Understanding Residual Plasmodium falciparum Transmission through Targeted Amplicon Deep Sequencing

Population genomic data are increasingly being used to improve our understanding of malaria epidemiology, for example by assessing connectivity between populations and providing key information on the genetic diversity of Plasmodium falciparum and the prevalence of antimalarial drug resistance. Parasite genetic data can inform malaria elimination strategies by helping to identify factors that contribute to residual transmission. This dissertation aimed to characterize the population structure of P. falciparum parasites, map antimalarial resistance profiles, and define parasite relatedness to provide new insights that will help inform malaria control policy in two pre-elimination settings in Zanzibar and Ethiopia.

First, to facilitate large-scale epidemiologic studies, I developed a novel high-throughput digital droplet PCR-based amplicon sequencing (AmpSeq) method targeting highly polymorphic microhaplotypes and drug resistance loci. To sequence low-density samples, I developed a sensitive method for DNA extraction from dried blood spots, which are routinely collected for molecular malaria surveys. I then applied my method to two pre-elimination settings, Zanzibar and Ethiopia.

Second, in Zanzibar, I elucidated fine-scale parasite population structure and inferred relatedness and connectivity of infections, and mapped antimalarial resistance profiles across five districts covering both main islands of Zanzibar. Using AmpSeq data from 290 P. falciparum infections, I observed strong fine-scale spatial and temporal structure of local parasite populations, despite high genetic diversity, providing evidence for ongoing local transmission. I found a high proportion of highly related parasites in individuals living closer together, including between clinical index cases and the mostly asymptomatic cases surrounding them. Further, I identified a substantial proportion of significantly related infection pairs between Zanzibar, and mainland Tanzania and Kenya, consistent with recent importation. Most parasites on the archipelago remained sensitive to the current first-line antimalarials. This study provided a high-resolution view of parasite genetic structure across the Zanzibar archipelago and revealed actionable patterns, including isolated parasite populations that may be prioritized for malaria elimination.

Third, using AmpSeq data from 187 P. falciparum samples from Ethiopia, I characterized the parasite population structure and transmission dynamics in the highlands. I identified a significant proportion of highly related infections with multiple clusters of clonal or near-clonal infections, even across transmission seasons, suggesting persistent local and focal transmission. I found that infections from travelers were frequently observed first in time, suggesting that parasites may have been imported and then transmitted locally. I identified high prevalence of drug-resistant parasites and ‘diagnostic-resistant’ pfhrp2/3-deleted parasites. Overall, these results corroborated local transmission and the importance of intensified control in the Ethiopian highlands.

Fourth, the need for advanced laboratory infrastructure, often unavailable in malaria-endemic sites, prevents the rapid generation and use of genotyping data. To overcome these limitations, I developed and validated two AmpSeq assays for P. falciparum nanopore sequencing in endemic sites using a mobile laboratory, targeting key antimalarial drug resistance markers and microhaplotypes. I field-tested the feasibility of rapid genotyping in Zanzibar in close collaboration with the local malaria elimination program. The assay produced actionable data with a turnaround time of a few days, and I identified current key challenges for implementing nanopore sequencing in endemic countries to accelerate malaria control and elimination.

In conclusion, my research contributes to our understanding of residual P. falciparum transmission in two pre-elimination settings in Zanzibar and Ethiopia using genomic epidemiology tools. My findings can support the development of better malaria control and elimination strategies.