Abstract
<jats:p>Histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) are central to malaria case management in Africa but fail when Plasmodium falciparum parasites lack the pfhrp2 or pfhrp3 genes. Widespread deletions have been reported in Eritrea, Ethiopia, and Djibouti, yet no systematic data have been available from Somalia. Between May and October 2023, we collected 7148 dried blood spot (DBS) samples from patients with suspected malaria attending eight health facilities across seven regions in Somalia. Field HRP2/pan-lactate dehydrogenase (LDH) RDTs and microscopy were performed, and DNA was extracted from 301 RDT-positive and 173 RDT-negative DBS samples. A multiplex quantitative PCR assay targeting pfldh, pfhrp2, and pfhrp3 was used to identify deletions in pfldh-positive samples lacking pfhrp2 or pfhrp3 amplification, with mixed infections inferred from delta cycle threshold (ΔCt) differences. Of 474 analysed samples, 301 (4.2%, 95% CI 3.7-4.7) were RDT or microscopy positive, and 159 (33.5%) were confirmed pfldh-positive by qPCR. Among these, six (3.8%, 95% CI 1.4-8.1) carried pfhrp2 deletions and 59 (37.1%, 95% CI 29.6-45.1) carried pfhrp3 deletions. Eleven infections (6.9%, 95% CI 3.5-12.1) produced discordant RDT outcomes, HRP-/LDH+ or RDT-negative despite pfldh positivity. Deletions were most frequent in Dolow, Luq, and Bosaso. A single isolate carried the pfk13 R622I mutation, confirming the first report of the emergence of an artemisinin partial resistance-associated in Dolow, Gedo region, Somalia. Pfhrp2/3 deletions causing false RDT results remain low in Somalia and the confidence interval overlaps with the 5% policy threshold for changing RDTs, indicating uncertainty that warrants larger-scale assessment. Pfhrp3 deletions are widespread and compromise the diagnostic redundancy of HRP2-based tests. Most deletion-carrying parasites remain detectable through the pan-LDH line, minimising immediate clinical risk but leading to systematic misclassification of P. falciparum as non-falciparum malaria. These findings support the continued use of HRP2/Pan-LDH RDTs but highlight high risk areas and emphasise the need for periodic and expanded molecular surveillance for prevalence trends to guide timely future diagnostic policy.</jats:p>