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8 recordsLinked to original sources
PubMed2022

High prevalence of Pfcrt 76T and Pfmdr1 N86 genotypes in malaria infected patients attending health facilities in East Shewa zone, Oromia Regional State, Ethiopia.

BACKGROUND: Plasmodium falciparum resistance to series of anti-malarial drugs is a major challenge in efforts to control and/or eliminate malaria globally. In 1998, following the widespread of chloroquine (CQ) resistant P. falciparum, Ethiopia switched from CQ to sulfadoxine-pyrimethamine (SP) and subsequently in 2004 from SP to artemether-lumefantrine (AL) for the treatment of uncomplicated falciparum malaria. Data on the prevalence of CQ resistance markers after more than two decades of its removal is important to map the selection pressure behind the targets codons of interest. The present study was conducted to determine the prevalence of mutations in Pfcrt K76T and Pfmdr1 N86Y codons among malaria-infected patients from Adama, Olenchiti and Metehara sites of East Shewa zone, Oromia Regional State, Ethiopia. METHODS: Finger-prick whole blood samples were collected on 3MM Whatman ® filter papers from a total of 121 microscopically confirmed P. falciparum infected patients. Extraction of parasite DNA was done by Chelex-100 method from dried blood spot (DBS). Genomic DNA template was used to amplify Pfcrt K76T and Pfmdr1 N86Y codons by nested PCR. Nested PCR products were subjected to Artherobacter protophormiae-I (APoI) restriction enzyme digestion to determine mutations at codons 76 and 86 of Pfcrt and Pfmdr1 genes, respectively. RESULTS: Of 83 P. falciparum isolates successfully genotyped for Pfcrt K76T, 91.6% carried the mutant genotypes (76T). The prevalence of Pfcrt 76T was 95.7%, 92.5% and 84.5% in Adama, Metehara and Olenchiti, respectively. The prevalence of Pfcrt 76T mutations in three of the study sites showed no statistical significance difference (χ2 = 1.895; P = 0.388). On the other hand, of the 80 P. falciparum samples successfully amplified for Pfmdr1, all carried the wild-type genotypes (Pfmdr1 N86). CONCLUSION: Although CQ officially has been ceased for the treatment of falciparum malaria for more than two decades in Ethiopia, greater proportions of P. falciparum clinical isolates circulating in the study areas carry the mutant 76T genotypes indicating the presence of indirect CQ pressure in the country. However, the return of Pfmdr1 N86 wild-type allele may be favoured by the use of AL for the treatment of uncomplicated falciparum malaria.

Antimalarials↗
PubMed1992

Selection for high-level chloroquine resistance results in deamplification of the pfmdr1 gene and increased sensitivity to mefloquine in Plasmodium falciparum.

A chloroquine resistant cloned isolate of Plasmodium falciparum, FAC8, which carries an amplification in the pfmdr1 gene was selected for high-level chloroquine resistance, resulting in a cell line resistant to a 10-fold higher concentration of chloroquine. These cells were found to have lost the amplification in pfmdr1 and to no longer over-produce the protein product termed P-glycoprotein homologue 1 (Pgh1). The pfmdr1 gene from this highly resistant cell line was not found to encode any amino acid changes that would account for increased resistance. Verapamil, which reverses chloroquine resistance in FAC8, also reversed high-level chloroquine resistance. Furthermore, verapamil caused a biphasic reversal of chloroquine resistance as the high-level resistance was very sensitive to low amounts of verapamil. These data suggest that over-expression of the P-glycoprotein homologue is incompatible with high levels of chloroquine resistance. In order to show that these results were applicable to other chloroquine selected lines, two additional mutants were selected for resistance to high levels of chloroquine. In both cases they were found to deamplify pfmdr1. Interestingly, while the level of chloroquine resistance of these mutants increased, they became more sensitive to mefloquine. This suggests a linkage between the copy number of the pfmdr1 gene and the level of chloroquine and mefloquine resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Amplification of the multidrug resistance gene pfmdr1 in Plasmodium falciparum has arisen as multiple independent events.

The multidrug resistance (MDR) phenotype in mammalian tumor cells can involve amplification of mdr genes that results in overexpression of the protein product termed P-glycoprotein. Chloroquine resistance (CQR) in Plasmodium falciparum has similarities with the MDR phenotype in tumor cells, and some isolates of P. falciparum have amplified levels of the pfmdr1 gene. To investigate the nature and origin of pfmdr1 amplicons, we have cloned large regions of a 110-kb amplicon from the CQR cloned isolate B8 by using the yeast artificial chromosome system. We have identified and sequenced the breakpoints of the amplicon by a novel method employing inverted polymerase chain reaction that is applicable to analysis of any large-scale repeat. We show that the five copies of the amplicon in this isolate are in a head to tail configuration. A string of 30 A's flank the breakpoints on each side of the amplified segment, suggesting a mechanism for the origin of the tandem amplification. Polymerase chain reaction analysis with oligonucleotides that cross the B8 breakpoint has shown in 26 independent CQR isolates, 16 of which contain amplified copies of pfmdr1, that amplification of the pfmdr1 gene in P. falciparum has arisen as multiple independent events. These results suggest that this region of the genome is under strong selective pressure.

Animals↗
PubMedDate not supplied

Chloroquine-resistant Plasmodium falciparum isolates from the Sudan lack two mutations in the pfmdr1 gene thought to be associated with chloroquine resistance.

Isolates of Plasmodium falciparum from 3 areas of the Sudan were recovered from cryopreservation in London and their chloroquine sensitivity was determined in vitro. Chloroquine resistance was detected in 6/6 isolates from Khartoum, 1/4 from Sennar and 3/3 from Gadarif, indicating that resistance is spreading. All the isolates were sensitive to mefloquine. Studies using blood spots on glass fibre discs and the polymerase chain reaction did not detect two mutations in the pfmdr1 gene, thought to be correlated with chloroquine-resistance, in any of the isolates studied.

Amino Acid Sequence↗
PubMed2026

Artemether-lumefantrine for the treatment of Plasmodium falciparum malaria in Laos: a therapeutic efficacy study coupled with genomic and in vitro phenotypic analyses.

BACKGROUND: Artemisinin-based combination therapies (ACTs) have played a crucial role in decreasing the impact of malaria worldwide. Since 2005, artemether-lumefantrine (AL) has been the main first-line treatment for uncomplicated Plasmodium falciparum malaria in Laos. Herein, we aimed to study the efficacy of AL in the context of malaria elimination in Laos. METHODS: Between Aug 1, 2019, and June 11, 2023, AL efficacy was evaluated in four provinces of southern Laos: Attapeu, Champassack, Salavan, and Savannakhet. Adults and children (aged 1-60 years) with microscopically confirmed P falciparum malaria received oral AL twice a day for 3 days, with follow-up on days 7, 14, 21, and 28. The primary outcome was PCR-adjusted adequate clinical and parasitological response (ACPR) by day 28. Resistance to dihydroartemisinin (DHA) and lumefantrine (LM) was assessed by an in vitro phenotypic analysis, and mutations in P falciparum kelch13 (pfkelch13), P falciparum multidrug resistance 1 (pfmdr1), P falciparum plasmepsin 2 (pfpm2), and P falciparum chloroquine resistant transporter (pfcrt) were characterised in parasites collected from enrolled patients. Safety outcomes included the frequency and nature of adverse events and serious adverse events. FINDINGS: A total of 198 patients (median age 16 years [IQR 10-28]; 124 [63%] male and 74 [37%] female) were initially enrolled, of whom three were lost to follow-up, resulting in 195 patients who received the 3-day AL regimen. At day 28, the PCR-adjusted ACPR was 96% (95% CI 92-98), with a treatment failure rate of 2% (1-5) and a reinfection rate of 2% (1-5). Among the four PCR-confirmed recrudescent isolates, one showed markedly reduced LM susceptibility (LM 50% inhibitory concentration [IC50] 59·9 nM, 2·5 times higher than the median IC50 of other isolates) and high artemisinin resistance in vitro (ring-stage survival survival rate 35·8%), which was associated with the pfkelch13 R539T mutation and day-3 microscopy-positive parasitaemia. Among 190 isolates with successfully determined pfkelch13 sequencing, nine (5%) carried the pfkelch13 mutation R539T and 43 (23%) carried the C580Y mutation, and both were associated with day-3 microscopy-positive parasitaemia (p=0·044). No amplification of pfmdr1 or pfpm2, nor any mutations in pfmdr1 and pfcrt, were associated with treatment failure. INTERPRETATION: Our findings indicate the potential emergence of LM resistance in Laos. Although AL remains efficacious, vigilance for decreasing efficacy and close monitoring of LM efficacy should be considered to support the country's goal of eliminating malaria by 2030. Importantly, none of the known pfmdr1 or pfcrt haplotypes were uniquely associated with treatment failure, including the isolate with the highest LM IC50, underscoring the need to identify reliable molecular markers for LM resistance. FUNDING: Bill and Melinda Gates Foundation and The Global Fund.

Humans↗
PubMed1990

Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum.

The lethal form of human malaria caused by Plasmodium falciparum is virtually uncontrollable in many areas because of the development of drug resistance, in particular chloroquine resistance (CQR). CQR is biologically similar to the multiple drug resistance phenotype (MDR) of mammalian tumour cells, as both involve expulsion of drug from the cell and both can be reversed by calcium channel antagonists. A homologue (pfmdr1) of the mammalian multidrug resistance gene has been implicated in CQR because it is amplified in some CQR isolates of P. falciparum as is an mdr gene in MDR tumour cells. We show here that the complete sequences of pfmdr1 genes from 2 CQ sensitive (CQS) P. falciparum isolates are identical. In 5 CQR isolates, 1-4 key nucleotide differences resulted in amino acid substitutions. On the basis of these substitutions, we have correctly predicted the CQS/CQR status of a further 34 out of 36 isolates. This is a paradox as CQR arises much less frequently than would be predicted if single point mutations were sufficient. We conclude that a mutated pfmdr1 gene is one of at least two mutated genes required for CQR.

Alleles↗
PubMed1991

A P-glycoprotein homologue of Plasmodium falciparum is localized on the digestive vacuole.

Resistance to chloroquine in Plasmodium falciparum bears a striking similarity to the multi-drug resistance (MDR) phenotype of mammalian tumor cells which is mediated by overexpression of P-glycoprotein. We show here that the P. falciparum homologue of the P-glycoprotein (Pgh1) is a 160,000-D protein that is expressed throughout the asexual erythrocytic life cycle of the parasite. Quantitative immunoblotting analysis has shown that the protein is expressed at approximately equal levels in chloroquine resistant and sensitive isolates suggesting that overexpression of Pgh1 is not essential for chloroquine resistance. The chloroquine-resistant cloned line FAC8 however, does express approximately threefold more Pgh1 protein than other isolates which is most likely because of the increased pfmdr1 gene copy number present in this isolate. Immunofluorescence and immunoelectron microscopy has demonstrated that Pgh1 is localized on the membrane of the digestive vacuole of mature parasites. This subcellular localization suggests that Pgh1 may modulate intracellular chloroquine concentrations and has important implications for the normal physiological function of this protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗