Correlation of Pfcrt, Pfmdr and PfK13 gene polymorphisms and in vitro drug susceptibility of Plasmodium falciparum isolates from China-Myanmar border region

doi:10.12140/j.issn.1000-7423.2020.05.009

Abstract

Abstract:

Objective To investigate correlation between the susceptibility to antimalarials and the polymorphisms of drug resistance genes of Plasmodium falciparum isolates from China-Myanmar border region, to provide basis for formulating rational strategy for using antimalarial drugs in the local areas. Methods In 2009, an investigation site was set up at the Nongriban Clinic in Lazan City, Myanmar, and the participant patients were selected based on the inclusion criteria: single infection of P. falciparum diagnosed by microscopy; and no chemotherapy history with antimalarials, sulfonamides, or tetracyclines within the past 2 weeks. For in vitro drug sensitivity test and genetic analysis, whole blood or filter paper blood samples were collected from the patients prior to drug treatment. The Rieckmann in vitro microassay method was used to assess the susceptibility of P. falciparum parasites to artemisinin, chloroquine, piperquine and pyronidine. Blood DNA was prepared to test examine the polymorphisms of chloroquine resistant transporter gene (Pfcrt), multiple-drug resistance (Pfmdr) and Kelch gene (PfK13). The associations between the susceptibility to antimalarial drugs and gene polymorphisms were analyzed with Spearman rank correlation coefficient test by SPSS 23.0 software. Results A total of 63 patients with single infection of P. falciparum examined by microscopy were included in this study, of them 51 samples underwent in vitro drug susceptibility test, with 42 samples were completed successfully, accounting for 82.4% (42/51). In the in vitro assay, P. falciparum showed higher mean inhibitory concentration, higher 50% inhibitory dose (ID₅₀) and higher resistance rate to chloroquine, which were 880 nmol/L, 320.5 nmol/L and 95.2%, respectively; and showed lower mean inhibitory concentration to pyronaridine (410 nmol/L), lower ID₅₀ to artemisinin (84.8 nmol/L) and lower resistant rate to piperaquine (7.1%). Of the 40 blood samples resistant to chloroquine, the cross resistance rate to pyronaridine was 52.5% (21/40), to artemisinin 37.5% (15/40) and to piperaquine 7.5% (3/40). Of the 3 blood samples resistant to piperaquine, all were cross-resistant to chloroquine (3/3), artemisinin (3/3) and pyronaridine (3/3). Among the samples displaying cross-resistance, 21 cases showed cross-resistance of chloroquine-pyronaridine, 15 of chloroquine-artemisinin, while 13 were artemisinin-pyronaridine cross-resistant, and respective 3 cases of piperaquine-resistance being cross-reactive with chloroquine, artemisinin and pyronaridine. Spearman rank correlation coefficient test revealed a significant correlation between artemisinin resistance and piperaquine or pyronaridine resistance (r = 0.354, 0.446; P < 0.05). The Pfcrt gene of 37 cases was sequenced and examined, indicative of triple-mutant genotype C₇₂V₇₃I₇₄E₇₅T₇₆ (mutation rate 100%, 37/37) at the loci 74-76. The mutation prevalence of Pfmdr gene at position N86Y and D1246Y were 2.1% (1/47) and 100% (49/49), respectively. The PfK13 gene was successfully sequenced from 20 isolates, and found to harbor 3 different mutations, including 10 cases with mutation at F446I, showing higher prevalence(50.0%, 10/20), one case with mutation at L492L/S and one case at C580Y showing low prevalence (5.0%, 1/20; 5.0%, 1/20; respectively). Among the drug resistant cases determined by in vitro drug test, the mutant prevalence of Pfcrt C₇₂V₇₃I₇₄E₇₅T₇₆, Pfmdr N₈₆Y₁₂₄₆, PfK13 I₄₄₆S₄₉₂C₅₈₀ and Pfcrt/Pfmdr I₇₄E₇₅T₇₆/Y₁₂₄₆ was 100% (37/37), 93.9% (46/49), 50% (10/20) and 69.0% (29/42) respectively. Spearman’s rank correlation coefficient analysis showed that there was no correlation of Pfcrt, Pfmdr and PfK13 gene mutation with the drug sensitivity in vitro. Conclusion The P. falciparum isolates from Laza City of China-Myanmar border region have developed resistance to chloroquine widely, and resistance to pyronaridine and artemisinin to certain extent, but remain usceptible to piperaquine.

Key words: Plasmodium falciparum, Drug sensitivity, In vitro microtest, Resistant gene, Laza City of Myanmar

CLC Number:

R382.312

ZHANG Cang-lin, NIE Ren-hua, XU Dan, LV Gao-wei, WANG Jian, YANG Ya-ming, DENG Yan, LIU Yan, ZHOU Hong-ning. Correlation of Pfcrt, Pfmdr and PfK13 gene polymorphisms and in vitro drug susceptibility of Plasmodium falciparum isolates from China-Myanmar border region[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(5): 580-588.

Figures/Tables 6

Table 1

Table 2

Table 3

Fig. 1

Fig. 2

Table 4

The frequency distribution of different genotypes of Pfcrt, Pfmdr and PfK13 in in vitro drug susceptibility test of P. falciparum isolates from China-Myanmar Border Region in 2009

基因类型^a Genotype^a	血样数量 No. isolates	百分比/% Percentage/%	体外检测对各药物产生抗性的血样数 Cases of in vitro resistance to drugs
基因类型^a Genotype^a	血样数量 No. isolates	百分比/% Percentage/%	青蒿素 Artemisinin		氯喹 Chloroquine		哌喹 Piperaquine	咯萘啶 Pyronaridine
Pfcrt基因（体外,n = 35） Pfcrt gene (in vitro, n = 35)
三重基因突变C₇₂V₇₃I₇₄E₇₅T₇₆ Triple-mutant genotype C₇₂V₇₃I₇₄E₇₅T₇₆	37	100	12	28		3			16
Pfmdr基因（体外,86位点n = 45,1246位点n = 47） Pfmdr gene (in vitro, n = 45 at codon 86, n = 47 at codon 1246）
单基因突变Y₁₂₄₆ Single-mutant genotype Y₁₂₄₆	2	4.1	0		2		0	1
单基因突变N₈₆Y₁₂₄₆ Single-mutant genotype N₈₆Y₁₂₄₆	46	93.9	16		34		3	21
双重突变Y₈₆Y₁₂₄₆ Double-mutant genotype Y₈₆Y₁₂₄₆	1	2.0	0		1		0	0
PfK13基因（体外, n = 20） PfK13 gene (in vitro, n = 20)
野生型F₄₄₆S₄₉₂C₅₈₀ Wild-type genotype F₄₄₆S₄₉₂C₅₈₀	8	40.0	3		5		2	3
单基因突变I₄₄₆S₄₉₂C₅₈₀ Single-mutant genotype I₄₄₆S₄₉₂C₅₈₀	10	50.0	4		9		1	8
单基因突变F₄₄₆L/S₄₉₂C₅₈₀ Single-mutant genotype F₄₄₆L/S₄₉₂C₅₈₀	1	5.0	1		1		0	1
单基因突变F₄₄₆S₄₉₂Y₅₈₀ Single-mutant genotype F₄₄₆S₄₉₂Y₅₈₀	1	5.0	1		1		0	1
Pfcrt/Pfmdr 多基因突变I₇₄E₇₅T₇₆/Y₁₂₄₆ Pfcrt/Pfmdr multi-mutant genotypes I₇₄E₇₅T₇₆/Y₁₂₄₆	29	69.0	8		21		2	10
Pfcrt/Pfmdr多基因突变I₇₄E₇₅T₇₆/Y₈₆Y₁₂₄₆ Pfcrt/Pfmdr multi-mutant genotypes I₇₄E₇₅T₇₆/Y₈₆Y₁₂₄₆	1	2.4	0		1		0	0
Pfcrt/Pfmdr/PfK13多基因突变I₇₄E₇₅T₇₆/Y₁₂₄₆/I₄₄₆ Pfcrt/Pfmdr/PfK13 multi-mutant genotypes I₇₄E₇₅T₇₆/Y₁₂₄₆/I₄₄₆	6	14.3	3		5		1	5
Pfcrt/Pfmdr/PfK13多基因突变I₇₄E₇₅T₇₆/Y₁₂₄₆/L/S₄₉₂ Pfcrt/Pfmdr/PfK13 multi-mutant genotypes I₇₄E₇₅T₇₆/Y₁₂₄₆/L/S₄₉₂	1	2.4	1		1		0	1
Pfmdr/PfK13多基因突变Y₁₂₄₆/I₄₄₆ Pfmdr/PfK13 multi-mutant genotypes Y₁₂₄₆/I₄₄₆	4	9.5	1		4		0	3
Pfmdr/PfK13多基因突变Y₁₂₄₆/Y₅₈₀ Pfmdr/ PfK13 multi-mutant genotypes Y₁₂₄₆/Y₅₈₀	1	2.4	1		1		0	1

Table 4

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基因Gene	引物Primer	引物序列（5′→3′） Primer sequence (5′→3′)	PCR轮次 PCR round	产物片段/bp Amplicon size/bp
Pfcrt	76A	GCGCGCGCATGGCTCACGTTTAGGTGGAG	第1轮	206
	76B	GGGCCCGGCGGATGTTACAAAACTATAGTTACC
	76D1	TGTGCTCATGTGTTTAAACTT	第2轮	145
	76D2	CAAAACTATAGTTACCAATTTTG
Pfmdr	86A	GCGCGCGTTGAACAAAAAGAGTACCGCTG	第1轮	450
	86B	GGGCCCTCGTACCAATTCCTGAACTCAC
	86D1	TTTACCGTTTAAATGTTTACCTGC	第2轮	291
	86D2	CCATCTTGATAAAAAACACTTCTT
	1246A	GGGGGATGACAAATTTTCAAGATTA	第1轮	295
	1246B	GGGGGACTAACACGTTTAACATCTT
	1246D1	AATGTAAATGAATTTTCAAACC	第2轮	203
	1246D2	CATCTTCTCTTCCAAATTTGATA
PfK13	PCR_F	CGGAGTGACCAAATCTGGGA	第1轮	2 181
	PCR_R	GGGAATCTGGTGGTAACAGC
	N1_F	GCCAAGCTGCCATTCATTTG	第2轮	849
	N1_R	GCCTTGTTGAAAGAAGCAGA

药物 Drug	测定血样 No. sample tested	抗性率/% Resistance rate/%	平均抑制浓度^a/nmol·L^-1 Mean inhibition concentration^a/nmol·L^-1	ID₅₀ （95%可信限） ID₅₀ (95%CI)	对其他抗疟药的抗性血样（交叉抗性率/%） No. resistance to other antimalarials (Proportion of cross resistance/%)
药物 Drug	测定血样 No. sample tested	抗性率/% Resistance rate/%		ID₅₀ （95%可信限） ID₅₀ (95%CI)	青蒿素 Artemisinin	氯喹 Chloroquine	哌喹 Piperaquine	咯萘啶 Pyronaridine
青蒿素 Artemisinin	42	40.5	640	84.8 (44.2~ 162.7)	-	15（88.2）	3（17.6）	13（76.5）
氯喹^[28] Chloroquine	42	95.2	880	320.5 (57.9~1775)	15（37.5）	-	3（7.5）	21（52.5）
哌喹^[28] Piperaquine	42	7.1	440	128.2 (125.1~ 131.4)	3（100）	3（100）	-	3（100）
咯萘啶^[28] Pyronaridine	42	54.8	410	96.0 (85.4~ 107.9)	13（56.5）	21（91.3）	3（13.0）	-