Introduction

Malaria is a parasitic disease caused by the genus Plasmodium, which is transmitted to humans through the bite of infected female Anopheles mosquitoes. This disease is still one of the leading causes of death worldwide. According to the World Health Organization (WHO), there were 249 million cases of malaria and 608,000 deaths due to malaria in 85 countries in 2022. This disease is most common in tropical countries. Symptoms can range from mild, such as fever, chills, and headache, to severe, including fatigue, confusion, seizures, and difficulty breathing. Infants, children under five, pregnant women, travelers, and people with Human Immunodeficiency Virus (HIV) or acquired immunodeficiency syndrome (AIDS) are at higher risk of developing severe infections. Currently, five Plasmodium species infect humans, namely P. falciparum, P. malaria, P. ovale, P. vivax, and P. knowlesi [1, 2].

A blood group is a classification of blood based on specific antigens located on the surface of erythrocytes, which are identified using an agglutination test. There are 45 blood group systems and 362 antigens, including ABO, Lewis, Ii, P, Rhesus, MNSs, Kell, Kidd, Duffy, Lutheran, etc. [3]. Among the 45 blood group systems, the Duffy blood group system is often associated with vivax malaria. This system consists of six antigens that differ based on amino acid sequence, with their prevalence varying between racial groups. The Duffy blood group system was identified in 1950 when anti-Fya was discovered in a suspected hemolytic transfusion reaction in a hemophilia patient who developed jaundice after receiving a blood transfusion. Duffy antigen is a glycoprotein consisting of 336 amino acids, with two possible Duffy messenger ribonucleic acids (mRNAs) translated into the Duffy antigen gene: a less abundant alpha form, and a significant beta form that differs with two amino acids at the N-terminus [4, 5].

The global distribution of Duffy-negative individuals indicates significant limitations in the frequency of the FY*B allele, with the highest prevalence in Europe and parts of the Americas and an increasing presence in regions bordering the FY*B dominant area in sub-Saharan Africa. The negative Duffy phenotype is highly restricted to sub-Saharan African populations, with a median frequency reaching 98–100% in the western, central, and southeastern regions stretching from the Gambia in the west to Mozambique in the east and a high median frequency of ≥90% covering 22 countries in this area. The prevalence of negative Duffy increases to c.10% south of the Sahara. The distribution of Duffy negatives is a crucial concern in the context of malaria, particularly regarding the susceptibility of Duffy-negative individuals to P. vivax infection [6]. The Duffy blood type raises the question of why Duffy negative does not hurt individuals who do not have the Fy (a–b–) phenotype. This suggests that the Duffy blood group determinant (FY*A or FY*B) is likely an erythrocyte receptor for P. vivax [7].

This article aims to provide a comprehensive overview of the current state of knowledge regarding the relationship between the Duffy negative blood type and vivax malaria, including its effects on resistance to vivax malaria infection.

Vivax malaria

Morphology and lifecycle

The lifecycle of P. vivax is similar to other malaria species because it requires both invertebrates and vertebrates as hosts, but there are differences in its biological characteristics (see Fig. 1). When a female Anopheles mosquito bites a human, sporozoites enter the human body. Within about 30 minutes, the sporozoites move to the liver through the blood vessels and develop into liver schizonts measuring 42–45 microns, which produce 10–12,000 merozoites. Hepatic schizonts, in the primary preerythrocyte or exoerythrocyte stage, reproduce asexually through hepatic schizogony. Hypnozoites can rest in liver cells for some time (3–9 months, or even several years) before becoming active again and starting the secondary exoerythrocytic cycle [8, 9].

Figure 1. Life cycle of P. vivax, causative agent of human malaria, which includes three main stages: mosquito stage, liver stage, and blood stage. In mosquito stage, sporozoites develop in Anopheles mosquito and are transmitted to humans through a bite. After entering human bloodstream, sporozoites travel to liver and develop into merozoites. Merozoites then enter red blood cells, develop through trophozoite and schizont forms, and produce gametocytes that are ready to be taken up by other mosquitoes, continuing infection cycle

Merozoites resulting from hepatic schizogony enter the bloodstream and invade erythrocytes, initiating the erythrocytic cycle for asexual reproduction (blood schizogony). Within the erythrocyte, the merozoites develop into young, ring-shaped trophozoites with basophil spots, about one-third the size of the erythrocyte. When stained with Giemsa, the cytoplasm appears blue, the nucleus red, and has large vacuoles. Young trophozoites then develop into very active old trophozoites, with the cytoplasm changing to amoeboid and containing red Schaffner spots. The pigment changes to a clear yellow color. Ripe schizonts in the erythrocytic cycle contain 12–18,000 merozoites that fill the entire erythrocyte, with pigment collected at the center or edges [8–10].

When erythrocytes rupture, the resulting merozoites infect new cells, and some develop into gametocytes. When a mosquito bites a human infected with malaria, gametocytes enter the mosquito’s body, continuing the Plasmodium life cycle to the sexual phase and producing sporozoites. The sexual phase (sporogony) in the mosquito’s body lasts 16 days at 20°C and 8–9 days at 27°C. Young oocysts in mosquitoes have 30–40 granules with yellow pigment and fine granules without a unique pattern. In the mosquito’s digestive tract, gametocytes fuse to produce zygotes which develop into sporozoites, which then move to the mosquito’s salivary glands [8–10].

Pathology and clinical symptoms

The intrinsic incubation period for malaria usually lasts 12–17 days, but in some strains, it can be 6–9 months or even more. The first attack begins with prodromal symptoms such as headache, backache, nausea, and general malaise. In the first 2–4 days, fever tends to be irregular but then becomes intermittent, with a clear difference between morning and evening, with a high temperature that then falls back to normal. The irregularity of the fever curve at the beginning of the disease is caused by groups of parasites with different sporulations so that the fever becomes irregular. Fever attacks in the morning or evening begin with a shivering stage, followed by a feverish phase, and then sweating. In the first attack, anemia may not be apparent, but it is more visible in chronic malaria. Malaria can cause complications such as respiratory problems, kidney failure, jaundice, severe anemia, spleen rupture, seizures, and impaired consciousness. Vivax malaria is important not because of its high death rate but because of the relapse phase, which causes weakness in sufferers [11].

Laboratory diagnostics and treatment

Laboratory diagnosis of vivax malaria involves microscopic examination of Giemsa-stained blood smears and a Rapid Diagnostic Test (RDT). Treatment consists of chloroquine to eliminate blood-stage parasites and primaquine to prevent recurrence, carefully considering the patient’s Glucose-6-Phosphate Dehydrogenase (G6PD) status to avoid potential complications [12, 13].

Duffy blood type

History of Duffy blood types

The investigation started with the identification of antibodies in a 43-year-old male patient who had hemophilia and frequently received blood transfusions. Duffy was the patient’s surname and was used to name the new blood group system. FY*A is used as an antigen response, anti-Fya as an antibody, and FY*B as an allele. Although anti-Fyb was not initially found in antibodies, it was expected to be discovered soon. Indeed, the following year, anti-Fyb was found in the serum of a woman from Berlin who was pregnant with her third child without having received a transfusion. Several other Duffy antigens were discovered two decades later, including Fy3, Fy4, Fy5, and Fy6, but only Fy3 has clinical significance [14].

DARC is encoded by the Duffy gene, which has two main alleles, namely FY*A and FY*B. These two alleles are codominant, whereby FY*A is inherited from one parent, and FY*B is inherited from the other parent like a gene product. The FY*A and FY*B antigens are expressed on erythrocytes. A critical minor Duffy phenotype is Fyx [Fy(b+x)], with the FY*X allele encoding the FY*B antigen. However, it is weak because it reduces the amount of Duffy protein and is not always detected by anti-Fyb. Duffy antigen is found on an acidic glycoprotein that spans the erythrocyte membrane seven-fold, with the N-terminal part forming the extracellular domain and the C-terminal part in the cytoplasm. The significance of the Duffy blood group increased substantially with recognition of its role as an entry barrier for malaria parasites in human erythrocytes [5].

P. falciparum attacks the host using a series of receptors on the surface of human erythrocytes. This differs from P. vivax and P. knowlesi, which rely on Duffy antigen interactions. This means that individuals lacking the Duffy antigen will resist merozoite invasion. Populations in Africa that do not express the DARC protein experience gaps in P. vivax distribution. Therefore, the lack of DARC protein in erythrocytes suggests an adaptive response to disease or selective pressure acting on the parasite [7].

Dose effects on DARC protein expression are related to susceptibility and resistance to infection. This indicates that erythrocytes heterozygous for the binding allele may provide significant protection and confer a selective advantage to Duffyneg heterozygotes in areas endemic to P. vivax. Although the mechanisms involved in invasion remain to be elucidated, the hypothesis proposes that copy number expansion of the Duffy Binding Protein (DBP) gene of P. vivax accounts for the use of alternative receptor-ligand pairs [7].

Duffy antigen determinants

The enzyme affects the FY*A and FY*B antigens on erythrocytes with sensitivity to papain, α chymotrypsin, pronase, and resistance to trypsin, sialidase, and 200 mM DTT. FY*A expression on fetal erythrocytes begins at six weeks of gestation, reaching a peak c.12 weeks after birth. The FY*A antigen is found in baboon erythrocytes, but not in many other primates such as chimpanzees, gorillas, gibbons, rhesus monkeys, cynomolgus macaques, squirrel monkeys, capuchins, and douroucouli. FY*B is present in the erythrocytes of primates such as chimpanzees, gorillas, gibbons, rhesus monkeys, cynomolgus macaques, and baboons, but not in squirrel monkeys, capuchins, or douroucouli [15].

The presence of Fy3 is always associated with FY*A or FY*B, except in erythrocytes with the Fy (a–b–) phenotype in black individuals. Although Fy3 is antigenically related to FY*A and FY*B, it is biochemically distinct due to the multimeric structure of the Duffy protein. Fy3 originates from Europe and Asia and is resistant to protease treatment on erythrocytes. Fy4 was first reported in 1973 and can react with all Fy phenotypes in black individuals except Fy (a+b+). Anti-Fy5 has been found in black women with the Fy (a–b–) phenotype who received transfusions, reacting with erythrocytes FY*A, FY*B, or both, but not with Fy (a–b–). The Fy6 antigen has been reported in some primates but not in cynomolgus macaques, baboons, or capuchins. Anti-Fy6 has been identified by peptide scanning and recognized as a linear epitope on the N-terminal extracellular domain of DARC (see Tab. I) [15, 16].

Molecular basis

The molecular basis associated with the FY*A and FY*B antigens is the presence of the amino acid Gly and the amino acid Asp located at residue 42. The FY*A allele has the essential protein guanine, while the FY*B allele has ade­nine, situated at bp 125 in exon two (see Tab. 1). This missense mutation causes a glycine codon in the FY*A allele and an aspartic acid codon in the FY*B allele at position 42 of the primary product (p.Gly42Asp), which determines the phenotypes Fy (a+b–), Fy (a–b+), and Fy (a+b+). Several other variants have been discovered that can produce weak (+w or *W) or no (0 or *N) Duffy FY*A or FY*B expression at all [7].

According to Höher et al. [7] located on chromosome 1q23.2, is the gene that encodes a glycoprotein expressing the Duffy blood group antigens. This gene is transcribed in two mRNA variants yielding two isoforms, encoding proteins with 338 and 336 amino acids. This review provides a general overview of the Duffy blood group to characterise and elucidate the genetic basis of this system. The Fya and Fyb antigens are encoded by co-dominant FY*A (FY*01, the genetic locus is located on chromosome 1q23.2 with the sequence DNA NC_000001.11 located in the region (159204013..159206500). This explains the presence of two exons spread over 1.521 kbp of genomic DNA. Duffy pro­duces two products, namely major β glycoprotein Duffy and minor α glycoprotein Duffy (see Fig. 2) [15].

Figure 2. Map of Duffy blood group gene with two exons (exon 1 and exon 2) separated by an intron. Nucleotide changes at specific positions are indicated by variations in restriction enzyme sites (Ban I, MspA1 I, Mwo I). Lower part illustrates FY*B and FY*A alleles with amino acid variations at key positions, affecting expression of Duffy blood group antigens [15]

The Fy (a–b–) phenotype, also known as silent erythrocytes (FY*ES), occurs in African lineages with a prevalence of almost 100%, especially in West Africa, and more than 80% frequency in Africa and the Americas. This phenotype is caused by homozygotes carrying the FY*B allele with the point mutation c.1-67T>C in the 5’ untranslated region, causing the formation of the FY*BES allele (Fy 02N.01), which impairs promoter activity in erythrocytes by disrupting the binding site of the GATA transcription factor-1. Similar mutations have been described previously, such as c.-33T>C and c.-46T>C, which were found in the FY*AES allele (Fy 01N.01) but only in a heterozygous form in Papua New Guinean and Sudanese populations [7]. Recently, a new mutation was discovered at position c.1-69 in the Duffyneg promoter, which also disrupts the GATA motif and is associated with suppression of the FY*A allele, causing the Fy (a–b–) phenotype [17].

Antigen carrier molecules

DARC, a receptor on erythrocytes, can bind various chemokines, including IL-8 and melanoma growth stimulatory activity (MGSA). The DARC structure crosses the membrane seven times and has 63 extracellular amino acids. The N-terminal portion has two potential N-glycosylation sites, while the C-terminal portion is located in the cytoplasm. This structure is typical of G protein-coupled receptors, including chemokine receptors (see Fig. 3).

Figure 3. DARC, an erythrocyte receptor for chemokines such as IL-8 and MGSA, spans membrane seven times, has 63 extracellular amino acids, two N-glycosylation sites in N-terminal domain, and a C-terminal cytoplasmic domain, characteristics of a G-protein coupled receptor [18]

Chemokines, which are divided into three main classes: Cysteine — any amino acid — Cysteine (C-X-C), Cysteine — Cysteine (CC), and Cysteine (C), are usually bound by class-specific chemokine receptors. However, DARC is a promiscuous receptor that can bind C-X-C and CC chemokines. Examples of C-X-C chemokines are IL-8 and MGSA, whereas CC chemokines are regulated upon activation, such as MCP-1 expressed by normal T cells. DARC does not bind class C chemokines such as lymphotactin. These chemokine receptors, which are Duffy blood group antigens on erythrocytes, have been shown to bind chemokines of classes C-X-C and CC and malaria parasites such as P. vivax and P. knowlesi. DARC is also detected as a transmembrane protein in endothelial cells and may play a role in receptor-mediated endocytosis, which may be important for generating the chemotactic gradient necessary to attract leukocytes [16, 18].

Relationship of negative Duffy mechanism to vivax malaria

P. knowlesi is used in malaria research to understand individual resistance mechanisms to P. vivax. In Africa and the Americas, there are significantly higher levels of resistance to P. vivax strains from different geographic regions. One study involved blood from individuals in Africa and the Americas infected with P. vivax and from individuals resistant to this infection for injection in the same groups. The results showed that individuals infected with P. vivax remained infected despite receiving 3–7 or more injections, whereas resistant individuals did not develop parasitemia in the blood. From these results, it can be concluded that P. vivax strains cannot adapt to infect-resistant individuals [19].

P. vivax DBP is a 140-kDa protein similar to the Duffy Binding-Like Erythrocyte Binding Protein (DBL-EBP) located in Plasmodium merozoite organelles. The 330-amino acid cysteine-rich portion of DBP is thought to be the protein domain responsible for Duffy binding to erythrocytes. The main similarity between DBL-EBPs is two cysteine-rich domains in regions II and VI. The gene encoding P. vivax DBPII is highly polymorphic, with distinct geographic variations from one area to another. This polymorphism suggests that high selection pressure on DBP and allelic variation function as immune response mechanisms [14].

Studies of multispecific chemokine receptors on erythrocytes show that IL-8 minimally binds to Fy (a–b–) and monoclonal antibodies to the Duffy antigen. Inhibition of IL-8 binding with the Duffy antigen also prevents erythrocyte binding and invasion by P. knowlesi. This proves that the Duffy blood group antigen functions as a chemokine receptor. In addition, it has been found that mutations in the promoter region of the DARC gene in erythrocyte precursor cells can prevent expression in erythrocytes, and cause complete resistance to P. vivax infection [18].

Merozoite invasion involves the uptake of proteins from organelles and their fusion with merozoites, which target erythrocytes. This process serves to reduce DBP exposure to the immune system. Humoral immune responses to DBP in human populations show that anti-DBP antibodies increase with exposure to P. vivax. This immune response involves antibodies that can inhibit the adhesion of DBPII to its receptor on erythrocytes. The same antibodies that blocked the DBPII-DARC interaction also inhibited erythrocyte invasion by P. vivax, proving that P. vivax antibodies (anti-FvDBP) can inhibit merozoite invasion. Children living in P. vivax endemic areas will develop anti-DBP antibodies that inhibit infection and protect at the blood stage of the disease. More than 90% of people in sub-Saharan Africa do not express DARC, so P. vivax has limited regional distribution [14].

The BPD protein is an antigen with excellent potential for malaria vaccine production, although its use as a vaccine model is rare. Given its crucial role in erythrocyte invasion by merozoites, the host immune response to P. vivax DBP is essential in eliciting immunity. This acquired immunity may be partly due to the production of anti-BPD antibodies that block binding to erythrocyte receptors. In support of this model, an increase in the proportion of antibodies recognizing recombinant Duffy Binding Protein (rDBP) has been observed, along with a decrease in the prevalence and intensity of P. vivax infections [14].

Antibody accumulation against linear epitopes correlates closely with rDBPII antibodies. This suggests that antibodies directed against some linear epitopes also recognize DBPs expressed on the merozoite surface. Based on current knowledge, innate resistance to malaria infection in humans is associated with blood group polymorphisms [14]. Erythrocytes with the Duffyneg blood group resist in vitro invasion by P. knowlesi. The ability of the malaria parasite P. vivax to infect erythrocytes depends on the expression of the Duffy blood group antigen on erythrocytes. In vitro studies with P. knowlesi show that invasive merozoites can bind and orient themselves apically with Duffyneg erythrocytes, but cannot form the junctions necessary for invasion as occurs with erythrocytes with the Duffy antigen. This suggests that the Duffy blood group is required for P. vivax invasion. In P. knowlesi parasite culture supernatants, the parasite ligand that binds the Duffy blood group antigen has been identified as DBP1 [20].

P. vivax DBP1 is known to have the ability to bind to the Duffy antigen but not to Duffyneg erythrocytes. The part of DBP1 that allows binding to Duffy is a cysteine-rich region. Duffy blood group antigen binds DBP1 via a sulfated tyrosine present in its first extracellular domain [20]. Miller’s research, as cited by Zimmerman [19], examined in vivo the susceptibility to malaria of individuals with the Duffyneg blood type and those with other Duffy antigens. The results showed that of 17 volunteers from Africa, America, and the Caucasus, none of the five individuals with the Duffyneg blood group developed blood parasitemia, even though daily blood smear evaluations were carried out for 90–180 days. In contrast, 12 individuals who had the Duffy antigen developed blood infections within 15 days.

In populations of individuals of African and American descent, expression of the Duffy antigen is stopped due to the presence of an single nucleotide polymorphism (SNP) in the GATA box sequence of the DARC gene in its promoter region, where cytosine replaces thymine 46 nucleotides before the erythrocyte cap site [21]. A single point mutation in the GATA1 binding sequence of the promoter region (T-42C, rs2814778) is responsible for the Duffyneg phenotype. This mutation leads to suppressed gene expression at the erythrocyte level, preventing the production of Duffy proteins on the surface of erythrocytes. Lack of DARC expression on erythrocytes is effective in preventing P. vivax infection. Additionally, recent research suggests that individuals heterozygous for this mutation may have reduced expression of the DARC gene, which provides partial protection against P. vivax infection [22].

The specific sequences of P. vivax form a phylogeny, likely due to relatively recent demographics. The Time to the Most Recent Common Ancestor (TMRCA) estimate for the P. vivax sequence is 70–250,000 years ago, which is in line with previous estimates of 50–500,000 years ago. Since the TMRCA of P. vivax previously overlapped with the TMRCA of Duffyneg, this supports the hypothesis that P. vivax may be the selective agent responsible for the increase in Duffyneg in Africa.

However, two possible hypotheses could explain the TMRCA estimates for P. vivax. Firstly, human TMRCA in P. vivax represents the beginning of the relationship between host and parasite, marking the onset of selective pressure on the host. And secondly, these estimates overlap with a period when individuals outside Africa currently infected with P. vivax may have originated from recent migrations [22].

Conclusions

Based on the results of the discussion regarding the mechanism of negative Duffy resistance to vivax malaria, it can be concluded that: the Fy (a–b–) phenotype is often found in native African populations; there is a relationship between FY and resistance to vivax malaria; the ability of the P. vivax parasite to attack erythrocytes depends on antigen expression Duffy in erythrocytes because mutations in the promoter region cause suppression of gene expression; FY does not produce Duffy protein on the surface of erythrocytes; and that a lack of gene expression in these erythrocytes is effective in preventing P. vivax infection.

Article information and declarations

Acknowledgments

Not applicable.

Authors’ contributions

BAS — conceived work, wrote initial draft, and edited work; YRT — conceived work and reviewed; HNM — reviewed and edited work.

Conflicts of interest

The authors declare no conflict of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Supplementary material

The online version does not contain supplementary material.