• View in gallery

    Map of Senegal showing the two NMCP sentinel sites covered in the 2018 therapeutic efficacy study (TES). This map was created using QGIS 3.8.3-Zanzibar (http://www.qgis.org/). This figure appears in color at www.ajtmh.org.

  • View in gallery

    PfCSP repeat region alleles with varying number of NANP (1) and NVDP (2) repeats (A) and count of each haplotypes across study sites (B). This figure appears in color at www.ajtmh.org.

  • View in gallery

    PfCSP C-terminal region highlighting the Th2R and Th3R T-cell epitope variations (A) and their frequency distributions in the two study sites (B). This figure appears in color at www.ajtmh.org.

  • View in gallery

    Logo plot based on a 21 SNP-based barcode showing the polymorphic patterns of the C-terminal region in Pf3k database Senegalese samples (2007–2011) (A) compared with our study samples (2018) (B). The positions of the amino acids across the C-terminal region are indicated above the Logo plot. The total height of the letters indicates the information content of the position, while the relative height of the letters indicates the relative frequency of the corresponding amino acid at that position, in bits. This figure appears in color at www.ajtmh.org.

  • View in gallery

    Temporal changes in the distribution of C-terminal haplotypes across study years. C2 to C23: parasites clustering in identical haplotypes with at least two members in a particular year. CU1 to CU6: haplotype with only one member in a particular year but the same haplotype, as singleton, is observed in other years. S: unique haplotypes observed in a particular year while at the same time those haplotypes are not found in other years. Shared haplotypes between early samples (2001–2011) versus late samples (2018) are shown in dotted boxes. This figure appears in color at www.ajtmh.org.

  • 1.

    WHO , 2019. World Malaria Report 2019. Geneva, Switzerland: World Health Organization.

  • 2.

    White NJ, Pukrittayakamee S, Hien TT, Faiz MA, Mokuolu OA, Dondorp AM, 2014. Malaria. Lancet 383: 723735.

  • 3.

    Meara WPO, Mangeni JN, Steketee R, Greenwood B, 2010. Changes in the burden of malaria in sub-Saharan Africa. Lancet Infect Dis 10: 545555.

  • 4.

    Seck MC et al., 2017. Malaria prevalence, prevention and treatment seeking practices among nomadic pastoralists in northern Senegal. Malar J 16: 413.

    • Search Google Scholar
    • Export Citation
  • 5.

    Trape J et al., 2014. The rise and fall of malaria in a west African rural community, Dielmo, Senegal, from 1990 to 2012: a 22 year longitudinal study. Lancet Infect Dis 14: 476488.

    • Search Google Scholar
    • Export Citation
  • 6.

    Sahel Malaria Elimination Initiative (SaME Initiative) | Mesa. Available at: https://mesamalaria.org/. Accessed July 13, 2020.

  • 7.

    Programme National de Lutte contre le Paludisme (PNLP), 2018. Bulletin Épidémiologique Annuel Du Paludisme Au Sénégal. Dakar. Dakar, Sensgal: PNLP.

  • 8.

    Ndiaye JLA et al., 2019. Seasonal malaria chemoprevention combined with community case management of malaria in children under 10 years of age, over 5 months, in south-east Senegal: a cluster randomized trial. PLoS Med 16: 124.

    • Search Google Scholar
    • Export Citation
  • 9.

    Moss WJ et al., 2015. Malaria epidemiology and control within the International Centers of Excellence for malaria research. Am J Trop Med Hyg 93: 515.

    • Search Google Scholar
    • Export Citation
  • 10.

    Hoffman SL, Vekemans J, Richie TL, Duffy PE, 2015. The march toward malaria vaccines. Vaccine 33 (Suppl 4): D13–D23.

  • 11.

    Goh YS, McGuire D, Rénia L, 2019. Vaccination with sporozoites: models and correlates of protection. Front Immunol 10: 118.

  • 12.

    Bell GJ et al., 2020. Case reduction and cost-effectiveness of the RTS,S/AS01 malaria vaccine alongside bed nets in Lilongwe, Malawi. Vaccine 38: 4079–4087.

    • Search Google Scholar
    • Export Citation
  • 13.

    Escalante AA, Grebert HM, Isea R, Goldman IF, Basco L, Magris M, Biswas S, Kariuki S, Lal AA, 2002. A study of genetic diversity in the gene encoding the circumsporozoite protein (CSP) of Plasmodium falciparum from different transmission areas - XVI. Asembo Bay Cohort Project. Mol Biochem Parasitol 125: 8390.

    • Search Google Scholar
    • Export Citation
  • 14.

    Plassmeyer ML et al., 2009. Structure of the Plasmodium falciparum circumsporozoite protein, a leading malaria vaccine candidate. J Biol Chem 284: 2695126963.

    • Search Google Scholar
    • Export Citation
  • 15.

    Keating C, 2020. The history of the RTS,S/AS01 malaria vaccine trial. Lancet 395: 13361337.

  • 16.

    Aragam NR, Thayer KM, Nge N, Hoffman I, Martinson F, Kamwendo D, Lin FC, Sutherland C, Bailey JA, Juliano JJ, 2013. Diversity of T cell epitopes in Plasmodium falciparum circumsporozoite protein likely due to protein-protein interactions. PLoS One 8: 62427.

    • Search Google Scholar
    • Export Citation
  • 17.

    Datoo MS et al., 2021. Efficacy of a low-dose candidate malaria vaccine, R21 in adjuvant Matrix-M, with seasonal administration to children in Burkina Faso: a randomised controlled trial. Lancet 397: 18091818.

    • Search Google Scholar
    • Export Citation
  • 18.

    Gowda DC, Wu X, 2018. Parasite recognition and signaling mechanisms in innate immune responses to malaria. Front Immunol 9: 3006.

  • 19.

    Pringle JC, Carpi G, Almagro-Garcia J, Zhu SJ, Kobayashi T, Mulenga M, Bobanga T, Chaponda M, Moss WJ, Norris DE, 2018. RTS,S/AS01 malaria vaccine mismatch observed among Plasmodium falciparum isolates from southern and central Africa and globally. Sci Rep 8: 18.

    • Search Google Scholar
    • Export Citation
  • 20.

    Riley EM, Stewart VA, 2013. Immune mechanisms in malaria: new insights in vaccine development. Nat Med 19: 168178.

  • 21.

    Bei AK et al., 2018. Dramatic changes in malaria population genetic complexity in Dielmo and Ndiop, Senegal, revealed using genomic surveillance. J Infect Dis 217: 622627.

    • Search Google Scholar
    • Export Citation
  • 22.

    Daniels RF et al., 2015. Modeling malaria genomics reveals transmission decline and rebound in Senegal. Proc Natl Acad Sci USA 112: 70677072.

  • 23.

    Tanabe K, Mita T, Palacpac NMQ, Arisue N, Tougan T, Kawai S, Jombart T, Kobayashi F, Horii T, 2013. Within-population genetic diversity of Plasmodium falciparum vaccine candidate antigens reveals geographic distance from a central sub-Saharan African origin. Vaccine 31: 13341339.

    • Search Google Scholar
    • Export Citation
  • 24.

    Neafsey DE et al., 2015. Genetic diversity and protective efficacy of the RTS,S/AS01 malaria vaccine. N Engl J Med 373: 20252037.

  • 25.

    Fofana M, Mitri C, Diallo D, Rotureau B, Diagne CT, Gaye A, Ba Y, Dieme C, Diallo M, Dia I, 2020. Possible influence of Plasmodium/Trypanosoma co-infections on the vectorial capacity of Anopheles mosquitoes. BMC Res Notes 13: 16.

    • Search Google Scholar
    • Export Citation
  • 26.

    Agence Nationale de la Statistique et de la Démographie (ANSD)/SRSD, 2015. Situation Economique et Sociale Regionale 2012. Dakar, Senegal: ANSD.

  • 27.

    Diouf I et al., 2017. Comparison of malaria simulations driven by meteorological observations and reanalysis products in Senegal. Int J Environ Res Public Health 14: 1119.

    • Search Google Scholar
    • Export Citation
  • 28.

    Lucchi NW, Narayanan J, Karell MA, Xayavong M, Kariuki S, DaSilva AJ, Hill V, Udhayakumar V, 2013. Molecular diagnosis of malaria by photo-induced electron transfer fluorogenic primers: PET-PCR. PLoS One 8: e56677.

    • Search Google Scholar
    • Export Citation
  • 29.

    Zeeshan M et al., 2012. Genetic VARIATION in the Plasmodium falciparum circumsporozoite protein in India and its relevance to RTS,S malaria vaccine. PLoS One 7: e43430.

  • 30.

    Van Den Berg M, Ogutu B, Sewankambo NK, Biller-Andorno N, Tanner M, 2019. RTS,S malaria vaccine pilot studies: addressing the human realities in large-scale clinical trials. Trials 20: 316.

    • Search Google Scholar
    • Export Citation
  • 31.

    Moorthy VS, Ballou WR, 2009. Immunological mechanisms underlying protection mediated by RTS, S: a review of the available data. 8: 312.

  • 32.

    Malaria Control and Elimination Partnership in Africa (MACEPA), 2021. Senegal - Charting the Path to Malaria Elimination. Available at: https://www.path.org/resources/senegal-charting-the-path-to-malaria-elimination/. Accessed July 13, 2021

  • 33.

    Seck MC et al., 2017. Malaria prevalence, prevention and treatment seeking practices among nomadic pastoralists in northern Senegal. Malar J 16: 413.

    • Search Google Scholar
    • Export Citation
  • 34.

    Bei AK et al., 2018. Dramatic changes in malaria population genetic complexity in Dielmo and Ndiop, Senegal, revealed using genomic surveillance. J Infect Dis 217: 622627.

    • Search Google Scholar
    • Export Citation
  • 35.

    Bei AK et al., 2015. Immune characterization of Plasmodium falciparum parasites with a shared genetic signature in a region of decreasing transmission. Infect Immun 83: 276285.

    • Search Google Scholar
    • Export Citation
  • 36.

    Daniels R et al., 2013. Genetic surveillance detects both clonal and epidemic transmission of malaria following enhanced intervention in Senegal. PLoS One 8: 410.

    • Search Google Scholar
    • Export Citation
  • 37.

    Long CA, Zavala F, 2016. Malaria vaccines and human immune responses. Curr Opin Microbiol 32: 96102.

  • 38.

    Kumkhaek C et al., 2005. Are extensive T cell epitope polymorphisms in the Plasmodium falciparum circumsporozoite antigen, a leading sporozoite vaccine candidate, selected by immune pressure? J Immunol 175: 39353939.

    • Search Google Scholar
    • Export Citation
  • 39.

    Gandhi K, Thera MA, Coulibaly D, Traoré K, Guindo AB, Ouattara A, Takala-harrison S, Berry AA, Doumbo OK, Plowe CV, 2016. Correction: variation in the circumsporozoite protein of Plasmodium falciparum: vaccine development implications. PLoS One 11: e0148240.

    • Search Google Scholar
    • Export Citation
  • 40.

    Tanabe K, Sakihama N, Kaneko A, 2004. Stable SNPs in malaria antigen genes in isolated populations. Science 303: 493.

  • 41.

    Patel P, Bharti PK, Bansal D, Raman RK, Mohapatra PK, Sehgal R, Mahanta J, Sultan AA, Singh N, 2017. Genetic diversity and antibody responses against Plasmodium falciparum vaccine candidate genes from Chhattisgarh, central India: implication for vaccine development. PLoS One 12: e0182674.

    • Search Google Scholar
    • Export Citation
  • 42.

    Kingston NJ, Kurtovic L, Walsh R, Joe C, Lovrecz G, Locarnini S, Beeson JG, Netter HJ, 2019. Hepatitis B virus-like particles expressing Plasmodium falciparum epitopes induce complement-fixing antibodies against the circumsporozoite protein. Vaccine 37: 16741684.

    • Search Google Scholar
    • Export Citation
  • 43.

    Langowski MD et al., 2020. Optimization of a Plasmodium falciparum circumsporozoite protein repeat vaccine using the tobacco mosaic virus platform. Proc Natl Acad Sci USA 117: 31143122.

    • Search Google Scholar
    • Export Citation
  • 44.

    Heppner DG et al., 2005. Towards an RTS, S-based, multi-stage, multi-antigen vaccine against falciparum malaria: progress at the Walter Reed Army Institute of Research. Vaccine 23: 22432250.

    • Search Google Scholar
    • Export Citation
  • 45.

    Draper SJ, Sack BK, King CR, Nielsen CM, Rayner JC, Higgins MK, Long CA, Seder RA, 2018. Malaria vaccines: recent advances and new horizons. Cell Host Microbe 24: 4356.

    • Search Google Scholar
    • Export Citation
  • 46.

    Ouattara A et al., 2013. Molecular basis of allele-specific efficacy of a blood-stage malaria vaccine. Vaccine Development Implications. 207: 511519.

    • Search Google Scholar
    • Export Citation
  • 47.

    King A, 2019. Building a better malaria vaccine. Nature 575: S51S54.

  • 48.

    Plowe CV, 2015. Vaccine-resistant malaria. N Engl J Med 373: 2082–2083.

  • 49.

    Ouattara A, Barry AE, Dutta S, Remarque EJ, Beeson JG, Plowe CV, 2015. Designing malaria vaccines to circumvent antigen variability. Vaccine 33: 75067512.

    • Search Google Scholar
    • Export Citation
  • 50.

    Greenwood B et al., 2017. Seasonal vaccination against malaria: a potential use for an imperfect malaria vaccine. Malar J 16: 182.

 
 

 

 

 

 

 

 

Spatiotemporal Dynamic of the RTS,S/AS01 Malaria Vaccine Target Antigens in Senegal

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  • 1 Department of Parasitology and Mycology, Cheikh Anta Diop University, Dakar, Senegal;
  • | 2 Aristide Le Dantec University Hospital, Dakar, Senegal;
  • | 3 Aix Marseille University, IRD, AP-HM, SSA, VITROME, Marseille, France;
  • | 4 IHU Méditerranée Infection, Marseille, France;
  • | 5 National Malaria Control Program (NMCP), Dakar, Senegal

ABSTRACT.

The RTS,S/AS01 malaria vaccine confers only moderate protection against malaria. Evidence suggests that the effectiveness of the RTS,S/AS01 vaccine depends upon the parasite population genetics, specifically regarding the circumsporozoite protein haplotypes in the population. We investigated Plasmodium falciparum circumsporozoite protein (PfCSP) gene sequences from two endemic sites in 2018 in Senegal. The PfCSP sequences were compared with those retrieved from the Pf3k genome database. In the central repeat region of PfCSP, the distribution of haplotypes differed significantly between the two study sites (Fisher’s exact test, P < 0.001). No 3D7 vaccine strain haplotype was observed in this locus. In the C-terminal region, there was no significant difference in haplotypes distribution between Kedougou and Diourbel (Fischer’s exact test, P = 0.122). The 3D7 haplotype frequency was 8.4% in early samples (2001–2011), but then it contracted in the subsequent years. The extensive plasticity of the P. falciparum genes coding the RTS,S/AS01 vaccine target antigens may influence the immune responses to circulating alleles. Monitoring the genetic diversity baseline and its dynamics over time and space would be instrumental in rationally improving the malaria RTS,S/AS01 vaccine and/or its implementation schedule.

INTRODUCTION

Over the last two decades, significant progress has been achieved by African countries in the fight against malaria. From 2010 to 2018, there was a 22% reduction in the incidence rate, but this fall slowed dramatically from 2014 to 2018.1 The massive scale-up of malaria interventions has led to the rapid decline in malaria morbidity and mortality in recent years. These interventions included insecticide-treated mosquito nets, indoor residual spraying, and a rapid diagnostic test combined with an effective treatment with artemisinin-based combination therapy.2

The impact of malaria control strategies in recent years has dramatically changed the epidemiological profiles of the disease in many countries.3 Some regions, such as northern Senegal, reported fewer than five indigenous cases per 1,000,4 reaching the pre-elimination stage as defined by WHO.5 These encouraging results prompted Senegal and other Sahelian countries to set a goal of eliminating malaria by 2030.6 However, in other areas where the same approaches have been implemented, the number of malaria cases and deaths remains high. This is the case in the southern part of Senegal where, despite a significant decrease over the past years, high seasonal transmission remains problematic, especially in children under 5 years old.7 In this regard, seasonal malaria chemoprevention has been seen as a promising approach in areas with high seasonal transmission. The deployment of seasonal malaria chemoprevention has already dramatically reduced the burden of the disease in many parts of Africa.8 However, the widespread resistance of Plasmodium falciparum to antimalarial drugs continue to undermine malaria control efforts.9 Thus, to achieve the vision of malaria elimination, complementary tools are needed.

One new intervention technology for malaria is the use of a vaccine. Advances in malaria vaccine development raise hope for effective use toward malaria elimination. Previous studies using irradiated sporozoites demonstrated the feasibility of producing an efficacious vaccine. Indeed, sterile protection against malaria has been achieved by attenuated sporozoite vaccination of animals and humans.10 On the other hand, the idea of a vaccine targeting the sporozoites form (the infecting form for humans) that completely blocks the establishment of infection in the liver make pre-erythrocytic stage vaccines attractive.11 In fact, this would totally prevent the transmission as well as clinical disease. Thus, the most advanced malaria vaccine, RTS,S/AS01, targets the P. falciparum circumsporozoite protein (PfCSP),12 which is the major protein found on the surface of P. falciparum sporozoites and is essential for liver cell invasion. The PfCSP can be segmented into three regions: the N-terminal region containing region I; the central repeat region, which contains tandem repeats; and the C-terminal region containing the region III and the thrombospondin-like type-I repeat.13 Initial circumsporozoite protein (CSP)-based vaccine development focused on the central repeat region that contains the immunodominant B cell epitope. The current RTS,S/AS01 target includes a portion of the central repeat and the C-terminal regions genetically fused to the hepatitis B virus surface antigen and a liposome-based adjuvant (AS01).14

RTS,S/AS01 is the world's first and only licensed malaria vaccine.15 This vaccine progressed beyond phase III clinical trials and is now undergoing large-scale pilot implementation in Malawi, Ghana, and Kenya under WHO coordination. Malaria vaccine has been proposed as a potential additional tool to the current WHO-recommended measures for the prevention, diagnosis, and treatment of malaria.12

However, despite the high expectations raised, the general consensus is that the RTS,S/AS01 achieves only moderate protection, with only a 55% and 31% reduction in clinical malaria during the first year among children 5– 7 months and 6–12 weeks of age, respectively.16 Recently, a modified version of RTS,S, called R21/Matrix M, demonstrated 77% efficacy over 1 year of follow-up (achieving the WHO goal of 75% efficacy against clinical malaria by 2030). These results provide a hope that this highly efficacious and safe malaria vaccine could substantially reduce the malaria disease burden in Africa and elsewhere.17

Unfortunately, despite decades of continuing scientific effort, we still lack a good understanding of the molecular mechanism of protective immunity against malaria.18 Challenges in developing an effective malaria vaccine are largely related to the complex life cycle of the Plasmodium parasites. Various antigens are expressed during each developmental stage of the parasite and distinct alleles are present at the same stage.10

Although the RTS,S/AS01 is derived from the 3D7 P. falciparum strain, recent studies reported a decrease in vaccine efficacy against non–vaccine strain parasites.19 Yet, parasites isolated from natural infections often do not match the vaccine strain, including in Africa where the phase III vaccine trial was conducted.19 The role of antigenic polymorphism as an immune evasion is well known in the field of vaccinology. The protection conferred by heterologous sporozoites illustrates that polymorphic antigens play a critical role in this phenomenon.20 Moreover, it has been demonstrated that parasites constantly change over time and according to the malaria transmission level.21,22 Geographic distance was significantly associated with target vaccine candidate antigenic variation.21,23 We hypothesized that the PfCSP sequence would also vary over time and space. Thus, surveilling the variation of amino acid sequences in naturally isolated parasites population could provide a better understanding of its temporal and spatial dynamics that might be helpful in enhancing vaccine efficacy or optimizing vaccine implementation schedule.24 This work aimed to assess the patterns of changes in B and T cell epitopes of PfCSP in two different sites in Senegal in the context of important decrease of malaria prevalence over the last two decades.

MATERIALS AND METHODS

Ethics statement.

This study was approved by the Ethics Committee of the Ministry of Health in Senegal. Samples in this study were obtained from a WHO therapeutic efficacy study for monitoring antimalarial drug efficacy. In this study protocol, it was expressly agreed that the collected samples can be used in future studies if the patient or his/her guardian has consented.

Study areas, participants, and samples collection.

In 2018, the therapeutic efficacy study was conducted in the health centers of Tomboronkoto (Kedougou region) and Keur Serigne Mbaye Sarr (Diourbel region) (Figure 1). Tomboronkoto is a village located in the southeast of Senegal in the region of Kedougou. It is located at the edge of the Niokolo Koba Park, about 660 km from the country capital of Dakar. It belongs to the Sudano-Guinean domain. Kedougou is situated in a junction zone between the savannah belt and the dry tropical forest. This region is characterized by a rainy season from June to November, and mean temperatures vary from 33°C to 39.5°C during the year.25 The population is predominantly rural and is estimated at 16,077 inhabitants in the commune of Tomboronkoto,26 mostly living in small-scattered villages. The incidence of malaria in this region is among the highest in Senegal (hyperendemic; > 300 cases per 1,000 inhabitants annually), with the highest prevalence of P. falciparum infection (14%) among children under 5 years of age.7

Figure 1.
Figure 1.

Map of Senegal showing the two NMCP sentinel sites covered in the 2018 therapeutic efficacy study (TES). This map was created using QGIS 3.8.3-Zanzibar (http://www.qgis.org/). This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 105, 6; 10.4269/ajtmh.21-0369

Diourbel, on the other hand, is located in central Senegal, 145 km southeast of Dakar, in the Groundnut Basin (“Bassin arachidier” or “Sine Saloum”). A shorter rainy season usually occurs between June and October. The central part of Senegal is characterized by a tropical Sudanese climate. High temperatures occur from April to June and from September to November (around 30°C).27 Diourbel is a malaria hypoendemic area with the greatest number of malaria cases occurring during the short rainy season.7

Participants enrolled in this study (N = 198) were patients over the age of 6 months admitted to the study sites with reported acute fevers within 24 hours of visiting the health post and no recent antimalarial use and were screened by microscopy and rapid diagnostic test to diagnose P. falciparum infection.

Finger-prick blood samples were taken from P. falciparum–infected patients prior to drug treatment and spotted on Whatman 3 MM filter paper (GE Healthcare, Maidstone, UK) for confirmation by photo-induced electron transfer fluorogenic primers polymerase chain reaction (PET-PCR)28 and for subsequent molecular analysis.

The Plasmodium PET-PCR reaction was performed in a 20 μL reaction containing 2X TaqMan Environmental Master Mix 2.0 (Applied BioSystems), 250 nM each forward and reverse primer for Plasmodium genus, and 2 μL of DNA template as previously described.28 The reactions were performed under the following cycling parameters: initial hot-start at 95°C for 15 minutes, followed by 45 cycles of denaturation at 95°C for 20 seconds and annealing at 60°C for 40 seconds. Samples with a cycle threshold value of 40.0 or below were considered positive.

PCR of PfCSP.

The region encoding PfCSP was amplified via nested PCR. The primers used in these experiments and PCR cycling programs have been described previously29 to amplify a 1,026-bp fragment encompassing the central repeat B cell epitope and the C-terminal T cell epitope regions. All PCR products were amplified with the following reaction mixture: 45 μL 1.1x Platinum Taq high-fidelity polymerase (Platinum PCR SuperMix High Fidelity; Invitrogen, Waltham, MA), 20 μM forward primer, and reverse primer to which DNA was added for each sample.

DNA sequencing.

DNA sequences were determined in both directions for each template using the dideoxynucleotide chain termination method with the Big Dye Terminator v3.1 Cycle Sequencing Kit (Perkin-Elmer Applied Biosystems, Foster City, CA) on an automated ABI3100 Genetic Analyzer (Applied Biosystems, Waltham, MA). The primers used to amplify and sequence the PfCSP fragment gene have been described previously.29

DNA and protein sequence analysis.

The chromatogram sequencing files were examined using SnapGene Viewer Version 5.0.8 (Biomatters Ltd, Auckland, New Zealand). Consensus sequences were prepared using Genious Prime version 2020.1.2 (Biomatters Ltd), and multiple sequence alignments were calculated using CLUSTALX algorithm. Only sequences that yielded good-quality sequences in both directions were included in the analysis. If the height of the secondary peak exceeded 50% of the primary peak height at any polymorphic position, the sample was classified as multiple allele infections and was excluded from further haplotype analysis.

A logo plot was constructed based on a 21–single-nucleotide polymorphism–based barcode for each PfCSP population to illustrate polymorphic patterns in the PfCSP C-terminal nonrepeat region with the WebLogo program (https://weblogo.berkeley.edu/logo.cgi).

DNA sequence submission.

The protein sequences described in this paper were submitted to GenBank using the National Center for Biotechnology Information (NCBI, Bethesda, MD) Bankit under accession numbers MT893052–MT893133.

Statistical analysis.

Allele frequencies were compared by using the Fisher's exact test, implemented in RStudio software Version 1.2.5001 (RStudio, Inc.). The simulie.p.value option (Fisher's exact test for count data with simulated P value, based on 2,000 replicates) was used to address small and unequal sampling between study sites. A 5% level of confidence was used, and statistical significance was set at P < 0.05.

Illumina paired-end short read sequence data.

Through the MalariaGEN (Genomic Epidemiology Network) website (https://www.malariagen.net/projects/pf3k), the Pf3k project provides an open access database of P. falciparum genome sequences from multiple countries of Africa and Asia using the latest sequencing technologies. In this database, P. falciparum samples from Senegal were collected between 2001 and 2011 in Thies (which is geographically close and shares similar malaria epidemiological patterns to Diourbel), Dakar (the capital city), and Velingara (south of Senegal, which is epidemiologically similar to Kedougou). Those samples were sequenced via various methods: either following culture adaption, directly from patient samples without WBC depletion, or from patient sample following hybrid selection. The data were in the form of paired-end short-read Illumina sequences, which were then mapped to the 3D7 reference genome to call variants. We downloaded the Variant Call Format file, which we visualized using Integrative Genomics Viewer software version 2.8.2. We examined the variants only in the C-terminal region as the ∼40 NANP repeats (which are much longer than Illumina short read lengths) were not accessible using short read data alone. The resulting dataset contained sequence reads from 137 isolates from the study sites in Senegal.

RESULTS

Study population.

A total of 198 P. falciparum isolates were collected from Diourbel and Kedougou.

Patient median age was 13 years, ranging from 3 to 64 years. In Diourbel the male to female ratio was 6.8; participants were predominantly young boys studying in Koranic school. In Kedougou the male to female ratio was 1.3.

Sequencing quality.

Of the 198 collected samples, we generated good-quality sequences for 101 samples in the central repeat region (with 86 as mono-genomic infections) and 99 samples in the C-terminal region (with 88 as mono-genomic infections). Thus, 15 samples in the central repeat region and 11 samples in the C-terminal regions were mixed infections and as such were excluded for haplotype analysis.

Variations in the central repeat region of PfCSP.

Among the 86 successfully sequenced PfCSP central repeat region samples (34 from Diourbel versus 52 from Kedougou), 38 unique haplotypes were identified at the amino acid level. As shown in Figure 2, these haplotypes differed by the number and arrangement of tetrapeptides asparagine–alanine– asparagine–proline (NANP) and asparagine–valine–aspartic acid–proline (NVDP) repeats, resulting in size polymorphisms. The number of NANP repeats varied from 16 to 44, with 39 NANP repeats being the most frequent pattern. Among the 38 unique haplotypes, only six (H6, H9, H10, H21, H24, and H26) were found both in Kedougou and Diourbel; among those, H6 (21.2%) and H9 (11.5%) were the major haplotypes in Kedougou, and H26 (14.7%) was predominant in Diourbel. The distribution of haplotypes differed significantly between the two study sites (Fisher’s exact test, P < 0.001). Moreover, shorter haplotypes (H31–H38, having < 34 NANP repeats) were exclusively found in Diourbel. Of note, no 3D7 haplotype was observed at this locus.

Figure 2.
Figure 2.

PfCSP repeat region alleles with varying number of NANP (1) and NVDP (2) repeats (A) and count of each haplotypes across study sites (B). This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 105, 6; 10.4269/ajtmh.21-0369

Variations in the C-terminal region of PfCSP.

In the C-terminal region of PfCSP, 99 samples were successfully sequenced. Of those, 11 consisted of mixed infection and were excluded for the haplotype analysis. As shown in Figure 3, 33 distinct haplotypes were identified among the 88 PfCSP C-terminal region successfully sequenced samples (37 from Diourbel and 49 from Kedougou). Eight haplotypes were found in both localities. There was no significant difference in haplotype distribution between Kedougou and Diourbel (Fischer’s exact test, P = 0.122). The most highly polymorphic residues were located in the Th2R and Th3R T-cell epitopes (Figure 3A).

Figure 3.
Figure 3.

PfCSP C-terminal region highlighting the Th2R and Th3R T-cell epitope variations (A) and their frequency distributions in the two study sites (B). This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 105, 6; 10.4269/ajtmh.21-0369

To further dissect the pattern of genetic variations regarding the temporal distance among Senegalese parasites, we compared the haplotype sequences from our study (2018) with those retrieved from the Pf3k database (2001–2011). For simplicity, we aggregated data from 2001 to 2007. We constructed a molecular signature, composed of 21 SNPs across the C-terminal regional of P. falciparum, including T cell epitopes (Th2R and Th3R; red boxes in Figure 3A).

An overall comparison showed amino acids in 2018 samples that were not present in the Pf3k Senegalese samples: lysine (K) in position 318, threonine (T) and arginine (R) in position 322, and lysine (K) in position 361; on the other hand, amino acid aspartate (D) in position 296, although relatively infrequent in Pf3k database, was not found in 2018 samples (Figure 4).

Figure 4.
Figure 4.

Logo plot based on a 21 SNP-based barcode showing the polymorphic patterns of the C-terminal region in Pf3k database Senegalese samples (2007–2011) (A) compared with our study samples (2018) (B). The positions of the amino acids across the C-terminal region are indicated above the Logo plot. The total height of the letters indicates the information content of the position, while the relative height of the letters indicates the relative frequency of the corresponding amino acid at that position, in bits. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 105, 6; 10.4269/ajtmh.21-0369

When samples were analyzed at the individual level, only three of the clustered parasites of the 2018 samples matched previously observed haplotypes in Pf3k database. Among those, three repeated barcode clusters, two of them (C2 and C11) included members from both Diourbel and Kedougou, and one cluster (CU3) had all of its members from Kedougou. The 3D7 haplotype (C2) frequency was 8.4% in early samples (2001–2011), but this contracted to 2.3% in 2018. The other nine clusters had not previously been observed, and none of the other remaining haplotypes in Pf3k matched the 2018 samples (Figure 5).

Figure 5.
Figure 5.

Temporal changes in the distribution of C-terminal haplotypes across study years. C2 to C23: parasites clustering in identical haplotypes with at least two members in a particular year. CU1 to CU6: haplotype with only one member in a particular year but the same haplotype, as singleton, is observed in other years. S: unique haplotypes observed in a particular year while at the same time those haplotypes are not found in other years. Shared haplotypes between early samples (2001–2011) versus late samples (2018) are shown in dotted boxes. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 105, 6; 10.4269/ajtmh.21-0369

Overall, the C-terminal region of PfCSP exhibited a complete change in just 7 years (between 2011 and 2018) in Senegalese P. falciparum isolates.

DISCUSSION

As the RTS,S/AS01 malaria vaccine is being tested at large-scale pilot implementation in Africa,30 its modest efficacy still remains problematic, in part due to the lack of understanding of the mechanism underlying the immune response to the vaccine.31 Because of the fast evolution and the changing nature of P. falciparum genome, polymorphisms of B and T cell epitopes should be analyzed over time in distinct geographic locations.23 Our study design took advantage of the current malaria epidemiological setting in Senegal, which is particularly suited to assess whether geographical patterns of malaria transmission may influence the diversity of malaria vaccine antigens in specific geographic regions. Indeed, following the deployment of malaria scale-up interventions, its global incidence dramatically decreased but is unevenly distributed: whereas the north is at pre-elimination stage, malaria transmission is moderate in the center and remains high in the south.32,33

A decrease in malaria incidence following malaria control measures has been associated with genetic diversity alteration of parasite populations.22,34 Particularly, the rapid expansion and contraction of genetically identical parasites, clustering genotypes, was reported in 2009 in Thies, a low malaria transmission area.3436 In this respect, our finding of a major temporal PfCSP genotype alteration was not unexpected. Yet, although low-frequency alleles may have been missed because of the relatively small parasite sample studied and the absence of available PfCSP sequences limits our understanding of the amino-acid sequences dynamics between 2011 and 2018, we describe for the first time an almost complete replacement of P. falciparum genotypes in such a short time period. Ten parasite clusters in the C-terminal region present in 2011 had disappeared by 2018. Essentially, our findings stress the capacity of P. falciparum to switch its vaccine target epitopes in a short period of time, even in the absence of vaccine selective pressure.

Plasmodium falciparum antigenic variability and host immune response plasticity in response to parasite antigenic alterations may drive a reciprocal selection pressure between the host immune system and the parasite. The hypothesis that this fluctuating selection process would result in a balanced polymorphism36,37 is inconsistent with the geographical and temporal stability of a particular PfCSP allelic variant observed in Asia, which suggests that other T cell epitopes may be involved in interacting with the immune system other than the well-known Th2R and Th3R T.38

At the very beginning of RTS,S/AS01 development, the choice of targeting the central repeat B cell epitopes was made because it contains conserved NANP motifs, which might overcome genetic variations issues.10 To date, little is known on the effect of repeat region sequence variations on the RTS,S/AS01 vaccine efficacy.39 It has been suggested that this repeat region provides structural stability to CSP through the type-I b turn and that its polymorphism is maintained by balancing selection.29 Moreover, rapid repeat length variations of this locus have been described in the southwestern Pacific.40 In addition, NANP and NVDP motifs count and their arrangements in the central repeat region are thought to influence the antibody response against PfCSP.41 For instance, a relative increase in repeat copy number has been associated with enhanced antibody-mediated complement fixation;42 on the other hand, a relative decrease in repeat copy number and looped presentation has been associated with enhanced immunogenicity and protection for the RTS,S/AS01 vaccine.43

Later, following the recognition of the potential role of cell-mediated immunity, the C-terminal region encompassing the T cell epitopes was included in the RTS,S/AS01 vaccine.44 However, and similarly to the repeat region, the role of these epitopes on the RTS,S/AS01 vaccine efficacy remains unclear.45

In areas where the malaria RTS,S/AS01 vaccine was tested, when large numbers of samples were analyzed via next generation sequencing, there was clear evidence that the genetic similarity of the C-terminal region of the field parasites to that of the 3D7 vaccine strain was associated with RTS,S/AS01 protective efficacy.24 This strain-associated efficacy has also been shown for other malaria vaccines targeting highly polymorphic blood-stage antigens.46 However, this cannot be the unique explanation of the vaccine efficacy because the vaccine clinical efficacy rate exceeds the 3D7 allele frequency among parasites. This suggests various levels of antibody cross-reactivity against polymorphic regions. Further investigations are warranted to understand the extent of this cross-recognition and its breadth of activity against circulating alleles. These findings have prompted effort over the past decades to improve the current RTS,S/AS01 to achieve more robust protection. For instance, an alternative vaccine, the R21/Matrix M, which is very similar to RTS,S/AS01 but with a larger portion of PfCSP, has been tested in a phase II clinical trial in Burkina Faso. R21/Matrix M combination could be less expensive and up to 77​% effective at preventing malaria over the course of 1 year.17,47

Overall, our findings call for reassessing the current malaria vaccine antigen formulation approach by considering co-formulating additional protein antigens and RTS,S/AS01 in a combined multi-antigen vaccine strategy.44 Our findings also stress the importance of taking into account both the circulating alleles in specific geographical locations and the right time to deploy the vaccine.48,49 For instance, seasonal malaria vaccination has been advocated to provide a high level of efficacy when deployed shortly before each malaria transmission season in highly seasonal malaria transmission areas.50 The strategy taking into account the parasite populations alleles diversity is also supported by other successful vaccines deployed against viruses or bacteria in which the inclusion of immunogen variants was guided by careful assessments of the target pathogen genetic diversity and strain-specific protective immunity.48

Limitations of this study include low sample size, reducing the statistical power. Also, the different sequencing techniques used for the previously collected samples (2007–2011) versus the technique used for the 2018 samples could have an impact on the results. The higher sequencing depth of next generation sequencing enables higher sensitivity than the Sanger method, allowing the detection of more variants and more mixed infections.

ACKNOWLEDGMENTS

The authors thank Sarah K. Volkman (Harvard T.H. Chan School of Public Health) and Amy K. Bei (Yale School of Public Health) for useful comments and for critical reading of the manuscript. Samples in this work were from a WHO therapeutic efficacy study (TES) in Senegal.

REFERENCES

  • 1.

    WHO , 2019. World Malaria Report 2019. Geneva, Switzerland: World Health Organization.

  • 2.

    White NJ, Pukrittayakamee S, Hien TT, Faiz MA, Mokuolu OA, Dondorp AM, 2014. Malaria. Lancet 383: 723735.

  • 3.

    Meara WPO, Mangeni JN, Steketee R, Greenwood B, 2010. Changes in the burden of malaria in sub-Saharan Africa. Lancet Infect Dis 10: 545555.

  • 4.

    Seck MC et al., 2017. Malaria prevalence, prevention and treatment seeking practices among nomadic pastoralists in northern Senegal. Malar J 16: 413.

    • Search Google Scholar
    • Export Citation
  • 5.

    Trape J et al., 2014. The rise and fall of malaria in a west African rural community, Dielmo, Senegal, from 1990 to 2012: a 22 year longitudinal study. Lancet Infect Dis 14: 476488.

    • Search Google Scholar
    • Export Citation
  • 6.

    Sahel Malaria Elimination Initiative (SaME Initiative) | Mesa. Available at: https://mesamalaria.org/. Accessed July 13, 2020.

  • 7.

    Programme National de Lutte contre le Paludisme (PNLP), 2018. Bulletin Épidémiologique Annuel Du Paludisme Au Sénégal. Dakar. Dakar, Sensgal: PNLP.

  • 8.

    Ndiaye JLA et al., 2019. Seasonal malaria chemoprevention combined with community case management of malaria in children under 10 years of age, over 5 months, in south-east Senegal: a cluster randomized trial. PLoS Med 16: 124.

    • Search Google Scholar
    • Export Citation
  • 9.

    Moss WJ et al., 2015. Malaria epidemiology and control within the International Centers of Excellence for malaria research. Am J Trop Med Hyg 93: 515.

    • Search Google Scholar
    • Export Citation
  • 10.

    Hoffman SL, Vekemans J, Richie TL, Duffy PE, 2015. The march toward malaria vaccines. Vaccine 33 (Suppl 4): D13–D23.

  • 11.

    Goh YS, McGuire D, Rénia L, 2019. Vaccination with sporozoites: models and correlates of protection. Front Immunol 10: 118.

  • 12.

    Bell GJ et al., 2020. Case reduction and cost-effectiveness of the RTS,S/AS01 malaria vaccine alongside bed nets in Lilongwe, Malawi. Vaccine 38: 4079–4087.

    • Search Google Scholar
    • Export Citation
  • 13.

    Escalante AA, Grebert HM, Isea R, Goldman IF, Basco L, Magris M, Biswas S, Kariuki S, Lal AA, 2002. A study of genetic diversity in the gene encoding the circumsporozoite protein (CSP) of Plasmodium falciparum from different transmission areas - XVI. Asembo Bay Cohort Project. Mol Biochem Parasitol 125: 8390.

    • Search Google Scholar
    • Export Citation
  • 14.

    Plassmeyer ML et al., 2009. Structure of the Plasmodium falciparum circumsporozoite protein, a leading malaria vaccine candidate. J Biol Chem 284: 2695126963.

    • Search Google Scholar
    • Export Citation
  • 15.

    Keating C, 2020. The history of the RTS,S/AS01 malaria vaccine trial. Lancet 395: 13361337.

  • 16.

    Aragam NR, Thayer KM, Nge N, Hoffman I, Martinson F, Kamwendo D, Lin FC, Sutherland C, Bailey JA, Juliano JJ, 2013. Diversity of T cell epitopes in Plasmodium falciparum circumsporozoite protein likely due to protein-protein interactions. PLoS One 8: 62427.

    • Search Google Scholar
    • Export Citation
  • 17.

    Datoo MS et al., 2021. Efficacy of a low-dose candidate malaria vaccine, R21 in adjuvant Matrix-M, with seasonal administration to children in Burkina Faso: a randomised controlled trial. Lancet 397: 18091818.

    • Search Google Scholar
    • Export Citation
  • 18.

    Gowda DC, Wu X, 2018. Parasite recognition and signaling mechanisms in innate immune responses to malaria. Front Immunol 9: 3006.

  • 19.

    Pringle JC, Carpi G, Almagro-Garcia J, Zhu SJ, Kobayashi T, Mulenga M, Bobanga T, Chaponda M, Moss WJ, Norris DE, 2018. RTS,S/AS01 malaria vaccine mismatch observed among Plasmodium falciparum isolates from southern and central Africa and globally. Sci Rep 8: 18.

    • Search Google Scholar
    • Export Citation
  • 20.

    Riley EM, Stewart VA, 2013. Immune mechanisms in malaria: new insights in vaccine development. Nat Med 19: 168178.

  • 21.

    Bei AK et al., 2018. Dramatic changes in malaria population genetic complexity in Dielmo and Ndiop, Senegal, revealed using genomic surveillance. J Infect Dis 217: 622627.

    • Search Google Scholar
    • Export Citation
  • 22.

    Daniels RF et al., 2015. Modeling malaria genomics reveals transmission decline and rebound in Senegal. Proc Natl Acad Sci USA 112: 70677072.

  • 23.

    Tanabe K, Mita T, Palacpac NMQ, Arisue N, Tougan T, Kawai S, Jombart T, Kobayashi F, Horii T, 2013. Within-population genetic diversity of Plasmodium falciparum vaccine candidate antigens reveals geographic distance from a central sub-Saharan African origin. Vaccine 31: 13341339.

    • Search Google Scholar
    • Export Citation
  • 24.

    Neafsey DE et al., 2015. Genetic diversity and protective efficacy of the RTS,S/AS01 malaria vaccine. N Engl J Med 373: 20252037.

  • 25.

    Fofana M, Mitri C, Diallo D, Rotureau B, Diagne CT, Gaye A, Ba Y, Dieme C, Diallo M, Dia I, 2020. Possible influence of Plasmodium/Trypanosoma co-infections on the vectorial capacity of Anopheles mosquitoes. BMC Res Notes 13: 16.

    • Search Google Scholar
    • Export Citation
  • 26.

    Agence Nationale de la Statistique et de la Démographie (ANSD)/SRSD, 2015. Situation Economique et Sociale Regionale 2012. Dakar, Senegal: ANSD.

  • 27.

    Diouf I et al., 2017. Comparison of malaria simulations driven by meteorological observations and reanalysis products in Senegal. Int J Environ Res Public Health 14: 1119.

    • Search Google Scholar
    • Export Citation
  • 28.

    Lucchi NW, Narayanan J, Karell MA, Xayavong M, Kariuki S, DaSilva AJ, Hill V, Udhayakumar V, 2013. Molecular diagnosis of malaria by photo-induced electron transfer fluorogenic primers: PET-PCR. PLoS One 8: e56677.

    • Search Google Scholar
    • Export Citation
  • 29.

    Zeeshan M et al., 2012. Genetic VARIATION in the Plasmodium falciparum circumsporozoite protein in India and its relevance to RTS,S malaria vaccine. PLoS One 7: e43430.

  • 30.

    Van Den Berg M, Ogutu B, Sewankambo NK, Biller-Andorno N, Tanner M, 2019. RTS,S malaria vaccine pilot studies: addressing the human realities in large-scale clinical trials. Trials 20: 316.

    • Search Google Scholar
    • Export Citation
  • 31.

    Moorthy VS, Ballou WR, 2009. Immunological mechanisms underlying protection mediated by RTS, S: a review of the available data. 8: 312.

  • 32.

    Malaria Control and Elimination Partnership in Africa (MACEPA), 2021. Senegal - Charting the Path to Malaria Elimination. Available at: https://www.path.org/resources/senegal-charting-the-path-to-malaria-elimination/. Accessed July 13, 2021

  • 33.

    Seck MC et al., 2017. Malaria prevalence, prevention and treatment seeking practices among nomadic pastoralists in northern Senegal. Malar J 16: 413.

    • Search Google Scholar
    • Export Citation
  • 34.

    Bei AK et al., 2018. Dramatic changes in malaria population genetic complexity in Dielmo and Ndiop, Senegal, revealed using genomic surveillance. J Infect Dis 217: 622627.

    • Search Google Scholar
    • Export Citation
  • 35.

    Bei AK et al., 2015. Immune characterization of Plasmodium falciparum parasites with a shared genetic signature in a region of decreasing transmission. Infect Immun 83: 276285.

    • Search Google Scholar
    • Export Citation
  • 36.

    Daniels R et al., 2013. Genetic surveillance detects both clonal and epidemic transmission of malaria following enhanced intervention in Senegal. PLoS One 8: 410.

    • Search Google Scholar
    • Export Citation
  • 37.

    Long CA, Zavala F, 2016. Malaria vaccines and human immune responses. Curr Opin Microbiol 32: 96102.

  • 38.

    Kumkhaek C et al., 2005. Are extensive T cell epitope polymorphisms in the Plasmodium falciparum circumsporozoite antigen, a leading sporozoite vaccine candidate, selected by immune pressure? J Immunol 175: 39353939.

    • Search Google Scholar
    • Export Citation
  • 39.

    Gandhi K, Thera MA, Coulibaly D, Traoré K, Guindo AB, Ouattara A, Takala-harrison S, Berry AA, Doumbo OK, Plowe CV, 2016. Correction: variation in the circumsporozoite protein of Plasmodium falciparum: vaccine development implications. PLoS One 11: e0148240.

    • Search Google Scholar
    • Export Citation
  • 40.

    Tanabe K, Sakihama N, Kaneko A, 2004. Stable SNPs in malaria antigen genes in isolated populations. Science 303: 493.

  • 41.

    Patel P, Bharti PK, Bansal D, Raman RK, Mohapatra PK, Sehgal R, Mahanta J, Sultan AA, Singh N, 2017. Genetic diversity and antibody responses against Plasmodium falciparum vaccine candidate genes from Chhattisgarh, central India: implication for vaccine development. PLoS One 12: e0182674.

    • Search Google Scholar
    • Export Citation
  • 42.

    Kingston NJ, Kurtovic L, Walsh R, Joe C, Lovrecz G, Locarnini S, Beeson JG, Netter HJ, 2019. Hepatitis B virus-like particles expressing Plasmodium falciparum epitopes induce complement-fixing antibodies against the circumsporozoite protein. Vaccine 37: 16741684.

    • Search Google Scholar
    • Export Citation
  • 43.

    Langowski MD et al., 2020. Optimization of a Plasmodium falciparum circumsporozoite protein repeat vaccine using the tobacco mosaic virus platform. Proc Natl Acad Sci USA 117: 31143122.

    • Search Google Scholar
    • Export Citation
  • 44.

    Heppner DG et al., 2005. Towards an RTS, S-based, multi-stage, multi-antigen vaccine against falciparum malaria: progress at the Walter Reed Army Institute of Research. Vaccine 23: 22432250.

    • Search Google Scholar
    • Export Citation
  • 45.

    Draper SJ, Sack BK, King CR, Nielsen CM, Rayner JC, Higgins MK, Long CA, Seder RA, 2018. Malaria vaccines: recent advances and new horizons. Cell Host Microbe 24: 4356.

    • Search Google Scholar
    • Export Citation
  • 46.

    Ouattara A et al., 2013. Molecular basis of allele-specific efficacy of a blood-stage malaria vaccine. Vaccine Development Implications. 207: 511519.

    • Search Google Scholar
    • Export Citation
  • 47.

    King A, 2019. Building a better malaria vaccine. Nature 575: S51S54.

  • 48.

    Plowe CV, 2015. Vaccine-resistant malaria. N Engl J Med 373: 2082–2083.

  • 49.

    Ouattara A, Barry AE, Dutta S, Remarque EJ, Beeson JG, Plowe CV, 2015. Designing malaria vaccines to circumvent antigen variability. Vaccine 33: 75067512.

    • Search Google Scholar
    • Export Citation
  • 50.

    Greenwood B et al., 2017. Seasonal vaccination against malaria: a potential use for an imperfect malaria vaccine. Malar J 16: 182.

Author Notes

Address correspondence to Mamadou Alpha Diallo, Department of Parasitology and Mycology, Cheikh Anta Diop University, Dakar, Senegal. E-mail: mamadoualpha.diallo@ucad.edu.sn

Financial support: Funding for this TES was provided by the US President's Malaria Initiative. Partial support also came from a PhD fellowship granted by the French Ministry or Foreign affairs (Ministère des Affaires Etrangères). PCR and sequencing were supported by the French Government under the Investissements d'Avenir (Investments for the Future) program managed by the Agence Nationale de la Recherche (ANR, fr: National Agency for Research), (reference: Méditerranée Infection 10-IAHU-03), the IHU-Méditerranée Infection Foundation.

Authors’ addresses: Mamadou Alpha Diallo, Khadim Diongue, Aida Sadikh Badiane, Mouhamad Sy, Mame Cheikh Seck, Mouhamadou Ndiaye, and Daouda Ndiaye, Department of Parasitology and Mycology, Cheikh Anta Diop University, Dakar, Senegal, and Aristide Le Dantec University Hospital, Dakar, Senegal, E-mails: mamadoualpha.diallo@ucad.edu.sn, khadimase@gmail.com, asbadiane@gmail.com, symouhamad92@gmail.com, mcseck203@yahoo.fr, mouhamadou.ndiaye@ucad.edu.sn, and daouda.ndiaye@ucad.edu.sn. Aly Kodio, Aix Marseille University, IRD, AP-HM, SSA, VITROME, Marseille, France, and IHU Méditerranée Infection, Marseille, France, E-mail: alkodio@icermali.org. Mamadou Lamine Tall, Aix Marseille University, IRD, AP-HM, SSA, VITROME, Marseille, France, E-mail: laminetall30@gmail.com. Doudou Sene, IHU Méditerranée Infection, Marseille, France, E-mail: drdocsene@yahoo.fr. Fatou Ba Fall, National Malaria Control Program (NMCP), Dakar, Senegal, E-mail: fall1fatou@yahoo.fr. Stéphane Ranque, Aix Marseille University, IRD, AP-HM, SSA, VITROME, Marseille, France, and IHU Méditerranée Infection, Marseille, France, E-mail: Stephane.RANQUE@ap-hm.fr.

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