Screening for plasmodium falciparum HRP2 and HRP3 gene deletions in Uganda using highly multiplexed deep sequencing, MAD4HatTeR assay
Résumé fourni par la source
Introduction Plasmodium falciparum histidine-rich protein 2/3 (pfhrp2/3) gene deletions threaten the effectiveness of HRP2-based malaria rapid diagnostic tests (RDTs), which are the primary malaria diagnostic tool in Uganda. Methods We collected 200 dried blood spot (DBS) samples from patients with uncomplicated malaria from 30 sentinel sites across Uganda in Jan-Apr 2023, Jul-Sep 2023, Feb-Apr 2024, and Aug-Oct 2024. From each site and round, 100 samples with parasitemia >= 1000 parasites/microliter by varATS qPCR were selected for targeted sequencing with the MAD4HatTeR assay. Data was analyzed with the MAD4HatTeR pipeline to generate allele calls data across diversity, drug and diagnostic resistance loci. Copy number variation (CNV) of pfhrp2 and pfhrp3 genes was estimated using a generalized additive model to correct for amplification bias and coverage, calculating fold-change in read depth relative to Pf3D7 controls lacking hrp2/hrp3 deletions. CNV fold change of <= 0.5 was used to screen samples for deletions in Pfhrp2&Pfhrp3 genes. Results 7,524 samples (3,999 samples in year 2023 and 3,525 samples in year 2024) passed quality filtering. Pfhrp3suspected deletions were detected in 61 samples representing 0.81% (95% CI: 0.63–1.04%) aggregated across all rounds of collections. The prevalence of Pfhrp3 single gene deletions remained very similar between 2023 and 2024 of 0.83% (95% CI: 0.59–1.16%) and 0.79% (95% CI: 0.55–1.15%) respectively. Site-level analysis revealed heterogeneous distribution, with low suspected Pfhrp3 gene deletion prevalence ranging from 0 to 4.7% across different sites and regions of the country. No suspected Pfhrp2 or Pfhrp2/3 gene deletions were observed in the samples at the set threshold. Conclusion Pfhrp2 and Pfhrp3 gene deletions remain rare in Uganda which makes the HRP2-based RDTs remain useful for diagnosis of malaria in Uganda currently. Digital PCR genotyping of samples with suspected deletions is ongoing to confirm and to estimate the true prevalences of the Pfhrp2&3 deletions.