Plasmodium falciparum, which can assist in designing and monitoring elimination efforts. However, malaria molecular epidemiology including understanding the genetic diversity of the parasite and performing molecular surveillance of transmission has been poorly documented in Senegal. Next Generation Sequencing (NGS) offers a practical, fast and high-throughput approach to understand malaria population genetics. This study aims to unravel the population structure of P. falciparum and to estimate the allelic diversity, multiplicity of infection (MOI), and evolutionary patterns of the malaria parasite using the NGS platform.Molecular epidemiology can provide important information regarding the genetic diversity and transmission of P. falciparum isolates from two epidemiologically different areas in the South and North of Senegal, was carried out.Multiplex amplicon deep sequencing of merozoite surface protein 1 (PfMSP1) and merozoite surface protein 2 (PfMSP2) in fifty-three Pfmsp1 and 116 Pfmsp2 clones were identified and 135 different alleles were found, 56 and 79 belonged to the pfmsp1 and pfmsp2 genes, respectively. K1 and IC3D7 allelic families were most predominant in both sites. The local haplotype diversity (Hd) and nucleotide diversity (π) were higher in the South than in the North for both genes. For pfmsp1, a high positive Tajima’s D (TD) value was observed in the South (D = 2.0453) while negative TD value was recorded in the North (D = − 1.46045) and F-Statistic (Fst) was 0.19505. For pfmsp2, non-directional selection was found with a highly positive TD test in both areas and Fst was 0.02111. The mean MOI for both genes was 3.07 and 1.76 for the South and the North, respectively, with a statistically significant difference between areas (p = 0.001).A total of 76 pfmsp1 and pfmsp2 genes and low genetic differentiation in P. falciparum population in Senegal. The MOI means were significantly different between the Southern and Northern areas. Findings also showed that multiplexed amplicon deep sequencing is a useful technique to investigate genetic diversity and molecular epidemiology of P. falciparum infections.This study revealed a high genetic diversity of  Plasmodium falciparum is the deadliest species of human malaria parasites (99.7%) and the most prevalent in Africa, responsible for 92% of malaria cases and 93% of death caused by malaria [ malaria . A bette malaria  and asse malaria –8.Plasmodium falciparum merozoite surface protein 1 (PfMSP1) and merozoite surface protein 2 (PfMSP2) are polymorphic antigens which have been extensively studied in malaria molecular epidemiology [Pfmsp1 and block 3 of Pfmsp2) can be used to assess the allelic diversity and to determine MOI [P. falciparum populations [Pfmsp1 from 222 samples collected in Ethiopia revealed 307 haplotypes out of which 99 were predominant haplotypes (the clone had the highest frequency within infection). In addition, a mean MOI of 2.68 was found by this study [emiology –11. Polymine MOI . Therefomine MOI –16. Howemine MOI –19. Micrmine MOI –22. Simimine MOI . Advancemine MOI . These tmine MOI , 26 and ulations . For exais study .P. falciparum genetic diversity and malaria molecular epidemiology [of Pfmsp1 and Pfmsp2 genes in P. falciparum isolates sampled from a malaria hyper-endemic area in the South (Kedougou region) and a pre-elimination areas in the North (Podor and Matam) was performed. This data was used to understand the population structure, to estimate allelic diversities and MOI, and to understand the population dynamics, gene flow and evolutionary pattern of the malaria parasite P. falciparum in Senegal.In Senegal, most of the studies about emiology , 28–30 hThis study was conducted in Kédougou region,  and Podor and Matam . Kédougou  Fig.  is hyperP. falciparum malaria with parasite density ≥ 1000 asexual forms per microlitre. Patients who presented signs or symptoms of severe malaria as defined by the World Health Organization (WHO) [P. falciparum positive rapid diagnostic tests (RDTs) were collected in health facilities (NMCP sentinel sites) in Matam and Podor.Samples were collected from patients with malaria symptoms attending the National Malaria Control Programme (NMCP) sentinel site health facilities during malaria transmission season between September and December 2016   and pregThe experimental workflow from amplicon preparation to data analysis are detailed in Fig. Plasmodium falciparum molecular identification was performed using the photo-induced electron transfer (PET)-PCR assay [P. falciparum strain) control. Samples with a cycle threshold (CT) of 40 or less were scored as positive [Pfmsp1 and Pfmsp2 polymorphic genes were amplified by multiplex PCR. All PCR reactions were conducted in a 20 μl reaction mixture containing 2 μl of template DNA, 4 μl Phusion high-fidelity (HF) PCR master mix, 0.2 μl HF Phusion Taq Polymerase, 250 μM deoxyribonucleotide triphosphate (dNTPs), 0.5 μM of each forward and reverse primer of each gene. Cycling conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 1 min and extension at 68 °C for 1 min 40 s; a final extension was done at 68 °C for 5 min [Pfmsp1 and Pfmsp2, respectively.Parasite genomic deoxyribonucleic acid (DNA), from filter paper and RDTs was extracted using QIAamp DNA Mini kit  according to the manufacturer's instructions. CR assay  on a Rocpositive , 34. Theor 5 min , 35. TheAmplicons from each sample were purified using a 0.6X DNA SPRI . Samples were normalized to 0.25 ng/μl concentration using the Qubit® 3.0 Fluorometer and Invitrogen™ Qubit® Quantitation Kit . The Nextera XT DNA Library Preparation kit  was used for library preparation of 1 ng of purified PCR product from each sample . SequencPfmsp1 or Pfmsp2 gene mapping using the Trinity software [Pfmsp1, and block 3 of Pfmsp2 using the Geneious software  was used to compare MOI between localities. For all tests, comparisons were considered statistically significant when P-values < 0.05.The MOI mean was calculated by dividing the total number of alleles detected by the total number of samples for each gene . The onlPfmsp1 and Pfmsp2. All Fastq sequencing data were submitted to the SRA/NCBI database with the following accession numbers: from SRX8632497 to SRX8632549 (https://www.ncbi.nlm.nih.gov/sra/PRJNA642844).Fifty-three (53) samples (28 from the South and 25 from the North) were successfully amplified and sequenced for the two polymorphic markers Pfmsp1 gene were successfully assembled from 53 P. falciparum isolates. A total of 76 clones were found of which 47 and 29 in the South and the North, respectively  of 0.93 and a nucleotide diversity (π) of 0.28306. The TD test was performed in each area in order to identify any deviation from the neutral evolution of parasites and a highly positive TD value was observed in the South (D = 2.0453) with a statistically significantly different (p < 0.05)  . The Fst between the two localities showed an almost panmictic population (Fst = 0.02111) (Table The observed genetic diversity was similarly elevated in both regions (Hd = 0.82 in the South and Hd = 0.843 in the North), and nucleotide diversity was also very similar (π = 0.48339 in the South and π = 0.42473 in the North). Non-directional selection was found with a highly positive TD test in the two areas, D = 5.21073 in the South and D = 3.46684 in the North with statistically different . For Pfmsp2, mean MOI was significantly different between the South and the North (p = 0.010). The number of clones per samples ranged from 1 to 7 in the South with a MOI = 2.71 while in the North a MOI = 1.6 and the average number of clones varying between 1 and 4. Mean MOI for both genes was 3.07 and 1.76 in the South and North, respectively, with statistically significant differences between areas (p = 0.001).The mean MOI for P. falciparum population structure could help to improve the local monitoring of parasite transmission, particularly in areas where P. falciparum genetic diversity has been poorly documented. Therefore, the aim of this study was to report the genetic diversity of P. falciparum and parasite population structure by performing multiplexed amplicon deep sequencing of Pfmsp1 and Pfmsp2 from two understudied areas in Senegal with significantly different endemicity, the Southern  and Northern  areas. A high degree of polymorphism of the genes Pfmsp1 and Pfmsp2 was found in Senegal. A total of one hundred thirty-five different alleles were identified; 56 Pfmsp1 alleles  and 79 Pfmsp2 alleles (54 IC3D7-like and 25 FC27-like). These results are consistent with a previous observation of high levels of polymorphism in both Pfmsp1  and Pfmsp2  using an amplicon deep sequencing technique in Myanmar [Pfmsp1  and Pfmsp2 (35 IC3D7-like and 18 FC27-like), respectively. Comparable results from elsewhere in sub-Saharan Africa (Gabon and Ivory-Cost) reported by Yavo et al. underlined the same diversity with 27 K1-like, 22 MAD20-like and 18 RO33-like alleles for Pfmsp1 and 28 IC3D7-like and 20 FC27-like for Pfmsp2 [Pfmsp1 and Pfmsp2, which are the main factors driving the increased allelic diversity [P. falciparum genetic diversity is indicative of the ability of malaria parasites to adapt to their hosts by selection of advantageous traits, such as drug resistance and antigenic variability [A better understanding of  Myanmar . Similar Myanmar . Most ofiversity , 46–48. iability .Pfmsp1, despite a haplotype diversity of Hd = 0.76 in the North, the negative value of TD suggests there is clonal expansion of the parasite. A similar finding was also reported by the team of Daniels et al. in 2013 in Thiès, using a 24 SNP molecular barcode approach [P. falciparum population exhibits a high Hd (Hd = 0.93) and a TD value significantly different from expected under the neutral model of molecular evolution (D = 2.0453). This variability in parasite population genetics observed across the two regions reflected the difference in malaria transmission intensity reported in these areas [P. falciparum is greater in high malaria transmission areas and decreases when transmission regresses [Pfmsp1 between parasite population from the South and the North indicates the presence of gene flow between these populations, likely facilitated by extensive human migration events between regions causing the vector’s displacement.For approach . This siPfmsp2, three clusters with a high Hd and π were observed associated with a balancing selection  in each area and an almost panmictic parasite population (low Fst and genetic relatedness) between the two studies sites. As widely known, malaria parasites use genetic diversity as an escape measure either from human immunity or treatment by anti-malarials [P. falciparum genetic diversity in the low malaria transmission region of Thies, as previously reported [p = 0.001), suggesting that malaria transmission remains still active in this area. Moreover, the low mean MOI found in the North (1.76), may suggest a decline in malaria transmission levels, underlining the effectiveness of the scale-up in malaria control measures since 2006 [For alarials , 51. Thealarials , 53. In reported . Such evreported . Allelicreported . Recombireported , 55. MOIreported –61. MOI nce 2006 .P. falciparum population in two malaria endemic areas in Senegal. In addition, this study argues that multiplexed amplicon deep sequencing represents an important advance for surveillance of parasites populations within the country. Although this approach is currently more expensive and longer per-sample that standard genotyping methods it offers several advantages, especially in the ability to scale to large numbers of samples as will be needed for large-scale disease surveillance. The costs of sequencing are also rapidly declining and methods are becoming faster and more portable. Finally, this method allows the sequencing of several genes of interest in one run and can be readily adapted to other genes as the sequencing of anti-malaria resistance markers.This study was carried out from malaria symptomatic patients. As known, in endemic areas asymptomatic malaria or sub-microscopic infections is thought to represent the majority of the infections . TherefoPfmsp1 and Pfmsp2 genes in Senegal. Findings also showed an almost panmictic P. falciparum population with an important gene flow and MOI disparities between a malaria hotspot  and a malaria pre-elimination area , a situation that may hamper malaria elimination in these Northern regions. Therefore, continuous molecular epidemiological surveillance to monitor the genetic diversity and parasite population structure in Senegal would be necessary for malaria control in Senegal. Additionally, this study showed that multiplexed amplicon deep sequencing is a useful technique to investigate genetic diversity and molecular epidemiology of P. falciparum parasite infections.This study revealed a high genetic diversity of Additional file 1. Multiplexed amplicon deep sequencing data of Pfmsp1 gene.Additional file 2. Multiplexed amplicon deep sequencing data of Pfmsp2 gene.


Genetic diversity of ABO blood, glucose-6-phosphate dehydrogenase (G6PD) deficiency and haemoglobin type and their ability to protect against malaria vary geographically, ethnically and racially. No study has been carried out in populations resident in malaria regions in western Kenya.A total of 574 malaria cases  seeking treatment at Vihiga County and Referral Hospital in western Kenya, were enrolled and screened for ABO blood group, G6PD deficiency and haemoglobin genotyped in a hospital-based cross-sectional study.P = 0.380, P = 0.183 and P = 0.464, respectively). Further regression analysis revealed that the carriage of the intermediate status of G6PD was associated with risk to SMA . There was, however, no association between AS and SS with severe malaria anaemia. Co-occurrence of both haemoglobin type and G6PD i.e. the AA/intermediate was associated with risk to SMA  while the carriage of the AS/normal G6PD was associated with protection against SMA .When compared to blood group O, blood groups A, AB and B were not associated with SMA (Results demonstrate that blood group genotypes do not have influence on malaria disease outcome in this region. Children in Vihiga with blood group O have some protection against malaria. However, the intermediate status of G6PD is associated with risk of SMA. Further, co-inheritance of sickle cell and G6PD status are important predictors of malaria disease outcome. This implies combinatorial gene function in influencing disease outcome. P. falciparum malaria [Despite the expanded use of proven malaria control strategies, over 216 million malaria cases and 0.5 million malaria related deaths have been reported worldwide [ malaria .P. falciparum-infected red blood cells to uninfected red blood cells to form clusters of cells that are thought to contribute to the pathology of falciparum malaria by obstructing blood flow in small blood vessels [P. falciparum form larger, stronger rosettes in non-O  blood groups than in group O red blood cells [falciparum malaria [Rosetting is characterized by the binding of  vessels . Rosettiod cells , 6 sugge malaria . On the  malaria , 6. Some malaria  while ot malaria .P. falciparum-infected HbAS and HbSS red blood cells contribute to protection against malaria [Impaired rosette formation due to increased sickling or reduced expression of erythrocyte surface adherence proteins in  malaria , 11. Cyt malaria . However malaria , 14.P. falciparum growth as well as accelerating ring stage erythrocyte senescence promoting phagocytic clearance and eryptosis of parasitized G6PD deficient cells [falciparum malaria. Some studies have shown that G6PD normal are more vulnerable to Plasmodium falciparum malaria than the G6PD deficiency and heterozygous individuals [Increased oxidative stress impairs nt cells –17 suppoividuals , whereasividuals .falciparum malaria is usually thought to act independently [P. falciparum malaria in children in Vihiga highland of western Kenya.Protection by AS haemoglobin genotype and G6PD deficiency against endently , 19. In endently . Heterozendently . To our endently , 22. Morendently –26, it iP. falciparum parasites [Anopheles mosquitoes are generally highly zoophilic, rather than anthropophilic, thus becoming efficient human malaria vectors in Vihiga highland [A cross-sectional study targeting children less than 3 years seeking treatment at Vihiga County Referral hospital, Vihiga, Western Kenya was carried out. Study participants were categorised as severe malaria anaemia  and non-severe malaria anaemia  There has been a marked increase in malaria in the Vihiga highland, nearly 1.3 times the overall rate in Kenya, largely due to the rise of drug-resistant strains of arasites , 28. Thearasites . Finallyhighland .n = Z2pq/d2 [The sample size was determined using the formula  Z2pq/d2 .Where:n = the sample size required,z = 1.96: confidence level test statistic at the desired level of significance,p = 52.0%: prevalence of malaria [ malaria .q = 1-p: proportion of malaria uninfected and d = 0.05: acceptable error willing to be committed.d = acceptable standard error of the mean.n = (1.96 × 1.96 × 0.52 × 0.48) ÷ 0.052.Therefore n = 384. Therefore, this study required at least 384 study participants.The optimum sample size estimated was Plasmodium falciparum malaria positive children who had received anti-malarial treatment within 48 h prior to the microscopical confirmation of their blood slides for malaria parasites and children co-infected with P. falciparum and other species of plasmodium, and Human Immunodeficiency virus type 1 (HIV-1), Hepatitis B virus (HBV) and Hepatitis C virus (HCV) were excluded from the study. Approximately, 2.0 mL of blood was collected in anticoagulant tube from each study participant and used for HIV-1/2, HBV and HCV serological testing, [Children were recruited via random sampling method and stratified into either severe malaria anaemia (SMA) and non-severe malaria anaemia (non-SMA)  or uninftesting,  haemogloThick and thin blood films were prepared from venous blood, stained with 10% Giemsa stain for 10 min and examined under a microscope. Parasite densities were calculated using the thick films by the WHO method (parasite count × 8000 divided by the number of WBCs counted which was 200) , and theABO blood groups typing was performed by forward grouping using commercial antisera  according to manufacturer’s protocol. Briefly, one drop of whole blood was placed in three different places on a grease-free clean glass slide labelled A, B and D. A drop of antisera A was added on the area labelled A, anti-B on B and anti-D on D. The blood cells and the antisera were mixed using a wooden applicator stick. The slide was then tilted to check for agglutination and result recorded accordingly.Haemoglobin genotypes were determined by cellulose acetate electrophoresis with Titan III plates according to the manufacturer’s protocols . Haemolysates prepared from blood samples and Hemo AFSC controls was dispensed onto the acetate paper, and haemoglobin variants separated by electrophoresis with an alkaline buffer at pH 8.6. The plates were then stained using Ponceau S stain, and haemoglobin genotypes scored using the Hemo AFSC control.+) was added, and samples excited with ultraviolet (UV) light at 340 nm. Based on the presence or absence of fluorescence emissions, the samples were scored as normal (high emission), intermediate (moderate emission), or deficient (no emission).Glucose-6-phosphate dehydrogenase (G6PD) deficiency was determined by a fluorescent spot test  as per the manufacturer’s protocol. Blood was hemolyzed and spotted onto a filter paper. Assay solution containing glucose-6-phosphate and oxidized Nicotinamide adenine dinucleotide phosphate . Proportions of sex between groups were determined by Chi-square test. Age, haemoglobin, parasitaemia, white blood cells (WBC), glucose and red blood cells (RBCs) levels between groups were determined using Mann-Whitney U test. Odds ratios were calculated with 95% confidence interval (CI) using logistic regression analyses. Statistical significance was set at P = 0.540). Those with the SMA, 11.3 (11.6) mos. Were comparatively younger than those with non-SMA 14.6 (10.5) mos., P = 0.004. Haemoglobin concentrations were lower in children with SMA 4.2 (1.3) relative to the non-SMA 8.5 (2.2), P < 0.001. The concentrations of the white blood cells were higher in the SMA group; 13.5 (8.8) relative the non-SMA group 12.1 (6.2), P < 0.001. Furthermore, RBC counts were lower in children with SMA; 2.4 (0.8) when compared to the non-SMA group 3.5 (0.7), P < 0.001. Glucose concentrations were however similar between the two groups, P = 0.584.The demographic and laboratory measurements of the study participants are presented in Table P = 0.331). To further determine the influence of ABO blood group on P. falciparum malaria in children, we performed a multinomial logistics regression using the common O blood group as the reference group. At 95%CI, the B, AB and A blood groups did not significantly influence outcome of malaria i.e. B; , AB;  and A; , respectively, Table The prevalence of the ABO blood groups in the study population were as follows O; 254/574 (42.7%), B; 152/574 (26.4%), AB; 22/574 (3.8%) and A; 156/574 (27.1%). We then determined the distribution of the various blood groups within the study groups. Results demonstrated no significant differences between the blood groups within the study stratifications (P = 0.040). Further, regression analysis revealed that the carriage of the intermediate status of G6PD was associated with risk to SMA . However, the carriage of the G6PD deficient status was not associated with SMA , Table Before determining the influence of the G6PD on malaria infection, we performed prevalence analysis. The data show that normal G6PD genotype were 360/574 (62.7%), intermediate genotype 201 (35.0%) and deficiency in G6PD was 13/574 (2.3%). Glucose-6-phosphate deficiency showed a significant differences between the two study groups. The carriage of intermediate status of the G6PD was higher in the SMA group relative to the non-SMA. Moreover, there were more individuals who were deficient of G6PD in the non-SMA group compared to SM (P = 0.001). When multinomial logistics regression was done with the AA as the reference group, the AS showed marginal association with protection against SMA . The carriage of the SS group could not run in the model to give a significant statistical meaning , AS 93/574 (16.2%) and SS 4/574 (0.7%), respectively. Assessment of carriage of the sickle cell genetic variations revealed that there were 123 (89.8%) in SMA group and 354 (81%) of the AA in non-SMA group. Of the AS variation, there were 11 (8.0%) in the SMA group while 82 (18.8%) were in the non-SMA group (Table P = 0.046). In addition, the AS/normal showed protection against SMA . These findings show important influence of co-inheritance in malaria disease outcome.The distributions of the coinheritance of the Hb and G6PD variations are as presented in Table Malaria remains one of the most important causes of morbidity and mortality in children leaving in endemic areas . HighestP. falciparum malaria infection among children in Nigeria [P. falciparum parasites prefer type O over type A erythrocytes [P. falciparum infection but showed that blood group O primiparous women appeared to be more susceptible to active placental P. falciparum infection [P. falciparum parasitaemia was not significant among individuals with blood group A or non-O compared with blood group O. Furthermore, the difference in haemoglobin level among P. falciparum infected individuals was also not significant between those with blood group A, B or AB versus those with blood group O [In the present study, we did not find a significant association between carriage of the blood group genotypes and severe malaria outcome in children. This finding is, however, inconsistent with previous studies involving Ghanaian children  in which Nigeria . Other phrocytes . These fnfection . Additio group O . These i group O .Our current study did not show a significant association between the haemoglobin genotypes and malaria disease outcome. The findings of this study are consistent with previous studies reporting no association between haemoglobin AA, AS and SS genotype with malaria infection among children in Nyando County, Kenya . HoweverThe findings of this study showed that intermediate status of G6PD was associated with SMA. In addition the carriage of other G6PD variations did not show any association with malaria disease outcome. It is also important to note that genetic impact on infectious diseases is multigenic and not a single gene might reveal the actual effect. This is partly consistent with previous studies in Kilifi, Kenya, and Tanzania which showed protection with this particular variant in females but not males , 25. HowWe show in the current study that the co-inheritance of haemoglobin AA variations and G6PD intermediate status is associated risk of severe malaria while the carriage of the sickle cell trait and normal G6PD is associated with protection against severe malaria. This reveal that the effect of one gene on the other and that genes act in combination. This is an important finding that its mechanisms are worth investigation.It is important to outline the limitations of our study. Several red blood cell polymorphisms, including those linked to pyruvate kinase, complement receptor-1 and haemoglobinopathies such as thalassaemia traits, have a role in the clinical outcome of malaria  and mighThis study reveals that blood groups do not have a significant influence on malaria disease outcome. Haemoglobin AS offers rather a marginal protection against severe compared to the normal haemoglobin. Carriage of the heterozygous G6PD type compared to the normal G6PD is associated with risk of severe malaria anaemia. The co-inheritance of Hb variations and G6PD variation are important predictors of malaria disease outcome in this region. The findings of this study underline the potential of the Genome Wide Association approach to provide candidates for the development of control measures against malaria in humans.Additional file 1.


Plasmodium falciparum and Plasmodium vivax infections. Herein, we aimed to identify the underlying mechanism and contribution of host erythrocytes toward depleted S1P levels in Plasmodium-infected patients vs. healthy individuals. The level and activity of SphK-1 were measured in vitro in both uninfected and cultured P. falciparum-infected erythrocytes. Infected erythrocytes demonstrated a significant decrease in SphK-1 level in a time-dependent manner. We found that 10–42 h post invasion (hpi), SphK1 level was predominantly reduced to ∼50% in rings, trophozoites, and schizonts compared to uninfected erythrocytes. We next analyzed the phosphorylation status of SphK-1, a modification responsible for its activity and S1P production, in both uninfected control and Plasmodium-infected erythrocytes. Almost ∼50% decrease in phosphorylation of SphK-1 was observed that could be corroborated with significant reduction in the production and release of S1P in infected erythrocytes. Serum S1P levels were studied in parallel in P. falciparum (N = 15), P. vivax (N = 36)-infected patients, and healthy controls (N = 6). The findings revealed that S1P concentration was significantly depleted in uncomplicated malaria cases and was found to be lowest in complicated malaria and thrombocytopenia in both P. falciparum and P. vivax-infected groups (∗∗p < 0.01). The lower serum S1P level could be correlated with the reduced platelet count defining the role of S1P level in platelet formation. In conclusion, erythrocyte SphK-1 and S1P levels were studied in Plasmodium-infected individuals and erythrocytes that helped in characterizing the complications associated with malaria and thrombocytopenia, providing insights into the contribution of host erythrocyte biology in malaria pathogenesis. Finally, this study proposes the use of S1P and its analog as a novel adjunct therapy for malaria complications.Sphingosine-1-phosphate (S1P), a bioactive lipid mediator is involved in an array of biological processes and linked to pathological manifestations. Erythrocyte is known as the major reservoir for S1P as they lack S1P-degrading enzymes (S1P lyase and S1P phosphohydrolase) and harbor sphingosine kinase-1 (SphK-1) essential for sphingosine conversion to S1P. Reduced S1P concentration in serum was correlated with disease severity in patients with  Plasmodium falciparum, being the most virulent causative agent of malaria, accounts for most of these cases , and subsequently metabolized by ceramidase to generate sphingosine (Sph), which in turn is phosphorylated by sphingosine kinases 1 and 2  to generate S1P, a pleotropic bioactive lipid mediator. Both ceramide and S1P regulate cellular responses to stress, with generally opposing effects -, P. vivax (N = 36)-infected patients, and healthy controls (N = 6) to understand the clinical relevance of SphK-1 activity linked to S1P production. Though the serum S1P level was found to be significantly reduced in uncomplicated malaria cases, it was found to be lowest in complicated malaria and thrombocytopenia in both P. falciparum and P. vivax malaria groups (∗∗p < 0.01). This depleted level of serum S1P can be correlated with reduced platelet count defining the possible role of S1P level in platelet formation, thus providing insights into the contribution of host-SphK-1 signaling in malaria pathogenesis. Taken together, we present the experimental proof of decreased S1P levels during malaria infection associated with thrombocytopenia and chronic malaria because of altered SphK-1 activity. Finally, this study suggests the use of S1P and its analog as a novel adjunct therapy for malaria complications.According to World Health Organization (WHO) report, an estimate of 219 million malaria cases have been detected with 435,000 number of deaths . Plasmodse cases . Elucidaradation . Synthesradation . Pathogeradation . Sphingoradation . S1P is radation , bone maradation , and celradation . Given tradation , plateleradation , endotheradation , mast ceradation , and macradation . Howeverradation . Severalradation . Surpris effects . Among t effects . Erythro effects . Phospho effects . Notably effects . Earlier effects . HoweverPlasmodium falciparum 3D7 strain was cultured using O+ human erythrocytes, under mixed gas environment  as described previously  for 30 min at −20°C. Smears were then blocked with 3% (w/v) bovine serum albumin (BSA) in phosphate buffer saline (PBS) blocking buffer (pH 7.4) for 30 min at room temperature (RT). After blocking, slides were incubated with anti-SphK-1  at RT for 1 h. Slides were washed with 0.05% Tween-20 in PBS (PBST) two times followed by washing with PBS and incubated with an Alexa Fluor 594 conjugated goat anti-rabbit IgG  at RT for 1 h. After removal of unbound antibodies with three PBS washes, slides were mounted with Pro Long Gold antifade reagent  with coverslips and viewed on a Nikon A1-R confocal microscope using a 100× (oil) objective, and acquired images were processed via NIS-Elements software. Mean fluorescent intensity (MFI) was determined for a single cell by measuring fluorescent intensity corresponding to Sphk-1  for uninfected erythrocyte and infected erythrocyte and plotted as bar graph showing the MFI at a single-cell level. All imaging parameters were kept the same during acquisitions.Immunofluorescence assay (IFA) was performed to detect the level of SphK-1 in Immunoblotting of SphK-1 and Phospho-SphK-1 (Ser225) was performed in parasite-infected and uninfected erythrocytes to analyze SphK-1 level and activity. Total cell pellets of parasite-infected and uninfected erythrocytes were re-suspended in RIPA buffer . Equal amounts of each sample was boiled with 2× Laemmli buffer and separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). The fractionated proteins were transferred from gel onto the PVDF membranes (Millipore) using the Trans-blot Turbo transfer system and blocked with 5% skim milk in Tris buffer saline  for 1 h at 4°C. The blots were washed twice with 0.1% Tween-20 in TBS (TBST) followed by TBS and incubated overnight at 4°C with anti-SphK-1  and anti-Phospho-SphK-1  rabbit antibodies in blocking buffer . GAPDH present in the lysates was used as a loading control and probed with anti-GAPDH  mouse antibody. Later, the blots were rinsed in TBST three times, 5 min each, and in Tris-buffered saline (TBS) for 5 min and incubated for 1 h with appropriate secondary antibodies anti-rabbit and anti-mouse  conjugated to HRP; after being rinsed in TBST three times, 5 min each, and in TBS for 5 min, target proteins were visualized using the Clarity Western ECL substrate (Bio-Rad). The intensity of each band was quantified with ImageJ software and defined as Arbitrary Unit (AU) and plotted as bar graph.2O and transferred to 900 μl of methanol. To quantify S1P in supernatant, a 1:15 ratio of methanol was taken. After vortexing and centrifugation at 10,000 × g for 5 min at RT, methanol extracts were removed to a new glass tube. After evaporation by N2, dried lipids were resuspended in 200 μl of methanol. Extracted lipid samples were subjected to liquid chromatography–mass spectrometry (LC/MS) analysis. We used a Waters Acquity H-Class UPLC system . Chromatographic separation was achieved on an Acquity BEH C18, 1.7 μm, 75 × 2.1 mm column . The mobile phase consisted of 0.1% formic acid solvent (A) and acetonitrile (B). The initial gradient condition was 90% A and 10% B, 80 and 20% for 2 min, 50 and 50% for 3 min, 20 and 80% for 1 min, 10 and 90% for 2 min, and then linearly changed to 34% B over 8 min and turned back to initial condition of 90% A and 10% B and washed up to 15 min. The column temperature was adjusted at 35°C. The flow rate was 0.3 ml/min, and the injection volume was 5 μl. Mass spectrometry was performed in negative electrospray mode using a high-resolution mass spectrometer SYNAPT G2 S HDMS  with a TOF-detector with linear dynamic range of at least 5,000:1. The desolvation gas  and the nebulizer gas (6.0 bar) were nitrogen. The cone gas had a flow of 52 l/h. The capillary voltage was 2.52 kV and the source temperature 90°C. The analyzer mode was set at “resolution” and the dynamic range at “extended.” The mass spectra were acquired over the range of 100–1000 Da with a spectral acquisition rate of 0.1 s per spectrum. For the S1P measurement by enzyme-linked immunosorbent assay  method, parasite-infected and uninfected erythrocytes were centrifuged to collect the supernatant. Pelleted erythrocytes were washed and lysed by multiple freeze–thaw cycles. EC (extra cellular) S1P was measured through the supernatant, whereas the lysed erythrocytes were used to measure IC (intra cellular) S1P level. Erythrocyte supernatant, lysate, and patient serum samples were added to the micro ELISA plate wells separately, pre-coated with S1P-specific antibody for 90 min at 37°C. A biotinylated detection antibody specific to human S1P was added and incubated for 1 h at 37°C. After washing, avidin–horseradish peroxidase (HRP) conjugate was added successively to each microplate well and incubated for 30 min at 37°C. The wells were washed to remove the unbound components followed by the addition of substrate solution to each well. Only those wells that contained S1P, biotinylated detection antibody, and avidin–HRP conjugate appeared blue in color. The enzyme–substrate reaction was terminated by the addition of stop solution turning the reaction color to yellow. The optical density (OD) proportional to the S1P level was measured spectrophotometrically at a wavelength of 450 nm.For S1P measurement, an equal number of synchronous parasitized erythrocytes at 7–8% parasitemia and uninfected erythrocytes were taken. Collected cell pellets and supernatant were employed for lipid extraction as reported previously . PelletsIntensity AU (IAU) for Sphk-1 and pSphk-1 in both uninfected and infected erythrocytes were normalized with band intensities of loading control GAPDH. The relative ratio of normalized band intensity for SphK-1 and pSphK-1 was determined. Finally, the ratio of pSphK-1 to SphK-1 intensity was plotted as AU.The collection of patient serum samples was done at the Department of Medicine, Sardar Patel Medical College and associated group of Hospitals, Bikaner, India. Bikaner is a part of the Thar Desert situated in the northwest part of India near the Indo–Pak border and is a hypoendemic region for malaria. This prospective study was conducted on admitted adult patients of malaria in whom the diagnosis was done by peripheral blood smears (PBFs) and rapid diagnostic tests (RDTs). After thorough clinical and laboratory investigations, the categorization of severe malaria and treatment was done according to the WHO guidelines . The finPlasmodium antigen, lactate dehydrogenase , and histidine-rich protein-2 . The final categorization of P. vivax, P. falciparum, or mixed infection was done by PCR examination of all the patients having severe manifestations. The PCR studies were targeted against the 18S ribosomal RNA gene of the parasite and used one genus-specific 5′-primer and two species-specific 3′-primers in the same reaction mixture or a nested PCR targeted against the 28S ribosomal RNA gene of the parasites  and microscopically examined under oil immersion. The slide was considered negative when there were no parasites in the 100 high-power field object. The RDTs were based on the detection of specific arasites , b. The Laboratory investigations done in all the patients of severe malaria included complete blood count, platelet count, bleeding time, clotting time, blood glucose, blood urea, serum creatinine, serum bilirubin (conjugated and unconjugated), serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), serum alkaline phosphatase, complete urine analysis, electrocardiogram, and appropriate blood test to rule out typhoid fever (typhi dot test), leptospirosis, dengue infection , and human immunodeficiency virus (HIV). Depending upon the clinical situation, other tests included skiagram of chest, serum electrolytes, and arterial blood gas analysis for acute respiratory distress syndrome (ARDS); fundus examination, cerebrospinal fluid (CSF) examination, computerized tomography (CT) of the head and electroencephalography (EEG) in patients having repeated convulsion and cerebral malaria (CM); ultrasonography of whole abdomen and specific test for hepatitis B and C in hepatic dysfunction and jaundice, and glucose-6-phosphate dehydrogenase (G6PD) enzyme level  for hemolysis. Blood culture was taken on brain–heart infusion broth in every patient who was having continuous high-grade fever >38.33°C for >24 h after admission. All the clinical symptoms were classified according to the WHO  criteriat-test was performed to calculate the p-values, where p < 0.05 was taken as significant. One-way ANOVA along with Tukey’s multiple comparisons test was performed to compare the three groups, with p < 0.05 was considered as significant.The data for all the assays are expressed as the mean ± standard deviation (SD) of three independent experiments done in triplicates. Student’s P. falciparum, culture at 5–6% parasitemia with highly synchronized parasites at ring stage (10–12 hpi) was incubated in complete medium for a period of 18 and 30 h, and the stages were monitored by counting. For IFA, synchronous population of ring-, trophozoite-, and schizont-infected erythrocytes were used  in the total cell lysates of parasite-infected and uninfected erythrocytes. For this experiment, the parasitemia was maintained at 12–15% at the trophozoite stage. Respective lysates were probed with specific anti-SphK-1 and phospho-SphK-1 (Ser225) antibodies. Probing with anti-GAPDH antibody was done for use as the loading control. In line with the immunofluorescence detection, the host SphK-1 level was found to be reduced in parasite-infected erythrocytes compared to the uninfected erythrocytes. The band intensities in individual lanes of immunoblot were quantified by Image J to confirm the relative level of SphK-1 and pSphK-1. Interestingly, the phosphorylated form of host SphK-1 (pSphK-1) was also relatively decreased in parasite-infected erythrocytes . Band in9 cells/200 μl of packed volume of uninfected and infected erythrocytes. Trophozoite stage was used with 10–12% of parasitemia. Lipid extracts were prepared from P. falciparum-infected and/or uninfected erythrocytes and subjected to LC/MS analysis, as earlier described . To further validate the difference in S1P levels, ELISA-based S1P detection method was employed, and the comparison was done between infected and uninfected erythrocytes. Supernatant and lysed erythrocytes were added to micro ELISA plate pre-coated with S1P-specific antibody followed by probing with biotinylated antibody specific to human S1P. The results suggested that in parasite-infected erythrocytes S1P levels were significantly decreased compared to uninfected erythrocytes (∗p < 0.05), strongly supporting the observation from S1P levels detected in LC/MS - as well as P. vivax (N = 36)-infected cases (N = 6), collected from the blood bank. Categorization of P. vivax and P. falciparum or mixed infection was done in all the patients as described in the section “Materials and Methods” and shown in P. falciparum (N = 9) and CM (N = 2/9) were found to be the lowest compared to the uncomplicated malaria (N = 4/9), thrombocytopenic cases (N = 3/9), and healthy controls (N = 6). Uncomplicated malaria and thrombocytopenic cases also showed lower levels of circulating S1P than the healthy controls. Student’s t-test was performed and the p-values detected as ∗p < 0.05, ∗p < 0.05, ∗∗p < 0.01  , wherein complicated malaria patients (N = 6/25) were found to have lowest circulating S1P concentration compared to uncomplicated malaria patients (N = 9/25), thrombocytopenia cases (N = 10/25), and healthy individuals (N = 6). Student’s t-test was performed and the p-values generated as ∗p < 0.05, ∗p < 0.05, ∗∗p < 0.01  (N = 35), irrespective of P. falciparum and P. vivax infection. The levels of S1P in all thrombocytopenia cases were found to be significantly lower than those of the uncomplicated malaria cases (N = 13). Student’s t-test was performed; ∗p < 0.05 , healthy control and malaria patients with thrombocytopenia (****p < 0.0001), and between uncomplicated malaria and malaria with thrombocytopenia (∗p < 0.05)  . Howeverviduals) . Furtherp < 0.05 . Further < 0.05) . Altoget earlier . Here, b earlier . This fi earlier . These fP. falciparum to human erythrocytes in blood stage progression involves multiple cycles of invasion and egress that modulates the host cell lipids  catalyzes the formation of the potent bioactive S1P that is produced and stored in various cell types including erythrocytes (1–5)] ] . It is ns (1–5)]  and its s (1–5)] . Additios (1–5)] . Indeed,s (1–5)] , and Sphs (1–5)] . Plausibs (1–5)] , sepsis s (1–5)] , chronics (1–5)] , obesitys (1–5)] , etc. Ins (1–5)] . Howevery ERK1/2  and PKC y ERK1/2 ; interesy ERK1/2  and PKC y ERK1/2  have beein vitro, from the IC and EC milieu of both infected and uninfected erythrocytes, authenticated that P. falciparum infection drastically reduced the S1P pool  proposing the severity of infection in correlation to altered S1P levels. Thus, S1P signaling has emerged as a novel therapeutic target, as many studies have acknowledged the fact that S1P-mediated signaling pathways play a critical role in numerous infectious disease manifestations  SPMC/2003/2395). The patients/participants provided their written informed consent to participate in this study.SS conceived and designed the research. RS performed the research. SS and SP analyzed the data. RS, SP, and SS conducted the lipid extraction and estimation experiments. MS and RS performed the microscopy experiments. SS, RS, and SP wrote the manuscript. AD was involved in the clinical study design. PB was involved in the sample processing. DK and SK were involved in classifying the clinical symptoms and coordinating sample collection.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.


Plasmodium antibodies and become semi-immune. Seasonal Malaria Chemoprevention (SMC) with sulfadoxine-pyrimethamine + amodiaquine (SPAQ) is a new strategy to reduce malaria morbidity in West African young children. However, SMC may impact on the natural acquisition of anti-Plasmodium immunity. This paper evaluates the effect of SMC with SPAQ on antibody concentration in young children from Niger.In endemic areas, children develop slowly and naturally anti-Plasmodium falciparum IgG antibody responses, the total antibody concentrations against two P. falciparum asexual stage vaccine candidate antigens, circumsporozoite protein (CSP) and glutamate-rich protein R2 (GLURP-R2), in children aged 3 to 59 months across the three areas were compared. Antibody concentrations are quantified using an enzyme-linked immunosorbent assay on the elution extracted from positive and negative malaria Rapid Diagnostic Test cassettes.This research was conducted in areas benefitting from SMC since 2014 (Zinder district), without SMC (Dosso district), and with 1 year of SMC since 2016 (Gaya district). To assess the relationship between SMC and The analysis concerns two hundred and twenty-nine children aged from 3 to 59 months: 71 in Zinder, 77 in Dosso, and 81 in Gaya. In Zinder (CSP = 17.5 µg/ml and GLURP-R2 = 14.3 µg/ml) median antibody concentration observed are higher than in Gaya (CSP = 7.7 µg/ml and GLURP-R2 = 6.5 µg/ml) and Dosso (CSP = 4.5 µg/ml and GLURP-R2 = 3.6 µg/ml) (p < 0.0001).P. falciparum infections in Niger. Increased antibody titres with increased SMC/SPAQ implementation. This contradicts hypothesis that SMC/SPAQ could reduce immunity to erythrocyte and liver-stage antigens. Further studies are necessary to provide better understanding of the SMC effect on malaria immunity.The research reveals some evidences which show that seasonal malaria chemoprevention with SPAQ has an effect on blood stage antibody responses and pre-erythrocytic stage of  Plasmodium falciparum remains the major cause of morbidity and mortality in children under 5 years in sub-Saharan Africa [Malaria caused by n Africa . It is tn Africa . The natn Africa –5. SMC in Africa . Withoutn Africa , 8.Plasmodium nucleotides (purines and pyrimidines). Pyrimethamine associated with sulfadoxine (SP) is a competitive inhibitor of dihydrofolate reductase (dhfr), a key enzyme in the redox cycle for the production of tetrahydrofolate, a cofactor necessary for the biosynthesis of DNA and proteins. SP acts on the asexual forms of the hepatic and erythrocytic stage of Plasmodium.Sulfadoxine is an antibacterial and anti-malarial drug of the chemical class of sulfonamides. It is a dihydropteroate synthetase (dhps) inhibitor, a key enzyme in the biosynthesis of folate. It acts by competitive inhibition of para amino benzoic acid (PABA) to block the synthesis of folic acid and Plasmodium.Amodiaquine is an anti-malarial with antipyretic and anti-inflammatory properties. It is a 4-aminoquinoline related to structure and activity with chloroquine. Amodiaquine is active on the erythrocyte form of Plasmodium immunity. In Senegal, Ndiaye et al. suggest that long-term SMC by SPAQ has limited impact on the development of acquired immunity [42 [42 and CSP titers [42 and CSP [Malaria immunity is partial, short-lived, and requires exposure to infected mosquitoes bites to be maintained . Monthlyimmunity . In the nity [42 . In MaliP titers . Other M and CSP .P. falciparum antibody responses, the total IgG concentrations against two P. falciparum asexual stage vaccine candidate antigens: circumsporozoite protein (CSP) and glutamate-rich protein R2 (GLURP-R2), of children aged 3 to 59 months across the three sites were compared. The CSP is a secreted antigen of the pre-erythrocyte stage of Plasmodium and GLURP-R2 is an antigen associated with mature schizont of blood stage. Antibody concentrations are quantified using an enzyme-linked immunosorbent assay (ELISA) on the elution extracted from positive and negative RDT cassettes.The hypothesis of this study is that SMC/SPAQ could reduce immunity to erythrocyte stage antigens and liver-stage, and malaria Rapid Diagnostic Tests (RDT) absorption filters could be used to measure IgG titers. To assess the relationship between SMC and The data presented here are generated from the malaria morbidity sentinel surveillance sites within the SMC programme in Zinder, Dosso and Gaya districts located in western Niger, where malaria transmission is seasonal , 16. ZinReport #1: 91%.Report #2: 73%.Report #3: 50% (Unpublished data)..In 2016, the reports on SMC Coverage from Zinder showed the following results: The coverage of Gaya district were 77.72%, 81.56%, 71.26%, and 69.47 respectively for round 1, 2, 3 and 4 (Unpublished data). All the 3 sites used Artemisinin-based Combination Therapy (ACT) as first line treatment for uncomplicated malaria cases and all received universal coverage of bed nets. The seasonality of malaria transmission in these 3 sites is the same.P. falciparum stage antigens, 6 health facilities in Zinder, Dosso and Gaya were selected. In these health facilities, malaria RDTs (SD-Bioline) of randomly selected children aged 3 to 59 months are collected from symptomatic cases (fever + positive or negative RDTs) for serological analysis. For all RDTs collected, the date of consultation, the age, the gender, whether the RDT test was performed and the result of RDT test P. falciparum circumsporozite protein synthesized by Sygma Genosys, while GLURP-R2  is an amino acid produced by the Statens Serum Institutes of Copenhagen (Denmark) and is expressed in Escherichia coli.The malaria antigens used in this study include a recombinant antigen circumsporozoite protein (CSP) and glutamate-rich protein R2 (GLURP-R2). CSP antigen is a 44-aa NANP repeat-sequence peptide of the Serum is eluted from filter paper inside RDTs cassette collected . RDTs ha2SO4 to stop the reaction. Standard curves were established using human IgG purified proteins  to determine the concentration of specific antibodies. Concentrations of the standard curve are as follows: 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 151 and 3.9 µg/mL. Each curve is run in duplicate on each plate. The ADAMSEL FLP b039 software [The standard operating procedure developed by the African Malaria Network Trust was used to assess total IgG concentrations by ELISA to CSP and GLURP R2, as described previously . Brieflysoftware  is used The Median test is used to analyse differences between IgG medians concentrations. The comparisons between IgG median concentrations in Zinder, Dosso and Gaya are performed to investigate the potential impact of SMC on antibody responses. Positive and negative cassette results as well as results between districts were compared. The comparison between IgG median concentrations are performed by Mann–Whitney test. Data are analysed with SPSS software version 16.0. P-values ≤ 0.05 are considered statistically significant.A total of 229 children aged 3 to 59 months were concerned by the analysis: 71 from Zinder, 77 from Dosso, and 81 from Gaya . The analysis of the difference between the median concentration of anti-CSP IgG and anti-GLURP-R2 in Zinder and Gaya was statistically significant (CSP: p = 0.008 and GLURP-R2: p = 0.017).In Zinder with SMC since 2014 (CSP = 17.5 µg/ml and GLURP-R2 = 14.3 µg/ml) and Dosso with no SMC (CSP = 4.5 µg/ml and GLURP-R2 = 3.6 µg/ml), median concentration of IgG antibody responses has been significantly different because of the two antigens by districts .The median concentrations of anti-CSP and GLURP-R2 IgG antibodies by RDTs results are in Table P. falciparum infections in Niger. An increased antibody titres are observed in SMC/SPAQ implementation areas. This contradicts hypothesis that SMC/SPAQ could reduce immunity to erythrocyte and liver-stage antigens.This study demonstrates that seasonal malaria chemoprevention with SPAQ has an effect on blood stage antibody responses and pre-erythrocytic stage of The total IgG antibodies to liver-stage vaccine candidate antigen CSP and blood stage antigen GLURP-R2 are significantly higher in Zinder where SMC has been implemented for 3 years, as opposed to Gaya, where SMC has been implemented for a year, and Dosso, as well, which has never benefitted from SMC. This is consistent with a previous intriguing finding that demonstrated sustained protection during one year of follow-up, in children who have received intermittent preventive malaria treatment .The concentrations of both antibodies against CSP and GLURP-R2 revealed an increase with SMC implementation probably as a result of the decrease of either liver-stage maturation or erythrocyte stage by SPAQ. This, contrasts with other studies –14, 23, Plasmodium yoelii models [Pfdhfr gene, known to be associated with resistance to pyrimethamine was observed [P. falciparum infections detected by PCR, consistent with SP affecting blood stage and not liver stage parasites.Others studies showed that sulfadoxine does not affect liver stages, pyrimethamine has some inhibitory effect on liver stages in i models , but thei models . In Nigeobserved . In vaccobserved , SP did This is the first use of antibody elution from RDT filter paper for the assessment of CSP and GLURPR2 antibody concentration in Niger. As demonstrated by Amrish Baidjoe et al. antibody elution from filter paper is an operationally attractive approach . RDT casp = 0.6813; GLURP-R2: p = 0.0760). No significant differences between positive and negative RDTs were observed, perhaps because of the small number of samples and the short period of observation, which, no doubt, limit the results of this study.The comparative analysis of antibodies concentration against CSP and GLURP-R2 antigens between age groups by district, observed no significant differences .Differences exist in RDT coverage between sites and collections.There is differences in endemicity between Zinder, which is mesoendemic, and Gaya and Dosso, which are hyperendemic. This suggests that differences in transmission intensity may be a major confounder, and future studies should aim to compare areas with the same transmission intensity.It is not possible to give the coating of the antigens concentration in order to directly compare them with other studies following the same protocol.Different antigens have different coating efficiencies, therefore, coating at the same concentration without optimization may result in a reduction in signal or specificity if over or under-coated, respectively.It is not possible to state that the quantity of RDT fragment eluted is the same per sample for antibody titres.It is not possible to interpret the immune responses in terms of implications for the risk of malaria. This approach might be useful for monitoring rates of acquisition of immunity in older children who have stopped receiving SMC.There are other important hurdles too: P. falciparum blood stage antigen GLURP-R2 and pre-erythrocytic stage antigen CSP. RDT filter paper serum elution methodology can significantly reduce the workload and cost in large-scale epidemiological and immunological studies.This data suggest that SMC by SPAQ have an effect on antibody responses against pre-erythrocytic stage and blood-stage antigens. However, other factors that have a significant influence on antibody titers, such as transmission intensity, may confound this. Future studies are necessary to provide a better understanding of the impact of SMC on malaria immunity in Niger. The duration of SMC administration may increase the antibody concentration of


Plasmodium species) that affects millions of people worldwide. The lack of effective malaria drugs and a vaccine contributes to this disease, continuing to cause major public health and socioeconomic problems, especially in low-income countries. Cell death is implicated in malaria immune responses by eliminating infected cells, but it can also provoke an intense inflammatory response and lead to severe malaria outcomes. The study of the pathophysiological role of cell death in malaria in mammalians is key to understanding the parasite–host interactions and design prophylactic and therapeutic strategies for malaria. In this work, we review malaria-triggered cell death pathways  and we discuss their potential role in the development of new approaches for human malaria therapies.Malaria is a parasitic disease (caused by different  Plasmodium genus—that is estimated to have infected around 228 million of people worldwide in 2018, representing a risk especially for residents of developing countries in tropical and subtropical regions [Malaria is a parasitic disease—caused by protozoa pathogens of the Plasmodium infection includes multiple stages, so immunity to malaria needs to be multifaceted and stage-specific [In the search for new methods to stem malaria, researchers have been studying intrinsic factors of the host, such as genetic profile and immunological mechanism ,4,5. Thespecific . The immspecific ,8,9.Plasmodium infection and the dual role of cell death in host immune protection and pathogenesis of severe malaria.Considering the above, comprehensive knowledge about the genetic, molecular, and biochemical mechanisms of the different cell death modalities has taken a prominent position in recent advances in immune response and the design of prophylactic and therapeutic methods against malaria. This infection has been reported to induce different forms of cell death: apoptosis, autophagy, necrosis, pyroptosis, NETosis, and ferroptosis. Here, we review what is currently known about the distinct modalities of cell death of host cells during Plasmodium are clinically relevant: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale , and Plasmodium knowlesi [P. falciparum can cause the most severe complications, such as anemia, cerebral, and placental malaria [P. vivax has a wider geographical distribution and, like P. malariae and P. ovale, it is associated with non-severe malaria [P. vivax, P. knowlesi, and P. malariae, allowing progression to severe malaria and leading to serious health problems, especially in immunocompromised patients [In humans, five species of knowlesi ,11,12,13 malaria ,15. P. v malaria ,17,18,19patients ,20,21. Spatients .Anopheles genus mosquitoes, and occasionally via blood transfusion or vertically [Plasmodium enters the host dermis [Transmission occurs primarily through the bites of infected female rtically ,24. The t dermis ; thus, tt dermis .P. vivax and P. ovale present an additional challenge to malaria elimination; they can develop into dormant forms during the liver stage, known as hypnozoites, so these forms may be reactivated, causing a relapse of the disease [In the liver, the sporozoites invade hepatocytes, where they differentiate into thousands of merozoites by schizogony process ,27,28. T disease .Plasmodium pathogen-associated molecular patterns ) and damage-associated molecular patterns ) [The innate immune system antigen-presenting cells (APCs) perform the first line of defense from the activation of pattern recognition receptors (PRRs) via recognition of sicles)) ,34.Plasmodium RNA via melanoma differentiation-associated gene 5/mitochondrial antiviral signaling protein (MDA5/MAVs) induces a type I interferon (IFN-I) response, which promotes the recruitment of cytokine-secreting cells and oxidative stress-inducing cells [In the liver stage, the detection of ng cells ,36. In tng cells ,39,40,41Upon these immune receptors’ activation, macrophages, neutrophils, natural killers (NK), T natural killers (NKT), dendritic cells (DCs), and then lymphocytes subsets readily produce pro-inflammatory cytokines—tumor necrosis factor α (TNF-α), Interferon γ (IFN-γ), interleukin-1 (IL-1), IL-6, and IL-12 ,44,45,46Plasmodium but also in the pathogenesis of severe malaria.Indeed, these components produced by immune cells in response to malarial infection act as activators of cell death pathways with unique genetic, biochemical, morphological, and physiological characteristics . SeveralCell death (CD) is a process of metabolic disorder that leads to the loss of the cell’s vital capacity. According to the Nomenclature Committee on Cell Death (NCCD), a cell can solely be considered as dead if it presents the following criteria: irreversible plasma membrane permeabilization and/or complete cellular fragmentation . CurrentCell death may occur in response to different stress conditions, especially oxidative stress . ResearcPlasmodium parasitemia, but also in the pathogenesis of severe malaria. In the next sections, we will characterize and highlight the main types of cell death in this context.Cell death and immune response have an interesting connection: while immune cells can secrete components capable of activating cell death, the death of an infected cell can release danger signals, such as DAMPs, which can provide immunostimulatory signals . That saApoptosis is the best-described form of PCD, and occurs in response to a wide range of physiological and pathological stimuli via two major signaling pathways: extrinsic or death receptors (DRs) and intrinsic or mitochondrial apoptosis, both resulting in activation of the caspase (CASP) cascade ,57 Figu.External stimuli such as extracellular stress and cellular immunity activate the extrinsic pathway by providing cytokines of the TNF family, which act as death ligands, namely: FAS-ligand (FASL), TNF-α, or TNF-related apoptosis-inducing ligand (TRAIL) . These dApoptotic signals transmitted by death receptors engage the following adapter proteins: Fas-associated death domain (FADD) in FASL/FAS and TRAIL/TRAILR interactions ; and TNFThe regulatory center of intrinsic or mitochondrial apoptosis is regulated by the B-cell lymphoma 2 (BCL-2) family gene, which includes both anti-apoptotic   and pro-Stress in the intracellular environment, such as damage to DNA or cellular organelles like mitochondria ,69 and eIn the apoptosis execution phase, the initiator caspases  will cleave and activate effector/executioner caspases  to carry out apoptosis . EffectoThe extrinsic and mitochondrial pathways are independent, but in certain types of cells, extrinsic apoptosis can be connected with mitochondrial apoptosis, through CASP-8-mediated cleavage of the BH3-interacting domain death agonist (BID) generating the truncated BID (tBID), which translocate to the mitochondria where it activates BAX and BAK to engage in mitochondrial apoptosis, and amplifying the caspase cascade to increase the efficiency of cell death .In addition to the FASL/FAS route, an altered cell may undergo cytotoxic lymphocyte-mediated apoptosis through the alternative perforin/granzyme pathway . This ocP. falciparum acute infection [P. falciparum infection [P. coatneyi [P. berghei [P. chabaudi [P. yoelii [In the 1990s, Baldé and colleagues ,87 were nfection ,87 as wenfection ,89,90 ancoatneyi , P. berg berghei , P. chabchabaudi , and P. . yoelii   and in FAS-deficient mice infected with P. yoelii, so researchers thought that the fate of T cells was partly defined by an IFN-γ-mediated role [−/− mice displayed reduced splenic cell death [P. vivax-infected patients, indicating that malaria infection induces apoptosis in CD4+ T cells by both extrinsic and intrinsic apoptosis [In malaria infections, there is an increase in FAS, FASL, and TNFR leading to specific and non-specific T cells death ,91,93,94ted role . This hyted role ,97 and Ill death . High lepoptosis .P. falciparum [P. vivax malaria [Plasmodium antigens; therefore, apoptosis became established as a modulator of the host’s immune response. However, we still do not fully understand the molecular details and physiological significance of activation-induced cell death (AICD) in immune cells [Furthermore, apoptosis is involved in the depletion not only of blood DCs during lciparum ,99 and P malaria ,96. The ne cells ,98,100. ne cells ,105,106.P. berghei-hepatocytes is independent of CTLs-mediated apoptosis either via FASL/FAS or perforin/granzyme [Plasmodium infection, thus activating NF-κB and MAPK pro-survival signaling [The death of infected granzyme , even ungranzyme  and Jo-2granzyme . Indeed,granzyme ,108,109.granzyme   infections, genetic apoptotic markers indicate that continued exposure to malaria-induced oxidative stress leads to extensive mitochondrial apoptosis, causing placental disorder in the maternal–fetal interface and the impairment of fetal development, the main consequences being low birth weight and fetal death ,120. In rocesses .Autophagy is a highly regulated and evolutionarily conserved mechanism that results in the lysosomal degradation of damaged cellular constituents and organelles with subsequent recycling of components of the degraded material, for intracellular clearance of potentially dangerous components in eukaryotic cells, to maintain cellular homeostasis in periods of nutrient starvations or stress ,126,127.There other types of autophagy described: (1) microautophagy, which comprises the lysosomal invagination of cytoplasmic entities for further degradation ; and (2)In viable cells, the nutrient-sensitive kinase mTOR  inhibits autophagy via phosphorylation of autophagy initiators ULK1 or ULK2 and ATG13, but under autophagic stimulus, mTOR is inhibited, allowing the formation of the ULK1/2 complex or preinitiation complex, which includes ULK1/2, ATG101, ATG13, and FIP200 ,134,135.The biogenesis of autophagosomes, double-membrane vesicles, initiates from the phagophore elongation followed by the closure of the edge of the membranes around the cytoplasmic material to be degraded ,139. PhaAlternatively, a cell can undergo autophagy through a non-canonical pathway, known as LC3-associated phagocytosis (LAP), in which LC3-II directly drives the fusion of the autophagosome to the lysosome, without the assembly of autophagy core machinery. Instead, LAP requires NADPH oxidase 2 (NOX2), which mediates ROS production, and Rubicon, a protein responsible for recruiting the PI3KC3 complex to internalize invading agents , so thatP. falciparum. For instance, these parasites lack kinase genes that sense nutrient deprivations and initiate autophagy, but conserved the P. falciparum ATG8 (PfATG8) gene [PfATG8 gene is expressed in all stages of human infection and functions as a human LC3 homologous for the autophagosome formation [P. falciparum survival in periods of moderate starvation in intraerythrocytic infection [Studies that explore malaria infection have analyzed the autophagy aspects in both the parasite and the host . ConcernG8) gene . The PfAormation ,148, whiormation ,149, andnfection .P. falciparum and P. vivax was reported in 1969 by De Brito and colleagues [Plasmodium from an immune response, but the host’s autophagic machinery can recognize PVM; therefore, autophagy is currently considered an immune cytosolic defense [As for the host, the first evidence of autophagic structures in liver parasitism by lleagues . Curiouslleagues ,153. It  defense ,143.P. berghei infections, it has been reported that this process is inverted, initially occurring with the recognition of PVM by LC3-II, followed by the ubiquitination and binding of autophagy receptors, which leads to direct degradation of parasites into autolysosomes [In general, during infections, the target pathogen is first ubiquitinated to then initiate xenophagy through autophagy receptors such as p62, NBR1, and NDP52, after being labeled with LC3-II . Intriguysosomes . In studysosomes ,156.P. vivax infection, the stimulation of IFN-γ to enhance the selective LAP-like process in infected hepatocytes is needed, supporting the protective role of LAP in the host’s antimalaria immunity [Plasmodium-associated autophagy-related (PAAR) response [Moreover, Walker and colleagues  demonstrimmunity , which iresponse .Plasmodium activity in infective sporozoites 3 (UIS3), a transmembrane protein that associates with LC3 on the PVM in a protein–protein intersecting to remove LC3, contributing to parasite development within hepatocytes. This explains why many parasites manage to escape autophagy, even after recruiting LC3 and treatment with rapamycin, an autophagy inducer [Despite the proven restriction of parasite replication by autophagy, evidence has shown that parasites can hijack the LAP pathway for their own benefit ,159,160.ULK1, BECN1, LC3, and mTOR were downregulated [This alteration in autophagic profile does not seem to be exclusive of the liver stage. In human PM, the autophagy-associated genes egulated ,164, andegulated ,165 and egulated ,148,162.Unlike apoptosis and autophagy, necrosis is lytic cell death, which is traditionally described as a form of ACD  . HoweverInitially, pyroptosis was described as an alternative form of apoptosis, since both involve caspase activation . HoweverThe canonical pyroptosis initiates when TLRs recognize a range of PAMPs and/or DAMPs, thus activating NF-κB, which transcriptionally regulates the expression of genes encoding pro-IL-1β, pro-IL18, and a member of the NOD-like receptor (NLR) family, NLRP3 . NLRP3 i2+ and K+) are also well-established activators of inflammasomes [NLRs detect “danger signals” of host or intracellular pathogens and recruit CASP-1 via apoptosis-associated speck-like protein containing a CARD (ASC), an adaptor protein, to trigger the assembly of inflammasomes ,174. Othmmasomes ,176. CASmmasomes .In non-canonical pyroptosis, Gram-negative bacterial lipopolysaccharides (LPS) activate human CASP-4, CASP-5 , and micYersinia-infected macrophages [Recently, a crosstalk between pyroptosis and other forms of cell death has been revealed. Wang and colleagues  therapeurophages . The invrophages  demonstrrophages ; the relrophages .P. vivax-infected patients [P. falciparum-infected patients [An important understanding is that excessive activation of immunity in malaria leads to a proinflammatory cytokines storm with a dual role in the host: on the one hand, it works to control parasites replication; on the other hand, it causes damage to organs and tissues . Much ofpatients  and the patients .P. chabaudi infection, necrosis in hepatic cells proved to be dependent on IL-α secreted by neutrophils [IL1A gene single nucleotide polymorphism (SNP) rs17561, has been implicated in higher risk of severe malaria [In experimental trophils . In addi malaria . Notably malaria   were resistant to lethal P. yoelii infection and severe malaria outcomes.The work of Gazzinelli’s group used genetic and molecular approaches to demonstrate that CASP-8, CASP-1, mouse CASP-11, and its human ortholog CASP-4 were upregulated in monocytes from malaria patients and mice malaria models . This waurine PM . Yu and urine PM  showed tNLRP1 gene are associated with malaria severity in Brazilian Amazonian patients with P. vivax [IL-1β (rs1143634) and IL-18 (rs5744256) SNPs in shaping malaria clinical outcomes [Although a role for NLRP1 in the pathogenesis of malaria has not yet been determined, one study of genetic polymorphisms suggests that SNPs  in the P. vivax . In thisoutcomes .P. berghei, thus reestablishing brain homeostasis and protecting it from neuroinflammation [Exploration of the pharmacological blockage of pyroptotic genes is of interest to avoid severe malaria. A recent report has demonstrated that immunotherapy with IL-33 along with antimalarial drugs treatment selectively inhibited the formation of NLRP3 and the release of IL-1β in microglia and intracerebral monocytes of mice infected with ammation . Certainammation  and the ammation . Taken tNETosis is a type of RN that integrates the set of strategies developed by neutrophils to combat pathogenic microorganisms . In thisNeutrophilic recognition of PAMPs and DAMPs  leads toFurthermore, histone modification and chromatin dispersion also depend on the direct activation of peptidyl-arginine deiminase 4 (PAD4) by ROS . PAD4 drAlternatively, neutrophils can release NETs in the extracellular environment through a non-conventional mechanism of NETosis. This mechanism requires factors like ROS, MPO, and NE activity, as well the destruction of neutrophils; instead, as mentioned in the subsection “Pyroptosis”, NETs are released in vesicles or neutrophil serine proteases via GSDMD cleavage ,211. It Plasmodium or tissue damage resulting in the pathogenesis of severe malaria has been brought to attention; therefore, knowledge on this relationship is gradually progressing. Neutrophils are the most abundant innate immunity cells and, differently from lymphocytes, their number generally increases in malaria infection [So far, NETosis is the only granulocyte RN described in malaria. Even though many details of NETosis mechanisms remain unknown, the role of this type of cell death in either protection against nfection ,215. Thenfection ,217, livnfection , lung innfection , and PM nfection .P. falciparum infection. The group proposed that the release of NETs would induce severe malaria pathogenesis in children, but it would confer immunoprotection in adults [P. falciparum, P. vivax, and P. malariae infections, while NETosis was correlated with parasite biomass reduction, associated with progression to severe malaria [Baker and co-workers  reportedn adults . However malaria .P. falciparum and P. berghei stimulated neutrophils to form NETs, which damaged the alveolar–capillary barrier, facilitating the infiltration of inflammatory cells and cytokines, ultimately contributing to the establishment of malaria-associated pulmonary pathologies [Data from independent groups further support the importance of NETosis in promoting inflammation during malaria infection. hologies . Boeltz hologies .However, the mechanism of NETosis activation and its pathophysiology in malaria became slightly clearer when Knackstedt and co-workers  revealedP. falciparum-iRBCs, the main inductor of NETs formation was the chemokine CXCR-4, being dependent on PAD4 histone citrullination and independent of MPO, NE, or ROS formation. Although both researchers have performed an in vitro analysis under similar conditions, the differences between them extend to the biological function of NETosis underlying malaria: while Knackstedt et al. did not observe changes in parasitemia but the increased in vivo susceptibility to severe malaria, Rodrigues et al. suggest that NETosis interfere in P. falciparum dissemination and improve host survival.Rodrigues et al.  also broPlasmodium infection.Despite controversial results, most studies agree that, in vivo, NETosis mainly presents a harmful role in the integrity of organs and tissues, whether in mice or in humans ,219,223.GPX4) gene activity, which encodes an enzyme that is indispensable for the degradation of lipid hydroperoxides [Ferroptosis is an RN form that depends on iron and amino acid metabolisms. It is characterized by overwhelming lipid peroxidation and dysregulation of homeostatic balance maintained by glutathione peroxidase 4  preferentially via transferrin receptor (TFR1) and, occasionally, divalent metal transporter 1 (DMT-1) absorbs ferrous iron (Fe2+) [3+ form is then reduced to Fe2+ in endosomes by six-transmembrane epithelial antigen of the prostate 3 (STEAP3), then transported to the cytoplasm to be used as a cofactor of cellular processes or stored as cytosolic and mitochondrial ferritin (FtMt) [2+ to Fe3+ [Iron is an essential nutrient for humans with clear biological functions in many cellular processes . Cells an (Fe2+) . The Fe3n (FtMt) . Ferriti to Fe3+ ,231  and SLC3A2 [C− ensures proper operation of GPX4 by regulating glutathione synthesis [C− [To reduce lethal accumulation of lipid peroxidation, the cell has antioxidant systems, especially in the cysteine-glutamate antiporter system Xd SLC3A2 ,236, as d SLC3A2 . Under nynthesis ,238. Theesis [C− . The majesis [C− . Besidesesis [C− ,240.At the level of transcriptional regulation, TP53 increases cell sensitivity to ferroptosis by upregulating GSL2 expression . This hiFerroptosis is suggested to be involved in cancer and neurodegenerative diseases, but its immunological functions are still poorly understood . PossiblPlasmodium needs hemoglobin to grow and develop, these parasites invade the iron-rich environment of RBCs [2+ and its liberation catalyzes the Fenton reaction [Iron is an essential nutrient for both humans and several pathogens, thereby iron status of human hosts regulates the susceptibility and the course of malaria infection ,247. Sinreaction ,249.Plasmodium species [P. falciparum than by P. vivax [Several studies have identified a substantial increase in lipid peroxidation in RBCs infected by different  species ,250,251, species ,251. In P. vivax ,250 (FigGLS2 and SLC7A11, leading to defects in ferroptotic cell death and iron accumulation. Mouse macrophages containing the S47 variant presented resistance to ferroptosis and elevated iron status, which decreased proinflammatory cytokines and increased IL-10-mediated anti-inflammatory response under exposition to malaria hemozoin [Jennis et al.  found thhemozoin  (Figure hemozoin ,253.Plasmodium development in both liver and blood stages [P. yoelii-infected hepatocytes directs these cells to lipid peroxidation and death via ferroptosis; the same occurs when cells are treated with Erastin and Sorofenib, pharmacological inhibitors of SLC7A11 (Plasmodium growth in hepatocytes, by depriving malaria parasites of iron and by the selective death of infected cells.Paradoxically, a large amount of iron provides the supply for d stages ,254. In d stages  showed t SLC7A11 D. In conCurrently, it is known that cell death pathways play critical roles in response to infections, given that the death of an infected cell is directly related to malaria immunopathology and the death of parasites, so these pathways are taken as inherent mechanisms of the immune system and a key process to understand host–parasite interaction. In this context, RCD pathways undoubtedly have the potential to become a new avenue for the development of preventive and therapeutic measures.Plasmodium infection. For instance, we know that the signaling of a specific death pathway takes place depending on different factors, such as the type of the dying cell, the species of pathogen, and the pathogen load [For such reasons, the study of cell death pathways in infectious diseases is a growing field, but there is still a lot to advance in the knowledge of the genetic, molecular, and biochemical mechanisms of cell death pathways triggered during gen load . CurrentPlasmodium that regulates cIAPs directly or indirectly? Why and how can these parasites modulate and resist extrinsic pathways, but cannot avoid mitochondrial apoptosis? In this sense, various works have pointed out cIAPs’ and BCL-2 antiapoptotic genes’ antagonists, as well as p53 agonists as potential targets for antimalarial drugs development [Apoptotic signaling is among the most common cell death pathways in malaria, although some gaps remain to be resolved in future studies. For instance, as we reviewed here, HGF/MET signaling ensures parasitized hepatocytes’ survival only at early stages of infection, which led to the idea that there is an additional mechanism that promotes cells survival at late stages. Recently, a study showed that cIAPs are upregulated during malaria infection, but how do cIAPs become upregulated in this infection? Is it elopment ,105,106.So far, differently from apoptosis, little is known about the mechanisms involved in the other RCD in malaria. Regarding autophagy, more investigations are necessary to explore the aspects related to the host–parasite interaction, such as how the autophagy machinery interact with immune response and its consequences for the host and the parasites. It is also not fully comprehended how the parasite evades the host’s autophagy, as well as how the host’s machinery recognizes the infection and prevents the growth of the parasite.P. vivax and P. ovale sporozoites. We cannot disregard other forms of cell death that have been coming up as potential therapy targets for infectious diseases. Since pyroptosis and NETosis did not have the expected effect on parasitemia control, causing serious damage to tissues and organs of the host, it would be more beneficial to inhibit them rather than to stimulate their execution. Although it has been shown that ferroptosis can limit parasitemia in the liver, we still do not know much about the immune potential of this type of RCD. It would be interesting for future investigations to consider which is the best method of prevention or treatment of malaria, whether it is by neutralizing the parasite through immunologically silent apoptosis or by inducing a setting of immunological responses through immunogenic apoptosis or ferroptosis, studying what would be the potential of these RCD forms in the elimination of liver hypnozoites in people exposed to Lastly, necroptosis—a form of RN mediated by RIPK1 and RIPK3—can be executed downstream of the same DRs that activate extrinsic apoptosis, particularly under FADD/CASP-8 deficiency or suppression, and can also be triggered by TLRs . WhereasMalaria is considered one of the major parasitic infections in humans, and different studies have added important insights in an attempt to develop new prophylactic methods and improve the already existing methods. In this scenario, studies on cell death pathways offer relevant information to achieve this goal, particularly RCD forms. Therefore, it is necessary to understand the unique characteristics of each cell death form and the factors that lead an infected cell to employ a certain pathway. In this sense, this review contributes to this major goal by providing the current state of research in this field, unraveling pathways and factors that are potential therapeutic targets for malaria, and recommending further approaches.


Plasmodium, and of the five species capable of infecting humans, infections with P. falciparum are the most severe. In addition to the parasites that infect people, there are hundreds of additional species that infect birds, reptiles, and other mammals, each exquisitely evolved to meet the specific challenges inherent to survival within their respective hosts. By comparing the unique strategies that each species has evolved, key insights into host-parasite interactions can be gained, including discoveries regarding the pathogenesis of human disease. Here, we describe the surprising observation that closely related parasites with different hosts have evolved remarkably different methods for repairing their genomes. This observation has important implications for the ability of parasites to maintain chronic infections and for the development of host immunity.Malaria remains one of the most prevalent and deadly infectious diseases of the developing world, causing approximately 228 million clinical cases and nearly half a million deaths annually. The disease is caused by protozoan parasites of the genus  Plasmodium species, we report the unexpected observation that malaria parasites that infect rodents do not display the same degree of antigen diversity as observed in Plasmodium falciparum and appear to undergo significantly less ectopic recombination. Using comparative genomics, we also identify key molecular components of the diversification process, thus shedding new light on how malaria parasites balance the maintenance of genome integrity with the requirement for continuous genetic diversification.The protozoan parasites that cause malaria infect a wide variety of vertebrate hosts, including birds, reptiles, and mammals, and the evolutionary pressures inherent to the host-parasite relationship have profoundly shaped the genomes of both host and parasite. Here, we report that these selective pressures have resulted in unexpected alterations to one of the most basic aspects of eukaryotic biology, the maintenance of genome integrity through DNA repair. Malaria parasites that infect humans continuously generate genetic diversity within their antigen-encoding gene families through frequent ectopic recombination between gene family members, a process that is a crucial feature of the persistence of malaria globally. The continuous generation of antigen diversity ensures that different parasite isolates are antigenically distinct, thus preventing extensive cross-reactive immunity and enabling parasites to maintain stable transmission within human populations. However, the molecular basis of the recombination between gene family members is not well understood. Through computational analyses of the antigen-encoding, multicopy gene families of different  Plasmodium infect a broad range of vertebrate hosts, enabling a comparative analysis of adaptations particular to each specific host. Such comparisons have revealed unexpected changes in basic aspects of cell biology, from components of transcriptional machinery , a fundamental mechanism responsible for repair of DNA double-strand breaks (DSBs). Malaria parasites depend almost entirely on homologous recombination (HR) to maintain genome integrity, despite spending most of their life cycle as haploid organisms and thus lacking the homologous chromosomes typically used for repair by HR . The loslciparum . Within lciparum . Furthere copies . In addie copies –17. This copies –, 16, 18. copies –, 20. TheP. falciparum, multigene family members in specific genomic positions were often nearly identical in different isolates of the rodent parasite Plasmodium chabaudi  with the highest-scoring alignment and then determined whether the paired sequences were located at comparable positions of their respective genomes. For single-copy housekeeping genes in both species, we found nearly universal synteny, as expected , the transcriptome data sets of Parahaemoproteus coatneyi and L. fringillinarum , and sequence data of a Hepatocystis parasite that were mined from the transcriptome of a Ugandan red colobus monkey  using the ContamFinder pipeline . Alignments were generated using the Constraint-based Multiple Alignment Tool  available through the National Library of Medicine, National Center for Biotechnology Information website. Alignments are displayed in the “compact” format to reduce space. Unaligned regions are displayed as [X] where X denotes the number of residues for a sequence in the unaligned range. Download FIG S1, PDF file, 0.2 MB.Amino acid sequence alignments for orthologs of the four components of the translesion polymerases described in the main text. Sequences for Copyright © 2020 Siao et al.2020Siao et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Taken together, our observations suggest a key role for translesion polymerases in diversification of malaria parasite antigens. In model eukaryotes, these enzymes interface with nucleotide excision , base ex31–


IntroductionPlasmodium vivax (P. vivax) malaria. Primaquine, a mainstay in the treatment of vivax malaria, may trigger episodes of acute hemolysis in patients with G6PD deficiency. The present study sought to delineate the frequency and genotypes of G6PD deficiency among patients suffering from vivax malaria infections. Glucose-6-phosphate dehydrogenase (G6PD) enzyme deficiency is the most common enzymopathy in humans, and its distribution has been historically described to be closely associated with that of malaria. North East India provides optimal conditions for transmission of malaria and bears a considerable burden of MethodsBlood specimens from 80 individuals diagnosed with vivax malaria underwent enzyme assay for G6PD deficiency. Samples with deficient phenotype underwent isolation of DNA using a genomic DNA isolation kit . The genomic DNA underwent amplification, serial denaturation, annealing, extension, final extension followed by digestion with restriction endonucleases Nla III and Fok I. The digested products were subjected to horizontal agarose electrophoresis for the separation of digested fragments. Samples without nucleotide 376 adenine→guanine (A→G) mutation were classified as G6PD B. Those with the mutation were further classified into G6PD A(+) and G6PD A(-) based on the presence of Nla III site.ResultsP. vivax malaria were found to have G6PD deficiency, of which a majority (n=24) had G6PD B genotype. Three individuals had Asparagine→Aspartic Acid mutation at position 376 (A→G), of which G6PD A(+) and G6PD A(-) were present in two and one cases, respectively.Twenty-seven out of 80 individuals (33.75%) with ConclusionG6PD deficiency was noted in about a third of patients with vivax malaria. Since primaquine therapy is contraindicated in this group of patients, there is a rationale for looking into screening patients with vivax malaria from the region prior to primaquine therapy. Further large scale studies may substantiate this and help in better genotypic and geographic characterization of G6PD deficiency in the region.  Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme deficiency, affecting an estimated 400 million people globally . G6PD is Plasmodium falciparum  invasion since the parasite requires the enzyme for its normal survival in the host cell [ P. falciparum [Plasmodium vivax (P. vivax) endemic areas have been less revealing, with a weak association between the two being reported from around the world [Approximately 160 genetic variants causing clinical deficiency of G6PD have been characterized . The geoost cell . This dehe world -9.Three G6PD genotypes are common: G6PD B, G6PD A(+), and G6PD A(-). G6PD A(+) is characterized by a mutation at nucleotide 376, resulting in an Asparagine→Aspartic Acid (Asn→Asp) mutation that increases electrophoretic mobility of the enzyme ,11. G6PDP. vivax remains the second most common cause of clinical malaria . Globally, P. vivax malaria burden has decreased from 24·5 million cases in 2000 to 14·3 million cases in 2017 on account of improved global health efforts and international funding to fight malaria [P. vivax malaria were reported in India [Plasmodium [ malaria . In 2014in India . North Ein India . In the  P. vivax malaria in this region. Considering the above, we undertook the present study to understand: (1) the frequency of G6PD deficiency in patients treated for P. vivax malaria in a tertiary care hospital in North East India, and (2) the most prevalent genotype of G6PD deficiency among the population seeking treatment for P. vivax malaria.To the best of our knowledge, no previous literature has reported the frequency and involved genotypes of G6PD deficiency in patients withThis hospital-based cross-sectional study was carried out between May 2014 and September 2016 after obtaining clearance from the institute's ethics committee (No. NEIGRIHMS/Micro/IEC-II/197/2012-13) dated 1st June 2012. P. vivax malaria through thin peripheral blood smear (PBS) using Leishman stain were included in the study. Individuals with a history of malaria or undiagnosed fever of more than 21 days duration were excluded. Patients with severe metabolic derangements and organ dysfunction evident in fasting blood glucose, serum creatinine, serum electrolytes, and liver function tests were also omitted from the study.Eighty individuals diagnosed withSample collection and processing Written consent was obtained from all participant subjects after communicating to them the exact nature of the study. Under aseptic & antiseptic precautions, 3 ml of venous blood was collected in ethylenediaminetetraacetic acid (EDTA) vial from the median cubital vein. A thin peripheral blood smear was also obtained. The EDTA mixed blood was sent to the Department of Biochemistry and a peripheral blood smear to the Department of Microbiology for further necessary diagnostic processing. After collection, samples underwent enzyme assay for G6PD deficiency by the kinetic method using reagents supplied by Coral Clinical System . G6PD in the red blood cells was released using a lysing agent . The released G6PD catalyzed the oxidation of glucose 6-phosphate with the reduction of nicotinamide adenine dinucleotide phosphate (NADP) to NADPH. The rate of reduction of NADP to NADPH was measured as an increase in absorbance at 340 nm, proportional to the G6PD activity in the sample.The EDTA samples with deficient phenotype were then centrifuged again to obtain the buffy coat and plasma. From the centrifuged sample, 50 µl buffy coat was used to isolate DNA. DNA was extracted with proteinase K digestion using a genomic DNA isolation kit . Approximately 1 μg of genomic DNA was amplified using primers 5′-GTCTTCTGGGTCAGGGAT-3′ (forward) and 5′-GGAGAAAGCTCTCTCTCC-3′ (reverse). Denaturation at 94°C for two minutes was followed by 45 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 60 seconds, and a final extension at 72°C for four minutes. The amplified products were digested with the restriction endonuclease Nla III & Fok I. The digested products were then subjected to horizontal agarose electrophoresis for the separation of digested fragments. Ethidium bromide-stained gels were then subjected to gel documentation apparatus  to study the fragments for polymorphism.The Fok I and Nla III sites were detected using the appropriate endonucleases. All the G6PD enzyme deficient samples were examined for the nucleotide 376 A→G mutation, which produces a new Fok I cleavage site. Samples without this site were classified as G6PD B. Those containing the nucleotide 376 mutation were further examined for the G6PD deficient A(-) mutation, guanine→adenine (G→A), at nucleotide 202 by restriction analysis with Nla III. The samples containing the Nla III site were designated A(-), and those without it as A(+).Data analysisData were recorded in Microsoft Excel  and were presented as counts or percentages. The prevalence of G6PD deficiency was calculated as the total number of subjects with deficient G6PD phenotype over the total of vivax malaria subjects in the study.P. vivax malaria, as diagnosed by thin PBS using Leishman stain, were recruited for the study. Out of 80 individuals, 27 (33.75%) had a phenotypic deficiency of G6PD , accounting for 88.88% of all cases with G6PD deficiency.Polymerase chain reaction (PCR) followed by agarose gel electrophoresis, as shown in Figures P. falciparum and P. vivax malaria [P. vivax malaria distribution are sparse [North East India bears a large case burden of malaria in India and is co-endemic for both  malaria -20. Viva malaria . G6PD dee sparse ,7-9,21.P. falciparum and P. vivax malaria [P. vivax malaria in our cohort had G6PD deficiency. G6PD deficiency in India has been reported to be higher in the East India zone, the Himalayan region, and populations belonging to scheduled tribes [One notable study by Kar et al. carried out among the Ao Naga tribe of North East India found that G6PD deficient female heterozygotes and male hemizygotes were protected against both  malaria . Saha et malaria . About ad tribes . Our stuG6PD B is the most common allele in all populations of G6PD deficiency, accounting for 60-80% of all cases ,21. UnliG6PD A(+) genotype corresponds to 90% enzymatic activity and is produced by mutation of adenine to guanine in position 376 resulting in the replacement of Asn by Asp . Its freP.vivax) [G6PD A(-) mutation results in 12% of normal G6PD activity and affects up to 25% of the population in Sub-Saharan Africa . In our P.vivax) ,27. DeveP.vivax) . A betteThere were certain limitations to our study. The small size recruited from a single center is the chief limitation of our study and may affect generalizability. Genotypic mapping based on demographic and geographic characteristics of the sample population would have likely yielded novel and informative results but could not be carried out due to logistic difficulties.In India, the national drug policy on malaria (2013) recommends treating vivax malaria with primaquine for 14 days but disallows its use in individuals with G6PD deficiency . AlthougPlasmodium. About a third of patients in our study with P. vivax malaria had underlying G6PD deficiency, which was observed to be quite high. G6PD B was the most common variant, although a few cases with G6PD A(+) and G6PD A(-) variants were also noted. Screening of individuals for G6PD deficiency prior to initiation of primaquine therapy may avoid potential episodes of acute hemolysis. Malaria has historically been a major cause of mortality and morbidity in the north-eastern region of India due to factors conducive to the transmission of P. vivax malaria and pave the way for consideration of guidelines for routine screening for G6PD deficiency in such patients.Further studies elucidating the geographic and genotypic characteristics of patients from the region may reveal or refute the association of G6PD deficiency and protection against severe