Introduction
Ghana comprises of three distinct ecological zones; Sahel zone (Northern Ghana), the Forest zone (Middle belt) and the Coastal zone (Southern Ghana). The prevalence of malaria varies across the three ecological zones, with the Forest zone having relatively higher parasite prevalence (22.8%) [
1] compared to other zones [
2]. Parasite prevalence peaks during the single rainy season (June–October) in the Sahel zone. However, malaria parasite prevalence peaks twice (May–June and October–November peaks) in a year and coincides with the bi-modal rainfall pattern in both the Forest and Coastal zones in Ghana [
3,
4].
Genetic diversity in
P. falciparum parasites primarily results from recombination between different clones in addition to within clone polymorphisms including chromosomal deletions, gene duplication, number of repeat sequences and point mutations at various genetic loci [
5]. Information on parasite diversity and population structure are highly relevant to the epidemiology of malaria and virulence of the parasite [
6]. The population structure of the parasite can help to determine the variations in malaria transmission between the different ecological zones as well as within the same ecological zone at different time points [
7] .
Microsatellite markers are simple sequence repeats found in the parasites genome that have proven to be selectively neutral except in instances such as when they are found near genes which confer drug resistance. These markers are extremely abundant in the genome of
P. falciparum [
8] and vital in determining diversity and distribution of various parasites genotypes [
9] across different transmission settings [
9,
10]. The number of microsatellite markers that have been used to determine the diversity and population structure of
P. falciparum ranges from six [
11] to as high as 26 [
12]. A complexity of the diversity of a parasite generally increases with the number of markers used in the analysis, however, some markers are more polymorphic than others, as such the use of a select few markers that have very high diversity can produce similar complexity as the use of a larger number of markers that have low to moderate diversity.
Many studies have shown reduced genetic diversity of the parasite populations as the result of intensified malaria control measures [
13,
14]. However, the use of certain anti-malarial drugs can also alter the genetic landscape of the parasite and how they spread in a specific geographic region as a consequence of the effect of selection in favor of certain genotypes and or phenotypes [
15].
Asymptomatic malaria infections present an opportunity for the mosquito vector to obtain a continuous source of parasites, which are subsequently transmitted to a new human host. The continued inoculation of genetically diverse malaria parasites into a host by different mosquitos can result in the generation of highly diverse parasites due to outcrossing and recombination events within the mosquito midgut when gametocytes of these genetically diverse parasites are picked up together by a feeding mosquito [
16]. This study identified the prevalence of asymptomatic
P. falciparum carriage by school children without any outward symptom of malaria living in five communities across three ecological zones of Ghana and evaluated the genetic diversity and population structure of the identified
P. falciparum parasites.
Discussion
Asymptomatic
P. falciparum infections are a hindrance to malaria control as they can serve as reservoirs for gametocyte carriage [
45] and at times can result in symptomatic infections weeks later [
46]. Investigating parasite population structure and gene flow across the three ecological zones of Ghana could help understand the impact of malaria control interventions that have been implemented in the country [
47]. In this study, relatively high prevalence of asymptomatic malaria parasite carriage by the school children were recorded in the Sahel and Forest zones than the Coastal zone. The rather low prevalence of asymptomatic
P. falciparum carriers in the Coastal zone compared to the Forest and Sahel zones might mean that ongoing malaria interventions might have worked better in the Coastal zone than the others. The higher prevalence of asymptomatic
P. falciparum carriers in the Sahel and Forest zones is despite additional malaria control interventions such as indoor residual spraying (IRS), which is not carried out in the Coastal zone.
This high prevalence of asymptomatic malaria parasite carriage by the children in both the peak and dry malaria season may mean that additional malaria control interventions that target asymptomatic parasite carriage such as mass test and treat exercises could be implemented to reduce parasite burden [
48].
The genetic diversity of the parasites identified across the three ecological zones of Ghana was generally moderate to high. In the rainy season, genetic diversity in the Sahel and Forest zones were higher than diversity in the Coastal zone (Ada), consistent with earlier findings that areas with the highest prevalence of asymptomatic parasite carriers would have the greatest genetic diversity and multiplicity of infection [
49]. Parasites identified in the Sahel zone (Tamale) has the least diversity relative to parasites from the two other zones. The low heterozygosity of
P. falciparum identified in the Coastal zone (Ada) relative to the Forest (Konongo) and Sahel zones (Tamale) could be due to relatively lower transmission intensity and consequently lower densities of mosquitoes in the Coastal zone relative to the Sahel and Forest zones (Afrane et al, unpublished). The low parasite diversity in the Coastal zone could also imply that the malaria control interventions implemented in the Coastal zone have been more effective and/or successful than in the Forest and Sahel zones as discussed already [
36]. The higher parasite diversity identified in the dry season relative to the peak season in all the three zones could be the result of a higher density of major parasite clones in the peak season that prevented the detection of a number of minor parasite isolates that were present at much lower frequencies.
The level of recombination, genetic variation and genetic structure from LD and clustering analyses reflect the patterns of gene flow and transmission intensity among the parasites circulating within the three ecological zones. In areas with high transmission, the frequent recombination among parasite strains, low genetic differentiation and large parasite gene pools result in high levels of genetic variation as seen in most malaria endemic areas in sub-Saharan Africa [
5,
30,
50,
51]. Similar genetic composition and phylogenetic closeness of parasites particularly between the Sahel and Forest zones suggest the likely carriage of diverse parasites by mobile population that end up mixing with the local parasite population and thereby reducing the genetic differentiation of the parasites [
34]. This suggestion is supported by the fact that movement from the Sahel zone of Ghana is very high and migration from the Sahel (North) to the Coastal zone of Ghana is mainly through the Forest zone, where most of the migrant population settle and some continue their migration to the Coast [
52,
53]. Similarities in the diversity of parasites from the Sahel and Forest zones relative to the Coastal zone also suggests that a recombinant vaccine based on the genetic background of a polymorphic antigen found in the Sahel zone would be equally effective in the Forest zone but may lack efficacy in the Coastal zone.
The mean MOI in samples from the Sahel zone (Tamale) was slightly lower than that identified in the Forest zone (Konongo) in the rainy season most likely due to the residents being exposed to a higher incidence of infectious mosquito bites [
54], with each bite likely to inoculate a genetically diverse parasite strain. The mean MOI for the forest and coastal zones were similar (mean MOI = 1.4) except the Sahel zone that recorded mean MOI of 1.0 during the off-peak season. This supports the fact that low transmission and the dry season are associated with reduced MOI in malaria endemic countries [
13,
28]. The low variations in MOI identified across the Sahel and Forest zones is contrary to an earlier report from Ghana where large differences in the MOI across different sites across the country were observed [
55]. High malaria parasite prevalence settings are usually characterized by infections containing high parasite multiplicity of infection and genetic diversity [
56,
57]. The multiplicity of infection identified in this study could have been higher than was reported due to the event that minor parasite clones are often undetected [
58].
High parasites diversity and MOI may imply high parasite survival and successful transmission in the midst of malaria control interventions [
13]. Asymptomatic cases with high MOI could progress towards symptomatic or severe form of infections. Also, genetically diverse clones may adapt better to existing interventions and increase the likelihood of developing antimalarial resistance. Most malaria control interventions are implemented without any recourse to the diversity of the parasites circulating within the implementation sites [
59], however, greater success can be achieved by reducing malaria incidence as well as parasite diversity [
59,
60]. A deeper investigation is needed to explore the association between polyclonality and anti-malarial drug resistance, given that malaria infections with high complexity could enhance the selection of drug resistant parasites than low complexity infections [
61]. Although microsatellites analysis is a cost-effective, rapid, and user-friendly tool for determining population structure and transmission [
30,
62], the genotype of each clone within a polyclonal sample cannot be easily distinguished. Amplicon deep sequencing offers an alternative means for inferring genetic relationships among clones within and between hosts [
63] that can be used in future studies to thoroughly investigate the association between the complexity of infections and genetic variation in high and low transmission areas.
Acknowledgements
The authors thank the parents who provided consent and the children who volunteered to be a part of this study. “The following reagent was obtained through BEI Resources, NIAID, NIH: Plasmodium falciparum, Strain 3D7, MRA-102, contributed by Daniel J. Carucci.”
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