Spatio-temporal analysis of the genetic diversity and complexity of Plasmodium falciparum infections in Kedougou, southeastern Senegal

Background Genetic analyses of the malaria parasite population and its temporal and spatial dynamics could provide an assessment of the effectiveness of disease control strategies. The genetic diversity of Plasmodium falciparum has been poorly documented in Senegal, and limited data are available from the Kedougou Region. This study examines the spatial and temporal variation of the genetic diversity and complexity of P. falciparum infections in acute febrile patients in Kedougou, southeastern Senegal. A total of 263 sera from patients presenting with acute febrile illness and attending Kedougou health facilities between July 2009 and July 2013 were obtained from a collection established as part of arbovirus surveillance in Kedougou. Samples identified as P. falciparum by nested PCR were characterized for their genetic diversity and complexity using msp-1 and msp-2 polymorphic markers. Results Samples containing only P. falciparum accounted for 60.83% (160/263) of the examined samples. All three msp-1 allelic families (K1, MAD20 and RO33) and two msp-2 allelic families (FC27 and 3D7) were detected in all villages investigated over the 5-year collection period. The average genotype per allelic family was comparable between villages. Frequencies of msp-1 and msp-2 allelic types showed no correlation with age (Fisher’s exact test, P = 0.59) or gender (Fisher’s exact test, P = 0.973), and were similarly distributed throughout the 5-year sampling period (Fisher’s exact test, P = 0.412) and across villages (Fisher’s exact test, P = 0.866). Mean multiplicity of infection (MOI) for both msp-1 and msp-2 was highest in Kedougou village (2.25 and 2.21, respectively) and among younger patients aged ≤ 15 years (2.12 and 2.00, respectively). The mean MOI was highest in 2009 and decreased progressively onward. Conclusion Characterization of the genetic diversity and complexity of P. falciparum infections in Kedougou revealed no spatio-temporal variation in the genetic diversity of P. falciparum isolates. However, mean MOI varied with time of sera collection and decreased over the course of the study (July 2009 to July 2013). This suggests a slow progressive decrease of malaria transmission intensity in Kedougou Region despite the limited impact of preventive and control measures implemented by the National Malaria Control Programme on malaria morbidity and mortality.


Background
According to the World Health Organization (WHO), the incidence of malaria and associated mortality respectively decreased by 30 and 47% globally between 2000 and 2013 [1]. Despite this significant achievement resulting from coordinated malaria preventive and control interventions, an estimated 198 million cases and about 584,000 deaths still occurred worldwide in 2014 [1].
In Senegal, malaria remains a public health concern even though malaria control interventions have successfully reduced the incidence rate nationwide over the last decade [2]. Nonetheless, the situation is particularly worrying in the Southeastern part of the country due to limited impact of applied preventive and control measures [2]. In Kedougou, a region situated in southeastern Senegal, malaria due to Plasmodium falciparum remains highly prevalent [2,3], particularly during the transmission season that coincides with the rainy season from July to November. In 2014, the National Malaria Control Program (NMCP) reported 25.55% of malaria incidence of which 2.73% turned into severe disease [2].
Extensive parasite genetic diversity and routine carriage of multiple parasite genotypes by malaria-infected individuals are generally observed in areas of intense malaria transmission [4,5]. The genetic diversity of P. falciparum parasites is therefore an indicator of malaria transmission intensity, thus serving as a tool to evaluate the effectiveness of malaria control interventions.
The merozoite surface protein-1 (msp-1) and -2 (msp-2) are polymorphic antigenic markers that have been extensively used for genetic characterization of parasite populations in malaria endemic areas but have also served to distinguish new from recrudescent infections during antimalarial drug trials and efficacy studies [6][7][8].
Despite previous investigations of the genetic diversity of P. falciparum in Senegal [9][10][11][12], further detailed studies are needed particularly in Kedougou Region where malaria transmission is still active. The present study examined the genetic diversity and complexity or multiplicity of infection (MOI) of P. falciparum infections among febrile patients in Kedougou Region. The spatial and temporal distributions of msp-1 and msp-2 allelic families of P. falciparum isolates were also analyzed.

Population and study design
The samples used in this study originated from Kedougou Region (Fig. 1), southeastern Senegal. A detailed description of the study area (climate, rainfall, landscape, fauna, population) has been provided elsewhere [3,13,14]. The Kedougou Region borders Guinea, Mali and Gambia between isohyets 1200 and 1300 mm. A total of 263 sera from patients with acute febrile illness (AFI) visiting healthcare facilities (Bandafassi, Kedougou, Ninefesha, Saraya, Khossanto) in Kedougou Region between July 2009 and July 2013 were obtained from a collection established during arbovirus surveillance in Kedougou Region [14]. Due to the similar clinical presentation between malaria and arboviral infection, malaria diagnostic screening was systematically conducted to differentiate arboviral to malaria disease.
AFI was defined as "any patient older than 1 year with a fever (temperature > 38°C) lasting for less than 2 weeks, exhibiting two or more of the following symptoms: headache, myalgia, eye pain, arthralgia, cough, nausea/vomiting, diarrhea, jaundice, bleeding and/or neurological signs". Sera were investigated for presence of only P. falciparum and subsequently analyzed for genetic diversity, temporal and spatial distribution of allelic families and complexity of malaria infection.

Characterization of malaria parasite species
To ensure that patients were infected with only P. falciparum, all 263 sera were subjected to Plasmodium species characterization following genomic DNA (gDNA) isolation using QIamp DNA Mini Kit (Qiagen, Hilden, Germany) as reported by others [15,16]. DNA extracted from blood samples of known microscopically confirmed P. falciparum, P. malariae, P. vivax and P. ovale-infected patients were used as positive controls [3] to discriminate P. falciparum-positive samples from other Plasmodiuminfected samples.
Qualitative detection of Plasmodium parasite DNA was based on a nested PCR approach with primers targeting the Plasmodium spp. 18S small subunit ribosomal RNA gene (18S ssrDNA) as described previously [17]. The primary and nested PCR reactions were performed using the GoTaq Green Master Mix protocol (Promega, Madison, USA), according to the manufacturer's recommendations, and amplification conditions were as described previously [3]. Nested PCR results were scored as a categorical variable (presence versus absence of amplification).

Genotyping of Plasmodium falciparum isolates
Genetic characterization of P. falciparum isolates was carried out by nested PCR amplification of the two highly polymorphic regions of msp-1 (block 2) and msp-2 (block 3) genes as described previously [18,19]. Primer sequences used for amplification of the three allelic families of msp-1 (K1, MAD20 and RO33) and two allelic families of msp-2 (FC27 and 3D7) have been reported elsewhere [11,19,20]. An initial amplification of the outer regions of the two genes was followed by a nested-PCR with allelic family specific primer pairs. In the primary PCR reaction, 2 μl of gDNA served as template while in the nested reaction, 1 μl of the outer PCR product was used as template. Amplification products were separated by electrophoresis on 1.5% agarose gel, and the fragments were visualized using ethidium bromide under UV light. The sizes of amplified products were determined using a molecular weight marker.
The frequency of each msp allelic family was calculated as the number of P. falciparum isolates containing at least one allele from that family out of the total number of samples. The multiplicity of infection (MOI) or number of genotypes per infection was calculated by dividing the total number of fragments detected in msp-1 or msp-2 by the number of samples positive for the same marker [21,22].

Statistical analysis
Data were analyzed using R statistical software version 3.1.1 (2014-07-10) [23]. Distributions of age and sex between villages were compared using Kruskall-Wallis rank sum test. All comparisons between frequencies were performed using Fisher's exact test. Comparisons were considered statistically significant when P-values were less than the Bonferroni-corrected threshold.

Characteristics of the study population
All 263 serum samples were screened for the presence of Plasmodium parasite DNA and characterization of Plasmodium species [3]. Samples containing only P. falciparum accounted for 60.83% (160/263) and were further characterized for genetic diversity and MOI using msp-1 and msp-2 polymorphic markers. The demographic characteristics of the 160 P. falciparum-positive patients are summarized in Table 1.

Genetic diversity of Plasmodium falciparum isolates
All three allelic families of msp-1 (K1, MAD20 and RO33) and two of msp-2 (3D7 and FC27) were detected among the 160 P. falciparum positive isolates genotyped in this study.
Although the total number of genotypes was highest in P. falciparum isolates obtained from Bandafassi (Table 2), the mean number of genotypes per allelic family was comparable between villages.
Frequency of msp-1 and msp-2 allelic types with respect to age and gender The frequencies of msp-1 and msp-2 allelic types were categorized within two arbitrarily defined age groups: ≤ 15 years and > 15 years ( Table 1). Frequencies of the three msp-1 allelic types (K1, MAD20 and RO33) and two msp-  2 allelic types (FC27 and 3D7) were similarly distributed with no significant differences between age groups (Fisher's exact test, P = 0.59) (Fig. 3a). Within a given age group, frequencies of the three msp-1 allelic types and the two msp-2 allelic types were also comparable. With respect to gender, frequencies of msp-1 and msp-2 allelic families showed similar distribution between males and females with no significant difference observed (Fisher's exact test, P = 0.973). The frequencies of all msp-1 and msp-2 allelic families were also comparable within a given gender (Fig. 3b).

Temporal and spatial distribution of msp-1 and msp-2 allelic families in Kedougou Region
Analysis of the spatio-temporal distribution of all msp-1 and msp-2 allelic families of P. falciparum isolates from 2009 to 2013 showed no significant difference (Fisher's exact test, P = 0.412) throughout the 5-year sampling period (Fig. 4a). Allelic frequencies were highest in 2009 and decreased progressively up to 2013 except for RO33 in 2011 (Fig. 4a). The distribution of allelic frequencies was also similar (Fisher's exact test, P = 0.866) between villages with a consistent low frequency of all allelic types in Ninefesha (Fig. 4b), probably related to the lower number of samples from this village.

Mean multiplicity of infection in relation to village, age and sampling period
Mean MOI was compared between villages (Kedougou, Bandafassi, Ninefesha and others), age groups (≤ 15 years and > 15 years), and sampling periods (2009 to 2013). The mean MOI for both msp-1 and msp-2 was found to be higher in P. falciparum isolates from patients from Kedougou village (MOI = 2.25 and 2.21), in patients aged ≤ 15 years (MOI = 2.12 and 2.00), and those diagnosed in 2009 (MOI = 2.21 and 2.50) than P. falciparum isolates originating from others villages, patients aged older than 15 years, and those diagnosed from 2010 onward, respectively (Table 3). Overall, the mean MOI varied between villages for both loci and decreased progressively Fig. 2 Frequency of unique (a) and combined (b) msp-1 and msp-2 allelic families of Plasmodium falciparum isolates from Kedougou Region. Alleles of the msp-1 and msp-2 genes were classified by the presence of fragment resulting from the PCR amplifications with allele specific primers. Pairwise Fisher's exact tests between allelic families showed only significant difference between FC27 and 3D7 Fig. 3 Distribution of frequencies of msp-1 and msp-2 allelic families according to age group (a) and gender (b). Frequencies were determined as the percentage of individuals within a given age group or gender among the positive samples. Statistical differences were determined using Fisher's exact test with increasing age and year of sample collection for both msp-1 and msp-2 loci (Table 2).

Discussion
The present study provides information about the spatial and temporal dynamics of the P. falciparum parasite population among febrile patients in Kedougou Region. This bridges an important gap in the understanding of the molecular characteristics of the parasite population in this region where Plasmodium transmission is still active and could inform efforts to monitor and control Plasmodium transmission in this region.
All msp-1 and msp-2 allelic families were represented in all villages in Kedougou Region where samples were collected and elevated frequencies of msp-1 and msp-2 genotypes were reported with MAD20 and 3D7 being the most prevalent, particularly in Bandafassi and Kedougou villages. However, the comparable average number of genotypes for each locus between villages suggested that the large number of genotypes in Bandafassi potentially reflects an elevated number of samples rather than higher genetic diversity of P. falciparum isolates in this village. FC27 allelic type has been shown to be more prevalent in asymptomatic rather than symptomatic Plasmodiuminfected individuals [24], suggesting a lower risk of developing symptomatic malaria with increasing carriage of isolates belonging to the FC27 allelic family. Such observations are supported by previous studies in Senegal [5,13] and Nigeria [25] but contrast with data from Benin and Gabon [26,27], emphasizing the need for multicentric studies using P. falciparum isolates from both asymptomatic and symptomatic individuals to comprehensively establish the potential relationship between specific allele carriage and disease outcome. High to moderate mean MOI was observed among P. falciparum isolates across all the studied villages with the highest mean MOI being observed in Kedougou village. A high MOI is a common feature in most malaria hyperendemic areas [28][29][30] and has been directly linked with malaria transmission intensity [22,28,30]. Thus, the slow decrease of the mean MOI for both msp-1 and To date, only a few studies have applied genetic surveillance tools to evaluate Plasmodium population structure in relation to changing malaria transmission in Senegal, thus precluding a meaningful comparison with the situation observed in Kedougou Region. The application of molecular barcoding genomic surveillance on P. falciparum parasites collected from Thies region (West Senegal) between 2006 and 2013 revealed reduced genetic diversity of P. falciparum population along with a decline of Plasmodium transmission from 2006 to 2010, a period transcending the introduction of insecticide-treated bednets in 2008 [31]. The mass distribution of insecticide-treated bednets in malaria-endemic countries was demonstrated to be a key player in the dramatic decline of Plasmodium transmission documented in Senegal [32,33] and elsewhere [34].
The finding that MOI decreased with age supports studies conducted in other countries [35,36] that have shown lower MOI in those aged 15 and above. The existence of a positive association between high childhood MOI and low adulthood MOI is in keeping with the concept that exposure to multiple strains of the parasite in early life is necessary to produce immunity in adults. Conflicting findings indicating a greater MOI in older individuals have also been reported [29,37]. The limited studies on P. falciparum genetic diversity conducted in Senegal failed to establish a link between MOI and age groups [10][11][12], thus depicting the complex nature of such an association. The lack of an association between MOI and age groups as reported in above-mentioned studies suggests that the MOI is not directly related to the period of acquisition of immunity, but rather reflects the exposure of subjects to malaria in an endemic area. The decrease of MOI over time despite the limited impact of control measures suggested a selection of clonal parasites populations, thus increasing the chance of an individual being re-infected with the same allele.

Conclusions
This study reports the spatial and temporal variation of the genetic diversity and complexity of P. falciparum infections in acute febrile patients in Kedougou, a Senegalese region with active malaria transmission. The findings revealed that P. falciparum isolates of Kedougou Region are highly diverse with limited spatio-temporal variation in the region. However, MOI decreased over time, which may be an indication of a progressive reduction in Plasmodium transmission in the region despite the limited impact of control measures on malaria morbidity and mortality in the area.
Additional studies that examine the dynamics of the genetic diversity of P. falciparum parasite populations, considering factors such as the malaria transmission intensity based on entomological inoculation rates and the immune status of P. falciparum-infected individuals would tremendously improve our understanding of P. falciparum parasite diversity in Kedougou Region and contribute to guide malaria interventions.