The Alere™ Malaria Ag P.f Ultra-sensitive RDT (UsmRDT) kit is an HRP2-based malaria rapid diagnostic test (RDT) with enhanced sensitivity relative to the SD Bioline Malaria Ag P.f RDT (mRDT) kit. However, the diagnostic performance of the UsmRDT kit has not been evaluated in Ghana.Plasmodium falciparum specific polymerase chain reaction (PCR) was performed on gDNA extracted from 100 µl of the whole blood.A total of 740 afebrile participants aged between 3 and 88 years old were recruited from the Central and Greater Accra Regions of Ghana during the off-peak malaria season. Axillary body temperature was measured, and a volume of 1 ml venous blood was drawn from each participant. Prior to separating the blood into plasma and packed cell pellets via centrifugation, the blood was spotted onto one UsmRDT and one mRDT kit and also used to prepare thick and thin blood smears as well as filter paper blood spots. The overall positivity rate for microscopy, PCR, UsmRDT and mRDT kit were 20.4%, 40.8%, 31.3% and 30.8%, respectively. Overall, the UsmRDT identified 9.3% (28/302) more PCR positive samples than the mRDT kits. All samples that were negative by the UsmRDT kit were also negative by the mRDT kit. Overall, the sensitivity and specificity of the UsmRDT was 73% (221/302) and 89% (388/436), respectively, while that for the mRDT kit was 58% and 90%, respectively.P. falciparum carriage, it correctly identified P. falciparum in 9.3% of the study participants that were not captured by the mRDT kit. In malaria endemic settings, the UsmRDT would provide an added advantage by identifying more asymptomatic P. falciparum carriers than the mRDT kit for targeted treatment interventions.Although the UsmRDT kit was not as sensitive as PCR at detecting asymptomatic  Global malaria eradication requires all malaria endemic countries to individually be free of all malaria parasites . AlthougHowever, asymptomatic malaria parasite carriage has been reported to be very high in SSA, with a study conducted in the Eastern Region of Ghana reporting a prevalence of asymptomatic participants to be 33.1% by rapid diagnostic tests (RDT) and 66% by molecular tests –9. AsympAsymptomatic parasitaemia is predominantly at densities below the detection limit of microscopy. These submicroscopic densities of parasites require enhanced detection tools, such as polymerase chain reaction (PCR) for detection. A variety of PCR techniques have been developed for the detection of malaria parasites. Advantages of PCR include the ability to detect parasite densities as low as 0.5 parasites per microlitre (p/µl) for real time PCR  and abouPlasmodium falciparum, the Plasmodium species which accounts for most (over 95%) of all malaria infections in SSA [Malaria RDTs were developed to enhance the diagnosis of malaria by providing rapid point of care diagnosis . The anas in SSA . An imprs in SSA  were ints in SSA –21. Amons in SSA . A previs in SSA . Using as in SSA . These cThis study set out to determine the performance of the UsmRDT kit at diagnosing asymptomatic malaria parasite carriage by afebrile children and adults in two communities where parasite carriage is high and pregnant women from a third community where parasite carriage is low in Ghana during the off-peak malaria season.This study was a pilot cross sectional study conducted between December 2019 and January 2020 in three communities in southern Ghana . The SD Bioline Malaria Ag P.f RDT kit  and the ultra-sensitive Alere™ Malaria Ag P.f RDT  were both used to test for the presence of HRP2 antigen as a proxy for the presence of P. falciparum according to manufacturer’s instructions. Briefly, both RDTs had their sample wells spotted with whole blood using the inverted cups supplied with the RDT package. Three drops of every kit’s accompanying assay diluent were dropped in the appropriate well immediately. The results of the RDT kits were read 15 min and 20 min after assay diluent addition for the mRDT and UsmRDT kit, respectively. All participants in this study were informed to visit the nearest health facility as soon as they develop any malaria related symptom. Thick and thin blood smears were prepared and stained according to standard protocols for identification and quantification of P. falciparum [The axillary temperature of each participant was measured and recorded prior to sample collection. Venous whole blood (1 ml) was collected from each participant into EDTA vacutainerrotocols . Four 50lciparum . A 100 µTwo independent certified malaria microscopists read the thick and thin blood smears. All discordant results were settled by an independent reading of the respective slides by a third microscopist. Parasite density was calculated based on the assumption that there are 8000 white blood cells (WBCs) in one microlitre of blood as the number of parasites observed per 200 white blood cells multiplied by 40. A slide was regarded as negative when no parasite was identified within a minimum of 200 fields.® DNA Kit  for whole blood samples according to manufacturer’s instructions. Briefly, 100 µl of whole blood was lysed with 400 µl of lysis buffer prior to running over the spin column. The DNA was eluted using 100 µl of elution buffer and stored at 4 °C for immediate use. The P. falciparum 18S rRNA gene was amplified from 20–40 ng of the extracted DNA in a 15 µl reaction volume. The PCR reactions were made up of 2.5 mM MgCl2, 200 nM deoxyribonucleoside triphosphate mix (dNTPs), 1 U of Onetaq™ DNA polymerase  and 250 nM each of forward and reverse primers (rPLU5 and rPLU1) for the primary reaction and rFAL1 (F) and rFAL2 (R) for the nested reaction [P. falciparum (MRA 102G) and double-distilled water were used as positive and negative control samples respectively for the PCR amplifications. The PCR amplification products were resolved on 2.0% agarose gels stained with 0.5 µg/ml ethidium bromide. After electrophoresis, the gels were visualized under UV and the image captured using a Vilber Lourmat Gel Dock System® .Genomic DNA was extracted from all the 740 samples using the Zymoreaction . GenomicAfter processing of the blood smears according to standard procedures , a samplt test was used to compare continuous numerical data from pregnant women and non-pregnant women in GraphPad Prism version 5. Linear correlation analysis was also used to assess possible associations between parasite density, age, haemoglobin levels and axillary temperature.The demographic and haematological data of all the participants from the three sites were compared using Kruskal–Wallis analysis and Dunn’s multiple comparison test was used as post hoc test to assess any observed differences between groups in GraphPad Prism version 5. Independent students The sensitivity and specificity as well as the positive and negative predictive values were determined using IBM SPSS version 21. IBM SPSS version 21 was also used to determine the level of agreement between the different tests were determined using the Cohen’s kappa inter-rater reliability test.Children aged between 3 and 17 years old and females represented 57.70% (427/740) and 67.20% (497/740) of the total study population respectively. The median age of the afebrile pregnant women from Ewim was significantly higher than participants from Obom and Simiw , whose median ages were not significantly different. The overall median axillary temperature for all the three study cohorts was about 36.6 °C (IQR: 36.20–36.90)  between the ages of non-pregnant women who were 18 to 40 years old from Simiw and Obom, both of which are high prevalence settings, their data was combined for comparison with similarly aged pregnant women of from Ewim. Haemoglobin levels and axillary temperatures of pregnant women were significantly lower than non-pregnant women of the same age group  , 20.4 151/740), 31.3 (231/738) and 30.8 (228/740) of participants were positive by PCR, Microscopy, UsmRDT and mRDT, respectively  among the pregnant women of Ewim, whereas among the participants  from Obom and Simiw, the UsmRDT recorded a higher prevalence than the mRDT kit in both Obom and Simiw (41.72% (121/290) versus 35.3% (103/292) and 39.62% (138/348) versus 33.91% (118/348), respectively) , older children (13–17 years old) and adults (18 years old and above) showed a general inverse relationship of age group with parasite prevalence; with adults having the lowest prevalence of Parasite prevalence in the pregnant women aged between 18 and 40 years old was lower than similarly aged non-pregnant women for all the different diagnostic tools assed in this study Fig. b. Among Overall, the UsmRDT was able to detect 95.23% (100/105) of all the samples that had parasite densities of ≥ 200 p/µl whilst the mRDT kit detected 88.57% (93/105) of these samples Tables . The senversus 42.9(9/21)) than the mRDT but s specificity was slightly higher for mRDT (96.4(53/55) versus 94.5 (52/55))  and 89% 389/435), respectively. The mRDT kit had sensitivity and specificity values of 65% (197/301) and 92% (404/438), respectively  in all three sites. Overall, the level of agreement between PCR and the RDT kit was higher for UsmRDT  than mRDT   of these mRDT negative/UsmRDT positive samples were identified to contain P. falciparum 18S rRNA gene to identify malaria parasite prevalence in afebrile study participants living in the south of Ghana. This study included pregnant women and children under the age of 10 years old as a high percentage of malaria-related deaths occur in these high risk population [Malaria parasite carriage is predominantly detected using either a malaria RDT or a combination of RDT and microscopy . Howeverpulation , and earpulation . Pregnanpulation . This maMicroscopy proved to be the least sensitive diagnostic tool in all the three cohorts. This is not surprising as a high proportion of asymptomatic infections are at submicroscopic densities . The obsPlasmodium falciparum species-specific PCR detected the highest number of asymptomatic malaria parasite carriers amongst the study participants attending antenatal checkup at Ewim (pregnant women) and from the Simiw community. This result is to be expected as PCR is known to have a lower limit of detection than the UsmRDT, the mRDT and microscopy. During pregnancy, malaria parasites are known to sequester in the placenta, making them unavailable for detection in the peripheral blood by less sensitive techniques such as microscopy [croscopy . The HRPcroscopy , 37. In croscopy .Although false positive test results were recorded for both the mRDT and the UsmRDT, the latter recorded about 4% more than the mRDT kit. False positive results were observed to be high in Obom and in children compared to adults. This has been observed in other studies , 21 espeObom has been reported as a high malaria transmission intensity setting for the past 5 years , 39, 40.Overall, the UsmRDT had a slightly higher sensitivity but lower specificity than the mRDT. Also, the performances of both RDTs were better in the high transmission settings than the low transmission settings. This could be attributable to persistent inoculation of human hosts in high transmission settings leading to higher parasite densities  and concThe UsmRDT and the mRDT kits exhibited similar specificities and positive predictive values, however the sensitivity and negative predictive values for the UsmRDT were higher than that of the mRDT kit. The level of agreement between the UsmRDT and PCR was substantial and higher than that for the mRDT kit and PCR, which was moderate  most likpfhrp2 gene deletions could also result in false negative RDT results [pfhrp2 gene deletions were not determined in this study.The high prevalence of false negative RDT results recorded by both the UsmRDT and the mRDT could have been due to the presence of low parasite densities, below their respective lower limit of detection. Low-density parasites are prevalent in asymptomatic parasite carriers during the off-peak malaria season . The pre results –49. HoweP. falciparum carriage, it correctly identified P. falciparum in 9.3% of the study participants that were not captured by the mRDT kit. In malaria endemic settings, the UsmRDT would provide an added advantage by identifying more asymptomatic P. falciparum carriers than the mRDT kit for the administration of targeted treatment.Although the UsmRDT kit was not as sensitive as PCR at detecting asymptomatic Additional file 1. Data analysis output files.


Plasmodium vivax to cause severe disease has prompted the need for a better characterization of the resulting clinical complications. India is making progress with reducing malaria, but epidemics of severe vivax malaria in Gujarat, one of the main contributors to the vivax malaria burden in the country, have been reported recently and may be the result of a decrease in transmission and immune development. Over a period of one year, we enrolled severe malaria patients admitted at the Civil Hospital in Ahmedabad, the largest city in Gujarat, to investigate the morbidity of severe vivax malaria compared to severe falciparum malaria. Patients were submitted to standard thorough clinical and laboratory investigations and only PCR-confirmed infections were selected for the present study. Severevivax malaria (30 patients) was more frequent than severe falciparum malaria (8 patients) in our setting, and it predominantly affected adults . This suggests a potential age shift in anti-malarial immunity, likely to result from the recent decrease in transmission across India. The clinical presentation of severe vivax patients was in line with previous reports, with jaundice as the most common complication. Our findings further support the need for epidemiological studies combining clinical characterization of severe vivax malaria and serological evaluation of exposure markers to monitor the impact of elimination programmes.The mounting evidence supporting the capacity of  Plasmodium vivax(Pv) is the most geographically prevalent human malaria parasite, with 2.5 billion individuals at risk of infection worldwide [Pv infection has been perceived as relatively benign, despite being debilitating and often life-threatening. As a result, clinical malaria research has mainly focused on Plasmodium falciparum(Pf) morbidity and mortality to date [Pvhas now become the predominant cause of malaria outside sub-Saharan Africa and is considered a key obstacle to malaria elimination [Pv burden globally [Pv to induce severe and potentially fatal malaria in recent years [orldwide . Histori to date . Howevermination . This isglobally , and whent years –11. Thisnt years .Pv burden in India, with a Pv/Pf ratio around 80:20 [Pv [Pvto induce a wide spectrum of severe malaria, with a risk of death similar to that observed for Pf infection [Pv invades primarily reticulocytes and uses the Duffy antigens expressed on the red blood cells as a receptor [Pv stages in the spleen, lungs, and bone marrow [Pv-infected reticulocytes may sequester and therefore play a role in the development of severe Pvmalaria.Gujarat is one of the major states contributing to the nd 80:20 . Remarka0:20 [Pv ,13. Thisnfection ,8,10,11.receptor ,15. It ie marrow –20, suggPvandPfmalaria infections.Endemic areas differ widely in terms of transmission, population immunity, and resulting severe disease frequency, prompting the need for a more comprehensive characterization of the spectrum of clinical complications in India. We conducted a prospective study to follow severe malaria patients with PCR-confirmed diagnosis admitted to the Ahmedabad Civil hospital in Gujarat, India, over a one-year period (December 2016 – November 2017), with the aim to meticulously differentiate and compare clinical and laboratory characteristicsbetweensevere Pv malaria burden in the country, accounting for 9% of the national total [Anopheles culicifacies, An. stephensi, and An. fluviatilis are the major malaria vectors in the region, with transmission closely following the monsoon, from mid-June to mid-October [The study was set up in the state of Gujarat, which has the third highest al total . Patient-October .Pf, Pvor Pf/Pv co-infections, and was conducted between December 2016 and November 2017. Microscopy-diagnosed Pf and Pv infections with or without WHO-defined severe malaria symptoms [Plasmodium species , and only PCR-confirmed cases were selected for the subsequent analyses.This prospective, hospital-based study focused on patients admitted with severesymptoms ,22 . A two-sided p < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software).All case record forms (CRFs) were anonymized, digitized, and stored using the REDCap (Research Electronic Data Capture) system, which allowed subsequent extraction of datasets . Chi-squhttps://clinepidb.org.The study data are available for download through the Clinical Epidemiology Database (ClinEpiDB)  at httpsPlasmodium positive and diagnosed using PCR amplifications either for Pf, Pv, or Pf/Pv co-infection. No study participants were found positives for P. malariae or P. ovale infections. Thirty-nine patients had WHO-defined severe malaria [Pv infection, with a median age of 33.5 years (± 23.5), and a male to female ratio of 16:14 , and a male to female ratio of 5:3. Only one patient had severe Pf/Pv co-infection ; p = 0.864. Pv patients had significantly higher sexual and asexual parasitemia compared to Pf, and Pf patients had wider variations in white blood cell counts . Thrombocytopenia, a common complication of Pf and Pv malaria [Pv and Pf infection when these demographic, socioeconomic, and epidemiological characteristics were compared. Ten severe malaria patients required blood transfusion, 7 out of 10 were infected with Pv parasites. Severe malaria Pf patients required assisted breathing (n = 3), dialysis (n = 1) and nasogastric tube (n = 2). The patient with both Pf/Pv co-infection presented complaints such as headache, chills, pains, vomiting, dizziness, elevated heart rate (96 beats/minutes) as well as increased bilirubin (24.5 mg/dL), urea (102 mg/dL), and creatinine (4.87 mg/dL) levels. Blood cultures were negative for all but one patient, who tested positive for Bacillus subtilis but was not categorized as having sepsis due to the individual’s clinical condition and immunocompetence. G6PD deficiency was only tested when deemed necessary by the physician, after asking the patient for any known blood disorders.6 Pv patients were tested for G6PD deficiency, and only one individual was positive.Forty-one out of 50 enrolled patients were  malaria and two pof 16:14 . Eight pnfection . The odd malaria , was obsPv cases, 16.7% (5/30) of the patients had history of fever or malaria in the previous 12 months and 20% (6/30) of them also had typhoid (n = 1), dengue (n = 1), chikungunya (n = 1), or tuberculosis (n = 1) co-infections; 23.3% (7/30) of patients came with fever (≥37.5°C). The median heart rate and body temperature were 88.0°C and 37.2°C, respectively. The demographic, socioeconomic, and epidemiological characteristics of these patients are presented in Pv cases, prostration (n = 27) was the most prevalent of the severe malaria signs  of the patients had a previous history of fever or malaria in the previous 12 months and no co-infections were found. Only one patient had a complaint of fever (≥37.5°C). The median heart rate and body temperature were 84.0°C and 37.2°C, respectively. The demographic, socioeconomic, and epidemiological characteristics of these patients are presented in Pfpatients, prostration (n = 8) was the most prevalent of the severe malaria signs  were admitted in hospital wards and were followed, treated, and evaluated until discharge, lasting for 3.39 days (± 2.13) in the hospital. No significant difference (p = 0.538) was observed for hospital admission duration between severe Pf (3.40 days ± 1.85) and Pv (3.86 days ± 3.14) patients; 78% (32/41) patients recovered, 14.6% (6/41) patients were discharged against the physician’s advice and 7.3% (3/41) patients died. Two of the fatal cases had Pf mono-infections and 1 Pf/Pv co-infection.All study participants  compared to 5% in Pv(16/323) in 2011.Jaundice was only observed in severe fections ,35,36. Hnfection –40. Howepatients ,41,42. SPv cases are treated with a 3-day course of chloroquine (25 mg/kg) followed by a 14-day course of primaquine (0.25 mg/kg). The use of primaquine prevents relapse by killing the dormant liver stages of Pv. However, the drug presents greater risks of hemolysis in G6PD-deficient patients [Pvpatients enrolled in this study underwent G6PD deficiency testing, and only one individual was found positive. In accordance with the Indian national policy, all G6PD-negative,Pv-infected patients were treated with primaquine [Pv and support future malaria elimination efforts [Severepatients ,43,43. Gpatients –46, withpatients ,47. Follimaquine , and no  efforts .Plasmodium infection [Socioeconomic factors have been shown to influence malaria case management, as they promote causal behaviors and choices ,50. In tnfection ,52. Thernfection .Pv-positive cases in this other site was 85.9% (61/71) and 83.3% (55/66) by microscopy and PCR, respectively [Pv cases reported in the present study. Collectively, these findings suggest that the recent decline in malaria transmission in India has not affected the overall ratio of Pv: Pf in Gujarat, which was reported to be 80:20 in 2014 [Pv was under 15 years in our cohort, a proportion of 3.3% that is ten-fold lower than the 30% of the patients under 14 with severe Pv reported across India in 2016 [Pv cases in Gujarat has declined steadily since 2004, and the lack of exposure may explain this shift in the age of the patients with severe disease [Pv malaria and serological evaluation of exposure markers to monitor the impact of elimination programmes in the future.India has the world’s largest national malaria control program ,53, whicectively , which a in 2014 . However in 2014 . Only on in 2016 . Inciden disease . Our finPv was more frequent than severe Pf infection among patients admitted at Ahmedabad Civil Hospital, and it affected predominantly adults. Their clinical presentation was in line with previous reports, and the overall fatality rate due to severe malaria was lower than average (~7%). The present report contributes to our understanding of the disease spectrum of severe Pvmalaria in India and is indicative of a high prevalence of severe Pv in Ahmedabad, which should be considered in local malaria elimination campaigns.In conclusion, severe malaria due to Click here for additional data file.


Plasmodium falciparum isolates with histidine-rich protein 2 (pfhrp2) gene deletion as one of the possible factors contributing to the failure of PfHRP2-based RDTs in detecting malaria. A total of 300 blood samples were collected from several health centres in Nyala City, Western Sudan. The performance of PfHRP2-based RDTs in relation to microscopy was examined and the PCR-confirmed samples were investigated for the presence of pfhrp2 gene. A total of 113 out of 300 patients were P. falciparum positive by microscopy. Among them, 93.81% (106 out of 113) were positives by the PfHRP2 RDTs. Seven isolates were identified as false negative on the basis of the RDTs results. Only one isolate  potentially has pfhrp2 gene deletion. The sensitivity and specificity of PfHRP2-based RDTs were 93.81% and 100%, respectively. The results provide insights into the pfhrp2 gene deletion amongst P. falciparum population from Sudan. However, further studies with a large and systematic collection from different geographical settings across the country are needed.In remote areas of malaria-endemic countries, rapid diagnostic tests (RDTs) have dramatically improved parasitological confirmation of suspected malaria cases, especially when skilled microscopists are not available. This study was designed to determine the frequency of  Late diagnosis and treatment of the disease can lead to life threatening conditions2. The World Health Organization (WHO) implements vector control, early diagnosis and prompt treatment strategies to eliminate malaria3. Accordingly, the early, rapid and accurate diagnosis is an essential component in malaria control and elimination programs. Microscopic detection of malaria parasites is still considered the gold standard for diagnosis of the disease; this method is relatively sensitive and allows parasitic quantitatification and species identification5. However, in remote areas, this procedure may not be easily available due to the requirement of high-quality equipment and the challenge in maintaining well-trained microscopists6. Alternatively, WHO recommends the use of rapid diagnostic tests (RDTs), which have later become an alternative way of establishing the rapid diagnosis of malaria infections7. Accordingly, RDTs were implemented as part of malaria case management along with microscopy for diagnosing malaria among suspected cases in Sudan8.Malaria remains a major public health problem particularly in sub Saharan Africa in which majority of the populations are at riskPlasmodium falciparum9. Recent reports indicate that PfHRP2-based RDTs may provide false negative results10. Though among the causes of false negative results are product quality, transportation, storage distribution, parasite density and due to the existence of P. falciparum isolates that lack the pfhrp2 gene. Such deletions have been reported in a broader range of malaria-endemic countries in Africa13, South America15 and Asia16.Most of the current malaria RDTs target the histidine-rich protein-2 (HRP2), which is a species-specific antigen of P. falciparum isolates with pfhrp2 gene deletion also circulate in the neighbouring countries18 with more than 80% of microscopically confirmed isolates found to carry pfhrp2 gene deletion in Eritrea19. Recently, P. falciparum isolates with deleted pfhrp2 was also reported in UK travellers from Sudan and south Sudan11. This condition highlights the importance of molecular surveillance to detect these deletions because they could lead to false-negative diagnoses when using PfHRP2-based malaria RDTs20.Recent evidence suggested that P. falciparum parasites lacking the pfhrp2 gene, sporadic data is available about the existence of P. falciparum parasites with pfhrp2 deletion in Sudan, which has high burden of malaria in the region. Screening of P. falciparum parasites with pfhrp2 deletions in Sudan is essential to asses RDTs performance and the impact on clinical case management and malaria elimination efforts. Therefore, the present study aimed to investigate the pfhrp2 gene deletion in P. falciparum isolates in Nayala City, Western Sudan.Despite the diagnostic threat posed by P. falciparum infection. Subsequently, the microscopically confirmed specimens were tested against HRP2-based malaria RDTs. Amongst the isolates, the PfHRP2 RDTs identified 106 (93.81%) positives. A total of 7 out of 113 of P. falciparum microscopy-confirmed cases were RDT negative. This finding resulted in overall 93.81% [95% confidence interval (CI) 87.65–97.47] and 100% (95% CI 98.05–100) sensitivities and specificities of the PfHRP2-based RDTs respectively  because all of them generated PCR products for P. falciparum specific ssrRNA primers. Therefore, the 106 RDT-positive isolates were found not eligible for further pfhrp2 deletion analysis. The remaining seven isolates were further investigated to examine whether pfhrp2 gene was lacking given the negative RDT results of these specimens.All the microscopy-confirmed Plasmodium falciparum ssrRNA gene was amplified in all seven RDT-negative isolates. The samples were subjected to another round of PCR to amplify the flanking regions of pfhrp2. The pfhrp2 gene was successfully amplified in most of the isolates , this exclude the deletion of the entire gene in all these samples. Amongst the seven samples, the entire pfhrp2 gene was deleted in one sample (14.3%). The pfcsp gene was successfully amplified to confirm this deletion. In addition the parasitic density of this sample was relatively high  is unknown, 0.9% (1/113) of the false negative can be explained by pfhrp2 deletion. The six samples determined as positive by microscopy and negative using HRP2 RDT were confirmed positive by PCR as indicated by the amplified pfhrp2 gene. This finding could be attributed to the sequence variability among the pfhrp2 region or low parasitaemia as some of the samples had parasitaemia below the detection limit of RDT23. Among the false negative isolates by RDT, the number of parasites density varies from 96 to 5,120 asexual parasites/µl. A very low parasite densities or target antigen concentrations could also contributed to false negative results. Another possible explanation is that other factors, including the product quality, transportation, storage and distribution, possibly affected the performance of RDTs. This is very unlikely as SD Bioline Pf/Pv/Mixed Combo cassettes used in this study was prequalified by WHO, and the shipment and storage conditions in the field were optimal and followed WHO recommendations. Thus, they are less likely to be the reasons for false negative results in this study. The most likely explanation could be low parasitaemia and/or sequence variation of the repeat region of the pfhrp2, which is targeted by the HRP2-based RDT.Our study reports, for the first time, the presence of pfhrp2 negative isolate may not reflect the actual number of the pfhrp2 negative P. falciparum among the study population. This is because the 106 RDT positive isolates were not screened for pfhrp2 gene deletion. The RDT positive signal could be generated from HRP3 due to the cross reactivity of HRP3 antigen with the HRP2. In this context, the actual number of pfhrp2 deletion could be higher than the estimated prevalence. Nevertheless, whether the deletion of pfhrp2 is compensated by the presence of HRP3 could not be determined and this is one of the limitations of the study.The only one pfhrp2 gene in samples obtained from Sudanese patients. A similar recent study with a small sample size conducted by our group in the centre of Sudan reported genetic variations in pfhrp2 and pfhrp2-negative samples but deletion was not confirmed21. Our current study, however, found one deletion that caused RDT negative result. Study area, sample size and collection period could explain the differences in findings. Other studies also reported genetic variation and suspected deletion of pfhrp2 gene22 as well as pfhrp2 deletions in malaria patients among UK travellers from Sudan11, though it is not clear which part of Sudan the UK patient travelled from.Limited information is available regarding the deletions of pfhrp2 gene have been unequivocally identified in many malaria endemic regions16. Despite the low prevalence of pfhrp2 deletion in the current study, the rates of deletions vary considerably in Africa and globally. Remarkably high rates of pfhrp2 gene deletions were reported from neighbouring Eritrea17. Moreover, findings from several countries indicated that P. falciparum isolate with pfhrp2 gene deletion is circulating in east Africa, including Kenya18, Tanzania and Uganda24, South Sudan11 and Rwanda25. By contrast, reports of presence of pfhrp2 gene deletions in West Africa have been limited, including in Mali12 and Senegal13. Recent studies provide evidence of deletion of the pfhrp2 gene as reported from Equatorial Guinea26 and Nigeria27. However, in many highly malaria-endemic countries, data about pfhrp2 deletion are missing. This underscores the importance of conducting comprehensive molecular surveillance to investigate the pfhrp2 gene deletion in all endemic countries.To date, parasites lacking the pfhrp2 deletion in western Sudan and the prevalence does not reflect the true prevalence in the whole region. However, the prevalence of pfhrp2 deletion is considerably lower than 5%, which is the minimum threshold needed to change the RDT types based on WHO guidelines. Evidently, malaria RDTs tests that rely on PfHRP2 for the detection of P. falciparum infections are still suitable for P. falciparum malaria diagnosis in Sudan.The study was not designed to systematically identify pfhrp2 gene across the whole country, this study was limited by the collection of samples from a single location in the western part of Sudan. The research needs to be expanded to cover other geographic regions given that this finding may not be representative of the situation in the whole country. Moreover, screening of pfhrp2 gene as well as the pfhrp3 gene among RDTs positive isolates could help in determining the actual number of HRP gene deletion among the study population, which is one of the limitations of the current study.Despite the importance of monitoring the deletion of P. falciparum isolates with pfhrp2 gene deletion are present in the parasite populations in western Sudan. The PfHRP2-based RDTs used in this study detected the majority of P. falciparum infections. Further studies, including a large and systematic collection from different geographical settings across the country, will be essential to estimate the pfhrp2 deletion prevalence and to better assess the performance of PfHRP2-based RDTs.In conclusion, this study revealed that 2. The city lies in a savannah zone between 9° 30′ 13″ N latitude and 15° 27′ 28″ E longitude. Patients presented with symptoms consistent with uncomplicated malaria attending the selected health centres were enrolled and blood smears for Malaria were obtained from each patient.A total of 300 suspected patients with malaria from 4 health centres in Nyala City were enrolled in a cross-sectional study conducted between July 2018 and April 2019. Nyala is the capital of the South Darfur State, Western Sudan and covers an area of about 127,300 kmA total of 300 venous blood samples (3 ml each) were collected from each participant for the preparation of thin and thick blood films and for rapid diagnostic tests (RDTs) and for DNA extraction. An aliquot of blood was saved on a Whatman 3MM filter paper  for parasite specimen preservation.P. falciparum and P. vivax, respectively. The RDT assays were performed, and the results were interpreted according to the instructions provided by the manufacturers.Thick and thin blood smears were prepared and stained with 10% Giemsa for 15 min. Subsequently, the stained slides were examined under a 100 × oil immersion microscope by two independent expert microscopists . Parasitaemia was calculated by counting the number of parasites observed per 200 leukocytes and assuming a total of 8,000 leukocytes/µl. Simultaneously, all blood samples were assayed with SD BIOLINE Malaria Ag P.f/P.v RDT, which is a qualitative and differential test for the immunological detection of HRP2 and parasite lactate dehydrogenase of For molecular analysis, genomic DNA was extracted from blood samples by using a Geneius™ Micro gDNA Extraction kit  in accordance with the manufacturer’s instructions. DNA was resuspended in 100 µl elution buffer and stored at − 20 °C.P. falciparum infection was further confirmed by species-specific nested PCR by using a small subunit ribosomal RNA (ssrRNA) gene via a previously described two-step procedure28. Plasmodium-specific primers were first used, and the PCR product of the positive samples was used as a template in a multiplex PCR system through species-specific primers.The presence of P. falciparum samples but RDT negative were subjected to pfhrp2 PCR amplification. Specific primers Pfhrp2-F (5′ ATTCCGCATTTAATAATAACTTG TGTAGC 3′) and Pfhrp2-R (5′ ATGGCGTAGGCAATGTGTGG 3′) targets exon 2 of pfhrp2 were used as described previously12.Microscopic and PCR positive pfhrp2 was amplified were subjected to P. falciparum circumsporozoite gene (pfcsp) PCR amplification, with the assumption that successful ssrRNA and csp gene amplification indicates reasonable quantity and quality of DNA that would allow pfhrp2 amplification. Thus, all the samples that were not amplified by pfhrp2 PCR may have pfhrp2 gene deletion.The PCR mixture had a total volume of 20 µl containing 4 µl of 5 × HOT FIREPol Blend Master Mix, 0.75 µM each primer and 2 µl DNA. The thermoprofile process consisted of an initial denaturation step at 95 °C for 4 min, followed by 35 cycles at 95 °C for 30 s, 58 °C for 45 s, 72 °C for 1 min and a final extension step at 72 °C for 5 min. Furthermore, the samples in which no This study protocol was reviewed and approved by the Institute of Molecular Biology, University of Nyala and the committee of the Research directorate, Federal Ministry of Health (fmoh/nhrc/rd/rec). Prior to blood samples collection, written informed consent was obtained from all subjects and also from a parent or guardian for children less than 18 years of age. All methods were performed in accordance with the relevant guidelines and regulations.


Colombia has officially adopted the parasite density levels of severe malaria established by the WHO . These values have been inferred from areas of high transmission in Africa and are not consistent with the dynamics of low and unstable transmission in Colombia. The objective of this study was therefore to determine the parasite density values observed in patients with severe malaria and their distribution in the different ecoepidemiological regions of Colombia.A retrospective and descriptive study of confirmed cases of severe malaria was conducted in endemic areas of malaria in Colombia over the period 2014–2017. Data were collected from secondary sources of the Subnational Programs of Malaria Prevention and Control. Person, place, and time variables were selected. The official definition of severe malaria was adopted, and compliance with these criteria was determined. Univariate and bivariate analyses were conducted with absolute and relative frequency measures, and the relevant statistical tests were applied.Plasmodium falciparum and 5,815  for Plasmodium vivax. The highest parasite density values were recorded in the Amazon ecoepidemiological region , and the lowest values were recorded in the Andean region .The overall parasite density values in Colombia showed a geometric mean of 5,919 parasites/μl . By parasite species, the values were 6,151  for In endemic areas of low and unstable malaria transmission in the Colombian territory, the parasite density levels observed in populations with severe malaria are lower than the officially established values. The parasite density criterion is not really a relevant criterion for the definition of severe cases in Colombia and it certainly not be used to make a clinical decision about the severity of the disease. Plasmodium falciparum or Plasmodium vivax, although traditionally, the progression to severe and lethal forms has been attributed mainly to infections by the former species [P. falciparum, P. vivax or mixed infection, and with one or more complication criteria based on the malaria clinical practice guideline, included in the national surveillance system. In Colombia between 2010 and 2018, the surveillance system was notified of a total of 6,684 cases of severe malaria, most of which were caused by P. vivax and more than 95% of the patients were hospitalized and managed in critical care units. The remaining small percentage, due to geographic accessibility problems and attention opportunities, are managed at the primary level [Despite marked progress in the control and prevention of malaria, the disease remains a major public health problem that contributes to morbidity and mortality, especially in children under 5 years of age ,2. Malar species . Early a species ,5. In thThere is evidence of a relationship between parasitemia density and variation in the prognosis of patients with severe malaria as a function of the level of disease transmission . High leThere is currently a gap in knowledge regarding parasite density thresholds in patients with severe malaria in endemic regions of Colombia. Although this threshold has been defined as ≥50,000 parasites/μl .Defining parasite density levels is important for evaluating the potential severity of the disease, the initiation of etiological treatment and the monitoring of the therapeutic response. Therefore, it is necessary to generate evidence to serve as a national reference for the diagnosis and management of severe malaria cases and to establish valid national parameters for surveillance protocols and clinical management guidelines in Colombia. All of the above efforts will contribute to and facilitate the implementation of diagnosis, treatment, investigation and response as a fundamental strategy within the elimination process that is currently being carried out in the country . The objA retrospective, observational and descriptive study observational was conducted based on the confirmed cases of severe malaria recorded in the endemic departments with the greatest burden of this event in the country from 2014–2017. Secondary information sources provided by the departmental and district programs for the prevention and control of malaria in Colombia were used.The definition of a case of severe malaria established by the national malaria epidemiological surveillance protocol was adopted . Cases tThe criteria contained in the malaria surveillance protocol for Colombia are as follows . A case Disorders of consciousness including coma, multiple seizures more than two episodes in 24 hours, prostration, or extreme weakness of the patient with difficulty or inability to sit, stand, walk without assistance, inability to feed.Serum creatinine >1.5 mg/dL, BUN >20 mg and or urine volume <400 ml in 24 hours (adults) or urine volume <12 ml/kg of weight in 24 hours (children).Presence of jaundice  and abnormal liver function tests (Transaminases >40 IU/L).Increased respiratory rate on admission, presence of pulmonary auscultation disorders such as wheezing, snoring, rales, and radiographic changes consistent with pulmonary edema.P. falciparum or in mixed malaria with P. vivax and schizontemia.Shock, hypoglycemia (<40 mg/dL), hyperemesis, hyperpyrexia (> 39.5°C), severe anemia (hemoglobin <7 g/dL), spontaneous abnormal bleeding or disseminated intravascular coagulation, metabolic acidosis , gross hemoglobinuria or hyperparasitemia ≥50,000 asexual forms/μl of Plasmodium spp. that was developed by the National Institute of Health. Microscopic examination of the blood smear is the gold standard in malaria diagnosis.The diagnosis of malaria and the level of parasite density in Colombia is made according to the guideline for laboratory surveillance of parasites of the genus The method used to determine the parasite density in all cases was a quantitative method that allows estimating the level of parasitemia in the thick blood smear examination, using this simple mathematical formula: Number of parasites x 8,000 / Number of WBCs (200).Count parasites in relation to a predetermined number of white blood cell (WBC) count and an average of 8000/μl is taken as standard.200 leucocytes are counted.All parasite species and forms including both sexual and asexual forms are counted together.If >10 parasites/100 fields are observed when counting 200 WBCs, the formula based on 200 WBCs can be applied.If <9 parasites/100 fields are observed when counting 200 WBCs, then the count should continue until reaching 500 WBCs and apply the formula based on 500 WBCs.If more than 500 parasites are observed without counting 200 WBCs, the count will be terminated when the reading of the last field is completed and the parasitemia must be calculated according to the previous formula.Ecoepidemiological regions were defined as receptive areas for malaria located below 1,600 meters above sea level, with geographic, ecological and entomological conditions favoring the endemic transmission of the disease . AccordiA database was designed in MS Excel  that contained the different study variables, and quality control of the data was performed. Data analysis was conducted using Stata  and ArcGIS version 10.5  was used to produce maps. The main person, place and time variables were count and type of parasitic species; patient age and sex; country, department, district, municipality and ecoepidemiological region; and endemic and epidemic years. In the data analysis, absolute and relative frequencies were used at the national and regional levels. The p75, p50 and p25 for parasitemia were determined, and the interquartile range was calculated. Measures of central tendency were applied, including the geometric mean with 95% confidence interval. Malaria prevalence for each age group were calculated as the number of malaria case events divided by the total number of people in that age group and considering the infecting parasite species.Graphical evaluation of the sampling distribution of the parasitemia values showed a data distribution skewed to the right. This was confirmed with the Kolmogorov-Smirnov hypothesis test . Continuous variables were analyzed using the Mann-Whitney U test. The level of significance adopted was p<0.05.The present study met the ethical requirements established in Resolution 8430 of 1993 of the Ministry of Health of Colombia, Article 11, which establishes that studies such as the present one are risk-free and do not require approval by the Ethics Committee. Confidentiality and anonymity of the data were guaranteed.Over the study period, 2,834 cases of severe malaria were recorded, of which 2,352 cases met the inclusion criteria. The mean age was 24 years, with a standard deviation of 18.2 (range 1–97 years). The female sex predominated with 55.7% as sex ratio of 0.79. In terms of ethnicity, 80% of the cases occurred in the Afromestizo population.P. vivax; 1,090 (46.3%) to P. falciparum; and the rest to mixed forms. In terms of the distribution by ecoepidemiological region, more than 90% of the cases of severe malaria were reported in the Pacific Region with 1,728 cases (73.5%), and in the region comprised by Sinú-San Jorge-Lower Cauca-Urabá-Middle Magdalena, with 469 (19.9%) cases. The remaining 6.6% were distributed among the Amazon , Orinoquía  and Andean  regions  corresponding to  regions .P. vivax was responsible for the largest proportion of cases, with 49.4% (1162).Regarding the distribution by year, it was observed that the largest number of cases was reported in 2016,  and the smallest number was reported in 2014 . On average, 588 cases of severe malaria were recorded per year. In terms of parasite species, P. falciparum and 5815 parasites/μl  for P. vivax. The interquartile range for parasitemia was 6,294. In other words, the intermediate 50% of the data was between 2,320 (Q1) and 14,909 (Q3).The geometric mean for overall parasitemia in severe malaria in Colombia was 5,919 parasites/μl . In terms of parasite species, this value was 6,151 parasites/μl  for P. vivax infection predominated, and in contrast, P. falciparum infection was more frequent in the age group from 5 to 14 years old , and the 15- to 29-year-old group had the lowest  . In the ears old .Regarding the distribution of parasitemia by ecoepidemiological region, the highest values were recorded in the Amazon region , and the lowest values were recorded in the Andean region  .P. falciparum in the Orinoquía region  and lower parasitemia was observed in the Amazon region . In turn, for P. vivax, higher parasitemia occurred in the Amazon region  and lower parasitemia occurred in the Andean region   .During the epidemic year studied (2016), the geometric mean of parasitemia recorded was 6122 . In contrast, densities during the nonepidemic year selected (2014) were lower at 4,101 . These differences in the observed values were significant between the epidemic and nonepidemic years (p<0.001) and among the ecoepidemiological regions (p = 0.045).No differences (p = 0.299) were observed in the geometric means of parasitemia between women  and men  or among the different age groups (p = 0.167). However, there was a significant difference between the parasite species (p = 0.039).In the present study, it was possible to establish that in the active malaria-endemic areas in the national territory, which have characteristics of low and unstable transmission, the parasite density levels that were observed in the population with severe forms of malaria are lower than the thresholds established and officially recommended in Colombia. Additionally, it was possible to demonstrate the existence of variability in the distribution of the geometric means of parasitemia density in cases of severe malaria with a trend toward a predominance of low values, both overall and in the different ecoepidemiological regions of the country.P. falciparum is hyperparasitemia, established by a cut-off point of >50,000 parasites/μl [P. falciparum and P. vivax in the present study. The definition of hyperparasitemia is variable and depends on the level of transmission in specific ecoepidemiological scenarios. In environments with low and unstable transmission, parasitemia >2%  has been recorded, and in areas of moderate-high transmission intensity and stability, parasitemia values >5%  have been reported [According to current guidelines and protocols applied in Colombia, one of the criteria applied to define severe malaria caused by sites/μl . These vreported . Neverthreported .P. falciparum in Colombia has changed in recent decades, from 250,000 parasites/μl at the end of the last century to 100,000 parasites/μl at the beginning of the first decade and up to 50,000 parasites/μl currently, according to the threshold established by the WHO [The criterion of hyperparasitemia for severe malaria caused by  the WHO ,16,17.P. falciparum had an average parasitemia <25000 parasites/μl, which was considered low at that time [P. vivax infections  than in infections by P. falciparum  [A study conducted 1988 and 1989 in the Antioquia regions of Urabá and Lower Cauca, with low and unstable transmission, in which hyperparasitemia was established as 250,000 parasites/μl, found that approximately 40% of the included cases of severe malaria caused by hat time . LikewisP. falciparum infections, and mean parasitemia of 2,282 parasites/μl in have been reported for P. vivax infections [P. vivax [The low parasitemia thresholds recorded in the present study coincide with those reported in other endemic countries in the Americas with transmission characteristics similar to ours. In studies carried out in communities of the Peruvian Amazon, geometric mean parasitemia of 4,011 parasites/μl (95% CI: 811–9746) and 3,976 parasites/μl have been reported for fections ,21. LikeP. vivax .P. falciparum was slightly higher than that in cases caused by P. vivax, although no significant differences were observed between these plasmodial species. One explanation is that P. falciparum merozoites penetrate indiscriminately and rapidly replicate in young or old red blood cells of affected patients, while those of P. vivax show a preference for young cells [P. vivax is generally characterized by a lower parasitemia rate than that observed in P. falciparum, but the pathogenesis related to this clinical form is not associated with significant microvascular obstruction of vital organs. Another possible explanation is that low parasitemia may mask parasite sequestration outside the vascular system, which could explain the development of severe syndromes in patients with low parasitemia levels [The present study showed that parasitemia in cases of severe malaria caused by ng cells . For thia levels .P. vivax that was very similar to the parameters observed in cases of P. falciparum. This is noteworthy because the WHO excludes this criterion from the definition of severe malaria caused by this species. In addition, the absence of this criterion reduces the specificity of the definition of severe cases caused by P. vivax [It is noteworthy that in this study, a defined and consistent pattern was observed in the parasitemia density of severe malaria caused by P. vivax .In terms of demographic variables, the highest parasite densities were recorded in the 15- to 29-year age group. Such individuals are part of the economically active group of susceptible migrants from nonendemic areas moving to territories with active disease transmission. According to the 2017–2018 malaria epidemiological report, individuals in this age group were those most affected by severe forms of the disease . This paSome heterogeneity was observed in the parasite density results by endemic ecoepidemiological region; the highest densities were found in the Amazon and Orinoquía regions. Possible explanations for this finding are related to the existence of receptive areas of very low transmission with low density and high dispersion of the native population, high migration of susceptible populations and difficulties accessing health services, all of which influence the dynamics of transmission in these areas ,29,30. IP. vivax  and P. falciparum  [Parasite densities during epidemic periods were higher than those during endemic periods. This finding could be explained by the dynamics inherent to the factors favoring epidemics, such as a concentration of susceptible individuals from nonendemic areas, a native population with low immunity to the infection, and a native or migrant population carrying parasites ,34. Howe3–7,943) .The results of this study show that the parasite density criterion is not really a relevant criterion for the definition of severe cases in Colombia, since the low levels of parasitemia observed in this large number of patients with severe malaria are similar to the levels found in patients with uncomplicated malaria , 37. TheThe main limitations of this study are related to the quality of the data and the secondary sources used, which may present information bias. However, the data were cleaned to ensure their reliability and validity in order to control for this bias. Another limitation was the definition of the lower limit of parasitemia of 250 parasites/μL, which was justified given that beyond this parasitemia level, cases were detected that met other criteria for severe malaria.P. vivax are similar to those recorded in cases caused by P. falciparum and could serve as a cut-off point for the definition of hyperparasitemia in severe malaria caused by this species. Further studies are needed to evaluate the trend of parasitemia density in severe malaria caused by P. vivax and to establish the validity of the cut-off point determined in the present study.The parasite density criterion is not really a relevant criterion for the definition of severe cases in Colombia and it certainly not be used to make a clinical decision about the severity of the disease. The parasitemia densities in severe malaria cases recorded in Colombia are low (<0.5%) and consistent with the expectation in settings with low and unstable transmission. It was found that in the different ecoepidemiological regions, the parasite densities were less than 10,000 parasites/μl, and this finding will allow the definition of specific parameters in the different endemic areas, taking into account that these parameters can vary over time and space. The parasitemia densities in severe malaria caused by


Plasmodium falciparum (Pf).The sickle‐cell trait phenotype is associated with protection from malaria. Multiple molecular mechanisms have been proposed to explain this protection, but the role of the host immune system has been poorly investigated. We hypothesised that cellular immunity to malaria may develop differently in sickle‐cell trait children (HbAS) and children with normal haemoglobin (HbAA) repeatedly exposed to Pf transmission season and during the first malaria episode of the ensuing transmission season from HbAS and HbAA children were analysed by multiplex bead‐based assay and comprehensive multi‐dimensional flow cytometry profiling.Paired samples collected prior to the Pf transmission season, with a distinct NK subset. These cells were identified as a novel subset of memory‐activated NK cells characterised by reduced expression of the ecto‐enzyme CD38 as well as co‐expression of high levels of HLA‐DR and CD45RO. The frequency of this NK subset before the transmission season was negatively correlated with parasite density quantified during the first malaria episode of the ensuing transmission season. Functional assessment revealed that these CD38dim CD45RO+ HLA‐DR+ NK cells represent a important source of IFN‐γ.Cellular immune profiles were enriched in HbAS relative to HbAA children before the start of the Our data suggest that this novel memory‐activated NK cell subset may contribute to an accelerated and enhanced IFN‐γ‐mediated immune response and to control of parasite density in individuals with the sickle‐cell trait. This distinct cellular immune profile may contribute to predispose HbAS children to a relative protection from malaria. Plasmodium falciparum. Our data suggest that these cells present before the start of the transmission season may contribute to an accelerated and enhanced IFN‐γ‐mediated immune response and to control of parasite density during the first malaria episode of the ensuing season.We present the first evidence for host cellular immunity in HbAS‐mediated relative protection from malaria. We identify the key cell population as a novel population of memory‐activated natural killer cells, which are a major source of IFN‐γ and associate with low parasitaemia in sickle‐cell trait children repeatedly exposed to  Plasmodium falciparum (Pf) infection,Pf transmission.The sickle‐cell trait phenotype results from the heterodimerisation of normal haemoglobin (Hb) A with Hb presenting the β6 Glu → Val single amino acid substitution known as HbS. Whereas the homodimerisation of mutated HbS is responsible for the sickle‐cell disease, which is characterised by a chronic haemolytic anaemia, no severe conditions are associated with the sickle‐cell trait phenotype (HbAS). On the contrary, it is well established that individuals carrying the heterozygous form HbAS are relatively protected from malaria.A number of molecular mechanisms have been proposed to explain the relative protection from malaria displayed by HbAS individuals, including parasite growth inhibition in hypoxic condition,Plasmodium burden. IFN‐γ, a key cytokine of the systemic immune response produced by both innate and adaptive immune cells, has been implicated in the protective immunity to Pf infection.Pf reinfection.Ex vivo studies have demonstrated that natural killer (NK) cells are the major source of IFN‐γ during the very early immune response following Pf infection.Pf infection, a memory‐like status of NK has been suggested based on their adaptive T‐cell‐dependent IFN‐γ responsePf‐exposed individuals, the frequency of an adaptive NK subset has been correlated with protection from malaria.In malaria‐endemic area, naturally acquired immunity is known to develop slowly with age and exposure.Pf. Accordingly, we compared IFN‐γ‐associated immune profiles of HbAS and HbAA children from a malaria‐endemic area of Mali by evaluating (1) the capacity of peripheral blood mononuclear cells (PBMCs) to respond to stimulation, and (2) the frequency of IFN‐γ‐producing cell subsets in total PBMCs, using paired samples collected prior to the Pf transmission season as well as during the first malaria episode of the ensuing transmission season. We then focused our work on NK cells, which were preferentially associated with sickle‐cell trait‐mediated protection, and identified a distinct subset of NK cells that was specifically enriched in HbAS children compared to HbAA children before the start of the Pf transmission season. We defined the phenotypic profile of these NK cells as CD38dim CD45RO+ HLA‐DR+ and characterised their functional profile.In this study, we hypothesised that cellular immunity to malaria may develop differently in HbAS and HbAA children repeatedly exposed to Pf to respond to stimulation. PBMCs collected before the start of the Pf transmission season (baseline) and during the first malaria episode of the ensuing transmission season  were stimulated in vitro, and the concentration of a broad range of cytokines and chemokines was measured in supernatants. Of all the cytokines and chemokines tested, only IFN‐γ was significantly different between HbAS and HbAA children before the start of the Pf transmission season , all subjects displayed a haemoglobin level > 7 g dL−1, an axillary temperature ≤ 37.5°C and were free of acute systemic illness and chronic disease. Malaria episodes were detected by passive  and active  surveillance. Malaria episodes were defined as parasitaemia ≥ 2500 parasites μL−1, an axillary temperature > 37.5°C and/or signs and symptoms consistent with malaria.Subjects included in the study presented herein were a subset from individuals aged 3 months to 25 years, enrolled in a previously described cohort study.Pf infection (as by PCR), with blood samples and clinical data available before the start of the Pf transmission season (Table P = 0.551), body weight (P = 0.460), age (P = 0.741) and haemoglobin concentration (P = 0.225)  was collected at baseline and at the time of the first malaria episode into sodium heparin tubes  and transported to the local laboratory for processing within 3 h of collection. PBMCs were isolated by standard density gradient centrifugation and stored at −80°C overnight before long‐term storage in liquid nitrogen.Haemoglobin typing for HbA, HbS and HbC was determined by high‐performance liquid chromatography using a D‐10 haemoglobin analyser .Pf asexual parasites extracted from dried blood spots as previously described.Pf 18S rRNA gene was amplified by 15‐cycle standard PCR amplification followed by a qPCR. The limit of detection of the nested qPCR was ~0.5 parasites μL−1.Parasite density was quantified from genomic DNA of −1 of PMA and 1000 ng mL−1 of Ionomycin  for 6 h at 37°C by using the Human ProcartaPlex Panel  following the manufacturer’s instructions. Detection was performed using a MagPIX analyser driven by xPONENT software .Concentrations of IFN‐γ, IL‐1β, IL‐6, IL‐12p70, IL‐17A, IP‐10, MCP‐1, MIP‐1α, MIP‐1β and TNF‐α were quantified in the supernatant of PBMCs stimulated with 20 ng mL−1 of PMA and 1000 ng mL−1 of Ionomycin (Sigma‐Aldrich) for 6 h at 37°C. Cells were then stained for 20 min with phenotype‐associated markers with anti‐human CD3‐APC‐H7 (SK7), CD4‐BUV495 (SK3), CD56‐PE (B159) , CD8‐Pe‐Cy7  and TCRγδ‐FITC ; and for activation/memory‐associated markers with anti‐human CD45RO‐BV510  and HLA‐DR‐PerCP‐Cy5.5 ; and with anti‐human CD38‐BV650 , monoclonal antibodies (mAbs). Samples were then analysed using a BD LSRFortessa X20 driven by FACSDiva software (BD Biosciences). FlowJo software version 10.4 was used for gating. Because of the limited availability of samples for some subjects at their first malaria episode, flow cytometry analyses could not be performed on the entire study population.Peripheral blood mononuclear cells isolated from peripheral blood samples from HbAS and HbAA children were stimulated with 20 ng mL−1 of PMA and 1000 ng mL−1 of Ionomycin (Sigma‐Aldrich) for 1 h at 37°C. Monensin A  was added, and cells were incubated for a further 5 h at 37°C. Stimulated cells were then stained with anti‐human CD3, CD56, HLA‐DR, CD45RO and CD38 mAbs (as described above) for 20 mins; fixed and permeabilised using the Cytofix/Cytoperm kit (BD Biosciences) following the manufacturer’s instructions; and then stained with either anti‐human IFN‐γ‐FITC  and TNF‐α‐BV785 , or perforin‐FITC (B‐D48) and Granzyme‐B‐AF647 (QA16A02) (both BioLegend) mAbs. Functional profile of NK cells was analysed on a BD LSRFortessa X20 driven by FACSDiva software (BD Biosciences). FlowJo software version 10.4 was used for gating.Peripheral blood mononuclear cells isolated from malaria‐naïve buffy coats were stimulated with 20 ng mL−1 of PMA and 1000 ng mL−1 of Ionomycin (Sigma‐Aldrich) for 6 h. Stimulated PBMCs were stained with CD3, CD56, HLA‐DR, CD45RO, CD38 (as described above) and NKG2C‐VioBright FITC  mAbs for 20 min. Cells were analysed as described above.Peripheral blood mononuclear cells isolated from malaria‐naïve buffy coats were stimulated with 20 ng mLn’ is too small. P‐values < 0.05 were considered statistically significant.Statistical analyses were performed with Prism 7 . Statistical tests used are indicated for each Table and Figure. Normality was assessed by either the d’Agostino–Pearson normality test or the Shapiro–Wilk normality test, if ‘http://www.clinicaltrials.gov (NCT01322581). Written informed consent was obtained from the parents or guardians of participating children. The laboratory study with samples collected during that study was approved by the James Cook University Human Research Ethics Committee (#H7735). Subjects with no reported history of malaria or malaria exposure  were provided by the Australian Red Cross under a protocol approved by the James Cook University Human Research Ethics Committee (#H6702); written informed consent was obtained from all subjects.The Malian cohort study was conducted in compliance with all applicable federal regulations governing protection of human subjects and was approved by the Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry at the University of Sciences, Technique and Technology of Bamako and the Institutional Review Board of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The study is registered on The authors have declared that no conflict of interest exists.CL, CP and DLD contributed to the project design. CL processed the samples, performed the experiments and analysed the data. OKD, BT and PDC recruited participants and provided samples. JLB organised import permits and assisted with the laboratory studies. KPDS, DLD and BT coordinated the shipment of samples. CL and DLD wrote the manuscript.Supplementary figure 1Click here for additional data file.Supplementary figure 2Click here for additional data file.Supplementary figure 3Click here for additional data file.Supplementary figure 4Click here for additional data file.Supplementary figure 5Click here for additional data file.Supplementary figure 6Click here for additional data file.Supplementary figure 7Click here for additional data file.Supplementary figure 8Click here for additional data file.


Plasmodium antigens and human IgG against Plasmodium parasites can be used as indicators to determine malaria infection status and exposure.Several refugee settlements in Bangladesh have provided housing and medical care for the forcibly-displaced Myanmar nationals  population. The identification of malaria infection status in the refugee settlements is useful in treating infected persons and in developing malaria prevention recommendations. Assays for Plasmodium antigen aldolase as well as Plasmodium falciparum histidine rich protein 2 (HRP2). A bead-based multiplex assay was also used to measure human IgG antibody response to P. falciparum, Plasmodium malariae, and Plasmodium vivax merozoite surface protein 1 antigen (MSP1) isoforms, and P. falciparum antigens LSA1, CSP, and GLURP-R0.Dried blood spot (DBS) samples (N = 1239) from a household survey performed April–May 2018 in three settlements in Cox’s Bazar district, Bangladesh were utilized for a sample population of children from ages 1–14 years of age. The samples were tested using a bead-based multiplex antigen assay for presence of the pan-Plasmodium antigens in any samples, suggesting no active malaria parasite infections in the tested children. IgG seroprevalence was highest to P. vivax (3.1%), but this was not significantly different from the percentages of children antibody responses to P. falciparum (2.1%) and P. malariae (1.8%). The likelihood of an anti-Plasmodium IgG response increased with age for all three malaria species. Evidence of exposure to any malaria species was highest for children residing 8–10 months in the settlements, and was lower for children arriving before and after this period of time.There were no detectable Plasmodium antigen in this population provides evidence that children in these three Bangladeshi refugee settlements did not have malaria at time of sampling. Higher rates of anti-malarial IgG carriage from children who were leaving Myanmar during the malaria high-transmission season indicate these migrant populations were likely at increased risk of malaria exposure during their transit.Absence of  Plasmodium are the causative agents of malaria, and a significant burden to humanity with nearly half of the human population at risk for infection. Children are particularly vulnerable, in part because they have not yet developed protective immunity carbodiimide hydrochloride) and 5 mg/mL sulfo-NHS (sulfo N-hydroxysulfosuccinimide) and incubating with rotation for 20 min at room temperature (RT) protected from light. After a wash with coupling buffer (50 mM 2-(4-morpholino)-ethane sulfonic acid, 0.85% NaCl at pH 5.0), antigens were coupled to beads in presence of coupling buffer for 2 h at an antigen concentration of 20 µg/mL for all antigens except for CSP and GLURP-R0 at 30 µg/mL and GST at 15 µg/mL. Beads were washed once with PBS, and suspended in PBS with 1% BSA with incubation for 30 min at RT by rotation. Beads were then resuspended in storage buffer  and stored at 4 °C.Six eviously ), and th1 , circum1 , and gl1 . The theviously , and the studies , 22. Bri® software. Median fluorescence intensity (MFI) signal was generated for a minimum of 35 beads/region, and background MFI from wells incubated with Buffer B was subtracted from each sample to give a final value of MFI-bg.For the IgG assay, all incubation steps were performed at RT in 50 µL reaction volumes protected from light as previously described . IncubatP. falciparum-specific antigens  considered that child positive to “Pf short-term” antibodies.To determine an assay signal  which would indicate positivity to antigen or antibody, a panel of 86 US resident blood samples were run by both the multiplex antigen assay (at 1:20 whole blood dilution) and multiplex antibody assay  to obtain the mean and standard deviation for a “malaria non-exposed” population. For each antigen or antibody, the mean + 3 standard deviations (s.d). MFI-bg value for this non-exposed population was used at the positivity threshold. For antigen detection, the MFI-bg thresholds were: pAldo (325) and HRP2 (100), which correspond to a limit of quantification of approximately 128 pg/mL for pAldolase and 8.8 pg/mL for HRP2. For the IgG antibody detection assay, the MFI-bg thresholds were: PfMSP1 (75), PvMSP1 (146), PmMSP1 (207), LSA1 (99), CSP (120), GLURP-R0 (62). Positivity of a sample to any of the three Plasmodium antigens, data was fit to logistic regression model in SAS v9.4  using PROC LOGISTIC with the MODEL statement. Wald OR estimates were obtained for camp of residence, age category, or duration at camp of residence. Duration categories were selected based on the seasonality of malaria for this region. Statistical significance was considered if 95% confidence interval did not include 1.0.To obtain unweighted odds ratios (ORs) for IgG carriage to Plasmodium antigens. Overall seroprevalence was low to each malaria species’ MSP1 antigen: P. falciparum at 2.1%, P. vivax at 3.1%, and P. malariae at 1.8%, and not statistically different from each other. None of the camp populations had a seroprevalence to any of the three species’ MSP1 antigens of greater than 3.9%. When generating seroprevalence estimates for any antibodies to any malaria species, the overall study population had an estimated seroprevalence of 7.3% with no single camp having a seroprevalence to any anti-malaria IgG above 8.9%.Figure Plasmodium species. Prevalence of IgG reliably increased with increasing age, but was only found to be statistically higher for the oldest age category (when compared to the youngest category) for the PfMSP1 and for antibodies against any malaria categories. Interestingly, the 5–10 year age category was significantly lower for PmMSP1 IgG in comparison to the 1–4 year age category. None of the 10 children residing in Bangladesh for less than 8 months was found to have IgG antibodies to malaria and, thus, OR regression estimates could not be generated for this group. Children residing in Bangladesh for 8–10 months were found to have higher seroprevalence to any species  in comparison to children residing in Bangladesh for more than 10 months, but this was only statistically significant for the categories of IgG to Pf short-term antigens, and IgG to any Plasmodium antigens.Table Plasmodium antigens as well as IgG antibodies against malaria. Presence of the pan-Plasmodium antigen aldolase and P. falciparum HRP2 antigens have both been shown to be indicative of active infection [P. falciparum infections within the past few months. The findings of no cases of active malaria infections in this survey are consistent with World Health Organization surveillance in 2018 showing a total of 83 confirmed cases among all FDMN camps among nearly a million persons living in these camps .In this study, blood samples were assayed from children residing in three refugee settlements in southeast Bangladesh for nfection , but HRPnfection . Since nP. falciparum, P. vivax, and P. malariae. This finding was not unexpected as this region of the world is known to be co-endemic for these three species, and this region represents higher transmission zones for both Bangladesh and Myanmar [P. falciparum-specific short-term antigens  gave strong evidence for a more recent exposure to P. falciparum malaria. Overall, low numbers of children were found to be positive for these short-term markers of P. falciparum exposure, and no significant differences were again seen in IgG prevalence to these P. falciparum markers among settlements.A proportion of the children of all ages and residing in all settlements were found to have IgG antibodies against all three species assayed for:  Myanmar , 25. Sin Myanmar , it is nP. falciparum antigens was also found to increase with increasing age, but with low overall seroprevalence , these increases were not statistically significant. Overall, seropositivity for IgG against any Plasmodium antigens was found to be significantly higher in the 11–14 age category when compared to the younger categories.Correlation of positivity to IgG among the different malaria antigens is displayed in Additional file P. falciparum antibodies (1.7% versus 0.3%) and antibodies to any malaria (8.9% versus 5.6%). Since the survey of the refugee settlements took place from April to May 2018, children travelling to settlements 8–10 months prior to this would have been in transit from July to September 2017, which is directly in the middle of the monsoon season for this region. Monsoon season in Bangladesh is from June to October [Anopheline species have been identified as vectors for Plasmodium in Bangladesh, and the period of peak transmittance corresponds directly to the monsoon season. Being in transit from Myanmar to the refugee settlements during the peak monsoon and malaria transmittance period could potentially render a mobile population more susceptible to malaria exposure during their time of passage. Children residing in the Bangladeshi settlements longer than 10 months would simulate a counterfactual population to compare malaria exposure since the monsoon still occurred in the refugee settlements (and mosquito breeding habitats still created), but it appears likelihood of malaria exposure was reduced for children living in these settlements with most pronounced differences for short-term markers of P. falciparum. Higher risk of malaria exposure during transit could be from a variety of factors including a lack of personal protection from mosquitoes during the monsoon season, or that during transit the refugees may not have had access to stable, consistent shelter. In contrast, residence in these three Bangladeshi refugee settlements appears to have a protective effect with respect to malaria exposure.Regardless of age or camp, no children residing in Bangladeshi settlements for less than 8 months were found to have antibodies against of the short-term malaria antigen targets. Additionally, children residing in settlements for longer than 10 months were found to have lower likelihood of IgG seropositivity to all antigens tested when compared with children residing in settlements for 8–10 months. In comparing these two durations of residence (8–10 months versus > 10 months), significant differences were seen for short-term  October , in whic October . The pea October , and thi October . Six AnoP. falciparum, P. vivax, and P. malariae is reduced with the strongest decreases in short-lived anti-P. falciparum IgG. Overall, multiplex bead assay has provided a simplified approach for testing current malaria parasite infection status and past exposure history in a refugee population and ruled out malaria as a major risk factor in their current settlement.Through serological testing of malaria antigen and anti-malaria IgG antibodies, active transmission of malaria does not appear to be a major threat in the Kutupalong Registered Camp, Nayapara Registered Camp, and Kutupalong Makeshift Settlements in Cox’s Bazar, Bangladesh. None of the 1239 children’s samples was found to be positive for malaria antigens, providing strong evidence for lack of active malaria infections at the time of sampling. Additionally, as the time spent in Bangladesh increases, the likelihood of antibody seropositivity to malaria species Additional file 1. Correlation of positivity for IgG against different malaria antigens.


Malaria has a negative impact on the activities of companies in endemic countries especially in Cameroon. In this regard, an increasingly growing number of companies have started to include management of malarious patients in their health policies. In the present study, we will evaluate the diagnostic performances of a fluorescence microscopy (FM), Cyscope® microscope, in the detection of malaria parasites.A cross-sectional study was conducted among employees of two companies of the town of Douala on 21 and 22 March 2017. Sociodemographic information of employees was collected using a questionnaire form. Blood samples of ~ 10 μL were collected by venipuncture for the diagnosis of malaria using FM and light microscopy (LM). Performances of FM with respect to sensitivity (Se), specificity (Sp), positive and negative predictive values (PPV and NPV), positive and negative likelihood rates (PLR and NLR), accuracy, reliability, and Kappa index were calculated using LM as gold standard.p < 0.01). Plasmodium falciparum and P. vivax were the two species involved in malaria infection cases. In terms of developmental stages, 68%, 45.3%, and 1.3% of employees carried gametocytes, trophozoites, and schizonts, respectively. Findings on diagnostic performances of FM were as follows: Se = 84%, Sp = 69.95%, PPV = 63.58%, NPV = 95.5%, accuracy = 89.36%, and reliability = 53.95%. Sensitivity of Cyscope® microscope increased as a function of parasitemia with values ranging from 76.92% at parasitemia between 1 and 500 parasites/μL to 91.11% at parasitemia between 501 and 5000 parasites/μL. The geometric mean parasite density was1850 parasites per μL of blood , and most of employees (60.8%) had moderate parasitemia. The performances of FM were similar between febrile and afebrile patients.In total, 442 employees, aged 37.8 ± 9.7 years old on average, were included in the study. Prevalence of malaria using FM and LM was 39.2% and 17%, respectively (This study showed good performances of Cyscope® microscope and outlines that this diagnostic tool could be used in management of malaria at workplace. Anopheles /(TP + FN) × (TN + FP) .▪ Kappa (k) values expressed the agreement beyond chance and were calculated with a 95% confidence interval. A kappa value of 0.21–0.60, 0.61–0.80, and > 0.80 was considered as moderate, good, and almost perfect agreement beyond chance, respectively .10-transformed before performing statistical analysis. Statistical significance was set at p value < 0.05.All data obtained were subjected to analysis by using the Statistical Package for Social Science (SPSS) software version 16 . McNemar’s chi-square test was used to compare the prevalence of malaria infection between LM and FM. Pearson’s chi-square and non-parametric Mann-Whitney tests were used to compare unpaired proportions and mean values of parasitemia, respectively. Parasitemia was logTable p < 0.01). Plasmodium falciparum and P. vivax were the two species identified in this study  was 1850 parasites per μL of blood  while most of the employees (60.8%) had moderate parasitemia. Regarding the three P. vivax cases recorded, two of them had moderate parasitemia while the remaining case had low parasitemia.The prevalence of malaria was 39.2% (173/441) and 17% (75/441) using FM and LM, respectively, and the difference was statistically significant between both the methods  . It was not possible to compute the sensitivity for group of patients with parasitemia above 5000 parasites/μL because of small sample size for this group (n = 3).Besides, sensitivity of Cyscope® microscope increased as a function of parasitemia with values ranging from 76.92% (95% CI 57.95–88.96%) in patients with parasitemia comprised between 1 and 500 parasites/μL to 91.11% (95% CI 79.27–96.49%) in patients with parasitemia comprised between 501 and 5000 parasites/μL. The difference between sensitivity values was not statistically significant  the production of dormant development stages (hypnozoites) for which all currently available antimalarial drugs, with the exception of primaquine, are inefficient, (ii) diagnosis-related problems as it may confuse with P. ovale, (iii) infection cases at low parasitemia that are generally revealed using molecular methods, and (iv) difficulty to cultivate this parasite [Most of infections cases were due to ern part , 18, 29 ern part . Briefly country , 40. Thi country . This faCameroon . This suparasite . In an aConcerning the performances of FM, we found a sensitivity value of 84% similar with the result of several other studies which also recorded values close to 95% in Southern Cameroon , 18, 29,The specificity that we found (69.9%) was less than that which was found by Hassan et al. (98.3%) . This diLM, a standard technique, has the advantage that can discriminate and quantify plasmodial species . HoweverSensitivity of Cyscope® has increased as a function of parasitemia with values ranging from 71 (1–500 parasites/μL) to 91% (501–5000 parasites/μL), and this is consistent with previous studies , 54. TheEscherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae [Toxoplasma gondii and Salmonella typhii were involved in fever cases among children [Surprisingly, sensitivity of Cyscope® microscope was better in afebrile individuals as compared to their febrile counterparts, although no statistically significant difference was found. This can be explained by differences in level of parasitemia in these two groups as level of parasitemia was on average higher in afebrile individuals. In addition, it should be noted that fever is not a pathognomonic sign of malaria infection as infections caused by virus and bacteria can also elicit fever. Studies outlined that a large proportion of febrile illness episodes were caused by urinary tract infection bacteria mainly eumoniae , 56. In children . Thus, sThe study has some limitations. First, polymerase chain reaction-based molecular method was not used as gold standard. Although this method is more sensitive than LM , it is nThe Cyscope® microscope has appreciable sensitive and specificity compared to the LM at the workplace. In this environment of workplace, operational characteristics of the Cyscope® will make it more adaptable at workplace than LM. The Cyscope® can therefore be recommended to all types of companies for a prompt and better diagnosis of malaria.


Plasmodium falciparum. These molecules cause infected erythrocytes to adhere to human receptors found on blood vessel and tissue surfaces. This protects the parasite within from clearance by the spleen and also causes symptoms of severe malaria. The PfEMP1 are exposed to the immune system during infection and are therefore excellent vaccine candidates for use in an approach to prevent severe disease. A key question, however, is whether their extensive diversity precludes them from forming components of the malaria vaccines of the future?Human infective parasites, such as those that cause malaria, are highly adapted to evade clearance by the immune system. In situations where they must maintain prolonged interactions with molecules of their host, they often use parasite surface protein families. These families are highly diverse to prevent immune recognition, and yet, to promote parasite survival, their members must retain the ability to interact with specific human receptors. One of the best understood of the parasite surface protein families is the PfEMP1 proteins of  Malaria is one of the most ancient diseases of humanity, evolving with its mammalian hosts for millennia. It still kills around half a million people each year and causes hundreds of millions of clinical cases . The quePlasmodium falciparum infections and have therefore not developed an antibody repertoire capable of managing the infection [Other possibilities on the target list are the proteins that determine whether a malaria infection develops severe and life-threatening symptoms. Severe malaria, which may include severe anaemia, respiratory distress or cerebral effects, is experienced among individuals who have not yet been exposed to repeated nfection . In malanfection , while anfection ,11.P. falciparum erythrocyte membrane protein 1, PfEMP1, a large protein family with ∼60 members encoded in each genome [The development of severe malaria symptoms occurs in the blood stage of infection and is linked to expression of parasite proteins found on infected erythrocyte surfaces, tethering parasites to human endothelium and tissues. This prevents infected erythrocytes, which have been swollen by parasites within, from being filtered from the blood by the human spleen. It also leads to inflammation, occlusion of blood vessels and to the symptoms of severe malaria, including cerebral malaria and pregnancy-associated malaria, which are characterized by marked sequestration of parasites in the brain and placenta . Endotheh genome ,14. Eachh genome . Differeh genome , intraceh genome  and clush genome .Figure In some regards, the PfEMP1s are excellent vaccine candidates. They are linked directly to the pathogenesis of malaria, are constantly exposed to the immune system and are the targets of many of the antibodies generated in response to infection ,20, and However, the PfEMP1 family is hugely diverse. As proteins that are constantly exposed to the immune system, selection pressure has caused them to expand into a large and antigenically variant protein family that varies significantly between different isolates ,14. The So to what degree is the PfEMP1 family too complex and too diverse to target therapeutically? Will it ever be possible to generate vaccine immunogens that induce a broadly inhibitory response against such a protein family? To answer these questions, we need to understand which PfEMP1s to target, due to their specific association with severe forms of malaria. We also need to understand their structures and to determine whether they retain the capacity to bind to specific endothelial receptors through conserved binding sites, and whether these sites can be mimicked by vaccine immunogens. If expression of specific PfEMP1s leads to the development of severe malaria, and if these PfEMP1s use conserved surfaces for ligand binding, then it may be possible to include their derivatives in future vaccines as components designed to reduce the incidence of death and severe disease.Only a fraction of malaria cases develop severe symptoms. With many varied PfEMP1s in the genome, a major question has therefore been whether specific PfEMP1s, with specific receptor-binding phenotypes, are associated with particular severe disease syndromes. Indeed, PfEMP1 domains have been suggested to interact with a wide range of different human protein and carbohydrate ligands ,25. ThisThe first PfEMP1 ligand-binding phenotype associated with a particular disease syndrome was found for pregnancy-associated malaria. Here, a single PfEMP1 in each parasite genome, known as VAR2CSA, was specifically expressed in parasites isolated from pregnant women suffering from malaria ,27. VAR2The association of specific PfEMP1s with severe malaria and the identification of their binding partners proved more challenging. Narrowing down the search required extensive sequence analysis and PfEMP1 domain classification . First, While expression of EPCR-binding PfEMP1s has been associated with all forms of severe malaria ,38–41, pThe links between these PfEMP1 types and severe or cerebral malaria are not absolute ,38,44. NThe identification of groups of PfEMP1s  associatHowever, VAR2CSA is not a classical PfEMP1 as it lies outside the normal organization of the protein family and is encoded by a single, relatively conserved gene in each parasite genome ,27,45. WWhile there is still more to discover about the PfEMP1 family, recent studies have cut through some of their complexity, identifying binding phenotypes associated with severe disease syndromes and characterizing conserved structural features that mediate receptor binding. This revealed important principles that are likely to be shared by other PfEMP1s or other parasite surface protein families such as the RIFINs, which are less well studied, but have been suggested to contribute to the pathogenesis of malaria . For exaA key question remaining is whether these insights can be used to design improved vaccine immunogens. Will knowledge of the chemically conserved surfaces that mediate receptor binding allow us to design molecules that mimic these regions? Will these molecules raise antibodies that specifically target the conserved chemistry of the receptor-binding sites and will these antibodies have broadly neutralizing potential?Recent advances in structure-guided vaccine design raise hope that the key receptor-binding sites of PfEMP1 domains can be mimicked in designed immunogens. This could be achieved by grafting critical epitopes from PfEMP1 domains onto much smaller, weakly immunogenic scaffolds, a method that has been used successfully in viral vaccine development ,50. In tMany believe that surface protein families, such as the PfEMP1s, are simply too challenging to target by vaccines, with their depth of diversity so extensive that no immunogen will be able to raise antibodies that neutralize function across the whole family. Indeed, there is no doubt that it will be easier to generate broadly neutralizing antibodies that target more conserved proteins, such as the blood stage malaria antigen RH5 or the liver stage antigen CSP. However, for specific PfEMP1s, associated with particular binding phenotypes, there is hope. To retain the capacity to bind to their conserved human ligands, these PfEMP1s must retain the shape and chemical properties of their receptor-binding surfaces. The identification of interactions associated with severe disease, and the characterization of critical conserved features therefore provides the foundation required for these immunogen design studies. It will be fascinating to see the degree to which the latest tools in protein design can now allow the production of such immunogens and the degree to which they generate broadly reactive immune responses. If successful, such immunogens may form a valuable part of the multi-stage malaria vaccines of the future.The malaria vaccines of the future are likely to be multi-component.Developing a component to prevent severe disease involves targeting parasite surface protein families such as PfEMP1.These are diverse and complex. However, recent studies have identified PfEMP1 associated with severe disease and have identified conserved ligand-binding surfaces.