Global analysis of Plasmodium falciparum histidine-rich protein-2 (pfhrp2) and pfhrp3 gene deletions using whole-genome sequencing data and meta-analysis

Abstract

Many rapid diagnostic tests (RDT) used on suspected malaria cases are based on the detection of the protein encoded by the Plasmodium falciparum histidine-rich protein-2 (pfhrp2) gene, which shares a high sequence homology with pfhrp3 in the 3D7 reference genome. Parasite isolates showing pfhrp2 and pfhrp3 gene deletions have been emerging over the years, but a comprehensive genetic analysis of these variants is still lacking. With this purpose, genomic data from experimental P. falciparum genetic crosses between different laboratory lines (3D7, HB3, DD2, 7G8 and GB4) were first analysed (n = 98). The frequency of pfhrp2 deletions was consistent with a Mendelian prediction in HB3 × DD2 (56.7%; 95%CI = (39.5%-72.9%)). Moreover, the pfhrp2 and pfhrp3 deletions segregated independently of each other in the same genetic cross. Analysis of 3D7 × HB3 and 7G8 × GB4 estimated the probability of spontaneously generating a pfhrp2 deletion during sexual recombination to be up to 6.2%. Next, whole genome sequence data from 1970 P. falciparum isolates collected globally were analysed. Nine samples displayed depth of coverage consistent with pfhrp2 deletions (0.5%), but the corresponding split-read analysis could not confirm deletions in seven of these samples. Twenty-eight isolates had evidence of pfhrp3 deletions (1.4%), which are widespread in Southeast Asia. Finally, a meta-analysis of published data revealed a positive mean association between the frequencies of pfhrp2 and pfhrp3 deletions in Africa and South America. This result suggested a shared selective pressure acting on these genetic variants. In conclusion, evidence of genetic selection on both pfhrp2 and pfhrp3 deletions was presented, but experimental crosses do not provide evidence of a fitness cost of these variants. Further work is urgently needed to accurately determine the prevalence and the degree of association between these genetic variants, and the respective impact on diagnostic accuracy of many in-use RDT.

An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples

“…Parasites with gene deletions of hrp2 and/or hrp3 have emerged as an important cause of RDT failure in a number of locations [75][76][77][78][79] . It is difficult to devise a simple genetic assay to monitor for risk of RDT failure because hrp2 and hrp3 deletions comprise a diverse mixture of large structural variations with multiple independent origins, and both genes are located in subtelomeric regions of the genome with very high levels of natural variation 29,[80][81][82][83] . In the absence of a well-validated algorithmic method, we visually inspected sequence read coverage and identified samples with clear evidence of large structural variants that disrupted or deleted the hrp2 and hrp3 genes.…”

“…These data have become a key resource for the epidemiology and population genetics of antimalarial drug resistance [9][10][11][12][13][14][15][16][17][18][19][20][21][22] and an important platform for the discovery of new genetic markers and mechanisms of resistance through genome-wide association studies [23][24][25][26][27] and combined genome-transcriptome analysis. 28 The data have also been used to study gene deletions that cause failure of rapid diagnostic tests 29 ; to characterise genetic variation in malaria vaccine antigens 30,31 ; to screen for new vaccine candidates 32 ; to investigate specific host-parasite interactions 33,34 ; and to describe the evolutionary adaptation and diversification of local parasite populations. 7,9,12,[35][36][37][38][39][40] The Pf Community Project data also provide an important resource for developing and testing new analytical and computational methods.…”

Deletions of pfhrp2 and pfhrp3 genes of Plasmodium falciparum from Honduras, Guatemala and Nicaragua

“…Some authors have stated that the deletion of those particular genetic sequences is the result of multiple independent events instead of the dispersion of strains originated from a single event [43, 44]. However, once they emerge, the rapid spread of double-negative populations could be caused by the selection of these parasites by the pressure acting on these variants [45], as demonstrated also by stochastic simulation when the use of RDTs is based on the exclusive detection of the PfHRP2 antigen [46]. Despite the reason behind the emergence and spread of this genotypes in Central America, the presence of double-negative parasites in this endemic region has direct implications on the use of RDTs for routine diagnosis, especially in areas where microscopy is not feasible.…”

A Novel Multiplex qPCR Assay for Detection ofPlasmodium falciparumwithHistidine-rich Protein 2 and 3 (pfhrp2 and pfhrp3)Deletions in Polyclonal Infections

“…Parasites with pfhrp2 and pfhrp3 gene (pfhrp2/3) deletions have emerged in regions of South America and since then, several studies have reported deletions in Africa and in India leading to false-negative RDT results (6)(7)(8). In some countries, a high proportion of RDT false negative results due to these gene deletions has led to the changes in diagnostic guidelines (9,10).…”

“…The various nPCR methods used differ in limit of detection and this can cause type I and type II errors. Further, the pfhrp2 and pfhrp3 PCR assays do not reliably detect P. falciparum DNA derived from dried bloodspots (DBS) particularly at low parasite density (5,12), and gel-electrophoresis approaches do not detect deletions masked in polyclonal infections (6).…”