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 pfhrp 2/ pfhrp 3 genes can lead to false-negative RDT results, which can sustain transmission and worsen clinical outcomes. This study assessed the prevalence and pattern of pfhrp 2, pfhrp 3 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 pfhrp 2, pfhrp 3 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 pfhrp 2 or pfhrp 3 deletions was low, at 0.3% (1/317) and 1.3% (4/317), respectively. All pre-RDT samples carried intact pfhrp 2 and pfhrp 3 genes. Among post-RDT samples, deletions were rare 0.4% (1/224) for pfhrp 2 gene only and 1.8% (4/224) for pfhrp 3 gene only . No sample exhibited a triple deletion of pfhrp 2 and both of its flanking genes or triple deletion of pfhrp 3 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 pfhrp 2 and pfhrp 3, suggesting that low parasitemia or other factors, rather than gene deletions, were the primary cause of diagnostic failure. Overall, pfhrp 2 and pfhrp 3 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.