Fifty-three (53) samples (28 from the South and 25 from the North) were successfully amplified and sequenced for the two polymorphic markers Pfmsp1 and Pfmsp2. All Fastq sequencing data were submitted to the SRA/NCBI database with the following accession numbers: from SRX8632497 to SRX8632549 (https://www.ncbi.nlm.nih.gov/sra/PRJNA642844)
Allelic polymorphisms and population structure of Pfmsp1
Fifty-one Pfmsp1 gene were successfully assembled from 53 P. falciparum isolates. A total of 76 clones were found of which 47 and 29 in the South and the North, respectively (Additional file 1). Of the detected Pfmsp1 clones, 56 different alleles were identified with 33 in the South area and 23 in the North area.
In the South, the 33 alleles found were classified into 3 different allele types: 18 of K1-like, 8 of MAD20-like, and 7 of RO33-like (Table 1). In the K1-like, each allele had different numbers and arrangements of amino acid tripeptide repeat unit SGASAQSGT, SGT or SGPSGT. The MAD20-like were varied by the number of SGGSVA amino acid tripeptide motif. Meanwhile RO33-like was less polymorphic, with only limited numbers of amino acid substitutions. The neighbor-joining tree of different allele types showed two parasite clusters (Fig. 3A). Plasmodium falciparum population presented high level of genetic diversity with high haplotype diversity (Hd) of 0.93 and a nucleotide diversity (π) of 0.28306. The TD test was performed in each area in order to identify any deviation from the neutral evolution of parasites and a highly positive TD value was observed in the South (D = 2.0453) with a statistically significantly different (p < 0.05) (Table 2).
Table 1
number of alleles per allelic family of Pfmsp1 and Pfmsp2 in the South (Kedougou) and the North (Podor and Matam).
Localities | | Kedougou | North area |
Gene /Allelic families | N |
Msp1 | | | |
K1-like | | 18 | 8 |
MAD20-like | | 8 | 6 |
RO33-like | | 7 | 9 |
Total alleles | | 33 | 23 |
Msp2 | | | |
IC/3D7-like | | 35 | 19 |
FC27-like | | 18 | 7 |
Total alleles | | 53 | 26 |
N: number of alleles |
Table 2
Genetic diversity of msp1 and msp2 genes of P. falciparum isolates from the South (Kedougou) and North (Podor and Matam).
Genes | Msp1 | | Msp2 |
| | Kedougou | North area | | Kedougou | North area |
No of Isolate | | 28 | 23 | | 28 | 25 |
Clones (N) | | 47 | 29 | | 76 | 40 |
Haplotype diversity (Hd) | | 0.93 | 0.761 | | 0.82 | 0.843 |
Nucleotide diversity | π | 0.28306 | 0.09457 | | 0.48339 | 0.42473 |
| θ | 0.24245 | 0.16236 | | 0.3534 | 0.37802 |
Tajima's D⁎ test of neutrality | | 2.0453* | -1.46045 | | 5.21073* | 3.46684* |
FSTs | | 0.19505* | | | 0.02111 | |
*indicates the significance at P,0.05 level. | | | | | |
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In the North, the 23 alleles found were classified into: 8 of K1-like, 6 of MAD20-like and 9 of RO33-like (Table 1). The K1-like were different based on the number of amino acid tripeptide repetition SGASAQSGT or SGPSGT. The MAD20-like varied with SGGSVA repeat unit and RO33-like was relatively well conserved sequences. The neighbor-joining tree of different allele types revealed two parasites clusters (Fig. 3B). Parasite population exhibited less genetic variation with π = 0.09457 and Hd = 0.76 and a negative TD value was recorded (D=-1.46045), which was not statistically significant (Table 2). A significant genetic differentiation between the South and the North P. falciparum populations was observed with Fst = 0.19505 (p < 0.05) (Table 2).
Allelic polymorphisms and population structure of Pfmsp2
All 53 Pfmsp2 gene of P. falciparum isolates were successfully assembled. A total of 116 clones were found, 76 in the South and 40 in the North (Additional file 2). Among these clones 79 different alleles were identified of which 53 and 26 in the South and the North, respectively.
In the South, among the 53 different alleles detected, 35 belonged to the IC3D7-like while the other 18 were classified into the FC27-like (Table 1). IC3D7-like showed different dimorphic structures based on GA or SG amino acid dipeptides repeat units and poly-threonine repeats. Meanwhile, FC27-like varied with (ADTIASGSQSSTNSASTSTTNNGESQTTTPTA) or its variant amino acid succession. The neighbour-joining tree of parasites showed four clusters (Fig. 4.A).
In the North, of the detected 26 different alleles, 19 were classified into IC3D7-like and 7 belonged to FC27-like (Table 1). IC3D7-like varied with GA or SG amino acid dipeptides repeat units and poly-threonine repeat. FC27-like were different based on the amino acid succession (ADTIASGSQSSTNSASTSTTNNGESQTTTPTA) and the typical non-synonymous substitution of 6 amino acids (SSGNAP) was found at the C-terminal region of 5 of the 7 FC27-like identified. The neighbour-joining tree of different alleles revealed three clusters (Fig. 4B).
The observed genetic diversity was similarly elevated in both regions (Hd = 0.82 in the South and Hd = 0.843 in the North), and nucleotide diversity was also very similar (π = 0.48339 in the South and π = 0.42473 in the North). Non-directional selection was found with a highly positive TD test in the two areas, D = 5.21073 in the South and D = 3.46684 in the North with statistically different (p < 0.05). The Fst between the two localities showed an almost panmitic population (Fst = 0.02111) (Table 2).
Multiplicity Of Infection (moi)
The mean MOI for Pfmsp1, Pfmsp2 and both genes together in each area are shown in (Fig. 5). The mean MOI for Pfmsp1 was 1.5 in the South and 1.24 in the North, the number of clones per isolate ranged from 1 to 5 in these two regions and no statistically significant difference was observed between regions (p = 0.159). For Pfmsp2, mean MOI was significantly different between the South and the North (p = 0.010). The number of clones per samples ranged from 1 to 7 in the South with a MOI = 2.71 while in the North a MOI = 1.6 and the average number of clones varying between 1 and 4. Mean MOI for both genes was 3.07 and 1.76 in the South and North respectively with statistically significant differences between areas (p = 0.001).