Plasmodium Falciparum Chloroquine Resistance Transporter Research Articles

Distribution patterns of molecular markers of antimalarial drug resistance in Plasmodium falciparum isolates on the Thai-Myanmar border during the periods of 1993–1998 and 2002–2008

BackgroundPolymorphisms of Plasmodium falciparum chloroquine resistance transporter (pfcrt), Plasmodium falciparum multi-drug resistance 1 (pfmdr1) and Plasmodium falciparum kelch 13-propeller (pfk13) genes are accepted as valid molecular markers of quinoline antimalarials and artemisinins. This study investigated the distribution patterns of these genes in P. falciparum isolates from the areas along the Thai-Myanmar border during the two different periods of antimalarial usage in Thailand.ResultsPolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to detect pfcrt mutations at codons 76, 220, 271, 326, 356, and 371 as well as pfmdr1 mutation at codon 86. The prevalence of pfcrt mutations was markedly high (96.4–99.7%) in samples collected during both periods. The proportions of mutant genotypes (number of mutant/total isolate) at codons 76, 220, 271, 326, 356 and 371 in the isolates collected during 1993–1998 (period 1) compared with 2002–2008 (period 2) were 97.9% (137/140) vs. 97.1% (401/413), 97.9% (140/143) vs. 98.8% (171/173), 97.2% (139/143) vs. 97.1% (333/343), 98.6% (140/142) vs. 99.7% (385/386), 96.4% (134/139) vs. 98.2% (378/385) and 97.8% (136/139) vs. 98.9% (375/379), respectively. Most isolates carried pfmdr1 wild-type at codon 86, with a significant difference in proportions genotypes (number of wild type/total sample) in samples collected during period 1 [92.9% (130/140)] compared with period 2 [96.9% (379/391)]. Investigation of pfmdr1 copy number was performed by real-time PCR. The proportions of isolates carried 1, 2, 3 and 4 or more than 4 copies of pfmdr1 (number of isolates carried correspondent copy number/total isolate) were significantly different between the two sample collecting periods (65.7% (90/137) vs. 87.8% (390/444), 18.2% (25/137) vs. 6.3%(28/444), 5.1% (7/137) vs. 1.4% (6/444) and 11.0% (15/137) vs. 4.5% (20/444), for period 1 vs. period 2, respectively). No pfk13 mutation was detected by nested PCR and nucleotide sequencing in all samples with successful analysis (n = 68).ConclusionsThe persistence of pfcrt mutations and pfmdr1 wild-types at codon 86, along with gene amplification in P. falciparum, contributes to the continued resistance of chloroquine and mefloquine in P. falciparum isolates in the study area. Regular surveillance of antimalarial drug resistance in P. falciparum, incorporating relevant molecular markers and treatment efficacy assessments, should be conducted.

Epitope specific antibodies to N- and C cytoplasmic domains of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) differentiate native and post-translationally modified variant

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (or PfCRT) were shown to be causative of decreased sensitivity to diverse quinoline-based antimalarials. In this report we describe the identification of a post-translational variant of PfCRT using highly characterized antibodies raised against its N- and C-terminal cytoplasmic domains (e.g., 58 and 26 amino acids, respectively). Western blot analyses of P. falciparum protein extracts with anti N-PfCRT antiserum revealed two polypeptides with apparent molecular masses of 52 kDa and 42 kDa, relative to the calculated molecular mass of PfCRT of 48.7 kDa. The 52 kDa polypeptide was detectable with anti C-PfCRT antiserum, only after alkaline phosphatase treatment of P. falciparum extracts. Detailed epitope mapping of anti N- and C-PfCRT antisera revealed epitopes covering two previously identified phosphorylation sites, Ser411 and Thr416, whereby substitution of these residues with Asp amino acid, to mimic phosphorylated residues, dramatically inhibited anti C-PfCRT binding. Consistently, alkaline phosphatase treatment of P. falciparum extract unmasked the binding of anti C-PfCRT to the 52 kDa polypeptide, suggesting that the 52 kDa but not 42 kDa polypeptide is phosphorylated at its C-terminal Ser411 and Thr416. Interestingly, Pfcrt expressed in HEK-293F human kidney cells showed the same reactive polypeptides with anti N- and C-PfCRT antisera, consistent with PfCRT origin of the two polypeptides (e.g., 42 kDa and 52 kDa), but lacking PfCRT phosphorylation at its C-terminal. Immunohistochemical staining of late trophozoite-infected erythrocytes with anti N-or C-PfCRT antisera showed both polypeptides are localized to the parasite's digestive vacuole. Moreover, both polypeptides are detected in chloroquine-susceptible and -resistant strains of P. falciparum. This is the first report describing a post-translationally modified variant of PfCRT. The physiologic role of the 52 kDa phosphorylated PfCRT in P. falciparum remains to be determined.

Structures of Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) Isoforms and Their Interactions with Chloroquine.

Using a recently elucidated atomic-resolution cryogenic electron microscopy (cryo-EM) structure for the Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein 7G8 isoform as template [Kim, J.; Nature 2019, 576, 315-320], we use Monte Carlo molecular dynamics (MC/MD) simulations of PfCRT embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane to solve energy-minimized structures for 7G8 PfCRT and two additional PfCRT isoforms that harbor 5 or 7 amino acid substitutions relative to 7G8 PfCRT. Guided by drug binding previously defined using chloroquine (CQ) photoaffinity probe labeling, we also use MC/MD energy minimization to elucidate likely CQ binding geometries for the three membrane-embedded isoforms. We inventory salt bridges and hydrogen bonds in these structures and summarize how the limited changes in primary sequence subtly perturb local PfCRT isoform structure. In addition, we use the "AlphaFold" artificial intelligence AlphaFold2 (AF2) algorithm to solve for domain structure that was not resolved in the previously reported 7G8 PfCRT cryo-EM structure, and perform MC/MD energy minimization for the membrane-embedded AF2 structures of all three PfCRT isoforms. We compare energy-minimized structures generated using cryo-EM vs AF2 templates. The results suggest how amino acid substitutions in drug resistance-associated isoforms of PfCRT influence PfCRT structure and CQ transport.

Pcr-rflp genotyping of pfcrt and pfmdr1 in plasmodium falciparum isolates from children in Vatomandry, Madagascar

RésuméContexteLa surveillance de l'efficacité thérapeutique et des marqueurs génétiques de la résistance aux antipaludiques est primordiale afin de détecter le plus tôt possible l’émergence des parasites potentiellement résistants. Dans ce contexte, notre étude a pour objectif de réaliser le typage du gène Plasmodium falciparum chloroquine resistance transporter ou Pfcrt et Plasmodium falciparum multidrug resistant gene 1 ou Pfmdr1 sur des isolats provenant des enfants du district de Vatomandry.MéthodologiesAu total, 142 isolats de P. falciparum collectés lors d'un dépistage actif du paludisme chez des enfants âgés de moins de 15 ans, entre février et mars des années 2016 et 2017 à Vatomandry, ont été analysés. Le typage du codon K76T du gène Pfcrt et du codon N86Y du gène Pfmdr1 a été ensuite effectué par la technique de polymérisation en chaîne suivie de digestion enzymatique (restriction fragment length polymorphism) ou PCR-RFLP.RésultatsLe taux de succès d'amplification des gènes Pfcrt et Pfmdr1 était faible, de l'ordre de 27 % et 39 % respectivement. La prévalence des isolats mutants pour le codon K76T de Pfcrt était de 2,6 % [intervalle de confiance à 95 % (IC95%) : 0,1 - 15,0 %] et de 36 % [IC95% : 23,7 - 49,7 %] pour le codon N86Y du gène Pfmdr1.ConclusionNotre étude a mis en évidence la présence d'isolats portant à la fois la mutation au niveau du codon K76T de Pfcrt et N86Y de Pfmdr1. Bien que le taux de mutation que nous avons observé soit faible, d'autres études méritent d’être effectuées afin de suivre l’évolution de ces marqueurs dans le temps et dans l'espace à Madagascar.

Mechanistic basis for multidrug resistance and collateral drug sensitivity conferred to the malaria parasite by polymorphisms in PfMDR1 and PfCRT.

Polymorphisms in the Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene and the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene alter the malaria parasite’s susceptibility to most of the current antimalarial drugs. However, the precise mechanisms by which PfMDR1 contributes to multidrug resistance have not yet been fully elucidated, nor is it understood why polymorphisms in pfmdr1 and pfcrt that cause chloroquine resistance simultaneously increase the parasite’s susceptibility to lumefantrine and mefloquine—a phenomenon known as collateral drug sensitivity. Here, we present a robust expression system for PfMDR1 in Xenopus oocytes that enables direct and high-resolution biochemical characterizations of the protein. We show that wild-type PfMDR1 transports diverse pharmacons, including lumefantrine, mefloquine, dihydroartemisinin, piperaquine, amodiaquine, methylene blue, and chloroquine (but not the antiviral drug amantadine). Field-derived mutant isoforms of PfMDR1 differ from the wild-type protein, and each other, in their capacities to transport these drugs, indicating that PfMDR1-induced changes in the distribution of drugs between the parasite’s digestive vacuole (DV) and the cytosol are a key driver of both antimalarial resistance and the variability between multidrug resistance phenotypes. Of note, the PfMDR1 isoforms prevalent in chloroquine-resistant isolates exhibit reduced capacities for chloroquine, lumefantrine, and mefloquine transport. We observe the opposite relationship between chloroquine resistance-conferring mutations in PfCRT and drug transport activity. Using our established assays for characterizing PfCRT in the Xenopus oocyte system and in live parasite assays, we demonstrate that these PfCRT isoforms transport all 3 drugs, whereas wild-type PfCRT does not. We present a mechanistic model for collateral drug sensitivity in which mutant isoforms of PfMDR1 and PfCRT cause chloroquine, lumefantrine, and mefloquine to remain in the cytosol instead of sequestering within the DV. This change in drug distribution increases the access of lumefantrine and mefloquine to their primary targets (thought to be located outside of the DV), while simultaneously decreasing chloroquine’s access to its target within the DV. The mechanistic insights presented here provide a basis for developing approaches that extend the useful life span of antimalarials by exploiting the opposing selection forces they exert upon PfCRT and PfMDR1.

Molecular identification and anti-malarial drug resistance profile of Plasmodium falciparum from patients attending Kisoro Hospital, southwestern Uganda

BackgroundThe evolution of malaria infection has necessitated the development of highly sensitive diagnostic assays, as well as the use of dried blood spots (DBS) as a potential source of deoxyribonucleic acid (DNA) yield for polymerase chain reaction (PCR) assays. This study identified the different Plasmodium species in malaria-positive patients, and the anti-malarial drug resistance profile for Plasmodium falciparum using DBS samples collected from patients attending Kisoro Hospital in Kisoro district, Southwestern Uganda.MethodsThe blood samples were prospectively collected from patients diagnosed with malaria to make DBS, which were then used to extract DNA for real-time PCR and high-resolution melting (HRM) analysis. Plasmodium species were identified by comparing the control and test samples using HRM-PCR derivative curves. Plasmodium falciparum chloroquine (CQ) resistance transporter (pfcrt) and kelch13 to screen the samples for anti-malarial resistance markers. The HRM-PCR derivative curve was used to present a summary distribution of the different Plasmodium species as well as the anti-malarial drug profile.ResultsOf the 152 participants sampled, 98 (64.5%) were females. The average age of the participants was 34.9 years (range: 2 months–81 years). There were 134 samples that showed PCR amplification, confirming the species as Plasmodium. Plasmodium falciparum (N = 122), Plasmodium malariae (N = 6), Plasmodium ovale (N = 4), and Plasmodium vivax (N = 2) were the various Plasmodium species and their proportions. The results showed that 87 (71.3%) of the samples were sensitive strains/wild type (CVMNK), 4 (3.3%) were resistant haplotypes (SVMNT), and 31 (25.4%) were resistant haplotypes (CVIET). Kelch13 C580Y mutation was not detected.ConclusionThe community served by Kisoro hospital has a high Plasmodium species burden, according to this study. Plasmodium falciparum was the dominant species, and it has shown that resistance to chloroquine is decreasing in the region. Based on this, molecular identification of Plasmodium species is critical for better clinical management. Besides, DBS is an appropriate medium for DNA preservation and storage for future epidemiological studies.

Investigation of <i>Plasmodium falciparum</i> Resistance Biomarkers among Primary School Children in Western Kenya

Background: A marked decrease in malaria-related deaths worldwide has been attributed to the administration of effective antimalarials against Plasmodium falciparum. However, the continuous spread of P. falciparum resistance to anti-malarial drugs is raising a serious problem in controlling Malaria to the vulnerable children’s immune system. In recent studies, Plasmodium falciparum Kelch 13 propeller gene (Pfk13) has been reported to develop resistance to artemisinin in South Asia. In this study, we checked Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt) involved in chloroquine (CQ) resistance. Method: In this study, archived 280 samples were collected from Alupe primary school children in Busia, Western Kenya from May, 2016 to November, 2016. Genomic DNA was extracted using the MightyPrep reagent. The samples were investigated for P. falciparum positivity out of which 67 of them tested positive giving a prevalence rate of 24%. The sixty-seven were subjected to PCR amplification for the molecular marker resistance to Pfcrt. After PCR amplification, the amplicons were purified and sequenced using Sanger Sequencing. The sequence data were analyzed using BioEdit software to identify point mutations. Results: 14 samples sequences were analyzed on Bioedit software giving the following amino acid changes F76C, Y66H, L70A, Y58C, T59V, V65I, P67L, T81L, Y60S, Y66S, P67T and I71F). New mutations have been reported at position 76 leading to an amino acid change, one of Pfcrt gold standard biomarkers. However, amino acid changes Y66H, L70A, Y58C, T59V, V65I, P67L, T81L, Y60S, Y66S, P67T and I71F are newly reported giving an increase in Pfcrt prevalence of concern from zero to 5.0%. A phylogenetic evolutionary relationship was constructed as shown below. Generally, the results showed a continuous resistance of P.falciparum to Pfcrt which calls for robust continuous monitoring and surveillance. Conclusion: Due to the increase of the resistant Pfcrt gene prevalence, continuous development of new mutants against chloroquine indicates that there is need to repurpose anti-malarial drugs for future partner drugs.

Substitution of Pro165 in Transmembrane 4 of PfCRT Abolishes Lysosome Acidification Function in Stably Transfected HEK-293F Cells

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is localised on the parasite digestive vacuole, an organelle that maintains an acidic lumen. Here we demonstrated the isolation of HEK-293F cells stably expressing wild type 3D7 and mutant Pfcrt alleles. Immuno-fluorescence staining of HEK-293F transfectants confirms the localization of Pfcrt alleles to the lysosomal vesicles. Moreover, cells expressing mut-PfCRTETSE showed greater lysosomal acidification as demonstrated by the dramatic increase in the accumulation of two weak bases, acridine orange and CytiPainter LysoOrange dyes. Furthermore, HEK-293 cells stably expressing mut-PfCRTETSE with a single substitution of proline 165 in transmembrane 4 completely inhibited the accumulation of weak bases. Taken together, our results demonstrate the role of Pro165 in PfCRT lysosomal acidification function in HEK-293F cells.

Detection of antimalarial drug resistance polymorphisms in Plasmodium falciparum chloroquine resistance transporter and Plasmodium falciparum multidrug resistance 1 genes of Plasmodium falciparum found in Kano State, Nigeria

Objectives: In 2018, malaria claimed an estimated 380,000 lives in African region, with Nigeria accounting for 24.0% (91,368) of malaria deaths from the region. Mutations in Plasmodium falciparum chloroquine resistance transporter (Pfcrt) and P. falciparum multidrug resistance 1 (Pfmdr-1) genes had reduced the effective use of artemisinin combination therapy through the development of resistance to these antimalarial agents. Our study set out to determine the antimalarial drug resistance polymorphisms in Pfcrt and Pfmdr-1 genes of P. falciparum isolates among patients in Kano State, Nigeria. Material and Methods: Malaria positive samples were collected across the three senatorial districts of Kano State. The samples were amplified using nested polymerase chain reaction to detect the Pfcrt and Pfmdr-1 genes. The amplicons were sequenced and bioinformatic analysis was done using CLC Sequence viewer 8.0 and BioEdit sequence alignment editor to detect the single-nucleotide polymorphisms. Results: In the Pfcrt gene, CVIET haplotype was seen in 26.2% of the samples while only two samples showed the 86Y mutation in the Pfmdr-1 gene. All the 86Y mutations and majority of the CVIET haplotypes were detected in the patients from rural settings where some of them noted that they consumed modern and traditional (herbs) antimalarial agents. One sample was observed to have the CVIET haplotype and N86Y mutation while the other five CVIET haplotypes were seen in five separate samples. A new mutation V62A was found in the Pfmdr-1 gene as observed in one of the sample. Conclusion: It is imperative to ensure the rational use of the right antimalarial agents and employ continuous resistance surveillance/mapping to ensure synergy in malaria containment and elimination strategies.

Epitope-specific IgG pools identify PfCRT monomer and homodimer polypeptides that are differentially phosphorylated at Ser411 in Plasmodium falciparum

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a phospho-protein with three identified phosphorylation sites (Ser33, Ser411 and Thr416) at its cytosolic N- and C-termini. In this study, we report on the characterization of PfCRT anti-serum and show the presence of three epitope-specific immunoglobulin (IgG) pools (i.e., IgG-P1, P2, and P3), each recognizing a different epitope in PfCRT cytoplasmic C-terminal. IgG-P2 bound the heptapeptide sequence (408NEDSEGE414), including Ser411. The effect of Ser411 phosphorylation on the binding specificity of IgG-P2 was confirmed using heptapeptides and full-length PfCRT with substitutions of Ser411 with aspartic acid (phospho-serine mimic) and alanine residues. Moreover, using purified IgG-P2, we show the presence of PfCRT homodimer that has un-phosphorylated Ser411 and migrates with an apparent molecular mass of 90 kDa on SDS-PAGE. In addition, parasite lysates showed PfCRT to be more phosphorylated at Ser411 in CQ-sensitive (3D7) than CQ–resistant (Dd2-H) strains of P. falciparum. Taken together, the findings of this study suggest a role for Ser411 phosphorylation in PfCRT structure-function.

Surveillance of Plasmodium falciparum pfcrt haplotypes in southwestern Uganda by high\u2010resolution melt analysis

Background Chloroquine (CQ) resistance is conferred by mutations in the Plasmodium falciparum CQ resistance transporter (pfcrt). Following CQ withdrawal for anti-malarial treatment, studies across malaria-endemic countries have shown a range of responses. In some areas, CQ sensitive parasites re-emerge, and in others, mutant haplotypes persist. Active surveillance of resistance mutations in clinical parasites is essential to inform treatment regimens; this effort requires fast, reliable, and cost-effective methods that work on a variety of sample types with reagents accessible in malaria-endemic countries.MethodsQuantitative PCR followed by High-Resolution Melt (HRM) analysis was performed in a field setting to assess pfcrt mutations in two groups of clinical samples from Southwestern Uganda. Group 1 samples (119 in total) were collected in 2010 as predominantly Giemsa-stained slides; Group 2 samples (125 in total) were collected in 2015 as blood spots on filter paper. The Rotor-Gene Q instrument was utilized to assess the impact of different PCR-HRM reagent mixes and the detection of mixed haplotypes present in the clinical samples. Finally, the prevalence of the wild type (CVMNK) and resistant pfcrt haplotypes (CVIET and SVMNT) was evaluated in this understudied Southwestern region of Uganda.ResultsThe sample source (i.e. Giemsa-stained slides or blood spots) and type of LCGreen-based reagent mixes did not impact the success of PCR-HRM. The detection limit of 10− 5 ng and the ability to identify mixed haplotypes as low as 10 % was similar to other HRM platforms. The CVIET haplotype predominated in the clinical samples (66 %, 162/244); however, there was a large regional variation between the sample groups (94 % CVIET in Group 1 and 44 % CVIET in Group 2).ConclusionsThe HRM-based method exhibits the flexibility required to conduct reliable assessment of resistance alleles from various sample types generated during the clinical management of malaria. Large regional variations in CQ resistance haplotypes across Southwestern Uganda emphasizes the need for continued local parasite genotype assessment to inform anti-malarial treatment policies.

Synthesis and biological evaluation of benzhydryl-based antiplasmodial agents possessing Plasmodium falciparum chloroquine resistance transporter (PfCRT) inhibitory activity.

Due to the surge in resistance to common therapies, malaria remains a significant concern to human health worldwide. In chloroquine (CQ)-resistant (CQ-R) strains of Plasmodium falciparum, CQ and related drugs are effluxed from the parasite's digestive vacuole (DV). This process is mediated by mutant isoforms of a protein called CQ resistance transporter (PfCRT). CQ-R strains can be partially re-sensitized to CQ by verapamil (VP), primaquine (PQ) and other compounds, and this has been shown to be due to the ability of these molecules to inhibit drug transport via PfCRT. We have previously developed a series of clotrimazole (CLT)-based antimalarial agents that possess inhibitory activity against PfCRT (4a,b). In our endeavor to develop novel PfCRT inhibitors, and to perform a structure-activity relationship analysis, we synthesized a new library of analogues. When the benzhydryl system was linked to a 4-aminoquinoline group (5a-f) the resulting compounds exhibited good cytotoxicity against both CQ-R and CQ-S strains of P.falciparum. The most potent inhibitory activity against the PfCRT-mediated transport of CQ was obtained with compound 5k. When compared to the reference compound, benzhydryl analogues of PQ (5i,j) showed a similar activity against blood-stage parasites, and a stronger invitro potency against liver-stage parasites. Unfortunately, in the invivo transmission blocking assays, 5i,j were inactive against gametocytes.

Distribution and Temporal Dynamics of Plasmodium falciparum Chloroquine Resistance Transporter Mutations Associated With Piperaquine Resistance in Northern Cambodia

Newly emerged mutations within the Plasmodium falciparum chloroquine resistance transporter (PfCRT) can confer piperaquine resistance in the absence of amplified plasmepsin II (pfpm2). In this study, we estimated the prevalence of co-circulating piperaquine resistance mutations in P. falciparum isolates collected in northern Cambodia from 2009 to 2017. The sequence of pfcrt was determined for 410 P. falciparum isolates using PacBio amplicon sequencing or whole genome sequencing. Quantitative polymerase chain reaction was used to estimate pfpm2 and pfmdr1 copy number. Newly emerged PfCRT mutations increased in prevalence after the change to dihydroartemisinin-piperaquine in 2010, with >98% of parasites harboring these mutations by 2017. After 2014, the prevalence of PfCRT F145I declined, being outcompeted by parasites with less resistant, but more fit PfCRT alleles. After the change to artesunate-mefloquine, the prevalence of parasites with amplified pfpm2 decreased, with nearly half of piperaquine-resistant PfCRT mutants having single-copy pfpm2. The large proportion of PfCRT mutants that lack pfpm2 amplification emphasizes the importance of including PfCRT mutations as part of molecular surveillance for piperaquine resistance in this region. Likewise, it is critical to monitor for amplified pfmdr1 in these PfCRT mutants, as increased mefloquine pressure could lead to mutants resistant to both drugs.

Polymorphism Analysis of pfmdr1 and pfcrt from Plasmodium falciparum Isolates in Northwestern Nigeria Revealed the Major Markers Associated with Antimalarial Resistance.

Suspicion of failure in the effectiveness of artemisinin-based combination therapies (currently the first-line treatment of malaria, worldwide) is leading to the unofficial use of alternative antimalarials, including chloroquine and sulfadoxine/pyrimethamine, across northern Nigeria. To facilitate evidence-based resistance management, antimalarial resistance mutations were investigated in Plasmodium falciparum multidrug resistance-1 (pfmdr1) and chloroquine resistance transporter (pfcrt), in isolates from Kano, northwestern Nigeria. Out of the 88 samples genotyped for pfmdr1 N86Y mutation using PCR/restriction fragment length polymorphism, one sample contained the 86Y mutation (86Yfrequency = 1.14%). The analysis of 610 bp fragments of pfmdr1 from 16 isolates revealed two polymorphic sites and low haplotype diversity (Hd = 0.492), with only 86 Y mutations in one isolate, and 184 F replacements in five isolates (184Ffrequency = 31.25%). The analysis of 267 bp fragments of pfcrt isolates revealed high polymorphism (Hd = 0.719), with six haplotypes and seven non-synonymous polymorphic sites. Eleven isolates (61.11%) were chloroquine-resistant, CQR (C72V73I74E75T76 haplotype), two of which had an additional mutation, D57E. An additional sequence was CQR, but of the C72V73M74E75T76 haplotype, while the rest of the sequences (33.33%) were chloroquine susceptible (C72V73M74N75K76 haplotype). The findings of these well characterized resistance markers should be considered when designing resistance management strategies in the northwestern Nigeria.

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (Pfcrt) and multidrug-resistant gene 1 (Pfmdr-1) in Nigerian children 10 years post-adoption of artemisinin-based combination treatments

The emergence and spread of Plasmodium falciparum parasites resistant to artemisinin derivatives and their partners in southeastern Asia threatens malaria control and elimination efforts, and heightens the need for an alternative therapy. We have explored the distribution of P. falciparum chloroquine resistance transporter (Pfcrt) and multidrug-resistant gene 1 (Pfmdr-1) haplotypes 10 years following adoption of artemisinin-based combination therapies in a bid to investigate the possible re-emergence of Chloroquine-sensitive parasites in Nigeria, and investigated the effect of these P. falciparum haplotypes on treatment outcomes of patients treated with artemisinin-based combination therapies. A total of 271 children aged <5 years with uncomplicated falciparum malaria were included in this study. Polymorphisms on codons 72–76 of the Pfcrt gene and codon 86 and 184 of Pfmdr-1 were determined using the high resolution melting assay. Of 240 (88.6%) samples successfully genotyped with HRM for Pfcrt, wildtype C72M74N75K76 (42.9%) and mutant C72I74E75T76 (53.8%) were observed. Also, wildtype N86Y184 (62.9%) and mutant N86F184 (21.1%), Y86Y184 (6.4%), and Y86F184 (0.4%) haplotypes of Pfmdr-1 were observed. Measures of responsiveness to ACTs were similar in children infected with P. falciparum crt haplotypes (C72I74E75T76 and C72M74N75K76) and major mdr-1 haplotypes (N86Y184, N86F184 and Y86Y184). Despite a 10 year gap since the malaria treatment policy changed to ACTs, over 50% of the P. falciparum parasites investigated in this study harboured the Chloroquine-resistant C72I74E75T76 haplotype, however this did not compromise the efficacy of artemisinin-based combination therapies. Should complete artemisinin resistance emerge from or spread to Nigeria, chloroquine might not be a good alternative therapy.

Changing Prevalence of Potential Mediators of Aminoquinoline, Antifolate, and Artemisinin Resistance Across Uganda.

In Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit efficacies. Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays. Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0-46% in 2018; 0-23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively). We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.

Persistence of Markers of Chloroquine Resistance in Plasmodium falciparum in Bangladesh.

The control of malaria, in terms of drug resistance, remains a significant global challenge, with Bangladesh, a malaria-endemic country, being no exception. The aim of this study was to explore antimalarial resistance in Bangladesh by molecular analysis of Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance transporter 1 (pfmdr1) genetic markers of P. falciparum. Samples were obtained from uncomplicated malaria patients between 2009 and 2014 from six malaria-endemic districts. Based on parasite transmission intensity, the endemic districts were divided into high-transmission (Chittagong Hill Tracts [CHT]) and low-transmission (non-CHT) regions. Falciparum malaria-positive isolates were genotyped for K76T of the pfcrt gene, and N86Y and Y184F of the pfmdr1 gene: in total, 262 P. falciparum clinical isolates were analyzed. In CHT areas, the prevalence of polymorphisms was 70.6% for 76T, 14.4% for 86Y, and 7.8% for 184F. In non-CHT areas, 76T and 86Y mutations were found in 78.0% and 19.5% of the samples, respectively, whereas no 184F mutations were observed. We compared our data with previous similar molecular observations, which shows a significant decrease in pfcrt 76T mutation prevalence. No pfmdr1 amplification was observed in any of the samples suggesting an unaltered susceptibility to amino alcohol drugs such as mefloquine and lumefantrine. This study provides an updated assessment of the current status of pfcrt and pfmdr1 gene mutations in Bangladesh, and suggests there is persistent high prevalence of markers of resistance to aminoquinoline drugs.

The impact of HIV-associated immunosuppression on the Plasmodium falciparum chloroquine resistance transporter gene (PfCRT) of HIV patients in Akure, Nigeria

BackgroundThe study evaluated the prevalence of malaria and Plasmodium falciparum chloroquine resistance transporter gene (PfCRT) in HIV patients attending Specialist Hospital, Akure. This study was carried out between April and June 2019. Three hundred and seventeen (317) patients attending the antiretroviral clinic (ART) were involved, out of which 89 (28.08%) were males and 228 (71.92%) were females. HIV test was done using the Unigold® HIV test kit, malaria test was done using thick and thin blood smear, CD4 test was done using the Partec® CD4 counter and PCR was used to detect the presence of plasmodium falciparum mutant gene. The data obtained from this analysis was subjected to Pearson’s Chi-square test.ResultsThe overall result showed low prevalence of malaria (23.03%) in the sampled patients. Highest malaria prevalence (31.0%) was recorded in HIV patients with CD4 count between 200–500 cells/μl of blood, with the males recording 24.7% malaria prevalence. The age group 20–29 years recorded the highest prevalence of 27.3%. A higher prevalence 91.1% of PfCRT gene in HIV-positive and (40.0%) in HIV-negative patients was recorded with 100% prevalence in patients with CD4 count ≤ 200. This shows that the low prevalence of malaria recorded in this study could be credited to good health-seeking attitude of HIV patients and the upscale of HIV care and treatment centres.ConclusionThe high prevalence of PfCRT gene shows that treatment of malaria with chloroquine is still being practised despite the availability of artemisinin-based combination therapy (ACTs) as the recommended regimen for malaria treatment.

The natural function of the malaria parasite\u2019s chloroquine resistance transporter

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a key contributor to multidrug resistance and is also essential for the survival of the malaria parasite, yet its natural function remains unresolved. We identify host-derived peptides of 4-11 residues, varying in both charge and composition, as the substrates of PfCRT in vitro and in situ, and show that PfCRT does not mediate the non-specific transport of other metabolites and/or ions. We find that drug-resistance-conferring mutations reduce both the peptide transport capacity and substrate range of PfCRT, explaining the impaired fitness of drug-resistant parasites. Our results indicate that PfCRT transports peptides from the lumen of the parasite’s digestive vacuole to the cytosol, thereby providing a source of amino acids for parasite metabolism and preventing osmotic stress of this organelle. The resolution of PfCRT’s native substrates will aid the development of drugs that target PfCRT and/or restore the efficacy of existing antimalarials.

Altered Drug Transport by Plasmodium falciparum Chloroquine Resistance Transporter Isoforms Harboring Mutations Associated with Piperaquine Resistance.

Patterns of multiple amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT, UniProtKB Q8IBZ9) have previously been shown to mediate chloroquine resistance in P. falciparum malarial parasites. Recent reports suggest that novel mutations in PfCRT may mediate resistance to piperaquine (PPQ), which is used extensively as a partner drug in one prominent artemisinin combination therapy. How these novel PfCRT isoforms might mediate PPQ resistance (PPQR) is not known. Using codon optimization and other previously perfected methods for PfCRT analysis in yeast, we have expressed all known PPQR-associated PfCRT isoforms in Saccharomyces cerevisiae yeast and tested whether these isoforms catalyze PPQ transport. Relationships between relative PPQ and CQ transport are analyzed for these isoforms versus other previously recognized drug resistance-associated PfCRT isoforms.