Polymorphism analysis of drug resistance genes in imported Plasmodium falciparum isolates from Equatorial Guinea in Henan Province

doi:10.12140/j.issn.1000-7423.2023.05.011

Abstract

Abstract:

Objectiv e To analyze the imported Equatorial Guinean Plasmodium falciparum drug resistance gene polymorphisms in Henan Province, and provide a reference for the treatment of imported P. falciparum infections. Methods The medical records and peripheral blood samples were collected from the imported P. falciparum malaria cases original from Equatorial Guinea in Henan Province from 2012 to 2019. The P. falciparum genomic DNA was extracted and the P. falciparum genes, including Kelch 13-propeller (PfK13), chloroquine resistance transporter (Pfcrt), multidrug resistance 1 (Pfmdr1), dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps), were amplified by nested PCR. Bidirectional sequencing of the secondary PCR products was performed after agarose gel electrophoresis. The obtained sequences were aligned with the corresponding reference P. falciparum 3D7 strain genomes by MEGA7 software. The reference genomes were obtained from the GenBank (GenBank accession numbers: PF3D7_1343700, PF3D7_0709000, PF3D7_0523000, PF3D7_0417200 and PF3D7_1324800 respectively). The data were analyzed by SPPS 21.0 software. Results A total of 1 522 imported malaria cases were reported in Henan Province during 2012 to 2019, including 117 cases imported from Equatorial Guinea. Among the 117 cases, 97 cases were infected with P. falciparum, 16 cases were infected with P. ovale, 1 case was infected with P. vivax, 1 case was infected with P. malariae, 1 case was mixed infected with P. falciparum and P. malariae and 1 case was mixed infected with P. falciparum and P. ovale. The PfK13 gene was successfully amplified from 91 P. falciparum samples and the mutant prevalence was 8.8% (8/91). The non-synonymous mutation sites were M476I mixed type (mixed), A481V mixed, A564E mixed, P574L mixed, A578S, V589I and N609I mixed respectively. The synonymous mutation sites were G625G, N664N and C469C respectively. The Pfcrt gene was successfully amplified from 91 P. falciparum samples and the mutant prevalence was 18.7% (17/91). Three Pfcrt haplotypes were identified, including wild-type C₇₂V₇₃M₇₄N₇₅K₇₆ (81.3%, 74/91), mutant C₇₂V₇₃I₇₄E₇₅T₇₆ (12.1%, 11/91) and mixed-type C₇₂V₇₃M/I₇₄N/E₇₅K/T₇₆ (6.6%, 6/91). The Pfmdr1 gene was successfully amplified from 92 P. falciparum samples and the mutant prevalence was 77.2% (71/92). Three mutant codons were detected, including N86Y (41.3%, 38/92), Y184F (75.0%, 69/92) and D1246Y (1.1%, 1/92). The mutant prevalence of N86Y decreased from 68.8% in 2012 to 11.1% in 2016 (χ² = 11.58, P < 0.05). Five Pfmdr1 haplotypes were identified, including wild-type N₈₆Y₁₈₄D₁₂₄₆ (22.8%, 21/92), single mutants Y₈₆Y₁₈₄D₁₂₄₆ (1.1%, 1/92), N₈₆F₁₈₄D₁₂₄₆ (34.8%, 32/92), N₈₆Y₁₈₄Y₁₂₄₆ (1.1%, 1/92) and double mutant Y₈₆F₁₈₄D₁₂₄₆ (40.2%, 37/92). The Pfdhfr gene was successfully amplified from 90 P. falciparum samples and the mutant prevalence was 96.7% (87/90). Three mutant codons were detected, including N51I (91.1%, 82/90), C59R (93.3%, 84/90) and S108N (96.7%, 87/90). Five Pfdhfr haplotypes were identified, including wild-type N₅₁C₅₉S₁₀₈ (3.3%, 3/90), single mutant N₅₁C₅₉N₁₀₈ (2.2%, 2/90), double mutants I₅₁C₅₉N₁₀₈ (1.1%, 2/90), N₅₁R₅₉N₁₀₈ (3.3%, 3/90) and triple mutant I₅₁R₅₉N₁₀₈ (90.0%, 81/90). The Pfdhps gene was successfully amplified from 90 P. falciparum samples and the mutant prevalence was 97.8% (88/90). Six mutant codons were detected, including I431V (8.9%, 8/90), S436A (27.8%, 25/90), A437G (92.2%, 83/90), K540E (3.3%, 3/90), A581G (1.1%, 1/90) and A613S (2.2%, 2/90). Eight Pfdhps haplotypes were identified, including wild-type I₄₃₁S₄₃₆A₄₃₇K₅₄₀A₅₈₁A₆₁₃ (2.2%, 2/90), single mutants I₄₃₁A₄₃₆A₄₃₇K₅₄₀A₅₈₁A₆₁₃ (5.6%, 5/90), I₄₃₁S₄₃₆G₄₃₇K₅₄₀A₅₈₁A₆₁₃ (66.7%, 60/90), double mutants I₄₃₁A₄₃₆G₄₃₇K₅₄₀A₅₈₁A₆₁₃ (10.0%, 9/90), I₄₃₁S₄₃₆G₄₃₇E₅₄₀A₅₈₁A₆₁₃ (3.3%, 3/90), triple mutants V₄₃₁A₄₃₆G₄₃₇K₅₄₀A₅₈₁A₆₁₃ (8.9%, 8/90), I₄₃₁A₄₃₆G₄₃₇K₅₄₀G₅₈₁A₆₁₃ (1.1%, 1/90) and I₄₃₁A₄₃₆G₄₃₇K₅₄₀A₅₈₁S₆₁₃ (2.2%, 2/90). The Pfdhfr and Pfdhps genes were simultaneously successfully amplified from 89 P. falciparum samples and 84 (94.4%) samples had mutations in both genes. The most frequent mutation was the quadruple mutant I₅₁R₅₉N₁₀₈-G₄₃₇, which accounting for 64.0% among the gene mutations of Pfdhfr and Pfdhps. Conclusion Multiple mutant codons of PfK13 gene were detected. M476I and P574L had been confirmed to be associated with artemisinin resistance. As the withdrawal of chloroquine, the mutant prevalence of Pfcrt and Pfmdr1 genes associated with artemisinin-compatible drug resistance gradually decreased. The resistance of P. falciparum to sulfadoxine-pyrimethamine were mostly “partial resistance”, with no “super-resistant” haplotype detected.

Key words: Plasmodium falciparum, Drug resistance, PfK13, Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, Equatorial Guinea

CLC Number:

R382.31

ZHOU Ruimin, JI Penghui, LI Suhua, YANG Chengyun, LIU Ying, QIAN Dan, DENG Yan, LU Deling, ZHAO Yuling, ZHAO Dongyang, ZHANG Hongwei. Polymorphism analysis of drug resistance genes in imported Plasmodium falciparum isolates from Equatorial Guinea in Henan Province[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2023, 41(5): 593-600.

Figures/Tables 5

Table 1

Fig. 1

Table 2

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Table 4

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基因 Gene	引物名称 Primer name	引物序列（5'→3'） Primer sequences (5'→3')	退火条件 Amplification conditions	产物长度/bp Product length/bp
PfK13	K13-1F	CGGAGTGACCAAATCTGGGA	60 ℃ 90 s	2 097
	K13-1R	GGGAATCTGGTGGTAACAGC	60 ℃ 90 s	2 097
	K13-2F	GCCAAGCTGCCATTCATTTG	60 ℃ 90 s	850
	K13-2R	GCCTTGTTGAAAGAAGCAGA	60 ℃ 90 s	850
Pfcrt	Pfcrt-1F	CCGTTAATAATAAATACACGCAG	56 ℃ 45 s	537
	Pfcrt-1R	CGGATGTTACAAAACTATAGTTACC	56 ℃ 45 s	537
	Pfcrt-2F	TGTGCTCATGTGTTTAAACTT	48 ℃ 45 s	145
	Pfcrt-2R	CAAAACTATAGTTACCAATTTTG	48 ℃ 45 s	145
Pfmdr1	Pfmdr86-1F	TTAAATGTTTACCTGCACAACATAGAAAAT T	50 ℃ 45 s	612
	Pfmdr86-1R	CTCCACAATAACTTGCAACAGTTCTTA	50 ℃ 45 s	612
	Pfmdr86-2F	TGTATGTGCTGTATTATCAGGA	50 ℃ 45 s	526
	Pfmdr86-2R	CTCTTCTATAATGGACATGGTA	50 ℃ 45 s	526
	Pfmdr1246-1F	AATTTGATAGAAAAAGCTATTGATTATAA	50 ℃ 45 s	880
	Pfmdr1246-1R	TATTTGGTAATGATTCGATAAATTCATC	50 ℃ 45 s	880
	Pfmdr1246-2F	GAATTATTGTAAATGCAGCTTTA	50 ℃ 45 s	799
	Pfmdr1246-2R	GCAGCAAACTTACTAACACG	50 ℃ 45 s	799
Pfdhfr	Pfdhfr-1F	TTTATGATGGAACAAGTCTGC	52 ℃ 45 s	650
	Pfdhfr-1R	CTAGTATATACATCGCTAACA	52 ℃ 45 s	650
	Pfdhfr-2F	TGATGGAACAAGTCTGCGACGTT	46 ℃ 45 s	594
	Pfdhfr-2R	CTGGAAAAAATACATCACATTCATATG	46 ℃ 45 s	594
Pfdhps	Pfdhps-1F	GATTCTTTTTCAGATGGAGG	51 ℃ 45 s	770
	Pfdhps-1R	TTCCTCATGTAATTCATCTGA	51 ℃ 45 s	770
	Pfdhps-2F	AACCTAAACGTGCTGTTCAA	51 ℃ 45 s	711
	Pfdhps-2R	AATTGTGTGATTTGTCCACAA	51 ℃ 45 s	711

基因 Gene	密码子 Codon	核苷酸突变 Nucleotide mutation	氨基酸突变 Amino acid mutation	血样数 No. sample	突变率/% Mutantion rate/%	病例报告年份 Year
PfK13	476^a，b	ATA→ATG	M→I	1	1.1（1/91）	2013
	481^a，c	GCT→GTT	A→V	1	1.1（1/91）	2019
	564^a	GCA→GAA	A→E	1	1.1（1/91）	2013
	574^a，b	CCT→CTT	P→L	1	1.1（1/91）	2013
	578	GCT→TCT	A→S	1	1.1（1/91）	2016
	589	GTC→ATC	V→I	1	1.1（1/91）	2013
	609^a	AAT→ATT	N→I	1	1.1（1/91）	2013
	469	TGC→TGT	C→C	1	1.1（1/91）	2017
	625	GGA→GGG	G→G	1	1.1（1/91）	2013
	664	AAT→AAC	N→N	1	1.1（1/91）	2015
Pfcrt	074^a	ATG→ATT	M→I	17	18.7（17/91）	2012—2016
	075^a	AAT→GAA	N→E	17	18.7（17/91）	2012—2016
	076^a	AAA→ACA	K→T	17	18.7（17/91）	2012—2016
Pfmdr1	086	AAT→TAT	N→Y	38	41.3（38/92）	2012—2018
	184	TAT→TTT	Y→F	69	75.0（69/92）	2012—2019
	1 246	GAT→TAT	D→Y	1	1.1（1/92）	2014
Pfdhfr	51^a	AAT→ATT	N→I	82	91.1（82/90）	2012—2019
	59	TGT→CGT	C→R	84	93.3（84/90）	2012—2019
	108	AGC→AAC	S→N	87	96.7（87/90）	2012—2019
Pfdhps	431^a	ATA→GTA	I→V	8	1.1（1/90）	2013—2016，2018
	436^a	TCT→GCT	S→A	25	27.8（25/90）	2012—2017
	437^a	GCT→GGT	A→G	83	92.2（83/90）	2012—2019
	540	AAA→GAA	K→E	3	3.3（3/90）	2012，2013，2015
	581	GCG→GGG	A→G	1	1.1（1/90）	2015
	613	GCC→TCC	A→S	2	2.2（2/90）	2015

基因 Gene	基因型 Genotype	血样数（占比/%） No. sample (Proportion/%)						合计 Total
基因 Gene	基因型 Genotype	2012	2013	2014	2015	2016	2017—2019	合计 Total
Pfcrt （n = 91）	C₇₂V₇₃M₇₄N₇₅K₇₆	14（87.5）	18（78.3）	10（62.5）	16（84.2）	8（88.9）	8（100）	74（81.3）
Pfcrt （n = 91）	C₇₂V₇₃I₇₄E₇₅T₇₆	2（12.5）	5（21.7）	6（37.5）	3（15.8）	1（11.1）	0	17（18.7）
Pfmdr1 （n = 92）	N₈₆Y₁₈₄D₁₂₄₆	3（18.8）	3（12.5）	7（43.8）	5（26.3）	3（33.3）	0	21（22.8）
	Y₈₆Y₁₈₄D₁₂₄₆	1（6.3）	0	0	0	0	0	01（1.1）
	N₈₆F₁₈₄D₁₂₄₆	2（12.5）	9（37.5）	3（18.8）	9（47.4）	5（55.6）	4（50.0）	32（34.8）
	N₈₆Y₁₈₄Y₁₂₄₆	0	0	1（6.3）	0	0	0	1（1.1）
	Y₈₆F₁₈₄D₁₂₄₆	10（62.5）	12（50.0）	5（31.3）	5（26.3）	1（11.1）	4（50.0）	37（40.2）
Pfdhfr （n = 90）	N₅₁C₅₉S₁₀₈	0	1（4.6）	1（6.3）	1（5.3）	0	0	3（3.3）
	N₅₁C₅₉N₁₀₈	0	0	2（12.5）	0	0	0	2（2.2）
	I₅₁C₅₉N₁₀₈	0	0	0	0	1（11.1）	0	1（1.1）
	N₅₁R₅₉N₁₀₈	2（12.5）	0	0	0	1（11.1）	0	3（3.3）
	I₅₁R₅₉N₁₀₈	14（87.5）	21（95.5）	13（81.3）	18（94.7）	7（77.8）	8（100）	81（90.0）
Pfdhps （n = 90）	I₄₃₁S₄₃₆A₄₃₇K₅₄₀A₅₈₁A₆₁₃	0	0	2（12.5）	0	0	0	2（2.2）
	I₄₃₁A₄₃₆A₄₃₇K₅₄₀A₅₈₁A₆₁₃	0	3（13.6）	0	1（5.3）	0	1（12.5）	5（5.6）
	I₄₃₁S₄₃₆G₄₃₇K₅₄₀A₅₈₁A₆₁₃	13（81.3）	13（59.1）	10（62.5）	13（68.4）	5（55.6）	6（75.0）	60（66.7）
	I₄₃₁A₄₃₆G₄₃₇K₅₄₀A₅₈₁A₆₁₃	2（12.5）	4（18.2）	1（6.3）	0	2（22.2）	0	9（10.0）
	I₄₃₁S₄₃₆G₄₃₇E₅₄₀A₅₈₁A₆₁₃	1（6.3）	1（4.6）	0	1（5.3）	0	0	3（3.3）
	V₄₃₁A₄₃₆G₄₃₇K₅₄₀A₅₈₁A₆₁₃	0	1（4.6）	3（18.8）	1（5.3）	2（22.2）	1（12.5）	8（8.9）
	I₄₃₁A₄₃₆G₄₃₇K₅₄₀G₅₈₁A₆₁₃	0	0	0	1（5.3）	0	0	1（1.1）
	I₄₃₁A₄₃₆G₄₃₇K₅₄₀A₅₈₁S₆₁₃	0	0	0	2（10.5）	0	0	2（2.2）

基因型 Genotype	血样数 No. sample	占比/% Proportion/%
I₅₁R₅₉N₁₀₈- V₄₃₁A₄₃₆G₄₃₇	8	9.0（8/89）
I₅₁R₅₉N₁₀₈- A₄₃₆G₄₃₇S₆₁₃	1	1.1（1/89）
I₅₁R₅₉N₁₀₈- A₄₃₆G₄₃₇G₅₈₁	1	1.1（1/89）
I₅₁R₅₉N₁₀₈- A₄₃₆G₄₃₇	6	6.7（6/89）
I₅₁R₅₉N₁₀₈- G₄₃₇E₅₄₀	3	3.4（3/89）
I₅₁R₅₉N₁₀₈- G₄₃₇^b	57	64.0（57/89）
I₅₁R₅₉N₁₀₈- A₄₃₆	4	4.5（4/89）
R₅₉N₁₀₈- A₄₃₆G₄₃₇	2	2.2（2/89）
R₅₉N₁₀₈- G₄₃₇	1	1.1（1/89）
I₅₁N₁₀₈- G₄₃₇	1	1.1（1/89）
A₄₃₆G₄₃₇S₆₁₃	1	1.1（1/89）
N₁₀₈	2	2.2（2/89）
A₄₃₆	1	1.1（1/89）
G₄₃₇	1	1.1（1/89）
合计 Total	89	100