Polymorphism of the Merozoite Surface Protein-1 Block 2 Region in Plasmodium Falciparum Isolates from Symptomatic Individual Living in Rural Area of Senegal

Background: Polymorphism and antigenic variation of malaria parasites determine malaria episode and its outcome. The aim of this study was to determine Plasmodium falciparum genetic diversity over time in population with uncomplicated malaria under ACT exposure in Senegal. Method: P. falciparum isolates collected from 300 individuals with uncomplicated malaria infection living in a rural area of Senegal from 2004 to 2008 were analyzed by a nested PCR amplification of msp1 and msp2 genes to compare P. falciparum allelic families’ diversity. Results: Allelic variation in both msp1 and msp2 genes were identified in the samples analyzed. For msp1 gene, 10 different alleles were found (3 msp1_K1, 4 msp1_Mad20 and 3 msp1_Ro33). Among them msp1_k1 allelic family was predominant (>70%) over year. Regarding msp2 gene, 7.0 different alleles were found (3 msp2_3D7, 4 msp2_FC27). However msp2_FC27 strain was predominant, especially in 2006 and 2007. Monoclonal infections were more frequent for msp1 gene, in 2004 (48.78%) and 2005 (45.05%) and for msp2 gene than polyclonal ones. Conclusion: This study demonstrated some differences in the P. falciparum diversity between symptomatic subjects over years living in rural area in Senegal and this should be taken into account when designing msp1 or msp2 malaria vaccine.


Background
Malaria remains a leading cause of ill health, causing an estimated 243 million cases of clinical malaria and 863 thousand deaths.More than 85% of malaria cases and 90% of malaria deaths occur in sub Saharan Africa [1].In Africa, the vast majority of cases and deaths occur in young children [1].Efforts to control this disease in Africa have been hindered by the spread of resistance to chloroquine and more recently, to sulfadoxine-pyrimethamine [2,3].Currently key interventions are recommended by World Health Organization (WHO) for the control of malaria such as: use of insecticidal treated nets (ITNs) or indoor residual spraying (IRS) for vector control, and prompt access to diagnosis and treatment with Artemisin Combination Therapy (ACT) for uncomplicated P. falciparum malaria.
Following the WHO recommendation, the National Malaria Control Programme (NMCP) of Senegal adopted ACT since 2006 as first line treatment of confirmed malaria.Furthermore, since 2004 several clinical trials using ACT derivate were performed in Senegal particularly in Kaolack region.As any drug ACT could have an impact in the msp1 and msp2 genes polymorphism over year.The relationship between parasites genotypes and clinical outcome has been reported in several studies [4][5][6].Genetic diversity of P. falciparum field isolates, the occurrence of variant forms of the parasite in different geographic areas, and occultation of multiple genotypes during a single mosquito, constitute one of the main obstacles to the design of a effective malaria vaccine [7,8].The objectives of this study was to determine the genetic diversity of P. falciparum parasites under ACT exposure and to compare the polymorphism of two highly polymorphic genes encoding the merozoite surface protein-1 (msp-1) and the merozoite surface protein-2 (msp-2) [9,10] in infected individuals living in rural areas in Senegal.

Study site
This study was conducted in rural area of Keur Soce, located from 200 km Southern-East of Dakar.The study was carried out in the health post of Lamarame belonging to the medical district of Ndoffane.Ndoffane is located at 17 km to the south of Kaolack.The terrain is generally flat with some depressions promoting the presence of water collection that are potential breeding sites.The climate is Sudano-Sahelien type with temperatures oscillating between 24°C and 40°C; extremes of 15°C in January and 44°C in July have been recorded.Rainfall is on average 600 mm per year.The district of Ndoffane host approximately 200 villages with a total population of nearly 75,000 inhabitants of which about 7% are aged up to 24 months.Malaria is the most important public health problem, accounting for 45% of general morbidity.Latest entomological surveys showed an entomological inoculation rate between nine and 12 bites per night during the period of high transmission.

Sample collection
This study was part of several studies assessing anti-malarial drug efficacy that were performed from 2004 to 2008 in Keur Soce area.Patients were enrolled in accordance with the World Health Organization (WHO) protocol for antimalarial assessment [11], with sligh modifications.The inclusion criteria are: 1) patient of any age; 2) fever (axillary temperature >= 37.5°C) or a history of fever within the past 48 hours; 3) P. falciparum mono infection with parasite density between 1,000 and 100,000 asexual forms per microliter, identified microscopically on blood smears; 4) no history of anti-malarial drug administration in the last two weeks; 5) no history of serious adverse events to the study drugs; 6) no evidence of a concomitant febrile illness; 7) no sign/symptoms of severe malaria as defined by WHO [12].Informed consent was obtained from parents or legal guardians of children and individual prior to their enrolment.A total of 300 positives samples were included in our study.At the time of the study, the studies protocols were reviewed and approved by the Ethical committee of Senegal.
Finger prick blood samples were collected from consenting thick and thin blood smears were prepared for microscopic identification of P. falciparum.Blood from a finger prick was blotted onto Whatman filter paper, labeled with patients' study numbers and stored dry at room temperature.Dry filter paper was individually placed into plastic bag protected with silica gel desiccant.

DNA extraction and analysis
P. falciparum DNA was extracted from blood collected on 3 mm Whatman filter paper by methanol method describe by Djimde et al. [13].Briefly, tree our four fragments of filter paper sample were incubated with 500 µl of methanol in 1.5 ml eppendorf tubes.The mixture was incubated 15 minutes at room temperature before removing the methanol.Eppendorf tubes with fragment of filter paper were left unmoved until they completely dry.Then 50-70 µl of distilled water were added in each tube with dry filter paper and heated at 95-100°C for 15 mn.During the incubation steps, tubes were vortexed and returned to the heat block every five minutes.Samples were centrifuged and extracted DNA was transferred into new, labeled tube and stored at +4°C for immediate use.PCR was performed following a 2-step amplification scheme, in which two microliter product of the first reaction (outer PCR) was used as the template for the second reaction (nested PCR) for both msp1 and msp-2.Outer and nested PCR reactions were performed in 25 µl final volume.For each reaction, 200 µM each of dNTP, 1 µM of each primer, 0.5 UI of Taq DNA polymerase and PCR Buffer 10X containing 100 mM Tris-HCl, pH 8.3 at 25°C, 500 mM KCl, 15 mM MgCl 2 and 0.01% gelatin were used.The Outer and the Nested amplification were performed on an Applied Biosystem 2720 and PTC-100 Peltier Thermal Cycler.For msp-1, the primers used in the first amplification reaction were conserved among all isolates.Allelic family-specific primers were used for the nested amplification reaction for block 2 of msp-1 corresponding to msp1_Mad 20, msp1_K1 and msp1_Ro33 allelic families.For msp2 gene, PCR amplifications of P. falciparum msp2 genes were done as

DNA visualization
The PCR products were subjected to electrophoresis on 2% agarose gels (Gibco-BRL) in Tris acetate-EDTA buffer for various lengths of time depending of the predicted size of the PCR products and visualized with ethidium bromide by transillumination with ultraviolet light.We used 100 base pair ladders (Pharmacia Biotech, Piscataway, NJ) as standards for estimation of the sizes of DNA fragments.

Allelic distribution and multiplicity of infection
The msp-1 and msp-2 alleles were categorized by their molecular weights and considered the same if their molecular weights were approximately within 10 bp [15].The multiplicity of infection (MOI), or complexity of infection, was estimated by the average number of PCR fragments per infected individual [15].The detection of a single PCR fragment for each allele was classified as a single genotype infection.Detection of more than one PCR fragment for either msp1, msp2 was considered as multiple P. falciparum genotype infection (MOI).

Statistical analysis
Data were entered in a Microsoft Excel sheet, and the haplotype of each positive sample was determined.Statistical analyses of data were performed with Epi info 6.04a (http://www.cdc.gov/epiinfo/Epi6/EI6dnjp.htm).A χ2-test was used to compare differences in proportions of parasites genotypes.Significance level of statistical tests was set at 0.05 with two sides.

PCR efficacy
Among the 300 samples analyzed, 247 (82.33%) and 198 (66%) were positives respectively for msp-1 and msp-2 genes.Failure amplification could be done by extraction efficiency.Negative PCR samples were excluded from further analysis.

Allelic polymorphism of msp-1 and msp-2 genes
A highly polymorphic nature of Senegalese P. falciparum isolates with respect to msp-1 and msp-2 genes were noted.For msp-1, the main allelic type (K1, Mad_20 and RO_33) were identified.Regarding msp-2, 3D7/IC and FC27 alleles were identified.
Considering allelic families, results showed that msp-2_FC27 strains were more frequent among our patients over year, especially in 2006 and 2007 except in 2008, where infections with msp-2_3D7 strains were predominant (Table 3).

Discussion
This study was undertaken to assess the polymorphism of the merozoite surface protein-1 block 2 allelic types in P. falciparum isolates from rural area in central Senegal.P. falciparum is thought to be responsible of nearly 95% of diagnosed malaria cases in the country.A better understanding of population structure of P. falciparum genotypes may be an important element for implementing malaria control strategies in the country.Data showed a relatively high polymorphic nature of K1, Mad_20 and RO_33 msp-1 allelic types according to the number of band sizes (10 different PCR products: 3 K1, 4 Mad20 and 3Ro33) and 3D7 and FC27 msp-2 allelic types among senegalese P. falciparum isolates.The number of different msp-1 alleles observed among P. falciparum isolates was lower than those found by Konate et al. [16] in the holoendemic area of Dielmo (Senegal) where 33 msp-1 alleles were identified.This study also revealed the predominance of K1 type alleles at study site level or throughout the parasite population compared to Mad20 and Ro33 over year.Similar results were found in several studies conducted in Gabon [17][18][19][20][21], in Central Africa or in Honduras [22] in Central America.Ours results showed also that the msp1_Mad20 family was less frequent except in 2008 where this pattern was more frequent than msp1_RO33.Our findings are supported by similar study conducted in Burkina Faso where authors found that msp1_Mad20 allelic family was less frequent in infected children from rural area than those from urban area [23].The difference in rural symptomatic malaria in 2008 may suggest a specific immune response against this allele (msp1_RO33), or a probable efficacy of ACTs introduced in Senegal in 2006 against this allele.ACT has been in use since 2006 but up scaling was done in 2008.RDTs were also introduced in 2007, and in 2008 following guideline of the Ministry of Health (MoH) through the National malaria Control Program (NMCP) ACTs are the first line treatment for uncomplicated P. falciparum malaria in Senegal.In Keur Soce area ACTs drug are highly effectives with an adequate clinical and parasitological cure rate over 95% [24].As any drug, ACT could result in genetic modification of the P. falciparum parasites.A random event due to a genetic drift can also explain the opposite we observed in 2008 [25].msp-2 allelic families diversity is also noted in Keur Soce area with a predominance of msp-2_IC/FC27 in 2006-2007 while in 2008, the frequency of msp2_3D7 was more frequent than msp2_3D7.During these two years, msp2_3D7 and msp2_IC/FC27 alleles were nearly equally frequent.Similar results were found in Tanzania [26] and more or less similar results were reported in Namawala, an area lying along the northern edge of flood plains of the Kililomero River [27].
Regarding the multiplicity of infections (MOI), our results showed that MSP1 single infection was more frequent from 2004 to 2005.From 2006 to 2008 MSP1 double infection become more frequent than single infection.Triple infection increased over time.Regarding MSP2 pattern, it appeared clearly that the single infection was more frequent than the double and triple infection was not found in our study.Differences in transmission level over time and anti-malarial immunity known to be strain specific [28], could partly explained the differences in the distribution of the different alleles over year.In a given locality, the parasite genetic pool may be relatively stable, perhaps related to the stable parasite life cycle; thus, the distribution of alleles may be determined randomly so that certain alleles will predominate by chance.Nevertheless, there are certain factors external to the natural life cycle that are known to affect allelic distribution, notably, antimalarial drug pressure and result in a higher prevalence of drug resistance markers, the proximity of populations to water reservoirs, as it has been observed in Keur Soce, the use of insecticide-treated nets, indoor insecticide spraying or certain human genetic factors (hemoglobin types, G6PD), that could explain the difference in the distribution of P. falciparum genotypes over years.

Conclusion
This study demonstrated that there were some differences in the P. falciparum diversity between symptomatic subjects over year living in rural area in Senegal and this should be taken into account when designing msp1 or msp2 malaria vaccine.The study also emphasizes the importance of evaluating the extent of parasite genetic variation in different part of Senegal and the factors affecting this variation.In this context, longitudinal studies examining the dynamics of the P.

Table 1 :
Sequence of oligonucleotide primers used to genotype P. falciparum and PCR conditions.
[14]ye JL, Ndiaye M, Sow D, Sylla K, Faye B, et al. (2017) Polymorphism of the Merozoite Surface Protein-1 Block 2 Region in Plasmodium Falciparum Isolates from Symptomatic Individual Living in Rural Area of Senegal.Malaria Contr Elimination 6: 151.Foley et al.[14]: the primary reaction was performed with 5 µl of DNA with S2 and S3 msp2 primers.Two microliters of the outer reaction was used for the nested PCR with S1 and S4 primers[14].

Table 2 :
Prevalence of P. falciparum K1, RO33 and Mad 20 allelic family over year.

Table 4 :
Overall Multiplicity of infection of the MSP1 genotypes of the P. falciparum K1, RO33 and Mad2O allelic family.

Table 5 :
Overall MOI of the MSP2 genotypes of the P. falciparum 3D7 and IC/FC27 allelic family.