Absence of false negative RDT results due to pfhrp2 deletion among symptomatic malaria infections in Kenya: findings from the 2022 Kenya national longitudinal surveillance of pfhrp2/3 deletions

Abstract

Background: Approximately 74% of malaria diagnoses worldwide rely on Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based rapid diagnostic tests (RDTs), whose accuracy can be compromised by deletions in its encoding pfhrp2 gene and its paralogous pfhrp3 genes. Estimating the burden of false-negative RDT results due to pfhrp2 deletion among individuals with malaria symptoms is critical for guiding the continued use of PfHRP2 tests.

Methods: This cross-sectional survey obtained 5394 samples from individuals presenting with symptoms of malaria at selected health facilities in ten counties, Kenya. Each participant was tested using both PfHRP2-based and P. falciparum lactate dehydrogenase (PfLDH)-based RDT. Samples that tested positive by PfLDH and negative by PfHRP2 were scored as discordant. All discordant samples, plus a random subset of the Plasmodium positive samples, were sequenced to characterize HRP2/3 gene deletion as well as polymorphisms in the Plasmodium falciparum kelch 13 (pfk13) propeller gene.

Results: The HRP2- and PfLDH-based RDTs showed similar positivity rates (44% vs. 45%), with 1.26% discordance. Discordance was highest in Nairobi, Trans Nzoia, and Kisumu, and minimal in Kwale and Tana River. None of the discordant samples carried pfhrp2 deletions, although 5.4% (3/56) had single pfhrp3 deletions, comparable to concordant infections. Among 598 samples that were sequenced, single pfhrp2 and pfhrp3 deletion prevalences were 4.5% and 12.4%, respectively, with no double-deletions detected. Nairobi exhibited the highest prevalence of pfhrp2-only deletions. Tana River, Garissa, Kirinyaga, and Kisii had the highest pfhrp3-only deletions. Among discordant samples, PfLDH-based RDTs identified P. ovale and P. malariae. Finally, sequencing of pfk13 in a subset of P. falciparum infections revealed 0.7% mutation prevalence, comprising A578S (n = 2) Kisumu and A675V (n = 2) each from Nairobi and Kirinyaga counties.

Conclusions: Although single pfhrp2 and pfhrp3 deletions were observed across Kenya, they do not appear to be the primary cause of false-negative HRP2-based RDT results. Continued absence of parasites with double pfhrp2/pfhrp3 deletions supports the ongoing reliability of HRP2-based RDTs for P. falciparum detection. Routine molecular surveillance remains critical to monitor emerging deletion trends. The detection of the A675V mutation, a WHO-validated marker of partial artemisinin resistance in two counties, suggests limited spread of artemisinin resistance.