Polygenomic Infections Are More Common Among Travelers to Richard Toll
To determine whether malaria infections obtained from travelers and non-travelers exhibited different levels of genetic diversity, we analyzed the genotyping data from all samples obtained from individuals who provided travel history information. Among infected individuals providing travel history information, 72% (401/555) indicated that they had traveled and spent at least one night outside Richard Toll district in the previous 14 days (Additional File 2). Genotyping data from infections derived from travelers were then compared to genotyping data from non-traveler malaria infections. When we evaluated the number of monogenomic and polygenomic infections for each of these two populations (travelers vs. non-travelers) we found a significant increase in number of polygenomic infections among individuals who reported recent travel outside Richard Toll (28.2%), compared to infections identified in non-travelers (20.1%) (one-tailed Fisher Exact Test, P = 0.032, Figure 1, Additional File 2).
A Large Proportion of Richard Toll Parasites are Genetically Related to Parasites from Thiès
To test whether malaria parasites detected in Richard Toll matched parasites from elsewhere in Senegal, we compared the genotyping data from Richard Toll parasites to a large existing genotyping database of Senegal parasites isolated from Thiès, a region of higher malaria transmission that has undergone dramatic declines in malaria burden since 2001. Pairwise analysis of relatedness, including identity by state (IBS), was performed on genotyping data from both Richard Toll samples and clinical samples collected during research conducted in Thiès. A total of 426 Richard Toll samples and 1,516 Thiès samples met the criteria of being monogenomic and missing either no or one single nucleotide polymorphism (SNP) assay (Additional Files 3-4). While the Thiès sample set represents only a fraction of malaria infections in Senegal, it provided pilot data for this proof of concept study. Genotype data were evaluated for relatedness by calculating the proportion of alleles that matched between pairs of samples to identify both identical and highly related sample sets (Additional File 5). This pairwise analysis revealed that a large proportion (21%) of Richard Toll infections (Additional File 3) shared an identical or highly related barcode with parasites from Thiès (Additional File 4), Senegal (Figure 2, Additional Files 3-4). For comparison, any two other populations within Senegal in the same time frame only share a small proportion of infections (<5%) sampled.
Persistence of Genetically Identical Infections Across Multiple Transmission Seasons
To ask whether genetically identical parasites from Richard Toll were detected in multiple transmission seasons, we examined barcode groups identified by the pairwise relatedness analysis. Transmission seasons in Senegal are seasonal and generally occur between September and December of the calendar year in northern and central Senegal. A barcode group contained identical or highly related genotypes from the pairwise analysis, and the genotype associated with barcode groups that contained parasites from multiple years represent a parasite lineage. From this analysis, we detected seven genetically identical parasite lineages and three highly related parasite lineages with these genotypes found in more than one transmission season between 2012 and 2015 (Figure 3, Additional File 6). Two of these barcode groups representing lineages contained parasites identical or highly related to genotypes from Thiès, while the remaining lineages contained only parasites detected in Richard Toll. In one of these, the genotype appeared in two cases in 2013, again in two cases in 2014, and expanded to nine cases in 2015.
Highly Related Parasites Found in Households of Non-Travelers
To ask whether infections from travelers contribute to a risk of local spread of these imported infections, we took advantage of the RACD program, in which all household members received an RDT,  and tested whether parasites within households share genetic relatedness. The index case’s infection was detected in the clinic, and the additional infections were found among household members of the index case. Only fifteen households (of 49 households in which RACD was conducted and genotyping data was available) had both an initial infection and at least one additional infection that yielded genotyping data for the comparison. These within-household infections were scored for genetic relatedness using pairwise analysis, then households that harbored highly related infections were classified according to whether the index case had travel history. We first scored these households based upon whether there was evidence of highly related infections and found that about half (7/15, 47%) of these households had at least one additional infection that genetically matched (i.e., identical or highly related) the initial infection (Figure 4, Additional File 7). Of seven households with at least one infected person in which there was no travel history, six contained at least two identical or highly related parasites, while of eight households with at least one infected person and travel history, only one household contained at least two identical or highly related parasites. Stratification of households based upon travel history revealed a significant increase of highly related parasites within households with no travel history (P = 0.01).