PfPHAST: Plasmodium falciparum Public Health Amplicon Sequencing Tool, a Streamlined Panel for Malaria Genomic Surveillance
Abstract
Genomic tools can support malaria control policy through surveillance of Plasmodium falciparum populations, tracking antimalarial drug resistance, pfhrp2/3 deletions that compromise rapid diagnostic tests, and selection at the circumsporozoite protein (PfCSP) vaccine target, as well as through molecular correction of therapeutic efficacy studies (TES). Multiplex Amplicons for Drug, Diagnostic, Diversity, and Differentiation Haplotypes using Targeted Resequencing (MAD4HatTeR), a comprehensive amplicon sequencing panel covering up to 276 targets, supports these applications but is tailored to research rather than routine programmatic use. We developed P. falciparum Public Health Amplicon Sequencing Tool (PfPHAST), a 56-target derivative of MAD4HatTeR spanning drug resistance loci, pfhrp2/3 deletion, PfCSP genotyping, non-falciparum species identification, and 20 high-heterozygosity microhaplotype loci for TES classification. We compared PfPHAST and MAD4HatTeR using laboratory strain controls, including two-strain dilution series and a five-strain mixture, across parasite densities of 100 to 10,000 parasites/L. At matched per-target depth, PfPHAST achieved a higher quality-control pass rate than MAD4HatTeR (94.4% versus 90.0%) and distributed reads more evenly across targets. The panels showed comparable recall and precision for drug resistance codons and microhaplotypes, reaching near-complete recall above 40% within-sample allele frequency (WSAF) at all densities, with reduced sensitivity for minor alleles below 10% WSAF at low parasite density in both panels. Observed and expected WSAF correlated strongly for both panels, and both resolved a five-strain polyclonal mixture, including a 5% minor strain. By concentrating sequencing capacity on targets of greatest programmatic relevance, PfPHAST offers a scalable, lower-cost alternative to comprehensive research panels without sacrificing performance on shared targets, complementing MAD4HatTeR for routine molecular malaria surveillance.
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BibTeX
@misc{semakuba2026pfphast,
title = {{PfPHAST: Plasmodium falciparum Public Health Amplicon Sequencing Tool, a Streamlined Panel for Malaria Genomic Surveillance}},
author = {F. D. Semakuba and B. Nsengimaana and T. Katairo and M. Mbabazi and K. Murie and N. Hathaway and I. Wiringilimaana and J. V. Muwanika and K. Lum and M. Davlieva and V. Asua and S. Kiyaga and S. Tukwasibwe and J. Nakasaanya and K. B. Achom and A. Ayitewala and J. Mulondo and E. Watyekele and S. L. Nsobya and M. R. Kamya and B. B. Agaba and P. J. Rosenthal and M. D. Conrad and I. Ssewanyana and B. Greenhouse and A. Aranda-Diaz and J. Briggs},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.26.754026},
url = {https://www.biorxiv.org/content/10.64898/2026.09.26.754026v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - PfPHAST: Plasmodium falciparum Public Health Amplicon Sequencing Tool, a Streamlined Panel for Malaria Genomic Surveillance
AU - F. D. Semakuba
AU - B. Nsengimaana
AU - T. Katairo
AU - M. Mbabazi
AU - K. Murie
AU - N. Hathaway
AU - I. Wiringilimaana
AU - J. V. Muwanika
AU - K. Lum
AU - M. Davlieva
AU - V. Asua
AU - S. Kiyaga
AU - S. Tukwasibwe
AU - J. Nakasaanya
AU - K. B. Achom
AU - A. Ayitewala
AU - J. Mulondo
AU - E. Watyekele
AU - S. L. Nsobya
AU - M. R. Kamya
AU - B. B. Agaba
AU - P. J. Rosenthal
AU - M. D. Conrad
AU - I. Ssewanyana
AU - B. Greenhouse
AU - A. Aranda-Diaz
AU - J. Briggs
PY - 2026
DA - 2026/09/26
PB - bioRxiv
DO - 10.64898/2026.09.26.754026
UR - https://www.biorxiv.org/content/10.64898/2026.09.26.754026v1
AB - Genomic tools can support malaria control policy through surveillance of Plasmodium falciparum populations, tracking antimalarial drug resistance, pfhrp2/3 deletions that compromise rapid diagnostic tests, and selection at the circumsporozoite protein (PfCSP) vaccine target, as well as through molecular correction of therapeutic efficacy studies (TES). Multiplex Amplicons for Drug, Diagnostic, Diversity, and Differentiation Haplotypes using Targeted Resequencing (MAD4HatTeR), a comprehensive amplicon sequencing panel covering up to 276 targets, supports these applications but is tailored to research rather than routine programmatic use. We developed P. falciparum Public Health Amplicon Sequencing Tool (PfPHAST), a 56-target derivative of MAD4HatTeR spanning drug resistance loci, pfhrp2/3 deletion, PfCSP genotyping, non-falciparum species identification, and 20 high-heterozygosity microhaplotype loci for TES classification. We compared PfPHAST and MAD4HatTeR using laboratory strain controls, including two-strain dilution series and a five-strain mixture, across parasite densities of 100 to 10,000 parasites/L. At matched per-target depth, PfPHAST achieved a higher quality-control pass rate than MAD4HatTeR (94.4% versus 90.0%) and distributed reads more evenly across targets. The panels showed comparable recall and precision for drug resistance codons and microhaplotypes, reaching near-complete recall above 40% within-sample allele frequency (WSAF) at all densities, with reduced sensitivity for minor alleles below 10% WSAF at low parasite density in both panels. Observed and expected WSAF correlated strongly for both panels, and both resolved a five-strain polyclonal mixture, including a 5% minor strain. By concentrating sequencing capacity on targets of greatest programmatic relevance, PfPHAST offers a scalable, lower-cost alternative to comprehensive research panels without sacrificing performance on shared targets, complementing MAD4HatTeR for routine molecular malaria surveillance.
ER -