Mutations In Pfcrt Gene Research Articles

Temporal evolution of the resistance genotypes of Plasmodium falciparum in isolates from Equatorial Guinea during 20\xa0years (1999 to 2019)

BackgroundMalaria is one of the deadliest diseases in the world, particularly in Africa. As such, resistance to anti-malarial drugs is one of the most important problems in terms of global malaria control. This study assesses the evolution of the different resistance markers over time and the possible influence of interventions and treatment changes that have been made in Equatorial Guinea.MethodsA total of 1223 biological samples obtained in the period 1999 to 2019 were included in the study. Screening for mutations in the pfdhfr, pfdhps, pfmdr1, and pfcrt genes was carried out by nested PCR and restriction-fragment length polymorphisms (RFLPs), and the study of pfk13 genes was carried out by nested PCR, followed by sequencing to determine the presence of mutations.ResultsThe partially and fully resistant haplotypes (pfdhfr + pfdhps) were found to increase over time. Moreover, in 2019, the fully resistant haplotype was found to be increasing, although its super-resistant counterpart remains much less prevalent. A continued decline in pfmdr1 and pfcrt gene mutations over time was also found. The number of mutations detected in pfk13 has increased since 2008, when artemisinin-based combination therapy (ACT) were first introduced, with more mutations being observed in 2019, with two synonymous and five non-synonymous mutations being detected, although these are not related to resistance to ACT. In addition, the non-synonymous A578S mutation, which is the most frequent on the African continent, was detected in 2013, although not in the following years.ConclusionsWithdrawal of the use of chloroquine (CQ) as a treatment in Equatorial Guinea has been shown to be effective over time, as wild-type parasite populations outnumber mutant populations. The upward trend observed in sulfadoxine-pyrimethamine (SP) resistance markers suggest its misuse, either alone or in combination with artesunate (AS) or amodiaquine (AQ), in some areas of the country, as was found in a previous study conducted by this group, which allows selective pressure from SP to continue. Single nucleotide polymorphisms (SNPs) 540E and 581G do not exceed the limit of 50 and 10%, respectively, thus meaning that SP is still effective as an intermittent preventive treatment (IPT) in this country. As for the pfk13 gene, no mutations have been detected in relation to resistance to ACT. However, in 2019 there is a greater accumulation of non-synonymous mutations compared to years prior to 2008.Graphical

Surveillance of Antimalarial Resistance Molecular Markers in Imported Plasmodium falciparum Malaria Cases in Anhui, China, 2012-2016.

Between 2012 and 2016, over 80% of registered malaria cases in Anhui province were Plasmodium falciparum returned from Africa. However, drug-resistance marker polymorphisms in imported P. falciparum cases have not been assessed. This study looked at the distribution of antimalarial-drug resistance by evaluating K13-propeller, pfmdr1, and pfcrt gene mutations. Fourteen synonymous and 15 nonsynonymous mutations in the K13-propeller gene were detected in samples from nine African countries, yet no candidate and validated K13 resistance mutations were found. The prevalence of pfcrt K76T and pfmdr1 N86Y mutants was 27.7% and 19.9%, respectively. Six different pfcrt genotypes were found, with C72V73M74N75T76 being the most common (89.2%). The pfcrt 76-pfmdr1 86 haplotype combination was evaluated in 173 isolates, and the N86T76 genotype was the most prevalent (50.3%). Notably, the prevalence of the N86Y mutation in Africa marked a decline from 31.0% in 2012 to 8.2% in 2016. Our findings suggest that there is no immediate threat to artemisinin efficacy in imported P. falciparum infections returned from Africa to Anhui province. Nevertheless, pfcrt K76T and pfmdr1 N86Y mutations were modestly prevalent, suggesting the presence of chloroquine resistance in these cases. Accordingly, dihydroartemisinin + piperaquine may be a better choice than artesunate + amodiaquine for the treatment of uncomplicated P. falciparum infections in Anhui province. In addition to, artemether-lumefantrine can be introduced as an alternative measure.

Prevalence of Multiple Drug-resistant Plasmodium falciparum Malaria Cases in Northeast India

Two numbers of Plasmodium falciparum field isolates from Gossingpara, Runikhata area in Chirang district of Assam had shown multiple mutations in Pfcrt-dhfr-dhps gene (up to seven mutations: One mutation in Pfcrt gene, three mutations in Pfdhfr gene and three mutations in Pfdhps gene). Similarly, two cases in Bat camp, Miao area under Changlang district of Arunachal Pradesh had shown a total of eight mutations, of which one mutation in Pfcrt gene, three mutations in Pfdhfr gene, three mutations in Pfdhps gene and one mutation in PfATPase6gene. One case in 3 Miles, Miao area of Changlang district has shown mutations in Pfcrt (one mutation), Pfdhfr (four mutations) and Pfdhps (three mutations) gene. These results indicated that there is an existence of multiple mutant P. falciparum malaria cases in northeastern region of India.

The detection of pfcrt and pfmdr1 point mutations as molecular markers of chloroquine drug resistance, Pahang, Malaysia

BackgroundMalaria is still a public health problem in Malaysia with chloroquine (CQ) being the first-line drug in the treatment policy of uncomplicated malaria. There is a scarcity in information about the magnitude of Plasmodium falciparum CQ resistance. This study aims to investigate the presence of single point mutations in the P. falciparum chloroquine-resistance transporter gene (pfcrt) at codons 76, 271, 326, 356 and 371 and in P. falciparum multi-drug resistance-1 gene (pfmdr1) at codons 86 and 1246, as molecular markers of CQ resistance.MethodsA total of 75 P. falciparum blood samples were collected from different districts of Pahang state, Malaysia. Single nucleotide polymorphisms in pfcrt gene (codons 76, 271, 326, 356 and 371) and pfmdr1 gene (codons 86 and 1246) were analysed by using mutation-specific nested PCR and restriction fragment length polymorphism (PCR-RFLP) methods.ResultsMutations of pfcrt K76T and pfcrt R371I were the most prevalent among pfcrt gene mutations reported by this study; 52% and 77%, respectively. Other codons of the pfcrt gene and the positions 86 and 1246 of the pfmdr1 gene were found mostly of wild type. Significant associations of pfcrt K76T, pfcrt N326S and pfcrt I356T mutations with parasitaemia were also reported.ConclusionThe high existence of mutant pfcrt T76 may indicate the low susceptibility of P. falciparum isolates to CQ in Peninsular Malaysia. The findings of this study establish baseline data on the molecular markers of P. falciparum CQ resistance, which may help in the surveillance of drug resistance in Peninsular Malaysia.

Mutant pfcrt "SVMNT" haplotype and wild type pfmdr1 "N86" are endemic in Plasmodium vivax dominated areas of India under high chloroquine exposure

BackgroundChloroquine resistance (CQR) phenotype in Plasmodium falciparum is associated with mutations in pfcrt and pfmdr-1 genes. Mutations at amino acid position 72-76 of pfcrt gene, here defined as pfcrt haplotype are associated with the geographic origin of chloroquine resistant parasite. Here, mutations at 72-76 and codon 220 of pfcrt gene and N86Y pfmdr-1 mutation were studied in blood samples collected across 11 field sites, inclusive of high and low P. falciparum prevalent areas in India. Any probable correlation between these mutations and clinical outcome of CQ treatment was also investigated.MethodsFinger pricked blood spotted on Whatman No.3 papers were collected from falciparum malaria patients of high and low P. falciparum prevalent areas. For pfcrt haplotype investigation, the parasite DNA was extracted from blood samples and used for PCR amplification, followed by partial sequencing of the pfcrt gene. For pfmdr-1 N86Y mutation, the PCR product was subjected to restriction digestion with AflIII endonuclease enzyme.ResultsIn 240 P. falciparum isolates with reported in vivo CQ therapeutic efficacy, the analysis of mutations in pfcrt gene shows that mutant SVMNT-S (67.50%) and CVIET-S (23.75%) occurred irrespective of clinical outcome and wild type CVMNK-A (7.91%) occurred only in adequate clinical and parasitological response samples. Of 287 P. falciparum isolates, SVMNTS 192 (66.89%) prevailed in all study sites and showed almost monomorphic existence (98.42% isolates) in low P. falciparum prevalent areas. However, CVIETS-S (19.51%) and CVMNK-A (11.84%) occurrence was limited to high P. falciparum prevalent areas. Investigation of pfmdr-1 N86Y mutation shows no correlation with clinical outcomes. The wild type N86 was prevalent in all the low P. falciparum prevalent areas (94.48%). However, mutant N86Y was comparably higher in numbers at the high P. falciparum prevalent areas (42.76%).ConclusionsThe wild type pfcrt gene is linked to chloroquine sensitivity; however, presence of mutation cannot explain the therapeutic efficacy of CQ in the current scenario of chloroquine resistance. The monomorphic existence of mutant SVMNT haplotype, infer inbreeding and faster spread of CQR parasite in areas with higher P. vivax prevalance and chloroquine exposure, whereas, diversity is maintained in pfcrt gene at high P. falciparum prevalent areas.

Alta frecuencia de mutaciones puntuales en pfcrt de Plasmodium falciparum y emergencia de nuevos haplotipos mutantes en Colombia

Studies on the molecular epidemiology of antimalarial resistance constitute a useful tool to understand the events underlying treatment failure and resistance in falciparum malaria in Colombia. Several authors have reported on the efficacy of some molecular markers to predict drug resistance in Plasmodium falciparum. The P. falciparum pfcrt gene has been widely characterized in this context. The frequency of pfcrt gene mutations in P. falciparum were associated with treatment failure to the antimalarials chloroquine, mefloquine, amodiaquine and sulfadoxine/pyrimethamine. A representative sample of 172 patients with non-complicated falciparum malaria was selected from two highly malaria-endemic areas of northeastern Colombia, the Turbo and Bajo Cauca regions. These patients were assessed for treatment response together with the status of codons 72, 74, 75 and 76 in the pfcrt gene using a PCR-RFLP approach. A high frequency of treatment failure to chloroquine (82%) and to amodiaquine (29%) was confirmed, whereas mefloquine and combined therapy remained effective. The presence of the T76 mutation in pfcrt was confirmed in all samples. The most common haplotype was CMNT (67%). No significant association was confirmed between specific haplotypes and the treatment response in any of the treatment groups. Two haplotypes, SMET and SMNT, were reported for the first time in Colombia. Twelve percent of the samples carried both mixed mutant and wild-type alleles.