Background
Methods
Study sites and parasite isolates
DNA extraction
Amplification by polymerase chain reaction
PCR primers named | PCR primers sequence 5' to 3' | Target gene | Amplicons size (bp) | GenBank accession number of reference sequences | |
---|---|---|---|---|---|
Nucleotide | Protein | ||||
PfHRP2-R | TTAATGGCGTAGGCAATGTG | P. falciparum histidine-rich protein II gene, exon 2 | 905 | M13986 | AAA51639 |
PfHRP2-F | TGTGTAGCAAAAATGCAAAAGG | ||||
HRP3-R1 | TGGTGTAAGTGATGCGTAGT | P. falciparum histidine-rich protein III gene | 546 | AF202093 | AAF14632 |
HRP3-F2 | AAATAAGAGATTATTACACGAAAG | ||||
Pf aldolase R1 | TTTCCTTGCCATGTGTTCAA | P. falciparum aldolase gene, exon 2 | 924 | J03084 | AAA29716 |
Pf aldolase F1 | AGCAGATGTTGCCGAAGAAT | ||||
PfLDH-R1 | TTTCAGCTATGGCTTCATCAAA | P. falciparum lactate dehydrogenase gene | 922 | DQ825436 | ABH03417 |
PfLDH-F1 | GCACCAAAAGCAAAAATCGT | ||||
Pvald PF | CCACTGGATCCGAATATAAA | P. vivax fructose 1,6-bisphosphate aldolase gene | 1091 | AF247063 | AAK43741 |
Pvald PR | CTTTTCGTAAAGGGATGCT | ||||
Pvald NF | CCTCAAACTACCCGCAGAAG | 994 | |||
Pvald NR | GGAGTTCGCTTCTGCTCTCT | ||||
Pvldh PF | GTAGAGGCAGGTGAACACTC | P. vivax L-lactate dehydrogenase gene | 1063 | DQ060151 | AAY59419 |
Pvldh PR | TCCTTTTAGTCTCCGCAAC | ||||
Pvldh NF | AACCCAAAATTGTGCTCGTC | 900 | |||
Pvldh NR | CGTCGAACTTGGTCTTCTCC |
Direct sequencing of PCR products
Data analysis
Statistical analysis
Results
Polymorphisms in PfHRP2
No. of amino acid repeats in PfHRP2 and PfHRP3 | Region and City | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
North | Centre | South | Total | ||||||||||||||
ATS | SBV | ATH | MJG | MAE | STM | TDD | MIA | MHT | MOR | FNR | MNK | IHO | TLR | EJE | |||
1
|
AHHAHHVAD
|
1–5
|
1–5
|
1–6
|
1–5
|
1–5
|
2
|
1–5
|
1–4
|
1–3
|
1–4
|
2–4
|
3
|
1–4
|
1–4
|
1–4
|
1–6
|
1
|
AHHAHHVAD
|
1
|
1–2
|
1–3
|
1–3
|
1
|
1
|
1–3
|
1–3
|
1–2
|
1
|
1–2
|
1
|
1–4
|
1
|
1
|
1–4
|
2
|
AHHAHHAAD
|
8–16
|
11–13
|
10–14
|
4–15
|
8–14
|
14
|
10–14
|
11–13
|
11–12
|
10–13
|
8–15
|
12
|
8–14
|
7–14
|
7–15
|
4–16
|
3
|
AHHAHHAAY
|
0–2
|
1–2
|
1–2
|
0–2
|
0–2
|
0
|
1–2
|
1–2
|
1–2
|
1–2
|
1–2
|
0
|
1–2
|
1–3
|
0–3
|
0–3
|
4
|
AHH
|
0–1
|
0–1
|
0–1
|
0–3
|
0–2
|
0
|
0–1
|
0–1
|
0–1
|
0–1
|
0
|
1
|
0
|
0–1
|
0–1
|
0–3
|
4
|
AHH
|
1
|
1
|
1–2
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1–2
|
5
|
AHHAHHASD
|
0–1
|
1
|
0–3
|
0–1
|
0–1
|
1
|
0–2
|
0–2
|
1
|
0–1
|
0–3
|
0
|
0–2
|
1–2
|
0–2
|
0–3
|
6
|
AHHATD
|
3–5
|
3–5
|
2–5
|
2–7
|
2–6
|
2
|
2–8
|
2–6
|
3–5
|
3–8
|
1–3
|
3
|
1–3
|
2–4
|
2–5
|
1–8
|
7
|
AHHAAD
|
2–10
|
4–7
|
4–11
|
3–8
|
4–8
|
6
|
3–9
|
3–8
|
5–8
|
3–10
|
4–13
|
6
|
5–10
|
4–9
|
3–8
|
2–13
|
7
|
AHHAAD
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
0–1
|
1
|
0–1
|
8
|
AHHAAY
|
1–2
|
1–2
|
1–2
|
1–2
|
1–2
|
1
|
0–2
|
0–2
|
1–2
|
0–2
|
0–2
|
3
|
0–2
|
1–2
|
1–2
|
0–2
|
9
|
AAY
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
10
|
AHHAAAHHATD
|
0–2
|
0–2
|
1–3
|
1–3
|
1–2
|
2
|
0–2
|
0–2
|
2–3
|
1–2
|
0–2
|
1
|
1–2
|
0–2
|
0–2
|
0–3
|
11
|
AHN
|
0
|
0
|
0
|
0–1
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0–1
|
12
|
AHHAAAHHEAATH
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
13
|
AHHASD
|
0–1
|
0–2
|
0–1
|
0
|
0–1
|
0
|
0–1
|
0–1
|
0
|
0–1
|
0
|
0
|
0–1
|
0
|
0–1
|
0–2
|
14
|
AHHAHHATD
|
0–2
|
0
|
0–1
|
0–1
|
0–1
|
0
|
0–1
|
0–1
|
0–1
|
0–1
|
0
|
0
|
0–1
|
0–1
|
0–1
|
0–2
|
15
|
AHHAAY
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
0–1
|
1
|
1
|
1
|
1
|
1
|
1
|
1
|
0–1
|
16
|
AAY
|
8–14
|
8–13
|
7–13
|
9–13
|
8–18
|
10–14
|
10–14
|
10–16
|
8–14
|
8–15
|
8–14
|
15
|
10–14
|
8–15
|
7–14
|
7–18
|
17
|
AHHAAAHHATD
|
1–8
|
5–9
|
5–8
|
4–7
|
5–8
|
4–8
|
5–7
|
3–7
|
5–10
|
0–7
|
4–10
|
6
|
4–8
|
4–6
|
3–8
|
0–10
|
18
|
AHN
|
1–3
|
1–4
|
1–3
|
1–4
|
1–3
|
2–3
|
1–3
|
1–3
|
2
|
0–3
|
2
|
2
|
1–2
|
1–3
|
2–3
|
0–4
|
PfHRP2 sequences | PfHRP3 sequences | |||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Type of repeats | Total (n = 229) | North (n = 90) | Centre (n = 81) | South (n = 58) |
P
| Total (n = 237) | North (n = 90) | Centre (n = 84) | South (n = 63) |
P
| ||||||||
mean | SD | mean | SD | mean | SD | mean | SD | mean | SD | mean | SD | mean | SD | mean | SD | |||
1
|
2.6
|
1.2
| 2.7 | 1.4 | 2.3 | 1.0 | 2.66 | 1.17 | 0.58 |
1.1
|
0.4
| 1.1 | 0.4 | 1.1 | 0.4 | 1.2 | 0.5 | 0.70 |
2
|
12.0
|
1.8
| 12.1 | 2.1 | 12.1 | 1.1 | 11.5 | 2.3 | 0.81 | |||||||||
3
|
1.4
|
0.6
| 1.2 | 0.7 | 1.5 | 0.5 | 1.5 | 0.6 | 0.15 | |||||||||
4
|
0.3
|
0.5
| 0.3 | 0.6 | 0.3 | 0.5 | 0.1 | 0.4 | 0.47 |
1.0
|
0.1
| 1.0 | 0.1 | 1.0 | 0 | 1.0 | 0 | 0.36 |
5
|
0.9
|
0.6
| 0.8 | 0.5 | 0.9 | 0.4 | 1.3 | 0.7 | 0.03 | |||||||||
6
|
3.4
|
1;2
| 3.5 | 1.1 | 3.8 | 1.4 | 2.7 | 0.8 | 0.01 | |||||||||
7
|
6.0
|
2.0
| 6.0 | 1.9 | 6.1 | 1.7 | 6.2 | 2.3 | 0.73 |
1.0
|
0.1
| 1.0 | 0 | 1.0 | 0 | 0.98 | 0.14 | 0.31 |
8
|
1.1
|
0.5
| 1.3 | 0.4 | 1.1 | 0.4 | 1.3 | 0.6 | 0.23 | |||||||||
10
|
1.5
|
0.8
| 1.6 | 0.7 | 1.6 | 0.8 | 1.4 | 0.8 | 0.71 | |||||||||
13
|
0.3
|
0.9
| 0.2 | 0.4 | 0.04 | 0.2 | 0,0 | 0,0 | 0.08 | |||||||||
14
|
0.1
|
0.3
| 0.1 | 0.4 | 0.1 | 0.3 | 0.1 | 0.3 | 0.8 | |||||||||
15
|
1.0
|
0.1
| 1.0 | 0 | 1.0 | 0.1 | 1.0 | 0 | 0.43 | |||||||||
16
|
11.4
|
1.8
| 11.2 | 1.5 | 11.5 | 1.9 | 11.4 | 1.8 | 0.73 | |||||||||
17
|
5.8
|
1.3
| 5.7 | 1.3 | 5.7 | 1.3 | 6.0 | 1.3 | 0.68 | |||||||||
18
|
2.1
|
0.5
| 2.1 | 0.6 | 2.1 | 0.5 | 2.0 | 0.4 | 0.61 |
Region of Madagascar | No. of type 2 × type 7 repeats goup (%) | ||||
---|---|---|---|---|---|
Group A | Group B | "Borderline" Group | Group C | Total | |
North
| 11 (12) | 65 (72) | 9 (10) | 5 (6) | 90 |
Centre
| 4 (5) | 57 (70) | 12 (15) | 8 (10) | 81 |
South
| 7 (12) | 36 (62) | 7 (12) | 8 (14) | 58 |
Total
| 22 (10) | 158 (69) | 28 (12) | 21 (9) | 229 |
Polymorphisms in the PfHRP3 gene
Polymorphisms in the aldolase gene
Genes | SNP (bp position and change) | Amino acid change (aa position and change) | Frequency No (%) |
---|---|---|---|
P. falciparum
aldolase
| 815 (CAC to CGC) | 104 (H to R) | 2 (0.8) |
1314 (CCA to CCC) | synonymous change | 1 (0.4) | |
1404 (CAC to CAG) | 300 (H to Q) | 2 (0.8) | |
1417 (ACC to GCC) | 305 (T to A) | 1 (0.4) | |
P. vivax
aldolase
| 510 (TCC to TCA) | synonymous change | 1 (0.9) |
651 (TTA to TTG) | synonymous change | 43 (39.1) | |
P. falciparum LDH
| 73 (CAG to AAG) | 25 (Q to K) | 1 (0.7) |
814 (GAT to AAT) | 272 (D to N) | 10 (7.3) | |
P. vivax LDH
| 168 (ATG to ATA) | 56 (D to N) | 1 (0.9) |
225 (AAG to AAA) | synonymous change | 84 (75.7) | |
405 (ATC to ATT) | synonymous change | 2 (1.8) | |
498 (GTC to GTT) | synonymous change | 1 (0.9) | |
544 (GTT to ATT) | 182 (V to I) | 1 (0.9) | |
627 (GTG to GTC) | synonymous change | 32 (28.8) | |
687 (GCC to GCT) | synonymous change | 23 (20.7) |