Tracking Plasmodium falciparum Histidine-rich protein 2/3 gene deletion in RDT-negative samples among febrile children in Nigeria

Abstract

Background: Nigeria, one of the most malaria-endemic regions globally, relies heavily on rapid diagnostic tests (RDTs) targeting Plasmodium falciparum histidine-rich protein 2/3 (Pfhrp2/3). However, emerging deletions in these genes can lead to false-negative results, undermining case management and surveillance. This study investigates the prevalence of Pfhrp2/3 gene deletions among false RDT-negative cases and assesses whether they exceed the 5% WHO threshold that would necessitate changes in diagnostic strategy.Methods: A cross-sectional study was conducted across five Nigerian states. Dried blood spots were collected from 824 febrile children (ages 3 months–12 years) with malaria-related symptoms, among whom 589 were RDT-negative. P. falciparum screening was performed on RDT-negative samples using nested PCR, and parasitaemia was assessed by Real-time qPCR. The gene deletion was detected using 4-plex qPCR targeting pfhrp2, pfhrp3, beta-tubulin, and cytochrome-b genes. The Mann-Whitney U test was used to analyse primary drivers of RDT negativity and the association between gene deletion and parasitaemia.Results: Among the RDT-negative samples, P. falciparum was detected in 134 (22.8%), of which the overall pfhrp2/3 deletion rate was 1.87%. Deletions of pfhrp2 and pfhrp3 in Kano were the highest with 16.0% and 12.0%, respectively, exceeding the WHO 5% threshold. The median parasitaemia in samples with deletions was 72.33 parasites/μL (IQR: 19.75-212.23), compared with 110.14 parasites/μL (IQR: 22.15-237.88) in those without deletions. (U = 606.50, p = 0.419).Interpretation: Pfhrp2/3 deletions are circulating in Nigeria and exceed WHO thresholds in an area. However, RDT false negativity is primarily driven by low parasitaemia.