Comparative analysis of molecular protocols for detecting pfhrp2 and pfhrp3 gene deletions in Plasmodium falciparum samples from Rondônia, Brazilian Amazon

BACKGROUND: Rapid diagnostic tests (RDTs) based on histidine-rich protein 2 (HRP2) detection are widely used for malaria diagnosis in remote settings. However, deletions in the pfhrp2 and pfhrp3 genes in Plasmodium falciparum compromise RDT performance, with studies reporting varying prevalences. This study compared multiple molecular approaches to assess their concordance and implications for deletion surveillance. METHODS: Sixty-three P. falciparum-positive clinical samples from Rondônia, Brazil, were analyzed using six PCR protocols: two conventional (cPCR), three quantitative (qPCR), and one droplet digital (ddPCR). Sanger sequencing was performed to investigate the genetic variability of pfhrp2. RESULTS: Discrepancies were observed across all methods, particularly at low parasite densities. For pfhrp2, non-amplification rates varied among protocols: 11.1-23.8% for cPCR and 0-9.5% for qPCR. ddPCR did not detect complete deletions. For pfhrp3, non-amplification ranged from 11.1% to 71.4% among qPCR protocols, while ddPCR detected deletions in 95.2% of samples. Sequencing revealed multiple insertion-deletion events in pfhrp2, suggesting local genetic variability contributes to assay discordance. CONCLUSION: Detection of pfhrp2/pfhrp3 deletions is highly dependent on the molecular protocol used. Genetic variability and low parasite density contribute to inconsistent results, potentially leading to incorrect estimation of deletion prevalence. Harmonization of molecular surveillance strategies is essential for reliable monitoring of pfhrp2/pfhrp3 deletions.