Background
Malaria is a leading cause of mortality and morbidity in tropical countries. In 2016, more than 212 million of clinical cases and 429,000 deaths were reported worldwide. Sub-Saharan African countries support 92% of the global malaria burden, with children younger than 5 years, pregnant women, and non-immune visitors to endemic areas being people the most at risk of developing severe or fatal malaria [
1]. In Senegal, 492,253 clinical cases and 526 deaths were reported in 2015. These figures represent a 12-times reduction compared to those from 2001, a substantial decrease that highlights the success of the malaria control programme that was implemented in Senegal in the beginning of the 2000s [
2]. This malaria control programme was based on several approaches including widespread use of insecticide-treated mosquito nets (ITNs), rapid diagnostic testing (RDTs), treatment with artemisinin-based combination therapy (ACT), indoor residual spraying of insecticides (IRS), intermittent preventive treatment in pregnancy (IPTp) and seasonal malaria chemoprevention (SMC) in children under 10 years of age with sulfadoxine–pyrimethamine plus amodiaquine in areas of high seasonal malaria transmission [
1]. However, despite this undisputable success, multiple factors can threaten malaria control efforts and compromise elimination including the emergence of drug-resistant parasite strains [
3], insecticide-resistant mosquito vectors [
4] as well as the weakness of the healthcare systems since malaria endemic countries are among the poorest in the world [
5].
Achieving malaria elimination requires the development of an effective vaccine, especially against
Plasmodium falciparum, which is the deadliest of the five human malaria parasites. A major obstacle toward this goal is the extensive genetic diversity of natural parasite populations that complicate the design of an effective vaccine against all
P. falciparum strains. The difficulty related to
P. falciparum genetic diversity has been clearly illustrated in clinical trials that tested different vaccine candidates. For example, the malaria vaccine combination B, designed as a three-component blood-stage product targeting the merozoite surface proteins MSP-1 (K1 parasite line) and MSP-2 (3D7), and the ring-infected erythrocyte surface antigen (RESA) was efficient against homologous parasites harboring the 3D7 allelic family of MSP-2, but failed to protect against those containing the FC27 allelic family, and was even associated with an increased rate of morbidity [
6]. More recently, the malaria vaccine FMP2.1/AS02 (A), a recombinant protein based on the apical membrane antigen 1 (AMA1) from the 3D7 strain of
P. falciparum did not provide significant protection against clinical malaria, but showed a strain-specific efficacy [
7]. Moreover, a phase III clinical trial with RTS,S, the most advanced malaria vaccine candidate, showed a greater activity against parasites carrying the matching allele of the circumsporozoite protein than against other strains. In this last trial, less than 10% of the 5–17 months old children who were infected harbored parasites carrying the vaccine allele [
8]. Collectively, these results point out the need to study
P. falciparum polymorphism as an important step toward the identification of efficient malaria vaccine candidates.
The study presented here falls within the framework of preliminary investigations aimed at establishing a malaria clinical trial site in the district of Toubacouta (Fatick region, Senegal). It follows epidemiological [
9], entomological [
10] and immunological investigations undertaken in the same area. The objective is to characterize the genetic diversity of
P. falciparum isolates from asymptomatic children in the selected study site by PCR-amplification of polymorphic regions of the two marker genes
msp-
1 and
msp-
2. The generated data are used to analyse the potential relationship between the levels of parasitaemia and specific parasite’s allelic types.
Discussion
Preliminary studies for a clinical trial site for malaria have been undertaken in the district of Toubacouta (Fatick region, Senegal). Eight villages were involved. The genetic diversity of
P. falciparum, the only species diagnosed, was analysed in samples from asymptomatic children collected through a cross-sectional survey. An important allelic diversity was observed with 15 and 21 different alleles, and 69% and 89% of multiple infections, for
msp-
1 and
msp-
2 genes, respectively. The high genetic diversity in our study is in agreement with previous findings on asymptomatic children in the mesoendemic malaria site of Niakhar in Senegal. In Niakhar, genotyping with the
msp-
2 polymorphic marker revealed a range from 2 to 7 different fragments per carrier with 64% of multiclonal infections [
16]. Similar results were observed 16 years ago in Ndiop in asymptomatic children [
14]. Comparison of genetic diversity of
P. falciparum isolates in Ndiop (1994) [
14] and our study area (2010) in the nearby locality shows a stable genetic diversity over time. A high level of diversity persists despite a 12 times difference in transmission levels (63 infected bites per person per 4 months in 1994 compared to four infected bites per person per 3 months in 2010) between the two periods as a result of malaria control interventions [
17]. This observation underlines that
P. falciparum genetic diversity does not only rely on parasite transmission rate. Thus, it has been demonstrated that the genotypic profile changes within hours or days in asymptomatic infections [
18].
Unlike genetic diversity, complexity of infection (COI) is more commonly associated with the level of malaria transmission. In line with this observation, Konate et al. [
19] found that COI was twice higher in the holoendemic area of Dielmo than in the mesoendemic village of Ndiop. Low COI was also associated with low transmission rate in Malaysia [
13]. Vafa et al. [
16] highlighted COI variation within the same year according to transmission period. In this study, COI is consistent with results from mesoendemic regions for both
msp-
1 and
msp-
2 markers [
14,
20,
21].
Association between the number of clones per sample and the level of parasitaemia has been investigated. The results show that polyclonality is likely to be associated with high parasitaemia. This is consistent with previous studies [
16,
21‐
23]. On the one hand, parasite density is attributed a major role in malaria physiopathology and some authors suggest that it might be used as a marker for morbidity and mortality associated with this disease [
24,
25]. On the other hand, it is considered as the centerpiece of malaria immunity. In this regard, it has been shown that asymptomatic polyclonal carriage may protect against clinical malaria [
26,
27]. Although the mechanism of this partial protection remains unclear, it is probably by maintaining protective immune responses to asexual blood stage parasites [
21,
28,
29].
Moreover, this study emphasizes the significant positive correlation between frequencies of trimorphic infections K1/MAD20/RO33 of
msp-
1 gene and parasitaemia (Fig.
3). Although the representative alleles of this combination have to be identified, these preliminary results point out the necessity of multivalent vaccine candidates to overcome antigenic diversity [
30].
This study has some technical limitations. Firstly, PCR cannot discriminate between alleles of differing sequences with similar size and can thus underestimate the number of distinct alleles [
30,
31]. Secondly, polymorphism of sub-microscopic infections is not taken into account in this study. Despite these limitations, the results provide an insight on the genetic diversity of
P. falciparum clones circulating in Toubacouta, Senegal.
Conclusions
Despite the general decline in malaria prevalence observed following the implementation of various malaria control strategies, P. falciparum strains are substantially circulating in this study site and display a high genetic diversity. The level of this diversity is comparable to that found in malaria endemic and mesoendemic areas, therefore, making the site interesting for potential vaccine and therapeutic clinical trials. The positive correlation between msp-1 trimorphic infection and parasitaemia suggests the use of field information on genetic diversity as starting blocks for designing new malaria vaccines.
Authors’ contributions
ATB conceived and coordinated this study, contributed to the methodology, to the revision of the manuscript and approved the final version. BD contributed to the conception of the study, performed molecular biology studies and data analysis and drafted the manuscript. FD contributed to the conception of the study, performed field studies, participated in the revision of the manuscript and approved the final version. ID contributed to data analysis, participated in drafting the manuscript and approved the final version. CL conducted the statistical analysis, and approved the final version. YD contributed to the analysis, participated in drafting and revising the manuscript and approved the final version. JF performed field recruitments, lead the management of the database and approved the final version. MS contributed to the methodology, participated in revising the manuscript and approved the final version. RP contributed to drafting and revising the manuscript and approved the final version. MN participated in the design and the conducts of the molecular biology studies, contributed to revising the manuscript and approved the final version. All authors read and approved the final manuscript.
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