Exploring the influence of alpha thalassemia on antibody responses to Plasmodium falciparum infections in Ghana

Exploring the influence of alpha thalassemia on antibody responses to Plasmodium falciparum infections in Ghana | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Exploring the influence of alpha thalassemia on antibody responses to Plasmodium falciparum infections in Ghana Augustine Boakye Donkor, Frank George Bernasko, Anisa Abdulai, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8231440/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Alpha thalassemia, a common inherited haemoglobin disorder in Africa, has long been associated with protection against Plasmodium falciparum malaria, but the exact mechanisms remain unclear. This study explored whether this protective effect is linked to antibody responses against key P. falciparum blood-stage antigens, including Apical Membrane Antigen-1 (P f AMA-1), Erythrocyte-Binding Antigen-175 (P f EBA-175), Glutamate-Rich Protein (P f GLURP), Merozoite Surface Protein-1 (P f MSP-1), and Reticulocyte-Binding Protein homologue 5 (P f RH5). We conducted a cross-sectional study of 220 malaria symptomatic individuals aged 1–80 years. Alpha thalassemia was genotyped using multiplex PCR, while Plasmodium falciparum infections were confirmed by microscopy and nested PCR. Using indirect Enzyme-Linked Immunosorbent Assay (ELISA) we measured antigen-specific IgG levels and analysed their patterns across genotypes, infection status, season, gender, and age categories. Heterozygotes consistently mounted the strongest antibody responses, with PfAMA-1 showing the highest median concentration (98.63 ng/mL) and homozygous recessive individuals had the lowest responses, particularly to PfRH5 (17.66 ng/mL). Across all five antigens, antibody levels differed significantly between genotypes (P < 0.0001). These findings reveal that heterozygous alpha thalassemia may enhance immune defense by boosting antibody production. These findings provide a deeper understanding of malaria protection and offer valuable clues for innovative malaria control strategies. Health sciences/Diseases Biological sciences/Immunology Biological sciences/Microbiology Alpha thalassemia Plasmodium falciparum Antibody response Antigens Malaria immunity Hemoglobinopathy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Despite decades of ongoing control efforts, malaria persists globally as a major public health burden. In 2023, malaria accounted for approximately 263 million clinical cases and nearly 597,000 fatalities across the world 1 . Plasmodium falciparum remains the deadliest malaria-causing species, escalating severe disease outcomes and high mortality, particularly in sub-Saharan Africa 2 . P. falciparum immunity is a multifaceted complex process involving the innate and adaptive arms of the immune system. Humoral immunity is particularly pivotal, orchestrating antibody-mediated mechanisms that are essential for the control and clearance of blood-stage infections 3 , 4 in natural and vaccine immunities. A number of host genetic variants have been reported to influence malaria humoral immunity. Investigated genetic variants include polymorphisms in Interleukin-4 promoter region polymorphism at position − 524 (IL4-524), Hemoglobin subunit beta (HBB), Interleukin-4 (IL-4), Interleukin-12 (IL-12), Tumor Necrosis Factor (TNF), Lymphotoxin alpha(LTA), Natural Cytotoxicity Triggering Receptor 3 (NCR3)and Fc Gamma Receptor IIA (FCGR2A ) 5 – 8 . Of relevance to the present study is alpha thalassemia. Alpha thalassemia is a hereditary hemoglobinopathy caused by deletions in one (1) or more α-globin genes. It is prevalent in malaria-endemic regions, suggesting a selective advantage against severe malaria 9 . The prevalences of heterozygous and homozygous α-thalassemia traits are 19.0–39.0 and 6.8–8.2 respectively in different regions of Ghana 10 , 11 . Previous studies have demonstrated that alpha thalassemia modifies red blood cell physiology, affecting parasite invasion and replication 1213 . However, beyond these known haematological effects, emerging evidence suggests that alpha thalassemia may also influence immune responses to Plasmodium infections; antibody production and function 1415 . Antibodies naturally elicited by the host in response to P. falciparum merozoite invasion proteins may mediate protection by inhibiting parasite invasion, promoting opsonic phagocytosis, or facilitating complement activation 16 . Despite this, the relationship between alpha thalassemia and humoral immunity to P. falciparum remains poorly understood. To protect the host against clinical malaria, alpha thalassemia may oppose blood stage infections by asexual parasites. Promising malaria vaccine candidate antigens associated with the asexual parasite include Apical Membrane Antigen-1 ( Pf AMA-1) 17 , Merozoite Surface Protein-1 ( Pf MSP-1) 18 , Reticulocyte-Binding Protein Homologue 5 ( Pf RH5) (limited antigenic diversity) 19 , Erythrocyte-Binding Antigen-175 ( Pf EBA-175) 20 , and Glutamate-Rich Protein ( Pf GLURP) 21 , for erythrocyte invasion 22 , 23 . This study investigated antibody responses against asexual P. falciparum antigens ( Pf AMA-1, Pf EBA-175, Pf GLURP, Pf MSP-1, and Pf RH5) in individuals from Ghana with varying alpha thalassemia genotypes. Understanding this relationship could provide valuable insights into the role host genetic factors play in shaping malaria immunity and the design of targeted therapies. RESULTS Demographic and Clinical Characteristics by α-Thalassemia Genotype Individuals with the heterozygous (−α/αα) genotype had a median age (range) of 16.0 years (3.0–80.0 years) compared to 13.0 years (1.0–33.0) for homozygous recessive (−α/−α) and 10.0 years (1.0–60.0) for wild-type (αα/αα). Median body temperatures (range) of participants were similar at 36.50 (35.90–37.10) for wild type and 36.60 (32.80–37.90) for heterozygous and 36.60 (36.20–37.10) homozygous recessive. The distribution of individuals across age categories varied by genotype. Females outnumbered males in all genotype groups: αα/αα: females; 28 / 53 total (52.8%) and males (Table 1 ). Table 1 Demographic and Clinical Characteristics of Study Participants by α-Thalassemia Genotype α-thalassemia genotypes αα/αα -α/αα -α/-α P value Age, yrs; median (range) 10.0 (1.0–60.0) 16.0 (3.0–80.0) 13.0 (1.0–33.0) Temperature, o C; median (range) 36.50 (35.90–37.10) 36.60 (32.80–37.90) 36.60 (36.20–37.10) Age categories, yrs; n/N (%) < 0.0001 20 yrs 6/53 (11.3) 48/144 (33.3) 4/23 (17.4) Malaria prevalence (microscopy),n/N (%) 18/53 (34.0) 52/144 (36.1) 10/23 (43.5) 0.7265 Malaria prevalence (PCR), n/N(%) 36/53(67.92) 109/144(75.7) 18/23(78.3) 0.4841 Gender, n/N (%) Male 25/53 (47.2) 60/144 (41.7) 9/23 (39.1) 0.7484 Female 28/53 (52.8) 83/144 (58.3) 14/23 (60.9) Female/Male ratio 28/25 (1.12) 83/60 (1.38) 14/9 (1.56) Seroprevalence of P. falciparum IgG, n/N (%) P f AMA1 5/53 (9.4) 138/144 (95.8) 16/23 (69.6) < 0.0001 P f EBA175 14/53 (26.4) 67/144 (46.5) 6/23 (26.1) 0.014 P f GLURP 28/53 (52.8) 105/144 (72.9) 15/23 (65.2) 0.028 P f MSP1 12/53 (22.6) 74/144 (51.4) 5/23 (21.7) < 0.0001 P f RH5 14/53 (26.4) 38/144 (26.4) 4/23 (17.4) 0.644 αα/αα = wildtype/homozygous dominant, -α/αα = heterozygote, -α/-α = homozygous recessive Microscopy-detected P. falciparum infections were found in 18/53 (34.0%) of αα/αα individuals, 52/144 (36.1%) of − α/αα individuals and 10/23 (43.5%) of − α/−α individuals. Malaria prevalence by PCR was 36/53 (67.9%) among the wild type(αα/αα) participants, 109/144 (75.7%) among heterozygous (−α/αα) individuals and 18/23 (78.3%) among the homozygous recessive (−α/−α) individuals. There were notable genotype-related differences in the proportion of individuals seropositive for P. falciparum antigens though not statistically significant (Table 1 ). Antibody Responses to P. falciparum Antigens and their Association with Alpha Thalassemia Genotypes Among P. falciparum -positive individuals, Pf AMA1-specific IgG levels were highest in individuals with the -α/αα genotype, 98.63 ng/mL (21.81–325.60 ng/mL), followed by -α/-α , 73.88 ng/mL (12.43–290.40 ng/mL), and αα/αα 60.77 ng/mL (28.62–91.86). Pf EBA175-specific IgG concentrations were similarly elevated, with the highest median values in αα/αα , 34.06 ng/mL (15.62–132.2 ng/mL), closely followed by -α/αα and -α/-α genotypes. Pf GLURP-specific IgG levels were highest in -α/αα individuals, 35.18 ng/mL (10.93–254.90), with a relatively lower response observed in -α/-α , 20.70 ng/mL (11.82–116.90 ng/mL). Pf MSP1-specific IgG concentrations showed a reversed pattern, with the -α/-α group having the highest median level, 57.59 ng/mL (31.64–96.83 ng/mL), followed by αα/αα , 51.97 ng/mL (20.09–153.4 ng/mL). Pf RH5-specific IgG concentrations were relatively comparable across genotypes among P. falciparum -positive individuals, with median values ranging narrowly from 17.66 ng/mL to 17.95 ng/mL. In contrast, among malaria-negative individuals, IgG levels specific to the relevant antigens were consistently lower, with minimal variation observed between genotypes. Statistical comparisons using the Kruskal-Wallis test demonstrated statistically significant differences in antigen-specific IgG concentrations across α-thalassemia genotypes for all antigens (p < 0.0001), indicating that both thalassemia genotype and malaria status significantly influence antibody levels (Table 2 ). Table 2 Antibody Responses to Plasmodium falciparum Antigens and their Association with Alpha Thalassemia Genotypes. P. falciparum IgG concentration, median, (Range) ng/mL α-thalassemia genotypes αα/αα -α/αα -α/-α Kruskal-Wallis test p-value P f AMA1 133.1 < 0.0001 Pos 60.77 (28.62–91.86) 98.63 (21.81–325.60) 73.88 (12.43–290.40) Neg 8.07 (0.69–20.31) 5.39 (1.87–18.39) 1.32 (0.68–8.49) P f EBA175 148.4 < 0.0001 Pos 34.06 (15.62–132.2) 30.89 (2.99–239.2) 19.73 (14.31-56.00) Neg 4.87 (2.49–13.47) 6.52 (1.88–13.81) 6.23 (2.99–12.63) P f GLURP 132.7 < 0.0001 Pos 26.71 (11.25–204.80) 35.18 (10.93–254.90) 20.70 (11.82–116.90) Neg 7.46 (5.28–10.36) 7.22 (3.58–10.44) 6.24 (4.53–9.07) P f MSP1 132.7 < 0.0001 Pos 51.97 (20.09–153.4) 36.46 (19.36–261.0) 57.59 (31.64–96.83) Neg 8.81 (3.13–18.45) 9.73 (2.02–18.20) 8.01 (4.29–17.49) P f RH5 128.4 < 0.0001 Pos 17.87 (15.97–24.86) 17.95 (15.71–34.77) 17.66 (15.80-17.83) Neg 10.09 (6.25–14.98) 11.26 (4.89–15.54) 11.61 (6.84–15.61) Pos = seropositive, Neg = seronegative, αα/αα = wildtype, -α/αα = heterozygote, -α/-α = homozygous recessive Distribution of IgG antibody concentrations stratified by α-thalassemia genotypes and P. falciparum infection status Pf AMA1 IgG levels were generally highest in P. falciparum -seropositive individuals across all genotypes (Fig. 2 a). P. falciparum -seropositive individuals had higher Pf EBA175 IgG responses across all genotypes compared to P. falciparum -seronegative individuals (Fig. 2 b). Similar trends were observed with higher Pf GLURP antibody levels in P. falciparum -seropositive individuals (Fig. 2 c). Pf MSP1 antibody responses remained higher in P. falciparum -seropositive individuals regardless of α-thalassemia genotype (Fig. 2 d). Pf RH5 antibody responses remained higher in P. falciparum -seropositive individuals regardless of α-thalassemia genotype (Fig. 2 e). No statistically significant genotype-associated differences in IgG concentrations were detected (Fig. 2 ). Antibody concentration distribution Relative to P. falciparum Detection by PCR . Pf AMA-1 (p = 0.6802) and Pf EBA175 (p = 0.7496) IgG levels were comparable between PCR⁺ and PCR⁻ groups but showed no significant variation between groups (Fig. 3 a and 3 b). Pf GLURP IgG levels showed no significant variation between diagnostic groups (p = 0.3822) (Fig. 3 c). Pf MSP1 IgG concentrations were similarly distributed among PCR⁺ and PCR⁻ individuals (p = 0.7196) (Fig. 3 d). Pf RH5 IgG levels also did not differ significantly between the two groups (p = 0.1817) (Fig. 3 e). (Fig. 3 ). Analysis of antibody concentrations and seroprevalence by sex Across all antigens, seropositive individuals exhibit markedly elevated IgG levels. Pf RH5 elicits the lowest IgG responses of all the antigens tested. Overall, males had a slightly higher IgG levels compared to females for all antigens, although this was not statistically significant. For Pf AMA-1 159/220 (72.27%) of the total population were seropositive, with 73.02% of males and 71.28% of females. For Pf EBA-175, 87/220 (39.55%) were seropositive, with 43.62% of males and 36.51% of females. For Pf GLURP, 148/220 (67.27%) of individuals were seropositive, with 68.09% of males and 66.67% of females. For Pf MSP-1, 91/220 (41.36%) were seropositive, with 43.62% of males and 39.68% of females. For Pf RH5, 56/220 (25.45%) participants were seropositive, with 25.53% of males and 25.40% of females testing positive. There was no statistical difference between the weighted antibody concentration between males and females ( Pf AMA1: p = 0.1520, Pf EBA175: p = 0.2706, Pf GLURP: p = 0.2247, Pf MSP1: p = 0.9863, Pf RH5: p = 0.8338) (Fig. 4 ). Variation in Antibody Concentration Across Different Age Categories Generally, age-dependent increase in IgG concentration was observed for Pf AMA1, P fEBA175, Pf GLURP, and Pf MSP1. For Pf AMA-1 (χ2 = 54.814, p = 0.0001, Dunn’s Pairwise Comparison), statistically significant differences existed between the IgG levels of participants less than 5 years and all other age groups (p < 0.01) and between participants 5–10 yrs and greater than 20 yrs (p = 0.0001); other comparisons are not statistically significant. For Pf EBA-175 antigen, χ2 = 14.398, p = 0.0061, Dunn’s Pairwise Comparison: 15–20 yrs with less than 5 years (p = 0.0227), more than 20 yrs with less than 5 years (p = 0.0308), Other comparisons are not statistically significant. For Pf GLURP (χ2 = 5.254, p = 0.2622, Dunn’s Pairwise Comparison), there were no statistically significant pairwise differences. For Pf MSP1 (χ2 = 8.147, p = 0.0864, Dunn’s Pairwise Comparison), there were no statistically significant pairwise differences. For Pf RH5 (χ2 = 13.702, p = 0.0083, Dunn’s Pairwise Comparison), there was a statistically significant difference between the IgG concentrations between individually 5–10 years and those greater than 20 years (p = 0.0123), as were as 5–10 years with 15–20 years (p = 0.0389), Other comparisons were not statistically significant (Fig. 5 ). Seasonal Variation in Antibody Concentrations Antibody levels are consistently higher in seropositive individuals across all antigens and seasons. IgG levels for all antigens in seropositive individuals were higher in the rainy season (high transmission period) than in the dry season. Pf EBA175 (p = 0.0024) showed significant seasonal variation. However, Pf AMA1 (p = 0.0998), Pf GLURP (p = 0.1395), Pf MSP1(p = 0.0892) and Pf RH5 (p = 0.4947) showed no significant seasonal variation. Seronegative individuals exhibit low IgG concentrations with no significant seasonal variation (Fig. 6 ). Discussion The analysis of IgG antibody levels against Plasmodium falciparum antigens reveals distinct patterns of immune responses. Plasmodium falciparum Apical Membrane Antigen 1 ( Pf AMA-1) elicited the highest median antibody concentration with a wide range. Pf AMA-1 is a highly immunogenic antigen expressed during the merozoite stage of the parasite. Its higher antibody response aligns with findings by Srinivasan et al 24 who demonstrated Pf AMA-1's role in inducing protective immunity through inhibition of merozoite invasion. The variability in antibody responses likely reflects differences in malaria exposure, immune history, and/ or genetic polymorphisms in the antigen 25 . Pf GLURP, Pf MSP-1, and Pf EBA-175 showed moderate antibody responses that are consistent with previous studies that emphasize these antigens' roles in immune recognition during merozoite invasion 26 . Pf RH5 recorded the lowest median antibody level, with varied individual responses but relatively stable levels among all genotypes. Pf RH5 specifically is a well-preserved antigen with less genetic variability, making it a potential target for vaccines, despite lower antibody responses compared to Pf AMA-1 27,28 . The findings also reaffirm Pf AMA-1’s strong immunogenicity supporting its vaccine candidacy. Pf GLURP, Pf MSP-1, and Pf EBA-175, while eliciting moderate responses, may complement a multi-antigen vaccine approach to enhance immune protection. Pf MSP-1 antibody levels were highest among the homozygous recessive individuals, aligning repeated exposure and the conserved capacity of this antigen and its peculiar role in merozoite invasion 29 . For all other antigens, consistently lower antibody levels in homozygous recessive individuals suggest reduced immune competence against malaria. Possible explanations include increased malaria susceptibility in these individuals that leads to higher parasite burdens, which can impair immune function and antibody production due to immune exhaustion 3031 . Additionally, homozygous recessive individuals have structural changes in their erythrocytes that interfere with parasite invasion and antigen presentation, leading to weaker immune responses 32 33 . However, Wild type individuals had intermediate antibody levels across most antigens. This suggests that while they may not have the enhanced immune advantages seen in heterozygous individuals, they also do not suffer from the impaired immunity observed in homozygous recessive individuals 34 . Heterozygous individuals consistently exhibited the highest antibody levels by genotype, across all antigens, except Pf MSP1, where the homozygous recessive group mounted a similar response. The higher antibody levels could be due to heterozygous individuals experiencing more controlled parasite replication, allowing for repeated immune stimulation and stronger antibody responses, and this suggests that heterozygosity may confer a selective immune advantage. Indeed, this aligns with previous studies on α-thalassemia that suggest that heterozygous individuals have a protective advantage against severe malaria 35 . Alternatively, alpha thalassemia could influence how these antigens are processed and presented by immune cells, leading to more robust B-cell activation and antibody production 36 . Again, the higher antibody responses in heterozygous individuals imply that host genetic factors could influence vaccine efficacy, and so future malaria vaccines should consider genetic diversity in target populations 37 . Genetic surveillance for alpha thalassemia mutations could improve malaria risk assessments by identifying populations at risk of severe disease 3839 . Furthermore, heterozygous individuals exhibiting the highest antibody levels, possibly could be due to a reduced parasite load, as depicted by Hamre et al 40 . This is further evidence that heterozygous mutation provides a survival advantage by enhancing immune-mediated clearance of malaria parasites 41 32 . However, despite Pf RH5 exhibiting a lower response, it was the most relatively stable antigen among all three genotypes. Indeed, it has been shown to be a promising vaccine candidate, as vaccine-induced Pf RH5 antibodies can block parasite invasion 42 , 43 , 44 . For all five antibodies, malaria prevalence among antibody-positive individuals was steadily high, suggesting that individuals who mounted antibody responses also had detectable active malaria infections by PCR. The strong association suggests that antibody positivity is a reliable marker of current malaria exposure and reflects their strong immunogenicity and potential use as reliable biomarkers of active and recent infection 45 . Notably, around 23–26% of antibody-positive individuals were P. falciparum -negative, implying past malaria exposure, with circulating antibodies persisting after parasite clearance or sub-microscopic infections potentially missed by standard diagnostics, especially microscopy, as well as immune memory responses due to repeated exposures in endemic regions 46 . Persistent antibody levels in P. falciparum -negative individuals highlight the challenge of distinguishing current from past infections, which is crucial for evaluating transmission dynamics 47 , reinforcing the utility of antigen-specific antibody detection as a proxy for malaria exposure and possibly recent infection 48 . However, the presence of antibodies in P. falciparum -negative individuals draw caution about using serology alone for malaria diagnosis. Seasonal variations impact participant availability and disease prevalence, particularly during the rainy season when access to study sites becomes difficult and malaria transmission increases 4950 . The study found that only the Pf EBA-175 antigen showed a significant seasonal difference, with higher antibody levels during the rainy season. This suggests that Pf EBA-175 antibody responses are influenced by malaria transmission intensity, which is higher during the rainy season when mosquito breeding increases. Similar studies have shown that Pf EBA-175 is associated with acute malaria infection, and higher transmission seasons lead to increased immune stimulation 51 . This also highlights Pf EBA-175's potential as a marker for recent infection and seasonal transmission. In contrast, no significant seasonal differences were observed for the other four antigens, suggesting these antibodies are long-lasting and less affected by seasonal transmission fluctuations 5253 . Again, P f EBA-175 could serve as a serological marker for recent malaria exposure and seasonal transmission intensity 5455 . The study found that the largest age group was children aged 5–10 years, followed closely by adults over 20 years, with children under five comprising the smallest group. Antibody analysis revealed that children under five had significantly lower levels of Pf AMA-1, Pf EBA-175, and Pf RH5, indicating that malaria immunity develops with age due to repeated exposure 36 , 56 .In contrast, Pf MSP-1 antibody levels were relatively stable across all age groups, likely due to frequent immune boosting of immunity with repeated infections 27 57 . Importantly, children under 5 years should remain a primary target for malaria prevention strategies, as their lower antibody levels put them at higher risk of severe disease 5859 . While the study offers meaningful insight into how α-thalassemia may influence antibody responses, a few limitations deserve mention. We were unable to include the –α⁴.² (4.2 kb) deletion, leaving part of the α-thalassemia landscape uncharacterized. Additionally, the distribution of genotypes was uneven, with a relatively small number of wild-type participants (n = 53) and homozygous individuals (n = 23) compared with the larger heterozygous group (n = 144). These differences in sample size naturally affect the precision of comparisons across genotypes and should be considered when interpreting the overall patterns observed. Conclusions This study demonstrates that all the five P. falciparum antigens studied were significantly associated with meaningful differences across the different thalassemia genotypes, highlighting the role of host genetics in shaping immune responses to malaria. Elucidating the complex interplay between thalassemia and malaria immunity offers valuable insights that could refine malaria control strategies. Such knowledge has the potential to inform the design of more targeted interventions for individuals with heightened susceptibility, optimize sero-surveillance approaches, and guide vaccine development. By integrating these findings into public health policies, malaria prevention efforts could be strengthened, particularly in vulnerable populations disproportionately affected by hemoglobinopathies. Methods Study design and study site In 2023, a cross-sectional study was undertaken in Anyakpor (5°46′51.96″N, 0°35′12.84″E), a rural community in the Ada East District of Ghana's Greater Accra Region, focusing on individuals of varying ages presenting with symptoms suggestive of malaria. Situated roughly 5 kilometers west of Ada Foah in southern Ghana, Anyakpor lies in a region with low malaria transmission. The community experiences a dry equatorial climate, with average temperatures ranging between 23 °C and 28 °C year-round, occasionally rising to 33 °C. Rainfall in the area is seasonal, typically occurring from April to June and again from September to November. The local landscape is dominated by coastal savannah vegetation (Figure 1). Study population and data collection Participants in the study had previously undergone thalassemia genotyping by multiplex PCR. A total of 220 individuals, aged 1 and 80 years and presenting with malaria symptoms, were enrolled during the dry season (January to March 2023, N=130 ) and the rainy/malaria transmission season (April to June 2023, N=90 ). Whole blood samples were collected into EDTA tubes and centrifuged at 720 g at 4 °C for 10 minutes; the plasma was promptly aliquoted and stored at –80 °C. Microscopic detection of P . falciparum Microscopic examination was carried out by WHO certified microscopists following standardized protocols 60 for malaria diagnosis. The procedure has previously been described by Amoah et al 61 . PCR detection of P . falciparum Detection of the mitochondrial cox 3 gene of P. falciparum was performed using nested PCR. In the initial amplification (nest 1), 2 µL of extracted DNA was added to a 10 µL reaction mixture containing 1X OneTaq standard buffer, 0.25 µM of each primer (mtu F and mtu R), 0.20 mM dNTPs, 2.08 mM MgCl₂, and 1 unit of OneTaq DNA polymerase (New England Biolabs, USA). For the second amplification (nest 2), 1 µL of the nest 1 product (1:10) was added to another 10 µL reaction mixture with 0.20 µM species-specific primers (MtNst_falF and MtNst_falR). Both PCR rounds (nests 1 and 2) began with an initial denaturation at 94 °C for 2 minutes, followed by 35 cycles of denaturation at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds in nest 1 and at 58 °C for 30 seconds in nest 2. Extension occurred at 68 °C, lasting 1 minute 40 seconds for the first round and 30 seconds for the second. Each reaction concluded with a final elongation at 68 °C for 5 minutes. To validate the assay, positive and negative controls were included in every run. The amplified fragments were then separated by electrophoresis on a 2% agarose gel stained with ethidium bromide, run at 120 volts for 40 minutes, and visualized using a UV transilluminator and gel documentation system. Determination of Alpha Thalassemia genotype Multiplex PCR was used to detect the 3.7 kb deletion in the α-globin gene, following the method described by Liu et al (2000) 62 . The assay employs a forward primer that binds to a conserved region of the template DNA, while multiple reverse primers anneal to distinct complementary sites on the opposite strand. The following primers were utilised: Forward primer (3.7F): AAGTCCACCCCTTCCTTCCTCACC Reverse primer 1 (3.7R1): ATGAGAGAAATGTTCTGGCACCTGCAC Reverse primer 2 (3.7R2): TCCATCCCCTCCTCCCGCCCCTGCCTT Measurement of Antibody Responses using Indirect Enzyme‑Linked Immunosorbent Assay (ELISA) Total immunoglobulin G (IgG) antibody responses against Apical Membrane Antigen-1 (P f AMA-1), Merozoite Surface Protein-1 (P f MSP-1), Reticulocyte-Binding Protein Homologue 5 (PfRH5), Erythrocyte-Binding Antigen-175 (P f EBA-175), and Glutamate-Rich Protein (P f GLURP) of P. falciparum were measured using indirect ELISA as previously described by 63 64 . Briefly, 96-well NUNC Maxisorp ELISA plates were coated with 1 µg of P f MSP-1, P f RH5, and P f GLURP antigen per well, 0.5 µg/well of P f AMA-1, and 20 µg/well of P f EBA-175 (100 µL/well) in phosphate-buffered saline (PBS, pH 7.2) and incubated overnight at 4 °C. Plasma samples were diluted (1:200). Polyclonal IgG - Purified 500 mg BP055 (The Binding Site, UK) was used as the standard. Positive controls (pooled plasma from seropositive individuals) and negative controls (malaria-naïve donors) were added. Plates were washed and blocked. Subsequently, 100 µL/well of goat anti-human IgG conjugated to horseradish peroxidase (HRP) (1:3000) was added and incubated for 1 hour. The enzymatic reaction was initiated with 3,3’5,5’-tetramethylbenzidine (TMB) substrate for 10 minutes. The reaction was stopped with 100 µL of 0.2 M sulfuric acid. Optical density (OD) was measured at 450 nm using a Multiskan FC plate reader (Thermo Scientific, USA). Statistical Analysis Raw data was entered into Microsoft Excel® 2019. Optical Density (OD) values were adjusted and antibody concentrations extrapolated based on ODs of recombinant purified polyclonal IgG (BP055, The Binding Site, UK) using ADAMSEL software (Malaria research, EU). Descriptive statistics were generated using STATA version 14 and GraphPad Prism version 9. Kruskal–Wallis and Dunn’s pairwise tests were used to compared antibody responses by α-thalassemia genotypes, P. falciparum infection status, sex, season, and age groups. Statistical significance was defined as p < 0.05. Ethical Considerations Ethical approval for this study was granted by the Ethical Protocol Review Committee of the College of Health Sciences, University of Ghana (EPRC-IRB 00006220), and the Ethics Review Committee of the Ghana Health Service (GHS-ERC 021/07/23). Prior to recruitment, written informed consent was obtained from all participants or their legal guardians. Participant’s data was always kept private. All methods were performed in accordance with relevant guidelines and regulations outlined in the Declaration of Helsinki. Availability of Data and Materials The data generated and/ or utilized in this study are accessible from the corresponding authors upon reasonable inquiry. Declarations Acknowledgements We thank the participants for their cooperation and participation. Author Contributions ABD conceived, designed and developed the study protocol with supervision and collaboration of YAA, LEA and KKA. SSK, EAB, RAA, RD, CKMA and ABD performed laboratory assays and assisted in the data analysis. FGB, FKA, KAK, and NASE performed the data management, statistical analysis and data interpretation. All authors read and approved the final manuscript. Competing interests The authors declare no competing interests. Consent for publication Not applicable Funding The authors were supported by grants from the West African Genetic Medicine Centre (WAGMC) of the University of Ghana and the National Institute of Health (D43 TW 011513). 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Rapid Detection of A-Thalassaemia Deletions and a-Globin Gene Triplication by Multiplex Polymerase Chain Reactions . (2000). Acquah, F. K. et al. Antibody responses to two new Lactococcus lactis-produced recombinant Pfs48/45 and Pfs230 proteins increase with age in malaria patients living in the Central Region of Ghana. Malar J 16 , (2017). Kwapong, S. S. et al. Mosquito bites and stage-specific antibody responses against Plasmodium falciparum in southern Ghana. Malar J 22 , (2023). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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16:09:36","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144791,"visible":true,"origin":"","legend":"","description":"","filename":"2837e86a38f0428db2298de7cbbb45de1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8231440/v1/424cc620332636ee2ba54276.xml"},{"id":98871373,"identity":"bf1ae10a-8d4b-4689-af43-19118f5c092d","added_by":"auto","created_at":"2025-12-23 12:01:01","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":168703,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8231440/v1/4a0f16c3eca8a70a0912d420.html"},{"id":99308971,"identity":"53e40875-93d8-4f3e-9b4c-b7d4e9684653","added_by":"auto","created_at":"2025-12-31 16:09:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":241607,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMap of Ghana Showing Study sites: Anyakpor, Kpalsogu, Pagaza and Konongo. The base map for the study site depiction was sourced from \u003c/strong\u003e\u003ca href=\"https://ghana-mission.co.in/mapofghana/\"\u003e\u003cstrong\u003ehttps://ghana-mission.co.in/mapofghana/\u003c/strong\u003e\u003c/a\u003e\u003cstrong\u003eand modified using Adobe Photoshop (Version 7.0.1)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe log-scaled scatter plots depict IgG antibody concentrations against the five malaria antigens, stratified by α-thalassemia genotype and serostatus. The analyses compare antibody response levels across different genotypes and examine the distribution of responses between seropositive and seronegative individuals within each genotype group. a: \u003cem\u003ePf\u003c/em\u003eAMA1 IgG, b: \u003cem\u003ePf\u003c/em\u003eEBA175 IgG, c: \u003cem\u003ePf\u003c/em\u003eGLURP IgG, d: \u003cem\u003ePf\u003c/em\u003eMSP1 IgG, e: \u003cem\u003ePf\u003c/em\u003eRH5 IgG. Blue circles = αα/αα (wild type), Red squares = -α/αα (heterozygous), Green triangles = -α/-α (homozygous), Sero\u003csup\u003e+\u003c/sup\u003e = seropositive individuals, and Sero\u003csup\u003e−\u003c/sup\u003e = Seronegative individuals. The absence of bars indicates no statistically significant difference\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8231440/v1/dfd4d98db5f2d60fbd92af06.png"},{"id":99309384,"identity":"56e36c79-9631-47f1-bd97-1af47f9ec8e2","added_by":"auto","created_at":"2025-12-31 16:10:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":129739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of IgG antibody concentrations stratified by α-thalassemia genotypes and serological status\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe scatter plots on a log scale showing IgG antibody concentrations to five \u003cem\u003eP. falciparum\u003c/em\u003e antigens, comparing PCR-positive and PCR-negative individuals. \u003cstrong\u003ea:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eAMA1 IgG, \u003cstrong\u003eb:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eEBA175 IgG, \u003cstrong\u003ec:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eGLURP IgG, \u003cstrong\u003ed:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eMSP1 IgG,\u003cstrong\u003e e:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eRH5 IgG. Blue dots = PCR\u003csup\u003e+\u003c/sup\u003e=Positive individuals, red dots = PCR\u003csup\u003e- \u003c/sup\u003e= PCR negative individuals, ns = not significant.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8231440/v1/686fea4adbb0308deeb4e897.png"},{"id":98871352,"identity":"1af030b4-dc18-413f-acfa-c7c7ce09f668","added_by":"auto","created_at":"2025-12-23 12:01:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":71891,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibody Concentration distribution based on PCR detection of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePlasmodium falciparum\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe log-scaled scatter plots compare IgG concentrations to the five \u003cem\u003eP. falciparum\u003c/em\u003e antigens, stratified by gender (male and female) and serostatus. \u003cstrong\u003ea:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eAMA1 IgG, \u003cstrong\u003eb:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eEBA175 IgG, \u003cstrong\u003ec:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eGLURP IgG, \u003cstrong\u003ed:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eMSP1 IgG,\u003cstrong\u003e e:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eRH5 IgG. Blue circles = Sero\u003csup\u003e+\u003c/sup\u003e = seropositive males, red squares = Sero\u003csup\u003e+ \u003c/sup\u003e= seropositive females, green triangles = Sero\u003csup\u003e− \u003c/sup\u003e=Seronegative males, Purple inverted triangles = Sero\u003csup\u003e−\u003c/sup\u003e =Seronegative females. The absence of bars indicates no statistically significant difference.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8231440/v1/650247760b8deb0b8f9c0dc1.png"},{"id":98871356,"identity":"223724e4-8e1d-4cff-b544-ceff01e8fbd1","added_by":"auto","created_at":"2025-12-23 12:01:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":70557,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibody concentration distribution across Gender\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe log-scaled scatter plots compare IgG concentrations to the five \u003cem\u003eP. falciparum\u003c/em\u003e antigens, stratified by different age categories. \u003cstrong\u003ea:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eAMA1 IgG, \u003cstrong\u003eb:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eEBA175 IgG, \u003cstrong\u003ec:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eGLURP IgG, \u003cstrong\u003ed:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eMSP1 IgG,\u003cstrong\u003e e:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eRH5 IgG, Blue circles: \u0026lt; 5 yrs, red squares: 5-10 yrs, green triangles: 11-15 yrs Purple inverted triangles: 16-20 yrs, orange diamonds: \u0026gt; 20 yrs. The absence of bars indicates no statistically significant difference\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8231440/v1/bd85e64da718bb3a77fd5b9d.png"},{"id":98871359,"identity":"7e2009d9-928d-4ee7-a79b-ed2d043e941a","added_by":"auto","created_at":"2025-12-23 12:01:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":152101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibody Concentration distribution across age categories\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese log-scaled scatter plots compare IgG concentrations to the five \u003cem\u003eP. falciparum\u003c/em\u003e antigens, stratified by different malaria seasons (rainy and dry) and serostatus. \u003cstrong\u003ea:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eAMA1 IgG, \u003cstrong\u003eb:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eEBA175 IgG, \u003cstrong\u003ec:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eGLURP IgG, \u003cstrong\u003ed:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eMSP1 IgG,\u003cstrong\u003e e:\u003c/strong\u003e \u003cem\u003ePf\u003c/em\u003eRH5 IgG. blue circles= Sero\u003csup\u003e+ \u003c/sup\u003eRainy: Seropositive individuals during the rainy season. Red squares= Sero\u003csup\u003e+\u003c/sup\u003e Dry = Seropositive individuals during the dry season. Green triangles: Sero\u003csup\u003e-\u003c/sup\u003e Rainy = Seronegative individuals during the rainy season. Purple diamonds: Sero\u003csup\u003e-\u003c/sup\u003e Dry Seronegative individuals during the dry season. The absence of bars indicates no statistically significant difference\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8231440/v1/4da72c4d6e11086cda0674d3.png"},{"id":99308866,"identity":"cc3e1d84-086a-41c5-b4e1-f838217bd235","added_by":"auto","created_at":"2025-12-31 16:09:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":115005,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibody concentration distribution across different seasons\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8231440/v1/b4ff3aeeeef3611c3c7bf0d1.png"},{"id":105444748,"identity":"cda3539c-6db4-4c39-9ed1-10ed866270c3","added_by":"auto","created_at":"2026-03-26 06:57:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2353345,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8231440/v1/390bc957-79b2-4dbe-a1c4-9cfd7a98c8e6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring the influence of alpha thalassemia on antibody responses to Plasmodium falciparum infections in Ghana","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDespite decades of ongoing control efforts, malaria persists globally as a major public health burden. In 2023, malaria accounted for approximately 263\u0026nbsp;million clinical cases and nearly 597,000 fatalities across the world\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePlasmodium falciparum\u003c/em\u003e remains the deadliest malaria-causing species, escalating severe disease outcomes and high mortality, particularly in sub-Saharan Africa \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eP. falciparum\u003c/em\u003e immunity is a multifaceted complex process involving the innate and adaptive arms of the immune system. Humoral immunity is particularly pivotal, orchestrating antibody-mediated mechanisms that are essential for the control and clearance of blood-stage infections\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e in natural and vaccine immunities. A number of host genetic variants have been reported to influence malaria humoral immunity. Investigated genetic variants include polymorphisms in Interleukin-4 promoter region polymorphism at position \u0026minus;\u0026thinsp;524 (IL4-524), Hemoglobin subunit beta (HBB), Interleukin-4 (IL-4), Interleukin-12 (IL-12), Tumor Necrosis Factor (TNF), Lymphotoxin alpha(LTA), Natural Cytotoxicity Triggering Receptor 3 (NCR3)and Fc Gamma Receptor IIA \u003cem\u003e(FCGR2A\u003c/em\u003e)\u003csup\u003e\u003cem\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Of relevance to the present study is alpha thalassemia.\u003c/p\u003e \u003cp\u003eAlpha thalassemia is a hereditary hemoglobinopathy caused by deletions in one (1) or more α-globin genes. It is prevalent in malaria-endemic regions, suggesting a selective advantage against severe malaria\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The prevalences of heterozygous and homozygous α-thalassemia traits are 19.0\u0026ndash;39.0 and 6.8\u0026ndash;8.2 respectively in different regions of Ghana\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Previous studies have demonstrated that alpha thalassemia modifies red blood cell physiology, affecting parasite invasion and replication \u003csup\u003e1213\u003c/sup\u003e. However, beyond these known haematological effects, emerging evidence suggests that alpha thalassemia may also influence immune responses to \u003cem\u003ePlasmodium\u003c/em\u003e infections; antibody production and function \u003csup\u003e1415\u003c/sup\u003e. Antibodies naturally elicited by the host in response to \u003cem\u003eP. falciparum\u003c/em\u003e merozoite invasion proteins may mediate protection by inhibiting parasite invasion, promoting opsonic phagocytosis, or facilitating complement activation \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Despite this, the relationship between alpha thalassemia and humoral immunity to \u003cem\u003eP. falciparum\u003c/em\u003e remains poorly understood.\u003c/p\u003e \u003cp\u003eTo protect the host against clinical malaria, alpha thalassemia may oppose blood stage infections by asexual parasites. Promising malaria vaccine candidate antigens associated with the asexual parasite include Apical Membrane Antigen-1 (\u003cem\u003ePf\u003c/em\u003eAMA-1)\u003csup\u003e17\u003c/sup\u003e, Merozoite Surface Protein-1 (\u003cem\u003ePf\u003c/em\u003eMSP-1)\u003csup\u003e18\u003c/sup\u003e, Reticulocyte-Binding Protein Homologue 5 (\u003cem\u003ePf\u003c/em\u003eRH5) (limited antigenic diversity)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, Erythrocyte-Binding Antigen-175 (\u003cem\u003ePf\u003c/em\u003eEBA-175)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and Glutamate-Rich Protein (\u003cem\u003ePf\u003c/em\u003eGLURP)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, for erythrocyte invasion \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study investigated antibody responses against asexual \u003cem\u003eP. falciparum\u003c/em\u003e antigens (\u003cem\u003ePf\u003c/em\u003eAMA-1, \u003cem\u003ePf\u003c/em\u003eEBA-175, \u003cem\u003ePf\u003c/em\u003eGLURP, \u003cem\u003ePf\u003c/em\u003eMSP-1, and \u003cem\u003ePf\u003c/em\u003eRH5) in individuals from Ghana with varying alpha thalassemia genotypes. Understanding this relationship could provide valuable insights into the role host genetic factors play in shaping malaria immunity and the design of targeted therapies.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDemographic and Clinical Characteristics by α-Thalassemia Genotype\u003c/h2\u003e \u003cp\u003eIndividuals with the heterozygous (\u0026minus;α/αα) genotype had a median age (range) of 16.0 years (3.0\u0026ndash;80.0 years) compared to 13.0 years (1.0\u0026ndash;33.0) for homozygous recessive (\u0026minus;α/\u0026minus;α) and 10.0 years (1.0\u0026ndash;60.0) for wild-type (αα/αα). Median body temperatures (range) of participants were similar at 36.50 (35.90\u0026ndash;37.10) for wild type and 36.60 (32.80\u0026ndash;37.90) for heterozygous and 36.60 (36.20\u0026ndash;37.10) homozygous recessive. The distribution of individuals across age categories varied by genotype. Females outnumbered males in all genotype groups: αα/αα: females; 28 / 53 total (52.8%) and males (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic and Clinical Characteristics of Study Participants by α-Thalassemia Genotype\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eα-thalassemia genotypes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eαα/αα\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-α/αα\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-α/-α\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge, yrs; median (range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.0 (1.0\u0026ndash;60.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16.0 (3.0\u0026ndash;80.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13.0 (1.0\u0026ndash;33.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature, \u003csup\u003eo\u003c/sup\u003eC; median (range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.50 (35.90\u0026ndash;37.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.60 (32.80\u0026ndash;37.90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36.60 (36.20\u0026ndash;37.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge categories, yrs; n/N (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;5 yrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7/53 (13.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3/144 (2.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5/23 (21.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u0026ndash;10 yrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23/53 (43.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e39/144 (27.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3/23 (13.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u0026ndash;15 yrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12/53 (22.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29/144 (20.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7/23 (30.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u0026ndash;20 yrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5/53 (9.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25/144 (17.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4/23 (17.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20 yrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6/53 (11.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e48/144 (33.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4/23 (17.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMalaria prevalence (microscopy),n/N (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18/53 (34.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e52/144 (36.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10/23 (43.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.7265\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMalaria prevalence (PCR), n/N(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36/53(67.92)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e109/144(75.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18/23(78.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.4841\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender, n/N (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25/53 (47.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60/144 (41.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9/23 (39.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.7484\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e28/53 (52.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e83/144 (58.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14/23 (60.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale/Male ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e28/25 (1.12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e83/60 (1.38)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14/9 (1.56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeroprevalence of \u003cem\u003eP. falciparum\u003c/em\u003e IgG, n/N (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003cem\u003ef\u003c/em\u003eAMA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5/53 (9.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e138/144 (95.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16/23 (69.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003cem\u003ef\u003c/em\u003eEBA175\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14/53 (26.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e67/144 (46.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6/23 (26.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003cem\u003ef\u003c/em\u003eGLURP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e28/53 (52.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e105/144 (72.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15/23 (65.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003cem\u003ef\u003c/em\u003eMSP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12/53 (22.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e74/144 (51.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5/23 (21.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003cem\u003ef\u003c/em\u003eRH5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14/53 (26.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e38/144 (26.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4/23 (17.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.644\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eαα/αα\u0026thinsp;=\u0026thinsp;wildtype/homozygous dominant, -α/αα\u0026thinsp;=\u0026thinsp;heterozygote, -α/-α\u0026thinsp;=\u0026thinsp;homozygous recessive\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMicroscopy-detected \u003cem\u003eP. falciparum\u003c/em\u003e infections were found in 18/53 (34.0%) of αα/αα individuals, 52/144 (36.1%) of\u0026thinsp;\u0026minus;\u0026thinsp;α/αα individuals and 10/23 (43.5%) of\u0026thinsp;\u0026minus;\u0026thinsp;α/\u0026minus;α individuals. Malaria prevalence by PCR was 36/53 (67.9%) among the wild type(αα/αα) participants, 109/144 (75.7%) among heterozygous (\u0026minus;α/αα) individuals and 18/23 (78.3%) among the homozygous recessive (\u0026minus;α/\u0026minus;α) individuals. There were notable genotype-related differences in the proportion of individuals seropositive for \u003cem\u003eP. falciparum\u003c/em\u003e antigens though not statistically significant (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibody Responses to\u003c/b\u003e \u003cb\u003eP. falciparum\u003c/b\u003e \u003cb\u003eAntigens and their Association with Alpha Thalassemia Genotypes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmong \u003cem\u003eP. falciparum\u003c/em\u003e-positive individuals, \u003cem\u003ePf\u003c/em\u003eAMA1-specific IgG levels were highest in individuals with the \u003cem\u003e-α/αα\u003c/em\u003e genotype, 98.63 ng/mL (21.81\u0026ndash;325.60 ng/mL), followed by \u003cem\u003e-α/-α\u003c/em\u003e, 73.88 ng/mL (12.43\u0026ndash;290.40 ng/mL), and \u003cem\u003eαα/αα\u003c/em\u003e 60.77 ng/mL (28.62\u0026ndash;91.86). \u003cem\u003ePf\u003c/em\u003eEBA175-specific IgG concentrations were similarly elevated, with the highest median values in \u003cem\u003eαα/αα\u003c/em\u003e, 34.06 ng/mL (15.62\u0026ndash;132.2 ng/mL), closely followed by \u003cem\u003e-α/αα\u003c/em\u003e and \u003cem\u003e-α/-α\u003c/em\u003e genotypes. \u003cem\u003ePf\u003c/em\u003eGLURP-specific IgG levels were highest in \u003cem\u003e-α/αα\u003c/em\u003e individuals, 35.18 ng/mL (10.93\u0026ndash;254.90), with a relatively lower response observed in \u003cem\u003e-α/-α\u003c/em\u003e, 20.70 ng/mL (11.82\u0026ndash;116.90 ng/mL). \u003cem\u003ePf\u003c/em\u003eMSP1-specific IgG concentrations showed a reversed pattern, with the \u003cem\u003e-α/-α\u003c/em\u003e group having the highest median level, 57.59 ng/mL (31.64\u0026ndash;96.83 ng/mL), followed by \u003cem\u003eαα/αα\u003c/em\u003e, 51.97 ng/mL (20.09\u0026ndash;153.4 ng/mL). \u003cem\u003ePf\u003c/em\u003eRH5-specific IgG concentrations were relatively comparable across genotypes among \u003cem\u003eP. falciparum\u003c/em\u003e-positive individuals, with median values ranging narrowly from 17.66 ng/mL to 17.95 ng/mL.\u003c/p\u003e \u003cp\u003eIn contrast, among malaria-negative individuals, IgG levels specific to the relevant antigens were consistently lower, with minimal variation observed between genotypes. Statistical comparisons using the Kruskal-Wallis test demonstrated statistically significant differences in antigen-specific IgG concentrations across α-thalassemia genotypes for all antigens (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating that both thalassemia genotype and malaria status significantly influence antibody levels (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibody Responses to \u003cem\u003ePlasmodium falciparum\u003c/em\u003e Antigens and their Association with Alpha Thalassemia Genotypes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP. falciparum\u003c/em\u003e IgG concentration, median, (Range) ng/mL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα-thalassemia genotypes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eαα/αα\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-α/αα\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-α/-α\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKruskal-Wallis test\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003cb\u003ef\u003c/b\u003e\u003cb\u003eAMA1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e133.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.77\u003c/p\u003e \u003cp\u003e(28.62\u0026ndash;91.86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.63\u003c/p\u003e \u003cp\u003e(21.81\u0026ndash;325.60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e73.88\u003c/p\u003e \u003cp\u003e(12.43\u0026ndash;290.40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.07\u003c/p\u003e \u003cp\u003e(0.69\u0026ndash;20.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.39\u003c/p\u003e \u003cp\u003e(1.87\u0026ndash;18.39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.32\u003c/p\u003e \u003cp\u003e(0.68\u0026ndash;8.49)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003cb\u003ef\u003c/b\u003e\u003cb\u003eEBA175\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e148.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.06\u003c/p\u003e \u003cp\u003e(15.62\u0026ndash;132.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.89\u003c/p\u003e \u003cp\u003e(2.99\u0026ndash;239.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.73\u003c/p\u003e \u003cp\u003e(14.31-56.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.87\u003c/p\u003e \u003cp\u003e(2.49\u0026ndash;13.47)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.52\u003c/p\u003e \u003cp\u003e(1.88\u0026ndash;13.81)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.23\u003c/p\u003e \u003cp\u003e(2.99\u0026ndash;12.63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003cb\u003ef\u003c/b\u003e\u003cb\u003eGLURP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e132.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.71\u003c/p\u003e \u003cp\u003e(11.25\u0026ndash;204.80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.18\u003c/p\u003e \u003cp\u003e(10.93\u0026ndash;254.90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.70\u003c/p\u003e \u003cp\u003e(11.82\u0026ndash;116.90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.46\u003c/p\u003e \u003cp\u003e(5.28\u0026ndash;10.36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.22\u003c/p\u003e \u003cp\u003e(3.58\u0026ndash;10.44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.24\u003c/p\u003e \u003cp\u003e(4.53\u0026ndash;9.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003cb\u003ef\u003c/b\u003e\u003cb\u003eMSP1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e132.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.97\u003c/p\u003e \u003cp\u003e(20.09\u0026ndash;153.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.46\u003c/p\u003e \u003cp\u003e(19.36\u0026ndash;261.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57.59\u003c/p\u003e \u003cp\u003e(31.64\u0026ndash;96.83)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.81\u003c/p\u003e \u003cp\u003e(3.13\u0026ndash;18.45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.73\u003c/p\u003e \u003cp\u003e(2.02\u0026ndash;18.20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.01\u003c/p\u003e \u003cp\u003e(4.29\u0026ndash;17.49)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003cb\u003ef\u003c/b\u003e\u003cb\u003eRH5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e128.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.87\u003c/p\u003e \u003cp\u003e(15.97\u0026ndash;24.86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.95\u003c/p\u003e \u003cp\u003e(15.71\u0026ndash;34.77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.66\u003c/p\u003e \u003cp\u003e(15.80-17.83)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.09\u003c/p\u003e \u003cp\u003e(6.25\u0026ndash;14.98)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.26\u003c/p\u003e \u003cp\u003e(4.89\u0026ndash;15.54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.61\u003c/p\u003e \u003cp\u003e(6.84\u0026ndash;15.61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003ePos\u0026thinsp;=\u0026thinsp;seropositive, Neg\u0026thinsp;=\u0026thinsp;seronegative, αα/αα\u0026thinsp;=\u0026thinsp;wildtype, -α/αα\u0026thinsp;=\u0026thinsp;heterozygote, -α/-α\u0026thinsp;=\u0026thinsp;homozygous recessive\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDistribution of IgG antibody concentrations stratified by α-thalassemia genotypes and\u003c/b\u003e \u003cb\u003eP. falciparum\u003c/b\u003e \u003cb\u003einfection status\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003ePf\u003c/em\u003eAMA1 IgG levels were generally highest in \u003cem\u003eP. falciparum\u003c/em\u003e-seropositive individuals across all genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). \u003cem\u003eP. falciparum\u003c/em\u003e-seropositive individuals had higher \u003cem\u003ePf\u003c/em\u003eEBA175 IgG responses across all genotypes compared to \u003cem\u003eP. falciparum\u003c/em\u003e-seronegative individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Similar trends were observed with higher \u003cem\u003ePf\u003c/em\u003eGLURP antibody levels in \u003cem\u003eP. falciparum\u003c/em\u003e-seropositive individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). \u003cem\u003ePf\u003c/em\u003eMSP1 antibody responses remained higher in \u003cem\u003eP. falciparum\u003c/em\u003e-seropositive individuals regardless of α-thalassemia genotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). \u003cem\u003ePf\u003c/em\u003eRH5 antibody responses remained higher in \u003cem\u003eP. falciparum\u003c/em\u003e-seropositive individuals regardless of α-thalassemia genotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). No statistically significant genotype-associated differences in IgG concentrations were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibody concentration distribution Relative to\u003c/b\u003e \u003cb\u003eP. falciparum\u003c/b\u003e \u003cb\u003eDetection by PCR\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePf\u003c/em\u003eAMA-1 (p\u0026thinsp;=\u0026thinsp;0.6802) and \u003cem\u003ePf\u003c/em\u003eEBA175 (p\u0026thinsp;=\u0026thinsp;0.7496) IgG levels were comparable between PCR⁺ and PCR⁻ groups but showed no significant variation between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). \u003cem\u003ePf\u003c/em\u003eGLURP IgG levels showed no significant variation between diagnostic groups (p\u0026thinsp;=\u0026thinsp;0.3822) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). \u003cem\u003ePf\u003c/em\u003eMSP1 IgG concentrations were similarly distributed among PCR⁺ and PCR⁻ individuals (p\u0026thinsp;=\u0026thinsp;0.7196) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). \u003cem\u003ePf\u003c/em\u003eRH5 IgG levels also did not differ significantly between the two groups (p\u0026thinsp;=\u0026thinsp;0.1817) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnalysis of antibody concentrations and seroprevalence by sex\u003c/h3\u003e\n\u003cp\u003eAcross all antigens, seropositive individuals exhibit markedly elevated IgG levels. \u003cem\u003ePf\u003c/em\u003eRH5 elicits the lowest IgG responses of all the antigens tested. Overall, males had a slightly higher IgG levels compared to females for all antigens, although this was not statistically significant. For \u003cem\u003ePf\u003c/em\u003eAMA-1 159/220 (72.27%) of the total population were seropositive, with 73.02% of males and 71.28% of females. For \u003cem\u003ePf\u003c/em\u003eEBA-175, 87/220 (39.55%) were seropositive, with 43.62% of males and 36.51% of females. For \u003cem\u003ePf\u003c/em\u003eGLURP, 148/220 (67.27%) of individuals were seropositive, with 68.09% of males and 66.67% of females. For \u003cem\u003ePf\u003c/em\u003eMSP-1, 91/220 (41.36%) were seropositive, with 43.62% of males and 39.68% of females. For \u003cem\u003ePf\u003c/em\u003eRH5, 56/220 (25.45%) participants were seropositive, with 25.53% of males and 25.40% of females testing positive. There was no statistical difference between the weighted antibody concentration between males and females (\u003cem\u003ePf\u003c/em\u003eAMA1: p\u0026thinsp;=\u0026thinsp;0.1520, \u003cem\u003ePf\u003c/em\u003eEBA175: p\u0026thinsp;=\u0026thinsp;0.2706, \u003cem\u003ePf\u003c/em\u003eGLURP: p\u0026thinsp;=\u0026thinsp;0.2247, \u003cem\u003ePf\u003c/em\u003eMSP1: p\u0026thinsp;=\u0026thinsp;0.9863, \u003cem\u003ePf\u003c/em\u003eRH5: p\u0026thinsp;=\u0026thinsp;0.8338) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eVariation in Antibody Concentration Across Different Age Categories\u003c/h3\u003e\n\u003cp\u003eGenerally, age-dependent increase in IgG concentration was observed for \u003cem\u003ePf\u003c/em\u003eAMA1, \u003cem\u003eP\u003c/em\u003efEBA175, \u003cem\u003ePf\u003c/em\u003eGLURP, and \u003cem\u003ePf\u003c/em\u003eMSP1. For \u003cem\u003ePf\u003c/em\u003eAMA-1 (χ2\u0026thinsp;=\u0026thinsp;54.814, p\u0026thinsp;=\u0026thinsp;0.0001, Dunn\u0026rsquo;s Pairwise Comparison), statistically significant differences existed between the IgG levels of participants less than 5 years and all other age groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and between participants 5\u0026ndash;10 yrs and greater than 20 yrs (p\u0026thinsp;=\u0026thinsp;0.0001); other comparisons are not statistically significant. For \u003cem\u003ePf\u003c/em\u003eEBA-175 antigen, χ2\u0026thinsp;=\u0026thinsp;14.398, p\u0026thinsp;=\u0026thinsp;0.0061, Dunn\u0026rsquo;s Pairwise Comparison: 15\u0026ndash;20 yrs with less than 5 years (p\u0026thinsp;=\u0026thinsp;0.0227), more than 20 yrs with less than 5 years (p\u0026thinsp;=\u0026thinsp;0.0308), Other comparisons are not statistically significant. For \u003cem\u003ePf\u003c/em\u003eGLURP (χ2\u0026thinsp;=\u0026thinsp;5.254, p\u0026thinsp;=\u0026thinsp;0.2622, Dunn\u0026rsquo;s Pairwise Comparison), there were no statistically significant pairwise differences. For \u003cem\u003ePf\u003c/em\u003eMSP1 (χ2\u0026thinsp;=\u0026thinsp;8.147, p\u0026thinsp;=\u0026thinsp;0.0864, Dunn\u0026rsquo;s Pairwise Comparison), there were no statistically significant pairwise differences. For \u003cem\u003ePf\u003c/em\u003eRH5 (χ2\u0026thinsp;=\u0026thinsp;13.702, p\u0026thinsp;=\u0026thinsp;0.0083, Dunn\u0026rsquo;s Pairwise Comparison), there was a statistically significant difference between the IgG concentrations between individually 5\u0026ndash;10 years and those greater than 20 years (p\u0026thinsp;=\u0026thinsp;0.0123), as were as 5\u0026ndash;10 years with 15\u0026ndash;20 years (p\u0026thinsp;=\u0026thinsp;0.0389), Other comparisons were not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eSeasonal Variation in Antibody Concentrations\u003c/h3\u003e\n\u003cp\u003eAntibody levels are consistently higher in seropositive individuals across all antigens and seasons. IgG levels for all antigens in seropositive individuals were higher in the rainy season (high transmission period) than in the dry season. \u003cem\u003ePf\u003c/em\u003eEBA175 (p\u0026thinsp;=\u0026thinsp;0.0024) showed significant seasonal variation. However, \u003cem\u003ePf\u003c/em\u003eAMA1 (p\u0026thinsp;=\u0026thinsp;0.0998), \u003cem\u003ePf\u003c/em\u003eGLURP (p\u0026thinsp;=\u0026thinsp;0.1395), \u003cem\u003ePf\u003c/em\u003eMSP1(p\u0026thinsp;=\u0026thinsp;0.0892) and \u003cem\u003ePf\u003c/em\u003eRH5 (p\u0026thinsp;=\u0026thinsp;0.4947) showed no significant seasonal variation. Seronegative individuals exhibit low IgG concentrations with no significant seasonal variation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe analysis of IgG antibody levels against \u003cem\u003ePlasmodium falciparum\u003c/em\u003e antigens reveals distinct patterns of immune responses. \u003cem\u003ePlasmodium falciparum\u003c/em\u003e Apical Membrane Antigen 1 (\u003cem\u003ePf\u003c/em\u003eAMA-1) elicited the highest median antibody concentration with a wide range. \u003cem\u003ePf\u003c/em\u003eAMA-1 is a highly immunogenic antigen expressed during the merozoite stage of the parasite. Its higher antibody response aligns with findings by Srinivasan et al \u003csup\u003e24\u003c/sup\u003e who demonstrated \u003cem\u003ePf\u003c/em\u003eAMA-1's role in inducing protective immunity through inhibition of merozoite invasion. The variability in antibody responses likely reflects differences in malaria exposure, immune history, and/ or genetic polymorphisms in the antigen \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePf\u003c/em\u003eGLURP, \u003cem\u003ePf\u003c/em\u003eMSP-1, and \u003cem\u003ePf\u003c/em\u003eEBA-175 showed moderate antibody responses that are consistent with previous studies that emphasize these antigens' roles in immune recognition during merozoite invasion \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePf\u003c/em\u003eRH5 recorded the lowest median antibody level, with varied individual responses but relatively stable levels among all genotypes. \u003cem\u003ePf\u003c/em\u003eRH5 specifically is a well-preserved antigen with less genetic variability, making it a potential target for vaccines, despite lower antibody responses compared to \u003cem\u003ePf\u003c/em\u003eAMA-1\u003csup\u003e27,28\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe findings also reaffirm \u003cem\u003ePf\u003c/em\u003eAMA-1\u0026rsquo;s strong immunogenicity supporting its vaccine candidacy. \u003cem\u003ePf\u003c/em\u003eGLURP, \u003cem\u003ePf\u003c/em\u003eMSP-1, and \u003cem\u003ePf\u003c/em\u003eEBA-175, while eliciting moderate responses, may complement a multi-antigen vaccine approach to enhance immune protection. \u003cem\u003ePf\u003c/em\u003eMSP-1 antibody levels were highest among the homozygous recessive individuals, aligning repeated exposure and the conserved capacity of this antigen and its peculiar role in merozoite invasion\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor all other antigens, consistently lower antibody levels in homozygous recessive individuals suggest reduced immune competence against malaria. Possible explanations include increased malaria susceptibility in these individuals that leads to higher parasite burdens, which can impair immune function and antibody production due to immune exhaustion \u003csup\u003e3031\u003c/sup\u003e. Additionally, homozygous recessive individuals have structural changes in their erythrocytes that interfere with parasite invasion and antigen presentation, leading to weaker immune responses\u003csup\u003e32 33\u003c/sup\u003e. However, Wild type individuals had intermediate antibody levels across most antigens. This suggests that while they may not have the enhanced immune advantages seen in heterozygous individuals, they also do not suffer from the impaired immunity observed in homozygous recessive individuals \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHeterozygous individuals consistently exhibited the highest antibody levels by genotype, across all antigens, except \u003cem\u003ePf\u003c/em\u003eMSP1, where the homozygous recessive group mounted a similar response. The higher antibody levels could be due to heterozygous individuals experiencing more controlled parasite replication, allowing for repeated immune stimulation and stronger antibody responses, and this suggests that heterozygosity may confer a selective immune advantage. Indeed, this aligns with previous studies on α-thalassemia that suggest that heterozygous individuals have a protective advantage against severe malaria \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Alternatively, alpha thalassemia could influence how these antigens are processed and presented by immune cells, leading to more robust B-cell activation and antibody production \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Again, the higher antibody responses in heterozygous individuals imply that host genetic factors could influence vaccine efficacy, and so future malaria vaccines should consider genetic diversity in target populations\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Genetic surveillance for alpha thalassemia mutations could improve malaria risk assessments by identifying populations at risk of severe disease \u003csup\u003e3839\u003c/sup\u003e. Furthermore, heterozygous individuals exhibiting the highest antibody levels, possibly could be due to a reduced parasite load, as depicted by Hamre \u003cem\u003eet al\u003c/em\u003e \u003csup\u003e40\u003c/sup\u003e. This is further evidence that heterozygous mutation provides a survival advantage by enhancing immune-mediated clearance of malaria parasites\u003csup\u003e41 32\u003c/sup\u003e. However, despite \u003cem\u003ePf\u003c/em\u003eRH5 exhibiting a lower response, it was the most relatively stable antigen among all three genotypes. Indeed, it has been shown to be a promising vaccine candidate, as vaccine-induced \u003cem\u003ePf\u003c/em\u003eRH5 antibodies can block parasite invasion\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, 44\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor all five antibodies, malaria prevalence among antibody-positive individuals was steadily high, suggesting that individuals who mounted antibody responses also had detectable active malaria infections by PCR. The strong association suggests that antibody positivity is a reliable marker of current malaria exposure and reflects their strong immunogenicity and potential use as reliable biomarkers of active and recent infection \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Notably, around 23\u0026ndash;26% of antibody-positive individuals were \u003cem\u003eP. falciparum\u003c/em\u003e-negative, implying past malaria exposure, with circulating antibodies persisting after parasite clearance or sub-microscopic infections potentially missed by standard diagnostics, especially microscopy, as well as immune memory responses due to repeated exposures in endemic regions \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Persistent antibody levels in \u003cem\u003eP. falciparum\u003c/em\u003e-negative individuals highlight the challenge of distinguishing current from past infections, which is crucial for evaluating transmission dynamics \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, reinforcing the utility of antigen-specific antibody detection as a proxy for malaria exposure and possibly recent infection \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. However, the presence of antibodies in \u003cem\u003eP. falciparum\u003c/em\u003e-negative individuals draw caution about using serology alone for malaria diagnosis.\u003c/p\u003e \u003cp\u003eSeasonal variations impact participant availability and disease prevalence, particularly during the rainy season when access to study sites becomes difficult and malaria transmission increases \u003csup\u003e4950\u003c/sup\u003e. The study found that only the \u003cem\u003ePf\u003c/em\u003eEBA-175 antigen showed a significant seasonal difference, with higher antibody levels during the rainy season. This suggests that \u003cem\u003ePf\u003c/em\u003eEBA-175 antibody responses are influenced by malaria transmission intensity, which is higher during the rainy season when mosquito breeding increases. Similar studies have shown that \u003cem\u003ePf\u003c/em\u003eEBA-175 is associated with acute malaria infection, and higher transmission seasons lead to increased immune stimulation \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. This also highlights \u003cem\u003ePf\u003c/em\u003eEBA-175's potential as a marker for recent infection and seasonal transmission. In contrast, no significant seasonal differences were observed for the other four antigens, suggesting these antibodies are long-lasting and less affected by seasonal transmission fluctuations \u003csup\u003e5253\u003c/sup\u003e. Again, P\u003cem\u003ef\u003c/em\u003eEBA-175 could serve as a serological marker for recent malaria exposure and seasonal transmission intensity\u003csup\u003e5455\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe study found that the largest age group was children aged 5\u0026ndash;10 years, followed closely by adults over 20 years, with children under five comprising the smallest group. Antibody analysis revealed that children under five had significantly lower levels of \u003cem\u003ePf\u003c/em\u003eAMA-1, \u003cem\u003ePf\u003c/em\u003eEBA-175, and \u003cem\u003ePf\u003c/em\u003eRH5, indicating that malaria immunity develops with age due to repeated exposure \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.In contrast, \u003cem\u003ePf\u003c/em\u003eMSP-1 antibody levels were relatively stable across all age groups, likely due to frequent immune boosting of immunity with repeated infections \u003csup\u003e27 57\u003c/sup\u003e. Importantly, children under 5 years should remain a primary target for malaria prevention strategies, as their lower antibody levels put them at higher risk of severe disease\u003csup\u003e5859\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile the study offers meaningful insight into how α-thalassemia may influence antibody responses, a few limitations deserve mention. We were unable to include the \u0026ndash;α⁴.\u0026sup2; (4.2 kb) deletion, leaving part of the α-thalassemia landscape uncharacterized. Additionally, the distribution of genotypes was uneven, with a relatively small number of wild-type participants (n\u0026thinsp;=\u0026thinsp;53) and homozygous individuals (n\u0026thinsp;=\u0026thinsp;23) compared with the larger heterozygous group (n\u0026thinsp;=\u0026thinsp;144). These differences in sample size naturally affect the precision of comparisons across genotypes and should be considered when interpreting the overall patterns observed.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates that all the five \u003cem\u003eP. falciparum\u003c/em\u003e antigens studied were significantly associated with meaningful differences across the different thalassemia genotypes, highlighting the role of host genetics in shaping immune responses to malaria. Elucidating the complex interplay between thalassemia and malaria immunity offers valuable insights that could refine malaria control strategies. Such knowledge has the potential to inform the design of more targeted interventions for individuals with heightened susceptibility, optimize sero-surveillance approaches, and guide vaccine development. By integrating these findings into public health policies, malaria prevention efforts could be strengthened, particularly in vulnerable populations disproportionately affected by hemoglobinopathies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy design and study site\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn 2023, a cross-sectional study was undertaken in Anyakpor (5\u0026deg;46\u0026prime;51.96\u0026Prime;N, 0\u0026deg;35\u0026prime;12.84\u0026Prime;E), a rural community in the Ada East District of Ghana\u0026apos;s Greater Accra Region, focusing on individuals of varying ages presenting with symptoms suggestive of malaria. Situated roughly 5 kilometers west of Ada Foah in southern Ghana, Anyakpor lies in a region with low malaria transmission. The community experiences a dry equatorial climate, with average temperatures ranging between 23 \u0026deg;C and 28 \u0026deg;C year-round, occasionally rising to 33 \u0026deg;C. Rainfall in the area is seasonal, typically occurring from April to June and again from September to November. The local landscape is dominated by coastal savannah vegetation (Figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy population and data collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants in the study had previously undergone thalassemia genotyping by multiplex PCR. A total of 220 individuals, aged 1 and 80 years and presenting with malaria symptoms, were enrolled during the dry season (January to March 2023, N=130 ) and the rainy/malaria transmission season (April to June 2023, N=90 ). Whole blood samples were collected into EDTA tubes and centrifuged at 720 g at 4 \u0026deg;C for 10 minutes; the plasma was promptly aliquoted and stored at \u0026ndash;80 \u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscopic detection of \u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e.\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;falciparum\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicroscopic examination was carried out by WHO certified microscopists\u0026nbsp;following standardized protocols\u003csup\u003e60\u003c/sup\u003e for malaria diagnosis. The procedure has previously been described by Amoah \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e\u0026nbsp;61\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR detection of \u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e.\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;falciparum\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetection of the mitochondrial \u003cem\u003ecox 3\u003c/em\u003e gene of \u003cem\u003eP. falciparum\u003c/em\u003e was performed using nested PCR. In the initial amplification (nest 1), 2 \u0026micro;L of extracted DNA was added to a 10 \u0026micro;L reaction mixture containing 1X OneTaq standard buffer, 0.25 \u0026micro;M of each primer (mtu F and mtu R), 0.20 mM dNTPs, 2.08 mM MgCl₂, and 1 unit of OneTaq DNA polymerase (New England Biolabs, USA). For the second amplification (nest 2), 1 \u0026micro;L of the nest 1 product (1:10) was added to another 10 \u0026micro;L reaction mixture with 0.20 \u0026micro;M species-specific primers (MtNst_falF and MtNst_falR). Both PCR rounds (nests 1 and 2) began with an initial denaturation at 94 \u0026deg;C for 2 minutes, followed by 35 cycles of denaturation at 94 \u0026deg;C for 30 seconds, annealing at 55 \u0026deg;C for 30 seconds in nest 1 and at 58 \u0026deg;C for 30 seconds in nest 2. Extension occurred at 68 \u0026deg;C, lasting 1 minute 40 seconds for the first round and 30 seconds for the second. Each reaction concluded with a final elongation at 68 \u0026deg;C for 5 minutes. To validate the assay, positive and negative controls were included in every run. The amplified fragments were then separated by electrophoresis on a 2% agarose gel stained with ethidium bromide, run at 120 volts for 40 minutes, and visualized using a UV transilluminator and gel documentation system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of Alpha Thalassemia genotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMultiplex PCR was used to detect the 3.7 kb deletion in the \u0026alpha;-globin gene, following the method described by Liu et al (2000)\u003csup\u003e62\u003c/sup\u003e. The assay employs a forward primer that binds to a conserved region of the template DNA, while multiple reverse primers anneal to distinct complementary sites on the opposite strand. The following primers were utilised:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eForward primer (3.7F):\u003c/strong\u003e AAGTCCACCCCTTCCTTCCTCACC\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse primer 1 (3.7R1):\u003c/strong\u003e ATGAGAGAAATGTTCTGGCACCTGCAC\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse primer 2 (3.7R2):\u003c/strong\u003e TCCATCCCCTCCTCCCGCCCCTGCCTT\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of Antibody Responses using Indirect Enzyme‑Linked Immunosorbent Assay (ELISA)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal immunoglobulin G (IgG) antibody responses against Apical Membrane Antigen-1 (P\u003cem\u003ef\u003c/em\u003eAMA-1), Merozoite Surface Protein-1 (P\u003cem\u003ef\u003c/em\u003eMSP-1), Reticulocyte-Binding Protein Homologue 5 (PfRH5), Erythrocyte-Binding Antigen-175 (P\u003cem\u003ef\u003c/em\u003eEBA-175), and Glutamate-Rich Protein (P\u003cem\u003ef\u003c/em\u003eGLURP) of \u003cem\u003eP. falciparum\u003c/em\u003e were measured using indirect ELISA as previously described by\u0026nbsp;\u003csup\u003e63\u003c/sup\u003e \u003csup\u003e64\u003c/sup\u003e.\u0026nbsp; Briefly, 96-well NUNC Maxisorp ELISA plates were coated with 1 \u0026micro;g of P\u003cem\u003ef\u003c/em\u003eMSP-1, P\u003cem\u003ef\u003c/em\u003eRH5, and P\u003cem\u003ef\u003c/em\u003eGLURP antigen per well, 0.5 \u0026micro;g/well of P\u003cem\u003ef\u003c/em\u003eAMA-1, and 20 \u0026micro;g/well of P\u003cem\u003ef\u003c/em\u003eEBA-175 (100 \u0026micro;L/well) in phosphate-buffered saline (PBS, pH 7.2) and incubated overnight at 4 \u0026deg;C. Plasma samples were diluted (1:200). Polyclonal IgG - Purified 500 mg BP055 (The Binding Site, UK) was used as the standard. Positive controls (pooled plasma from seropositive individuals) and negative controls (malaria-na\u0026iuml;ve donors) were added. Plates were washed and blocked. Subsequently, 100 \u0026micro;L/well of goat anti-human IgG conjugated to horseradish peroxidase (HRP) (1:3000) was added and incubated for 1 hour. The enzymatic reaction was initiated with 3,3\u0026rsquo;5,5\u0026rsquo;-tetramethylbenzidine (TMB) substrate for 10 minutes. The reaction was stopped with 100 \u0026micro;L of 0.2 M sulfuric acid. Optical density (OD) was measured at 450 nm using a Multiskan FC plate reader (Thermo Scientific, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw data was entered into Microsoft Excel\u0026reg; 2019. Optical Density (OD) values were adjusted and antibody concentrations extrapolated based on ODs of recombinant purified polyclonal IgG (BP055, The Binding Site, UK) using ADAMSEL software (Malaria research, EU). Descriptive statistics were generated using STATA version 14 and GraphPad Prism version 9. Kruskal\u0026ndash;Wallis and Dunn\u0026rsquo;s pairwise tests were used to compared antibody responses by \u0026alpha;-thalassemia genotypes, P. falciparum infection status, sex, season, and age groups. Statistical significance was defined as p \u0026lt; 0.05.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Considerations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for this study was granted by the Ethical Protocol Review Committee of the College of Health Sciences, University of Ghana (EPRC-IRB 00006220), and the Ethics Review Committee of the Ghana Health Service (GHS-ERC 021/07/23). Prior to recruitment, written informed consent was obtained from all participants or their legal guardians. Participant\u0026rsquo;s data was always kept private. \u0026nbsp;All methods were performed in accordance with relevant guidelines and regulations outlined in the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated and/ or utilized in this study are accessible from the corresponding authors upon reasonable inquiry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the participants for their cooperation and participation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eABD conceived, designed and developed the study protocol with supervision and collaboration of YAA, LEA and KKA. SSK, EAB, RAA, RD, CKMA and ABD performed laboratory assays and assisted in the data analysis. FGB, FKA, KAK, and NASE performed the data management, statistical analysis and data interpretation. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors were supported by grants from the West African Genetic Medicine Centre (WAGMC) of the University of Ghana and the National Institute of Health (D43 TW 011513). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWHO. \u003cem\u003eWorld Malaria Report 2023\u003c/em\u003e. (World Health Organization, 2023).\u003c/li\u003e\n \u003cli\u003eZekar, L. \u0026amp; Sharman, T. \u003cem\u003ePlasmodium Falciparum Malaria Continuing Education Activity\u003c/em\u003e. https://www.ncbi.nlm.nih.gov/books/NBK555962/ (2023).\u003c/li\u003e\n \u003cli\u003eLanghorne, J., Ndungu, F. M., Sponaas, A. M. \u0026amp; Marsh, K. Immunity to malaria: More questions than answers. \u003cem\u003eNature Immunology\u003c/em\u003e vol. 9 725\u0026ndash;732 Preprint at https://doi.org/10.1038/ni.f.205 (2008).\u003c/li\u003e\n \u003cli\u003eBarry, A. \u0026amp; Hansen, D. 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S. \u003cem\u003eet al.\u003c/em\u003e Mosquito bites and stage-specific antibody responses against Plasmodium falciparum in southern Ghana. \u003cem\u003eMalar J\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alpha thalassemia, Plasmodium falciparum, Antibody response, Antigens, Malaria immunity, Hemoglobinopathy","lastPublishedDoi":"10.21203/rs.3.rs-8231440/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8231440/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlpha thalassemia, a common inherited haemoglobin disorder in Africa, has long been associated with protection against Plasmodium falciparum malaria, but the exact mechanisms remain unclear. This study explored whether this protective effect is linked to antibody responses against key \u003cem\u003eP. falciparum\u003c/em\u003e blood-stage antigens, including Apical Membrane Antigen-1 (P\u003cem\u003ef\u003c/em\u003eAMA-1), Erythrocyte-Binding Antigen-175 (P\u003cem\u003ef\u003c/em\u003eEBA-175), Glutamate-Rich Protein (P\u003cem\u003ef\u003c/em\u003eGLURP), Merozoite Surface Protein-1 (P\u003cem\u003ef\u003c/em\u003eMSP-1), and Reticulocyte-Binding Protein homologue 5 (P\u003cem\u003ef\u003c/em\u003eRH5). We conducted a cross-sectional study of 220 malaria symptomatic individuals aged 1\u0026ndash;80 years. Alpha thalassemia was genotyped using multiplex PCR, while \u003cem\u003ePlasmodium falciparum\u003c/em\u003e infections were confirmed by microscopy and nested PCR. Using indirect Enzyme-Linked Immunosorbent Assay (ELISA) we measured antigen-specific IgG levels and analysed their patterns across genotypes, infection status, season, gender, and age categories. Heterozygotes consistently mounted the strongest antibody responses, with PfAMA-1 showing the highest median concentration (98.63 ng/mL) and homozygous recessive individuals had the lowest responses, particularly to PfRH5 (17.66 ng/mL). Across all five antigens, antibody levels differed significantly between genotypes (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). These findings reveal that heterozygous alpha thalassemia may enhance immune defense by boosting antibody production. These findings provide a deeper understanding of malaria protection and offer valuable clues for innovative malaria control strategies.\u003c/p\u003e","manuscriptTitle":"Exploring the influence of alpha thalassemia on antibody responses to Plasmodium falciparum infections in Ghana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-23 12:00:56","doi":"10.21203/rs.3.rs-8231440/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6504a2ac-6aef-45d5-8b45-77aef9348393","owner":[],"postedDate":"December 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59988499,"name":"Health sciences/Diseases"},{"id":59988500,"name":"Biological sciences/Immunology"},{"id":59988501,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-03-26T06:56:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-23 12:00:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8231440","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8231440","identity":"rs-8231440","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}