Abstract

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

Akala, H. M. Kandie, R. Githuka, G. N. Kiplagat, R. Ahmeddin, O. Keitany, K. Kiarie, J. Wamari, A. Machini, B. Oyugi, E. Mahugu, S. Otieno, J. D. Egbo, T. E. Juma, D. W. Sang, L. P. Haynes, R. C. Osoti, V. Vesely, B. A. Smith, D. Travis, J. Cunningham, J. Cheng, Q. Oyier, I. Ogutu, B.
Malar J. 2026;

Permanent descriptor

https://doi.org/10.1186/s12936-026-05942-9

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.