Background
Malaria is still a major public health burden in Kenya, despite the intensification of control measures that have resulted in recent reductions in morbidity and mortality. About 70% of the population is at risk of malaria infection with the Coastal and Lake Victoria endemic regions bearing the highest community prevalence of around 4.5% and 18.9%, respectively, based on microscopy [
1]. Malaria control and eventual elimination is threatened by the emergence of drug resistant parasites and insecticide resistance by mosquitoes, the perennial presence of asymptomatic
Plasmodium falciparum infections and highly diverse parasite populations [
2,
3]. Asymptomatic infections harbour distinct parasite sub-populations, also termed clones/variants that normally undergo recombination in the mosquito mid gut during zygote formation resulting in genetically diverse parasites [
4]. An individual may thus be infected with parasites of multiple genotypes from a single mosquito bite inoculation or multiple mosquito inoculations [
5]. These number of distinct parasite genotypes in an individual is referred to as complexity of infection (COI).
The genetic diversity of
P. falciparum and COI are correlates of malaria transmission intensity and can be used in assessing the impact of malaria control strategies [
6]. Generally, studies have shown that
P. falciparum parasites have a higher within-host genetic diversity in high transmission settings than in low transmission settings [
7,
8]. This has led to the notion that a reduction in transmission intensity translates to a reduction in genetic diversity due to decreased chances of recombination between genetically distinct variants [
9,
10]. The extensive genetic diversity of
P. falciparum vaccine targets is a major hinderance in malaria vaccine development as the host immune responses may fail to recognize all the variants of an antigen. The merozoite surface protein-2 (MSP2) has been shown to be highly polymorphic and informative in genotyping parasite populations [
11]. It is a glycoprotein encoded by the
msp2 gene that is located on chromosome 2. It is divided into five blocks that include a highly polymorphic central block 3 and is flanked by unique variable domains and conserved N- and C-terminal domains [
12,
13]. The polymorphic block 3 contains repeats that vary in number, length and sequence that are grouped into two allelic families i.e. IC/3D7 and FC27 [
14] that are associated with different malaria outcomes [
15,
16].
Asymptomatic infections constitute the biggest proportion of
P. falciparum infections in endemic regions [
17]. They result from partial immunity developed after repeated exposure to the parasite especially in endemic areas [
18]. These individuals act as a reservoir for infectious parasites. They may be associated with either increased or reduced risk of symptomatic malaria [
19,
20] depending on several factors, such as age, transmission intensity, COI, parasitaemia and acquisition of new clones [
21].
This study investigated the temporal genetic diversity and complexity of P. falciparum infections in asymptomatic and first-febrile follow-up samples. In addition, the msp2 genetic diversity between asymptomatic and first-febrile pairs was examined. The samples were collected during the period of decline in malaria transmission in a moderate to high transmission region of Kilifi, Kenya, and msp2 gene polymorphisms assessed.
Discussion
Despite the decline in malaria positivity prevalence over 12 years in the community cohort, malaria is still characterized by highly genetically diverse
P. falciparum infections, the stability of
msp2 alleles and a high complexity of infection. This corresponds with the sustained moderate-high transmission in the study area. There was no temporal change in
msp2 genetic diversity or COI, suggesting that in this moderate to high transmission area though malaria positivity rate significantly declined between 2007 and 2018, it was not substantial enough to result in a change in the parasite genetic profile. In the wider study area, Kilifi County, a significant decline in county referral hospital malaria admissions was described between 2002 and 2009 [
30,
31]. The decline was not sustained and thereafter from 2009 there was an increase in hospital admission malaria positivity in older children [
30]. The decline in the localized community population observed in this study, during a period of an overall rise in malaria hospital admissions [
30], highlights the differences in the surveillance populations. The hospital surveillance data provides a better representation of the population since it covers a wider catchment area of the county, compared to the local community cohort analysis that is a subset of the wider county population and includes asymptomatic infections in the counts. It is possible that a genetically diverse parasite population was maintained by the sustained transmission in the county despite the local decline in the Junju area. This hypothesis is consistent with findings at a household level, where serological surveys showed evidence of diverse populations in homesteads at low malaria risk where the surrounding area was at high transmission, and vice versa evidence of less diverse populations in homesteads at high malaria risk where surrounding areas were at low transmission [
32].
Thus, the extensive parasite genetic diversity is maintained. In great contrast, a dramatic reduction in malaria transmission as observed in Grande Comore Island, Union of Comoros, from 108,260 cases in 2006 to 1072 in 2015, was followed by a commensurable decline in MOI based on
msp2 genotyping from 2.75 to 1.35 in healthcare facility samples obtained from 2006/2007 and 2013–2016 [
9]. Furthermore, there was a significant reduction in
msp2 alleles between the two time-points [
9]. Altogether the
msp2 genetic profile corresponded to the decline in malaria transmission, indicating COI as a marker of assessing the changes in transmission. Similarly, intensification of malaria control interventions in Senegal between 2006 and 2011 resulted into a reduction in genetic diversity of parasite populations [
33]. On the contrary, reduction in malaria transmission in the Kingdom of Eswatini did not result into low parasite genetic diversity mainly due to malaria importation from neighbouring countries with high malaria transmission intensity [
34]. Thus, inferring malaria transmission intensity from parasite genetic data ought to consider the impact of external factors affecting the parasite population genetics.
The apparent preference for the
msp2 FC27 alleles was a significant feature of first-febrile infections in the asymptomatic-first-febrile paired analysis. This observation that has been made before in Congo and Tanzania, FC27 alleles were associated with severity of disease and were more predominant in children who had two or more febrile malaria episodes [
16,
35]. Interestingly, in a case-control study conducted in Papua New-Guinea, the FC27 genotypes were twice as likely to be found in symptomatic than asymptomatic individuals [
36]). The FC27 allelic family is potentially an important set of genetic variation to interrogate further to determine their impact on immunity. The IC/3D7 family has been associated with asymptomatic infections and is thought to protect against clinical malaria [
16,
37,
38]. However, contradictory findings have reported that parasites carrying FC27 like alleles are more prevalent among asymptomatic carriers [
15,
39]. There is no clear consensus on whether the two
msp2 allelic families are likely to be found in asymptomatic or symptomatic infections. Larger studies in regions with different transmission intensities are needed to gain more insights into the effect of each allelic family on clinical outcome.
The high COI and large proportion of polyclonal asymptomatic infections is a result of the frequent and repeated exposure to genetically distinct malaria parasites in endemic areas, as described in previous studies [
40]. This leads to the development of partial immunity that results in a reduction in clinical symptoms and carriage of low-level parasitaemia [
41,
42]. The paired samples revealed the rapid turnover of alleles between asymptomatic and first-febrile infections, which is expected given ongoing malaria transmission in the study area. Asymptomatic
P. falciparum infections can act as precursors to symptomatic malaria [
43]. Genotyping of
msp2 has previously been used to assess whether the development of symptoms is due to persistence of an existing clone or due to infection with a new clone [
44]. In this study, the febrile infections were characterized by more monoclonal infections, an overall lower COI and new alleles unobserved in the prior asymptomatic infection. The new alleles likely escape immune responses, rapidly increasing parasitaemia thereby causing massive tissue damage that manifests as symptoms. Similar findings have been reported in other studies, implicating the lack of protective immune responses against the new clones [
44‐
47]. Although the study used the more sensitive capillary electrophoresis to determine fragment sizes, a strict inclusion criterion was used to define true peaks during data analysis, which may have underestimated the fragment numbers impacting the estimation of COI. The presence of stutter peaks in the capillary electrophoresis data also presented technical challenges in the definition of true peaks. Future studies should consider using more sensitive methods like targeted amplicon deep sequencing (TADS) to define COI.
The high
msp2 genetic diversity maintained across the study period was expected as Kilifi is a region of moderate to high malaria transmission. The 291 bp, 327 and 411 bp FC27 and 555 bp IC/3D7 fragment sizes were common in both asymptomatic and first-febrile infections. Strikingly, some of these genotypes have been reported in other countries, such as Mali [
48], as the most common genotypes, suggesting that they can be selected as candidates for malaria vaccine development. However, identical fragment lengths may not always represent identical sequence lengths and sequencing is required for confirmation.
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