A Computational Study of Molecular Mechanism of Chloroquine Resistance by Chloroquine Resistance Transporter Protein of Plasmodium falciparum via Molecular Modeling and Molecular Simulations

The molecular mechanism of chloroquine resistance by the chloroquine resistance transporter protein of Plasmodium sp. is explored using molecular modeling and computational methods. The key mutation, lysine(K)-76 to threonine(T) (LYS76THR) in the transporter protein pertains to increased recognition of the protonated forms of the antimalarial drug. Such enhanced affinity can promote drug efflux from host digestive vacuole, rendering aminoquinoline-based treatment ineffective.

The return of chloroquine-sensitive Plasmodium falciparum parasites in Jazan Region, Southwestern Saudi Arabia over a decade after the adoption of artemisinin-based combination therapy: analysis of genetic mutations in the pfcrt gene

This study investigated the polymorphism in the P. falciparum chloroquine resistance transporter (pfcrt) gene 11 years after chloroquine (CQ) cessation in Jazan region, southwestern Saudi Arabia. Two hundred and thirty-five P. falciparum isolates were amplified to detect mutations in the pfcrt gene. The pfcrt 76T molecular marker for CQ resistance was detected in 66.4% (156/235) of the isolates, while the K76 CQ-sensitive wild type was detected in 33.6%. The pfcrt 74I and pfcrt 75E point mutations were each found to be present in 56.2% of isolates, while only four isolates (1.7%) were found to carry the pfcrt 72S mutation. Moreover, four pfcrt haplotypes were identified: the CVIET triple-allele (56.2%), SVMET double-allele (1.7%), and CVMNT single-allele (8.5%) mutant haplotypes, and the CVMNK wild haplotype (33.6%). The analysis also revealed significant associations between the prevalence of mutant pfcrt alleles and haplotypes and the age group, governorate, and nationality of the patients as well as the parasitaemia level (P < 0.05). The findings provide evidence of the potential re-emergence of CQ-susceptible P. falciparum strains in Jazan region over a decade after CQ discontinuation, with about one third of the isolates analysed carrying the pfcrt K76 CQ-sensitive wild allele and the CVMNK ancestral wild haplotype. Although the reintroduction of CQ cannot be recommended at present in Saudi Arabia, these findings support the rationale for a potential future role for CQ in malaria treatment. Therefore, continuous molecular and in-vitro monitoring mutations of pfcrt polymorphism in Jazan region is highly recommended.

The return of chloroquine-sensitive Plasmodium falciparum parasites in Jazan Region, Southwestern Saudi Arabia over a decade after the adoption of artemisinin-based combination therapy: analysis of genetic mutations in the pfcrt gene

This study investigated the polymorphism in the P. falciparum chloroquine resistance transporter (pfcrt) gene 11 years after chloroquine (CQ) cessation in Jazan region, southwestern Saudi Arabia. Two hundred and thirty-five P. falciparum isolates were amplified to detect mutations in the pfcrt gene. The pfcrt 76T molecular marker for CQ resistance was detected in 66.4% (156/235) of the isolates, while the K76 CQ-sensitive wild type was detected in 33.6%. The pfcrt 74I and pfcrt 75E point mutations were each found to be present in 56.2% of isolates, while only four isolates (1.7%) were found to carry the pfcrt 72S mutation. Moreover, four pfcrt haplotypes were identified: the CVIET triple-allele (56.2%), SVMET double-allele (1.7%), and CVMNT single-allele (8.5%) mutant haplotypes, and the CVMNK wild haplotype (33.6%). The analysis also revealed significant associations between the prevalence of mutant pfcrt alleles and haplotypes and the age group, governorate, and nationality of the patients as well as the parasitaemia level (P < 0.05). The findings provide evidence of the potential re-emergence of CQ-susceptible P. falciparum strains in Jazan region over a decade after CQ discontinuation, with about one third of the isolates analysed carrying the pfcrt K76 CQ-sensitive wild allele and the CVMNK ancestral wild haplotype. Although the reintroduction of CQ cannot be recommended at present in Saudi Arabia, these findings support the rationale for a potential future role for CQ in malaria treatment. Therefore, continuous molecular and in-vitro monitoring mutations of pfcrt polymorphism in Jazan region is highly recommended.

P. falciparum and Its Molecular Markers of Resistance to Antimalarial Drugs

The use of molecular markers of resistance to monitor the emergence, and the spread of parasite resistance to antimalarial drugs is a very effective way of monitoring antimalarial drug resistance. The identification and validation of molecular markers have boosted our confidence in using these tools to monitor resistance. For example, P. falciparum chloroquine resistance transporter (PfCRT), P. falciparum multidrug resistance protein 1 (PfMDR1), P. falciparum multidrug kelch 13 (pfk13), have been identified as molecular markers of resistance to chloroquine, lumefantrine, and artemisinin respectively. The mechanism of resistance to antimalarial drugs is mostly by; (1) undergoing mutations in the parasite genome, leading to expelling the drug from the digestive vacuole, or (2) loss of binding affinity between the drug and its target. Increased copy number in the pfmdr1 gene also leads to resistance to antimalarial drugs. The major cause of the widespread chloroquine and sulfadoxine-pyrimethamine resistance globally is the spread of parasites resistant to these drugs from Southeast Asia to Africa, the Pacific, and South America. Only a few mutations in the parasite genome lead to resistance to chloroquine and sulfadoxine-pyrimethamine arising from indigenous parasites in Africa, Pacific, and South America.

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.

Selection of pfcrt K76 and pfmdr1 N86 Coding Alleles after Uncomplicated Malaria Treatment by Artemether-Lumefantrine in Mali

Background: Artemether-lumefantrine is a highly effective artemisinin-based combination therapy that was adopted in Mali as first-line treatment for uncomplicated Plasmodium falciparum malaria. This study was designed to measure the efficacy of artemether-lumefantrine and to assess the selection of the P. falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multi-drug resistance 1 (pfmdr1) genotypes that have been associated with drug resistance. Methods: A 28-day follow-up efficacy trial of artemether-lumefantrine was conducted in patients aged 6 months and older suffering from uncomplicated falciparum malaria in four different Malian areas during the 2009 malaria transmission season. The polymorphic genetic markers MSP2, MSP1, and Ca1 were used to distinguish between recrudescence and reinfection. Reinfection and recrudescence were then grouped as recurrent infections and analyzed together by PCR-restriction fragment length polymorphism (RFLP) to identify candidate markers for artemether-lumefantrine tolerance in the P. falciparum chloroquine resistance transporter (pfcrt) gene and the P. falciparum multi-drug resistance 1 (pfmdr1) gene. Results: Clinical outcomes in 326 patients (96.7%) were analyzed and the 28-day uncorrected adequate clinical and parasitological response (ACPR) rate was 73.9%. The total PCR-corrected 28-day ACPR was 97.2%. The pfcrt 76T and pfmdr1 86Y population prevalence decreased from 49.3% and 11.0% at baseline (n = 337) to 38.8% and 0% in patients with recurrent infection (n = 85); p = 0.001), respectively. Conclusion: Parasite populations exposed to artemether-lumefantrine in this study were selected toward chloroquine-sensitivity and showed a promising trend that may warrant future targeted reintroduction of chloroquine or/and amodiaquine.

Distribution and temporal dynamics of P. falciparum chloroquine resistance transporter mutations associated with piperaquine resistance in Northern Cambodia

Background 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 2009-2017. Methods The sequence of pfcrt was determined for 410 P. falciparum isolates using PacBio amplicon sequencing or whole genome sequencing. Quantitative PCR was used to estimate pfpm2 and pfmdr1 copy number. Results Newly emerged PfCRT mutations increased in prevalence after the change to dihydroartemisinin-piperaquine in 2010, with &gt;98% of parasites harboring these mutations by 2017. After 2014, the prevalence of PfCRT F145I declined, being out-competed 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. Conclusions 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 pfmdr1N86Y 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.