Background
Methods
Study site and sample collection
Plasmodium species identification, and Pfcrt, Pfmdr1, Pfatp6, Pfk13 genotyping
Genus and species | Primer name | Primer sequence (5′-3′) | PCR conditions | Reference |
---|---|---|---|---|
Plasmodium genus
| rPLU 1 rPLU 5 | TCAAAGATTAAGCCATGCAAGTGA CCTGTTGTTGCCTTAAACTTC | 95 °C for 5 min; 25× [94 °C for 1 min; 58 °C for 1 min; and 72 °C for 2 min]; 72 °C for 5 min | Snounou et al. [22] |
P. falciparum
| rFAL1 rFAL2 | TTAAACTGGTTTGGGAAAACCAAATATATT ACACAATGAACTCAATCATGACTACCCGTC | 95 °C for 5 min; 30× [94 °Cfor 1 min; 58 °C for 1 min; and 72 °C for 2 min]; 72 °C for 5 min | |
P. malariae
| rMAL1 rMAL2 | ATAACATAGTTGTACGTTAAGAATAACCGC AAAATTCCCATGCATAAAAAATTATACAAA | ||
P. vivax
| rVIV1 rVIV2 | CGACTTCCAAGCCGAAGCAAAGAAAG TCCTTACTTCTAGCTTAATCCACATAACTGATAC | ||
P. ovale
| rOVA1WC rOVA2WC | TGTAGTATTCAAACGCAGT TATGTACTTGTTAAGCCTTT | Fuehrer et al. [21] |
Loci | Primer name | Primer (5′-3′) | PCR conditions | Reference |
---|---|---|---|---|
Pfcrt SNPs at codons 72 and 76 | OF P1 OR P2 | 5′-CCGTTAATAATAAATACACGCG-3′ 5′-CGGATGTTACAAAACTATAGTCC-3′ | 35 cycles of 94 °C for 30 s; 56 °C for 30 s; and 62 °C for 1 min | Mekonnen et al. [11] |
NF P3 NR P4 | 5′-AGGTTCTTGTCTTGGTAAATTTGC-3′ 5′-CAAAACTATAGTTACCAATTTTG-3′ | 30 cycles of 94 °C for 30 s; 56 °C for 30 s; and 65 °C for 1 min | ||
Pfmdr1 SNPs at codon 86 and 184 | OF P5 OR P6 | 5′-AGGTTGAAAAAGAGTTGAAC-3′ 5′-ATGACACCACAAACATAAAT-3′ | 30 cycles of 94 °C for 30 s; 55 °C for 30 s; and 65 °C for 1 min | |
NF P7 NR P8 | 5′-ACAAAAAGAGTACCGCTGAAT-3′ 5′-AAACGCAAGTAATACATAAAGTC-3′ | 30 cycles of 94 °C 30 s; 60 °C for 30 s; and 65 °C for 1 min | ||
Pfmdr1 SNPs at codon 1034,1024, and 1246 | OF P9 OR P10 | 5′-GTGTATTTGCTGTAAGAGCT-3′ 5′-GACATATTAAATAACATGGGTTC-3′ | 34 cycles of 94 °C for 30 s; 55 °C for 1 min and 72 °C for 2 min | |
NF P11 NR 12 | 5′-CAGATGATGAAATGTTTAAAGATC-3′ 5′-TAAATAACATGGGTTCTTGACT-3′ | 29 cycles of 94 °C for 30 s; 60 °C for 30 s; and 65 °C for 1 min | ||
Pfatp6 at codon 243, 263 and 431 | NF P13 NR P14 | 5′-TCATCTACCGCTATTGTATG-3′ 5′-TCCTCTTAGCACCACTCC-3′ | 35 cycles of 94 °C for 45 s; 55 °C for 1 min; 72 °C for 1 min | Zakeri et al. [24] |
Pfatp6 SNPs at codon 623 and 769 | NF P15 NR P16 | 5′-TGGAGACAGTACCGAATTAGC-3′ 5′-TCTTCCTACATATTTACGTGGTG-3′ | ||
Pfk13 | OF P17 OR P18 | 5′-CGGAGTGACCAAATCTGGGA-3′ 5′-GGGAATCTGGTGGTAACAGC-3′ | 30 cycles of 95 °C for 30 s; 58 °C for 2 min; 72 °C for 2 min | Ariey et al. [16] |
NF P19 NR P20 | 5′-GCCAAGCTGCCATTCATTTG-3′ 5′-GCCTTGTTGAAAGAAGCAGA-3′ | 40 cycles of 95 °C for 30 s; 60 °C for 1 min; 72 °C for 1 min |
Results
Plasmodium species identification
Pfcrt polymorphisms
Locus | Mutation | Cohort 2010 (%) | Cohort 2015 (%) |
---|---|---|---|
Pfcrt
| M74I, N75E and K76T | 39/40 (98) | 20/28 (71) |
Pfmdr1
| N86Y | 30/41 (73) | 7/26 (27) |
S113R | 1/41 (2) | 0/26 (0) | |
Y184F | 20/41 (49) | 14/26 (54) | |
T1069T | 8/41 (20) | 3/26 (12) | |
G1113G | 0/41 (0) | 1/26 (4) | |
D1127D | 1/41 (2) | 0/26 (0) | |
T1157T | 2/41 (5) | 2/26 (8) | |
V1225A | 1/41 (2) | 0/26 (0) | |
D1246Y | 9/41 (22) | 0/26 (0) |
Pfmdr1 polymorphisms
Pfatp6 polymorphisms
Locus | Mutation | Cohort 2010 (%) | Cohort 2015 (%) |
---|---|---|---|
Pfatp6
| H243Y | 2/8 (25) | 0/16 (0) |
I291V | 0/8 (0) | 1/16 (6) | |
L402V | 1/8 (12) | 2/16 (12) | |
E431K | 1/8 (12) | 2/16 (12) | |
N569K | 5/8 (63) | 2/16 (12) | |
A630S | 3/8 (38) | 0/16 (0) | |
G632E | 0/8 (0) | 1/16 (6) | |
G639D | 0/8 (0) | 1/16 (6) | |
F646F | 1/8 (12) | 0/16 (0) | |
H747Y | 0/8 (0) | 1/16 (6) | |
Pfk13
| D464N | 1/41 (2) | 0/25 (0) |
S466I | 0/41 (0) | 1/25 (4) | |
Q467H | 0/41 (0) | 1/25 (4) | |
N489N | 0/41 (0) | 1/25 (4) | |
G496G | 0/41 (0) | 1/25 (4) | |
R539R | 1/41 (2) | 0/25 (0) | |
S549P | 1/41 (2) | 0/25 (0) | |
I552M | 0/41 (0) | 1/25 (4) | |
L598L | 1/41 (2) | 0/25 (0) | |
E602E | 1/41 (2) | 0/25 (0) | |
N609N | 0/41 (0) | 1/25 (4) | |
F628L | 0/41 (0) | 1/25 (4) | |
K669K | 1/41 (2) | 0/25 (0) |