Demographics of P. falciparum infection
A total of 425 P. falciparum blood samples were involved in this study. Among all the participants, 418 (98.4%) were male. The patients were mainly found in the age group of 40–50 years old (n = 182, 42.8%) and the median age was 42 years old. The samples were mainly collected in Huancui (n = 21, 4.9%), Feicheng (n = 20, 4.7%), and Zhifu (n = 20, 4.7%) (Fig. 1). The most common occupation was farmers (n = 196, 46.1%) (Table 1). The imported P. falciparum cases were mainly reported in January (n = 52, 12.2%) and October (n = 50, 11.8%). The median (range) cases reported per month was 35 (20–52). The median interval from onset to confirmed diagnosis was 3.4 d, and the median interval from confirmed diagnosis to report was 1 d, respectively. A total of 5 deaths attributing to the P. falciparum were reported in the timeframe but no mutation alleles were detected in pfK13 and pfcrt. The P. falciparum cases were mainly imported from Nigeria (n = 88, 20.7%), Equatorial Guinea (n = 63, 14.8%), and the Democratic Republic of the Congo (n = 61, 14.4%).
Table 1
Characteristics of study participants with P. falciparum by study sites in Shandong Province, 2015–2019.
General
|
Participants
|
Proportion (%)
|
Sex
|
|
|
Male
|
418
|
98.4
|
Female
|
7
|
1.6
|
Age
|
|
|
< 20
|
2
|
0.5
|
20–30
|
73
|
17.2
|
30–40
|
102
|
24.0
|
40–50
|
183
|
43.1
|
50–60
|
59
|
13.9
|
60–70
|
5
|
1.2
|
> 70
|
1
|
0.2
|
Occupation
|
|
|
Farmer
|
196
|
46.1
|
Worker
|
117
|
27.5
|
House servant
|
30
|
7.1
|
Business
|
16
|
3.8
|
Students
|
3
|
0.7
|
Others
|
63
|
14.8
|
Molecular analysis of pfK13 polymorphisms
Out of all successful sequenced 425 P. falciparum isolates, a total of 31 (7.3%) isolates harboring the pfK13 polymorphisms were identified in the returners. Among them, 17 (54.8%) were nonsynonymous polymorphisms, whereas the other 14 (45.2%) were synonymous polymorphisms, the ratio of nonsynonymous isolates to synonymous isolates was 1.2. The mutant allele A578S, Q613H, C469C, and S549S were the more frequently detected allele, and the prevalence was all the same as 0.7% (3/425). (Table 2). The pfK13 mutation rate was found as 8.9% (4/45), 8.1% (5/62), 10.6% (10/94), 6.3% (7/111), and 4.4% (5/113) in 2015–2019, respectively (Fig. 2). Refer to the geographical distribution of pfK13 alleles, Western Africa was the most reported region (n = 14) with Nigeria (n = 8), Guinea (n = 4) and Côte d'Ivoire (n = 2) (Fig. 2). In addition, 2 isolates of C580Y were identified among the returners from Cambodia back to Shandong (Fig. 3).
Table 2
Nonsynonymous and synonymous SNPs in the pfK13 in Shandong Province, 2015–2019.
Type of polymorphisms
|
Genotype
|
Subtotal
|
Amino acid reference
|
Nucleotide reference
|
Amino acid mutation
|
Nucleotide mutationa
|
Prevalence (%)
|
Nonsynonymous (n = 17)b
|
F439S
|
1
|
F
|
TTT
|
S
|
TCT
|
0.2
|
F442 L
|
2
|
F
|
TTC
|
L
|
TTA
|
0.5
|
P443 A
|
1
|
P
|
CCA
|
A
|
GCA
|
0.2
|
C473 S
|
1
|
C
|
TGT
|
S
|
TCT
|
0.2
|
A578 S
|
3
|
A
|
GCT
|
S
|
TCT
|
0.7
|
C580 Y c
|
2
|
C
|
TGT
|
Y
|
TAT
|
0.5
|
S588 C
|
1
|
S
|
AGT
|
C
|
TGT
|
0.2
|
Q613 H
|
3
|
Q
|
CAA
|
H
|
CAT
|
0.7
|
S626 R
|
2
|
S
|
AGC
|
R
|
AGA
|
0.5
|
G665 S
|
1
|
G
|
GGC
|
G
|
GGA
|
0.2
|
a Mutations are in boldface. |
b The total samples for tested was 425, while 31 of them harboring the pfK13 polymorphisms, and 17 were nonsynonymous polymorphisms. |
c Those mutations have been validated as molecular markers associated with in vitro ACT resistance. |
Mutation prevalence of pfcrt polymorphisms
A total of 13 different haplotypes were identified in 77 isolates (18.1%), including T76T356 found in 20 isolates (4.7%), and T76 in 18 isolates (4.2%) (Table 3). Refer to the codon 72–76, we have identified the mutation alleles with CVIET and CVIKT exhibited a prevalence of 3.5% and 0.7%, respectively (Table 3). The CVIET were mainly distributed in Congo (5.2%, 4/77) and Mozambique (5.2%, 4/77). In addition, one isolate carrying S93 was identified in Cambodia in 2015, and no mutations was found at loci 97, 101 and 145. For the mutation alleles at 323–355, we have identified one isolate harbouring S323R334 in Ethiopia, and 2 isolates carrying T355 and T76T355, all were from Nigeria. For polymorphisms at locus 356, a total of 24 isolates were identified and most of them were from Congo (n = 7, 29.2%). The geographical distribution of pfcrt haplotypes showed that 30 isolates and 26 isolates harbouring pfcrt haplotypes were found in Central Africa (39.0%, 30/77) and Western Africa (33.8%, 26/77) (Fig. 4). Interestingly, a total of 2 isolates simultaneously harbouring pfK13 and pfcrt were found in the patients returned from Cambodia (C580Y in pfK13 and K76TT93SI356T in pfcrt) and Congo (G690G in pfK13 and K76I356T in pfcrt) in this study.
Table 3
Genetic haplotypes of pfcrt with P. falciparum in Shandong Province, 2015–2019.
Haplotypes a
|
Subtotalb
|
Proportionc (%)
|
T76T356
|
20
|
4.7
|
T76
|
18
|
4.2
|
I74E75T76
|
15
|
3.5
|
E75K76
|
8
|
1.9
|
I74
|
5
|
1.2
|
I74K75T76
|
3
|
0.7
|
T356
|
2
|
0.5
|
T355
|
1
|
0.2
|
Q56H58T76T356
|
1
|
0.2
|
T76T355
|
1
|
0.2
|
T76S93T356
|
1
|
0.2
|
S323
|
1
|
0.2
|
S323R334
|
1
|
0.2
|
a Mutations are in boldface. |
b The total samples for tested was 425, while 77 of them harboring the pfcrt polymorphisms. |
c the proportion refers to the mutated genotype samples accounting for the whole samples collected at the study site. |