目的 分析河南省自赤道几内亚输入性恶性疟原虫抗药性基因多态性，了解恶性疟原虫基因突变情况，评估其流行特征。 方法 收集河南省2012—2019年自赤道几内亚输入性恶性疟病例信息和外周血样，提取血样中恶性疟原虫基因组DNA，巢式PCR扩增恶性疟原虫Kelch 13螺旋体（PfK13）基因、恶性疟原虫氯喹抗性转运蛋白（Pfcrt）基因、恶性疟原虫多药物抗性基因1（Pfmdr1）、恶性疟原虫二氢叶酸还原酶（Pfdhfr）基因和恶性疟原虫二氢蝶酸合酶（Pfdhps）基因，琼脂糖凝胶电泳后进行双向测序。获得的基因序列通过MEGA7软件与参考基因组进行比较获得突变位点信息，参考基因组来自GenBank的恶性疟原虫3D7野生虫株（GenBank登录号分别为PF3D7_1343700、PF3D7_0709000、PF3D7_0523000、PF3D7_0417200和PF3D7_1324800）。使用SPPS 21.0软件对数据进行统计学分析。 结果 2012—2019年，河南省共报告输入性疟疾病例1 522例，其中自赤道几内亚输入病例117例，包括恶性疟病例97例、卵形疟病例16例、间日疟和三日疟病例各1例、恶性疟原虫/三日疟原虫混合感染和恶性疟原虫/卵形疟原虫混合感染病例各1例。测序获得91份恶性疟原虫样品的PfK13基因序列，基因突变率为8.8%（8/91）；突变样品中共检测出7个非同义突变位点和3个同义突变位点，其中非同义突变位点分别为M476I混合型（混）、A481V混、A564E混、P574L混、A578S、V589I和N609I混，同义突变位点分别为G625G、N664N和C469C。测序获得91份恶性疟原虫样品的Pfcrt基因序列，基因突变率为18.7%（17/91）；检测出3种Pfcrt基因型，分别为野生型C₇₂V₇₃M₇₄N₇₅K₇₆（81.3%，74/91）、突变型C₇₂V₇₃I₇₄E₇₅T₇₆（12.1%，11/91）和混合型C₇₂V₇₃M/I₇₄N/E₇₅K/T₇₆（6.6%，6/91）。测序获得92份恶性疟原虫样品的Pfmdr1基因序列，基因突变率为77.2%（71/92）；检测出3个突变位点，分别为N86Y（41.3%，38/92）、Y184F（75.0%，69/92）和D1246Y（1.1%，1/92），其中N86Y突变率从2012年的68.8%（11/16）下降到2016年的11.1%（1/9）（χ² = 11.58，P < 0.05）；检测出5种Pfmdr1基因型，分别为野生型N₈₆Y₁₈₄D₁₂₄₆（22.8%，21/92），单基因突变型Y₈₆Y₁₈₄D₁₂₄₆（1.1%，1/92）、N₈₆F₁₈₄D₁₂₄₆（34.8%，32/92）、N₈₆Y₁₈₄Y₁₂₄₆（1.1%，1/92）和双重突变型Y₈₆F₁₈₄D₁₂₄₆（40.2%，37/92）。测序获得90份恶性疟原虫样品的Pfdhfr基因序列，基因突变率为96.7%（87/90）；检测出3个突变位点，分别为N51I（91.1%，82/90）、C59R（93.3%，84/90）和S108N（96.7%，87/90）；检测出5种Pfdhfr基因型，分别为野生型N₅₁C₅₉S₁₀₈（3.3%，3/90），单基因突变型N₅₁C₅₉N₁₀₈（2.2%，2/90），双重突变型I₅₁C₅₉N₁₀₈（1.1%，2/90）、N₅₁R₅₉N₁₀₈（3.3%，3/90）和三重突变型I₅₁R₅₉N₁₀₈（90.0%，81/90）。测序获得90份恶性疟原虫样品的Pfdhps基因序列，基因突变率为97.8%（88/90）；检测出6个突变位点，分别为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）和A613S（2.2%，2/90）；检测出8种Pfdhps基因型，分别为野生型I₄₃₁S₄₃₆A₄₃₇K₅₄₀A₅₈₁A₆₁₃（2.2%，2/90），单基因突变型I₄₃₁A₄₃₆A₄₃₇K₅₄₀A₅₈₁A₆₁₃（5.6%，5/90）、I₄₃₁S₄₃₆G₄₃₇K₅₄₀A₅₈₁A₆₁₃（66.7%，60/90），双重突变型I₄₃₁A₄₃₆G₄₃₇K₅₄₀A₅₈₁A₆₁₃（10.0%，9/90）、I₄₃₁S₄₃₆G₄₃₇E₅₄₀A₅₈₁A₆₁₃（3.3%，3/90），三重突变型V₄₃₁A₄₃₆G₄₃₇K₅₄₀A₅₈₁A₆₁₃（8.9%，8/90）、I₄₃₁A₄₃₆G₄₃₇K₅₄₀G₅₈₁A₆₁₃（1.1%，1/90）和I₄₃₁A₄₃₆G₄₃₇K₅₄₀A₅₈₁S₆₁₃（2.2%，2/90）。89份恶性疟原虫样品的Pfdhfr和Pfdhps基因同时测序成功，其中84例（94.4%）同时发生双基因突变，四重突变体I₅₁R₅₉N₁₀₈-G₄₃₇占比最高（64.0%，57/89）。 结论 自赤道几内亚输入的恶性疟原虫样品中检出多个PfK13基因突变位点，其中M476I和P574L已被证实与青蒿素类药物抗性相关；随着氯喹的停用，与青蒿素配伍药物抗性相关的Pfcrt和Pfmdr1基因突变率呈下降趋势；恶性疟原虫对磺胺多辛-乙胺嘧啶药物抗性水平多为“部分抗性”，未检出“超级抗性”样品。

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.