Fitness Costs of pfhrp2 and pfhrp3 Deletions Underlying Diagnostic Evasion in Malaria Parasites

[[[ Background: ]]] Rapid diagnostic tests based on detection of histidine-rich proteins (HRPs) are widely used for malaria diagnosis, but parasites carrying pfhrp deletions can evade detection and are increasing in frequency in some countries. Models aim to predict conditions under which pfhrp2 and/or pfhrp3 deletions will increase, but a key parameter-the fitness cost of deletions-is unknown. [[[ Methods: ]]] We removed pfhrp2 and/or pfhrp3 from a Malawian parasite clone using gene editing approaches) and measured fitness costs by conducting pairwise competition experiments. [[[ Results: ]]] We observed significant fitness costs of 0.087 ± 0.008 (1 standard error) per asexual cycle for pfhrp2 deletion and 0.113 ± 0.008 for the pfhrp2/3 double deletion, relative to the unedited progenitor parasite. Selection against deletions is strong and comparable to that resulting from drug resistance mutations. [[[ Conclusions: ]]] Prior modeling suggested that diagnostic selection may drive increased frequency of pfhrp deletions only when fitness costs are mild. Our experiments show that costs of pfhrp deletions are higher than these thresholds, but modeling and empirical results can be reconciled if the duration of infection is short. These results may inform future modeling to understand why pfhrp2/3 deletions are increasing in some locations (Ethiopia and Eritrea) but not in others (Mekong region).