Background
Significant progress has been made in reducing
Plasmodium falciparum malaria transmission since the early 2000s, due in part to massive distributions of insecticide-treated bed nets (ITNs), increased coverage with indoor residual spraying (IRS), and the introduction of artemisinin-based combination therapy (ACT) [
1,
2]. In fact, it is estimated that the global burden of malaria declined by 40% between 2000 and 2015, leading 35 countries to establish malaria elimination targets as of September 2015 [
1,
3]. While the current arsenal of malaria control tools has been broadly effective, it has not been sufficient to reduce transmission everywhere [
4]. Despite malaria control programmes, the ten countries with the highest malaria burden in Africa experienced increases in malaria cases between 2015 and 2017 [
5,
6]. Acknowledging that the success of malaria control in these and other high burden regions is critical for the attainment of malaria elimination targets, the World Health Organization (WHO) renewed its focus on high transmission settings as a key component of malaria elimination programmes in the recent High Burden to High Impact (HBHI) response plan [
6]. Achieving malaria control in regions where transmission has previously been refractory to interventions will require an enhanced understanding of the unique mechanisms that perpetuate transmission in different settings and data-driven approaches that target them [
6].
Plasmodium falciparum molecular epidemiology has emerged as a tool to genetically track transmission patterns [
7‐
10]. Population genetic diversity and multiplicity of infection (MOI) have been used to monitor changes in transmission intensity and evaluate whether programmes or interventions have altered transmission patterns [
11‐
19]. Parasite genotyping may allow more refined discrimination of parasite locality of origin than travel surveys alone, possibly enabling gene flow between populations to be estimated [
20‐
22] and source populations of on-going transmission to be identified [
23]. Parasite genetic approaches have demonstrated their value in complementing routine epidemiological and entomological surveillance to inform control programmes in moderate to low transmission settings where elimination efforts have been concentrated [
7]. With renewed attention to malaria control in high burden regions as a component of elimination planning, it is important to define the potential role of molecular epidemiology in such settings.
Zambia, a malaria-endemic country in southern Africa, aims to eliminate malaria from every district by 2021. As a country, Zambia has made great strides towards achieving this goal. In fact,
P. falciparum prevalence by microscopy among children under 5 years old declined from 19.4% in 2015 to 9.1% in 2018 [
24]. Unfortunately, the success in malaria control has been heterogeneous across the country. Luapula Province, located in northern Zambia along the international border with the Democratic Republic of the Congo (DRC), continues to experience holoendemic transmission despite use of control interventions for over a decade [
4]. In Nchelenge District, located in Luapula Province, ITN distributions were carried out in 2007, 2011, and 2014, and IRS has been conducted annually, using pyrethroids between 2008 and 2010, carbamates between 2011 and 2012, and the organophosphate, pirimiphos-methyl (Actellic
® 300CS), since 2014 [
4,
25]. IRS with Actellic
® led to a moderate decrease in
P. falciparum prevalence by 25% in the sprayed regions within Nchelenge District; however, the effect was short-lived, lasting only for 6 months after spraying, and did not impact unsprayed areas [
25]. Despite control measures, Luapula Province, including Nchelenge District, continues to experience the highest malaria prevalence among children under 5 years in the country at 30.4% by RDT [
24,
26].
Across Lake Mweru from Nchelenge, situated along the international border, are two villages, Kilwa and Kashobwe in Haut-Katanga Province, the DRC. The most recent Demographic and Health Survey (DHS) from the DRC conducted in 2013–2014 reported that 23.1% of children ages 6 months to 5 years tested positive for malaria by microscopy in Haut-Katanga Province [
27]. This Province has received fewer malaria control interventions historically compared to neighboring Nchelenge District in Zambia. Four cross-sectional surveys in Kilwa and Kashobwe conducted between August 2016 and July 2017 revealed that very few households (range: 0.2–2.5%) reported ever receiving IRS in these villages (Kobayashi, unpublished). During September and October 2016, a large-scale ITN distribution took place in Haut-Katanga Province. Epidemiological survey data revealed that following the ITN campaign, there was a significant increase in the proportion of individuals who reported sleeping under a bed net and a decline in parasite prevalence by microscopy in Kilwa and Kashobwe from 32% in 2016 to 18% in 2017 (Kobayashi, unpublished).
In high transmission settings including Nchelenge District, Zambia and Kilwa and Kashobwe, the DRC, individuals are typically infected with multiple genetically distinct parasites. Classic population genetic approaches that utilize neutrally-evolving SNPs or microsatellites across the genome inadequately address high polyclonality. Amplicon deep sequencing enables each parasite clone in an infection to be enumerated, but typically only at specific, high-diversity and non-neutral amplicons in the genome. Given the interest in molecular epidemiology for malaria control in high burden regions and the limitations of classic population genetic analysis in these settings, the performance of amplicon deep sequencing to enhance the understanding of malaria epidemiology in a key region for malaria elimination and control was evaluated. P. falciparum genetic diversity at two time points in June-July 2016 and January-March 2017 in Nchelenge District, Zambia and Kilwa and Kashobwe, the DRC is described. Genetic relatedness of parasites from northern Zambia and southern DRC were compared in order to assess whether this border region represents one contiguous transmission foci. The impact of declining parasite prevalence in Haut-Katanga Province on parasite population genetics was evaluated over the two time periods of the study. Finally, methods for detecting epidemiologically relevant genetic signals amidst high genetic diversity in a holoendemic transmission setting are discussed.
Discussion
The utility of parasite genotyping to enhance malaria epidemiology has been well-demonstrated in low to moderate transmission settings. Although parasite genotyping has been useful in assessing the prevalence of drug resistance mutations in high transmission settings [
42], it remains to be proven as a tool for evaluating control interventions or enhancing the understanding of transmission epidemiology in this context. In a holoendemic transmission region along the international border between Luapula Province, Zambia and Haut-Katanga Province, the DRC, high genetic
Pfama1 and
Pfcsp diversity was observed across two different seasons, indicating that these loci are diverse and unstructured in high transmission settings. Given the benefit of using amplicon deep sequencing in such regions with high polyclonality, this observation highlights the limitations of utilizing parasite population genetic analysis to understand transmission epidemiology in high-burden areas. Although multiple studies in moderate to low transmission settings reported decreases in genetic diversity and MOI following decreased transmission [
11‐
19], no signatures of a parasite population bottleneck were detected in this study despite an ITN distribution campaign between sampling timepoints that reduced the parasite prevalence by microscopy in Kilwa and Kashobwe, the DRC using amplicon deep sequencing of two highly diverse antigens. This reflects the fact that transmission remains high in this region even after the observed decrease in parasite prevalence, and the parasite population remains sufficiently large and, therefore, shielded from a genetic bottleneck. Further, the choice of non-neutral genetic loci, which enabled haplotypic characterization of polyclonal infections, may have hindered the ability of this study to detect changes in population genetic diversity, underscoring the current limitations of implementing molecular epidemiologic approaches in high-burden transmission settings. It is also possible that genetic signatures of a population bottleneck may take longer than 6 months to become apparent, and were missed in this study. A much larger decline in transmission is likely required to bottleneck the parasite population. Further, although parasite genotyping has been touted as a tool for monitoring changes in transmission intensity and evaluating control interventions [
7,
8], these utilities may be limited to moderate and low transmission settings, and may be less useful in high transmission regions, where high polyclonality necessitates analysis of non-neutral markers.
No evidence of population structure was detected comparing
Pfama1 and
Pfcsp isolates between Zambia and the DRC. While clear signatures of population differentiation are easily interpretable, it is more challenging to attribute cause for observations with no discernable population structure. A lack of population structure could either reflect the true underlying biology of an admixed population or may be an artifact of the use of genetic markers that are under selection and alone not ideal for assessing population structure on a small spatial scale in high transmission zones [
43,
44]. Although the same
Pfcsp amplicon that was examined in this study revealed parasite population genetic structure on a continental scale [
20], additional research is merited to assess the utility of these
Pfama1 and
Pfcsp amplicons to detect population structure on smaller geographic scales. While these data suggest
P. falciparum parasites from Nchelenge, Zambia and Kilwa and Kashobwe, the DRC, exist as a single panmictic population, increasing either the number of neutral SNPs characterized or the number of isolates sequenced could reveal finer scale population structuring.
It is typical to analyse neutral, unlinked SNPs in population genomic analysis. This study characterizes two highly variable
P. falciparum genes,
Pfama1 and
Pfcsp which are known to be under balancing selective pressure [
45]. If balancing selection were to occur independently at geographically separated sites, then the isolation by distance signal could be attenuated, which would lead to the inability to correctly identify population structure when it truly exists. Further, since
Pfama1 and
Pfcsp are under balancing selection, changes in their diversity do not necessarily reflect changes in transmission. Finally, the SNPs within each of these two amplicons are in linkage disequilibrium in the
P. falciparum genome. It is possible that the use of non-neutral, linked loci biased these analyses such that true population differentiation was not detected between Nchelenge District, Zambia and Haut-Katanga Province, the DRC or failed to detect genetic signatures of a population bottleneck. However, in regions where most infections are comprised of multiple, genetically distinct parasite clones, amplicon deep sequencing is perhaps the most cost-effective method capable of preserving parasite haplotypes, bypassing the need to invoke potentially biased haplotype reconstruction methods or discarding polyclonal infections prior to analysis. In
P. falciparum genetics, it has been common practice to exclusively analyse monoclonal infections [
11,
46,
47] or disregard loci where two or more alleles are present in polyclonal infections [
48,
49]. While such practices may be appropriate in some settings where MOI is low, they are not an option in high burden regions like Luapula and Haut-Katanga Provinces, where restricting an analysis to monoclonal infections would require discarding close to 80% of the data (Pringle, unpublished). As methods for handling polyclonal genetic data continue to improve, it may eventually be possible to select unlinked, neutral loci for additional analyses to assess parasite population structure in border regions and detect signatures of population bottlenecks in moderate to high transmission settings.
Despite the use of non-neutral, linked SNPs, the data suggesting a contiguous
P. falciparum population are consistent with whole genome sequencing analyses from
Anopheles funestus mosquitoes that did not detect population structure of vectors between Nchelenge, Zambia and Haut-Katanga Province, the DRC ([
50], Lee, unpublished). These data supporting a single and large primary vector population suggest a possible mechanism that might drive the regular genetic crossing and lack of population differentiation among
P. falciparum isolates from across the country border. The observation of a contiguous parasite population across the border between Zambia and DRC suggest that collaborative malaria control efforts targeting both regions together may enhance intervention success. Border regions of a country frequently experience higher malaria transmission than non-border regions and often harbor the final transmission foci prior to elimination [
51]. The observation that Luapula Province, Zambia and Haut-Katanga Province, the DRC together comprise a contiguous high transmission foci along an international border highlights the importance of expanding existing regional partnerships [
52] like Elimination 8 (E8) in southern Africa that can facilitate the coordination of elimination efforts across multiple nations. A study which looked at how frequently the Global Fund funded malaria projects aiming to establish multi-national control efforts [
53] found that these proposals are rarely funded, and that there is little guidance for what makes these projects successful. Developing new strategies to guide, fund, and support regional initiatives that encourage international cooperation towards malaria elimination may enhance current and future efforts. As efforts to eliminate malaria across the globe continue to expand, addressing the unique challenge of controlling border malaria is essential.
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