IFN-γ is vital for developing severe malaria, particularly cerebral malaria, by affecting endothelial integrity76. During cerebral malaria, IFN—producing CD8 + T-cells are recruited to the brain and cause cerebral pathology by destroying the blood–brain barrier in perforin- and granzyme-dependent manner78. IFN-γ production is modulated by several cytokines such as IL-12 and IL-18 or broadly reactive antigen receptors79. In the study by Wroczyńska et al.25, increased IFN-γ accompanied by increased IL-18 levels were observed in patients with severe malaria, indicating that excessive production of both cytokines is associated with severe malaria infections. IL-18-dependent IFN-γ overproduction was reported to relate to decreased IL-12 levels25. Therefore, these data suggested that severe malaria is associated with increased IFN-γ and decreased IL-12 levels, indicating the occurrence of immunoregulation in resolving malaria infection25. Furthermore, reduced IL-12 levels were associated with suppression of Th1 cytokine activation by NK cells or CD8 cells56. The previous studies also showed that IFN-γ was associated with IL-10 and IL-6, indicating a balance between these cytokines80. IFN-γ and IL-10 were markedly increased in patients with severe malaria81.IFN-γ contributes to the activation and differentiation of B lymphocytes, T lymphocytes and macrophagesThis study had some limitations. First, the degree of heterogeneity was extreme in the meta-analysis. Although meta-regression and subgroup analyses were conducted to identify the source(s) of heterogeneity, the heterogeneity remained in the subgroup analysis, showing that other factors confound the association between IFN-γ levels and malaria severity. Second, publication bias among the studies included in the meta-analysis was noted. Therefore, the pooled effect estimate (MD of IFN- γ levels) after applying the trim-and-fill method should be considered.In conclusion, patients with severe malaria present higher IFN-γ levels than those with uncomplicated malaria, although the heterogeneity of the outcomes is yet to be elucidated. To confirm whether alteration in IFN-γ levels of patients with malaria may indicate disease severity and/or poor prognosis, further studies are warranted.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.


Plasmodium falciparum is the predominant species. Molecular diagnosis is essential for malaria surveillance, but requires additional blood samples for DNA extraction. Used RDTs is an attractive alternative that can be used as a source of DNA. Plasmodium falciparum genetic diversity and multiplicity of infection, usually determined by the genotyping of polymorphic regions of merozoite surface proteins 1 and 2 genes , and the repeated region RII of the glutamate-rich protein gene (glurp) have been associated with malaria transmission levels and subsequently with the impact of the deployed control strategies.Rapid diagnostic tests (RDT) are widely used for malaria diagnosis in Madagascar, where Plasmodium species, to characterize Plasmodium falciparum genetic diversity and determine the multiplicity of infection.Thus, the study aims to use RDT as DNA source to detect Plasmodium DNA was extracted by simple elution with nuclease free water. Nested-PCR were performed to confirm Plasmodium species and to analyse P. falciparum msp1, msp2 and glurp genes polymorphisms.A pilot study was conducted in two sites with different epidemiological patterns: Ankazomborona (low transmission area) and Matanga (high transmission area). On May 2018, used RDT  were collected as DNA source. Plasmodium positivity rate was 23.5% (40/170) [95% CI 17.5–30.8%] by nested-PCR with 92.2% (37/40) positive to P. falciparum, 5% (2/40) to Plasmodium vivax and 2.5% (1/40) to P. falciparum/P. vivax mixed infection. Results showed high polymorphisms in P. falciparum msp1, msp2 and glurp genes. Multiple infection rate was 28.6% [95% CI 12.2–52.3%]. The mean of MOI was 1.79 ± 0.74.Amongst the 170 obtained samples (N = 74 from Ankazomborona and N = 96 from Matanga), This pilot study highlighted that malaria diagnosis and molecular analysis are possible by using used malaria RDT. A large-scale study needs to be conducted to assess more comprehensively malaria parasites transmission levels and provide new data for guiding the implementation of local strategies for malaria control and elimination.Trial registration Retrospectively registered Plasmodium species infecting humans, Plasmodium falciparum is the most prevalent malaria species in sub-Saharan Africa and the most virulent species leading severe malaria msp2, con in 2016 .msp2, out of the 9 positive samples, 6 distinct alleles were detected. Regarding to glurp gene, 8 distinct alleles were observed in the 20 positive samples. Polymorphism analysis showed high genetic diversity of P. falciparum populations in Ankazomborona and Matanga. Majority of alleles detected during in this study were single with a high polymorphism level. No significant difference of polymorphism level was detected in two study sites. Parasite genetic diversity is one of key elements to consider for strategies implementation to fight against malaria [Analysis based on the PCR product fragments showed 4 distinct alleles for MAD 20 allelic family (11 samples), 1 allele for RO33 10 samples) and 6 alleles for K1 (6 samples). The K1 allelic family was found at low frequency (28.6%) compared to MAD20 and RO33, but this allelic family was more polymorphic . For FC27 allelic family of  malaria . It has  samples  malaria , 25. HenP. falciparum msp1 gene not for msp2 and glurp. These results indicate that polyclonal infection frequency is low compared to those found in other countries [P. falciparum populations circulating in Madagascar showed a high level of genetic polymorphism. That high polymorphism reflects transmission intensity. Consequently, it would be interesting to increase samples number in order to continue genetic diversity study over time and space for carrying out a cartographic study about Plasmodium allelic families’ distribution in Madagascar.This study showed 28.6% multiple infection for ountries , 47. Theountries , 37, 40.ountries , is not Plasmodium using blood samples collected on RDT as DNA source. Preliminary data from this study showed high genetic diversity of P. falciparum populations in Ankazomborona and in Matanga but multiple infection rate was low in the both study sites.The present study highlighted the possibility of performing malaria diagnosis and molecular genetic diversity analysis of P. falciparum genetic diversity and allow to update available data. The results of this study can be used as a baseline information for future studies on parasite transmission dynamics and to evaluate effectiveness malaria prevention and control strategies.This study provides fundamental information on


Plasmodium falciparum infections remain among the leading causes of morbidity and mortality in holoendemic transmission areas. Located within region 5q31.1, the colony-stimulating factor 2 gene (CSF2) encodes granulocyte–macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor that mediates host immune responses. Since the effect of CSF2 variation on malaria pathogenesis remains unreported, we investigated the impact of two genetic variants in the 5q31.1 gene region flanking CSF2:g-7032 G > A (rs168681:G > A) and CSF2:g.64544T > C (rs246835:T > C) on the rate and timing of malaria and severe malarial anemia  episodes over 36 months of follow-up. Children  were recruited from a holoendemic P. falciparum transmission area of western Kenya. Decreased incidence rate ratio (IRR) for malaria was conferred by inheritance of the CSF2:g.64544 TC genotype (P = 0.0277) and CSF2 AC/GC diplotype (P = 0.0015). Increased IRR for malaria was observed in carriers of the CSF2 AT/GC diplotype (P = 0.0237), while the inheritance of the CSF2 AT haplotype increased the IRR for SMA (P = 0.0166). A model estimating the longitudinal risk of malaria showed decreased hazard rates among CSF2 AC haplotype carriers (P = 0.0045). Investigation of all-cause mortality revealed that inheritance of the GA genotype at CSF2:g-7032 increased the risk of mortality (P = 0.0315). Higher risk of SMA and all-cause mortality were observed in younger children (P < 0.0001 and P = 0.0015), HIV-1(+) individuals (P < 0.0001 and P < 0.0001), and carriers of HbSS (P = 0.0342 and P = 0.0019). Results from this holoendemic P. falciparum area show that variation in gene region 5q31.1 influences susceptibility to malaria, SMA, and mortality, as does age, HIV-1 status, and inheritance of HbSS. Plasmodium falciparum  infections    .The pathophysiology of SMA is complex, and includes the destruction of malaria parasite-infected and non-infected erythrocytes, as well as decreased erythropoiesis . We haveGranulocyte monocyte-colony stimulating factor (GM-CSF) is a hematopoietic growth factor that facilitates the differentiation of progenitor cells into three lineages in the bone marrow, namely the lymphoid, myeloid, and erythroid progenies –11. GM-CP. falciparum malaria . We selected rs246835:T > C since this SNP is part of a potential distal enhancer with the loss of TFBS for USF2 and creation of TFBS for GATA-1 by switch of T allele to C allele  a MAF of 0.20 (1000 Genome Project) in the AFR population and a MAF of 0.18 in the YRI population (HapMap), and (2) potential functional effects, where the wild-type G allele creates TFBS for ER-alpha and ZEB1, and transition to the minor A allele ablates such binding and creating a TFBS for NF-X3. These TFBSs are part of a potential distal enhancers , and rs246835:T > C (P = 0.2202) were comparable across the groups. The allele frequencies for the study population is comparable to those from the International HapMap Project and 1000 Genomes Project , and diplotypes (P = 0.3255) were not significantly different across the study groups.The distribution of CSF2 variants on malaria episodes over a follow-up period of 36 months. Factors associated with the rate of malaria episodes were determined using a generalized linear model (Poisson regression). Results for the covariates that emerged for the 6029 recorded malaria episodes over 36 months of follow-up are shown in Table P < 0.0001)]. Children who were HIV-1 positive had a lower incidence rate of malaria episodes (P = 0.0023), relative to HIV-1 negative participants, as did female study participants (P = 0.0013), compared to their male counterparts. Carriage of the homozygous double deletion (-α3.7/-α3.7) for α+-thalassemia also conferred a protective effect against malaria episodes (P = 0.0068) relative to homozygous αα/αα carriers. Protection against multiple bouts of malaria over time was also associated with co-inheritance of homozygosity for the sickle cell disease  and heterozygosity for the sickle cell trait [Hb AS, (P < 0.0001). Protective effects against the longitudinal risk of malaria were also present in heterozygous carriers of the rs246835 TC genotype (P = 0.0277) relative to the wild type genotype TT. Similarly, the positional interaction of the CSF2 AC/GC diplotype was associated with decreased malaria episodes (P = 0.0015) compared to wild-type diplotype GT/GT. Conversely, co-inheritance of the CSF2 AT/GC diplotype increased the rate of malaria episodes (P = 0.0237). There was also a trend towards more malaria episodes in carriers of the CSF2 GC/GT diplotype, however, the trend was not significant (P = 0.0663) . Similarly, children who were recruited into cohort 2 (2007–2012) had lower incidences of SMA (P = 0.0013), relative to those enrolled in study cohort 1 (2003–2005). Unlike the protection observed against malaria in HIV-1-positive children, being positive for HIV-1 markedly increased the risk of SMA (P < 0.0001). Inheritance of the double mutation that confers sickle cell disease (Hb SS) increased the rate of SMA (P = 0.0024), whereas carriage of sickle cell trait (Hb AS) protected against SMA episodes (P = 0.0026). The only CSF2 variant found to influence the rate of SMA episodes was carriage of the CSF2 AT haplotype, which increased the risk of SMA episodes (P = 0.0166) compared to the wild-type GT haplotype.The effect of the CSF2 variants on the rate of malaria and SMA episodes, we then determined the effect of the variants on the time between events using an ordered-events, generalized Cox proportional hazard model. Shown in Table P < 0.0001). Although retained in the model, cohort (P = 0.1159), HIV-1 positivity (P = 0.1711), sex (P = 0.0580) and -α3.7/-α3.7 (P = 0.0874) were not significant predictors for the time between malaria infections (Table P < 0.0001) or the Hb AS variant (P = 0.0001) decreased the longitudinal hazard for malaria infections. Carriage of the CSF2 AC haplotype was also protective against malaria infections (P = 0.0045) relative to wild-type haplotype (GT), while co-inheritance of the CSF2 GC/GT diplotype showed a non-significant trend towards a lower risk of malaria (P = 0.1108) compared to wild type (GT/GT)  as did children enrolled in cohort 2 (P = 0.0036). Co-infection with malaria and HIV-1 was a strong predictor of increased susceptibility to SMA (P < 0.0001), as was carriage of the Hb SS genotype (P = 0.0342). Conversely, inheritance of the Hb AS variant decreased the longitudinal risk of SMA (P = 0.0196). Although non-significant, there was a trend towards increased risk of SMA in children with the CSF2 AT haplotype (P = 0.0809).Cox proportional hazard model was also used to examine the influence of P = 0.0015), whereas children with HIV-1 had a 16-times higher risk of mortality (P < 0.0001). Children with Hb SS also had a marked increase in the risk of mortality (P = 0.0019). Conversely, carriage of sickle cell trait (Hb AS) conferred a lower hazard risk for mortality (P = 0.0427). Inheritance of the rs168681 GA genotype significantly increased the risk of dying during the follow-up period (P = 0.0315), as did carriage of the CSF2 GC/GC diplotype, although not significant (P = 0.0592).The influence of covariates on the risk of all-cause mortality was investigated using a Cox proportional hazard model Table . Older cP. falciparum transmission [CSF2 (rs168681:G > A and rs246835:T > C) on the rate and timing of malaria and SMA over a 36-month longitudinal follow-up period during the developmental phase of naturally acquired malarial immunity. The impact of CSF2 gene variants on all-cause mortality was also investigated. Since other factors can influence the development of malaria, SMA, and mortality, we also determined the influence of important covariates in all statistical models.Advances in human gene mapping, along with an increased understanding of the molecular mechanisms of protective immunity, illustrate that susceptibility to malaria and its clinical outcomes is conditioned by genotypic variation . The worsmission . In suchsmission , 24. Howsmission , 8. To bCSF2 on susceptibility to malaria and its severe disease manifestations  in children as they progressively develop immunity to clinical malaria.Previous studies from our laboratories have shown that the host releases both pro- and anti-inflammatory cytokines, chemokines, growth factors, and effector molecules as part of the innate immune response to malaria infections , 8. AmonCSF2 has a TFBS for USF2 in the presence of the T allele and a TFBS for GATA-1 in carriers of the C allele. Previous studies have confirmed that CSF contains consensus elements for ZEB1 and GATA [D′ = 0.416 and r2 = 0.01 indicate that the two variants are not strongly linked [Selection of the polymorphic variants was based on interrogating SNPs (and their combinations) that are previously unexplored in malaria and have the potential to impart functional changes on GM-CSF production. For example, the presence of the wild-type G allele at the rs168681:G > A locus within one distal enhancer produce TFBSs for ER-alpha and ZEB1, whereas transition to the A allele creates a binding site for NF-X3. Variation at rs246835:T > C locus within another distal enhancer of and GATA , 28. Sinand GATA . The alland GATA . The rs1and GATA , there wy linked .+-Thalassemia and/or sickle cell traits influenced longitudinal susceptibility to malaria and SMA infections and mortality outcomes. These results parallel those of previous studies that reported variability in the degree of protection conferred by hemoglobinopathies against mild and severe malaria  altered the incidence rate and hazard ratios to malaria and SMA over the follow-up period. Results presented here complement previous studies illustrating that children living in regions with high transmission rates for P. falciparum experience multiple infections prior to developing malarial immunity, with the greatest burden of severe disease manifesting in children less than 5 years of age [Analysis of the interacting covariates revealed that the children’s age, cohort (2003–2005 vs 2007–2012), sex , and HIV-1 status (+ vs −) altered the incidences and risk of malaria and SMA over the longitudinal follow-up of 36 months. Further, co-inheritance of genetic variants; αiewed in ). In adds of age , 50–52. CSF2 AC/GC diplotype decreased the incidence of malaria, while the CSF2 AT/GC diplotype increased the incidence of malaria episodes. Additional Poisson analysis identified significantly more episodes of SMA among children who inherited the CSF2 AT haplotype. To further elucidate the impact of the CSF2 genetic variants on the longitudinal risk of malaria and SMA, a Cox proportional hazard model was fit with independent increments according to the Anderson–Gill method. Carriage of the CSF2 AC haplotype was associated with a lower hazard risk for malaria infections. Additionally, inheritance of the CSF2 AT haplotype that significantly increased the incidence rate for SMA was associated with an elevated longitudinal hazard for SMA, albeit non-significant. Results obtained in this study support our previous findings that showed innate immune response genes influence longitudinal susceptibility to malaria and subsequent development of SMA in this P. falciparum holoendemic region [CSF2 genetic variants are associated with both the rate and timing of malaria and SMA episodes during the development of naturally acquired malarial immunity in children living in a high malaria transmission region. Although transcript and protein levels in circulation for CSF2/GM-CSF were not measured in the current study due to sample availability, we hypothesize that the variants alter the gene products and, thereby, influence susceptibility to malaria and SMA, as demonstrated in our previous studies for other genetic variants [A Poisson regression model estimating the rate of malaria episodes over 36 months revealed that the presence of the rs246835 TC heterozygote genotype and c region –52. Morevariants , 61.CSF2 variants were also associated with increased susceptibility to all-cause mortality, including carriage of the rs168681 GA genotype (1.8 times higher) and the CSF2 GC/GC diplotype (2.6 times higher). Interestingly, neither of these variants significantly impacted susceptibility to either malaria or SMA, suggesting that their impact on mortality may be due to non-malaria-related causes.We also investigated predictors of all-cause mortality across 36 months using Cox proportional hazard modeling and found that younger children and those with HIV-1 had an increased risk of death. In addition, carriage of the Hb AS trait protected against mortality, whereas inheritance of the Hb SS homozygous mutant imparted 6.5 times higher risk of dying. Several CSF2, and the soluble inflammatory mediator it encodes, GM-CSF, on susceptibility to malaria, and the subsequent development of severe disease once an individual becomes infected.In summary, although the two variants investigated correspond to distal enhancer regions within the 5q31.1, and are associated with altered susceptibility to malaria, SMA, and all-cause mortality, the underlying molecular mechanism(s) for the disease associations remain largely unknown. To our knowledge, this is the first study examining the impact of two genetic variants flanking the 5q31.1 gene region on longitudinal malaria disease outcomes and all-cause mortality. It will be important for future studies to establish the precise role of