Background
For over 50 years various strategies have been developed to reduce the burden of
Plasmodium falciparum, the most virulent malaria species and the primary cause of malaria-related mortality across the globe [
1]. International funding programmes have first tasked the eradication of this disease and, as a follow-up for regional elimination, have provided tools such as insecticide-treated bed nets (ITNs), indoor residual spraying (IRS), artemisinin-based combination therapy (ACT), and rapid diagnostic tests (RDTs) to many malaria-endemic regions. This report focuses on the implications of genetic diversity in the
pfhrp2 gene that may compromise the use of RDTs for the diagnosis of malaria.
In 2006, the Senegalese National Malaria Control Programme (NMCP) scaled up the use of malaria RDTs at all health facilities in Senegal for case management [
2] with the ultimate goal being to treat all (and only) true malaria infections, as misdiagnosis of malaria can contribute to drug resistance [
3]. The number of RDTs currently used in Senegal is between 900,000 and 1,200,000 annually [
4]. RDTs are ideal for rural conditions in developing countries where well-equipped health facilities and expert microscopists are lacking, as they give rapid results, require no electrical equipment or temperature-sensitive reagents, and can be performed without extensive training. The technical performance of these tests has been described previously [
5], and although they are sensitive and specific to detect most cases of malaria at parasitaemia greater than 200 parasites/μl, product performance can vary widely at low parasitaemia [
6], and these tools may miss individuals harbouring a very low parasitaemia.
RDTs differ in the antigens they detect. Some are based on detection of the
P. falciparum specific histidine-rich protein-2 (PfHRP2), or
P. falciparum specific lactate dehydrogenase (PfLDH), while others recognize antigens common to
P. falciparum,
Plasmodium vivax, Plasmodium ovale, and
Plasmodium malariae (pan-species pLDH and aldolase) [
7].
Plasmodium aldolase and lactate dehydrogenase (pLDH) are highly conserved RDT targets, [
8,
9] in contrast to the
P. falciparum PfHRP2 which is considered to be more variable [
10,
11]. In Senegal, HRP2-based RDTs are most commonly used in health facilities because of their previously demonstrated sensitivity and specificity and because
P. falciparum represents the major
Plasmodium species (more than 98%) in Senegal [
12], with
P. malariae and
P. ovale comprising less than 2% of malaria cases
, and no
P. vivax (0%) observed in 2013 [
4].
The
pfhrp2 gene of the
P. falciparum 3D7 is localized on chromosome 8 (PF3D7_0831800, PlasmoDB Version 9.3) and contains two exons separated by an intron. The first exon encodes a signal peptide and the second exon contains a protein-export motif followed by specific histidine-rich repeats, which have been described previously [
10,
11]. PfHRP2 is synthesized throughout the asexual life cycle and in early sexual stages of
P. falciparum[
13]. The protein is characterized by the presence of a number of variable tandem repeats, each one named with a Type based on the motif being repeated [
10,
11].
Specific amino acid repeats AHHAAD (Type 7), AHHAHHAD (Type 2) have been described as possible epitopes targeted by the monoclonal antibodies used to detect HRP2 in some RDTs [
14]. Many studies of the genetic diversity of this antigen show extensive variation within isolates of the same country and between isolates of different countries [
10,
11,
15]. In Senegal, the genetic diversity of the gene target by the RDT has not yet been investigated in depth. The goal of this study is to determine the extent of genetic polymorphism in the
pfhrp2 gene in a Senegalese population in comparison with two other diverse African populations: Mali and Uganda. Indeed, previous reports from Mali have indicated the loss of the HRP2 locus from some parasites, and this study investigates whether changes in this locus among Senegalese parasites may compromise the performance of the RDTs so widely used for malaria diagnosis.
To accomplish this goal, the pfhrp2 gene was sequenced using patient samples from Senegal, and along with available sequencing data from Mali and Uganda (three endemic African countries with different malaria ecologies and epidemiology), was used to assess the nucleotide polymorphism as well as the distribution of specific PfHRP2 amino acid repeats.
Discussion
Plasmodium falciparum tests targeting the PfHRP2 antigen have demonstrated high detection rates [
26], however test performance showed variability between lots and between similar products. Recently
P. falciparum genetic factors have been investigated as a possible cause of variability in RDT detection rates [
15,
25,
27]. As genetic diversity in
P. falciparum is associated with transmission intensity, such findings could significantly interfere with the ability to accurately detect all malaria cases, and therefore have great potential to impact control strategies and efforts to eliminate or eradicate the disease [
28]. For this reason, it has become increasingly important to characterize the diversity of target antigens used in RDTs to predict the impact genetic variation has on diagnosis, since it has the potential to affect the sensitivity of these diagnostic tests. In this study, the genetic diversity (π and π
NS) in the
pfhrp2 gene was described and polymorphisms in the PfHRP2 protein among parasites obtained from human infections in three African countries with distinct patterns of malaria transmission were analyzed, to provide additional information about the diversity of this locus to that described previously [
10,
11,
25].
Here, the global nucleotide diversity π values calculated for
pfhrp2 in the Senegal, Mali and Uganda isolates are similar to highly polymorphic genes families exposed to strong immune pressure, such as
msp1 and
msp6, since these genes are highly polymorphic [
29]. In addition, the π
NS for the
pfhrp2 is high, similar to π
NS for genes encoding the polymorphic SURFIN antigens expressed on
P. falciparum merozoite and infected erythrocytes [
30]. The
pfhrp2 gene is located in the subtelomeric region of chromosome 8 within a dynamic area where recombination events frequently contribute to the high genetic variation observed among genes from these regions [
31]. In addition, the
pfhrp2 gene reveals differential polymorphism by geography, with distinct population profiles of synonymous and non-synonymous SNP variants, with over half these variants being synonymous SNPs. This is particularly important because non-synonymous SNP may destabilize protein structure or interfere with either ligand binding or the formation of domain-domain interfaces [
32] and these changes may affect the ability of monoclonal antibodies to detect PfHRP2, thus compromising the sensitivity of the RDT.
Insertions and deletions of nucleotides observed in this study provided similar level of polymorphisms as SNPs in
pfhrp2. Previous studies have reported deletion of the
pfhrp2 gene associated with false-negative HRP2-based RDTs in Mali, suggesting that spontaneous
hrp2 deletions could be one mechanism of genetic variation responsible for RDT failures [
27]. Such deletions have been reported before in South America, where 15/275 parasites (5.5%) were found with deletions of the
pfhrp2 gene, generating concerns over the reliability of PfHRP2-based RDTs in these regions [
33]. Furthermore, genetic deletions were not restricted to the
pfhrp2 alone, but were found to extend into neighbouring genes such as Hsp70 (PF3D7F_0831700) in the 3’ region of
pfhrp2 and in 5’ a pseudo-gene encoding a
Plasmodium exported protein (PF3D7F_0831900) [
34]. Such data emphasize the importance of precisely mapping deletions in
pfhrp2 in parasites from Senegal and other geographic regions.
It is important to note that the genomic DNA samples used in this study were obtained from culture-adapted
P. falciparum isolates (and additionally from single clone infections). Further studies should focus on DNA obtained directly from patients as many infections are polygenomic, and also culturing parasites can lead to artifactual differences resulting from the process of culture adaptation, such as breakage of
P. falciparum chromosomes occurring frequently in the
pfhrp2, a subtelomeric gene [
35]. However, the number of isolates was quite small for Mali and Uganda to allow more stringent comparison. Nevertheless, these findings suggest that the parasite differences based upon different geography, and consequently ecology and epidemiology, are important to consider.
At the amino acid level, the PfHRP2 protein showed significant differences, in the organization and the number of repeats, likely due to chromosome breakage and healing occurring randomly in a site-unspecific manner during culture
in vitro[
35]. The composition of the PfHRP2 sequence repeats in Senegalese isolates is similar to Mali and Uganda isolates where the Type 2 (AHHAHHAAD), Type 4 (AHH) and the Type 7 (AHHAAD) are the mostly common repeats (Table
1). The antigen PfHRP2 differs in the composition and the number of repeat types compared to parasites from global endemic countries previously reported [
10,
11,
15]. Specifically, Senegalese PfHRP2 isolates differ from Cameroon isolates (presence of Type 11, and Type 14 repeats), Thailand (presence of Type 14 repeat), Philippines (presence of Type 11 repeat), and Papua New Guinea (presence of Type 14 repeat). In this analysis Type 11 and Type 14 repeats were not observed. Additionally, Type 20 (SHHDD), Type 21 (AHHAHHATY), Type 22 (AHHAHHAGD), Type 23 (ARHAAD), and Type 24 (AHHTHHAAD) repeats reported previously [
10] were not found.
These results agree with previously published studies [
10,
25], which found a high frequency of Type 2 and Type 7 repeats. Ideally, for a universal and sensitive RDT, the target epitope should be present in all parasites, regardless of specific population, and should be present in multiple copies [
14]. For RDT sensitivity, a major factor is the binding specificity and affinity of the monoclonal antibodies for the target epitopes, thus the expression level and number of these epitopes could potentially influence the binding affinity. From these data and that of other studies, the high frequency of Type 2, Type 4 and Type 7 repeats observed in
P. falciparum isolates could contribute to the sensitivity of the RDTs in the African populations studied.
The study of the amino acid composition of the PfHRP2 protein showed that different factors can contribute to polymorphism at the protein level: both the organization of the repeats within sequences and the position of repeats in the antigen. In fact, the frequency or distribution of the target epitopes present in a particular parasite population may have an impact on the efficiency of antigen detection in this population and the sensitivity of RDTs [
10,
15]. These parameters should be taken into account in the choice and the design of monoclonal antibodies that could be used for PfHRP2 based RDTs.
Conclusion
To counter increasing malaria drug resistance and to achieve the goal of malaria eradication, the need for reliable diagnostic tools is critical. This study provides information about the genetic diversity of the pfhrp2 gene and the predicted polymorphism in the PfHRP2 antigen from sequences obtained from three African parasite populations Senegal, Mali and Uganda. For all three populations of parasites, the higher rate of synonymous SNPs in pfhrp2 suggests evolutionary selection of this SNP. However, for Senegal isolates, the π and πNS warrant deeper follow-up investigations as they may represent functional variation, which would potentially interfere with RDT detection. Pfhrp2 is polymorphic at the nucleotide level, shown by both synonymous and non-synonymous SNPs; nucleotide (π) and non synonymous (πNS) diversity similar to highly polymorphic genes such as msp1 and gene encoding SURFIN antigen. PfHRP2 showed extensive repeat length polymorphisms with an occurrence of the Type 2, Type 4 and Type 7 repeats. Similar patterns were observed in the number, organization and the type of amino acid repeats in the protein among the three African populations. However, the PfHRP2 showed variation within isolates of the same country and between isolates of different countries, addressing concern in the efficacy of the HRP2 based RDT test. Since the polymorphism described in the target antigen is a recognized concern, comprehensive studies of possible factors influencing the sensitivity of these diagnostic tools, including genetic deletions and protein expression level, are critical in predicting the efficacy of RDTs in global populations.
Competing interests
The authors declare that they have no any competing interests.
Authors’ contributions
Data analysis, manuscript writing, and PCR re-sequencing were performed by ABD. DP provided the sequencing data for analysis and reviewed the manuscript. AKB gave constructive advice on analysis, assisted with writing and revision of the manuscript. OS, ASB, POG, and AA reviewed the manuscript. SKV performed culture-adaptation of the Senegal and Mali parasites and prepared DNA for sequencing from these parasite populations, and assisted with the writing and revision of the manuscript. DN led the Senegal field collection sites for samples collected. DP and SKV guided the analysis performed by ABD. All authors have read and approved the final version of manuscript.