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This study on severe malarial anemia (SMA: Hb < 6.0 g/dL), a leading global cause of childhood morbidity and mortality, compares the entire expressed whole blood host transcriptome between Kenyan children (3-48 mos.) with non-SMA (Hb ≥ 6.0 g/dL, n  = 39) and SMA ( n  = 18). Differential expression analyses reveal 1403 up-regulated and 279 down-regulated transcripts in SMA, signifying impairments in host inflammasome activation, cell death, and innate immune and cellular stress responses. Immune cell profiling shows decreased memory responses, antigen presentation, and immediate pathogen clearance, suggesting an immature/improperly regulated immune response in SMA. Module repertoire analysis of blood-specific gene signatures identifies up-regulation of erythroid genes, enhanced neutrophil activation, and impaired inflammatory responses in SMA. Enrichment analyses converge on disruptions in cellular homeostasis and regulatory pathways for the ubiquitin-proteasome system, autophagy, and heme metabolism. Pathway analyses highlight activation in response to hypoxic conditions [Hypoxia Inducible Factor (HIF)−1 target and Reactive Oxygen Species (ROS) signaling] as a central theme in SMA. These signaling pathways are also top-ranking in protein abundance measures and a Ugandan SMA cohort with available transcriptomic data. Targeted RNA-Seq validation shows strong concordance with our entire expressed transcriptome data. These findings identify key molecular themes in SMA pathogenesis, offering potential targets for new malaria therapies. Here, the authors analyze the blood transcriptome of Kenyan children with severe malarial anemia and observe impaired immune responses and molecular activation of hypoxia and reactive oxygen species networks, providing insight into disease pathogenesis.

This prospective cohort study explored the association between two upstream IFN-γ variants (rs2069709: G > T and rs2069705: A > G) and hazard factors for malaria outcomes in a longitudinal cohort of children ( n  = 941, 3–36 mos.), followed for three years. The impact of age, sex, previous malaria exposure, HIV1 infection, and sickle-cell genotypes (HbAA, HbAS, and HbSS) was also investigated. Reduced malaria episodes were associated with older age at enrollment [HR = 0.957 (95% CI = 0.953–0.961) per month, P  < 2.2e-16], HIV1 infection [0.687 (0.545–0.866), P  = 0.001], being female [0.910 (0.859–0.964), P  = 0.040], and HbAS [0.823 (0.754–0.898), P  = 0.005]. The GA/TA diplotype [0.376 (0.230–0.614), P  = 0.002] also reduced the hazard of malaria, while TA haplotype increased susceptibility [1.749 (1.159–2.640), P  = 0.029]. Factors protecting against the development of SMA [Hemoglobin (Hb < 6.0 g/dL)] included older age [0.927 (0.913–0.942) per month, P  < 2.2e-16], previous malaria episodes [0.576 (0.542–0.614, P  = 9.5e-32)], HbAS [0.553 (0.400-0.766), P  = 0.015]. The rs2069705AG genotype increased the hazard of SMA [1.697 (1.002–2.875), P  = 0.042]. Reduced hazard of mortality was observed for older children [0.898 (0.857–0.941), P  < 2.2e-16], while a higher hazard was present in HIV-infected children [12.475 (6.380-24.392), P  < 2.2e-16], and in those with HbSS [6.341 (1.944–20.686), P  = 0.007]. The GG haplotype increased the mortality hazard [1.817 (0.936–3.527), P  = 0.078]. The results here highlight critical factors influencing the hazard of malaria, SMA, and mortality.

Background Epstein Barr virus (EBV)-associated endemic Burkitt’s Lymphoma pediatric cancer is associated with morbidity and mortality among children resident in holoendemic Plasmodium falciparum regions in western Kenya. P. falciparum exerts strong selection pressure on sickle cell trait (SCT), alpha thalassemia (-α 3.7 /αα), glucose-6-phosphate dehydrogenase (G6PD), and merozoite surface protein 2 (MSP-2) variants (FC27, 3D7) that confer reduced malarial disease severity. The current study tested the hypothesis that SCT, (-α 3.7 /αα), G6PD mutation and (MSP-2) variants (FC27, 3D7) are associated with an early age of EBV acquisition. Methods Data on infant EBV infection status (< 6 and ≥ 6–12 months of age) was abstracted from a previous longitudinal study. Archived infant DNA (n = 81) and mothers DNA (n = 70) samples were used for genotyping hemoglobinopathies and MSP-2. The presence of MSP-2 genotypes in maternal DNA samples was used to indicate infant in-utero malarial exposure. Genetic variants were determined by TaqMan assays or standard PCR. Group differences were determined by Chi-square or Fisher’s analysis. Bivariate regression modeling was used to determine the relationship between the carriage of genetic variants and EBV acquisition. Results EBV acquisition for infants < 6 months was not associated with -α 3.7 /αα (OR = 1.824, P  = 0.354), SCT (OR = 0.897, P  = 0.881), or G6PD [Viangchan (871G > A)/Chinese (1024 C > T) (OR = 2.614, P =  0.212)] and [Union (1360 C > T)/Kaiping (1388G > A) (OR = 0.321, P  = 0.295)]. There was no relationship between EBV acquisition and in-utero exposure to either FC27 (OR = 0.922, P =  0.914) or 3D7 (OR = 0.933, P =  0.921). In addition, EBV acquisition in infants ≥ 6–12 months also showed no association with -α 3.7 /αα (OR = 0.681, P  = 0.442), SCT (OR = 0.513, P  = 0.305), G6PD [(Viangchan (871G > A)/Chinese (1024 C > T) (OR = 0.640, P  = 0.677)], [Mahidol (487G > A)/Coimbra (592 C > T) (OR = 0.948, P  = 0.940)], [(Union (1360 C > T)/Kaiping (1388G > A) (OR = 1.221, P  = 0.768)], African A (OR = 0.278, P  = 0.257)], or in utero exposure to either FC27 (OR = 0.780, P  = 0.662) or 3D7 (OR = 0.549, P  = 0.241). Conclusion Although hemoglobinopathies (-α 3.7 /αα, SCT, and G6PD mutations) and in-utero exposure to MSP-2 were not associated with EBV acquisition in infants 0–12 months, novel G6PD variants were discovered in the population from western Kenya. To establish that the known and novel hemoglobinopathies, and in utero MSP-2 exposure do not confer susceptibility to EBV, future studies with larger sample sizes from multiple sites adopting genome-wide analysis are required.

Background Plasmodium falciparum malaria is a leading cause of pediatric morbidity and mortality in holoendemic transmission areas. Severe malarial anemia [SMA, hemoglobin (Hb) < 5.0 g/dL in children] is the most common clinical manifestation of severe malaria in such regions. Although innate immune response genes are known to influence the development of SMA, the role of natural killer (NK) cells in malaria pathogenesis remains largely undefined. As such, we examined the impact of genetic variation in the gene encoding a primary NK cell receptor, natural cytotoxicity-triggering receptor 3 (NCR3), on the occurrence of malaria and SMA episodes over time. Methods Susceptibility to malaria, SMA, and all-cause mortality was determined in carriers of NCR3 genetic variants (i.e., rs2736191:C > G and rs11575837:C > T) and their haplotypes. The prospective observational study was conducted over a 36 mos. follow-up period in a cohort of children ( n  = 1,515, aged 1.9–40 mos.) residing in a holoendemic P. falciparum transmission region, Siaya, Kenya. Results Poisson regression modeling, controlling for anemia-promoting covariates, revealed a significantly increased risk of malaria in carriers of the homozygous mutant allele genotype (TT) for rs11575837 after multiple test correction [Incidence rate ratio (IRR) = 1.540, 95% CI = 1.114–2.129, P  = 0.009]. Increased risk of SMA was observed for rs2736191 in children who inherited the CG genotype (IRR = 1.269, 95% CI = 1.009–1.597, P  = 0.041) and in the additive model (presence of 1 or 2 copies) (IRR = 1.198, 95% CI = 1.030–1.393, P  = 0.019), but was not significant after multiple test correction. Modeling of the haplotypes revealed that the CC haplotype had a significant additive effect for protection against SMA (i.e., reduced risk for development of SMA) after multiple test correction (IRR = 0.823, 95% CI = 0.711–0.952, P  = 0.009). Although increased susceptibility to SMA was present in carriers of the GC haplotype (IRR = 1.276, 95% CI = 1.030–1.581, P  = 0.026) with an additive effect (IRR = 1.182, 95% CI = 1.018–1.372, P  = 0.029), the results did not remain significant after multiple test correction. None of the NCR3 genotypes or haplotypes were associated with all-cause mortality. Conclusions Variation in NCR3 alters susceptibility to malaria and SMA during the acquisition of naturally-acquired malarial immunity. These results highlight the importance of NK cells in the innate immune response to malaria.

Background Sulfadoxine-pyrimethamine (SP) is the only anti-malarial drug formulation approved for intermittent preventive treatment in pregnancy (IPTp). However, mutations in the Plasmodium falciparum dhfr ( Pfdhfr ) and dhps ( Pfdhps ) genes confer resistance to pyrimethamine and sulfadoxine, respectively. Here, the frequencies of SP resistance-associated mutations from 2005 to 2018 were compared in samples from Kenyan children with malaria residing in a holoendemic transmission region. Methods Partial sequences of the Pfdhfr and Pfdhps genes were amplified and sequenced from samples collected in 2005 (n = 81), 2010 (n = 95), 2017 (n = 43), and 2018 (n = 55). The frequency of known mutations conferring resistance to pyrimethamine and sulfadoxine were estimated and compared. Since artemisinin-based combination therapy (ACT) is the current first-line treatment for malaria, the presence of mutations in the propeller domain of P. falciparum kelch13 gene ( Pfk13 ) linked to ACT-delayed parasite clearance was studied in the 2017/18 samples. Results Among other changes, the point mutation of Pfdhps S436 H increased in frequency from undetectable in 2005 to 28% in 2017/18. Triple Pfdhfr mutant allele (C IRN I) increased in frequency from 84% in 2005 to 95% in 2017/18, while the frequency of Pfdhfr double mutant alleles declined (allele C I C N I from 29% in 2005 to 6% in 2017/18, and CN RN I from 9% in 2005 to undetectable in 2010 and 2017/18). Thus, a multilocus Pfdhfr / Pfdhps genotype with six mutations ( HGE AA/C IRN I), including Pfdhps S436 H , increased in frequency from 2010 to 2017/18. Although none of the mutations associated with ACT-delayed parasite clearance was observed, the Pfk13 mutation A578S, the most widespread Pfk13 SNP found in Africa, was detected in low frequency (2.04%). Conclusions There were changes in SP resistance mutant allele frequencies, including an increase in the Pfdhps S436 H . Although these patterns seem consistent with directional selection due to drug pressure, there is a lack of information to determine the actual cause of such changes. These results suggest incorporating molecular surveillance of Pfdhfr / Pfdhps mutations in the context of SP efficacy studies for intermittent preventive treatment in pregnancy (IPTp).

Cyclooxygenase-2 [(COX-2) or prostaglandin endoperoxide H2 synthase-2 (PTGS-2)] induces the production of prostaglandins as part of the host-immune response to infections. Although a number of studies have demonstrated the effects of COX-2 promoter variants on autoimmune and inflammatory diseases, their role in malaria remains undefined. As such, we investigated the relationship between four COX-2 promoter variants (COX-2 −512 C > T, −608 T > C, −765 G > C, and −1195 A > G) and susceptibility to malaria and severe malarial anemia (SMA) upon enrollment and longitudinally over a 36-month follow-up period. All-cause mortality was also explored. The investigation was carried out in children (n = 1081, age; 2–70 months) residing in a holoendemic Plasmodium falciparum transmission region of western Kenya. At enrollment, genotypes/haplotypes (controlling for anemia-promoting covariates) did not reveal any strong effects on susceptibility to either malaria or SMA. Longitudinal analyses showed decreased malaria episodes in children who inherited the −608 CC mutant allele (RR = 0.746, P = 1.811 × 10−4) and -512C/-608T/-765G/-1195G (CTGG) haplotype (RR = 0.856, P = 0.011), and increased risk in TTCA haplotype carriers (RR = 1.115, P = 0.026). Over the follow-up period, inheritance of the rare TTCG haplotype was associated with enhanced susceptibility to both malaria (RR = 1.608, P = 0.016) and SMA (RR = 5.714, P = 0.004), while carriage of the rare TTGG haplotype increased the risk of malaria (RR = 1.755, P = 0.007), SMA (RR = 8.706, P = 3.97 × 10−4), and all-cause mortality (HR = 110.000, P = 0.001). Collectively, these results show that SNP variations in the COX-2 promoter, and their inherited combinations, are associated with the longitudinal risk of malaria, SMA, and all-cause mortality among children living in a high transmission area for P. falciparum.

Background Naturally-acquired immunity to Plasmodium falciparum malaria develops after several episodes of infection. Fc gamma receptors (FcγRs) bind to immunoglobulin G (IgG) antibodies and mediate phagocytosis of opsonized microbes, thereby, linking humoral and cellular immunity. FcγR polymorphisms influence binding affinity to IgGs and consequently, can influence clinical malaria outcomes. Specifically, variations in FcγRIIA -131Arg/His, FcγRIIIA-176F/V and FcγRIIIB-NA1/NA2 modulate immune responses through altered binding preferences to IgGs and immune complexes. Differential binding, in turn, changes ability of immune cells to respond to infection through production of inflammatory mediators during P. falciparum infection. Methods We determined the association between haplotypes of FcγRIIA-131Arg/His, FcγRIIIA-176F/V and FcγRIIIB-NA1/NA2 variants and severe malarial anemia (SMA; hemoglobin < 6.0 g/dL, any density parasitemia) in children ( n  = 274; aged 6–36 months) presenting for their first hospital visit with P. falciparum malaria in a holoendemic transmission region of western Kenya. FcγRIIA-131Arg/His and FcγRIIIA-176F/V genotypes were determined using TaqMan® SNP genotyping, while FcγRIIIBNA1/NA2 genotypes were determined using restriction fragment length polymorphism. Hematological and parasitological indices were measured in all study participants. Results Carriage of FcγRIIA-131Arg/FcγRIIIA-176F/FcγRIIIBNA2 haplotype was associated with susceptibility to SMA (OR = 1.70; 95% CI; 1.02–2.93; P =  0.036), while the FcγRIIA-131His/ FcγRIIIA-176F/ FcγRIIIB NA1 haplotype was marginally associated with enhanced susceptibility to SMA (OR: 1.80, 95% CI; 0.98–3.30, P =  0.057) and higher levels of parasitemia ( P =  0.009). Individual genotypes of FcγRIIA-131Arg/His, FcγRIIIA-176F/V and FcγRIIIB-NA1/NA2 were not associated with susceptibility to SMA. Conclusion The study revealed that haplotypes of FcγRs are important in conditioning susceptibility to SMA in immune-naive children from P. falciparum holoendemic region of western Kenya.

Plasmodium falciparum infections remain among the leading causes of morbidity and mortality in holoendemic transmission areas. Located within region 5q31.1, the colony-stimulating factor 2 gene ( CSF2 ) encodes granulocyte–macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor that mediates host immune responses. Since the effect of CSF2 variation on malaria pathogenesis remains unreported, we investigated the impact of two genetic variants in the 5q31.1 gene region flanking CSF2 :g-7032 G > A (rs168681:G > A) and CSF2 :g.64544T > C (rs246835:T > C) on the rate and timing of malaria and severe malarial anemia (SMA, Hb < 5.0 g/dL) episodes over 36 months of follow-up. Children ( n  = 1654, aged 2–70 months) were recruited from a holoendemic P. falciparum transmission area of western Kenya. Decreased incidence rate ratio (IRR) for malaria was conferred by inheritance of the CSF2 :g.64544 TC genotype ( P  = 0.0277) and CSF2 AC/GC diplotype ( P  = 0.0015). Increased IRR for malaria was observed in carriers of the CSF2 AT/GC diplotype ( P  = 0.0237), while the inheritance of the CSF2 AT haplotype increased the IRR for SMA ( P  = 0.0166). A model estimating the longitudinal risk of malaria showed decreased hazard rates among CSF2 AC haplotype carriers ( P  = 0.0045). Investigation of all-cause mortality revealed that inheritance of the GA genotype at CSF2 :g-7032 increased the risk of mortality ( P  = 0.0315). Higher risk of SMA and all-cause mortality were observed in younger children ( P  < 0.0001 and P  = 0.0015), HIV-1(+) individuals ( P  < 0.0001 and P  < 0.0001), and carriers of HbSS ( P  = 0.0342 and P  = 0.0019). Results from this holoendemic P. falciparum area show that variation in gene region 5q31.1 influences susceptibility to malaria, SMA, and mortality, as does age, HIV-1 status, and inheritance of HbSS.

Background Severe malarial anemia (SMA) is a leading cause of malaria-related morbidity and mortality in children. The genetic factors that influence development of SMA and inefficient erythropoiesis, a central pathogenic feature of SMA, are only partially understood. Methods We performed a pilot Genome-wide Association Study (GWAS) on children with Plasmodium falciparum . The GWAS was performed using the Illumina® Infinium® HD Super Assay in conjunction with Illumina’s® Human Omni2.5-8v1 BeadChip (with > 2.45 M markers). Data were analyzed using single SNP logistic regression analysis with an additive model of inheritance controlling for covariates. Results from our pilot global genomics study identified that variation in interleukin (IL)-7 was associated with enhanced risk of SMA. To validate this finding, we investigated the relationship between genotypes and/or haplotypes of two single nucleotide polymorphisms (SNPs) in IL7 [72194 T/C and − 2440 A/G] and susceptibility to both SMA and inefficient erythropoiesis [i.e., reticulocyte production index (RPI) < 2.0 in anemic children (Hb < 11.0 g/dL). Children presenting with P. falciparum malaria (< 3 years, n  = 883) were stratified into two groups: Uncomplicated malaria (UM, n  = 718) and SMA ( n  = 165). Results Regression modeling, controlling for anemia-related confounders, revealed that carriage of the TC genotype at position 72194 T/C was associated with enhanced susceptibility to inefficient erythropoiesis (OR = 1.90; 95% CI 1.09–3.30; P  = 0.02) as was homozygous CC (OR 5.14; 95% CI = 1.20–21.99; P  = 0.03). Consistent with this finding, individuals with the CA (72194C/−2440A) haplotype had an increased risk of inefficient erythropoiesis (OR = 1.90; 95% CI = 1.10–3.30; P  = 0.02), whereas TA haplotype carriers had marginal protection against inefficient erythropoiesis (OR = 0.24; 95% CI = 0.06–1.21; P  = 0.05). These observations were supported by Cochran-Armitage trend test for inefficient erythropoiesis (CA > TA > CG; P  < 0.01). Although none of the genotype and/or haplotypic variants were significantly associated with SMA, the direction of the risk profiles were consistent with the erythropoiesis results. Conclusion Taken together, variation in IL7 is associated with erythropoietic responses in children with falciparum malaria, a central physiological feature contributing to development of SMA.

Severe malarial anemia (SMA, Hb < 6.0 g/dL) is a leading cause of childhood morbidity and mortality in holoendemic Plasmodium falciparum transmission zones. This study explored the entire expressed human transcriptome in whole blood from 66 Kenyan children with non-SMA (Hb ≥ 6.0 g/dL, n = 41) and SMA (n = 25), focusing on host immune response networks. RNA-seq analysis revealed 6862 differentially expressed genes, with equally distributed up-and down-regulated genes, indicating a complex host immune response. Deconvolution analyses uncovered leukocytic immune profiles indicative of a diminished antigenic response, reduced immune priming, and polarization toward cellular repair in SMA. Weighted gene co-expression network analysis revealed that immune-regulated processes are central molecular distinctions between non-SMA and SMA. A top dysregulated immune response signaling network in SMA was the HSP60-HSP70-TLR2/4 signaling pathway, indicating altered pathogen recognition, innate immune activation, stress responses, and antigen recognition. Validation with high-throughput gene expression from a separate cohort of Kenyan children (n = 50) with varying severities of malarial anemia (n = 38 non-SMA and n = 12 SMA) confirmed the RNA-seq findings. Proteomic analyses in 35 children with matched transcript and protein abundance (n = 19 non-SMA and n = 16 SMA) confirmed dysregulation in the HSP60-HSP70-TLR2/4 signaling pathway. Additionally, glutamine transporter and glutamine synthetase genes were differentially expressed, indicating altered glutamine metabolism in SMA. This comprehensive analysis underscores complex immune dysregulation and novel pathogenic features in SMA.