PfHRP-2), Plasmodium lactate dehydrogenase (pLDH), and Plasmodium aldolase (pALDO). pfHRP-2 is produced exclusively by Plasmodium falciparum and is very Pf sensitive, but an alternative antigen or antigen combination is required for regions like Asia with significant Plasmodium vivax prevalence. RDT sensitivity also decreases with low parasitemia (<100 parasites/uL), genetic variability, and prozone effect. Thus, proper RDT selection and understanding of test limitations are essential. The Center for Disease Control recommends confirming RDT results by microscopy, but this is challenging, due to the utilization of clinical laboratory standards, like the College of American Pathologists (CAP) and the Clinical Lab Improvement Act (CLIA), and limited recourses. Our focus is to provide quality assurance and quality control strategies for resource-constrained environments and provide education on RDT limitations.Malaria rapid diagnostic tests (RDTs) have had an enormous global impact which contributed to the World Health Organization paradigm shift from empiric treatment to obtaining a parasitological diagnosis prior to treatment. Microscopy, the classic standard, requires significant expertise, equipment, electricity, and reagents. Alternatively, RDT’s lower complexity allows utilization in austere environments while achieving similar sensitivities and specificities. Worldwide, there are over 200 different RDT brands that utilize three antigens: Plasmodium histidine-rich protein 2 ( Rapid diagnostic tests (RDTs) have had an enormous impact on the global impact on malaria diagnostics since their emergence in the 1990s . Asia acThese improvements are part of a comprehensive elimination and prevention program involving vector control, improved treatments, and better diagnostics . The traThere are over 200 brands of RDTs ,8, and tPlasmodium-specific proteins identified are histidine-rich protein 2 [HRP2), Plasmodium lactate dehydrogenase (pLDH), and aldolase. The HRP2 assay utilizes a specific protein that only detects P. falciparium. LDH has both a non-specific PpanLDH for all species, as well as more specific. P. falciparum (PfLDH) and P. vivax (PvLDH) assays. Aldolase is a protein present in all malaria species, and thus, this assay is utilized to diagnose the presence of malaria without speciation [HRP2 based RDTs have reasonable sensitivity for P. falciparum, but RDTs, in general, have had variable performance and variable sensitivity. Attributed causes include issues with product design, including difficulty reading the colors on the test bars, operator error, storage issues with the card itself, parasite factors, such as HRP2 gene deletions, and low parasitemia below the level of detectability and high parasitemia known as the prozone effect [The RDT card is a lateral flow device that uses immunochromatography to detect antigens associated with Malaria ,12,13. Teciation ,14,15. Ie effect ,13.HRP2 is a protein that is produced only by P. falciparum, and thus, RDTs that utilize HRP2 provide the benefit of Pf specificity and with its high sensitivity. Thus, over 80% of all RDTs utilize HRP2, and it is a common choice in Africa, with 99.7% of malaria cases being P. falciparum [Plasmodium falciparum produced higher levels of proteins containing histidine than other amino acids, such as methionine or isoleucine [Plasmodium Glutamate Dehydrogenase, which was not present in human erythrocytes [HRP, now exist. The premise for HRP based RDTs is that Plasmodium falciparum produces a family of multiple histidine-rich proteins in HRP2 and HRP3. Thus, the development of HRP2 monoclonal antibodies to detect the HRP2 antigen resulted in a cost-effective technology for RDT malaria diagnostics. The first operational RDT cards for HRP2 were available in the 1990s [lciparum ,15. Infeoleucine . The inihrocytes . Howeverhttps://www.who.int/malaria/areas/diagnosis/rapid_diagnostic_tests/en/ (Accessed on 11 February 2021) [HRP-RDTs has often outperformed regional microscopy with sensitivities of 94.1% compared to local hospital microscopic diagnostic capability at 71.8% [The WHO and the Foundation for Innovative New Design (FIND) interactive guide provides a report of RDTs, including false positive and false negative rates, which can be accessed through a spreadsheet at ry 2021) ,19. Thisat 71.8% . Highly at 71.8% .HRP2. In fact, HRP2 based RDTs have outperformed pLDH and even regional microsocopy at low parasitemia. For example, in a two-center study in Mozambique and Tanzania, 1898 febrile children were evaluated with 94% sensitivity identified for HRP2, but this decreased to 69.9% when parasitemia was <1000 parasites/uL. pLDH was 88% sensitive, but its sensitivity was affected even more profoundly at 45.7% [HRP2 minimum detection varied from 62–500 parasites/uL, but sensitivity is dramatically higher with increased parasitemia > 500 parasites/uL [At low parasitemia, however (<1000 parasites/uL), the test line is often faint which can be interpreted as a false negative ,13,14,20at 45.7% . HRP2 misites/uL .HRP2 based RDTs have shown excellent sensitivity in Africa, but variable sensitivity in other regions, with one cause being related to genetic variability causing deletions in the pHRP2 and pHRP3 genes [Plasmodium gene encoding the HRP2 protein is a single copy subtelomeric gene on chromosome 7 [pHRP2 proteins, HRP3 cross-reacts with HRP2. False negatives occur with gene deletions of HRP2. However, there have been identified cases of HRP2 RDT positive tests with high parasitemia patients despite having molecular evidence of an HRP2 gene deletion [HRP3, and thus, cross-reactivity was triggered, resulting in a positive HRP2 RDT test at high parasitemia [HRP2 and HRP3 genes occurred, the test line was not detectable by HRP2 RDTs, which further supports the cross-reactivity of these proteins on RDTs [P3 genes ,13. The deletion . These pasitemia . However on RDTs ,22.HRP2 and HRP3 deletions appear to occur independently of each other [HRP2 gene deletions were also more prevalent at lower parasitemia [pHRP2 variability has been seen initially in Asia and Oceana, including Papua New Guinea, Thailand, Philippines, and the Solomon Islands [HRP2 deletions were also identified in Honduras and Peru with a 40.6% deletion rate identified in Iquitos, Peru, which makes HRP2 a poor option in that region [HRP2 and HRP3 mutations were reported in 15 countries and confirmed in 11 countries, including: China, Equatorial Guinea, Ethiopia, Ghana, Myanmar, Nigeria, Sudan, Uganda, United Kingdom (imported), Tanzania, Zambia [PfHRP2/3 deletions and neighboring countries should “conduct baseline surveys among suspected malaria cases” to determine whether there is a greater than 5% HRP2 deletion rate resulting in false negatives [HRP2 combined with either aldolase or pLDH as the second assay, which are not subject to HRP gene deletion issues [HRP2 combination test as being able to distinguish P. falciparum from P. vivax [Mutations resulting in ch other . False nasitemia . pHRP2 v Islands ,13. HRP2t region . From 20, Zambia . As suchn issues . To dateHRP2 does have an issue with persistent positivity for weeks after effective treatment and resolution of clinical symptoms [HRP2 RDT has limited utility as a test of cure, due to persistent antigenemia. Utilizing a PfHRP2 assay, 14 day and 21 day false positive rates have been as high as 98.2% and 94.6%, respectively [HRP2 rates at 26.4%, with effective clearance finally occurring at day 35 [HRP2 RDTs are not effective as a test of treatment effectiveness. Additionally, they have limited utility in detecting reinfection within the one month timeframe, due to persistent antigenemia.symptoms . Thus, tectively ,27. Othet day 35 . Thus, HHRP2 sensitivities at 94% compared to 72–78% for microscopy [HRP2 RDTs for various symptoms of severe malaria has ranged from 94–100% with the symptoms of reduced consciousness being on the lower end to hemoglobinuria and jaundice being on the higher end of sensitivity [HRP2 tests with parasitemia greater than 4% secondary to the prozone effect. Prozone has not been seen with pLDH [Although RDTs have supported the paradigm shift from empiric treatment to parasitological diagnosis prior to treatment , the guicroscopy ,21. WHO croscopy ,29. Howecroscopy ,21. The ith pLDH ,25. Diluith pLDH . Thus, tHRP2 and LDH had sensitivities of 94% and 88%, respectively. However, at low parasitemia defined as <1000 parasites/uL, the sensitivity decreased for both assays to 69.9% and 45.7%, respectively [HRP2 RDT 80% sensitive based on malaria PCR as the gold standard [Malaria RDT sensitivity decreases with low parasitemia . In one ectively . The faiectively . One speectively . In one standard . This isstandard . AlthougPlasmodium parasite, and its epitopes are uniquely different from human LDH [P. ovale, P. vivax, or P. knowlesi LDH assays [HRP2 [HRP2, which has a twoday specificity of 17.3%, seven day of 29.9%, and still only 73.6% specificity at day 28 [P. knowlesi [P. knowlesi is exquisitely affected by parasite counts with a sensitivity of 97% when counts >1000 parasites/uL, but only 25% when parasite count was <1000 parasites/uL [Plasmodium vivax with a sensitivity of 94% except at low parasitemia (<1000 parasites/uL), sensitivity decreased to 60%, which further emphasizes the major effect of parasite count on pLDH sensitivity [The pLDH assays identify enzymes specific to the malaria glycolytic pathway produced by the uman LDH ,14. Beneys [HRP2 . Furtherys [HRP2 ,28. On aknowlesi . Howeversites/uL . In the sitivity .Plasmodium falciparum LDH sensitivities have been variable compared to HRP2 at 82.6–88% compared to 93.4–98.5%, respectively [HRP2 to pLDH [HRP2. [Pf-LDH may be favored over pHRP2 with sensitivities of 98.7% compared to 71.6% associated with pHRP2 gene deletions [HRP2 with either PpanLDH or Pf/PvLDH have shown higher sensitivity than HRP2 alone [Overall, ectively ,35. Othe to pLDH . HoweverH [HRP2. ,20. In reletions . AdditioP2 alone ,35.P. falciparum than HRP2. Sensitivity of aldolase has been comparable to pLDH [P. vivax, including 37.5% sensitivity in a combined study of Africa and the Caribbean [P. knowlesi, P. falciparum, and P. vivax, respectively, in a 129 patient study in Malaysia [® assay [Aldolase is an enzyme in the malaria glycolytic pathway that is found in all species of malaria ,34. The  to pLDH , but sevaribbean , and 62%aribbean . Additio[® assay .TM), which is a combination HRP2/Aldolase card. It has reported a sensitivity of 95.3% for P. falciparum, and 94.2% specificity [Plasmodium vivax; the sensitivity ranged from 68.9–74.6%, and specificity was 99.8% [TM RDT has a P. falciparum (T1) line that is linked to HRP2, and a pan-malaria T2 line  that is linked to aldolase. If both T1 and T2 lines are present, it cannot be distinguished by RDT alone whether this is a multi-species infection involving P. falciparum plus a non-falciparum species or high Pf parasitemia as the aldolase enzyme is preserved in all malaria species, including P. falciparum. However, the T2 bar is frequently not present in lower parasite counts as aldolase sensitivity is low with parasite counts <1000 parasites/uL [TM. Moreover, RDTs effectiveness has a significant regional variability. Thus, BinaxNOWTM is a reasonable augment when used for travelers returning from Africa, with 99.7% of cases being P. falciparum [The United States Food and Drug Administration has approved only one RDT  systems. Training of microscopists and establishing effective QA/QC in malaria diagnosis are key tools for malaria eradication programs. According to Breman (2007), microscopy with a functional QA system was the mainstay of malaria diagnosis during the malaria eradication era in the malaria-eradicated countries . MicroscP. falciparum malaria continues to emerge and as new regimens are developed for differential treatment of P. falciparum and other species, accurate species determination becomes critical, and the importance of competency in microscopic diagnosis assumes substantial new weight [Training of microscopists and regular competency assessment is critical in malaria diagnosis to ensure the required microscopist skills are not lost over time. External competency assessment and/or retraining for certified competent microscopists is recommended by the WHO at three-year intervals to ensure the accuracy and reliability of malaria microscopy results . This isw weight .A quantitative readout is absolutely required to detect emerging drug resistance, as parasite clearance times lengthen. More so, mRDTs are the most basic tools for parasite-based confirmation of malaria in primary health care settings, and require adequate training and competency in addition to validation against microscopy to ensure the reliability of results. Thus, a national QA/QC program for the training and certification of malaria microscopists is urgently required both for better microscopy and to assure a safe and effective RDT program. Such a program would involve the generation of a large bank of malaria positive stained blood films to use for both initial training, refresher courses, and certification exams.http://www.who.int/diagnostics_laboratory/evaluations/en/ Accessed on 24 February 2021) [P. falciparum and P. vivax) and heat stability  and WHO/FIND further offer a lot testing program [Substandard malaria RDTs are widespread in resource-limited settings, and lot-to-lot variations may affect the performance of RDTs ,149,150.ry 2021) . Further program ,152.http://www.finddiagnostics.org/programs/malaria-afs/ Accessed on 24 February 2021) and have developed positive controls (freeze dried recombinant parasite antigen) that are currently under implementation and evaluation [Some countries have a national reference laboratory with services and levels of expertise that exceed the minimum standards. The national laboratory can provide higher levels of microscopy, RDTs, training, reference, quality control/assurance, research and evaluation, standard operating procedures, data management, surveillance, equipment maintenance, and laboratory supervision . In the aluation . Pendingaluation .The new QA/QC programs should be prioritized and thoroughly evaluated in routine implementation sites to ensure that healthcare workers can identify problems with RDT performance using these tools. In the meantime, periodic supervision and comparison to reference microscopy may be the best currently available option for quality control at the health facility level . The natOver the past few years, the Division of Microbiology and Infectious Disease (DMID) of the National Institutes of Health (NIH) in the United States has been working toward improving the performance of clinical research laboratories of institutions conducting NIH-sponsored clinical trials to ensure that results generated from studies will be reliable and acceptable to regulatory bodies. The ultimate goal of the Quality Assurance/Quality Control (QA/QC) activities is to achieve compliance with the College of American Pathologists (CAP) and WHO-AFRO checklists in preparation for accreditation through the implementation of GCLP and the improvement of PT performance .Technology can also play a role in developing good QA/QC activities. The Fionet system uses a device called Deki Reader™, which combines standard mobile devices with custom software to gather demographic patient data, provide guidance to health care workers on conducting testing, taking pictures of completed RDT assays, and transmits data over commercially available cell phone services. The system also contains a web portal for uploading processed RDT images, the transmission of patient demographic information, and remote storage and access of the data. This mobile health technology platform has been successfully used in small programs for quality assurance and quality improvement of malaria diagnosis by community health workers in Kenya . See TabPlasmodium infection results in hemoglobin breakdown that liberates the iron-containing organic crystal called hemozoin [P. vivax (87% sensitivity and 88% specificity), which could be incorporated into a comprehensive malaria strategy in non-falciparum regions where RDT assays have lower sensitivity. Magnetic resonance relaxometry (MRR) is a tool that uses the relaxation time of protons after magnetic excitement for various diagnostic correlations, including the diagnosis of malaria. Previously, MRR had poor sensitivity at low parasitemia, but cell enrichment techniques have improved its level of detection [HRP2, LDH, aldolase), hemozoin, or other malaria biomarkers to provide a readout in a manner analogous to a glucometer [In addition to microscopy, RDTs and PCR, there are several emerging diagnostic technologies that will likely have a role in future comprehensive malaria programs. These technologies include novel photacoustics that utilizes a sensor, which has shown promising results through detection of a specific frequency corresponding to the malaria ring stage . Anotherhemozoin . RMOD haetection . Spectroetection . Howeverucometer . These tHRP2 remains the predominant assay in RDTs, and the WHO still only endorses combination RDTs that contain HRP2—in part, due to quality, but also related to P. falciparum being a clear misdiagnosis. However, regions, such as Central and South America and the Indian subcontinent that have a high P. vivax, should consider combinations that also include PvLDH/PpanLDH or aldolase. Combinations utilizing HRP2/PfLDH/PpanLDH can also be beneficial in Africa, which is predominately P. falciparum, but the LDH can increase the sensitivity of HRP2 alone as it does not have the same gene deletion or prozone effects as HRP2. There are, however, cost and storage considerations to each RDT, and utilization of the WHO Malaria FIND resource is an appropriate way for a health ministry and hospital to select the most appropriate agent. Furthermore, we cannot emphasize enough the importance of developing and implementing a QA/QC program based on high-quality microscopy training and outside verification. RDTs are a great tool for diagnosing and managing malaria, but monitoring its limitations with a QA/QC program and educating clinicians on results can dramatically improve a nation’s malaria program.The use of RDTs has greatly expanded the ability to diagnose malaria, particularly in resource-limited regions. There are, however, limitations, including variable sensitivity, regional variation secondary to gene deletions, and decreased detection, due to the degree of non-falciparum malaria in a region.


Plasmodium falciparum knob-associated histidine-rich proteins (PfKAHRPs) play an important role in cerebral malaria pathophysiology as well as pathogenesis of P. falciparum infections. The repeat region of PfKAHRP C-terminal interaction domain has been found to bind to the infected red blood cells and the vascular endothelium. Thus, this study aimed to assess the allelic variations, genetic diversity, and natural selection acting at the C-terminal PfKAHRP between parasite isolates from Saudi Arabia.  Malaria is still a public health problem in Saudi Arabia specifically in the Jazan region. genetic diversity, polymorphism, haplotype, and natural selection. Haplotype network analysis was constructed by using the median-joining method in the NETWORK version 5.0.0.1 software.  The PfKHARP C-terminal interaction domain was successfully PCR-amplified and sequence data from 441 clinical isolates from Saudi Arabia were obtained. The DnaSP v5.10 software was used to determine the KEA, TKEASTSKGA, TKEASTTEGA, and TKEASTSKRA). Among the repeat types, Type I  was the most abundant in Saudi Arabia, followed by Type II . Overall, the nucleotide diversity in the PfKHARP C-terminal region was found to be low in Saudi Arabia (π = 0.00142); however, natural selection tests indicated positive selection  which was due to the variations within the repeat motifs. Genealogical relationship haplotype network of PfKAHRP from 4 different countries  revealed 1 major shared haplotype cluster (H_1) with samples representative from all 4 countries .  Alignment and analysis of 441 C-terminal PfKAHRP-deduced amino acid sequences identified 5 genotypes (I–V) based on the decapeptide repeat arrangements (TKEASTSP. falciparum malaria. Since this is the first study to report on genetic diversity of C-terminal PfKAHRP interaction domain and the repeat motifs from clinical samples in Saudi Arabia, it will contribute towards the rational design of antiadhesion drug therapies for  Plasmodium falciparum [Anopheles arabiensis is the dominant vector for malaria in the region which usually peaks following the rainy season [An. dthali, An. stephensi, An. superpictus, An. culicifacies, and An. fluviatilis [P. falciparum-infected patients. The pathophysiology involves the cytoadherence of parasite knob-associated histidine-rich proteins (KAHRPs) and infected red blood cells (RBCs) within the brain cells of infected patients. These knob-associated proteins within P. falciparum parasites remodel the host cell membrane to assure ionic homeostasis, thereby increasing nutrient uptake for their own survival [Saudi Arabia has made significant progress in reducing the annual number of malaria cases thereby inching closer towards malaria elimination; however, it still remains a public health problem in the Jazan region . A recenlciparum . Though lciparum , 3. Anopy season ; howeverviatilis –7. Cerebsurvival , 9. Knobsurvival .P. falciparum. Structural analysis of PfKAHRP and PfEMP-1 indicated formation of complexes with host erythrocyte membranes for formation of knobs and facilitating cytoadherence [P. falciparum infections.One more key parasite protein family known as erythrocyte membrane protein-1 (EMP1) members play a vital role during cytoadherence in association with KAHRP in dherence . Targetedherence . The PfKP. falciparum knob structures are unique, all Plasmodium species that infect primates have KAHRP-like proteins [P. falciparum malaria.Structurally, the PfKAHRP protein constitutes 3 domains, the N-terminal histidine-rich domain, the central domain, and the C-terminal repeat domain, which has decapeptide repeats  and the proteins . The genproteins . Thus, tP. falciparum patients attending health centres in Jazan province. Informed consent was obtained from all patients before blood sampling. Thick and thin blood smears were also obtained from the patients for microscopic detection using Giemsa stain. Detailed information on parasite counts, age, and sex of 441 patients enrolled in the study are given in Supplementary Finger-prick blood samples on filter paper were collected from RDT-positive DNA extraction was carried out from filter papers using QIAamp DNA blood mini kit according to manufacturer's instructions. The C-terminal domain of PfKAHRP gene  was amplified using nested PCR (KAHRPFOR-1 and KAHRPREV-1 for Nest 1 PCR and KAHRPFOR-2 and KAHRPREV-2 for Nest 2 PCR amplification). To facilitate the PCR product sequencing, the inner primers were linked to M13 forward and reverse primers . The folPfKAHRP sequence data originating from Iran [All raw sequence generated in this study were initially analysed and trimmed using the SeqMan software (DNA Star) and best quality sequences were converted to fasta format for analysis. Published rom Iran , Burundirom Iran , and Indrom Iran  were alsπ), number of polymorphic sites, number of synonymous and nonsynonymous substitutions, haplotype diversity (Hd), and number of haplotypes (H) within the PfKAHRP sequences was determined by using the DnaSP v5.10 software [Z-test (P < 0.05) in MEGA software v5 with 1000 bootstrap replications [D, Fu and Li's D∗, and F∗ neutrality tests were performed as implemented in the DnaSP v5.10 software.Sequence diversity . Sequences obtained in this study were submitted to NCBI.Genealogical relationships between the KEA)n and its arrangement with 3 decapeptide motifs TKEASTSKGA, TKEASTTEGA, and TKEASTSKRA and two nonapeptide motif KEASTSKEA and KEASTTEGA  out of which, 5 sites were nonsynonymous. There were 4 parsimony informative sites and 2 singleton variable sites. The overall nucleotide diversity was marginally higher (π = 0.00158 ± SD 0.00029) when compared to sequences from Saudi Arabia (π = 0.00142 ± 0.00004). The haplotype diversity of PfKAHRP was higher (Hd = 0.477 ± SD 0.013) compared to overall combined diversity of all 4 countries (Hd = 0.106 ± SD 0.019).Alignment and comparison of 487 nucleotide sequences  of the C-terminal region of PfKAHRP gene, the average difference of (dN-dS) was evaluated. We found positive values (dN-dS = 1.64) at the C-terminal domain  (P < 0.03). However, Tajima's D, Li and Fu's F∗, and D∗ statistics were found to be negative for pftrap C-terminal domain  . We alsol domain .PfKAHRP haplotypes from 4 different countries . The network tree revealed 2 shared haplotypes (H_1 and H_2) . Haplotype 2 (H_2) was shared between Burundi and Iran. Haplotype (H_3) was a unique cluster with samples originating only from Burundi, East Africa. Two unique haplotypes (H_4 and H_5) were observed with samples from India.A haplotype network tree was constructed to understand the phylogeography and genealogical relationships with and H_2)  among thKEA)n. Type I  and Type II  were the major genotypes observed within Saudi Arabia. It would be interesting to know whether these genotypes have varying degree of binding to host erythrocytes and endothelium leading to severe disease in some patients. Antibody response to these tandem repeat types needs to be explored too. Higher nucleotide diversity was observed within the repeat region with evidence of positive natural selection indicating possible parasite-host interaction to evade host immunity. Similar higher levels of nucleotide diversity with strong positive selection within the repeat regions were also observed in P. ovale CSP [P. falciparum and P. knowlesi merozoite surface proteins (MSPs): MSP4, MSP7, MSP1, Pk41, and MSP1P show varying levels of polymorphisms and strong natural selection due to exposure to host immune response [PfKAHRP from Saudi Arabia as well as all 4 countries together were of similar levels indicating that it is the SNPs associated with the variations in the repeat region was responsible for diversity. These findings of the present study will contribute to the knowledge of KAHRP variants from Saudi Arabia and contribute towards the rational design of antiadhesion therapies for P. falciparum malaria.Peptide repeats are very common in malarial antigens and thus are the major source of antigenic variations which helps the parasite to evade host immunity –21. Paravale CSP . These sresponse –32. Amonresponse . HaplotyPfKAHRP genotypes from Saudi Arabia and shared major haplotypes circulating within the region. Since PfKAHRP is a key participant in severe malaria pathology and include repeat motifs that are recognised by the immune system, understanding these sequences is critical for understanding severe disease, treatment, and prevention. More such studies are necessary to understand the major parasite variants within and to understand the functional aspects for designing antiadhesion therapies against P. falciparum.This is the first study to report on the


Background: The cytokines interferon gamma (IFN-γ), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF- α) play an important role in malaria infection. The aim of this study was to determine the prevalence of malaria and to evaluate cytokine responses to malaria infection in patients from the East Nile locality of Khartoum State.Methods: This study was carried out from May to July 2018 in the East Nile Locality, Khartoum State. Blood samples were collected from 384 randomly selected patients for blood film analysis. Of these, 39 were selected for cytokine level analysis (10 control and 29 patient samples), determined using enzyme-linked immunosorbent assays.Results: The malaria prevalence rate among 384 patients was 18.5%.Plasmodium falciparum was the most prevalent (13%), while the prevalence ofPlasmodiumvivax was 4.6%. The rate of mixed infection was 0.8%. There was a higher prevalence rate (22.7%) in males than females (15.6%). However, we found no significant correlation between cytokine levels and parasitemia in the study group. Nevertheless, our study demonstrated a significant correlation between cytokine levels and recurrent infections.Conclusions: Together, our data show that malaria remains a public health problem in East Nile locality with a high prevalence. Additionally, serum levels of IFN- γ and TNF- α were significantly higher in malaria-infected individuals compared to non-infecting individuals and cytokine levels were found to be correlated with recurrent malaria infection. Plasmodium. Malaria symptoms include fever and headache, which result from parasite invasion of red blood cells. In severe cases, malaria infection may progress to coma or death . The species that causes the most severe cases isP. falciparum. Malaria control efforts in Sudan began in 1904, when Dr. A. Balfour succeeded in eradicating malaria from Khartoum  are said to be involved in malaria pathogenesis. Severe malaria has been associated with low serum levels of IL-12 and low IL-10 to TNF- α serum concentration ratios in a few studies of childhood malaria in holoendemic areas.In order to explore the effect of the immune response to malaria and the development of clinical immunity, this study aimed to measure and determine the prevalence of malaria and quantify cytokine levels in patients with malaria infection in the East Nile locality.st May to 23rd of July 2018.This cross-sectional study was carried out in the East Nile locality, which is located in the eastern part of Khartoum State, Sudan. This study was conducted in different pre-urban areas during the period from 1The study population included participants of all ages and genders from a population admitted to Elbanjadeed Hospital, Aldebaba Medical Health Center, Eid Babekir Medical Health Center, Helat Koko Medical Health Center and Omdom Medical Health Center. In total, 384 participants were asked to participate in this study, all of whom were admitted for malaria diagnosis. Participants were selected using quota sampling and all patients admitted for malaria diagnosis were eligible to be included in the study. The age groups were categorized as follows: less than 10 years, 11–49 years and over 50 years old. After participants signed an informed consent form for participation in the study, a questionnaire was used by expert laboratory technician to collect demographic data  and medical history of chronic disease  from patients enrolled in the study.In total, 384 samples were collected from patients admitted for malaria infection at hospitals and health centers. The sample size was calculated on the following formula :where N = sample size, Z = statistic for a level of confidence (1.96), P = prevalence in study area (50%) and d = precision (5%).Plasmodium species. Thin films were fixed with methanol and slides were placed face down on a drying rack for five minutes to allow the methanol to fix. Thick and thin blood films were stained with Giemsa stain at a concentration of 10% for 10 minutes. The stain was flushed from the slides by adding drops of buffered water until all the stain has been washed away. Where the blood film analysis was positive for malaria, 5 ml of venous blood was collected from each patient into a sterile container. For cytokine analysis, 5ml of venous blood was also collected from 10 participants who tested negative for malaria and agreed to participate further in the study. These participants were selected using stratified random sampling, with two participants being randomly selected from each of the five health centers. Following collection, blood samples were centrifugated at 3000 rpm for 10 minutes. After centrifugation, the serum was separated and transferred to another labeled sterile container and stored in refrigerator at 4oC until use.From each patient, thick and thin blood films were prepared using finger prick blood samples taken as part of the routine diagnosis of infection withPlasmodium and the thick film to classify parasitemia as follows:After the films dried, they were examined microscopically by experienced personal to determine the parasite stages , using the thin blood film to identify the species of+: 1–10 parasites per 100 thick film fields+ +: 11–100 parasites per 100 thick film fields+ + +: 1–10 parasites per one thick film field+ + + +: more than 10 parasites per one thick film fieldFor cytokine analysis, 29 of the patient serum samples were selected using simple stratified sampling, with five positive samples randomly selected from each health center and nine randomly selected from the hospital. Cytokine analysis was not performed for all samples due to financial restrictions. The stored serum was brought to the laboratory and was allowed to thaw. Serum concentrations of IFN-y, IL-10, TNF-α were determined using an enzyme-linked immunosorbent assay (ELISA) according to manufacturer’s instructions  for the patient samples and 10 control samples.Briefly, 100µL of diluted capture antibody solution was added to each well and the sealed plate was incubated overnight between 2–8°C. The plates were washed four times and then blocked by adding 200µL assay diluents to each well, then were sealed and incubated for one hour with shaking on a palate shaker at 500 rpm with a 0.3cm circular orbit. The plates were washed four times and then 100μl diluted standards and samples were added to each well. The plate was sealed and incubated at room temperature for two hours with shaking. The plate was washed four times, then 100µL diluted detection antibody solution was added to each well. Plates were sealed and incubated at room temperature for one hour with shaking. The plate was washed four times, then 100μl diluted avidin–HRP solution was added to each well. The plate sealed and was incubated at room temperature for 30 minutes with shaking. The plate was washed five times and then soaked for 30 seconds to one minute per wash. Then, 100µL fresh TMB substrate solution was added to each well and incubated in the dark for 20 minutes. Finally, 100 µL of stop solution was added to each well and the absorbance was read with the SPECTROstar Nano Microplate Reader at 540 nm and 570 nm within 15 minutes.P-value of less than 0.05 was considered statistically significant.Data were analyzed using SPSS version-20. The Chi-squared test was performed to determine statistical significance and aEthical clearance for this study was obtained from Committee of Scientific Research Deanship, Sudan University of Science and Technology, ethical approval number (DSR – IEC – 12 – 07). Written informed consent for participation and publication of the data was obtained from all participants included in this study or for children, from their guardian.A total of 384 patients were enrolled in this study, of which 154 were male and 230 were female. Their ages were grouped into three categories: less than 10 years old (134), 11 – 49 years old (198) and more than 50 years old (52).First, we sought to determine the prevalence of malaria in our study population. To this end, we collected blood samples from our patients and examined them using blood films. Out of 384 blood samples collected from different pre-urban areas  in the East Nile locality during the period from May to July 2018 , 71 (18.5%) were found to be positive and 313 (81.5 %) were negative for malaria  Abd Al. MoreovePlasmodium species in our study population. We observed thatP. falciparum had the highest prevalence rate (13%), followed byP.vivax (4.6%). However, mixed infection byP. falciparum andP. vivax had the lowest prevalence rate (0.8%). We failed to detect any positive results forP. malariae orP. ovale infection in our study population  for INF – γ , TNF-α and IL-10 respectively.Our data reveal that ,there was no significant statistical association between P.falciparum andNext, we determined the distribution of severe malaria in the studied population. Our data showed that most (58%) of the study population had a low parasite count (mild parasitemia), while 13% had a moderate parasitemia (++). We detected no cases that exhibited severe parasitemia (+++ and ++++)  and no sP value = 0.026). However, TNF-α serum levels were comparable between patients and non-infected individuals . Mean serum levels of IL-10 were higher in patients compared to non-infected individuals, although this difference was not statistically significant   .Next, we sought to determine whether there is a correlation between the cytokine profiles of individuals enrolled in our study and severity of malaria and/or recurrent infection. However, we found no correlation between levels of IFNγ, TNF-α, or IL-10 and level of parasitemia . IntriguThe findings of our study revealed a prevalence rate of malaria of 18.5%. This rate was greater than the rate reported in Khartoum byP. falciparum andP. vivax). However, we observed no cases ofP. malariae andP. ovale infection. Moreover, males had higher prevalence rate (22.7%) of malaria infection than females (15.6%). Our study findings agree with a study in Khartoum byOur study show that Falciparum malaria is the most prevalent and constitutes about 13% of all infections, benign tertian Vivax malaria has prevalence of 4.6%, and the lowest prevalence rate of 0.8% is observed for mixed infection  of moderate parasitemia was in the under 10 years age group. Although children are more susceptible to malaria infection due to a slow developing immune system, a high prevalence rate (34.5%) of mild parasitemia was reported among the 11–49 years age group. A lower prevalence rate of 2% and mild parasitemia 12.1% was reported among the over 50 age group. This finding was closer to the finding ofP. falciparum andP. vivax infection and IFN-γ was found to be significantly higher in patients than in non-infected individuals. This finding is in line with a study in Poland byIn this study, serum levels IFN-γ, TNF- α and IL-10 were measured in healthy controls and in patients withThe present study reported no association between parasite density and levels of IFN-γ, TNF-α and IL-10. These findings are in line with study done byhttps://doi.org/10.6084/m9.figshare.8986178.v2 - samle and control.sav - Data dictionary_FL.docxCreative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).Data are available under the terms of the The authors have attended to all the comments satisfactorily.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Immunotherapeutic target for malaria infection.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I confirm that I have read the revised article carefully. The authors have already corrected some points according to my suggestion. I approve the revised article. However, I feel that Table 2 and 3 (with only 2 columns) are not necessary. If possible, the authors should describe completely using 2-3 sentences.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?NoAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Pathology of malariaI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. It was mentioned that patients serum samples were selected randomly for cytokines analysis. How does this random selection carry out? Does it take into consideration the age of patients since they are categorized according to age at the beginning? There would be differences in cytokine release profile between child, adult and elderly as the level of immune response between the three groups towards invading organisms would be different. So how does the selection carry out among the three groups of patients?The symbol for gamma for IFN must be presented properly with the right symbol… not “y”.What is the age profile or category of the 29 patients in whom the cytokine profile was carried out? It would be interesting to see if the cytokines release is influenced by age.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Immunotherapeutic target for malaria infection.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The conclusion in the abstract is not clear. The authors did not indicate any cytokines.The rationale and the benefit of study is not clear.P. falciparum andP. vivax infected patients.The author did not show the difference in any cytokine levels betweenIt seems that there are too many tables. Is it possible to merge Table 1 and 2; 3 and 6?Correlation between parasitemia and cytokines in Table 8 is not reliable. Sample size (n=4) is too small for determining correlation.The authors did not mentioned about detail of patients in ELISA results. Why sample size was 10 control and 29 patients?As per my knowledge, there have been several studies in roles of cytokine profiles in human and animal malaria. This study aimed to determine important cytokines in malaria. These included IFN-γ, IL-12. TNF- α. They used rather appropriate sample size. However, as follows, it was found some points are unclear and should be clarified. Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?NoAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Immunopathology in malaria.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


Identifying preimmunization biological characteristics that promote an effective vaccine response offers opportunities for illuminating the critical immunological mechanisms that confer vaccine-induced protection, for developing adjuvant strategies, and for tailoring vaccination regimens to individuals or groups. In the context of malaria vaccine research, studying preimmunization correlates of protection can help address the need for a widely effective malaria vaccine, which remains elusive. In this study, common preimmunization correlates of protection were identified using transcriptomic data from four independent, heterogeneous malaria vaccine trials in adults. Systems-based analyses showed that a moderately elevated inflammatory state prior to immunization was associated with protection against malaria challenge. Functional profiling of protection-associated genes revealed the importance of several inflammatory pathways, including TLR signaling. These findings, which echo previous studies that associated enhanced preimmunization inflammation with protection, illuminate common baseline characteristics that set the stage for an effective vaccine response across diverse malaria vaccine strategies in adults. Identifying preimmunization correlates of protection, therefore, offers opportunities for illuminating the immunological pathways that promote or attenuate protective vaccine responses, for identifying avenues whereby the immune system can be influenced to respond effectively, and for developing personalized approaches that predict vaccine efficacy on an individual or group basis. These opportunities can potentially benefit malaria vaccine development efforts: Although substantial progress has been made developing anti-malaria vaccines, a vaccine that is widely effective has remained elusive5, and it remains unclear why malaria vaccines induce a protective response in some individuals but not in others. Exploring the preimmunization correlates of protection in malaria vaccine trials presents an opportunity to identify the immunological conditions that set the stage for vaccine-induced responses that are protective against future infection and to help guide the rational design of more effective vaccines. While the list of studies in this research area is not extensive, some preimmunization correlates of protection have been identified for vaccines against influenza9, hepatitis B12, yellow fever13, and malaria21.Whether a vaccine generates an immune response that results in protection from infection or disease depends in part on the preimmunization status and capacities of the vaccinated individual’s immune systemMosquito Bite with Radiation-Attenuated P. falciparumSporozoites (IMRAS) trial22, the Bagamoyo SPoroZoite Vaccination 1 (BSPZV1) trial23, the MAL68 RTS,S vaccine trial24, and a Chloroquine Prophylaxis and Sporozoites (CPS) trial26. These trials varied in the vaccines administered , the route of administration  the country of trial subject recruitment , whether subjects were malaria-naive prior to immunization  or experienced (Tanzania), and the source of transcriptomic material . Each trial included a challenge phase, wherein subjects were infected with infectious P. falciparum to determine whether the subject mounted a vaccine response protective against malaria. Despite the wide-ranging clinical trial conditions, our results indicate that protection against post-immunization malaria challenge is associated with innate immune system activation and a moderately elevated inflammatory state prior to immunization, including higher expression in genes associated with TLR signaling and other inflammatory pathways. These results echo molecular- and pathway-level preimmunization correlates of malaria protection derived from previous analyses on narrower sets of clinical trials21. They highlight specific immunological pathways that may play critical roles in the development of effective malaria vaccine-elicited responses and offer potential targets for manipulating the immune system into a state that promotes such responses.To identify common preimmunization correlates of protection across various malaria vaccine trials, we performed transcriptomic analysis of four independent trials: the Immunization via n = 38) and all non-protected participants (n = 46) pooled from the trials revealed no genes with statistically significant differences in transcript abundance using an FDR-adjusted P-value cutoff of 0.1. To test for differences among functionally related groups of genes, we performed Gene Set Enrichment Analysis (GSEA)27 to identify statistically significant overrepresentation of predefined gene sets including immunological blood transcriptional modules29 and the MSigDB Hallmark gene sets30. GSEA results on genes ranked according to their DESeq2 Wald test statistic revealed significant differences in transcript abundances  among 207 gene sets. Gene sets enriched for higher abundance transcripts in protected subjects were predominantly associated with inflammation and inflammatory signaling pathways, myeloid lineage cells including monocytes and neutrophils, coagulation, antigen presentation, cell death and apoptosis, the endoplasmic reticulum, the extracellular matrix, and the complement system  between protected and non-protected subjects. Our analysis revealed that, when comparing protected and non-protected subjects, differences in transcript abundance of gene sets showed statistical significance, whereas differences at the individual gene level did not.Preimmunization transcriptomes were pooled from the four malaria vaccine trials and consisted of 84 samples Table , MethodsN = 2 for IMRAS, N = 0 for BSPZV1 and MAL68, N = 14 for CPS; FDR-adjusted P-value < 0.1). To identify differences at the level of gene sets, we then performed the same type of GSEA as performed with the pooled samples. From the GSEA results on each trial  for 28 IPA Canonical Pathways ays Fig. . Reflectz-scores (≥2.0) showing significant FDR-adjusted P-values in this analysis included cytokines with proinflammatory activity , cytokine groups , lipopolysaccharides, colony-stimulating factors , transcription regulators , TLRs  as well as adapter proteins that participate in their signaling pathways , other immunomodulators , the protein synthesis regulator LARP1, thrombin, APP, signaling pathway enzymes and ligands , the PDGF-BB complex, the growth hormone protein group, the C11orf95-RELA gene fusion product, and ZBTB10. Based on the IPA knowledge base, increased activity of these molecules would shift expression of the 98 leading-edge genes in a manner consistent with their mean expression differences between protected and non-protected subjects across trials. The upstream molecules with low IPA activation z-scores (≤−2.0) showing significance included the p38 MAPK inhibitor SB203580, the progesterone antagonist mifepristone, miR-155-5p, the alpha catenin protein group, the transcription factor MLXIPL, the transcription inhibitor actinomycin D, DIO3 and DUSP1, the gene fusion product ETV6-RUNX1, the immunosuppressant cyclosporin A, and the corticosteroid budesonide. Decreased activity of these molecules is predicted to induce expression changes in the 98 leading-edge genes consistent with those observed between protected and non-protected subjects. The full set of Upstream Analysis results are provided in Supplementary Table In addition to the Canonical Pathways analysis, we also performed an IPA Upstream Analysis, which provides hypotheses about which molecular perturbations might cause the expression differences observed in the 98-gene list. In the context of this study, this analysis can help identify pathway activators and inactivators  with the potential to shift an individual’s immunological state in the direction that is more consistent with a protective response to immunization. The molecules with high IPA activation -scores ≥.0 showinHALLMARK TNFA SIGNALING VIA NFKB gene set to that of the DC.M4.3 Protein synthesis gene set produced a score with the highest discriminatory power  and seven from the DC.M4.3 Protein synthesis set .The protection-associated genes and gene set common across trials suggest there are transcriptomic features that might be used in classifiers that discriminate, on an individual basis, which vaccinees will mount a protective immune response to immunization and which will not. To assess the discriminatory power of these features, we developed and tested various scores based on transcript abundances (counts per million) of the 98 protection-associated, leading-edge genes described above as well as mean transcript levels corresponding to those genes within protection-associated gene sets. We also tested the discriminatory power of scores based on transcript abundance ratios between protection-associated genes and between gene sets. For all scoring strategies, individual scores were computed for each trial participant based on their preimmunization transcriptomic profile. We then assessed the discriminatory power of the score by varying the threshold used to classify individuals as protected or non-protected and then generating a receiver operating characteristic (ROC) curve from the sensitivity and specificity of the classifiers. We quantified the discriminatory power of each score based on the area under the curve (AUC) of the ROC results. We found that the ratio of mean leading-edge gene expression in the To test whether the discriminatory power of the best-performing score might be biased by specific trials in our study, we computed separate ROC curves for each trial using only their participating subjects and their corresponding scores. We found that the score showed comparable discriminatory power across trials Fig. . The sco6, and our results also link this finding to the malaria vaccination strategies analyzed here. Our findings contrast with studies investigating correlates of protection following hepatitis B immunization where innate immune activation prior to immunization has been linked to non-protection in adults12.Our findings indicate that a moderately elevated preimmunization inflammatory state, as revealed by RNA-seq transcriptomic measurements on whole blood or PBMCs, is associated with a protective response to malaria challenge following a variety of immunization strategies in adults. A heightened preimmunization inflammatory state has previously been linked to vaccine-induced protection against influenza in non-elderly adultsAcross the four trials we analyzed, transcripts from several inflammatory and innate response gene sets trended higher in protected subjects, and this trend was significant when treating gene sets as the unit of analysis. Analyses at the level of individual transcripts did not reveal significant differences between protected and non-protected subjects across trials. Thus, the expression differences we observed between these groups link protection to a moderately higher activation of inflammatory and innate response processes. While moderate, these differences are, collectively, substantial enough to have potential utility in metrics that predict whether an individual subject will mount a protective vaccine response prior to immunization Fig. . The bes19 who identified TLR4 as a member of several gene signatures predictive of protection in RTS,S-vaccinated children. These signatures were derived from expression changes in preimmunization PBMCs that were stimulated with CSP and/or P. falciparum-infected erythrocytes. Their results suggested that higher preimmunization TLR4 expression may help the immune system respond more effectively to the RTS,S vaccine. As noted by these authors, the LPS-derived chemical monophosphoryl lipid A (MPL) used as an adjuvant in combination with the subunit RTS,S vaccine is a TLR4 agonist33. Our results, which link higher preimmunization expression of TLR1, TLR4 and TLR8 to protection, also suggest a role for TLR agonists as adjuvants in the development of a protective vaccine response.The set of common, protection-associated genes we gathered from our GSEA results showed enrichment for several signaling pathways Fig. , most ofMoncunill et al. also found preimmunization signatures of protection in a separate trial where adults were immunized with fresh sporozoites delivered via mosquito bite under chloroquine prophylaxis. The best-performing preimmunization signature derived from CSP-stimulated PBMCs for this trial linked protection with proinflammatory activity of participants in the canonical and non-canonical NFκB pathways. These pathway participants included CSF2 , and our IPA Upstream Analysis identified CSF2, RELA, and NFκB complex activation as potential upstream perturbations contributing to the increased expression and transcript abundance differences we observed between protected and non-protected subjects. Also included in the CPS signature is ITGA2, an integrin that contributes to p38 MAPK signaling which in turn controls the activity of the RELA NFκB subunit. The p38 MAPK signaling pathway was also enriched for leading-edge genes associated with protection across trials in our IPA Canonical Pathways analysis Fig. . The lea21 provides evidence suggesting preimmunization correlates of protection may be driven by actively decreased inflammatory functions in non-protected subjects. The authors report that higher baseline expression of T cell inhibitory ligands CTLA-4 and TIM-3 in CD4+ T cells is associated with a “slow-responder” phenotype in adults that is less likely to result in sterile protection following immunization via controlled human malaria infection under chloroquine prophylaxis (CHMI-CPS). Increases in these inhibitory ligands can result from mechanisms that protect against inflammation-induced damage . The authors argue that these inhibitory ligands may interfere with NFκB signaling, which is preferentially activated in subjects who mount a protective response to this type of immunization, and this leads to insufficient production of IFN-γ. Rapid induction of IFN-γ following immunization is a characteristic of a “fast-responder” phenotype in CHMI-CPS trials that is more likely to result in sterile protection. Consistent with these findings, our IPA Upstream Analysis identified increased IFN-γ as a perturbation that would generate expression changes consistent with those observed between protected and non-protected individuals. IFN-γ had the highest z-score in this analysis, indicating strong agreement between a hypothetical increase in IFN-γ and our observed expression patterns showing higher inflammation in protected subjects prior to immunization. Given this consistency between our findings and those of Yap et al., it may be that non-protected individuals in our study had a less successful immunization response due to protective inflammatory inhibition. However, the group of individuals we analyzed was heterogeneous; therefore, we cannot rule out attributing protection to higher-than-average innate responses among protected subjects. Furthermore, these two interpretations are not mutually exclusive: our results may simultaneously reflect immuno-suppressive factors in non-protected subjects and immuno-activating factors in protected subjects.An important difference between the Moncunill et al. study and ours is that our transcriptomic measurements were taken from unstimulated tissues and therefore present snapshots of subjects’ preimmunization immunological states whereas the Moncunill, et al. measurements reflect immunological responses to antigen stimulation. Taken together, these results associate protection with an innate immune system that is simultaneously more activated prior to immunization and more capable of responding to immunization. These results may be driven by higher-than-average innate immune functionality in protected subjects or lower-than-average functionality in non-protected subjects, or a combination of both. A recent report by Yap et al.Children, due to their increased vulnerability to malaria, are the primary target for malaria vaccines, and further studies will be needed to determine whether the preimmunization correlates of protection we identified generalize to younger populations. As mentioned above, preimmunization signatures of protection in children identified by Moncunill et al. included TLR4, which is a participant in many of the pathways associated with protection in our study. This suggests that the pathways critical for mounting an effective malaria vaccine response in adults may be similar to those in children. We also note that all vaccines used in the studies we analyzed target the sporozoite stage and further studies will be needed to generalize our results to malaria vaccines that target other stages.The results presented here suggest that assessing malaria vaccinee’s preimmunization status may lead to important correlates of protection and tailoring immunization regimes accordingly may emerge as an important element to ensuring a protective response to immunization. Moreover, our results also offer potential pharmacological interventions that may coax an individual’s immune state into one that is primed to respond effectively to immunization.P. falciparum following immunization. Transcriptomic and challenge data were obtained for the IMRAS and BSPZV1 trials through direct collaboration with trial researchers. The MAL68 and CPS trial data are publicly available (see Data availability). The trials, described below, are summarized in Table Trials selected for use in the study represent all publicly available studies, to our knowledge, that included preimmunization transcriptomics analyzed via RNA-seq to allow a more direct comparison across a wider dynamic range, and which also included an assessment of protection via controlled human challenge with infectious 22  was performed in malaria-naive participants recruited from the United States. The trial assessed the safety, tolerability, and biomarkers of protection of immunization via radiation-attenuated sporozoites delivered by infectious mosquito bites. Preimmunization transcriptomics data were obtained from whole blood samples collected on the day of the first immunization from 11 immunized participants in the trial’s Cohort 1, each of whom received five doses of radiation-attenuated P. falciparum via mosquito bite. Three weeks after the fifth immunization, participants were challenged via P. falciparum-infected mosquito bites. To facilitate the identification of correlates of protection, the IMRAS immunization regimen was designed to achieve ~50% sterile protection in immunized participants: 6 out of the 11 immunized participants (54%) in Cohort 1 were protected following challenge.The IMRAS trial23  was performed in male, malaria-exposed recruits from Tanzania. The trial was designed to determine if protective immune responses against P. falciparum infection could be generated in a relatively non-immune population within a malaria-endemic country through immunization with aseptic, cryopreserved, live, radiation-attenuated P. falciparum sporozoites delivered intravenously (IV). Preimmunization transcriptomic data from 22 immunized participants were obtained from whole blood samples collected on the day of the first immunization. Each participant received five immunizations and, three weeks after the last, were challenged with infectious, aseptic, cryopreserved P. falciparum sporozoites delivered IV.The BSPZV1 trial24  compared the ability of two malaria vaccination regimens to elicit protection against malaria challenge in malaria-naive recruits from the United States. The study’s first vaccine regimen consisted of three doses of the RTS,S/AS01 vaccine, which combines a recombinant protein-encoding part of the malaria parasite’s circumsporozoite (CSP) protein, the hepatitis B surface antigen, and the AS01 adjuvant. This adjuvant includes the Toll-like receptor 4 (TLR4) agonist 3-O-desacyl-4′-monophosphoryl lipid A (MPL) and the QS-21 saponin33. The second regimen was the same as the first except a different vaccine, Ad35.CS.01, was used for the first immunization. Ad35.CS.01 consists of replication-deficient adenovirus 35 expressing recombinant CSP. Preimmunization transcriptomics from 45 immunized participants  were obtained from PBMCs collected on the day of the first immunization. Participants were challenged with P. falciparum-infected mosquitos two weeks following the third immunization.The MAL68 trial26  investigated the influence of dosing level on the development of protection against malaria challenge when immunizing malaria-naive participants using P. falciparum-infected mosquito bites obtained under chloroquine prophylaxis. Participants were recruited in The Netherlands and received three immunizations separated by 4-week intervals. They were challenged 19 weeks after the last immunization by exposure to bites from five mosquitoes infected with a homologous P. falciparum strain. Preimmunization transcriptomics were obtained from whole blood samples collected one week prior to the first immunization and were from seven members of the study group immunized with 15 mosquito bites total.The CPS trial34 version 2.3.1d. Gene count tables were generated with HTSeq35 version 0.6.0 using the intersection-strict setting and including strand information. Gene annotations used to generate count tables were obtained from the Ensembl database (GRCh37.74).RNA-seq FASTQ files were preprocessed to adjust base calls with phred scores <5 to ‘N’. Read pairs for which either end has fewer than 30 unambiguous base calls were removed, a method that indirectly removes pairs containing mostly adaptor sequences. Read pairs were aligned to the human genome (hg19) using STAR36 version 1.28.0. Principal Component Analysis (PCA) was used to identify outliers among transcriptomic samples: If a sample’s first or second principal component value was more than three standard deviations from the corresponding principal component’s mean, they were excluded from downstream analyses. One sample from the CPS trial was identified as an outlier based on PCA. To account for batch effects among trials, we used a DESeq2 design formula that included a trial term as well as a protected/non-protected classification term. Differential expression analyses performed on samples from the same trial only included the latter term.Differential expression analyses comparing preimmunization sample counts from challenge-protected trial subjects to non-protected subjects were performed using DESeq227 was performed on gene lists ranked by the DESeq2 Wald test statistic using the fgsea package37 version 1.14.0 in R. Gene sets used for GSEA were obtained through the tmod R package38 version 0.46.2 and the MSigDB Hallmark gene set30 download site (http://www.gsea-msigdb.org/gsea/msigdb/genesets.jsp?collection=H). IPA content version 62089861 was used to generate all IPA-based results. ROC analyses were performed using the pROC package39 version 1.17.0.1 in R. For all statistical tests, FDR-adjusted P-values (Benjamini-Hochberg method40) <0.05 were considered significant unless otherwise indicated.GSEAAll transcriptomic data processing and analyses were performed using publicly available software; no custom algorithms or software packages played a central role in the study. Supplementary Fig. Further information on research design is available in the Supplementary InformationREPORTING SUMMARY


Plasmodium falciparum-infected erythrocytes express a unique variant surface antigen, VAR2CSA, that mediates sequestration in the placenta. This process may initiate a range of host responses that contribute to placental inflammation and dysregulated placental development, which affects placental vasculogenesis, angiogenesis and nutrient transport. Collectively, these result in the impairment of placental functions, affecting fetal development. In this review, we provide an overview of malaria in pregnancy and the different pathological pathways leading to malaria in pregnancy-associated low birthweight. We also discuss current prevention and management strategies for malaria in pregnancy, and some potential therapeutic interventions that may improve birth outcomes. Lastly, we outline some priorities for future research that could bring us one step closer to reducing this health burden.Pregnant women in malaria-endemic regions are susceptible to malaria in pregnancy, which has adverse consequences on birth outcomes, including having small for gestational age and preterm babies. These babies are likely to have low birthweights, which predisposes to infant mortality and lifelong morbidities. During malaria in pregnancy,  Plasmodium spp. parasite, can result in severe morbidity and mortality. Worldwide, infection with Plasmodium falciparum contributes to the highest burden, and it will be the main focus of this review  is their ability to adhere to endothelial and placental receptors. The latter allows them to sequester in the placenta, leading to placental infection and inflammation. Histological sections of P. falciparum-infected placentas reveal the presence of IEs, particularly at the surface of the syncytiotrophoblast, which is the main site of exchange for nutrient, waste and other metabolites between maternal and fetal circulations. Within the placental intervillous spaces, which are occupied by maternal blood, increased infiltrates of phagocytic cells with substantial amounts of ingested parasites can be observed; this is known as intervillositis  was associated with a higher risk of preterm delivery , primigravidity, long-term iron supplementation and severe anemia –35. A reP. falciparum-infected children and adults with uncomplicated malaria illness  and vascular development. An ed women . In the ed women . There aed women . In a Bred women . Often,  protein , 37. In  protein , 52. The protein , 37, whi protein , 53. TakPlasmodium infection during pregnancy increases the risk of intervillositis . These spaces are occupied by maternal blood that surrounds the chorionic villi, serving as the materno-fetal exchange surface. Importantly, intervillositis, but not PM without intervillositis per se, was more commonly linked to adverse birth outcomes. This is particularly true in areas of high malaria transmission such as Tanzania and Malawi, where intervillositis was associated with abnormalities in the placenta and significantly higher rates of LBW (up to 50%) (Untreated  to 50%) , 54, 55.Plasmodium-infected placentas revealed that compromised nutrient transport is one of the molecular mechanisms contributing to growth restriction and reduced birth weight. In women with PM-associated intervillositis, reduced expression of glucose transporter isoform 1 (GLUT-1) on the syncytiotrophoblast basal membrane was reported , IPTp and effective case management of malaria disease. Additionally, there are encouraging data to suggest that indoor residual spraying insecticide (IRS) may also be effective in reducing MiP and improving birth outcomes , 63. ITNliveries –66. On tliveries , 63.Currently, all pregnant women residing in areas with high malaria transmission are recommended to receive three or more treatment courses of IPTp with SP, where each course should be administered at least one month apart, starting in the second trimester . This reDespite these preventive strategies, the prevalence of PM and LBW remain a huge threat to pregnancies in malarious regions due to several reasons. Given that IPTp is only administered from the second trimester onwards, pregnant women are not protected during the first trimester and the later weeks of third trimester. This inadequate IPTp coverage throughout pregnancy, poor population coverage, development of drug and insecticide resistance, and the use of suboptimal drugs which results in the failure to clear submicroscopic infection, are likely to contribute to increased risk of preterm birth, SGA and LBW , 74, 75.The exact mechanisms by which SP leads to improved birth outcomes in the treated individuals remain elusive. In addition to malaria prevention, SP also serves as a broad-spectrum antibiotic that can resolve or prevent other infections of bacterial origin  that have also been associated with adverse birth outcomes , 82. In Given the limitations associated with current drugs, there is an urgent need to evaluate the safety and efficacy of new antimalarials or a combination of preventive strategies that are suitable for wider coverage and at earlier time points in pregnancy to protect against MiP in the first trimester.Another major contributor to LBW is maternal undernutrition, a condition that is also highly prevalent in many areas with high malaria transmission. There is a growing number of studies suggesting that malaria and maternal undernutrition may worsen pregnancy outcomes , 87. MalIn a comprehensive study that analyzed 23 systematic reviews on nutritional interventions during pregnancy, a few factors including provision of vitamin A, low-dose calcium, zinc and multiple micronutrients were shown to be associated with reduced risk of LBW . Similarvia the parasites' VAR2CSA protein. These binding-inhibition antibodies have been associated with protection against preterm birth, increased gestational age, the delivery of heavier infants and importantly, reduced risk of LBW delivery , thus it may resolve infections other than malaria to improve adverse birth outcomes . Future Malnutrition is an important contributor to LBW, and nutrient supplementations during pregnancy appear to be an attractive and feasible intervention to minimize the risk of LBW deliveries. However, the beneficial effect of nutrient supplementation may be masked if administered together with IPTp, thus undermining its impact on birth outcomes. Hence, future trials of nutrient supplementations require careful study designs such as having intermittent-screening and treatment with nutrient supplementations, or choosing a trial site in a lower transmission setting where IPTp is not a first line policy.As of 2019, 38 countries and territories were granted the “malaria-free” status by WHO, while transmission continues in other regions. Hence, pregnant women remain at risk of MiP, which leads to increased mortality and morbidity in both the mother and her infant. MiP-associated LBW is preventable, but there needs to be a concerted effort in the adoption and implementation of current prevention strategies in malarious areas. Continuous efforts in research are needed, to better understand the molecular mechanisms associated with poor birth outcomes, and ultimately to enable design of specific targeted approaches for improving birth outcomes. In the face of increased parasite resistance, the effectiveness of IPT with SP in preventing this health burden may be at threat in the near future. Therefore, there is an urgent need to design and evaluate other therapeutic options that can be used to protect against MiP and its deleterious consequences.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors SR.