Low prevalence of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions in archived samples collected before and after nationwide malaria-RDT rollout in Kenya.

Rapid and accurate malaria diagnosis is essential for effective treatment and malaria control. In Kenya, most malaria testing relies on P. falciparum histidine-rich protein 2 (PfHRP2)-based rapid diagnostic tests (RDTs). However, deletions in the pfhrp2/pfhrp3 genes can lead to false-negative RDT results, which can sustain transmission and worsen clinical outcomes. This study assessed the prevalence and pattern of pfhrp2, pfhrp3 and their respective flanking genes in archived samples collected before and after the nationwide rollout of PfHRP2-based RDTs in Kenya in 2012. A total of 350 archived whole-blood samples were analysed, comprising 95 pre-RDT samples (collected before the nationwide rollout of RDTs in Kenya) from 2003 to 2005 and 255 post-RDT samples (collected after the nationwide rollout of RDTs in Kenya) from 2013 to 2017 from multiple malaria-endemic regions of Kenya. Samples were first screened by quantitative Real-Time PCR to identify those with adequate parasite density for genotyping. Nested PCR assays were then used to genotype pfhrp2, pfhrp3 and their flanking genes. Where available, PfHRP2-based RDT results were compared with molecular genotyping outcomes to investigate potential causes of false-negative RDT results. The overall prevalence of single pfhrp2 or pfhrp3 deletions was low, at 0.3% (1/317) and 1.3% (4/317), respectively. All pre-RDT samples carried intact pfhrp2 and pfhrp3 genes. Among post-RDT samples, deletions were rare 0.4% (1/224) for pfhrp2 gene only and 1.8% (4/224) for pfhrp3 gene only. No sample exhibited a triple deletion of pfhrp2 and both of its flanking genes or triple deletion of pfhrp3 and both of its flanking genes. The sensitivity of the PfHRP2-based RDT was 89.2% (174/195). Notably, 95.2% of false-negative RDT results occurred in samples with intact pfhrp2 and pfhrp3, suggesting that low parasitemia or other factors, rather than gene deletions, were the primary cause of diagnostic failure. Overall, pfhrp2 and pfhrp3 deletions remained rare in the samples collected up to five years after the nationwide rollout of PfHRP2-RDTs in Kenya. However, continuous molecular surveillance is essential to identify emerging deletion hotspots and to ensure the sustained reliability of PfHRP2-based diagnostics as testing practices and parasites continue to evolve.