Spatiotemporal Analysis of Plasmodium falciparum Erythrocyte Binding Antigen (Pf EBA) 175 Gene Dimorphism in Ghana

doi:10.21203/rs.3.rs-4920439/v1

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Spatiotemporal Analysis of Plasmodium falciparum Erythrocyte Binding Antigen (Pf EBA) 175 Gene Dimorphism in Ghana

https://doi.org/10.21203/rs.3.rs-4920439/v1

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Background

Malaria remains a leading cause of death worldwide, claiming over 600,000 lives each year. Over 90% of these deaths, mostly among children under five years, occur in sub-Saharan Africa and are caused by Plasmodium falciparum. The merozoites stage of the parasite, crucial for asexual development invade erythrocytes through ligand-receptor interactions. Erythrocyte binding antigen (EBA)-175 is one of the key ligands facilitating invasion via interaction with glycoprotein A (GpA) receptors on the erythrocytes. EBA-175 is known to exist in two dimorphic allelic (F and C) forms with each found to infer different virulence. There is paucity of data on the prevalence of these alleles and their epidemiology in the Ghanaian malaria landscape and hence this study.

Methods

Parasite gDNA was extracted from archived Dried Blood Spots (DBS) prepared from 700 confirmed malaria-infected individuals and analysed for Pf EBA-175 dimorphism. Selective EBA-175 gene amplification via nested PCR and allele scoring using agarose gel electrophoresis for F, C and F/C alleles.

Results

Of the total 632 successfully genotyped samples, we found a prevalence of F, C, and F/C allelic forms were 61.2% (n = 387), 20.7% (n = 131), 18.0% (n = 114) respectively. Seasonality analysis did not reveal a statistically significant difference in the prevalence of dimorphic forms between the wet (n = 475) and dry (n = 157) seasons (p = 0.051). The prevalence ratio (wet/dry) for C, F and F/C were determined to be 1.0, 1.1 and 1.4 respectively. Between 2019 and 2022, the prevalence of the alleles changed significantly (χ² = 6.5427, p = 0.03). Geometric mean parasite density for the C, F, and F/C alleles were 21477.1 [95%CI: 15749.2 -29288.1], 18308.0 [95%CI: 15149.9-22124.5] and 22690.4[95% CI: 16891.9–30479.2] respectively.

Conclusion

The F-allele was the most prevalent form across all age groups, followed by the C allele and mixed F/C alleles. No significant difference in allele prevalence was observed between the high malaria season (wet) and low malaria season (dry). However, we observed a statistically significant difference (p = 0.03) in the temporal prevalence of pure alleles (F & C) between two time points.

Malaria

EBA-175 dimorphism

Plasmodium falciparum

merozoites

erythrocytes

glycoprotein A (GpA)

alleles

Plasmodium falciparum is responsible for the most severe form of malaria (1); it causes fever, chills, and potentially life-threatening complications such as cerebral malaria and multiorgan failures (2). The parasite has a complex life cycle that involves both human and mosquito hosts (1): with the merozoite stage being crucial for invasion of human red blood cells (3). The process of merozoites invasion is aided by receptor-ligand interactions involving parasite proteins such the 175-kDa erythrocyte binding antigen (EBA-175) expressed on the parasite's merozoite surface (4). The binding of EBA-175 to erythrocytes is specific to glycophorin A (GpA), and requires the presence of N-acetylneuraminic acid (Neu5Ac), a sialic acid, on the host cell surface (5).

EBA-175 is a member of the Duffy-binding-like erythrocyte-binding protein (DBL-EBP) family, which include key adhesion molecules required for malaria parasites invasion (6). The EBA-175/GpA pathway is the primary chymotrypsin-resistant invasion pathway (7). Antibodies against EBA-175 can inhibit binding to GpA and block invasion of merozoites in vitro (8). The protein is shown to have an N-terminal signal peptide, cysteine-rich DBL domains, a carboxyl cysteine-rich region (C-Cys), a transmembrane domain, and a short cytoplasmic tail (9, 10). EBA-175 comprises of three domains: an N-terminal ligand binding domain (LBD), a central cysteine-rich domain (CRD), and a C-terminal transmembrane domain (TMD). The LBD binds to GpA on erythrocytes with high affinity, and the CRD helps to stabilize the binding(11). This interaction triggers re-orientation of the merozoite surface, leading to the formation of a tight junction between the merozoite and erythrocyte surface (5, 12). Merozoites then secrete additional proteins, which aid in the entry process (13). The role of EBA-175 in the invasion of erythrocytes is critical for the survival of the malaria parasite (7, 8, 14).

The EBA-175 gene is located on chromosome 7 and comprising four exons and with seven distinct regions(15); region III is notably dimorphic, containing an insertion of 423 base-pair segment (F-allele) in Fajara, Gambia, strain 3 (FCR3) or a 342 base-pair segment (C-allele) in the (Malayan Camp strain) CAMP (5). In a resolved agarose gel, Fig. 3, the C-allele show 714 bp whiles the F-allele shows 795 bp (16–20). Since Plasmodium merozoites are haploid, this dimorphism is conserved across strains, with only one of the two segments present in uniclonal infections (12, 17, 21). The interaction of the dimorphic segments of the region III with the glycophorin backbone is thought to facilitate the smooth entry of the parasite through the erythrocyte membrane (13, 17). Some studies report that this dimorphism, F- and C-alleles, infer different parasite virulence (19, 22, 23). C-allele allele has been found in absolute linkage disequilibrium with specific single nucleotide polymorphisms (SNPs) (23). This could imply that the C-allele allele is favored under specific environmental or immunological conditions, potentially impacting the virulence and adaptability of the parasite (17, 22, 23). However, in a high malaria transmission region in Uganda, the F-allele was more prevalent in children under 5 years old compared to those over 10 years (24).

Therefore, understanding this nuanced distribution of the dimorphic alleles and their association with different eco-epidemiological parameters in Ghana will contribute to the understanding of EBA-175, and aid in therapeutic interventions that target the disruption of EBA-175 interaction.

This study aimed to assess the prevalence and distribution of the dimorphic alleles (F- and C-alleles) of the EBA-175 in malaria-infected individuals in Ghana. The focus was on the three malaria transmission areas in Ghana, identified as ecological zones: the Northern Savannah Zone, Middle Transitional/Forest Zone, and Coastal Savannah Zone (25). We examined the prevalence of these alleles and their seasonal patterns, while also investigating potential correlations between EBA-175 dimorphism and the parasite densities. Furthermore, we analysed how age groups might influence allelic variation to determine whether there are notable differences in allele distribution among different age brackets, as well as gender.

Study Area and Subjects

This spatiotemporal study as part of a larger study was carried out with samples collected all year round in 2019 (n = 311) and 2022 (n = 321). These samples were collected from 32 sentinel health facilities located in fourteen of the sixteen administrative regions in Ghana. The samples collected were further grouped into three ecological zones: the Northern Savannah Zone, Middle Transitional/Forest Zone, and The Coastal Savannah Zone to reflect the malaria transmission pattern in Ghana.

Malaria transmission differs among the three ecological zones in Ghana due to varying climatic and environmental conditions that influence the propagation of Anopheles mosquitoes and malaria parasites transmission patterns. The Coastal Savannah and Forest zones exhibit a bimodal rainfall pattern, allowing for two peaks of malaria transmission, while the Northern Savannah zone experiences seasonal malaria transmission due to a unimodal rainfall pattern (25, 26).

A total of 632 dried blood spots samples from malaria positive patients were collected from 32 different health centres across Ghana with their biodata well documented were successfully genotyped.

Sample collection, processing, and DNA extraction.

This study is part of a larger malaria molecular surveillance (MMS) study across 32 health facilities used by the Ghana National Malaria Elimination Programme (NMEP) as sentinel sites for drug efficacy studies. Out-patients reporting fever (or history of fever) within the past 24 hours at these health facilities were screened with OnSite^® Malaria Rapid Diagnostic Test (mRDT) kits. Individual informed consent was documented for patients who tested positive and were willing to participate in the larger MMS studies. In accordance with the WHO guidelines (27–29), approximately 5 mL of whole blood was collected from consented participants into an EDTA tube, two 50 µL drops was spotted on a filter paper (Whatman No. 2) to make a Dried Blood Spots (DBS) with 6 µL and 2 µL to prepare thick and thin malaria blood smears respectively for parasite identification and quantification. Plasmodium falciparum density was determined by standard microscopy and asexual parasites were scored against 200 white blood cells (WBCs). Malaria smears were considered negative after examining 100 fields without detecting asexual or sexual parasites. The parasite density per 200 WBCs was converted to density per microliter assuming 8000 WBCs.

The parasite DNA was extracted from DBS using the QIAamp® DNA Investigator Kit (QIAGEN Catalogue No. 56504), following the manufacturer's instructions with minor modifications. The incubation time at 56°C was extended to 17 hours at 600rpm. The speed of the final centrifugation step at 14000 rpm for 3 instead of 1 minute to dry the membrane. These modifications were aimed at optimizing DNA yield and purity. The DNA concentration was determined using a QuBit Flex 4 fluorometer.

Genotyping of EBA-175 alleles

To confirm genotypes of Plasmodium falciparum infection, nested PCR was conducted using a set of oligonucleotide primers as described in (16) with modifications. For the outer PCR, the following primers were used: forward primer (EBA-175 F1) 5'-ATTAACGCTGTACGTGTGTCTAG-3', and reverse primer (EBA-175 R1) 5'-TCTCAACATTCATATTAACAATTC-3'. For the inner PCR, forward primer (EBA-175 F2) 5'-AAGAAATACTTCATCTAATAACG-3', and reverse primer (EBA-175 R2) 5'-CAATTCCTCCAGACTGTTGAACAT-3'. The lyophilized primers were reconstituted to 100 µM

The first round of nested PCR constituted a mixture of 0.25 µL of each forward and reverse primer, 5.5 µL of one Taq standard buffer, 3 µL of nuclease-free water, and 2 µL of DNA template. The second round of PCR used a similar mixture but with 4 µL of nuclease-free water and 1 µL of the amplicon from the first round. The cycling conditions for the first round included an initial denaturation at 96ºC for 3 minutes, 30 cycles of denaturation at 96ºC for 10 seconds, annealing at 57ºC for 10 seconds, extension at 72ºC for 50 seconds, followed by a final extension at 72ºC for 10 minutes.

The second round of PCR had similar cycling conditions, with an initial denaturation at 95ºC for 5 minutes, 30 cycles of denaturation at 95ºC for 10 seconds, annealing at 60ºC for 8 seconds, and extension at 72ºC for 50 seconds. The final extension was also 72ºC for 10 minutes.

The PCR products were resolved on 2% agarose gel electrophoresis, with 3.5 µL of the PCR product and 1.5 µL of loading dye (purple 6x) and a low weight molecular DNA marker (100 to 1500 bp, New England Biolabs) for allele analyses. The gel was run at 75 V for 90 minutes, and the band separations were visualized with an Amersham Imager 600. The weights of the alleles were used to categorize them into either a C or an F allele.

Statistical analysis

Statistical analyses were conducted using R (version 4.4.0). The frequency of F and C alleles was determined for the entire sample set and by ecoepidemiological zones delineated as the Northern Savannah, the Middle Transitional/Forest belt, and the Coastal Savannah Zones.

To assess potential significant correlation between the alleles’ counts in different seasons, chi-squared analysis was used. The geometric mean parasite density (GMPD) was calculated with 95% CI in the different ecological zones, age groups, gender, and seasons. Kruskal–Wallis and one-way ANOVA tests were also employed to examine whether there was a significant correlation between parasitemia and the other parameters (sex, age, year). Further descriptive statistical measures were used to evaluate seasonal patterns of allele prevalence and explore possible relationships between the dimorphic alleles and parasitemia levels.

Clinical and demographic characteristics of patients

A total of 632 samples were successfully genotyped for the different alleles of the EBA-175 allele types at two time points, 311 in 2019 and 321 in 2022. Of the 632 patients, 554 had gender recorded, of whom, 46.8% (259/554) were males and 53.2% (295/554) were females. The mean age (completed years) was 20.8 years (SD = ± 17.1). Overall, the geometric mean parasite density (GMPD) was 19670.87 [95%CI: 17052.5-22691.2]. (Table 1).

Allele prevalence between eco-epidemiological zones.

In the coastal ecological zone where malaria transmission intensity is relatively lower, the observed prevalence of the EBA-175 alleles was 23.8% for the C-allele, 55.6% for the F-allele and 20.5% mixed infections (F/C-alleles). The GMPD for the coastal zone was 20838.5 [95%CI: 17260.6-25158.1]. The observed prevalence in the middle transitional/forest zone was 17.9% C-allele, 63.7% for F-allele and 18.0% mixed infection (F/C-alleles). The GMPD was 14884.6 [95% CI:11083.8-19989.0]. In contrast, the prevalence in the northern Sahelian savannah zone was 17.9% for C-allele, 69.1% for F-allele and 13.0% for mixed infections (F/C-alleles). The GMPD was 23204.1[95%CI: 171.7–31412.3]. Females showed higher prevalence of all alleles compared to males; mean age are also provided for each allele within these zones. Further details may be reviewed in Table 1 and Fig. 1.

Table 1

Prevalence of EBA-175 alleles, mean ages, gender distribution and GMPD by eco-epidemiological zones
Transmission-zone	n	Prevalence (%)	Mean Age (yrs)	^aGMPD [95% CI]	^bGender dist. (M/F)
Overall, n	632	100	20.8	19670.9 [17052.5-22691.2]	554
C-allele	131	20.7	22.0	21477.1 [15749.2-29288.1]	M: 55
C-allele	131	20.7	22.0	21477.1 [15749.2-29288.1]	F: 62
F-allele	387	61.2	20.1	18308.0 [15149.9-22124.5]	M: 157
F-allele	387	61.2	20.1	18308.0 [15149.9-22124.5]	F: 184
Mixed allele	114	18.0	21.8	22690.4 [16891.9-30479.2]	M: 47
Mixed allele	114	18.0	21.8	22690.4 [16891.9-30479.2]	F: 49
Coastal Savannah	302	100	23.7	20838.5 [17260.6-25158.1]	226
C-allele	72	23.8	23.7	23801 [16610.8-34104.9]	M: 31
C-allele	72	23.8	23.7	23801 [16610.8-34104.9]	F: 27
F-allele	168	55.6	23.7	19520.2 [14990.6-25418.5]	M: 67
F-allele	168	55.6	23.7	19520.2 [14990.6-25418.5]	F: 56
Mixed allele	62	20.5	23.3	21317.1 [14309.4-31756.7]	M: 26
Mixed allele	62	20.5	23.3	21317.1 [14309.4-31756.7]	F: 19
Middle transitional/forest	162	100	20.1	14884.6 [11083.8-19989.0]	161
C-allele	29	17.9	21.7	14858.2 [7356.9-30007.6]	M: 14
C-allele	29	17.9	21.7	14858.2 [7356.9-30007.6]	F: 15
F-allele	112	63.7	19.9	13663.8 [9517.1-19617.4]	M: 49
F-allele	112	63.7	19.9	13663.8 [9517.1-19617.4]	F: 62
Mixed allele	21	18.5	18.5	23552.36 [11419.4-48576.7]	M: 6
Mixed allele	21	18.5	18.5	23552.36 [11419.4-48576.7]	F: 15
Northern Savannah	168	100	16.4	23204.1 [171.7-31412.3]	167
C-allele	30	17.9	18.1	23963.0 [10767.9-53327.7]	M: 10
C-allele	30	17.9	18.1	23963.0 [10767.9-53327.7]	F: 20
F-allele	107	69.1	14.7	22486.9 [15238.8-33182.4]	M: 41
F-allele	107	69.1	14.7	22486.9 [15238.8-33182.4]	F: 66
Mixed allele	31	13.0	20.8	25066.7 [14222.3-44180.0]	M: 15
Mixed allele	31	13.0	20.8	25066.7 [14222.3-44180.0]	F: 15
^aGMPD is the geometric mean parasite density and CI is the 95% confidence interval; ^bGender distribution across: M is male, and F is Female

Allele prevalence by transmission season.

The prevalence of the EBA-175 alleles during the high malaria season (wet: May-October) was 19% (91/475) for the C-allele, 61% (290/475) for the F-allele, 20% (94/475) mixed infections with an overall GMPD of 1850.89 [95%CI:15680.3- 22018]. Prevalence in the low malaria season (dry: November-April) was 25% (40/157) for the C-allele, 62% (97/157) for the F-allele, 13% (20/157) for mixed infections with overall GMPD of 23373.54.2 [95%CI: 18063.2–30245.1]. However, seasonal differences in allele prevalence were not statistically significant (Chi-squared p = 0.06).

Prevalence of EBA-175 alleles by age group

The bar chart illustrates the prevalence (%) of the F, C, and F/C alleles across different age groups. The F-allele consistently shows the highest prevalence across all groups, peaking at 70% in the 0–5 years age group and remaining above 50% in all other groups. The C-allele exhibits a varying pattern, with increased prevalence in older age groups, particularly 12–18 years (25%) and 25–35 years (28.3%). The combined F/C allele remains relatively stable, with the highest prevalence (21.7%) observed in the 36 + years group as shown in Fig. 2.

Temporal Prevalence of EBA-175 alleles

A total of 49.2% (311/632) of the samples were collected in 2019 and the remaining 50.8% (321/632) were collected in 2022. Of the samples collected in 2019, the F-allele remained the most prevalent at 66.0% followed by C-allele and mixed C/F-allele at 17.0% each. On the one hand, in 2022 (n = 321) prevalence of the F-allele was 57.0% followed by the C-allele and mixed C/F-alleles was 24.0% and 19.0% respectively. The temporal difference in the occurrence of F, C and F/C alleles between the two time points, 2019 and 2022, was statistically significant (χ² = 6.54, p = 0.03).

The need for candidates to be used in vaccine development call for in-depth genetic analysis of all possible candidates. In this light, many candidates with vaccine potential are studied for their suitability. Considering Ghana with unique malaria transmission dynamics, we sort to study the EBA-175 gene dimorphic forms circulating among patients. The study assessed spatio-temporal variation in the allelic dimorphism of EBA-175 (C-allele (714 bp) and F-allele (795 bp), Fig. 3) across different malaria transmission zones in 2019 and 2022.

Overall, F-allele was observed in higher frequency than C-allele, but a significant proportion of infections also carried mixed F/C alleles, which may be due to the multiplicity of infection in these high malaria transmission regions. In high transmission settings, mosquitoes are more likely to acquire and transmit a diverse array of parasite strains during blood meals, leading to co-infections in humans (22, 30, 31). Our data agree well with reports from previous studies in Ghana, neighboring Burkina Faso, Gambia, Nigeria, Gabon, and Equatorial Guinea (16, 17, 23, 32, 33) which also confirm the high prevalence of the F-allele across sub-Saharan Africa. The EBA-175 allelic distribution amongst the Ghanaian population highlights the genetic makeup of P. falciparum infections, with multiple alleles co-circulating within the same geographic area. The spatial and temporal prevalence of the F-allele across the population screened with eco-epidemiological variation suggests that the parasite population is largely panmictic with significant gene flow across time and space, which can be important for public health strategies. Thus, the high proportion of infections which carried CAMP/FCR-3 alleles further underscores the significant levels of transmission intensity resulting in high outcrossing in the mosquito midgut. Previous studies have also suggested immunological selection (17), and human demography and/or genetic background as key drivers of EBA-175 allelic dimorphism (34, 35). The high prevalence of F-allele in African populations and high prevalence of C-allele in Latin American populations such as Brazil (17) suggests that malaria transmission, type of vectors and host factors may be structuring the diversity and distribution of these alleles. Our study did not observe any clear trend of rapidly decreasing frequency of F-allele with age, even though its frequency was highest among children under 5 years (Fig. 2). The behavior of the F-allele from our data is consistent with data from Uganda where the F-allele was found to be predominant among children below 5 years (24). The frequency of C-allele however generally increased with age across all age groups and is likely to be under much stronger immune selection than the F-allele (17, 22).

Malaria transmission in northern Ghana remains unimodal with the highest transmission season in occurring during the rainy season. These transmission seasons however did not have any significant effect on the prevalence of the EBA-175 alleles. Thus, the observed pattern of dominance of the F-allele in clinical infections may be reflective of the malaria reservoir of asymptomatic infections that continues to fuel transmission during the low season, further studies need to confirm this or otherwise.

The Geometric mean parasite density (GMPD) of children, 0 to 5 years showed higher parasite density compared to adults from 36 years and above (Table 1). The high GMPD infer increased susceptibility which can be attributed to their developing immune systems which may not effectively respond to the diverse array of Pf genotypes. This highlights how children continue to stand a risk of high parasite density infection and severe malaria (36). Interestingly, the GMPD for mixed (F/C) infections was highest. This could be due to synergistic effects where different strains complement each other’s growth dynamics or evade the host's immune response more effectively; this was followed by the C-allele and the lowest was the F-allele. We opine that, parasites carrying the F-allele though common, may be slow growers as compared to strains carrying the C-allele.

The study found the F-allele to be the most predominant form across all age groups, followed by the C-allele and mixed F/C alleles. No significant difference in allele prevalence was observed between the high malaria season (wet) and low malaria season (dry). Furthermore, no pattern was found in the distribution of geometric mean parasite densities between the different allelic forms, with the highest densities observed in mixed allele infections. The current study highlights the co-circulation of alleles in these high malaria endemic areas in Ghana.

Ethics approval and participant consent

Scientific and ethical approval for the use of human subjects was obtained from the Ghana Health Service Ethics Review Committee (GHS-ERC 005/12/17). All participants provided informed consent prior to inclusion in the study.

Consent for publication

Not applicable

Availability of data and materials

The datasets used and analysed in the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Funding

This study was supported by the Bill & Melinda Gates Foundation grant (INV-050873) to LA and the National Institute of Health and Care Research (grant number: NIHR134717) to LA & GA.

Authors' contributions

Abraham Y. Kpirikai: conducted laboratory work and drafted the manuscript.

Belinda Ofosu: assisted with statistical analysis.

Abdul Rashid Kassim, Esther Donkor, Frederica Malm, Ahmed Osumanu, Mona-Liza E. Sakyi, Josie Okai, Victor Kornu: assisted with sample preparation, DNA extraction, and other laboratory work.

Charles Mensah, Albert Yao Kudakpo: fieldwork coordination of samples, selection and processing of DBS samples.

Francis Dzabeng: Metadata curation and data management.

Samirah Saiid, Collins Morang’a, Gordon Awandare, Yaw Aniweh: contributed to the study design and provided supervisory support. Reviewed the manuscript and offered critical feedback.

Lucas N. Amenga-Etego: conceptualized the study, supervised the entire project, reviewed the manuscript, and provided critical revisions.

Acknowledgements

The samples for this study were drawn from a larger study integrated malaria molecular surveillance (iMMS) project, which is conducted in collaboration with the Ghana National Malaria Elimination Programme (NMEP). We extend our gratitude to the 32 health facilities that served as sentinel sites for surveillance work. We are deeply grateful to the patients who participated in these studies and provided informed consent, as well as to the medical staff at these facilities for their invaluable support in patient screening.

Authors' information

Corresponding author: Amenga-Etego N. Lucas^1* [email protected]

¹West African Centre for Cell Biology of Infectious Pathogens, Volta Rd, University of Ghana, Accra, Ghana.

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No competing interests reported.

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Spatiotemporal Analysis of Plasmodium falciparum Erythrocyte Binding Antigen (Pf EBA) 175 Gene Dimorphism in Ghana

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Version 1

Abstract

Figures

Introduction

Methods

Study Area and Subjects

Genotyping of EBA-175 alleles

Statistical analysis

Results

Clinical and demographic characteristics of patients

Prevalence of EBA-175 alleles by age group

Temporal Prevalence of EBA-175 alleles

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1