DOI: https://doi.org/10.21203/rs.3.rs-45491/v1
Background
The therapeutic efficacy study (TES) of Dihydroartemisinin-Piperaquine (DHA-PIP) for uncomplicated P.falciparum patients had implemented during 2012-2016 along China (Yunnan province) -Myanmar border. The study focusing on the genetic features of falciparum parasites based on in vivo parasite clearance time (PCT) was investigated to explore if they had produced potential resistance to DHA and PIP at molecular level.
Methods
The genetic features were invested based on K13 propeller genotypes, Copy numbers of genes PfPM2 and Pfmdr1 and 9 microsatellite loci (Short Tandem Repeats, STR) flanking the K13 gene on chromosome 13. The (PCTs), copy numbers, expected heterozygosity ( He ), coefficient of gene differentiation (F st ) were calculated, compared and analyzed in Graph Prim5.0 and SPSS 23 with method of One-way Anova (Post Hoc test:LSD) or Nonparametric tests. Genetic diversity ( He and F st ) of parasites was estimated in Excel 2016 with method of GenAlEx 6.501.
Results
In NW (North-West Yunnan province bordering with Myanmar) area, F446I prevalence was 58.96% (79/134) and no significant different PCT50 presented between any two K13 groups (Chi-Square=2.35, P=0.31, df=2). But the PCT50 of parasite isolates in this area (12.10, 10.72-13.47, n=136 hrs) was significantly shorter than that in SW (South-West Yunnan province bordering with Myanmar) area (P=0.036, t=-2.11, df=174) where isolates all showed wild K13 genotype. For the copy numbers of Pfmdr1 gene, 14.63% (18/123) and 62.5% (10/16) parasite isolates showed amplification (≥ 1.6) in NW and SW areas, but for those of PfM2 gene, none of them did. Based on STR data, He values of parasite isolates of 4 groups lined as ML group (Menglian County in SW), F446I group, Others (Non-F446I K13 mutation) group and W (wild K13 genotype) group from low to high. All of them showed significant with one another (Chi-Square=25.20, df=3, P=0.000). The mean F st value of 4 groups at 9 different loci (0.410±0.067) were significant higher than that of 3 NW groups (0.238±0.041) (Paired-T Test: t=-3.220,df=8,P=0.009). Highest mean F st (0.320±0.053) presented between ML group and W group when all mean F st values between W group and each of other 3 groups were compared.
Conclusions
According to our study, we are inclined to conclude that no confirmed resistance of P.falciparum parasites to DHA and PIP occurred, although parasite isolates with prolonged PCTs were observed. Potentially resistant parasite isolates are widely spread along the Yunnan-Myanmar border based on the genetic index of K13, PfM2/3 and Pfmdr1, along with great genetic differentiation based on different areas and K13 mutations. F446I is the domain K13 mutation allele in NW area and seem to have emerged newly in those years.
Trial registration
ISRCTN, ISRCTN 11775446. Registered 13 April 2020 - Retrospectively registered, http://www.isrctn.com/ISRCTN11775446.
This preprint is available for download as a PDF.
Table 1 K13 mutations in different sites in different years
|
year |
Sites |
K13 test |
Successful K13 Test |
||
|
Wild |
Mutations |
Total |
|||
|
2012 |
Tengchong |
28 |
18 |
8 |
26 |
|
Yingjiang |
29 |
10 |
19 |
29 |
|
|
2013 |
Ruili |
7 |
2 |
2 |
4 |
|
Yingjiang |
54 |
0 |
43 |
43 |
|
|
2014 |
Menglian* |
40 |
40 |
0 |
40 |
|
Yingjiang |
26 |
2 |
24 |
26 |
|
|
2015 |
Tengchong |
1 |
0 |
1 |
1 |
|
Yingjiang |
1 |
0 |
1 |
1 |
|
|
2016 |
Yingjiang |
4 |
3 |
1 |
4 |
|
Total |
180 |
75 |
99 |
174 |
|
*There was an outbreak of P.falciparum malaria there in that year.
Table 2 K13 genotypes in different sites
|
|
Yingjiang |
Tengchong |
Ruili |
Total |
|
C447S |
1 |
0 |
0 |
1 |
|
F446I |
70 |
8 |
1 |
79 |
|
N458Y |
1 |
1 |
0 |
2 |
|
P574L |
7 |
0 |
0 |
7 |
|
C447R |
1 |
0 |
0 |
1 |
|
G495C |
1 |
0 |
0 |
1 |
|
F483S |
1 |
0 |
0 |
1 |
|
A676D |
2 |
0 |
0 |
2 |
|
C580Y |
2 |
0 |
0 |
2 |
|
C469Y |
2 |
0 |
0 |
2 |
|
S466I |
0 |
0 |
1 |
1 |
|
Total |
88 |
9 |
2 |
99 |
Table 3 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.
Table 4 K13 genotypes in different years
|
|
Total No. of Cases with CFUD |
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 |
Table 5 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.