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Objective: To investigate the prevalence of single nucleotide polymorphisms (SNPs) of artemisinin resistance-related Pfubp1 and Pfap2mu genes in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea, so as to to provide baseline data for the formulation of malaria control strategies in Bioko Island., Methods: A total of 184 clinical blood samples were collected from patients with P. falciparum malaria in Bioko Island, Equatorial Guinea from 2018 to 2020, and genomic DNA was extracted. The Pfubp1 and Pfap2mu gene SNPs of P. falciparum were determined using a nested PCR assay and Sanger sequencing, and the gene sequences were aligned., Results: There were 159 wild-type P. falciparum isolates (88.83%) from Bioko Island, Equatorial Guinea, and 6 SNPs were identified in 20 Pfubp1 -mutant P. falciparum isolates (11.17%), in which 4 non-synonymous mutations were detected, including E1516G, K1520E, D1525E, E1528D. There was only one Pfubp1 gene mutation site in 19 Pfubp1 -mutant P. falciparum isolates (95.00%), in which non-synonymous mutations accounted for 68.42% (13/19). D1525E and E1528D were identified as major known epidemic mutation sites in the Pfubp1 gene associated with resistance to artemisinin-based combination therapies (ACTs). At amino acid position 1525, there were 178 wild-type P. falciparum isolates (99.44%) and 1 mutant isolate (0.56%), with such a mutation site identified in blood samples in 2018, and at amino acid position 1528, there were 167 wild-type P. falciparum isolates (93.30%) and 12 mutant isolates (6.70%). The proportions of wild-type P. falciparum isolates were 95.72% (134/140), 79.25% (126/159) and 95.83% (161/168) in the target amplification fragments of the three regions in the Pfap2mu gene ( Pfap2mu -inner1, Pfap2mu -inner2, Pfap2mu -inner3), respectively. There were 16 different SNPs identified in all successfully sequenced P. falciparum isolates, in which 7 non-synonymous mutations were detected, including S160N, K199T, A475V, S508G, I511M, L595F, and Y603H. There were 7 out of 43 Pfap2mu -mutant P. falciparum isolates (16.28%) that harbored only one gene mutation site, in which non-synonymous mutations accounted for 28.57% (2/7). For the known delayed clearance locus S160N associated with ACTs, there were 143 wild-type (89.94%) and 16 Pfap2mu -mutant P. falciparum isolates (10.06%)., Conclusions: Both Pfubp1 and Pfap2mu gene mutations were detected in P. falciparum isolates from Bioko Island, Equatorial Guinea from 2018 to 2020, with a low prevalence rate of Pfubp1 gene mutation and a high prevalence rate of Pfap2mu gene mutation. In addition, new mutation sites were identified in the Pfubp1 (E1504E and K1520E) and Pfap2mu genes (A475V and S508G).

Objective: To investigate the genetic polymorphisms of Plasmodium falciparum multidrug resistance protein 1 ( PfMDR1 ), chloroquine resistance transporter ( PfCRT ) and Kelch 13 ( PfK13 ) genes in Bioko Island, Equatorial Guinea, so as to provide insights into the development of the malaria control strategy in local areas., Methods: A total of 85 peripheral blood samples were collected from patients with Plasmodium falciparum infections in Bioko Island, Equatorial Guinea in 2018 and 2019, and genomic DNA was extracted. The PfMDR1 , PfCRT and PfK13 genes were amplified using a nested PCR assay. The amplification products were sequenced, and the gene sequences were aligned., Results: There were no mutations associated with artemisinin resistance in PfK13 gene in Bioko Island, Equatorial Guinea, while drug-resistant mutations were detected in PfMDR1 and PfCRT genes, and the proportions of PfMDR1_N86Y, PfMDR1_Y184F and PfCRT_K76T mutations were 35.29% (30/85), 72.94% (62/85) and 24.71% (21/85), respectively., Conclusions: There are mutations in PfMDR1 , PfCRT and PfK13 genes in P. falciparum isolates from Bioko Island, Equatorial Guinea.

Objective: To investigate the drug-resistant gene polymorphisms in Plasmodium falciparum imported from Equatorial Guinea to Shandong Province., Methods: From 2015 to 2016, blood samples were collected from imported P. falciparum malaria patients returning from Equatorial Guinea to Shandong Province, and genome DNA of the malaria parasite was extracted. The drug-resistant Pfcrt , Pfmdr1 , Pfdhfr , Pfdhps , and K13 genes of P. falciparum were amplified using a PCR assay, followed by DNA sequencing, and the sequences were aligned., Results: The target fragments of all 5 drug-resistant genes of P. falciparum were successfully amplified and sequenced. There were 72.8%, 18.6%, and 8.6% of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfcrt gene, respectively, and all mutant haplotypes were CVI ET (the underline indicates the mutation site). There were 20.0%, 61.4% and 18.6% of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfmdr1 gene, respectively, and the mutant haplotypes mainly included YF and NF (the underlines indicate the mutation sites). There were 1.4%, 98.6%, and 0 of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfdhfr gene, respectively, and A IRN I was the predominant mutant haplotype (the underline indicates the mutation site). There were 1.4%, 94.3%, and 4.3% of P. falci parum parasites carrying the wild-, mutant-, and mixed-type Pfdhps gene, respectively, and S G KAA was the predominant mutant haplotype (the underline indicates the mutation site). The complete drug-resistant IRNGE genotype consisted of 8.6% of the Pfdhfr and Pfdhps genes, and the K13 gene A578S mutation occurred in 1.4% of the parasite samples., Conclusions: There are mutations in the Pfcrt , Pfmdr1 , Pfdhfr , Pfdhps , and K13 genes of P. falciparum imported from Equatorial Guinea to Shandong Province, with a low frequency in the Pfcrt gene mutation and a high frequency in the Pfmdr1 , Pfdhfr , and Pfdhps gene mutations, and the K13 gene A578S mutation is detected in the parasite samples.

Among 41 people returned from Equatorial Guinea, 16 persons were infected with Plasmodium falciparum (39.02%) in Longlin County, Guangxi Zhuang Autonomous Region from October 2011 to February 2012. Among the 16 cases, Plasmodiumfalciparum was found in their blood smears of 15 cases, and 1 patient was diagnosed clinically as falciparum malaria. All the 41 people were once suffered from malaria and they received antimalarial drugs when they were in Equatorial Guinea. These imported malaria cases received the standard treatment and the foci were sprayed with insecticides. No secondary malaria cases were found. In conclusion, the measures of imported malaria control are effective. The persons returned from Equatorial Guinea have a high risk of malaria; therefore, it is necessary to strengthen the monitoring of malaria for the floating population from high malaria prevalent areas.