The research, led by scientists at the Kenya Medical Research Institute in collaboration with the Walter Reed Army Institute of Research–Africa and the University of Nairobi, examined 350 archived whole-blood samples collected from multiple malaria-endemic regions of Kenya. Ninety-five samples dated from 2003 to 2005, before the nationwide rollout of rapid diagnostic tests in 2012, while 255 came from 2013 to 2017, the years immediately following widespread adoption. The hospitals contributing samples spanned Kisumu, Kombewa, Kericho, Malindi, and Alupe, capturing high-transmission lake-endemic zones, epidemic-prone highland areas, and coastal malaria regions.

The diagnostic target in question is Plasmodium falciparum histidine-rich protein 2, or PfHRP2, an antigen that is abundant and stable in peripheral blood and therefore an excellent marker for infection. More than 90 percent of rapid diagnostic tests used in sub-Saharan Africa are built around this protein. A related paralog, PfHRP3, shares epitopes with PfHRP2 and can sometimes be cross-detected, but its expression is generally too low to be reliably picked up at low parasite densities. Parasites that lose the pfhrp2 gene, and sometimes the pfhrp3 gene alongside it, can therefore slip past the most common point-of-care test entirely, resulting in false-negative results that allow untreated infections to sustain transmission and worsen clinical outcomes.

Kenya sits in a region of particular concern. Neighboring countries in the Horn of Africa have reported alarmingly high deletion prevalences: Eritrea documented 80 percent of samples producing false-negative results, while Djibouti reported double pfhrp2 and pfhrp3 deletions in 86.5 percent of tested parasites. These findings have prompted Ethiopia, Eritrea, and Djibouti to shift national diagnostic policy away from PfHRP2-based tests. Kenya, by contrast, has previously recorded deletion rates below the World Health Organization’s 5 percent action threshold, though global modeling work has classified it as a country at high risk for the potential expansion of deletion-bearing parasites.

To establish whether selection pressure from routine diagnostic use has begun reshaping the Kenyan parasite population, the team first screened all samples by quantitative real-time PCR targeting the 18S ribosomal RNA gene. Of the 350 samples, 327, or 93.4 percent, were confirmed as P. falciparum positive. Ten samples fell below the 30 parasites per microliter threshold corresponding to the approximate detection limit of rapid diagnostic tests and were excluded, leaving 317 samples for genotyping. Nested PCR assays were then used to determine the presence or absence of pfhrp2, pfhrp3, and the flanking genes on either side of each locus, with laboratory strains 3D7, Dd2, and HB3 serving as positive and negative controls.

The results were strikingly reassuring. Overall, single pfhrp2 deletions were found in just 0.3 percent of samples, one of 317, while single pfhrp3 deletions appeared in 1.3 percent, four of 317. Every single pre-rollout sample, collected between 2003 and 2005, carried intact copies of both genes. Among the 224 post-rollout samples, deletions remained rare: 0.4 percent for pfhrp2 alone and 1.8 percent for pfhrp3 alone. Critically, no sample exhibited a triple deletion of pfhrp2 and both of its flanking genes, or of pfhrp3 and both of its flanking genes, the pattern that would suggest a coordinated chromosomal loss of the kind seen in parts of South America.

That said, the flanking regions told a more nuanced story. For pfhrp2, 41 samples, 12.9 percent, had lost the 5′ flanking gene while retaining the diagnostic gene itself, and four samples lacked the 3′ flanking gene. At the pfhrp3 locus, 10 samples showed upstream flanking deletions and 16 showed downstream flanking deletions, all with the gene intact. The authors interpret these independent flanking-gene losses as distinct genetic events rather than the coordinated multi-gene deletion mechanisms reported in Honduras, Colombia, and Peru, where deletion of the antigen gene typically occurs together with loss of one or both neighboring regions. This pattern suggests that in Kenya the parasite population has not yet undergone the concerted genomic rearrangements that would signal strong selection for immune evasion of diagnostics.

The study also examined why rapid diagnostic tests sometimes fail despite intact genes. Among 195 PCR-confirmed samples with adequate parasite densities that were positive on the pan-Plasmodium lactate dehydrogenase band, 21, or 10.8 percent, were negative on the PfHRP2-based test, giving an overall test sensitivity of 89.2 percent. Notably, 20 of these 21 false-negative samples, 95.2 percent, carried intact pfhrp2 and pfhrp3 genes, and none lacked pfhrp2 at all. Firth-corrected logistic regression found no significant association between false-negative results and deletion of either gene. Low parasitemia, which reduces antigen concentration below the test’s detection threshold, is the most likely explanation, along with possible sample degradation over long-term storage.

False-negative rates varied considerably by site and year, with Malindi recording the highest cumulative prevalence at 31.6 percent, followed by Kericho at 13.7 percent and Kombewa at 4.9 percent, but no consistent temporal or spatial trend emerged across the study period. Interestingly, the lone pfhrp2-deleted sample was actually RDT-positive, most likely because an intact pfhrp3 gene produced enough cross-reactive antigen to generate a signal, or because a mixed infection contained parasites still expressing PfHRP2. Such observations underscore the complexity of interpreting single-locus results in polyclonal infections, where dominant strains may mask minor deletion-bearing clones that nested PCR cannot reliably detect.

The findings carry clear implications. PfHRP2-based rapid diagnostic tests remain reliable for malaria diagnosis in the regions surveyed, and the near-absence of deletions both before and up to five years after the 2012 rollout suggests that diagnostic-driven selection has not yet taken hold in Kenya, likely owing to high transmission intensity that sustains parasite genetic diversity, frequent polyclonal infections that limit the spread of mutants, and comparatively modest selection pressure. The authors caution, however, that the situation in the Horn of Africa demonstrates how quickly this can change under sustained exclusive reliance on a single antigen target. Continuous molecular surveillance, they argue, is essential to identify emerging deletion hotspots early and to safeguard the long-term effectiveness of the diagnostics on which Kenya’s malaria control program depends.

Subject of Research: Prevalence of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions affecting malaria rapid diagnostic tests in Kenya

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

Article References: Low prevalence of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions in archived samples collected before and after nationwide malaria-RDT rollout in Kenya. (n.d.). https://doi.org/10.3389/fepid.2026.1817539

Image Credits: AI Generated

DOI: 10.3389/fepid.2026.1817539

Keywords: malaria, Plasmodium falciparum, pfhrp2, pfhrp3, gene deletions, rapid diagnostic tests, Kenya, molecular surveillance, diagnostics, public health, KEMRI, parasite genomics