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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 Pfubp1gene 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).
RESUMO
Objective To establish a fluorescent assay for rapid detection of Plasmodium falciparum based on recombinaseaided amplification (RAA) and CRISPR-Cas12a system,and to preliminarily evaluate the diagnostic efficiency of this system.. Methods The 18S ribosomal RNA (rRNA) gene of P. falciparum was selected as the target sequence, and three pairs of RAA primers and CRISPR-derived RNA (crRNA) were designed and synthesized. The optimal combination of RAA primers and crRNA was screened and the reaction conditions of the system were optimized to create a fluorescent RAA/CRISPR-Cas12a system. The plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 was generated, and diluted into concentrations of 1 000, 100, 10, 1 copy/μL for the fluorescent RAA/CRISPR-Cas12a assay, and its sensitivity was evaluated. The genomic DNA from P. vivax, P. malariae, P. ovum, hepatitis B virus, human immunodeficiency virus and Treponema pallidum was employed as templates for the fluorescent RAA/CRISPR-Cas12a assay, and its specificity was evaluated. Fifty malaria clinical samples were subjected to the fluorescent RAA/CRISPR-Cas12a assay and nested PCR assay, and the consistency between two assays was compared. In addition, P. falciparum strain 3D7 was cultured in vitro. Then, the culture was diluted into blood samples with parasite densities of 1 000, 500, 200, 50, 10 parasites/μL with healthy volunteers’ O-positive red blood cells for the RAA/CRISPR-Cas12a assay, and the detection efficiency was tested. Results The Pf-F3/Pf-R3/crRNA2 combination, 2.5 μL as the addition amount of B buffer, 40 min as the RAA reaction time, 37 °C as the reaction temperature of the CRISPR-Cas12a system were employed to establish the fluorescent RAA/CRISPR-Cas12a system. Such a system was effective to detect the plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 at a concentration of 1 copy/μL, and presented fluorescent signals for detection of P. falciparum, but failed to detect P. ovum, P. malariae, P. vivax, T. pallidum, hepatitis B virus or human immunodeficiency virus. The fluorescent RAA/CRISPR-Cas12a system and nested PCR assay showed completely consistent results for detection of 50 malaria clinical samples (kappa = 1.0, P < 0.001). Following 6-day in vitro culture of the P. falciparum strain 3D7, 10 mL cultures were generated and the fluorescent RAA/CRISPR-Cas12a system showed the minimal detection limit of 50 parasites/μL. Conclusion The fluorescent RAA/CRISPR-Cas12a system is rapid, sensitive and specific for detection of P. falciparum, which shows promising value for rapid detection and risk monitoring of P. falciparum.