Vivax malaria diagnosis remains a challenge in malaria elimination, with current point of care rapid diagnostic tests (RDT) missing many clinically significant infections because of usually lower peripheral parasitaemia. Haemozoin-detecting assays have been suggested as an alternative to immunoassay platforms but to date have not reached successful field deployment. Haemozoin is a paramagnetic crystal by-product of haemoglobin digestion by malaria parasites and is present in the food vacuole of malaria parasite-infected erythrocytes. This study aimed to compare the diagnostic capability of a new haemozoin-detecting platform, the Gazelle™ device with optical microscopy, RDT and PCR in a vivax malaria-endemic region.A comparative, double-blind study evaluating symptomatic malaria patients seeking medical care was conducted at an infectious diseases reference hospital in the western Brazilian Amazon. Optical microscopy, PCR, RDT, and Gazelle™ were used to analyse blood samples. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Kappa values were calculated.Out of 300 patients, 24 test results were excluded from the final analysis due to protocol violation (6) and inconclusive and/or irretrievable results (18). Gazelle™ sensitivity was 96.1 % (91.3–98.3) and 72.1 % (65.0–78.3) when compared to optical microscopy and PCR, respectively whereas it was 83.9 % and 62.8 % for RDTs. The platform presented specificity of 100 % (97.4–100), and 99.0 % (94.8–99.9) when compared to optical microscopy, and PCR, respectively, which  was the same for RDTs. Its correct classification rate was 98.2 % when compared to optical microscopy and 82.3 % for PCR; the test’s accuracy when compared to optical microscopy was 98.1 % (96.4–99.7), when compared to RDT was 95.2 % (93.0–97.5), and when compared to PCR was 85.6 % (82.1–89.1). Kappa (95 % CI) values for Gazelle™ were 96.4 (93.2–99.5), 88.2 (82.6–93.8) and 65.3 (57.0–73.6) for optical microscopy, RDT and PCR, respectively.The Gazelle™ device was shown to have faster, easier, good sensitivity, specificity, and accuracy when compared to microscopy and was superior to RDT, demonstrating to be an alternative for vivax malaria screening particularly in areas where malaria is concomitant with other febrile infections . Plasmodium infection and, to an extent, differentiation of species, with the capacity for rapid screening becoming increasingly important for elimination. While malaria caused by Plasmodium falciparum, the principal cause of malaria mortality worldwide, is relatively well diagnosed in most areas using antigen-detecting rapid diagnostic tests (RDTs), other species are less well detected, particularly in remote settings [Plasmodium vivax, the second most prevalent species of the six human malaria-causing Plasmodium species, is concentrated in Asia, Central and South America, the Western Pacific and the Horn of Africa [P. falciparum and Plasmodium vivax cause disease, malaria control efforts have effectively reduced the number of P. falciparum infections, but there has been an increase in the proportion of infections attributed to P. vivax [Malaria is the leading cause of death from parasitic infection worldwide . Achievisettings . Plasmodf Africa  where itf Africa . In 2019f Africa . In manyP. vivax .P. vivax is complicated by hard-to-detect low densities of circulating blood-stage parasites in many symptomatic patients and by its undetectable latent liver stage (hypnozoites) that results in relapses if not specifically targeted with 8-aminoquinolone drugs. Parasite densities of P. vivax are commonly much lower than P. falciparum in symptomatic infections [Diagnosis, control and elimination of fections , limitinPlasmodium species [Because RDTs have poor sensitivity and molecular-based diagnosis are expensive for detecting some  species , light m species . However species .P. falciparum, and the parasite’s metabolic enzymes, lactate dehydrogenase (pLDH) and aldolase, which are common to all species. HRP2-detecting tests are of limited utility in some regions because of their inability to detect P. falciparum parasites with hrp2 and hrp3 gene deletions [P. vivax infection is generally lower than that of PfHRP2-based RDTs for P. falciparum [P. vivax, for successful case management and in achieving malaria elimination [Similarly, the performance of an RDT can be affected by multiple host and parasite factors . Most RDeletions . The senlciparum , 13. Altlciparum , it requlciparum . For thelciparum , 17. Howlciparum . Faster,mination .Plasmodium species but in no other circulating pathogens, its detection is a promising approach to accurate diagnosis, especially in low parasitaemia. Attempts to develop portable, magneto-optical, point-of-care diagnostic technologies that detect haemozoin have been made but no commercial product has yet emerged. Earlier efforts to use haemozoin as a biomarker for malaria were largely unsuccessful [P. falciparum endemic area [Haemozoin is a paramagnetic crystal by-product of haemoglobin digestion by malaria parasites . Becauseccessful , 22. In ccessful . Other mccessful –22, 24. mic area .Here, are reported the results from a study that assessed the accuracy of Gazelle™ , in detecting vivax malaria cases. The device is a late-stage portable prototype using magneto-optic technology for malaria diagnosis developed to detect haemozoin. The performance of Gazelle™ was compared with three current diagnostic methods: microscopy, RDTs and PCR.Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD). The hospital is a reference centre for infectious disease care in Manaus, western Brazilian Amazon. Approximately 30 % of all cases of malaria in Manaus municipality are diagnosed at the FMT-HVD.A comparative, double-blind study evaluating symptomatic individuals with suspected malaria, comparing optical microscopy, PCR, RDT, and Gazelle™ was carried out between June and October 2019 at Both male and female individuals aged ≥ 18 years seeking care at the FMT-HVD outpatient clinic were considered for enrolment. Per protocol, subjects were recruited, screened and enrolled by the investigator’s team at the study site, after the hospital’s routine malaria screening. All patients were informed of the study objectives and risks of participation. Participants were given adequate time to read the informed consent form (ICF) after which they were requested to sign it. Exclusion criteria were severe malaria, pregnancy and use of anti-malarials in the 30 days prior to study enrolment.The sample size was calculated based on the assumption of 95 % confidence interval, an estimated 10 % accuracy (95 % CI width ≤ 10 %) and allowed for up to 10 % of subjects to be excluded because of incomplete or missing data. This translated to a minimum of 100 positive and 200 negative cases of malaria.On study admission, immediately before drug administration, trained laboratory technicians collected whole blood samples in EDTA. Microscopy was the method used to confirm vivax malaria status of the participants before recruiting them into the study. Blinded study microscopists analysed all samples after slide preparation (thick and thin smears). Collected blood samples were stored at 4 °C and tested by RDT and Gazelle™ within utmost 4 h of collection. Aliquots of the whole blood samples were stored at − 20 °C until needed for DNA extraction for the subsequent molecular diagnosis of malaria by PCR analysis. All testers were blinded to the malaria status of the samples.P. falciparum and P. vivax haemozoin. When haemozoin, which is partly composed of iron, is subjected to an intense magnetic field the haemozoin crystals align in a manner that increases the opacity of the blood sample  and 15 µL of blood into a cuvette Fig. . The cuvVenous blood samples were tested using the Malaria Ag Pf/Pan RDT  according to the manufacturer’s instructions. The RDT kits were stored at the recommended temperatures and were used before the expiration date.Thick blood smears were prepared, stained by Giemsa  and examPlasmodium species by targeting a conserved region of the 18 S rRNA gene in the extracted DNA samples. The following probes and primers were used to analyse the extracted DNA samples: Fw-TTA GAT TGC TTC CTT CAG TRC CTT ATG; Rev-GT TGA GTC AAA TTA AGC CGC AA; FAM - TCA ATT CTT TTA ACT TTC TCG CTT GCG CGA - BHQ1 (wobble R = A/G) [2, 104, 106 copies/µL). Briefly, 4 µL of DNA samples and plasmids were used in a total reaction volume of 12 µL to screen for malaria cases.Extraction of total DNA from 200 µL of whole blood was performed using the QIAamp DNA Blood Mini Kit , according to the manufacturer’s protocol. QMAL Taqman qPCR was used to detect R = A/G) . To quanAll qPCR assays were run in the 7500 Fast Real-Time PCR System . The primer and probe sequences, the composition of reaction mixes, PCR profiles and the detection limit for each assay used were as described by Almeida et al. [STARD (Standards for Reporting of Diagnostic Accuracy Studies) shown in Fig. Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD) approved the study . Subjects were informed of the study objectives and gave their consent for participation through written and signed informed consent forms before study procedures. Subjects diagnosed with malaria were treated according to the Brazilian Ministry of Health guidelines [The Ethics Review Board of the idelines .P. vivax population (PVP) excludes all subjects with P. falciparum or mixed infections. The PVP is the primary population analysed in this study.Table Gazelle™ diagnostic performance was measured by determining its sensitivity, specificity, likelihood ratios (LR), positive predictive value (PPV), negative predictive value (NPV), and detection accuracy. Gazelle™ was compared to RDT, to optical microscopy and PCR. Likelihood ratios for positive (LR+) and negative (LR−) test results were considered good when LR + was > 10, and LR− <0.1 . Diagnosp < 0.05. Analyses were performed on STATA .The two-graph ROC analysis enables the visualizing of sensitivity and specificity curves on a single graph, according to the range of values of the new test given the reference test. This provides a visualization for the specificity-sensitivity ‘optimal’’ trade-off in determining the best and acceptable cut-off for the new test to be used in practice against the already established gold standards available. The parasite detection parameters for the Gazelle™ test against microscopy and PCR were determined by a two-graph ROC analysis . ResultsP. falciparum; and, 3 with mixed infections of P. vivax and P. falciparum ; 3 were infected with P. vivax data analysis, 168 (60.9 %) reported one or more previous episodes of malaria prior to the current infection. The mean parasite density was 6,220 parasites/µL . Patient demographic and malaria infection characteristics are presented in Table Of the 276 subjects whose test results were analysed per protocol, 163 (59.1 %) were male. The study participants were aged between 18 and 79 years with a mean age of 40.4 (± 18.8) years. Out of the total number of subjects included in the Malaria detection characteristics such as mean parasite density, range and density distribution are shown in Table When microscopy was positive for malaria, Gazelle™ produced a positive result for 96.2 % of the samples (125 of 130 tests). On the other hand, when microscopy was negative for malaria, the Gazelle™ result was 100 % consistent (146 of 146 tests). A ROC analysis of Gazelle™ against microscopy showed an accuracy of 98.3 % . When PCR was negative for malaria, the Gazelle™ was negative 99.0 % of the time (103 of 104 tests). Further ROC analysis of Gazelle™ against PCR showed an AUC of 0.837, meaning that the Gazelle™ test was slightly less sensitive than PCR but very specific and with very good accuracy Fig. b. Of theSensitivity and specificity of Gazelle™ test results compared with PCR were 72.1 and 99 %, whereas, for RDTs they were 62.8 and 99 %, and for microscopy, they were 75 and 99 %, respectively Table . The oveFor the sub-set of 48 tests where the PCR result was positive and Gazelle™ was negative, the average PCR count was 326.65 DNA copies/µL (or only 0.014 % of the average PCR count) with a median of 19.73 DNA copies/µL.P. vivax infections in endemic regions.Accuracy is essential for rapid and timely diagnosis and treatment, identifying and interrupting malaria transmission in the community. Low parasite density infection continues to challenge diagnostic accuracy and obstruct malaria elimination efforts. Highly sensitive, rapid and affordable diagnostic tools are urgently needed to detect and improve the clinical management of Gazelle™, a novel point-of-care, haemozoin-based malaria diagnostic device was evaluated, and compared to microscopy, RDT, and PCR. Sensitivity and specificity of Gazelle™ was nearly as sensitive and was as specific as expert microscopy and considerably more sensitive than RDTs, the most used diagnostic tools at community health centres. In addition, Gazelle™ had advantages in terms of speed, with a turn-around time of 90 s. Although relatively low cost, microscopy is rarely used in remote areas because infrastructure and skilled personnel are lacking, and electrical power supplies are often erratic or unreliable . FurtherP. vivax parasite densities down to 72 parasites/µL. These results affirmed the superiority of the test against widely used conventional diagnostic methods. Studies by Kumar et al. [P. falciparum parasites/µL with 95 % accuracy; as low as 35 P. vivax parasites/µL from Indian patients could be detected with 100 % accuracy by the device. The difference in the limit of detection (LOD) between this study and the Indian study may be due to differences in parasite burden and parasite characteristics  of the P. vivax strains prevalent in the two regions. Dilution experiments elsewhere, applying the magneto-optical concept used in this device, suggest a LOD for Gazelle™ could go to as low as potentially < 20 parasites/µL with higher blood volume and trophozoite/schizont parasite stages [From this field trial study, the Gazelle™ outperformed RDTs, detecting r et al.  demonstre stages . These Le stages , 37. Thee stages .TM test use was determined to be slightly lower than 100 parasites/µL and 1,000 copies/µL relative to microscopy and PCR, respectively. Further studies in different malaria transmission settings are necessary to validate the proposed “optimal cut-off” and LOD.An attempt to determine the device cut-off limit relative to microscopy and PCR diagnostic techniques was also done. The parasitaemia and PCR copies raw data obtained from laboratory analysis ranged into thousands (parasitaemia) and millions (PCR copies). The identification of a precise cut-off using a cut-off graph plot was however based on too many points with little gain in accuracy. Subsequently, intervals of 500 were used, making 500,000 PCR copies/µL as the maximum value. With this modification, the “optimal cut-off” for GazelleP. falciparum demonstrated elsewhere [P. vivax have potential to address the inadequacies of current RDTs in community-based vivax management and may prove useful in scenarios such as airports or borders in elimination settings where large numbers of people must be rapidly screened each day.The current Gazelle™ prototype provides only qualitative malaria diagnosis without species differentiation, with detection of lsewhere . In its lsewhere . PerhapsFor operational purposes, the samples used for all diagnostic tests were venous blood. For malaria diagnosis in the field peripheral capillary blood is used, which could be a limitation of the study’s current sample collection and analysis design in that malaria parasite concentrations in capillary and venous blood differ. However, the diagnostic device can use small volumes of blood such as those obtained from finger prick (15 µL) for analysis. Despite all tests having been performed  with venous samples, future studies will need to address this issue by comparing the diagnostic potential of Gazelle™’s device  using blood from both collection sites. Expanding the capacity to diagnose and differentiate between species should increase  the Gazelle™ use as a POC testing device in field settings where mixed infections occur, or in areas where several species are in circulation. The results obtained in this study could be restricted to the malaria strains present in the Brazilian Amazon, and the application of this device in other countries should be addressed. Despite these limitations, results from this study support the use of Gazelle™ in areas with limited qualified human resources and electrical power supply, and thereby increase capacity of malaria diagnosis in areas where transmission mostly occurs.P. vivax. Gazelle™ demonstrated a limit of detection below that of the comparator rapid test. Its ease of use, short time to result, and accuracy hold potential for rapid screening for P. vivax in remote resource-poor locations where PCR and microscopy are not feasible and cannot immediately access to modern medical attention, but further development is required to differentiate species where necessary to guide treatment. In addition, its speed, storage capacity, rechargeable battery, cost-efficiency, and alternative to lack of microscopists makes it very practical for use in field settings. Even so, more studies are required to determine the device’s performance against different malaria species (and co-infections), using peripheral blood samples and in the field where energy and cold chains are not available.This study demonstrated that Gazelle™, the first commercially developed haemozoin-detecting instrument for the diagnosis of malaria, outperformed RDTs and was nearly as sensitive and as specific as expert microscopy for


Plasmodium falciparum infection in sub‐Saharan Africa. Successes are largely attributed to malaria prevention strategies, such as insecticide‐treated mosquito nets and indoor spraying, as well as improved access to existing treatments. One important hurdle to new approaches for the treatment and prevention of malaria is our limited understanding of the biology of Plasmodium infection and its complex interaction with the immune system of its human host. Therefore, the elimination of malaria in Africa not only relies on existing tools to reduce malaria burden, but also requires fundamental research to develop innovative approaches. Here, we summarize our discoveries from investigations of ethnic groups of West Africa who have different susceptibility to malaria.There are an estimated over 200 million yearly cases of malaria worldwide. Despite concerted international effort to combat the disease, it still causes approximately half a million deaths every year, the majority of which are young children with  According to the World Health Organization (WHO), there are over 200 million yearly cases of malaria worldwide, with the heaviest burden of malaria due to P falciparum infections in sub‐Saharan Africa.Malaria is an age‐old scourge of humankind and also has a significant impact on the economic and social development of affected communities.Plasmodium infection and its complex interactions with the human immune system.After a period of success in globally reducing the number of malaria cases since the start of the 21st century, the rate of improvement has slowed dramatically over the last five years. This is partly because of increased resistance against existing drugs and insecticides, as well as a lack of new interventions.Plasmodium sporozoites.How could one identify immunological mechanisms and correlates of protection to guide next‐generation malaria vaccine development? One could, of course, study samples from clinical trials, where people are protected or not following vaccination. This, however, requires large study groups. Alternatively, one could study samples from controlled human malaria infections, where people are injected with defined numbers of What are sympatric ethnic groups? They are groups of different ancestries that live under similar conditions, including similar socio‐economic factors and in this case similar malaria inoculation rates. Furthermore, they maintain their genetic heritage, for example they do not intermarry between different ethnic groups. For many years, we have been studying such groups living in malaria endemic regions in sub‐Saharan Africa. Particularly, we have studied the Fulani non‐negroid nomadic pastoralists, compared to their negroid sympatric ethnic groups, the Dogon of Mali, or the Mossi and Rimaibé of Burkina Faso.2P falciparum malaria than other sympatric ethnic groups. There are over 30 million Fulani distributed across West and Central Africa, particularly in the Sahel region. Fulani populations coincide with regions of very high incidence of P falciparum malaria. Since the first report regarding the different responses of Fulani to P falciparum infection in 1996,P falciparum infection rates and lower P falciparum densities in infected individuals.The Fulani ethnic group has relatively better protection from Examination of inter‐ethnic genetic differences has shown that the Fulani have a distinct genetic background.The Fulani have a long history as nomadic herders, seasonally moving with the needs of their livestock. These cultural traditions of the Fulani have resulted in striking lifestyle differences compared to other ethnic groups. For example, Fulani typically have milk cultures, milk products and couscous as their staple foods, while sympatric ethnic groups usually have at least three well‐cooked meals a day.P falciparum malaria.Thus, the underlying cause for the lower susceptibility to malaria in the Fulani, genetic or environmental, has been the focus of several studies. But, so far, no conclusive data have been obtained. However, whatever the basis, there is strong evidence that Fulani have different immunological responses to 3P falciparum antigens, with higher levels of P falciparum‐specific IgG, IgM and IgE antibodiesP falciparum infection, driving more effective adaptive immunity.P falciparum malaria in other ethnic groups,Studies examining specific characteristics of the immune response to malaria in the Fulani have established, despite similar exposure to the parasite, a number of differences from other sympatric ethnic groups. Fulani are more responsive to 4Epigenetic changes underlie the development, differentiation and activation of immune cells, which are regulated by precise spatial and temporal control of gene expression.5P falciparum infection in the Fulani to genetic differences have not been successful. Several nucleotide polymorphisms (SNPs) have been identified in immune genes but none of these were associated with the better protection seen in the Fulani.Attempts to link the better protection from P falciparum belonging to the Fulani, or the Mossi sympatric ethnic group, living in the same area and thus exposed to the same level of the parasite. Our results show that Fulani monocytes, specifically, were more transcriptionally reactive to P falciparum infection. This was not related to differences in DNA methylation. Rather, several genes involved in chromatin remodelling and epigenetic regulation of gene expression in immune cell lineages are differently expressed, suggesting that the underlying cause is a change in epigenetic regulation in these innate immune cells.For this purpose, we performed genome‐wide transcriptome and DNA‐methylome analysis in CD14+ (monocytes) and CD14‐ (predominantly lymphocytes) from the same individuals, either uninfected or infected with P falciparum. On the other hand, epigenetic mechanisms may contribute to the suppression of host immune responses imposed by P falciparum. We are investigating both of these scenarios.Currently, there are very few studies directly addressing the role of epigenetics in the response to malaria.5.1P falciparum infection can interfere with the functions of innate immune cells.The malaria parasite has a number of strategies to evade the immune system, and one such method is that We have recently shown that monocyte‐derived dendritic cells (DCs) exposed to nHz only partially matured, as indicated by production of high levels of the inflammatory chemokine MCP1, secreted by immature dendritic cells, and a sustained expression of the inflammatory chemokine receptor CCR5, together with an increased expression of maturity markers, such as major histocompatibility complex (MHC) class II and CD86.P falciparum.To elucidate in more detail how nHz affected the DC maturation process, we investigated the binding of transcription factors as well as histone modifications at the promotor region of these genes important in DC maturation. We choose to study the binding of factors downstream of TLR signalling, including NF‐κB and interferon regulatory factors (IRF), as well as the chromatin remodelling factor Brahma‐related gene‐1 (BRG1) in the SWI/SNF complex, which is involved in gene activation. We could not detect any recruitment of NF‐κB subunits (p105/p50 or p65) or IRF3, neither at the transcriptional start site (TSS) nor at the NF‐κB sites following exposure to nHz, in contrast to what usually is seen following stimulation with LPS. These findings suggest an inability of nHz‐exposed DC to recruit certain transcription factors to the promoter regions of genes important for the maturation process. In addition, BRG1 was absent from the promoter region of these genes after nHz exposure. Based on this, we suggest that the necessary remodelling events are also hampered after nHZ exposure. We then investigated the enrichment of various histone modifications, both activating and silencing, at the promoter regions. Our data show that nHz did not enrich for activating histone modifications at these genes. On the contrary, we observed a possible enrichment for the silencing mark H3K27me3 at the TSS of CD83 following nHZ exposure. Thus, it is tempting to speculate that nHZ has the capacity to actively inhibit recruitment of certain transcription factors to the promoter region of genes important in DC maturation via epigenetic mechanisms. In summary, our observations might help to shed light on the molecular mechanisms and stability of suppression of innate immune cell functions by 5.2Plasmodium can induce either trained or tolerized innate immune responses. Hyper‐responsiveness has been reported in peripheral blood mononuclear cells (PBMCs) from patients with malaria and correlated with lower rates of reinfection.The innate immune system can develop short‐term memory, where a previous challenge results in increased (‘trained’) or decreased (‘tolerized’) response to a second later challenge.P falciparum‐infected red blood cells to capillary veins of vital host organs and excessive production and release of pro‐inflammatory cytokines.High levels of pro‐inflammatory innate immune responses in malaria could be either pathological or protective. Severe pathophysiological events during malaria infection involve erythrocyte destruction and ineffective erythropoiesis, adhesion of P falciparum, or due to other mechanisms, is still unclear. For example, epigenetic differences that mediate immune responses may be established in response to environmental factors, or as a result of escape from the suppression of host immune response imposed by P falciparum.In our genome‐wide study of the Fulani, we observed that the differently expressed genes in monocytes were enriched in immune response , metabolism, RNA metabolism, and chromatin and transcriptional regulation. Importantly, these differences were not observed in the Mossi individuals, nor were significant differences observed in the non‐monocyte cell fraction in Fulani and Mossi individuals. The Fulani had increased levels of pro‐inflammatory cytokines when uninfected , as well as increased levels of IL‐1β and IL‐18 upon infection, indicative of a higher inflammasome activity.6Plasmodium parasite similar to ‘trained immunity’. We observe more reactive innate immune cells, with expression of genes involved in innate immunity, metabolism and chromatin remodelling, as well as higher levels of pro‐inflammatory cytokines. However, we do not yet know when or how the changes underlying the transcriptional response seen in the Fulani are established. Our further studies performing genome‐wide analysis of chromatin states in the Fulani will shed light on these questions. Nevertheless, our data indicate that monocytes of uninfected Fulani are already set in a ‘high alert’ state, enabling a stronger reaction upon P falciparum infection, suggesting this phenomenon is correlated with protection from malaria. These findings may provide a key to new/improved vaccine candidates and anti‐malarial drugs.Our studies in the Fulani have identified differences in innate immune responses to malaria. Cells of the innate immune system, such as monocytes, macrophages and dendritic cells, can develop epigenetic states that provide ‘immune tolerance’ or ‘trained immunity’. Recent work has provided support for both suppressive and activating epigenetic changes occurring following malaria infection in different contexts. However, it is so far unclear how this complex interaction affects the pathology of the disease. Our results show that the Fulani, in comparison with the sympatric Dogon and Mossi ethnic groups, exhibit immune responses to the The authors agree on the contents of the manuscript and declare that the research was conducted in the absence of any commercial or financial relationship that could be considered as a potential conflict of interest.MTB conceived and outlined this review manuscript. AÖF, CA, JQ and IB assisted in writing and editing this manuscript and approved the final version.


Plasmodium falciparum infections. This study aimed to compare the diagnostic performance of the PfHRP2-based Abbott Malaria Ag P. falciparum ultrasensitive RDT (uRDT) to that of the conventional SD-Bioline Malaria Ag P. falciparum RDT (cRDT) when performed under field conditions.An ultrasensitive malaria rapid diagnostic test (RDT) was recently developed for the improved detection of low-density Plasmodium lactate dehydrogenase (pLDH) antigens was evaluated in a subset from dried blood spots by a quantitative antigen assay. pfhrp2 and pfhrp3 gene deletions were assessed in samples positive by RT-qPCR and negative by both RDTs.Finger-prick blood samples were collected from adults and children in two cross-sectional surveys in May of 2017 in southern Mozambique. Using real-time quantitative PCR (RT-qPCR) as the reference method, the age-specific diagnostic performance indicators of the cRDT and uRDT were compared. The presence of histidine-rich protein 2 (HRP2) and P. falciparum infections at similar geometric mean parasite densities (112.9  parasites/μL for uRDTs and 145.5 parasites/μL for cRDTs). The presence of HRP2 antigen was similar among false positive (FP) samples of both tests (80.5% among uRDT-FPs and 84.4% among cRDT-FPs). Only one false negative sample was detected with a partial pfhrp2 deletion.Among the 4,396 participants with complete test results, the sensitivity of uRDTs  was marginally better than that of cRDTs  , while the specificities were similar . While the performance of both RDTs was lowest in ≥ 15-year-olds, driven by the higher prevalence of low parasite density infections in this group, the sensitivity of uRDTs was significantly higher in this age group  compared to the sensitivity of cRDTs  . Both RDTs detected Pf malaria RDTs in the community and are still not comparable to molecular methods to detect P. falciparum infections in this study setting.This study showed that the uRDTs developed by Abbott do not substantially outperform SD-Bioline  Plasmodium parasites accounted for an estimated 228 million cases and 405,000 deaths in 2018, most of them circumscribed to sub-Saharan Africa and concentrated in the under-five years of age and pregnant populations ) antigens were estimated at PATH’s laboratories  through a Q-plex enzyme-linked immunosorbent assay (ELISA) from a subset of 223 samples comprised of samples positive by RT-qPCR and either cRDT or uRDT (N = 82), negative by all diagnostic forms (N = 45), as well as all samples positive by cRDT or uRDT but negative by RT-qPCR (N = 44) and samples positive by RT-qPCR but negative by both RDTs (N = 52). A small disc (6-mm) was punched out of each dried blood spot (DBS) and transferred to a well in a low-binding V-bottom 96-well plate (Thermo Scientific) with calibrator diluent . The plate was incubated overnight in 4 °C and shaken for 1 h at room temperature. The eluates were tested in two replicates on Q-Plex Human Malaria Array  according to the manufacturer’s instructions  = 3–10). Women accounted for 55.2% (2402/4353) of the study sample, among whom 21 reported to be pregnant. Only 0.5% (19/4128) of participants presented a body temperature ≥ 37.5 °C during testing. However, 8.5% (371/4387) reported having a febrile episode in the preceding 24 h. Within the previous month, 14.6% (640/4394) reported having a febrile episode and 6.5% (284/4342) reported taking anti-malarials. Additional file 8.4%, XinThe all-age weighted malaria prevalence by cRDT was 6.1%  4.2% to 8.7%) in Manhiça, 1.9% (95% CI 0.9% to 3.9%) in Xinavane and 2.6% (95% CI 2.0% to 3.4%) in Magude, while the weighted prevalence by uRDT in these same settings was 6.5% (95% CI 4.6% to 9.1%), 2.8% (95% CI 1.4% to 5.8%) and 3.2% (95% CI 2.3% to 4.0%), respectively. The same indicator measured by RT-qPCR was 7.9% (95% CI 5.5% to 11.3%) in Manhiça, 6.1% (95% CI 3.4% to 10.8%) in Xinavane and 4.5% (95% CI 3.6% to 5.6%) in Magude.P. falciparum infections detected by cRDT, 166 (3.8%) by uRDT, and 179 (4.1%) by RT-qPCR  CR Table . When stCR Table . In all Considering RT-qPCR as the gold standard, cRDT misclassified 34 RT-qPCR-negative samples as positive (FP) and 69 RT-qPCR-positive samples as negative (FN), while uRDT misclassified 44 RT-qPCR-negative samples as positive and 57 RT-qPCR-positive samples as negative Table . Figure The sensitivity of uRDTs was slightly higher  than that of cRDTs  , while the specificity of both RDTs was very similar: 99.0 (95% CI 98.6 to 99.2) for uRDT and 99.2 (95% CI 98.9 to 99.4) for cRDT . Table The GMPD of infections detected by RT-qPCR was 77.5 parasites/µL (95% CI 50.2 to 119.7) and decreased with increasing age. The GMPD was 112.9 parasites/µL (95% CI 65.3 to 195.0) among TP uRDT samples and 145.5 parasites/µL (95% CI 82.0 to 258.0) among TP cRDT samples. FN samples by uRDT or cRDT had GMPDs of < 35 parasites/µL   of the study population had fever at the time of the study or reported a febrile episode during the preceding 24 h. Using RT-qPCR, malaria was detected in 13.9% (53/382) of these febrile individuals and in 3.1% of afebrile (126/4011) ones. Fever was detected or reported within preceding 24 h in 29.6% (53/179) of the individuals with a positive RT-qPCR, 33.7% (56/166) of individuals with a positive uRDT, and 35.4% (51/144) of individuals with a positive cRDT.The GMPD of febrile participants was 346.3 parasites/µL (95% CI 152.0 to 788.7) (n = 53) and 41.3 parasites/µL (95% CI 25.6 to 66.5) (n = 126) for afebrile ones. Consequently, the sensitivity of cRDTs was higher among febrile individuals  than among afebrile individuals  . Likewise, the sensitivity of uRDTs was higher among febrile individuals  than among afebrile ones  . Only the sensitivity among afebrile individuals varied significantly between RDTs.Two infections were detected by RT-qPCR among the 21 women who reported being pregnant in this study. One infection had a parasite density of 1904 parasites/µL by RT-qPCR and was diagnosed by microscopy, cRDT and uRDT; and had detectable HRP2 antigenaemia. The other infection had a parasite density of 2.9 parasites/µL by RT-qPCR, but was not detected by any other diagnostic tool despite having detectable HRP2. Neither presented with a fever during the survey nor reported having had a fever or taking anti-malarials in the preceding 30 days.Pf malaria uRDT (68.2) was marginally higher compared to the SD-Bioline Pf malaria cRDT (61.5), while specificities were very similar (99.0 for uRDT vs. 99.2 for cRDT). This translated into very few differences in absolute numbers. The performance of both types of RDTs was affected by age in a study population exposed to malaria since a very young age [pfhrp2 or pfhrp3 deletions, as only one FP sample was detected with a partial pfhrp2 deletion, in agreement with previous findings from Mozambique [This study evaluated the performance of a recently developed ultrasensitive malaria RDT in field conditions among individuals in a malaria endemic community of southern Mozambique. The study showed that the sensitivity of the Abbott oung age . In thiszambique , 32.Overall, these results raise the question of the added value of the uRDTs evaluated in this study, in Mozambique. In fact, the utility of these uRDTs remains unclear, partly as a result of the emergent body of evidence in line with the performance outcomes presented here , 27, 29.uRDTs have been proposed as a tool to detect malaria infections even at low-density infections (below 100–200 parasites/µL), thus triggering their treatment in order to interrupt transmission in areas aiming for elimination, through mass test and treat (MTAT) campaigns, or proactive or reactive testing and treating. MTAT strategies evaluated throughout Africa and Asia have to date shown not to have a strong impact on malaria transmission when using cRDTs , 48–52, This study showed that the sensitivity of both RDTs significantly dropped among individuals older than 15 years-old, particularly for cRDTs (39.2) compared to uRDTs (54.9) due to the high prevalence of lower density infections in this age group likely as a result of the cumulative malaria exposure . MoreoveuRDTs have also been proposed to improve malaria case management at the health facility level . This stOne of the commonly reported concerns of using a more sensitive HRP2-based RDT is that it may provide a higher number of FP in post-treated individuals, due to its capacity to detect remaining concentrations of HRP2 in the blood for a longer period of time . This isThis study is subject to several limitations. First, the diagnostic performance analysis was conducted using RT-qPCR as the gold standard. In the younger age groups, the proportion of positive RT-qPCR samples was very similar or even lower to the proportion of positive uRDT samples. This can be partly explained by the high number of FP samples that were HRP2-positive in younger age groups. Children may be more likely to remain RDT positive for a longer time after anti-malarial treatment than adults, as a result of less acquired immunity and higher parasite densities leading to higher HRP2 circulating concentrations , 59. HowPf malaria uRDTs do not substantially outperform SD-Bioline Pf malaria RDTs in the community or among febrile cases, and are still not comparable to molecular methods like RT-qPCRs. Therefore, the use-cases for the currently available uRDTs should be reviewed before malaria endemic countries with a long experience using the standard RDTs decide to switch to a different yet similarly performing test.Overall, these results contributed to the growing evidence from field-based studies that show that the Abbott Additional file 1: Table S1. Definitions of performance indicators used. TP = True positive, FP = False positive, TN = True negative, FN = False negative. Table S2. Study population characteristics, overall and by regionAdditional file 2: Figure S1. Flow-chart of the parasite pfhrp2 and pfhrp3 gene deletions analysis


Plasmodium falciparum candidate RTS,S was favorably reviewed by the European Medicines Agency and subsequently introduced into national pilot implementation programs, marking the first human anti-parasite vaccine to pass regulatory scrutiny. Since the first trials published in 1997, RTS,S has been evaluated in a series of clinical trials culminating in Phase 3 testing, while testing of other pre-erythrocytic candidates (that target sporozoite- or liver-stage parasites), particularly whole sporozoite vaccines, has also increased. Interest in blood-stage candidates (that limit blood-stage parasite growth) subsided after disappointing human efficacy results, although new blood-stage targets and concepts may revive activity in this area. Over the past decade, testing of transmission-blocking vaccines  advanced to field trials and the first generation of placental malaria vaccines (that clear placenta-sequestering parasites) entered the clinic. Novel antigen discovery, human monoclonal antibodies, structural vaccinology, and improved platforms promise to expand on RTS,S and improve existing vaccine candidates.Malaria vaccine development entered a new era in 2015 when the pre-erythrocytic  TM) received a favorable opinion from the European Medicines Agency (EMA) in 2015 after review of its safety and efficacy to reduce clinical Plasmodium falciparum malaria episodes in young African children. This was a milestone in vaccine development as the first human parasite vaccine passed the highest level of regulatory scrutiny (referred to as WHO-listed authority maturity level 4 (WLA ML4))1. RTS,S/AS01E pilot implementation programs requested by WHO were launched in 2019 to assess safety and benefits during delivery through standard public health mechanisms. Meanwhile, novel malaria vaccine candidate clinical development has continued apace. Some new vaccine candidates seek to improve on the efficacy of RTS,S/AS01E to prevent clinical malaria in African children, while other candidates in the clinic will pursue different indications such as to protect pregnant women from malaria, or to interrupt the parasite’s cycle of transmission and thereby contribute to regional elimination of malaria by blocking P. falciparum infection or transmission to mosquitoes.The malaria vaccine RTS,S/AS01E , has remained steady at ~10 trials each year  that target parasite sexual stages to prevent parasite transmission to mosquitoes. Further, the first vaccine candidates to protect women from placental malaria entered the clinic in the past 5 years, and trials of blood-stage vaccines (BSV)  decreased in frequency from 2001–2010 to 2011–2020. Interest has increased in the use of vaccines for malaria elimination, or a so-called vaccine to interrupt malaria transmission (VIMT), that could include antigens expressed during pre-erythrocytic, blood-stage and/or mosquito-sexual stage development in order to reduce or halt the spread of parasites in the community2. P. vivax vaccine trials were registered sporadically, reflecting the dearth of resources dedicated to this neglected disease that afflicts millions each year. Notably, some promising P. vivax candidates induced functional activity in Phase 1 trials.Over the past 20 years, the rate of new malaria vaccine trials registered at ClinicalTrials.gov, a major venue to register clinical trials that launched in 2000  antibodies against surface antigens that clear sporozoites from skin or bloodstream or block their invasion of hepatocytes, or (2) T cell responses that attack infected hepatocytes. Protective efficacy of PEV was first demonstrated in a human in the 1970s using radiation-attenuated WSV delivered through hundreds of mosquito bites; the vaccinee was protected from subsequent challenge with homologous 3 and heterologous4P. falciparum sporozoites (PfSPZ) but not from challenge with homologous blood-stage parasites3. PEV with high activity can completely clear pre-erythrocytic parasites before release into the bloodstream, and these have also been referred to as anti-infection vaccines (AIV).Pre-erythrocytic vaccines (PEV) target antigens from 5, which continues to be a major focus of vaccine development. RTS,S, the most advanced PEV, incorporates a P. falciparum CSP fragment comprising central repeat (hence “R”) and C-terminal regions  fused to hepatitis B surface antigen (“S”), or altogether “RTS”. RTS is expressed in yeast that also carry hepatitis B “S” expression cassettes, and thus synthesize S and RTS polypeptides that spontaneously co-assemble into mixed lipoprotein particles  with the CSP fragment on their surface6.The demonstration that WSV induce sterilizing immunity in humans coincided with the development of genetic engineering tools. The first malaria gene to be cloned encodes the major surface antigen of sporozoites called circumsporozoite protein or CSP7. The phase III trial enrolled 15,459 children at 11 centers in seven African countries, and delivered 3 doses at 1-month intervals to coincide with the Extended Program for Immunization schedule, with a booster dose 18 months after the third dose. Clinical malaria episodes (the primary efficacy endpoint) were reduced by ~36% in young children and ~26% among infants who received four vaccine doses , with statistically significant efficacy against severe malaria in young children but not infants. Efficacy waned over time, with 68% reduction in the incidence of clinical malaria in the first 6 months8. The vaccine prevented an estimated 1774  clinical malaria episodes per 1000 children that received four vaccine doses and 1363 (95% CI 995–1797) per 1000 children that received 3 doses9. Although vaccine efficacy tended to have a higher point estimate in lower transmission settings, this difference was not significant, and the highest numbers of cases averted were noted in areas of high malaria incidence.RTS,S formulated in GSK’s proprietary AS01 adjuvant completed trials in adults, children, and young infants in sub-Saharan AfricaPlasmodium falciparum parasite, and against hepatitis B”10. In 2019, a pilot implementation program of RTS,S was launched in Malawi, Ghana, and Kenya to assess protective benefits and safety during routine use in real-life settings. The program will enroll more than a million children over a period of 3 years in selected areas of medium-to-high malaria burden11. Children will be randomized to unvaccinated or vaccinated clusters that are offered 4 doses of RTS,S. While WHO views the program as a pilot introduction into national childhood immunization schedules, the program is registered as a study , prompting ethical concerns over the absence of informed consent, which was considered by WHO to be “implied” as part of the children’s routine vaccination schedule12.In 2015, the Committee for Medicinal Products for Human Use (CHMP) of the EMA adopted a positive scientific opinion for use of RTS,S outside the European Union “in areas where malaria is regularly found, for the active immunisation of children aged 6 weeks to 17 months against malaria caused by the 13.Progress with RTS,S represents a historic milestone, but its partial efficacy leaves room for improvement. RTS,S is administered to children according to their age, in order to coincide with other routine EPI vaccinations. The period of peak vaccine-induced antibody levels (and presumed maximum protection) often does not coincide with the malaria parasite transmission season. An ongoing trial in West Africa is using RTS,S for seasonal vaccination with annual booster doses scheduled to maximize antibody responses during the peak malaria parasite transmission season . Other field trials are examining fractional dosing regimens that deliver 1/5th of the full dosage for last vaccine administration, a strategy that has increased efficacy against homologous CHMI as well as antibody somatic hypermutation and avidity in malaria-naïve adult subjects14. In mice, R21 was immunogenic at low doses using human-use adjuvants, and unlike RTS,S, induced minimal antibody responses to the HBsAg fusion partner. Vaccinated mice displayed sterile protection against challenge with transgenic sporozoites expressing homologous PfCSP, and R21 has advanced to Phase 1/2 testing in Africa . Viral-vectored PEV candidates (including those that target CSP) have also been assessed in human trials, particularly as part of heterologous prime-boost approaches. These have induced strong CD8 T cell proliferation among other responses, but thus far have failed to exceed the protective efficacy against sporozoite challenge seen with RTS,S (reviewed in ref. 15).Another approach is to improve immunogenicity of CSP-based vaccines. At Oxford’s Jenner Institute, a “next-generation RTS,S-like vaccine” called R21 dispenses with the unfused “S” and generates particles solely comprised of CSP-HBsAg fusion proteinP. falciparum antigens, CSP displays high sequence diversity including in the C-terminal region targeted by RTS,S vaccine. In sieving analysis of the Phase 3 trial, RTS,S showed greater protection against parasites that matched its C-terminal sequence16, indicating that parasite variation can limit efficacy and that escape variants could spread. Speculatively, CSP-based vaccines that eliminate or reduce responses to variant epitopes could improve overall efficacy. Further, the N terminal region is not included in RTS,S, but is critical to hepatocyte attachment and invasion by sporozoites17, and naturally acquired antibodies to an N-terminal peptide are associated with protection from disease in Tanzanian children18. Full-length CSP candidates have been developed20 and some have recently entered the clinic [ClinicalTrials.gov ID NCT03589794].As with many 27. Most anti-CSP human mAbs react to the repeat region, and a subset of these recognize or cross-react to an epitope at the junction of N-terminal and repeat regions. Antibodies to the C-terminal region have been infrequent: only 4 of 215 monoclonal antibodies derived from PfCSP-specific B cells after whole sporozoite vaccination bound the C terminal region specifically25. Although the sieving analysis of the RTS,S trial indicated differential efficacy against parasites with C terminal regions that did or did not match the vaccine, the few C-terminal-specific human mAbs tested failed to show functional activity in vitro or in vivo in mice after passive transfer against parasites carrying the homologous CSP sequence25. Functional assessment of additional C-terminal-reactive human mAbs may be useful to understand the differential efficacy of RTS,S.Structural vaccinology approaches are being applied to design improved CSP-based vaccines by defining epitopes of functional human monoclonal antibodies. Monoclonal antibodies to CSP have been prepared from humans after PEV administration or malaria parasite exposure28. In 2010, the company Sanaria introduced a platform technology that entails harvesting PfSPZ from the salivary glands of aseptic mosquitoes infected by cultured laboratory parasites, followed by purification, vialing, and cryopreservation in liquid nitrogen vapor phase29. PfSPZ are attenuated by different approaches to prepare the vaccine candidate product: radiation attenuation , chemoattenuation achieved in vivo by concomitant administration of antimalarial drugs , or genetic attenuation by deletion of genes required to complete liver-stage development30 )31. PfSPZ Vaccine has required direct venous inoculation to confer sterile immunity against challenge with sporozoites32. The logistical and potential cost challenges to implementing WSV will include (1) liquid nitrogen cold chain, (2) intravenous inoculation, (3) scale-up of manufacture.Despite evidence since the 1970s that WSV confer sterilizing immunity against sporozoite challenge of humans, WSV were not pursued as a product owing to the perception that manufacture of irradiated sporozoites was impractical for a vaccine34. In malaria-naive adults, the level and duration of protection from homologous or heterologous sporozoite challenge depend on dose and regimen with either PfSPZ Vaccine or PfSPZ-CVac, and these have achieved high levels of sterile homologous immunity37. Protection against heterologous CHMI and protection beyond a few months have not yet been studied systematically. In an area of intense malaria transmission in Mali, five administrations of PfSPZ Vaccine (2.7 × 105 PfSPZ dosage) to adult residents reduced the risk of new P. falciparum infection by 52% in time-to-event analysis over the 24 weeks after last dose, and reduced the proportion infected across the transmission season by 29%34. The time-to-event efficacy achieved appears greater than that reported for RTS,S in adults using AS02 or AS01 adjuvants39. Additional field efficacy trials of PfSPZ Vaccine with 3-dose regimens have been completed in adults and infants  egress from host erythrocytes and within seconds each merozoite has invaded a new erythrocyte to initiate another round of multiplication .BSV target the asexual parasite forms that undergo repeated multiplicative cycles in erythrocytes and cause disease and death. Cycle duration varies between malaria parasite species and determines the period between fevers, or periodicity: 1 day for 46 and later in Thai adults47. Of note, the studies in Africa included children with malaria who did not receive antimalarial chemotherapy as the standard of care46. and hence would not now pass ethical scrutiny. In subsequent studies, immunization with whole parasite preparations rich in merozoites protected monkeys from P. falciparum infection48, focusing attention of vaccine developers on merozoite invasion over the ensuing years.Blood-stage parasites are an attractive target because this is the disease-causing stage of development, and also because passive transfer of IgG purified from semi-immune African adults was shown to clear parasitemia from African children 6 decades ago49. Ultimately, the results showed scant evidence of protection against controlled human infection or against naturally occurring infection. In particular, AMA-1 candidates induced high titer antibody that was functional by in vitro assays in two trials but failed to show efficacy against controlled infection with the homologous parasite51. Among all BSV candidates, only GMZ2 (consisting of conserved domains of GLURP and MSP3) showed statistically significant albeit low (14%) efficacy in a pre-specified analysis against naturally acquired infection52.The challenges to developing anti-merozoite vaccines include 1) the brief time (seconds) when merozoites pass between erythrocytes and are accessible to antibodies, (2) antigenic polymorphism, (3) redundant invasion pathways, and (4) the large number of parasites that need to be targeted compared with the numerical bottlenecks attacked by PEVs and TBVs. Between 2000-2015, over 30 BSV trials registered in ClinicalTrials.gov were completed  binds the essential red cell receptor basigin and shows limited polymorphism53, and entered clinical trials using a viral-vectored prime-boost immunogen54. PfRH5 is the first highly conserved merozoite antigen shown to induce broadly neutralizing antibody in preclinical studies55. In monkeys, different combinations of PfRH5 viral-vectored and/or adjuvanted protein immunogens conferred protective immunity that controlled parasitemia after challenge with virulent heterologous parasites56.58 and PfRH5 studies in monkeys showed good protection against virulent blood-stage parasite challenge but modest or no boosting of vaccine-induced antibody by infection56. This may limit the duration of protection conferred by a vaccine. In addition, protection in monkeys required an estimated 200 µg/mL of anti-PfRH5 IgG56, a high level to achieve and sustain by vaccination. Efforts to improve RH5 vaccine candidates include presentation in virus-like particles (VLP) and production of a protein vaccine in Drosophila (Schneider 2) cells60.Notably, natural infections induce modest or no antibody against PfRH561. This knowledge will inform the design of improved Rh5 immunogens that focus the antibody response on neutralizing and potentiating epitopes.In addition, scientists at Jenner Institute have generated human mAbs from PfRH5 vaccinees and used these in structural studies to identify epitopes targeted by neutralizing, non-neutralizing and potentiating antibodies 62. In monkeys, AMA1-RON2 showed significantly greater protection against heterologous blood-stage challenge versus AMA1 alone, and conferred sterile protection in half the animals62. As with Rh5, AMA1 vaccines may be improved by structural studies of antigen-antibody complexes to determine epitopes to include or exclude in re-designed immunogens. Unlike Rh5, AMA1 displays extensive sequence variation, and therefore future studies will need to assess the number of alleles or chimeric sequences that will be required for AMA1-RON2 to confer broadly effective immunity.Despite its poor efficacy in previous trials, AMA1 is an essential protein for blood-stage parasite growth. The recognition that AMA1 binds to the rhoptry neck protein RON2 at the merozoite-erythrocyte interface to initiate invasion has revived interest in AMA1 as an immunogen in complex with RON2. When complexed with RON2 peptide, AMA1 antigenicity is altered to generate more potent anti-invasion antibodies than monomeric AMA1 antigenP. falciparum, and is a target of naturally acquired protective antibody63. However its highly polymorphic sequence, large size, and cysteine-rich conformational structure have impeded vaccine development and no trials of PfEMP1-based vaccines have been reported. An exception to this is VAR2CSA, a distinctly structured PfEMP1 family member used by the parasite to sequester in the placenta, as discussed in the next section on placental malaria vaccines (PMV).The search for novel BSV antigens has also moved beyond merozoite targets. Parasite antigens are exported to the surface of infected erythrocytes where they are accessible to antibody for hours. Among these, the variant surface antigen family PfEMP1 is immunodominant, mediates parasite sequestration and hence virulence of 64. Antibodies to PfGARP induced programmed cell death of intraeythrocytic trophozoites in vitro and naturally acquired PfGARP antibodies were related to control of P. falciparum parasitemia and protection from severe malaria. In monkey studies, PfGARP vaccines conferred partial protection against P. falciparum challenge.Interestingly, a non-PfEMP1-infected erythrocyte surface protein called PfGARP has just been described as the target of protective antibodiesP. falciparum Schizont Egress Antigen 1 (PfSEA-1)65. Antibodies bind to intraeythrocyic PfSEA-1 and arrest P. falciparum schizont rupture in vitro, and vaccination of mice with recombinant P. berghei SEA-1 reduced parasitemia and delayed mortality after challenge with lethal P. berghei.Parasite egress from erythrocytes has also been identified as a target of protective antibody. A differential screen of sera from children that did or did not control parasite density during infection associated protection to antibody against 68 and persisted after CD8+ T cell depletion67. In Aotus nancymaae monkeys, a single CAP dose did not delay patent parasitemia after blood-stage parasite challenge but may have delayed drug treatment and induced CD8 T cell responses69. In humans, CAP were well-tolerated in malaria-naïve volunteers and induced T cell but not antibody responses70. A human trial of a 3-dose CAP regimen has been registered to assess efficacy against challenge with homologous blood-stage parasites [ACTRN12618001314213]. As with PfSPZ-CVac, CAP will need to be convincingly shown to be safe and implementable to be viewed as a viable vaccination strategy.Given the disappointing record of subunit BSV in human trials, scientists at Griffith University in Australia are exploring whole blood-stage parasite vaccines attenuated by incubation with a DNA-binding drug . Unlike the PfSPZ-CVac approach (described above) that chemoattenuates parasites in vivo, chemically attenuated blood-stage parasites (CAP) are prepared in vitro before administration. In mice, CAP (but not lysed parasites) induced homologous and heterologous immunity; protection was CD4+ T cell-dependent71. Placental parasites uniformly express the distinctive PfEMP1 family member VAR2CSA that binds CSA72; recombinant VAR2CSA induces antibodies that block parasite binding to CSA (reviewed in ref. 73). VAR2CSA is a complex target that has a large (>300 kD) extracellular domain with six DBL domains and additional interdomain regions, and a recent report identified atypical VAR2CSA with seven or eight DBL domains in some field isolates that can be functional74.PMV target chondroitin sulfate A (CSA)-binding parasites that uniquely sequester in the placenta; hence PMV represent a distinct BSV approach. While vaccines such as PEV and BSV candidates that protect the general population may also benefit pregnant women, naturally acquired protection against placental malaria offers a focused vaccine approach. Natural antibodies to CSA-binding parasites are associated with protection from placental malaria and are acquired over successive pregnancies as women in endemic areas become resistant to placental malariaDrosophila cell-expressed PAMVAC was tested in different human adjuvants and proved to be safe, well-tolerated, and induced functionally active antibodies against homologous parasites75. PAMVAC will be tested in malaria-experienced nulligravidae next. A second subunit VAR2CSA candidate, PRIMVAC, has completed a first-in-human trial in France and Burkina Faso, which showed the vaccine was safe, immunogenic, and induced functional antibodies against the homologous VAR2CSA variant expressed by NF54-CSA infected erythrocytes. However, cross-reactivity against heterologous VAR2CSA variants was limited and only observed in the higher dose group76. Researchers hypothesized that an alternate schedule of immunization, antigen dose, and combinations with other VAR2CSA-based vaccines could improve the cross-reactivity against heterologous VAR2CSA variants.The first trials of VAR2CSA-based vaccines have been conducted over the past 5 years. Owing to its large size, VAR2CSA vaccine development has focused on individual domains or domain combinations. Two candidates based on N-terminal VAR2CSA fragments that have high binding affinity for CSA have completed first-in-human trials. The 78. Target antigens were identified with monoclonal antibodies that were raised in rodents against gamete/zygote preparations and blocked infection of mosquitoes. The four leading candidates have been grouped as gamete surface proteins first expressed by gametocytes in human blood79 such as Pfs230 and Pfs48/45 of P. falciparum, and zygote surface proteins expressed only post-fertilization in the mosquito host81 such as Pfs25 and Pfs28. These antigens are cysteine-rich with multiple 6-cys or epidermal growth factor (EGF)-like domains that have been challenging to prepare as properly folded recombinant protein. Pfs25 was the first TBV candidate prepared as a recombinant protein82. In animal studies, Pfs25 candidates have induced equal or greater serum transmission-blocking activity as other antigens or antigen combinations84 and hence Pfs25 has been the focus of clinical trials published to date. Ongoing trials are now examining the activity of Pfs230 vaccine candidates . Pfs230 antibodies raised in animals show lytic activity against P. falciparum gametes in the presence of complement85, which might similarly enhance activity of human Pfs230 antisera.TBV incorporate surface antigens of mosquito/sexual-stages (gametes and zygotes) in order to induce antibodies that kill parasites in the mosquito bloodmeal and interrupt parasite transmission through the vectorPichia-expressed Pfs25 to carriers such as ExoProtein (EPA) to generate nanoparticles, and formulate these in adjuvants86. While several previous trials of Pfs25 candidates failed to induce adequate antibody responses or were overly reactogenic in human vaccinees, Pfs25-EPA conjugate formulated with Alhydrogel® was reported in 2016 to be well-tolerated and to induce functional antibodies in humans that block transmission of P. falciparum to mosquitoes in membrane feeding assays87, and this activity correlated with titers. However, functional activity in most vaccinees required 4 doses and antibody titers and activity waned rapidly.Both Pfs25 and Pfs230 recombinant antigens have shown poor immunogenicity as monomers. To enhance immunogenicity, our group prepares protein-protein conjugate vaccines by chemically coupling Pichia-expressed Pfs230 domain 188. These studies are also assessing the benefits of alternative adjuvants, including the GSK adjuvant AS01 used in the RTS,S vaccine .Ongoing studies  are comparing and combining Pfs25 and Pfs230 vaccine antigens using 89, where it forms a complex with Pfs23090. Pfs48/45 and Pfs230 play a role in male gamete fertility91. Pfs48/45 comprises three 6-cys domains, of which the C-terminal domain contains a conformational epitope targeted by potent transmission-blocking mAbs92.Additional TBV candidates will enter the clinic in the coming years and can be compared or combined with the current candidates. Gamete surface antigen Pfs48/45 is likely to be the next target tested in humans. Like Pfs230, Pfs48/45 expression occurs during the later stages of gametocyte development in the human red cell. Once ingested by mosquitoes, gametocytes egress from red cells as gametes. Pfs48/45 appears as GPI-anchored antigen on both male and female gametesLactococcus lactis93. The resulting antigen, called R0.6 C, reacts to conformation-dependent functional monoclonal antibodies and induces transmission-blocking antibodies in animals. Further, a chimeric protein comprising the pro-domain of Pfs230 (upstream of domain 1) and the C-terminal domain of Pfs48/45 induced significantly higher serum functional activity than did R0.6C, suggesting an additive effect of antibody to Pfs230 and Pfs48/4594.Pfs48/45 vaccine development has been hindered by difficulty in recombinant expression of properly folded protein. Progress has recently been reported for the C-terminal 6-Cys domain as the downstream partner in a fusion with the R0 region of asexual stage Glutamate Rich Protein by expression in Current challenges of TBV development include achieving sufficient adaptive responses that maintain high levels of antibodies over time, as well as widespread coverage to accomplish herd immunity. Furthermore, TBVs must have an exceptional safety profile since they do not confer direct benefit to the individual. TBVs could be implemented in combination with a PEV to prevent both infection in humans and transmission to mosquitoes, and could similarly be combined with BSV that reduce transmission to assess additive or synergistic activity.P. vivax causes an estimated 14.3 million malaria episodes each year and is the leading cause of malaria in Asia and Latin America95. Although it has been historically designated as benign tertian malaria, P. vivax is increasingly recognized as a public health threat causing severe morbidity and mortality96. Further, sterile heterologous immunity against P. vivax has been demonstrated97. Despite this, P. vivax research suffers from a dearth of resources since the funds dedicated to malaria research—which are not commensurate to the scope of the problem in any case—are predominantly allocated to P. falciparum research. This inadequate investment is particularly short-sighted, since vaccines may disproportionately benefit P. vivax control: dormant liver forms called hypnozoites produced by P. vivax  allow the parasite to relapse repeatedly over months or years and thwart efforts to control or eliminate this species, hence the benefit of durable immunological protection conferred by vaccines.P. vivax vaccine development has generally followed P. falciparum efforts. Vivax vaccines tested in humans include orthologues  of the PEV (PfCSP) and TBV (Pfs25) candidates that have commanded greatest attention for P. falciparum. However, PvCSP vaccine prepared as a monomer formulated in GSK’s AS01 adjuvant failed to induce sterile protection against challenge with P. vivax sporozoites98, and Pvs25 expressed in S. cerevisiae formulated in Montanide ISA 51 caused systemic reactogenicity that prompted termination of the clinical trial99. Notably, when formulated in Alhydrogel®, Pvs25 was well-tolerated, and the antibody responses, though modest, showed functional transmission-blocking activity in mosquito feeding assays that correlated to antibody concentration100. Based on clinical progress with the P. falciparum candidate Pfs230D1-EPA, the P. vivax candidate Pvs230D1-EPA is currently being manufactured in anticipation of trials that may launch in 2021.P. vivax BSV trials have focused on Duffy-Binding Protein (PvDBP) which binds the Duffy Antigen Receptor for Chemokines (DARC) on erythrocytes and is required for merozoite invasion. Two DBP candidates have completed Phase 1 trials, including a viral-vectored101 and a recombinant protein candidate102. Both candidates induced strain-transcending functional antibodies measured in vitro. Using human mAbs generated through vaccination or natural vivax exposure, structural studies have identified functional and non-functional epitopes that will provide a rational basis to improve the design of PvDBP immunogens104.The landscape for malaria vaccines in 2020 is very different from that in the year 2000. The pre-erythrocytic vaccine (PEV) product RTS,S/AS01E has proven to be safe and efficacious for reducing clinical malaria in African children. Upon completion of ongoing implementation programs in 2022 in three African countries, the results will be reviewed by international bodies including WHO and RTS,S/AS01E will be considered by national policy decision-makers for broader use in Africa.P. falciparum vaccine candidates that have demonstrated efficacy or activity in human trials justifies increased investment in P. vivax vaccines to pursue similar goals.While RTS,S reduces clinical malaria risk in African children, newer PEV candidates such as R21/Matrix M, PfSPZ whole sporozoite vaccines, and full-length CSP immunogens seek to improve on its efficacy. In parallel, TBV have advanced to Phase 2 clinical trials over the past decade. Efficacious TBV can be combined with the most effective pre-erythrocytic vaccines to pursue malaria elimination programs in combination with other malaria control tools. The substantial progress made with BSV that target merozoite invasion proteins have delivered disappointing efficacy results in clinical trials over the past 20 years. Novel or improved immunogens that target non-redundant merozoite invasion pathways may improve on these dismal results. Meanwhile, vaccines against other BSV targets such as infected red cell surface proteins, schizont egress antigens, or intact infected erythrocytes that have been attenuated, are progressing in preclinical and clinical studies.PMV represent a distinct type of BSV by targeting the surface antigens of CSA-binding infected erythrocytes that sequester in intervillous spaces and cause placental malaria. Two vaccine candidates that target VAR2CSA, the immunodominant surface antigen of CSA-binding infected erythrocytes, have completed first-in-human trials. An initial report suggests that these vaccines can induce functional activity against homologous parasites. Future studies will determine whether they can induce heterologous activity that is boosted during naturally occurring pregnancy malaria infections to confer durable protection over successive pregnancies.Malaria vaccine candidates are progressing in clinical trials and RTS,S has advanced to implementation. The question remains how well can malaria vaccines work, and how can we best deploy them to the advantage of the communities devastated by malaria. Scientists are pursuing antigen discovery, structural vaccinology studies, and improved platforms to expand on or improve our existing portfolio of candidates. As the portfolio advances in development, adequate resources are needed to develop promising candidates; as more candidates transition to products, we must ensure these valuable new interventions are optimally deployed to maximize their benefits in the fight against this ancient scourge.supplementary-materialsSupplementary Data Set 1


Plasmodium falciparum and Plasmodium vivax infections were investigated in different transmission settings in Ethiopia.As countries move to malaria elimination, detecting and targeting asymptomatic malaria infections might be needed. Here, the epidemiology and detectability of asymptomatic A total of 1093 dried blood spot (DBS) samples were collected from afebrile and apparently healthy individuals across ten study sites in Ethiopia from 2016 to 2020. Of these, 862 were from community and 231 from school based cross-sectional surveys. Malaria infection status was determined by microscopy or rapid diagnostics tests (RDT) and 18S rRNA-based nested PCR (nPCR). The annual parasite index (API) was used to classify endemicity as low (API > 0 and < 5), moderate (API ≥ 5 and < 100) and high transmission (API ≥ 100) and detectability of infections was assessed in these settings.Plasmodium infections by microscopy/RDT, nPCR and all methods combined was 12.2% (105/860), 21.6% (183/846) and 24.1% (208/862), respectively. The proportion of nPCR positive infections that was detectable by microscopy/RDT was 48.7% (73/150) for P. falciparum and 4.6% (2/44) for P. vivax. Compared to low transmission settings, the likelihood of detecting infections by microscopy/RDT was increased in moderate  and high endemic settings . After adjustment for site and correlation between observations from the same survey, the likelihood of detecting asymptomatic infections by microscopy/RDT  declined with age.In community surveys, the overall prevalence of asymptomatic Plasmodium reservoir detected by nPCR. The detectability of infections was particularly low in older age groups and low transmission settings. These findings highlight the need for sensitive diagnostic tools to detect the entire parasite reservoir and potential infection transmitters.Conventional diagnostics missed nearly half of the asymptomatic  Plasmodium falciparum and Plasmodium vivax : 11–35). As expected, participants from the school surveys were younger  than community surveys . Results are presented separately for community and school surveys, focusing on community surveys for the main comparisons (Table Plasmodium infections was 12.2% (105/860) by microscopy/RDT and 21.6% (183/846) by nPCR  of samples were P. falciparum positive; 3.7% (31/846) were P. vivax and 1.5% (13/846) were mixed P. vivax and P. falciparum. Although the school surveys were from high and moderate transmission sites, there was overall lower Plasmodium infection prevalence in the school surveys than in the community surveys as measured by all methods combined .In the community surveys, the overall prevalence of asymptomatic P. falciparum, 5.2% (12/231) were due to P. vivax, and 3.5% (8/231) were due to mixed P. falciparum and P. vivax species infections  by microscopy/RDT whilst 11.3% (26/231) were parasite positive either by nPCR or both methods combined. Of these nPCR positive malaria infections from the school surveys, 2.6% (6/231) were due to /231 by mAcross the community surveys, in high transmission settings, nPCR-based prevalence of malaria infection ranged from 17.6% (n/N) at Meng to 46.1% (47/102) at Lare district. In the moderate transmission sites, the nPCR-based prevalence was 29.3% (34/116) at Mao-Komo and 12.4% (20/161) at Arbaminch Zuria district. In low transmission sites, the overall nPCR infection prevalence was 9.4% (22/234). The overall microscopy/RDT based prevalence was 23.1% (81/351), 7.9% (22/277), and 0.9% (2/232), in high, moderate, and low transmission settings, respectively  compared to moderate  and low transmission settings . Moreover, the burden of asymptomatic Plasmodium infection was higher in the 5–15 age groups as measured by microscopy/RDT  and nPCR   were detected by RDT was increased for individuals living in higher transmission settings  and individuals living in moderate transmission  compared to low transmission settings  of nPCR detected Plasmodium species detected in the community samples, the majority were attributable to P. falciparum  when all samples were combined. Of the nPCR detected P. falciparum-mono species infections (n = 139) and mixed-species infections (n = 13), microscopy/RDT successfully detected P. falciparum in 48.7% (73/150) of infections (Table P. vivax-mono infections (n = 31) and mixed-species infections (n = 13), microscopy/RDT successfully detected P. vivax in 4.6% (2/44) of infections . More asymptomatic infections were detected in high transmission settings by both methods. The detectability of asymptomatic Plasmodium infections using microscopy/RDT relative to nPCR increased as transmission intensity increases. As a result, most infections in low transmission settings were not detectable by microscopy/RDT.This study describes the prevalence and detectability of asymptomatic Plasmodium infections in the current study was in the same range as other reports from high [In Ethiopia, several cross-sectional studies have documented asymptomatic parasite carriage using conventional and molecular methods , 33, 34.rom high , 34 and rom high  transmisrom high , 36, 37.Plasmodium infections that was detectable by microscopy/RDT increased with increasing in transmission intensity. Whilst this trend has been reported in meta-analyses for P. falciparum [P. falciparum and P. vivax. Moreover, the effect size was comparatively large with approximately 5-fold higher detectability of infections in high endemic settings compared to low endemic settings. The trend of increasing detectability with increasing transmission intensity may be attributable to the fact that asymptomatically infected individuals have higher average parasite densities in high transmission settings [Consistent with other studies , 38, 39,lciparum , 36, 40,settings , 41. Morsettings , furthersettings .P. vivax compared to P. falciparum [P. vivax infections by microscopy/RDT. This low density in P. vivax is mainly attributable to the parasite’s preference to infect reticulocytes [P. falciparum and P. vivax infections. The introduction of sensitive molecular tools may thus improve the detection of P. vivax infections substantially. Since treatment strategies differ for P. falciparum and P. vivax, this is relevant for public health interventions.Lower parasite densities in lciparum , 45 alsoulocytes , 47 thatulocytes  and alsoAlthough RDT and microscopy were used separately in the study sites due to logistics reasons, the prevalence measured by conventional RDT and microscopy was assumed to be comparable .P. falciparum positive by RDT were P. vivax positive by nPCR. False RDT positivity might be due to the presence of parasite antigens after adequate clearance of parasites which might explain the variation between RDT positivity and PCR negative detection among asymptomatic malaria infections [P. falciparum while missing the low-density P. vivax infection from the same patient.Nine samples that were declared microscopy/RDT positive were negative by nPCR while seven samples that were detected fections , 50. HenPlasmodium infections in all endemic sites might reflect the long persistence of these infections from weeks up to months in high [Conventional diagnostics missed nearly half of the asymptomatic malaria reservoir detected by nPCR. Moreover, the detectability of asymptomatic  in high  as well  in high , 53 even in high –15, a deAdditional file 1: Table S1. School based prevalence of asymptomatic malaria in selected sites from different transmission settings using nPCR and microscopy/RDT from 2016 to 2018, Ethiopia.Additional file 2: Table S2. Concordance of RDT and Microscopy detected samples compared to nPCR among the study participants, 2016–2020.Additional file 3: Table S3. GEE model for association of malaria infection prevalence using all methods combined (nPCR and/or microscopy/RDT) among community survey samples with sample characteristics such as gender, age category, level of endemicity from 2016 to 2020, Ethiopia.