Genetic polymorphism of Merozoite Surface Protein 1 (msp1) and 2 (msp2) genes and multiplicity of Plasmodium falciparum infection across various endemic areas in Senegal

Introduction Despite a significant decline in Senegal, malaria remains a burden in various parts of the country. Assessment of multiplicity of Plasmodium falciparum infection and genetic diversity of parasites population could help in monitoring of malaria control. Objective To assess genetic diversity and multiplicity of infection in P. falciparum isolates from three areas in Senegal with different malaria transmissions. Methods 136 blood samples were collected from patients with uncomplicated P. falciparum malaria in Pikine, Kedougou and Thies. Polymorphic loci of msp1 and 2 (Merozoite surface protein-1 and 2) genes were amplified by nested PCR. Results For msp1gene, K1 allelic family was predominant with frequency of 71%. Concerning msp2 gene, IC3D7 allelic family was the most represented with frequency of 83%. Multiclonal isolates found were 36% and 31% for msp1et msp2 genes respectively. The MOI found in all areas was 2.56 and was statistically different between areas (P=0.024). Low to intermediate genetic diversity were found with heterozygosity range (He=0,394–0,637) and low genetic differentiation (Fst msp1= 0.011; Fst msp2=0.017) were observed between P. falciparum population within the country. Conclusion Low to moderate genetic diversity of P.falciparum strains and MOI disparities were found in Senegal.


Introduction
Malaria control interventions showed a significant progress worldwide 1 . However, malaria remains a burden in sub-Saharan Africa despite scaling up malaria interventions 1 . Senegalese national malaria control program (NMCP) intensified malaria control strategies for more than ten years including large distribution of long-lasting insecticide-treated bed nets (LLINs), use of histidine rich protein 2-based rapid diagnostic tests (HRP2-RDT) and artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated Plasmodium(P) falciparum malaria 2 . Nevertheless in 2015, 492253 cases and 526 deaths due to P. falciparum malaria had been registered in the country 3 .
In Senegal, the overall population is exposed to malaria with a gradient of transmission increasing from North to South due to malaria epidemiology that occurs as a result of intensified control efforts in these areas. Indeed, malaria is hyperendemic in the South (annual incidence is greater than 100/1000 inhabitants) and hypoendemic in the North (annual incidence rates are now less than 5/1000 inhabitants) 3 . Malaria transmission pattern affects the multiplicity of infection (MOI) and genetic diversity of parasite populations, the latter's increase when transmission is high and decrease when transmission is low 4,5 . Thus, assessment of genetic diversity and multiplicity of infection (MOI) provide insight on malaria transmission and genetic differentiation measurements such as heterozygosity (He) and fixation index (Fst) which are important data in monitoring of malaria control and elimination strategies 4,6,7,8,9,10 .
The polymorphic loci of merozoite surface proteins (msp1 and msp2) are now well established to assess the genetic diversity of P. falciparum population and multiplicity of infection (MOI) which is an indicator of malaria transmission intensity in endemic areas 9,11,12,8,13,14,15 . Additionally, many studies demonstrated that msp1 and msp2 genes are enough robust polymorphism markers to characterize the parasite population structure 12,16 and are the gold standards to evaluate the MOI17 Msp1 gene has three allelic families K1, MAD20 and RO3318 and msp2 gene two allelic families, IC3D7 and FC27 19 .
The characterization of genetic diversity of P. falciparum through different areas with various endemicities within a country has been extensively looked into in different parts of the world 8,9,13,16,20,21 . However, a few studies about this topic have been performed in Senegal by Ahouidi et al 22 and Konaté et al 23 . Hence, it is important to have current data on genetic diversity on parasite populations through several areas in the country to better inform malaria control programs. In addition, the monitoring of malaria transmission intensity is a clear priority to malaria elimination. As MOI is an important metric of malaria transmission 24,25,26,27,28 , it is a robust measure of changing malaria transmission and remains one of the most accurate molecular approaches to evaluate malaria parameters 29 in areas moving towards elimination like Senegal. It is important to have insight of malaria transmission intensity using the MOI within the country to know which area need more interventions in order to achieve the malaria elimination target in Senegal. This study aimed to investigate the genetic diversity and the multiplicity of P. falciparum infection of circulating parasites strains from three areas in Senegal having different endemicities by genotyping the highly polymorphic loci of msp1 and msp2 genes. Some genetic measurements such as He and Fst were also analyzed.  This map was generated using online website (http://www.d-maps.com)

Study sites
This study was carried out in three areas of Senegal with different endemicity of malaria: Pikine, Thies and Kedougou. Pikine is a sub-urban area which is 15 km the capital center (Dakar) with a heterogeneous endemicity (related to recurrent floods) with an entomological inoculation rate (EIR) ranging from 0 to 20 infectious bites per years and a malaria incidence greater than 15 malaria cases per 1000 habitants. Thies, in the west of the country at 70 km from Dakar, is a hypo-endemic area with a low EIR varying from 0 to 20 infectious bites per years and an average of 5 to 15 malaria cases per 1,000 habitants. Kedougou, located in the South-East, at 685 Km from Dakar, is hyper-endemic with an incidence higher than 15 malaria cases per 1000 habitants. In Kedougou, EIR is high ranging from 20 to 100 infectious bites/person/ year 2 .

Sample collection
This study was approved by Ethics Committee of the Ministry of Health of Senegal. Febrile patients visiting the NMCP sentinel site in these three areas during malaria transmission season were enrolled in this study after giving informed consent or guardians consent for children depending on the age of the patient. Blood samples were collected on filter-paper from patients who met the following criteria: Living in a 15-km radius of health facilities, having fever (axillary temperature ≥ 37.5 C) or history of fever in the previous 48 h, age ranging from 6 months to 75 years and uncomplicated P. falciparum malaria with parasite density ≥ 1000 asexual forms per microliter. Patients who presented signs or symptoms of severe malaria as defined by World Health Organization (WHO) 30 and pregnant women were not included.

DNA extraction and PCR genotyping
Parasite DNA, from filter paper was extracted using QIAamp DNA Mini kit (Qiagen  , QIAGEN, USA) according to the manufacturer's instructions. The polymorphic loci of msp1 block 2 (K1, MAD20 and RO33 allelic families) and msp2 central region (IC3D7 and FC27 allelic families) were amplified by nested PCR as described previously 31,32 . The primer sequences are as shown in Table 1. All PCR reactions were performed out in a total volume of 20 μl containing 6 μl GoTaq Green Master Mix, 0.5 μM of each primer, and 11 μl reagent grade water. In the first round reaction (nest 1), 1 μl of genomic DNA was added as a template. In the second nested reaction (nest 2), 1 μl of the nest 1 PCR product was used as DNA template. Cycling conditions for primary PCR were as follows: initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 2 min and extension at 72°C for 2 min; a final extension was done at 72°C for 3 min. The cycling conditions for secondary PCR were, initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 61°C for 2 min, extension at 72 °C for 2 min, with a final extension cycle of 72°C for 3 min. Positives (3D7 and Dd2) and negative (reagent grade water) controls were systematically incorporated in each PCR run. The nested PCR product were revealed by electrophoresis on 2% agarose gels stained with ethidium bromide and visualized under UV trans-illumination (VersaDoc®, BIORAD, Hercules, USA). The size of PCR fragments were estimated using 100 bp molecular weight.

Statistical analysis
The online Biostatgv was used for statistical analysis. For all tests, the significance level was p < 0.05. Isolates presenting more than one allele were considered as multiclonal isolates. The chi-square test (X2) was used to compare the frequencies of multiclonal isolates between localities. The multiplicity of infection (MOI) was calculated by dividing the total number of alleles detected for msp1and msp2 genes by the total number of samples 11 . Student's t test was used to compare MOI between localities. We used the expected heterozygosity (He) and genetic differentiation (FST) to assess population structure of parasites. Heterozygosity was calculated using the following formula He = n/(n-1) (1-ΣPi2), where n = sample size, Pi = allele frequency as described by Nei et al. 33 and Fst was calculated as described previously 22,34 .

Results
The study population characteristics A total of 13 P. falciparum infected patients were enrolled from three Senegalese localities. Among these patients, 64.7% were male and 35.3% were female with a sex ratio of 1.83. Patients' age ranged from 4 to 75 years with a mean age of patients was 22.3. The parasite density ranged from 1000 to 404000 asexual forms per microliter.

Allelic distribution of msp1and msp2 genes
Overall, the three allelic families (K1, MAD20 and RO33) of msp1gene and two (3D7 and FC27) of msp2 gene were observed in this study.
A total of forty-two alleles types were detected for the two genes in all localities: twenty-two for msp1 (Fig 2) and twenty for msp2 (Fig 3).  In msp1 gene, nine K1 alleles types (range from 100 bp to 350 bp), eight MAD20 alleles types (100 bp-300 bp) and five RO33 alleles types (160 bp-250 bp) were observed while for msp2 gene, eleven IC3D7 (300 bp-800 bp) and nine FC27 (200 bp -600 bp) alleles types were identified. Distribution of the different allelic families of msp1 and msp2 genes and their combinations in different localities are shown in table 2.

Multiplicity of infection, heterozygosity and genetic differentiations
The multiplicity of infection (MOI) and heterozygosity (He) in different localities are shown in table 3. The number of msp1 and msp2 genotypes per isolates ranged from 1 to 5. A higher value of MOI was found in Pikine 2.97. MOI found in all localities was 2.56 with statistically different between areas (p=0.024).  (27) 42 (31) n: number of isolates  For msp1 gene, heterozygosity (He) and genetic differentiation (Fst) observed in all study areas were 0.588 and 0.011 respectively. We found approximately the same value of He in Kedougou (0.637) and Pikine (0.632).
Regarding msp2 gene, He and Fst found in whole study sites were 0.453 and 0.017 respectively. A low He value was observed in Thies (0.394) while a high value was noticed in Pikine with 0.509.

Discussion
The present study aimed to provide a current overview on P. falciparum population structure in Senegal by analyzing the most polymorphic regions of msp1 and msp2 genes on isolates from areas presenting different malaria transmission patterns. This recent information on the molecular epidemiology of the most virulent plasmodial species in this country could inform malaria program of monitoring, control and to adapt if necessary the interventions to local malaria epidemiology settings.
In this study, K1 and IC3D7 allelic families were most predominant in all study sites. The strong presence of both allelic families has been already reported by studies carried out in West Africa 8,15,35 , Western Uganda 36 and Iran 37 . Twenty-two alleles for msp1 gene and twenty alleles for msp2 gene were identified. Similar data were found in areas where malaria transmission is low 13,38 . However, a larger allele's numbers were observed in high malaria transmission areas in Kedougou, Senegal 23 and in Burkina Faso 14 suggesting that malaria endemicity affects the circulating strains numbers. P. falciparum multiclonal isolates for msp1 and msp2 allelic families found in all study areas were relatively high with the higher in Kedougou. Similar findings were reported in hyperendemic areas 13,14,33,39 . The lower multiclonal isolates were found in Thies reflecting the low endemicity level of this study area which is agreement with other studies performed in hypoendemic areas 16,40,41 . These differences show that multiclonal isolates are more common in high malaria transmission area 16 .
MOI is an indicator of malaria transmission level because it has been to be higher in high malaria transmission areas and decreased when this latter decline 16,42 . Our results show moderate to high MOI values and a significant difference on MOIs between these localities (p=0.024). Therefore, our findings found in Thies and Kedougou are in phase with this hypothesis. In this last locality, Niang et al, found the same results in 2017 43 . However, our most important MOI value was obtained in Pikine, an area with particular malaria epidemiological characteristics linked to floods during raining season (artificial water collection reservoirs) which increase opportunities for mosquito breeding growth 2 . These urban characteristics, reported by many studies performed in African cities 44,45,46,47,48,49 , could explain the high MOI found in this locality.
Also, low to moderate genetic diversity with heterozygosity were found in our study, ranging from 0.394 to 0.637. The lowest He value was found in Thies (He=0,394) and the highest He values were found in Kedougou (He=0.637) and Pikine (He=0.632). This variability between genetic diversity levels observed within country could be explained by local malaria epidemiology settings. Similar results were reported in Mali where the He values increase with malaria transmission gradient north to South 8 . The low genetic diversity of P. falciparum population found in Thies is consistent with clonal expansion reported previously in this area 50 suggesting a high self-fertilization rate between genetically identical parasites during the sexual stages in mosquito. This low genetic diversity was also reported in countries where malaria decline, due to scale up of interventions 9,51 . However, moderate heterozygosity was found in Kedougou and Pikine indicating a reduction in clonal expansion and an increase of genetic diversity in these localities. Likewise, it suggest that genetic diversity of P. falciparum is greater in high malaria transmission areas and decreases when transmission regresses 4,5,52 .
Concerning Fst, we found a very low genetic differentiation (Fst msp1= 0.011; Fst msp2= 0.017) between P. falciparum population within country. Ahouidi et al, 22 had already reported this little genetic divergence (Fst msp2 = 0.012) in parasites populations in Senegal. These data reveal a gene flow between parasite populations facilitated by extensive human migration events between endemic regions and then causing the vector's displacement. Likewise, a lack of genetic differentiation was reported by others studies, on sites not far as ours studies areas, located approximately from 61 km to 685km 8,22 . Similar trends have been observed in Mali 8 and elsewhere of the world such as in Latin American countries, Malaysia and South-East Asia where malaria transmission declines 16,53,54,55,56 . Overall, the low to moderate genetic diversity and little genetic differentiation in P. falciparum population found in Senegal shows that the effectiveness of the malaria controls scaling up since 2006 2 . Nonetheless, disparities exist on malaria transmission levels in different areas.
The main limitation of this study was the use of msp genotyping which, as others marker based on DNA fragment size, could reduce the genetic diversity evaluation of the parasites strains. Nevertheless, msp1 and msp2 genes are robust polymorphism markers and can be used successfully to characterize genetic P. falciparum strains populations 12,13,14,15 .

Conclusion
This study gives current data on genetic diversity of P. falciparum population in Senegal. Low to moderate genetic diversity was found on parasite strains reflecting the decline of malaria transmission as well as interventions effectiveness. A high gene flow of parasites between localities shows rapid genes diffusion on P. falciparum population in this country. Our findings also confirm the disparities on malaria transmission level within Senegal. Thus, to achieve the malaria elimination objectives, Senegalese national malaria control program (NMCP) should readjust the malaria control strategies linked to the malaria epidemiological local patterns of sentinel sites like Pikine and Kedougou due to their endemicities.