Conference Abstract | Volume 8, Abstract NACNDC/19JASH065 (Oral) | Published: 25 Nov 2025
Bienvenu Nsengimaana1,2,&, Sneha Verma3, Thomas Katairo1,2, Francis Ddumba Semakuba1,2, Stephen Tukwasibwe1,4,5,2, Innocent Wiringilimaana1,2, Caroline Mwubaha1,2, Brian Asiimwe Kagurusi1,2, Jackline Nakasanya1,2, Trevor Edgar Esilu1, Kylie Hilton4, Victor Asua1, Shahiid Kiyaga1,2, Monica Mbabazi1,2, Kisakye Diana Kabbale1,2, Alisen Ayitewala2, Jerry Mulondo1, Eric Watyekele1, Samuel Lubwama Nsobya1, Moses Joloba4, Grant Dorsey3, Moses Kamya1,4,, Isaac Sewanyana1,2, David Patrick Katete4, Daudi Jjingo5, Bosco Bekiita Agaba6, Catherine Maiteki-Sebuguzi6, Jimmy Opigo6, Eric Neubauer Vickers3, Manuel Garcia3, Aranda-Diaz Andres3, Bryan Greenhouse3, Philip Rosenthal3, Melissa Conrad3, Jessica Briggs3
1Infectious Diseases Research Collaboration, Kampala, Uganda, 2Central Public Health Laboratories, Kampala, Uganda, 3University of California, San Francisco, USA, 4Makerere University, Kampala, Uganda, 5Uganda Christian University, Mukono, Uganda, 6National Malaria Elimination Division, Ministry of Health, Kampala, Uganda
&Corresponding author: Bienvenu Nsengimaana, Infectious Diseases Research Collaboration, Kampala, Uganda. Email: bienvenunsengimaana@gmail.com, ORCID: https://orcid.org/0009-0002-9786-7294
Received: 14 Sept 2025, Accepted: 20 Oct 2025, Published: 25 Nov 2025
Domain: Public Health
Keywords: Malaria, Diagnostic resistance, RDTs, Pfhrp2, Pfhrp3
©Bienvenu Nsengimaana et al. Journal of Interventional Epidemiology and Public Health (ISSN: 2664-2824). This is an Open Access article distributed under the terms of the Creative Commons Attribution International 4.0 License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Cite this article: Bienvenu Nsengimaana et al., Screening for Plasmodium falciparum hrp2 and hrp3 gene deletions in Uganda using highly multiplexed deep sequencing, MAD4HatTeR assay. Journal of Interventional Epidemiology and Public Health. 2025;8(ConfProc6):00065. https://doi.org/10.37432/JIEPH-CONFPRO6-00065
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.
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.
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.
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