Abstract:bSoutheast Asia (the Thailand-Cambodia border) has been considered the primal epicenter for most antimalarial drug resistance; however, numerous molecular epidemiological studies have successively reported multiple independent origins of sulfadoxinepyrimethamine (SP) resistance-associated Plasmodium falciparum dhfr (pfdhfr) and pfdhps alleles in other areas. To better understand the origin and evolutionary pathway of the SP resistance in Southeast Asia, a total of 374 P. falciparum field isolates from the Yunn… Show more
“…The quadruple mutant (C IRN I-S G KAA) was predominant, with a prevalence of 65.38% (Table 3 ), which is higher than reports from mainland EG (54%) [ 4 ]. The saturation of the Pfdhfr triple mutants could further induce the Pfdhps mutants, and thus, the presence of quadruple mutants (C IRN I-S G KAA) was common [ 40 ]. Although quintuple mutant genotypes (C IRN I-S GE AA) are highly linked to SP failure [ 34 ], this mutant was detected at a rate of 4.62% (Table 3 ).…”
BackgroundSulfadoxine–pyrimethamine (SP) is recommended for intermittent preventive treatment of malaria in Africa. However, increasing SP resistance (SPR) affects the therapeutic efficacy of the SP. As molecular markers, Pfdhfr (dihydrofolate reductase) and Pfdhps (dihydropteroate synthase) genes are widely used for SPR surveillance. This study aimed to assess the prevalence of Pfdhfr and Pfdhps genes mutations and haplotypes in Plasmodium falciparum isolates collected from Bioko Island, Equatorial Guinea (EG).MethodsIn total, 180 samples were collected in 2013–2014. The single nucleotide polymorphisms (SNPs) of the Pfdhfr and Pfdhps genes were identified with nested PCR and Sanger sequencing. The genotypes and linkage disequilibrium (LD) tests were also analysed.ResultsSequences of Pfdhfr and Pfdhps genes were obtained from 92.78% (167/180) and 87.78% (158/180) of the samples, respectively. For Pfdhfr, 97.60% (163/167), 87.43% (146/167) and 97.01% (162/167) of the samples carried N51I, C59R and S108N mutant alleles, respectively. The prevalence of the Pfdhps S436A, A437G, K540E, A581G, and A613S mutations were observed in 20.25% (32/158), 90.51% (143/158), 5.06% (8/158), 0.63% (1/158), and 3.16% (5/158) of the samples, respectively. In total, 3 unique haplotypes at the Pfdhfr locus and 8 haplotypes at the Pfdhps locus were identified. A triple mutation (CIRNI) in Pfdhfr was the most prevalent haplotype (86.83%), and a single mutant haplotype (SGKAA; 62.66%) was predominant in Pfdhps. A total of 130 isolates with 12 unique haplotypes were found in the Pfdhfr and Pfdhps combined haplotypes, 65.38% (85/130) of them carried quadruple allele combinations (CIRNI-SGKAA), whereas only one isolate (0.77%, 1/130) was found to carry the wild-type (CNCSI-SAKAA). For LD analysis, the Pfdhfr N51I was significantly associated with the Pfdhps A437G (P < 0.05).ConclusionBioko Island possesses a high prevalence of the Pfdhfr triple mutation (CIRNI) and Pfdhps single mutation (SGKAA), which will undermine the pharmaceutical effect of SP for malaria treatment strategies. To avoid an increase in SPR, continuous molecular monitoring and additional control efforts are urgently needed in Bioko Island, Equatorial Guinea.
“…The quadruple mutant (C IRN I-S G KAA) was predominant, with a prevalence of 65.38% (Table 3 ), which is higher than reports from mainland EG (54%) [ 4 ]. The saturation of the Pfdhfr triple mutants could further induce the Pfdhps mutants, and thus, the presence of quadruple mutants (C IRN I-S G KAA) was common [ 40 ]. Although quintuple mutant genotypes (C IRN I-S GE AA) are highly linked to SP failure [ 34 ], this mutant was detected at a rate of 4.62% (Table 3 ).…”
BackgroundSulfadoxine–pyrimethamine (SP) is recommended for intermittent preventive treatment of malaria in Africa. However, increasing SP resistance (SPR) affects the therapeutic efficacy of the SP. As molecular markers, Pfdhfr (dihydrofolate reductase) and Pfdhps (dihydropteroate synthase) genes are widely used for SPR surveillance. This study aimed to assess the prevalence of Pfdhfr and Pfdhps genes mutations and haplotypes in Plasmodium falciparum isolates collected from Bioko Island, Equatorial Guinea (EG).MethodsIn total, 180 samples were collected in 2013–2014. The single nucleotide polymorphisms (SNPs) of the Pfdhfr and Pfdhps genes were identified with nested PCR and Sanger sequencing. The genotypes and linkage disequilibrium (LD) tests were also analysed.ResultsSequences of Pfdhfr and Pfdhps genes were obtained from 92.78% (167/180) and 87.78% (158/180) of the samples, respectively. For Pfdhfr, 97.60% (163/167), 87.43% (146/167) and 97.01% (162/167) of the samples carried N51I, C59R and S108N mutant alleles, respectively. The prevalence of the Pfdhps S436A, A437G, K540E, A581G, and A613S mutations were observed in 20.25% (32/158), 90.51% (143/158), 5.06% (8/158), 0.63% (1/158), and 3.16% (5/158) of the samples, respectively. In total, 3 unique haplotypes at the Pfdhfr locus and 8 haplotypes at the Pfdhps locus were identified. A triple mutation (CIRNI) in Pfdhfr was the most prevalent haplotype (86.83%), and a single mutant haplotype (SGKAA; 62.66%) was predominant in Pfdhps. A total of 130 isolates with 12 unique haplotypes were found in the Pfdhfr and Pfdhps combined haplotypes, 65.38% (85/130) of them carried quadruple allele combinations (CIRNI-SGKAA), whereas only one isolate (0.77%, 1/130) was found to carry the wild-type (CNCSI-SAKAA). For LD analysis, the Pfdhfr N51I was significantly associated with the Pfdhps A437G (P < 0.05).ConclusionBioko Island possesses a high prevalence of the Pfdhfr triple mutation (CIRNI) and Pfdhps single mutation (SGKAA), which will undermine the pharmaceutical effect of SP for malaria treatment strategies. To avoid an increase in SPR, continuous molecular monitoring and additional control efforts are urgently needed in Bioko Island, Equatorial Guinea.
“…The study locations, sample size (n), types of patient blood collected and years of collection were as follows: Hainan province (N = 136), with venous blood samples treated with EDTA from Sanya city (SY, n = 13) collected in 2005,2007; finger-prick blood spot samples collected on filter paper from Dongfang city (DF, n = 106) collected in 2004, 2007, and 2008; and Ledong county (LeD, n = 17) collected in 2004; and Yunnan province (N = 289), with finger-prick blood spot samples collected on filter paper from Dehong region (DH, n = 59) collected in 2007, venous blood samples treated with EDTA from Tengchong county (TC, n = 106) collected in 2006 and Lazan county (LZ, n = 71) collected in 2006, 2007; and blood smears from Xishuangbanna region (BN, n = 53) collected in 2006. Most samples (n = 349) were previously genotyped as part of a survey on pyrimethamine resistance in South China [ 27 ]. Figure 1 A schematic map showing the distribution of sampling sites for Plasmodium falciparum.…”
Section: Methodsmentioning
confidence: 99%
“…In other studies, microsatellite markers were used to analyse the population genetics of P. falciparum samples collected from a single region or country, including Thailand [ 20 ], Philippines [ 21 ], Malaysian Borneo [ 22 ], Papua New Guinea [ 23 ], Western Kenya [ 24 , 25 ] and the Republic of the Congo [ 26 ]. In China, molecular biological studies on drug-resistant malaria were recently reported [ 27 , 28 ], although studies using neutral microsatellite markers to analyse the basic genetic diversity of the parasite are limited.…”
BackgroundYunnan and Hainan provinces are the two major endemic regions for Plasmodiumfalciparum malaria in China. However, few studies have investigated the characteristics of this parasite. Therefore, this study aimed to evaluate the genetic diversity and population structure of P. falciparum to predict the geographic origin of falciparum malaria.MethodsThirteen highly polymorphic microsatellite loci were studied to estimate the genetic diversity and population structure of 425 P. falciparum isolates obtained from blood samples collected from Yunnan and Hainan provinces of South China. The isolates were analysed for genetic diversity, linkage disequilibrium, and population structure. The parasite populations were clustered into two subgroups (i.e., Yunnan and Hainan) and a classification algorithm was used to identify molecular markers for classifying the P. falciparum populations.ResultsAll 13 microsatellite loci were highly polymorphic, with the number of alleles per locus varying from 5 to 20. The mean expected heterozygosity (He) in Yunnan and Hainan was 0.766 ± 0.036 and 0.677 ± 0.039, respectively, revealing a moderate high level of genetic diversity. Significant linkage disequilibrium was found for some regions of Yunnan (Lazan county and Xishuangbanna region) and Hainan (Dongfang city and Sanya city) province. According to the classification algorithm, a combination of three microsatellites could be used as a discriminatory marker to identify the origin of P. falciparum isolates.ConclusionsThe results on the genetic structure of P. falciparum populations from South China provide a basis for developing a genetic marker-based tool to trace the source of the parasite infections and consequently improve malaria control and elimination strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0786-0) contains supplementary material, which is available to authorized users.
“…First, we did not detect pfCRT mutations from Malawi samples, consistent with the return of chloroquine-susceptible malaria in Malawi after chloroquine use was abandoned ( 41 ). The pfDHFR-pfDHPS mutant patterns IRNL -SG EG A and IRNL - A G E AA (mutations are underlined), which are linked to high-level sulfadoxine-pyrimethamine (SP) resistance ( 42 ), were observed as predominant alleles in Thailand and China samples, respectively. The pfMDR1 Y184F was the most common mutation observed in this study and has been reported as a common mutation found in Asia and Africa ( 43 – 45 ).…”
Antimalarial drug resistance exacerbates the global disease burden and complicates eradication efforts. To facilitate the surveillance of resistance markers in countries of malaria endemicity, we developed a suite of TaqMan assays for known resistance markers and compartmentalized them into a single array card (TaqMan array card, TAC). We included 87 assays for species identification, for the detection of Plasmodium falciparum mutations associated with chloroquine, atovaquone, pyrimethamine, sulfadoxine, and artemisinin resistance, and for neutral single nucleotide polymorphism (SNP) genotyping. Assay performance was first optimized using DNA from common laboratory parasite lines and plasmid controls. The limit of detection was 0.1 to 10 pg of DNA and yielded 100% accuracy compared to sequencing. The tool was then evaluated on 87 clinical blood samples from around the world, and the malaria TAC once again achieved 100% accuracy compared to sequencing and in addition detected the presence of mixed infections in clinical samples. With its streamlined protocol and high accuracy, this malaria TAC should be a useful tool for large-scale antimalarial resistance surveillance.
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