General briefing of samples testing
Totally, 198 recruited volunteers were completed followed-up during 2012–2016 and 3 were lost after D7. In NW area, 8 patients (5.00%, 8/160) showed D3 positive, while 2.44% (1/41) in SW area (See the appendix).
K13 genotypes of 174 isolates including 169 with PCTs data and 5 without were successfully identified, in which 134 from NW area and 40 from SW area. Among the 169 isolates, copy numbers of pfM2 from 163 and pfmdr1 from 159 and STR data from 139 isolates were successfully detected and were applied in analysis.
Genotypes of K13 mutations
Among 174 parasite isolates which obtained K13 genotypes, 46.90% (99/174) present K13 mutations (See Table 1). All 99 isolates with positive K13 mutations were collected from NW area with a total K13 mutation rate of 73.88% (99/134) and F446I prevalence of 58.96% (79/134). Based on comparison, prevalence of K13 mutations in different years in NW area showed dramatic increases from 2012 (49.09%, 27/55) to 2013 (95.74%, 45/47) and 2014 (92.31%, 24/26). Most of the isolates in NW area were collected from Yingjiang county (59.20%, 103/134), which showed a K13 mutation prevalence of 85.44% (88/103). Forty isolates from SW area showed K13 wild genotype.
Table 1
Distribution of K13 genotypes in different years and different sites
K13 genotypes
|
2012
|
2013
|
2014
|
2015
|
2016
|
Sum
|
Yingjiang
|
Tengchong
|
Ruili
|
Yingjiang
|
Yingjiang
|
Menglian
|
Yingjiang
|
Tengchong
|
Yingjiang
|
C447S
|
1
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
1
|
F446I
|
9
|
6
|
1
|
35
|
24
|
0
|
1
|
1
|
1
|
79
|
N458Y
|
1
|
1
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
2
|
P574L
|
2
|
0
|
0
|
4
|
1
|
0
|
0
|
0
|
0
|
7
|
C447R
|
1
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
1
|
G495C
|
1
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
1
|
F483S
|
1
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
1
|
A676D
|
1
|
0
|
1
|
0
|
1
|
0
|
0
|
0
|
0
|
2
|
C580Y
|
2
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
2
|
C469Y
|
0
|
0
|
0
|
0
|
2
|
0
|
0
|
0
|
0
|
2
|
S466I
|
0
|
0
|
1
|
0
|
0
|
0
|
0
|
0
|
0
|
1
|
Wild
|
10
|
18
|
2
|
0
|
2
|
40
|
0
|
0
|
3
|
35
|
Total
|
29
|
25
|
5
|
39
|
30
|
40
|
1
|
1
|
4
|
174
|
The composition structure of all K13 mutations was shown in Fig. 2. A total of 11 mutation alleles were found, in which most of them were F446I (80%, 79/99) and followed by P574L (7%, 7/99). The other mutations alleles showed very low prevalence of 1–2%, such as C469Y (2%, 2/99), C580Y (2%, 2/99), N458Y (2%, 2/99). Most of the mutation alleles were found in Yingjiang County. In Tengchong county and Ruili city, only 2 K13 mutations alleles were detected in each site (Table 1).
Based on the K13 genotypes, 129 isolates with PCTs data in NW area were divided into 3 groups. In detail, 34 isolates with K13 wild genotype were classified as W group, 75 with F446I as F446I group, 20 with other K13 mutational genotypes as Others group. Forty isolates with K13 wild genotype in Menglian county in SW area were defined as ML group.
Comparison of PCT 50 based on K13 genotypes
The average H, PCT50 and PCT99 in NW area were 4.96 (4.46–5.46, N = 129), 12.85 (11.54–14.16, N = 129) and 34.22 (31.75–36.69, N = 129) hours, respectively. The PCT data of 4 groups are shown in Table 2. The average H value of W and F446I groups were 4.26 (3.06–5.47, N = 34) hours and 4.51 (3.87–5.14, N = 71) hours, respectively, while those of Others and ML groups were a little more than 5 hours. However, all of the average PCT99 were less than 49 hours.
Table 2
PCTs data based on K13 genotypes
|
|
H
|
PCT50
|
PCT75
|
PCT90
|
PCT95
|
PCT99
|
W Group
(n = 34)
|
Mean values
|
4.26
|
10.36
|
14.75
|
19.28
|
22.23
|
28.14
|
Lower 95% CI
|
3.06
|
7.69
|
11.41
|
15.41
|
18.09
|
23.59
|
Upper 95% CI
|
5.47
|
13.03
|
18.09
|
23.15
|
26.37
|
32.68
|
F446I Group
(n = 45)
|
Mean values
|
4.51
|
12.63
|
18.01
|
23.50
|
27.04
|
34.12
|
Lower 95% CI
|
3.87
|
10.94
|
15.84
|
20.89
|
24.17
|
30.71
|
Upper 95% CI
|
5.14
|
14.33
|
20.18
|
26.11
|
29.92
|
37.53
|
Others Group
(n = 20)
|
Mean values
|
5.72
|
13.98
|
19.66
|
25.62
|
29.57
|
37.75
|
Lower 95% CI
|
3.78
|
8.65
|
12.92
|
17.50
|
20.56
|
26.93
|
Upper 95% CI
|
7.66
|
19.31
|
26.40
|
33.74
|
38.57
|
48.58
|
ML Group
(n = 40)
|
Mean values
|
6.23*
|
15.41*
|
21.38*
|
27.42*
|
31.29**
|
38.86**
|
Lower 95% CI
|
5.02
|
12.02
|
17.28
|
22.66
|
26.15
|
33.07
|
Upper 95% CI
|
7.44
|
18.80
|
25.49
|
32.19
|
36.43
|
44.64
|
PCT: Parasites Clearance Time; |
*P ≤ 0.05 when compared with the value in W group. |
**P ≤ 0.01 when compared with the value in W group. |
The PCT 50 of different groups were compared and the results are shown in Fig. 3. The mean PCT 50 of 3 groups in NW area were 10.36 (7.69–13.03, N = 34) hours in W group, 12.63(10.94–14.3, N = 75) hours in F446I group, and 13.98 (8.65–19.31, N = 20) hours in Others group. No significant different PCT 50 presented between any two groups in this area (Chi-Square = 2.35, P = 0.31, df = 2). For the parasite isolates in ML group, their mean PCT 50 was 15.41 hours (12.02–18.80, N = 40) and significantly higher than those in W group and F446I group. (Z=-3.09, P = 0.002; Z=-2.24, P = 0.025).
Comparison of PCT 50 based on period
Details of K13 mutations of isolates collected in 2012 and 2013–2014 were shown in Table 3. Among parasites collected in 2012, 48.15% (26/54) showed K13 mutations and 57.69% (15/26) of the mutations were F446I. The total F446I prevalence were 27.78% (15/54) in 2012. For isolates in 2013 and 2014 (2013–2014 isolate), 95.65% (66/69) of them showed K13 mutation and 86.36% (57/66) of the mutations were F446I. The total F446I prevalence was 82.61% (57/69) in these 2-year period.
Table 3
K13 genotypes in different years
|
Total No. of Cases with PCT50 data
|
No. of Cases with K13 mutations
|
No. of Cases with F446I mutation
|
F446I prevalence
(%)
|
2012
|
54
|
26
|
15
|
27.78
|
2013–2014
|
69
|
66
|
57
|
82.61
|
The comparison of mean PCT 50 between isolates of 2012 (11.43, 9.44–13.41, N = 54) and 2013–2014 (13.61, 11.53–15.70, N = 69) in NW area showed no difference (P = 0.14, t=-1.49, df = 121).
Comparison of PCT 50 based on sites
The PCT 50 of parasite isolates in NW area (12.10 hours, 10.72–13.47 hours, N = 134) was significantly shorter than that in SW (ML group) area (P = 0.036, t=-2.11, df = 174). The mean PCT 50 of isolates in Tengchong and Yingjiang were 11.87 (9.31–14.43, N = 28) and 12.32 (10.69–13.95, N = 102) separately. However, no significant difference presented between each of these 3 surveillance sites (F = 2.11, P = 0.13). Data of those in Ruili site were not analyzed because only 6 cases were included.
Amplification of pfM2 and pfmdr1 gene
For the pfM2 gene, no multiple copies present in all isolates, though the mean copy number of isolates in NW area (0.95, 0.93–0.96, N = 126) was significantly lower than that in SW area (1.08, 1.00-1.16, N = 15) (Z=-3.36, P = 0.001) (See Fig. 4).
The tested copy numbers of pfmdr1 gene from 159 samples ranged from 0.97 to 2.15. Among them, 19.35% (6/31) isolates in W group, 14.63% (18/123) in NW area and 62.5% (10/16) in ML group contained multiple copies, with mean values of 1.69 (1.61–1.77, N = 6), 1.73 (1.64–1.81, N = 18) and 1.79 (1.69–1.88, N = 16) separately. The mean copy numbers of pfmdr1 gene based on K13 genotypes were also calculated and compared (see Fig. 5). They were 1.41 (1.34–1.48, N = 31) in W group, 1.37 (1.31–1.42, N = 69) in F446I group and 1.40 (1.34–1.47, N = 20) in Others group, with 19.35% (6/31), 13.04% (9/69) and 10% (2/20) isolates showing copy numbers of more than or equaling to 1.6 in each group. The average copy numbers were not significantly different between every two groups in NW area (F = 0.61, P = 0.55, df = 2), but they were significant lower than that in SW area (Z=-3.21, P = 0.001).
There was no significant difference on PCT 50 between all isolates with single and double copies of the gene (P = 0.50, t=-0.67, df = 137), neither between the W isolates (including both of W group and ML group) with single and double copies of the gene (P = 0.091, t = 1.73, df = 45).
Correlation coefficient (r value) between PCT 50 and copy numbers of pfmdr1 gene was estimated. However, no strong correlations were presented (r = 0.056, P = 0.52).
Reduced genetic He of the mutational K13 propeller allele and increased Fst between parasites of ML group and W group
He values of parasite isolates were calculated basing on STR data and grouped according to different K13 genotypes and areas were shown in Fig. 6. According to their mean He values, the four group were arranged as ML group, F446I group, Others group and W group from low to high. In ML group from SW area, He values of parasites at all loci sites was 0 (N = 8). In NW area, average He value of F446I isolates was 0.15 (0.014–0.28, N = 71) with each individual value of less than 0.30 at all loci except of one which was 0.52 and located at upstream − 56Kb of K13 gene. The He values in Others group fluctuated greatly from 0 to 0.65 with a mean value of 0.39 (0.20–0.58, n = 7). The mean He value in W group was the highest among 4 groups which was 0.65 (0.52–0.78, n = 34). All the mean Hes showed significant difference (Chi-Square = 25.20, df = 3, P = 0.000) with one another.
Fst values of parasite isolates were also calculated basing on STR data and grouped according to different K13 genotypes and areas. Nearly all Fst values at 9 different loci (See Table 4 [47]) between different groups were higher than 0.05, except of that at the site of -56Kb, which presented 0.048 between Others group and W group. The mean Fst values among 4 groups at 9 different loci (0.410 ± 0.067) were significantly higher than that among 3 NW groups (0.238 ± 0.041) (Paired-T Test: t=-3.220,df = 8༌P = 0.009). The highest difference between them presented at the sites of -0.15Kb (0.743 and 0.143) and 1.7Kb (0.474 and 0.213) which were closest to K13 genes among 9 loci along chromosome 13. The mean Fst values between W and each of other 3 groups at 9 loci were also showed in Table 4. The Fst values between ML and W groups were significantly higher than other 2 pairs (Paired-T Test: t=-2.659༌df = 8༌P = 0.029; t=-4.966, df = 8༌P = 0.001). However, Fst of F446I group and W group at site of 1.7 kb closing to K13 gene did not show lower value than that of ML group and W group.
Table 4
Fst values at different loci and between different groups
Loci
|
Among all 4 Groups
|
Among 3 NW Groups
|
F446I + W
|
Others + W
|
ML + W
|
-56.0Kb
|
0.240
|
0.149
|
0.082
|
0.048
|
0.190
|
-50Kb
|
0.237
|
0.179
|
0.170
|
0.051
|
0.189
|
-6.36Kb
|
0.552
|
0.417
|
0.152
|
0.260
|
0.468
|
-0.15Kb
|
0.743
|
0.143
|
0.112
|
0.112
|
0.562
|
1.7Kb
|
0.474
|
0.213
|
0.304
|
0.076
|
0.258
|
8.6Kb
|
0.542
|
0.432
|
0.311
|
0.198
|
0.387
|
11Kb
|
0.371
|
0.274
|
0.197
|
0.104
|
0.312
|
15.1Kb
|
0.087
|
0.077
|
0.057
|
0.063
|
0.063
|
31.5Kb
|
0.442
|
0.256
|
0.066
|
0.184
|
0.449
|
Mean values
|
0.410 d
|
0.238 ac
|
0.161 bc
|
0.122 b
|
0.320 a
|
SE
|
0.067
|
0.041
|
0.032
|
0.025
|
0.053
|
a, b, c, d: No difference present if the letters on up-right of mean values are same according to paired T-Test; Significant different if the letters are different. |
Differentiation Degree[40]: 0 ≦ Fst < 0.05, little genetic differentiation; |
0.05 ≦ Fst < 0.15, moderate differentiation; |
0.15 ≦ Fst < 0.25, great differentiation; |
0.25 ≦ Fst, very great genetic differentiation. |