Background
Malaria is the most prevalent communicable disease in Ethiopia, with 75% of the country’s landmass classified as malaria-endemic [
1]. This disease has caused tremendous human suffering and major negative effects on economic productivity. From 2007 to 2008, malaria accounted for 10% of all hospital admissions and for ~15% of the overall disability adjusted life years (DALYs) lost in the country [
1,
2]. The malaria morbidity reported by the Ethiopian Government and World Health Organization (WHO) may underestimate the actual burden due to the lack of epidemiological data, in addition to poor health infrastructure and accessibility in the country [
3,
4]. The problem is compounded by the presence of multiple malaria parasite species [
4,
5], increasing drug resistance in the parasites [
6,
7] and insecticide resistance in the mosquito vectors [
8,
9]. Across the country,
Plasmodium falciparum and
Plasmodium vivax account for approximately 60 and 40%, respectively, of infected cases [
3-
5]. Nonetheless, information on epidemiological significance, i.e., the distribution and clinical prevalence of
P. falciparum and
P. vivax malaria in endemic areas is still insufficient.
Natural selection in malaria-endemic regions may have favoured individuals who lack the Duffy blood group antigen on the surface of their red blood cells because of the conferred resistance to certain malaria parasites [
10-
13]. The Duffy antigen receptor for chemokines (
DARC), also known as Fy glycoprotein, belongs to a family of silent heptahelical chemokine receptors [
10].
Plasmodium vivax and
Plasmodium knowlesi require this protein to infect red blood cells during their asexual blood stage, while
P. falciparum uses a different set of receptors to gain access to the cell [
14,
15]. A point mutation, T-33C, located in a GATA-1 transcription factor-binding site of the
DARC gene promoter can lead to failure of Duffy antigen expression on the surface of red blood cells in humans [
10]. The absence of a receptor for the pathogen confers resistance to
P. vivax malaria [
10,
16]. The rare presence of
P. vivax malaria in western or central Africa is likely attributed to high Duffy-negativity among African blacks (88-100%) [
17,
18]. However, this interpretation is challenged by recent findings of
P. vivax infection in Duffy-negative people in different parts of Africa [
19-
24] and the Brazilian Amazon region [
25,
26]. These data support the hypothesis that
P. vivax may have evolved the capability to infect Duffy-negative red blood cells and that the parasites are more prevalent and widespread than reported previously.
There has been a number of population-based studies of
P. vivax infections in Duffy-negative individuals among clinical and community samples [
19-
21,
23-
27]. Accurate information on the distribution and clinical prevalence of
P. vivax and
P. falciparum malaria in endemic areas, as well as in Duffy-negative populations, is essential to develop integrated control strategies and to more broadly evaluate the magnitude of the ‘derived’
P. vivax invasion. The present study defines the epidemiology of
P. vivax and
P. falciparum malaria in large areas of Ethiopia with three specific questions: (1) whether there are variations in the geographical distribution of
P. vivax and
P. falciparum malaria across Ethiopia; (2) is there a difference in the prevalence of
P. vivax and
P. falciparum malaria between age groups in local communities; and, (3) what is the frequency of
P. vivax infection in the Duffy-negative populations? Furthermore, the parasite gene copy number between symptomatic and asymptomatic infections of
P. vivax and
P. falciparum were compared with the goal to evaluate the performance of a quantitative real-time PCR (qPCR) method for detecting high and low parasite density samples. This is of key relevance in providing accurate epidemiological data in local communities with mostly asymptomatic infections.
Discussion
Approximately 35% (139/390) and 22% (94/416) of the community and clinical samples analysed in this study are negative for the Duffy antigen. The proportion of Duffy-negatives observed in the clinical samples is similar to an earlier study that reported an average of 20% homozygous Duffy-negative patients in Harar and Jimma [
23]. In contrast, the proportion of Duffy-negatives observed in the community is over one-third more than that in the clinical samples. However, these findings are still lower than the proportion of Duffy-negatives documented in West and Central Africa (>97%) [
17,
18]. Four
P. vivax infections were identified among the Duffy-negative samples, one of which was from Jimma, one from Mankush, and the other two from Asendabo. Two of these infected samples were obtained from individuals with malaria symptoms and had mixed infections; and qPCR showed that all these samples had a relatively low number of parasite gene copy. These findings confirm previous studies that documented a number of
P. vivax infections in Duffy-negative individuals in Cameroon [
19], Madagascar [
20], Angola [
21], Equatorial Guinea [
21], Ethiopia [
23], Mauritania [
24], as well as the Brazilian Amazon region [
25,
26]. Although these studies collectively are consistent with the conclusion that Duffy-negative individuals are not completely resistance to
P. vivax infection, the observation of low
P. vivax GCN in the present Duffy-negative samples supports the hypotheses that the infectivity of the parasite to human erythrocyte is reduced in the absence of the Duffy antigen. It is worth mentioned that the identification of Duffy phenotypes was inferred solely based on the
DARC genotypes but without direct measure of antigen expression phenotypes of the erythrocytes. Thus it is not entirely impossible for a Duffy receptor to be present on the erythrocyte surfaces of a genotypically Duffy-negative individual and that the
P. vivax strains use such to invade the erythrocytes of Duffy negatives in this study.
The mechanism of
P. vivax erythrocyte invasion in Duffy-negatives is not yet fully understood [
11,
13,
32]. Apart from the Duffy antigen, there are several tryptophan-rich antigens that play important role to the survival and growth of malarial parasites in the host [
33-
35]. For example, one of the
P. vivax tryptophan-rich antigens PvTRAg33.5 has been previously shown to induce immune responses in humans and binds to host erythrocytes [
36]. Recently, 10 of 36 PvTRAgs of the Pv-fam-a family were reported to possess erythrocyte-binding activity [
35]. Based on transcriptome data, a number of the erythrocyte-binding PvTRAgs including PvTRAg, PvTRAgs, PcTRAg36.6, and PvTRAg69.4 were found in the early stage of the parasite and involve in the rosetting phenomenon [
35]; while others including PvTRAg35.2, PvTRAg38, PvTRAg36, and PvTRAg34 were found to express at the late merozoite stage of the parasite that can recognize more than one erythrocyte receptor and help the parasite to invade the host erythrocytes [
35]. It appears that the binding of each of the antigens/ligands to different receptors as well as the recognition of each receptor by more than one parasite ligand could be advantageous to
P. vivax. The parasite can use the redundancy in the receptor-ligand interaction as an alternate invasion pathway or for tightly binding to its host cell during the invasion or rosetting process even in the absence of a Duffy receptor on the erythrocyte surfaces. It merits further investigations on whether the alternate receptor-ligand interactions that allow erythrocyte invasion evolve independently among the
P. vivax lineages and whether the newly derived
P. vivax strain has spread among endemic regions subsequent to its emergence.
The geographical distribution of the two
Plasmodium species varies among sites in Ethiopia. Sites in the southwest (Halaba and Jimma) had a greater proportion of
P. vivax than
P. falciparum infections, whereas sites in the north (Mankush and Shewa Robit), with the exception of Bure, had a greater proportion of
P. falciparum than
P. vivax infections
. The predominance of either
Plasmodium species was reported previously in other parts of the country [
23,
37-
39], and this appears to be dependent on the study population and the season of sampling. Samples of this study were collected from September-November 2013, during the peak season of malaria transmission in Ethiopia. It is unlikely that the difference in the distribution of the two
Plasmodium species is due to seasonal characteristics of
Plasmodium infection in Ethiopia, but rather to the variation in climatic conditions among sites [
3,
40,
41]. Despite the fact that the studied area was set in the highlands, sites at lower latitudes (Halaba and Jimma) may experience warmer and more humid weather than sites at higher latitudes (Mankush and Shewa Robit); such climatic variations may influence transmission and distribution of the
Plasmodium species. Another possible explanation is that difference in the age distribution of the infected individuals may in part influence the distribution of the two
Plasmodium species. While such an effect is demonstrated by the community samples that indicated a greater prevalence of
P. vivax infection in children and adolescents than in adults, the prevalence among the clinical samples were not significantly different between the two age groups.
Complicated and severe clinical malaria as well as the prevalence of asymptomatic infections have been previously shown to be highest in young children [
42-
44]. The acquisition of immunity is age-dependent and young children are most represented among malaria-diagnosed deaths in many African countries [
45]. Consistent with previous findings, the present study shows that children aged from 0–5 years old have the highest
P. vivax (13.9%) and
P. falciparum prevalence (18.1%) than in adolescents (6.3% and 7%, respectively, for
P. vivax and
P. falciparum) and adults (2.6% and 14.2%, respectively, for
P. vivax and
P. falciparum). Children and adolescents appear to be more prone to
P. vivax infection than adults, and the reason for this is not known. One possible explanation is that
P. vivax can stay dormant in the liver of the adults for a longer time and remain undetected at the time of sample collection. Also, the difference in the level of immunity between children/adolescents and adults may in part influence the invasive capability of the malaria parasite species [
45].
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
EL, DY, EZ, ML, and GY designed the research and collected the samples. EL, DZ, TD, KT, and MH collected the data and performed the analysis. EL, DY, and AJ drafted and edited the manuscript. All authors were involved in the interpretation and discussion of the results, and provided comments and approved the manuscript.