Background
Malaria diagnosis with rapid diagnostic tests (RDTs) has increased dramatically since the 2010 World Health Organization (WHO) recommendation that all suspected malaria cases be confirmed with a parasite-based test [
1]. Globally, over 340 million malaria RDTs were sold by manufacturers in 2019 [
2]. Due to the high prevalence of
Plasmodium falciparum in sub-Saharan Africa, a majority of RDTs used in this region target that parasite, with most detecting the
P. falciparum-specific antigen histidine rich protein 2 (HRP2). HRP2 is abundantly produced by the parasite [
3,
4] and is highly stable within and outside the host, including retention in previously infected RBCs [
5,
6]. Therefore, RDTs targeting HRP2 are more sensitive than RDTs targeting lactate dehydrogenase (pLDH) or aldolase, the other parasite antigens used in malaria RDTs [
7]. As a result, and in accordance with global malaria guidance, most countries in sub-Saharan Africa with predominantly
P. falciparum malaria prioritize HRP2-targeting RDTs either as a single antigen test or in combination with other antigens. Consequently, HRP2-targeting RDTs account for most of all global malaria RDT procurements [
8].
Previous reports from South America, Asia, and recent reports from sub-Saharan Africa [
9‐
14] suggest there are
P. falciparum parasites with deletions of either or both genes coding for HRP2 and the related protein HRP3, rendering these parasites incapable of producing these antigens. Parasites with deletions of both these genes produce false negative results on HRP2-based RDTs and are therefore a threat to the use of HRP2-based RDTs [
9,
14]. The genes coding for HRP2 and HRP3 are located on different chromosomes in the
P. falciparum genome [
15]. However, due to conserved repeating epitopes between both antigens, some monoclonal antibodies to HRP2 cross-react with HRP3 [
16]. Consequently, HRP2-based RDTs can produce positive results when tested with samples from infections with parasites with
hrp2 deletions, but intact
hrp3 [
17‐
19].
Plasmodium falciparum parasites have been identified with deletions in both
hrp2 and
hrp3 or singly deleted for one of these genes. This cross-reactivity has been reported using a highly sensitive HRP2-based RDT [
17] and models using
hrp2−/
hrp3+ data from Kenya predict high density infections with such parasites could produce positive results on conventional RDTs [
18]. However, better understanding of this interaction is needed for conventional RDTs that are more commonly used and expected to play a role in
hrp2 deletion surveillance. In addition, analyses of how HRP2 and HRP3 interact to produce a test result on HRP2-based RDTs is necessary to better design studies that determine the effects of
hrp2 deletions on the effectiveness of HRP2-based RDTs. In this study, the reactivity of serial dilutions of four culture-adapted
P. falciparum strains with intact
hrp2 and
hrp3 or with one or both genes deleted on three different RDT products was determined. Extensive HRP3 cross-reactivity on HRP2 detecting RDTs is described.
Discussion
The presence in endemic regions of
P. falciparum parasites lacking either or both
hrp2 and
hrp3 is recognized as a threat to malaria case management especially in sub-Saharan Africa, where the WHO recommends the use of HRP2 only RDTs. Exceptions to this recommendation are Ethiopia and Madagascar where
P. vivax transmission occurs. The extent of these deletions is unclear because of inadequate surveillance. The WHO recommends a method of surveillance [
25] that uses results from a combination of HRP2-based RDT with a pan-LDH RDT, Pf-LDH RDT or smear microscopy on clinical malaria cases with HRP2 false negative RDT result as an initial trigger for widespread
hrp2/hrp3 deletion surveillance. This surveillance protocol depends on phenotypic expression of HRP2 as the most important initial trigger rather than an initial investigation into costly molecular testing for
hrp2/hrp3 deletions. Because HRP2-specific antibodies can cross-react with HRP3, malaria patient sample reactivity on HRP2-based RDTs would be expected to be influenced by HRP3 when the gene is present in the infecting parasite. Understanding how this cross-reactivity influences HRP2 RDT reactivity is critical for conducting
hrp2/
hrp3 deletion surveys and how policy changes away from HRP2-based RDTs are made.
Results presented here show that at parasite densities of ≥ 1000 parasites/µL, often associated with symptomatic malaria infection in sub Saharan-Africa,
P. falciparum parasites with either
hrp2 only or
hrp3 only deletions may not be distinguishable phenotypically from parasites with both genes intact when tested on HRP2 RDTs and that HRP2 RDTs remain useful except when both
hrp2 and
hrp3 are deleted. Indeed, for RDTs used in this study, cross-reactivity is seen even at lower parasite densities typically associated with asymptomatic infections in sub-Saharan Africa. These data imply that genotyping for
hrp2 only in a parasite population is insufficient to provide information needed to advocate for changes in testing policy in favor of non-HRP2-based tests. Complete loss of HRP2 reactivity at all parasite densities was only observed for the
hrp2/
hrp3 double-deleted parasite 3BD5. An important observation that also needs to be considered for
hrp2/
hrp3 deletion surveillance and which is emphasized by the WHO
hrp2/hrp
3 deletion surveillance protocol [
25] is that samples used for deletion surveillance by necessity should be high density infections. At 100 parasites/µL, parasites with
hrp2 or
hrp3 only deletions (Dd2 and HB3) showed similar reactivity patterns on HRP2 test bands. However, RDTs have decreased sensitivity at low densities and false negative RDT tests observed at such densities may reflect the lowered test sensitivity rather than a deletion of the relevant genes.
Plasmodium falciparum 3D7 with both
hrp2 and
hrp3 intact was observed on the bead assay to have a higher measured HRP2 concentration per parasite biomass at ≥ 1000 parasites/µL than the other parasites with single gene deletions. This observation is likely due to a combination of the parasite strain producing both HRP2 and the cross-reacting HRP3 as well as an inherent ability to produce more of the protein. In addition, HB3 (
hrp2+/
hrp3−) had higher HRP2 concentration than Dd2 (
hrp2−/
hrp3+) per parasite biomass due to the greater number of antibody epitopes on HRP2 or greater avidity for the homologous antigen compared to HRP3 [
26]. While the distinction in HRP2 concentration of each parasite strain and dilution was clear on the quantitative bead assay, assay saturation point on the qualitative RDT means that any differences in HRP2 concentration between
hrp2 only or
hrp3 only parasites is lost at high (≥ 1000 parasites/µL) parasite densities. Therefore, when surveying for
hrp2/
hrp3 deletions, false negative HRP2 RDT reactivity at higher parasite densities is likely to be more informative than false negative reactivity at low (~ 100 parasites/µL) densities. The cross-reactive pattern described here also means single
hrp2 or
hrp3 deletions are likely underestimated in parasite populations since these parasites, especially in high density clinical cases, will produce positive HRP2 RDT reactions and are, therefore, unlikely to be selected for genotyping. The pattern of pLDH reactivity largely reflected expectations with all parasites producing parasite density and antigen concentration-dependent reactivity on the pan-pLDH RDT and no reactivity on the Pv-LDH RDT.
A limitation of this study is that RDTs from only two manufacturers were used and products from other manufacturers could produce different results due to the use of different HRP2 antibodies. It remains to be tested how product-specific antibody types influence reactivity. However, this potential cross-reactivity requires that hrp2 deletion surveys are not conducted in isolation but always in conjunction with hrp3 deletion with the knowledge that cross-reactivity is extensive enough to mask the effects of hrp2 only deletions and that HRP2 production phenotype as measured by RDTs is influenced by both genes. In addition, this potential cross-reactivity needs to be understood and addressed for any RDT being used as part of hrp2/hrp3 deletion surveillance. An HRP3-specific antibody with no HRP2 cross-reactivity in the bead assay could also help with better resolution of cross-reactivity between the two antigens. Another limitation of the study is that, parasites with different genetic backgrounds have inherent differences in antigen production capabilities and although higher HRP2 antigen levels were measured for 3D7 compared to the same parasite concentration of the other parasites, the differences may be in part due to that parasite’s ability to produce more HRP2 and not necessarily only due to hrp2 and hrp3 being intact. Also, at the lower parasite densities, RDT reactivity did not always reflect the presence of antigen as measured in the bead assay, demonstrating the lower sensitivity of RDTs closer to their limits of detection. However, except for HRP2 for the 3BD5 parasite, there was a high positive correlation between antigen concentration and parasite density for all parasite strains (r range 0.83–0.99).
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