PfHRP2 is the target antigen for many RDTs detecting P. falciparum. In this study, pfhrp2 from 458 P. falciparum isolates collected from 38 countries, all major malaria endemic areas, were sequenced and evaluated. It was shown that this gene exhibits extensive diversity both within and between countries and regions. A similar protein, PfHRP3, also exhibited extensive diversity between parasite isolates examined. This comprehensive examination and analysis of the extent of the diversity and geographic variation in these genes provides important information for laboratory and field evaluation of malaria RDTs. It also provides an indication of whether the genetic diversity in the antigen contributes to the variability in RDT sensitivity.
Both pfhrp2 and pfhrp3 are located in the subtelomeric regions of chromosomes. In general, genes located in telomeric and subtelomeric regions of Plasmodium have vast genetic diversity, and are highly susceptible to changes during recombination events [28–33]. Subtelomeric regions from different malaria species appear to have undergone rapid evolution, with significant sequence variation generated in the complex repeats in these regions [32, 33]. Molecular mechanisms contributing to the generation of tandemly repeated regions, changes in the length of repeat blocks and other variation events include slipped strand mispairing followed by DNA replication or repair, unequal reciprocal combination and gene conversion [34–39]. The different organization and varying number of repeats observed in both pfhrp2 and pfhrp3 are likely the result of frequent recombination of the chromosomes. Therefore, a correlation between malaria transmission intensity and pfhrp2 diversity should be expected, as malaria infections in high transmission settings often involve co-infection of multiple strains which increase the probability of recombination during sexual reproduction in the mosquito vector. Indeed, this general trend was observed in this study with the ratio of different pfhrp2 sequence types to total sequences being higher in countries with high transmission intensity such as in Africa, and lower in South American and Asian countries. However, this may also reflect differences in the geographical spread of collections within countries, which varied between sites.
The function of PfHRP2 still remains to be determined. Early theories suggested that PfHRP2 may be involved in detoxification of free haem by converting it to inactive haemozoin [40, 41]. Other theories suggest that PfHRP2 may be involved in remodeling the infected erythrocyte cytoskeleton  and in modulating host immune responses . The extensive diversity in the pfhrp2 sequence provides evidence that the function(s) of the molecule is not affected by the sequence diversity in exon 2 and that parasites with a particular pfhrp2 sequence do not appear to have a significant fitness or survival advantage. The host immune responses may also contribute to the maintenance of diversity by selecting for immunologicly different types.
A mechanism of chromosome breakage and healing is believed to contribute to generation of deletions in various parasite chromosomes . Chromosome deletions at the pfhrp2 locus of chromosome 7 and pfhrp3 locus of chromosome 13 have been observed in laboratory-adapted lines [17, 20, 27], and in field isolates from the Amazon region of Peru . This observation of gene deletions in field isolates suggests that PfHRP2-detecting RDTs may not be reliable for detecting P. falciparum infections in this region of South America, and also raised a serious question as to whether parasites lacking PfHRP2 and PfHRP3 may exist in other parts of the world and what are possible fitness or survival advantages. In the 485 isolates, collected in over 30 malaria endemic countries, that we examined, we did not observe any deletions of pfhrp2 or pfhrp3 in any field isolates, suggesting that the parasites with gene deletions may not be widespread outside of South America. However, it should be noted that the sampling did not include some endemic regions (e.g. South and Western Asia) and the number of samples examined for some countries was quite small, not coming from different areas within the country. To ensure the performance of RDTs, efforts should be taken to monitor the existence and spread of parasites with gene deletions, especially when false negative results associated with a high parasitaemia are reported.
Pfhrp2 and pfhrp3 variability appears to occur independent of the other, as no correlation between the lengths of the two genes was evident. However, both proteins share several repeats, such as type 1 and 2 repeats. These shared repeats are probably the basis for observed cross reactivity between PfHRP2 and PfHRP3 by monoclonal antibodies against PfHRP2 [8, 18], which may contribute to the detection sensitivity of PfHRP2-detecting RDTs and reduce the effect of PfHRP2 variability on RDT sensitivity, particularly at high parasite densities.
The organization and the number of repeats in PfHRP2 vary extensively between parasite isolates. Theoretically, the presence and absence, as well as the number of repeats could affect the binding affinity of the antibodies used in RDTs to the parasites' antigen. Indeed, this preliminary analysis based on 16 cultured lines and tested on two earlier malaria RDTs, Paracheck Pf (Orchid Biomedical Systems, India) and ICT Malaria (ICT Diagnostics, South Africa), yielded a binary logistic regression model that was able to predict detection sensitivity of these two RDTs based on sequence structure . However, the number of isolates used was limited and cultured parasites were used. In this study, the analysis was repeated using the recently completed WHO product testing of malaria RDTs (Round 1) results that included the testing of 34 PfHRP2-detecting RDTs tested against 79 isolates at 200 parasites/μl. This much larger sample size enabled more stringent analysis. The regression analysis this time did not show a correlation between PfHRP2 structure and the overall RDT detection rates. This result suggests that the performance of this group of RDTs is not greatly affected by the diversity of PfHRP2 at parasite densities of 200 parasite/μL or above and, therefore, should increase the confidence in the performance of the devices in various geographic locations. However, this does not exclude an effect of sequence diversity on RDT detection rates at lower parasite densities, or by RDTs employing other monoclonal antibodies.