Parasite Population Dynamics Shape pfhrp2 and pfhrp3 Deletion Patterns in the Peruvian Amazon

Plasmodium falciparum parasites with deletions of the pfhrp2 and pfhrp3 (pfhrp2/3) genes, involved in rapid diagnostic test (RDT) failure, have been increasingly predominant in the Peruvian Amazon since 2012. However, the evolutionary factors underlying this phenomenon remain unclear since HRP2-based RDTs have not been commonly used in this region. Here, we characterized the P. falciparum population in Peru (2006-2018) to identify genomic regions with evidence of recent positive selection. For this purpose, we PCR-genotyped 159 samples from the Loreto region, finding 60% with double pfhrp2/3 deletions, 22% without deletions and 16% with single pfhrp2 deletion. Then we performed whole genome sequencing (WGS) to a subset of the PCR-genotyped samples (n = 42) and integrated these results with existing genome data (n = 60). We revealed a significant reduction in the parasite population structure complexity from Period 1 (2006-2011) to Period 2 (2012-2018), suggesting a bottleneck event likely caused by the PAMAFRO control program. No selection signal was found on pfhrp2/3 genes, supporting a population expansion of parasites carrying pfhrp2/3 deletions due to genetic drift. Despite the clear change in phenotype (RDT evasion due to pfhrp2/3 deletions), these results point to a different evolutionary direction than positive selection forces. This study also advocates the use of continuous surveillance using untargeted genomic approaches such as WGS to track emerging adaptations impacting malaria diagnostics and other control strategies.