Background
Malaria is a major public health problem in sub-Saharan Africa, and is responsible for over half a million deaths annually, especially in children under the age of 5 years [
1]. Four major species of the protozoan parasite,
Plasmodium, (
Plasmodium
falciparum,
Plasmodium vivax,
Plasmodium malariae,
Plasmodium ovale) cause human malaria in sub-Saharan Africa. In malaria-endemic countries, there is an overlap in the geographic distribution of the different
Plasmodium species and the
Anopheles mosquito vectors that transmit these parasites, and that individuals may be exposed to, and harbour multiple
Plasmodium species [
2]. However, the epidemiology of the different
Plasmodium species in endemic human populations is not well documented [
3]. Diagnosis of malaria in endemic clinical settings is predominantly by the ‘gold standard’ blood film microscopic examination, and rapid diagnostic tests (RDT), both of which lack sensitivity in differentiating the species of
Plasmodium causing malaria. Microscopic examination has several limitations such as the inability to detect low levels of parasitaemia, and the difficulty in species differentiation owing to subtle differences in the morphology of blood stage parasites [
4]. This results in the species of
Plasmodium causing disease being rarely reported, and almost all cases of malaria are therefore attributed to
P.
falciparum, the species causing the most serious form of malaria [
3]. This has led to underestimates of the prevalence of both mixed-species and non-falciparum species infections [
5]. These non-falciparum species are of significant clinical importance; for example,
P.
vivax and
P.
ovale which form latent liver stage ‘hypnozoites’ are capable of causing disease several months or years after the primary infection [
6]. Incidences of the diagnosis of systemic diseases caused by
P.
malariae several months or years after people have returned from malaria-endemic regions have been reported [
7]. In some cases, drug treatment failure attributable to the misdiagnosis of primary infections caused by the non-falciparum species or as co-infecting species with
P.
falciparum have been observed [
8]. While PCR typing of infecting
Plasmodium species is not frequently available or applicable in many African field settings, most RDTs may not differentiate non-falciparum species [
3]. There is an urgent need for additional diagnostic tools [
2‐
4] capable of rapid detection of all four infecting
Plasmodium species for effective treatment and control of malaria.
In this study, a new assay has been developed that detects exposure to all four human
Plasmodium species based on serum antibody responses to merozoite surface protein 1 (MSP-1). The surface of the invasive merozoite is coated in MSP-1 that constitutes 31 % of the GPI-anchored proteins on
P. falciparum merozoites [
9]. MSP-1 is expressed by all four human
Plasmodium species. In
P. falciparum, MSP-1 undergoes two proteolytic cleavages resulting in a C-terminal MSP-1
19 fragment that is carried into the erythrocyte during merozoite invasion [
10,
11]. Until recently, only the MSP-1 genes of
P.
falciparum and
P.
vivax had been characterized. Recently, the sequences of the MSP-1
19 gene fragments for
P.
malariae and
P.
ovale have been determined with limited characterization of the responses to these parasite proteins [
12]. Although the gene sequences of MSP-1
19 antigens are unique to each of these four
Plasmodium species, extensive homology can be found among them. The number and relative positions of cysteine residues within the C-terminus fragments of MSP-1
19 are comparable in all four
Plasmodium species [
12]. For example, there are about 32 amino acid sites within the MSP-1
19 gene where all four parasite species share the same amino acid, and about 30 sites where the same amino acid is conserved in two or three species (Additional file
1: Figure S1). To date, there has been no field study using MSP-1
19 antigens from all four malaria parasite species to characterize the epidemiology of exposure to
Plasmodium in any African population.
In Zimbabwe over half of the population are exposed to malaria, with
P.
falciparum being the predominant species, accounting for almost all cases of the disease [
13]. There is little epidemiological data of exposure to non-falciparum species and/or mixed
Plasmodium infections in Zimbabwe. The aim of this study is to determine the species specificity of IgG antibody responses to recombinant
Plasmodium MSP-1
19 antigens in three meso-endemic villages of Zimbabwe: Burma Valley, Mutoko and Chiredzi. Using these antigens as diagnostic tools, this study describes the sero-epidemiology of multiple
Plasmodium species infections in these study sites.
Discussion
In this current study, IgG responses to recombinant MSP-1
19 antigens (an indication of prior exposure to
Plasmodium antigens) from the four major human
Plasmodium species were evaluated in three Zimbabwean villages with meso-endemic malaria transmission dynamics. Individuals living in malaria-endemic regions may harbour multiple
Plasmodium species owing to the geographical overlap of the four major human
Plasmodium species [
4,
19,
20]. Malaria diagnosis in most African field settings is largely by microscopy of blood films, which reports the presence or absence of
Plasmodium parasites without cognisance to the species causing disease. Low-level parasitaemia of the non-falciparum species in mixed infection with
P.
falciparum accounts for the misdiagnosis of these species. There have been case study reports of treatment failures [
21] and acute renal injury [
22] attributable to undiagnosed
P.
malariae infection or co-infection. Knowledge of the type of infecting species is therefore essential for effective treatment as well as the implementation of control programmes. IgG responses to
P.
falciparum MSP-1
19 antigens have been shown to rise following clinical episodes of malaria and decline in the absence of the disease [
15]. In the current study, the antibody response to
Plasmodium species recombinant MSP-1
19 antigens in humans was seen to be highly species-specific. Furthermore, the study showed that the responses were not cross-reactive, despite the amino acid sequence similarities between the four
Plasmodium MSP-1
19 antigens. In experimental monkey and human studies utilizing all four
Plasmodium species MSP-1
19 antigens, a superior sensitivity was seen when compared to commercially available antibody assays which only utilize MSP-1
19 antigens from
P.
falciparum and
P.
vivax and depend on cross-reactivity in detecting the other two species [
23]. The specificity of antibodies to these antigens supports the evidence that these antigens could be used in pan-malaria diagnostic assay to enable the rapid detection of the type of
Plasmodium species causing malaria [
23].
In many malaria-endemic countries in sub-Saharan Africa,
P.
falciparum is the predominant species that causes malaria, thus it was not surprising that the antibody response to
P.
falciparum MSP-1
19 antigens was predominant in all three study sites. Since
P.
falciparum infections have higher parasitaemias than the other malaria parasite species [
20,
24], it is likely that individuals will have a stronger immune response to
P.
falciparum infection. The novel results from this study were the indication that the exposure prevalence of
P.
malariae and
P.
ovale is higher, as previous reports have attributed about 98 % of malaria in Zimbabwe to be caused by
P.
falciparum. More importantly, the observed higher exposure prevalence of
P.
malariae in the Burma Valley district was striking, as reports suggest that this species only accounts for between 1 and 2.6 % of all malaria cases by light microscopy [
25‐
27].
Microscopy has long been known to underestimate the prevalence of the non-falciparum species owing to difficulties in distinguishing the subtle differences in the morphology of the different species as well as the challenge posed in detecting minority species in a blood film with high density
P.
falciparum parasitaemia. It is therefore not surprising that these assays detected a higher sero-prevalence of these species, as this also reflects recent and concurrent parasite exposure. Studies employing nucleic acid based techniques for
Plasmodium parasite detection and species identification in some African countries have reported prevalence of the non-falciparum species to be between 1 and 17 % [
20,
24].
While antibody responses to single species
P.
falciparum antigens were common, single species responses to
P.
malariae and
P.
ovale antigens were infrequently detected. A significant proportion of individuals with IgG responses to
P.
malariae and/or
P.
ovale MSP-1
19 almost always had responses to
P.
falciparum MSP-1
19. This results support the findings of a recent study in Ghana, which reported frequent detection of
P.
malariae and
P.
ovale in individuals who are also PCR positive for
P.
falciparum [
28]. The reasons for this co-occurrence of the non-falciparum species with
P.
falciparum may be both epidemiological and biological. Of the epidemiological reasons, it has been suggested that the same
Anopheles mosquito circulating in a population might be responsible for the simultaneous or sequential inoculation of the different species [
4], thereby increasing the likelihood of multiple species infections. Biological reasons may include selective advantages for these minor species when co-infecting with
P.
falciparum. For example, due to density-dependent regulation of immune responses directed against the majority species (
P.
falciparum), these non-falciparum species may be able to evade host immune responses and establish disease [
29,
30]. There are parallels in other infectious diseases, such as the obligate satellite virus hepatitis D, which is unable to establish disease independent of hepatitis B virus [
31]. Hepatitis D virus co-infection in Hepatitis B-infected individuals worsens hepatic damage and inflammation, and is more likely to lead to hepatocellular carcinoma [
32,
33]. The results show some single species
P.
malariae responses, indicating that this species is capable of establishing infection independent of other
Plasmodium. However, the significant proportion of individuals with co-occurrence of antibody responses to
P.
falciparum suggests a possible dependency on
P.
falciparum receptors or proteins for successful disease by
P.
malariae. These non-falciparum species, which usually exist as part of a complex mixed-infections with
P.
falciparum [
2,
34] may cause chronic, sub-clinical disease with potential health consequences, including treatment failure, disease relapse and long-term systemic consequences [
5‐
7]. A recent study in Indonesia found
P.
malariae to be associated with a lower mean haemoglobin, nephrotic syndrome and death [
27].
Antibody responses to
P. vivax MSP-1
19 were rarely observed in this study.
Plasmodium vivax requires the Duffy antigen to establish a successful infection [
35], and is predominantly endemic in Asian and Latin American countries. It has long been known that the Duffy antigen is absent in most African populations [
35]; it was therefore not surprising to observe a low frequency of responses to
P.
vivax MSP-1
19. In recent years however, there have been reports of
P.
vivax infections in both Duffy positive and negative individuals in Cameroon [
36] suggesting that this species might have evolved and adapted to using other receptors to invade erythrocytes and establish disease.
Serological responses generally increase with age. In this present study, age was not a confounding factor in Burma Valley and Mutoko, while is Chiredzi responders 10 years old and above had a higher overall exposure prevalence to parasite antigens. Although all three villages are described as meso-endemic, the observed differences in age responses could be due to the respective transmission dynamics of the different seasons in which sampling was done. In very low and unstable malaria transmission areas such as Daraweesh in eastern Sudan, reports suggest that the age dynamics associated with malaria and serological responses are not apparent, as malaria affects all age groups [
37,
38].
Authors’ contributions
FM, DRC and SAA conceived, designed and performed the experiments: FM, NM and TM participated in the fieldwork: FM, DRC, SAA, NM, and TM contributed to draft manuscript editing/reviewing. All authors contributed to the revisions. All authors read and approved the final manuscript.