Background
Methods
Study area
Patients and blood sample collection
Genetic characterization of Plasmodium falciparum isolates
Pfcrt single-nucleotide polymorphisms
Gene | Protocol step | Primers | Primers sequences |
---|---|---|---|
Pfdhfr
| PCR I | F | 5′-TTC TCC TTT TTA TGA TGG AAC AAG T-3′ |
R | 5′ ATA TTT GAA AAT CAT TTG GAT GTA TAG-3′ | ||
PCR II | F | 5′-GTT GAA CCT AAA CGT GCT GT-3′ | |
R | 5′-TTC ATC ATG TAA TTT TTG TTG TG-3′ | ||
Multiplex |
Pfdhfr51F
| 5′-AGG AGT ATT ACC ATG GAA ATG TA-3′ | |
Pfdhfr16R
| 5′-GAC TGA CTC TCA TTT TTG CTT TCA ACC TTA CAA CAT-3′ | ||
Pfdhfr108F
| 5′-GAC TGA CTA CAA AAT GTT GTA GTT ATG GGA AGA ACA A-3′ | ||
Pfdhfr164R
| 5′-CTG ACT GAC TGA CTG ACT AAT TCT TGA TAA ACA ACG GAA CCT CCT A-3′ | ||
Pfdhfr59R
| 5′-CTG ACT GAC TGA CTG ACT GAC TTG ATT CAT TCA CAT ATG TTG TAA CTG CAC-3′ | ||
Pfdhps
| PCR I | F | 5′-TGC TTA AAT GAT ATG ATA CCC GAA TAT AAG-3′ |
R | 5′ TCC ACC TGA AAA GAA ATA CAT AAA T-3′ | ||
PCR II | F | 5′-GTT GAA CCT AAA CGT GCT GT-3′ | |
R | 5′-TTC ATC ATG TAA TTT TTG TTG TG-3′ | ||
Multiplex |
Pfdhps613R
| 5′-TTG ATC ATT CAT GCA ATG GG-3′ | |
Pfdhps540F
| 5′-GAC TGA GGA AAT CCA CAT ACA ATG GAT-3′ | ||
Pfdhps581R
| 5′-TAA GAG TTT AAT AGA TTG ATC ATG TTT CTT C-3′ | ||
Pfdh436-1F
| 5′-GAC TGA CTA GTG TTA TAG ATA TAG GTG GAG AAT CC-3′ | ||
Pfdhps436-2R
| 5′-GAC TGA CTG ACT GAC TTG GAT TAG GTA TAA CAA AAA GGA ICA-3′ | ||
Pfdhps437R
| 5′-GAC TGA CTG ACT GAC TGA CTT TTT TGG ATT AGG TAT AAC AAA AGG A-3′ | ||
Pfmdr1
| PfMDR1- 1 | F | 5′-AGA GAA AAA AGA TGG TAA CCT CAG-3′ |
R | 5′-ACC ACA AAC ATA AAT TAA CGG-3′ | ||
PfMDR1- 2 | F | 5′-CAG GAA GCA TTT TAT AAT ATG CAT-3′ | |
R | 5′-CGT TTA ACA TCT TCC AAT GTT GCA-3′ | ||
Pfcrt
| PCR I | F | 5′-GTT CTT GTC TTG GTA AAT GT-3′ |
R | 5′- CGG ATG TTA CAA AAC TAT AGT T-3′ | ||
PCR II | F | 5′-GTT CTT GTC TTG GTA AAT GT-3′ | |
R |
6-FAM-5′- TAA ATG TGC TCA TGT GTT TA -3′ |
Pfmdr1 SNPs
Pfdhps and Pfdhfr SNPs
Pfmdr1 gene copy number
Statistical analysis
Results
Pfcrt polymorphisms
Gene | Codons | Aa | n (%) | |||
---|---|---|---|---|---|---|
Aioun | Kobeni | Tintane | Total | |||
Pfcrt
| K76T
| K | 12 (21) | 42 (31) | 33 (38) | 87 (31) |
K/T
| 8 (14) | 15 (11) | 10 (11) | 33 (12) | ||
T
| 36 (64) | 80 (58) | 44 (51) | 160 (57) | ||
Pfmdr1
| N86Y
| N | 35 (70) | 102 (77) | 66 (80) | 203 (77) |
Y
| 15 (30) | 30 (23) | 16 (20) | 61 (23) | ||
Y184F
| Y | 14 (33) | 46 (36) | 20 (26) | 80 (32) | |
F
| 29 (67) | 81 (64) | 57 (74) | 167 (68) | ||
S1034C
| S | 29 (100) | 67 (96) | 53 (98) | 149 (97) | |
C
| 0 | 3 (4) | 1 (2) | 4 (3) | ||
N1042D
| N | 29 (100) | 76 (99) | 49 (100) | 154 (99) | |
D
| 0 | 1 (1) | 0 | 1 (1) | ||
D1246Y | D | 37 (100) | 81 (98) | 70 (100) | 188 (99) | |
Y
| 0 | 2 (2) | 0 | 2 (1) |
Pfdhps and Pfdhfr polymorphisms
Gene | Codon | Aa | n (%) | |||
---|---|---|---|---|---|---|
Aioun | Kobeni | Tintane | Total | |||
Pfdhps
| S436A/F
| S | 17 (32) | 33 (26) | 32 (38) | 82 (31) |
S/A
| 10 (19) | 16 (13) | 10 (12) | 36 (14) | ||
A
| 26 (49) | 75 (60) | 43 (51) | 144 (55) | ||
F
| 0 | 2 (2) | 0 | 2 (1) | ||
A437G
| A | 33 (62) | 96 (76) | 43 (51) | 172 (65) | |
A/G
| 10 (19) | 12 (10) | 9 (11) | 31 (12) | ||
G
| 10 (19) | 18 (14) | 32 (38) | 60 (23) | ||
K540E/A
| K | 52 (98) | 122 (97) | 85 (100) | 259 (98) | |
K/A
| 0 | 1 (0.8) | 0 | 1 (0.4) | ||
K/E
| 0 | 2 (2) | 0 | 2 (1) | ||
E
| 1 (2) | 1 (1) | 0 | 2 (1) | ||
Pfdhfr
| N51I
| N | 31 (58) | 73 (55) | 39 (47) | 143 (53) |
N/I
| 10 (19) | 16 (12) | 11 (13) | 37 (14) | ||
I
| 12 (23) | 44 (33) | 33 (40) | 89 (33) | ||
C59R
| C | 32 (60) | 68 (51) | 40 (48) | 140 (52) | |
C/R
| 9 (17) | 18 (14) | 9 (11) | 36 (13) | ||
R
| 12 (23) | 47 (35) | 34 (41) | 93 (35) | ||
S108N/T
| S | 31 (58) | 68 (52) | 42 (51) | 141 (53) | |
S/T
| 1 (2) | 3 (2) | 1 (1) | 5 (2) | ||
S/N
| 7 (13) | 14 (11) | 6 (7) | 27 (10) | ||
T/N
| 2 (4) | 1 (1) | 6 (7) | 9 (3) | ||
T
| 2 (4) | 0 | 1 (1) | 3 (1) | ||
N
| 10 (19) | 46 (35) | 27 (33) | 83 (31) | ||
I164L
| I | 52 (98) | 131 (99) | 73 (88) | 256 (96) | |
I/L
| 1 (2) | 1 (1) | 10 (12) | 12 (4) |
Haplotypes | n (%) | |||
---|---|---|---|---|
Aioun | Kobeni | Tintane | Total | |
SAKAAa
| 10 (19) | 18 (14) | 6 (7) | 34 (13) |
AA/GKAA | 2 (4) | 2 (2) | 3 (4) | 7 (3) |
AAKAA | 22 (42) | 69 (55) | 36 (42) | 127 (48) |
AGKAA | 2 (4) | 4 (3) | 4 (5) | 10 (4) |
FAKAA | 0 | 2 (2) | 0 | 2 (1) |
S/AA/GKAA | 6 (11) | 9 (7) | 4 (5) | 19 (7) |
S/AAK/EAA | 0 | 1 (1) | 0 | 1 (0.4) |
S/AAKAA | 1 (2) | 6 (5) | 1 (1) | 8 (3) |
SA/GKAA | 2 (4) | 1 (1) | 2 (2) | 5 (2) |
SGEAA | 1 (2) | 0 | 0 | 1 (0.4) |
SGK/AAA | 0 | 1 (1) | 0 | 1 (0.4) |
SGK/EAA | 0 | 1 (1) | 0 | 1 (0.4) |
SGKAA | 4 (8) | 11 (9) | 23 (27) | 38 (14) |
Total | 53 (100) | 126 (100) | 85 (100) | 264 (100) |
Haplotype | n (%) | |||
---|---|---|---|---|
Aioun | Kobeni | Tintane | Total | |
ANCSIa
| 28 (53) | 64 (48) | 35 (42) | 127 (47) |
ANCS/TI | 1 (2) | 2 (2) | 0 | 3 (1) |
ANC/RSI | 0 | 1 (1) | 0 | 1 (0.4) |
ANC/RS/NI | 0 | 3 (2) | 1 (1) | 4 (1) |
ANCSI/L
| 1 (2) | 0 | 2 (2) | 3 (1) |
AN/IC/RS/NI | 6 (11) | 11 (8) | 4 (5) | 21 (8) |
AN/IC/RS/NI/L
| 0 | 0 | 1 (1) | 1 (0.4) |
AN/IC/RS/TI | 0 | 1 (1) | 0 | 1 (0.4) |
AN/IC/RSI | 1 (2) | 2 (2) | 2 (2) | 5 (2) |
AN/IC/RSI/L
| 0 | 0 | 1 (1) | 1 (0.4) |
AN/ICS/TI/L
| 0 | 0 | 1 (1) | 1 (0.4) |
AN/ICSI | 1 (2) | 1 (1) | 0 | 2 (1) |
AN/ICSI/L
| 0 | 0 | 2 (2) | 2 (1) |
AN/IRNI | 2 (4) | 1 (1) | 0 | 3 (1) |
ANRNI | 1 (2) | 3 (2) | 1 (1) | 5 (2) |
AIC/RS/NI | 1 (2) | 0 | 0 | 1 (0.4) |
AIC/RT/NI | 1 (2) | 0 | 0 | 1 (0.4) |
AICNI | 1 (2) | 1 (1) | 0 | 2 (1) |
AIRNI | 6 (11) | 41 (31) | 25 (30) | 72 (27) |
AIRNI/L
| 0 | 0 | 1 (1) | 1 (0.4) |
AIRT/NI | 1 (2) | 0 | 4 (5) | 5 (2) |
AIRT/NI/L
| 0 | 1 (1) | 2 (2) | 3 (1) |
AIRTI | 2 (4) | 0 | 1 (1) | 3 (1) |
Total | 53 (100) | 133 (100) | 83 (100) | 269 (100) |
Pfmdr1 polymorphisms
Pfmdr1 copy number
Number of copies | n (%) | |||
---|---|---|---|---|
Aioun | Kobeni | Tintane | Total | |
One | 6 (100) | 78 (88.6) | 8 (100) | 92 (90.2) |
Two | 0 | 9 (10.3) | 0 | 9 (8.8) |
Three | 0 | 1 (1.1) | 0 | 1 (1.0) |