Background
Malaria remains the most important parasitic infection in the world, with 228 million cases in 2018 (95% confidence interval [CI] 206–258 million) [
1], caused by infection with one or more of the six species of
Plasmodium parasites. Two species,
Plasmodium falciparum and
Plasmodium vivax, are responsible for most of the morbidity and mortality due to malaria globally [
2,
3]. However,
P. vivax malaria does not attract as much attention in almost every aspect as does the deadlier
P. falciparum malaria because, traditionally, the infection was thought to be benign and self-limiting [
4,
5]. Recent evidence is however challenging this long-held notion of the benign nature of
P. vivax malaria, demonstrating that infection with
P. vivax can also result in severe illness and death [
6]. Globally, 53% of the
P. vivax burden is in the WHO South-East Asia Region and
P. vivax is the predominant parasite (75% of malaria cases) in the WHO Region of the Americas [
1]. In 2018, an estimated 704,000 (95% CI 91,000–1,813,000)
P.
vivax malaria cases were reported in Africa [
1].
An important biological difference between
P. vivax and
P. falciparum is that only
P. vivax merozoites use the Duffy (Fy) antigen receptor for chemokines (DARC) to invade erythrocytes [
7,
8]. The DARC-coding gene is polymorphic with multiple alleles as the codominant FY*A and FY*B, which encode for the two antigens—Fya and Fyb. Four genotypes are possible as a result of the combination of the major alleles, Fy(a+b+), Fy(a+b−), Fy(a−b+) and Fy(a−b−) [
9]. The first three correspond to a Duffy-positive phenotype, mostly prevalent in Asian and in Caucasian populations and the last one corresponds to the Duffy-negative phenotype, mainly prevalent in African people, who are consequently deemed to be refractory to
P. vivax infection. The Fy(a−b−) genotype results from a point mutation, − 33 T → C, in the promoter region of allele FY*B, in the GATA box region [
10], preventing transcription and resulting in the null ‘erythrocyte silent’ (ES) phenotype.
Until now, the Duffy-negative phenotype was viewed as giving almost total protection against infection with
P. vivax because it prevents
P. vivax from invading host erythrocytes and completing its complex life cycle [
11]. Field observations indicate that the conclusion of the absolute dependence on the presence of Duffy on the red cell for
P. vivax infection and development in the red cell no longer holds true because of a number of reports concerning findings of
P. vivax in the blood of Duffy-negative persons in Brazil [
12], Ethiopia [
13,
14], Madagascar [
15], Kenya [
16], Equatorial Guinea and Angola [
2] including West African countries, such as Mauritania [
17], Cameroon [
18,
19], Mali [
20], and Benin [
21]. Thus, contrary to expectation, there is evidence of
P. vivax transmission even in areas mapped with highest Duffy-negativity frequencies [
20,
22,
23].
The exact frequency of Duffy blood group is poorly documented across Africa, as indeed few populations have been surveyed and there are large gaps in the documentation on Duffy genotypes and phenotypes across Africa [
24], with Ghana being no exception. Culleton et al. [
25] have concluded that there are sufficient numbers of Duffy-positive individuals in some areas in Africa to maintain
P. vivax transmission in areas where the majority of the population is Duffy-negative. The first objective of the present study was to evaluate the
P. vivax circulation among both symptomatic and asymptomatic outpatients seeking medical care in various parts of Ghana. The second objective was to explore the Duffy antigen genotype frequency among the study population.
Discussion
Until relatively recently,
P. vivax was rarely studied across most of sub-Saharan Africa and malaria diagnostics frequently remained limited to
P. falciparum. Since 2010, however, evidence of the presence
P. vivax in West Africa has emerged [
21,
23,
36,
37] despite the high prevalence of Duffy-negative red blood cell phenotype. Nevertheless, no
P. vivax infections were found in this molecular based study conducted in nine sites across Ghana.
Both symptomatic and asymptomatic outpatients were involved in this study. Malaria is hyperendemic in Ghana and 44% of outpatient visits at the various health facilities are attributed to malaria [
38]. At the community level, fever or a history of fever is presumptively treated as malaria. However, according to [
39], ≥ 75% of infections in malaria endemic areas are asymptomatic. This has been attributed to the development of protective immunity in adult populations against high parasitaemia and clinical disease due to the long-term continuous exposure to mosquito bites [
40]. In Ghana, studies in both low and high transmission areas have found evidence of asymptomatic malaria in adult residents [
41] as well as children [
42] and pregnant women [
43]. Asymptomatic malaria cases have been found to be higher than symptomatic cases in some studies [
42,
44]. Most asymptomatic malaria infections are linked to submicroscopic parasite densities, and require the use molecular diagnostics methods [
40,
45], since conventional microscopy and rapid diagnostic tests (RDTs) are of limited sensitivity.
Though
P. vivax infections cannot be entirely ruled out in Ghana, it is important to note that an earlier study from China [
46] reported a case of a 39-year-old Chinese man who had stayed in Ghana, for 6 months in 2012, for whom a microscopic examination of Giemsa-stained thin and thick blood smears initially indicated
P. vivax infection. However, the results of a thrice conducted rapid diagnostic test were not in agreement with
P. vivax and standard PCR analysis of the SSU rRNA gene, followed by gene sequencing, pointed to a variant
P. ovale wallikeri. Microscopic identification of
P. ovale and
P. vivax due to their morphological similarities [
47] may be unreliable since
P. vivax can be misdiagnosed for
P. ovale infections and conversely [
48]. There is also potential for cross-reactivity between
P. ovale- and
P. vivax-specific antigens in serological screening [
49].
In a 2019 case report also from China [
50], a 49-year-old Chinese man was diagnosed by both microscopy and PCR as having uncomplicated
P. vivax malaria on December 19, 2016. This was 39 days after he returned from Ghana after a stay of one and a half years. However, the Duffy genotype of the Chinese man was not given. The presence of the Fy(a−b−) phenotype outside the African continent and the Arabian Peninsula has been estimated to be at frequencies not exceeding 10% [
22]. It is, therefore, highly likely that the Chinese man is Duffy-positive since the frequency of Fya among the Chinese has been estimated to exceed 97% [
51].
Evidence relating to
P. vivax transmission across Africa appears inconsistent [
49]. In the West African countries were
P. vivax infections have been recorded, Nigeria [
52,
53], Mauritania [
54], Mali [
55], Cameroon [
19], and Benin [
21], the prevalence has been very low from these studies. These studies have varied in terms of sample size and diagnostic methods [
56] and in some reports the Duffy antigen status of the patients was not determined [
36,
52,
54]. As in this present study, extensive surveys using high-sensitivity molecular methods have repeatedly failed to diagnose
P. vivax [
25,
57].
The low prevalence of
P. vivax infection in West Africa has been attributed to the high frequency of the Duffy-negative phenotype in this region [
7,
22,
58]. In this study, 90.5% (862/952) of the malaria patients had the
FYES allele and were classified as Fy(a−b−) in agreement with the report by Howes et al. [
49]. It is clearly obvious that Duffy-negativity provides significant protection against
P. vivax blood-stage infection, particularly in symptomatic patients presenting for treatment, though this protection is not absolute. This is in agreement with long-prevailing thinking that for
P. vivax invasions to occur an interaction between the parasites and antigens of the Duffy blood group system is necessary [
59,
60]. However, several other host cell receptors have recently been identified as being involved in the parasite invasion pathway of RBCs. Gruszczyk et al. [
61] identified host transferrin receptor 1 (TfR1 or CD71) as an alternative receptor, critical for
P. vivax entry into reticulocytes. CD98 has also been shown to be involved in entrance of the parasite into the host cell [
62]. Lack of the Duffy antigen thus seems to places a certain restriction on the invasion mechanism, but not completely. A better understanding of the mechanisms that allow interaction between
P. vivax and the Fya and Fyb host antigens may allow more specific assessments of the risks of
P. vivax infection and clinical disease across the Duffy-negative populations previously considered fully protected, as well as identifying potential vaccine targets.
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