Malaria cases in Tanzania comprise 3% of globally reported cases, but transmission is heterogeneous, with the coastal mainland witnessing declining but substantial transmission of Plasmodium falciparum (Alegana et al., 2021; World Health Organization, 2022). On the other hand, the archipelago of Zanzibar is a pre-elimination setting, with low level seasonal transmission (Björkman et al., 2019). This is largely due to the routine implementation of a combination of effective control measures, including robust vector control and routine access to effective antimalarials (Björkman et al., 2019). Despite these efforts, malaria has been difficult to eliminate from the archipelago. There are several reasons this may be the case: (1) frequent importation of malaria from moderate or high transmission regions of mainland Tanzania and Kenya (Björkman et al., 2019; Le Menach et al., 2011; Lipner et al., 2011; Monroe et al., 2019; Morgan et al., 2020; Tatem et al., 2009); (2) ongoing local transmission due to residual vector capacity despite strong vector control (Björkman et al., 2019); and (3) a reservoir of asymptomatic infections (Björkman et al., 2019; Björkman & Morris, 2020).

Parasite genomics has the potential to help us better understand malaria epidemiology by uncovering population structure and gene flow, providing insight into changes in the parasite population including how parasites move between regions (Neafsey et al., 2021). Genomics has previously been used to study importation and transmission chains in other low transmission settings in Africa and elsewhere (H. H. Chang et al., 2019; Morgan et al., 2020; Moser et al., 2021; Patel et al., 2014; Roh et al., 2019; Sane et al., 2019). Previously, we had investigated the importation of malaria into Zanzibar from the mainland using whole genome sequencing, showing highly similar populations within the mainland and within the archipelago, but also identifying highly related parasite pairs between locations suggesting a role for importation (Morgan et al., 2020). However, this work lacked sufficient samples to assess transmission of parasites within Zanzibar. The larger and spatially rich sample set analyzed in this manuscript offers an opportunity for more refined analyses of transmission across Zanzibar and how parasites are related to those from coastal mainland.

A panel of molecular inversion probes (MIPs), a highly multiplexed genotyping assay, were designed in a previous study to target single nucleotide polymorphisms (SNPs) throughout the P. falciparum genome (Aydemir et al., 2018). We leveraged this assay to investigate the genetic epidemiology of parasites in the coastal mainland and Zanzibar utilizing 391 samples collected from cross-sectional surveys of both asymptomatic infections and symptomatic, uncomplicated malaria cases during 2016-2018. Specifically, we use identity by descent analyses to compare the genetic relatedness of mainland and Zanzibari parasites, and to investigate the geography/spatial relationships of genetically related parasites on the archipelago. We further characterize how the genetic complexity of infections differ by clinical status and describe patterns of antimalarial drug resistance polymorphisms in the parasite populations. In this low transmission setting, these analyses characterize fine-scale local parasite populations that contribute to continued transmission within the region, highlighting a key barrier to malaria elimination in the Zanzibar archipelago.

Samples from coastal Tanzania (178) and Zanzibar (213) were previously sequenced through multiple studies (Table 1, Supplemental Figure 1). These samples include 213 dried blood spots (DBS) collected in Zanzibar between February 2016 and September 2017, coming from cross-sectional surveys of asymptomatic individuals (n = 70) and an in vivo efficacy study of artesunate-amodiaquine (ASAQ) with single low dose primaquine (SLDP) in pediatric uncomplicated malaria patients in the western and central districts of Unguja island and Micheweni District on Pemba island (n = 143) (Msellem et al., 2020). These samples were geolocalized to shehias, the lowest geographic governmental designation of land in Zanzibar, across its two main islands, Unguja and the northern region of Pemba (Supplemental Figure 1). Mainland Tanzania samples were collected in rural Bagamoyo District, where malaria transmission persists, and residents frequently travel to Dar es Salaam, the major port from where travelers depart for Zanzibar. Of the mainland Bagamoyo samples, 138 were whole blood collected from 2015-2017 as part of an in vivo efficacy study of artemether-lumefantrine (AL) in pediatric uncomplicated malaria patients (Topazian et al., 2022), and the remaining 40 samples were leukodepleted blood collected in 2018 from asymptomatic but RDT-positive children who participated in a study investigating the transmission of P. falciparum to colony reared mosquitos. This project leveraged molecular inversion probe (MIP) data from SRA including PRJNA926345, PRJNA454490, PRJNA545345, and PRJNA545347.

In order to place coastal Tanzanian and Zanzibari samples in the context of African P. falciparum population structure across multiple regions, MIP data from 147 whole blood samples collected in Ahero District, Kenya from the same parasite clearance study were used (Topazian et al., 2022) in conjunction with a subset of data from 2,537 samples genotyped for a study of the 2013 Demographic Health Survey of the Democratic Republic of the Congo which included samples from DRC, Ghana, Tanzania, Uganda and Zambia (Verity et al., 2020) (see Supplemental Figure 2).

In this study, we leverage high-throughput targeted sequencing using molecular inversion probes (MIPs) to characterize the populations and the relationships of P. falciparum isolates in Zanzibar and coastal mainland Tanzania. The parasite populations appear to be highly related to each other (Figure 1A) when evaluated using SNPs in the core genome. Interestingly, within Zanzibar, structure could be observed, with parasites closer to the main ferry terminal in Zanzibar town clustered more closely with coastal mainland parasites (Figure 1B, Supplemental Figure 1) compared to parasites that were more geographically distant. This, in combination with the evidence of rapid decline of genetic relatedness with distance on the archipelago (Figure 3), is consistent with population microstructure within the island chain. This microstructure within the archipelago is supported by K-means clustering where Zanzibari isolates show higher within cluster than between cluster IBD (Figure 2). It is also consistent with isolates with higher IBD (Figure 4A and B) in Unguja and Pemba compared to a maximum IBD of 0.20 between Zanzibar and coastal mainland Tanzania (Supplemental Figure 8) or between Unguja and Pemba (Supplemental Figure 9). Parasite populations within the very low transmission region of Zanzibar may be more isolated than expected, allowing them to differentiate from each other. This may be indicative of very effective local malaria control, yet with continued micro-transmission remaining. Thus, directly targeting local malaria transmission, including the asymptomatic reservoir which contributes to sustained transmission (Barry et al., 2021; Sumner et al., 2021), may be an important focus for ultimately achieving malaria control in the archipelago (Björkman & Morris, 2020).

Despite the overall genetic similarity between archipelago populations, we did not find parasite pairs with high levels of IBD between the coastal mainland and Zanzibar, with the highest being 0.20. While this level still represents a significant amount of genetic sharing, similar to a cousin, the lack of higher levels does not allow us to identify specific importation events. This is largely due to the study design, which is based on convenience sampling, the relatively low numbers of samples and lack of sampling from all mainland travel hubs (Bisanzio et al., 2023). Sampling was also denser in Unguja compared to Pemba. On the other hand, we see clear transmission of highly related parasites within each population (IBD > 0.99). In Zanzibar, we see this both within and between shehias, suggesting that parasite gene flow occurs over both short and long distances. These results are similar to our previous work using whole genome sequencing of isolates from Zanzibar and mainland Tanzania, showing increased within population IBD compared to between population IBD (Morgan et al., 2020). 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). Less frequent longer distance transmission events also occur, likely due to longer range human migration within the islands.

Asymptomatic parasitemia has been shown to be common in falciparum malaria around the globe and has been shown to have increasing importance in Zanzibar (Lindblade et al., 2013; Morris et al., 2015). What underlies the biology and prevalence of asymptomatic parasitemia in very low transmission settings where anti-parasite immunity is not expected to be prevalent remains unclear (Björkman & Morris, 2020). Similar to a few previous studies, we found that asymptomatic infections had a higher COI than symptomatic infections across both the coastal mainland and Zanzibar parasite populations (Sarah-Matio et al., 2022). Potential reasons for this phenomenon include low level immune response resulting in chronic infection with multiple clones over time in asymptomatic individuals (Felger et al., 2012), and dominance of more virulent clones in shorter symptomatic infections resulting in lower COI (Collins et al., 2022). In Zambia, one study suggested that infections that cause asymptomatic infection may be genetically different than those that cause symptomatic infection (Searle et al., 2017). However, this study included samples collected over different time periods and relied on a low-density genotyping assay which only investigated the diversity of 24 single nucleotide polymorphisms across the genome. Here, based on SNPs throughout the core genome, we did not see differential clustering of asymptomatic or symptomatic infections in Zanzibar or the mainland (Figure 1A), suggesting that these parasite populations remain similar when comparing clinical status. However, this genotyping approach does not address potential variation in the many hypervariable gene families that encode genes known to be associated with pathogenesis (e.g. var, rifin and stevor genes) and does not address differences in expression of genes associated with pathogenesis that may reflect differences in the populations. Investigation with other methods, such as long-read genome sequencing and transcriptional profiling, would be needed to address these differences. Finally, any collection of “asymptomatic” isolates is likely to be heterogeneous, including those that are pre-symptomatic, those who may have been previously treated, and detection of multiple strains is also reliant on the ability of the genotyping method or sampling strategy to sensitively capture minor strains (minority genomes).

While mutations for partial artemisinin resistance were not observed in K13, other antimalarial resistant mutations of concern were observed. Validated drug resistance mutations linked to sulfadoxine/pyrimethamine resistance (Pfdhfr-N51I, Pfdhfr-C59R, Pfdhfr-S108N, Pfdhps-A437G, Pfdhps-K540E) were found at high prevalence (Table 2). Prevalence of polymorphisms associated with amodiaquine resistance (Pfcrt-K76T, Pfmdr1-N86Y, Pfmdr1-Y184F, Pfmdr1-D1246Y) were seen at similar proportions as previous reports (Msellem et al., 2020). The wild-type Pfmdr1-N86 was dominant in both mainland and archipelago populations, concerning for reduced lumefantrine susceptibility. Polymorphisms associated with artemisinin resistance did not appear in this population. However, continued surveillance is warranted given emergence of these mutations in East Africa and reports of rare resistance mutations on the coast consistent with spread of emerging Pfk13 mutations (Moser et al., 2020).

Overall, parasites between Zanzibar and coastal mainland Tanzania remain highly related, but population microstructure on the island reflects ongoing low level transmission in Zanzibar, partially driven by asymptomatic infections that potentially constitute a long-term reservoir. This is likely the result of the continued pressure on the population through the implementation of effective control measures. In this study, parasite genomics allows us to parse differences in parasite populations and reveals substructure in an area of low transmission intensity. A recent study identified “hotspot” shehias, defined as areas with comparatively higher malaria transmission than other shehias, near the port of Zanzibar town and in northern Pemba (Bisanzio et al., 2023). These regions overlapped with shehias in this study with high levels of IBD, especially in northern Pemba (Figure 4). These areas of substructure represent parasites that differentiated in relative isolation and are thus important locales to target intervention to interrupt local transmission (Bousema et al., 2012). A potential strategy is targeting these focal regions through both mass drug administration and vector control to eliminate these sources of transmission (Morris et al., 2018; Okell et al., 2011). Such strategies and measures preventing imported malaria could accelerate progress towards zero malaria in Zanzibar.