Highly multiplexed ddPCR-amplicon sequencing reveals strongPlasmodium falciparumpopulation structure and isolated populations amenable to local elimination efforts in Zanzibar

Abstract

Over the past 15 years, Zanzibar has made great strides toward malaria elimination; yet progress has stalled. Parasite genetic data of Plasmodium falciparum may inform strategies for malaria elimination by helping to identify contributory factors to parasite persistence. Here we elucidate fine-scale parasite population structure and infer relatedness and connectivity of infections using an identity-by-descent (IBD) approach. We sequenced 518 P. falciparum samples from 5 districts covering both main islands using a novel, highly multiplexed droplet digital PCR (ddPCR)-based amplicon deep sequencing method targeting 35 microhaplotypes and drug-resistance loci. Despite high genetic diversity, we observe strong fine-scale spatial and temporal structure of local parasite populations, including isolated populations on Pemba Island and genetically admixed populations on Unguja Island, providing evidence of ongoing local transmission. We observe a high proportion of highly related parasites in individuals living closer together, including between clinical index cases and the mostly asymptomatic cases surrounding them, consistent with isolation-by-distance. We identify a substantial fraction (2.9%) of related parasite pairs between Zanzibar, and mainland Tanzania and Kenya, consistent with recent importation. We identify haplotypes known to confer resistance to known antimalarials in all districts, including multidrug-resistant parasites, but most parasites remain sensitive to current first-line treatments. Our study provides a high-resolution view of parasite genetic structure across the Zanzibar archipelago and reveals actionable patterns, including isolated parasite populations, which may be prioritized for malaria elimination.

Plasmodium falciparumtransmission in the highlands of Ethiopia is driven by closely related and clonal parasites

“…These barriers may also explain why pfhrp2/3 deletions appear to be disproportionately prevalent in the Horn of Africa (31)(32)(33)(34)(35)(36)(37)(38). The genetic diversity of P. falciparum infections in Gondar and Ziway was markedly lower (mean HE: 0.54) than previously observed in another low transmission area in Zanzibar (mean HE: 0.73), using the same microhaplotype panel (39), suggesting a smaller parasite population size in Gondar. The direct comparison between two elimination sites corroborates the usefulness of malaria genomic surveillance to understand transmission intensities in different sites.…”

“…Multiplexed amplification in microdroplets was performed as previously described (39). The final library at a concentration of 1.6pM, including 25% PhiX v3 control spiked in, was sequenced on a NextSeq 500 instrument (Illumina) using 150 bp paired-end sequencing with dual indexing using NextSeq at the University of Notre Dame Genomics and Bioinformatics Core Facility.…”

“…Genetic data on P. falciparum parasites from Ethiopia remain scarce, and no data are available on parasite diversity, connectivity between highland and lowland populations, drug resistance, and pfhrp2/3 deletion status from the highland area of Gondar, and Ziway. We used a highthroughput multiplexed amplicon deep sequencing (AmpSeq) method targeting highly diverse microhaplotypes and drug resistance loci previously used in a low-transmission setting in Zanzibar (39). Here, we characterize for the first time the parasite population structure across the high and low transmission seasons, map antimalarial and diagnostic resistance profiles, and define parasite genetic relatedness.…”

Strong isolation by distance and evidence of population microstructure reflect ongoingPlasmodium falciparumtransmission in Zanzibar

“…The network of highly related P. falciparum parasites from 6 shehias in North Pemba provides an excellent example of likely recent near clonal transmission, consistent with an outbreak (Figure 4C). A recent study investigating population structure in Zanzibar also found local population microstructure in Pemba (Holzschuh et al, 2023).…”

Strong isolation by distance and evidence of population microstructure reflect ongoing Plasmodium falciparum transmission in Zanzibar

“…The network of highly related P. falciparum parasites from 6 shehias in North Pemba provides an excellent example of likely recent near clonal transmission, consistent with an outbreak (Figure 4C ). A recent study investigating population structure in Zanzibar also found local population microstructure in Pemba (Holzschuh et al, 2023 ). Overall, given the findings of microstructure with significant local sharing of highly related strains, these small clusters still potentially drive much of the malaria transmission that occurs within the archipelago through routine human movement or mosquito travel between locales (Huestis et al, 2019 ).…”