Fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum clinical isolates collected from unrest tribal agencies of Pakistan

Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.

Single Nucleotide Polymorphism Analysis of Pvmdr-1 in Plasmodium Vivax Isolated from Military Personnel of Republic of Korea in 2016 and 2017

Background: Malaria chemoprophylaxis using chloroquine (CQ) and primaquine (PQ) has been administered to resident soldiers in the 3rd Army of Republic of Korea (ROK) to prevent malaria infection since the year 1997. Due to mass chemoprophylaxis against malaria, concern exists about occurrence of chloroquine resistance (CQR). Herein, we investigated the single nucleotide polymorphisms (SNPs) of the Plasmodium vivax multi-drug resistance protein-1 (pvmdr-1) gene to monitor the risk of CQR. Methods: SNPs of the pvmdr-1 gene were analyzed in 73 soldiers of the 3rd Army of ROK diagnosed with infection by Plasmodium vivax (P. vivax). Results: Quintuple mutations (G698S, L845F, M908L, T958M, and F1076L) were detected in 73 soldiers. Mutation in the Y541C position was firstly detected in soldiers at a frequency of 1.3% (1/73). In addition, synonymous mutations were detected at positions K44, L493, T529, and E1233. Based on these SNPs, pvmdr-1 sequences of ROK were classified into 6 haplotypes. The phylogenetic analysis closed to Type of North Korean showed that P. vivax malaria of ROK could be a reason of influx from North Korea. In this study, there was no therapeutic resistance (CQ-mediated parasite clearance within 72 hours) for clinical samples that possessed various SNPs of pvmdr-1. Various SNPs including a newly identified non-synonymous mutation (Y541C) had been introduced into P. vivax malaria-endemic areas in ROK. Conclusions: Our study showed that synonymous and non-synonymous mutations of pvmdr-1 were introduced to the malaria chemoprophylaxis-executed regions of ROK from 2016 to 2017. Thus, to prevent the emergence of CQR, continuous surveillance for SNPs of pvmdr-1 related with CQR in the malaria-endemic regions of ROK is essential.

Population genomics and evidence of clonal replacement of Plasmodium falciparum in the Peruvian Amazon

Previous studies have shown that P. falciparum parasites in South America have undergone population bottlenecks resulting in clonal lineages that are differentially distributed and that have been responsible for several outbreaks different endemic regions. In this study, we explored the genomic profile of 18 P. falciparum samples collected in the Peruvian Amazon Basin (Loreto) and 6 from the Peruvian North Coast (Tumbes). Our results showed the presence of three subpopulations that matched previously typed lineages in Peru: Bv1 (n = 17), Clonet D (n = 4) and Acre-Loreto type (n = 3). Gene coverage analysis showed that none of the Bv1 samples presented coverage for pfhrp2 and pfhrp3. Genotyping of drug resistance markers showed a high prevalence of Chloroquine resistance mutations S1034C/N1042D/D1246Y in pfmdr1 (62.5%) and K45T in pfcrt (87.5%). Mutations associated with sulfadoxine and pyrimethamine treatment failure were found on 88.8% of the Bv1 samples which were triple mutants for pfdhfr (50R/51I/108N) and pfdhps (437G/540E/581G). Analysis of the pfS47 gene that allows P. falciparum to evade mosquito immune responses showed that the Bv1 lineage presented one pfS47 haplotype exclusive to Loreto and another haplotype that was present in both Loreto and Tumbes. Furthermore, a possible expansion of Bv1 was detected since 2011 in Loreto. This replacement could be a result of the high prevalence of CQ resistance polymorphisms in Bv1, which could have provided a selective advantage to the indirect selection pressures driven by the use of CQ for P. vivax treatment.

Intolerable Burden of Malaria among Primitive Tribal Community in Odisha: Examining the Fundamental Cause

This study seeks to examine the living conditions, working conditions, and health seeking behaviour for malaria among Kondho community after one is infected with malaria. The residential surroundings of those diagnosed with malaria positive cases were extremely conducive for mosquito breeding. For instance, the majority of households threw garbage near their house, went for open defecation, the cowshed was beside their houses, and above all the houses were mostly situated in the jungle or near thick forest. Sub-centre followed by the community health centres was the first point of contact in most cases but medical care was sought only after routine life was affected. While malaria treatment plans are changing towards administering more powerful drugs as a result of chloroquine resistance but not as much has been done in the ground to prevent malaria at the first place. Therefore, together with continuing curative care for malaria—more emphasis is needed on its prevention. Community, civil society and the government need to work in tandem to improve the living and working conditions of backward communities particularly those living in malaria endemic zone so as to be able to take effective preventive measures for malaria.

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.

Evolution of multidrug resistance in Plasmodium falciparum : a longitudinal study of genetic resistance markers in the Greater Mekong Subregion

Increasing drug resistance in Plasmodium falciparum to artemisinins and their ACT partner drugs jeopardises effective antimalarial treatment. Resistance is worst in the Greater Mekong Subregion. Monitoring genetic markers of resistance can help to guide antimalarial therapy. Markers of resistance to artemisinins ( PfKelch mutations), mefloquine (amplification of P. falciparum multidrug resistance-1, PfMDR1, ), and piperaquine ( PfPlasmepsin2/3 amplification and specific P. falciparum chloroquine resistance transporter, PfCRT, mutations) were assessed in 6,722 P. falciparum samples from Vietnam, Lao PDR, Cambodia, Thailand, Myanmar between 2007 and 2019. Against a high background prevalence of PfKelch mutations, PfMDR1 and PfPlasmepsin2/3 amplification closely followed regional drug pressures over time. PfPlasmepsin2/3 amplification preceded piperaquine-resistance associated PfCRT mutations in Cambodia, and reached a peak prevalence of 23/28 (82%) in 2015. This declined to 57/156 (38%) after changing first-line treatment away from dihydroartemisinin-piperaquine to artesunate-mefloquine (ASMQ) between 2014 and 2017. PfMDR1 amplification increased from 0/293 (0%) between 2012 and 2017 to 12/156 (8%) in 2019. Amplification of PfMDR1 and PfPlasmepsin2/3 in the same parasites was extremely rare (4/6,722; 0.06%) and dispersed over time. Mechanisms conferring mefloquine and piperaquine resistance may be counterbalancing. This supports the development of ASMQ plus piperaquine as a triple artemisinin combination therapy.

Ten-Year Molecular Surveillance of Drug-Resistant Plasmodium spp. Isolated From the China–Myanmar Border

Antimalarial drug resistance has emerged as a major threat to global malaria control efforts, particularly in the Greater Mekong Subregion (GMS). In this study, we analyzed the polymorphism and prevalence of molecular markers associated with resistance to first-line antimalarial drugs, such as artemisinin, chloroquine, and pyrimethamine, using blood samples collected from malaria patients in the China–Myanmar border region of the GMS from 2008 to 2017, including 225 cases of Plasmodium falciparum and 194 cases of Plasmodium vivax. In artemisinin resistance, only the C580Y mutation with low frequency was detected in pfk13, and no highly frequent stable mutation was found in pvk12. In chloroquine resistance, the frequency of K76T mutation in pfcrt was always high, and the frequency of double mutations in pvmdr1 of P. vivax has been steadily increasing every year. In pyrimidine resistance, pfdhfr and pvdhfr had relatively more complex mutant types associated with drug resistance sites, and the overall mutation rate was still high. Therefore, artemisinin-based combination therapies are still suitable for use as the first choice of antimalarial strategy in the China–Myanmar border region in the future.

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.

Decreased prevalence of the Plasmodium falciparum Pfcrt K76T and Pfmdr1 and N86Y mutations post-chloroquine treatment withdrawal in Katete District, Eastern Zambia

Background In 2002, Zambia withdrew chloroquine as first-line treatment for Plasmodium falciparum malaria due to increased treatment failure and worldwide spread of chloroquine resistance. The artemisinin combination regimen, artemether–lumefantrine, replaced chloroquine (CQ) as first choice malaria treatment. The present study determined the prevalence of CQ resistance molecular markers in the Pfcrt and Pfmdr1 genes in Eastern Zambia at 9 and 13 years after the removal of drug pressure. Methods Samples collected from Katete District during the drug therapeutic efficacy assessments conducted in 2012 and 2016 were assayed by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) to determine the prevalence of genetic mutations, K76T on the Pfcrt gene and N86Y on the Pfmdr1 gene. A total of 204 P. falciparum-positive DBS samples collected at these two time points were further analysed. Results Among the samples analysed for Pfcrt K76T and Pfmdr1 N86Y in the present study, 112 (82.4%) P. falciparum-infected samples collected in 2012 were successfully amplified for Pfcrt and 94 (69.1%) for Pfmdr1, while 69 (65.7%) and 72 (68.6%) samples from 2016 were successfully amplified for Pfcrt and Pfmdr1, respectively. In 2012, the prevalence of Pfcrt 76K (sensitive) was 97.3%, 76T (resistant) was 1.8%, and 0.8% had both 76K and 76T codons (mixed). Similarly in 2012, the prevalence of Pfmdr1 86N (sensitive) was 97.9% and 86Y (resistant) was 2.1%. In the 2016 samples, the prevalence of the respective samples was 100% Pfcrt 76K and Pfmdr1 86N. Conclusion This study shows that there was a complete recovery of chloroquine-sensitive parasites by 2016 in Katete District, Eastern Zambia, 13 years following the withdrawal of CQ in the country. These findings add to the body of evidence for a fitness cost in CQ-resistant P. falciparum in Zambia and elsewhere. Further studies are recommended to monitor resistance countrywide and explore the feasibility of integration of the former best anti-malarial in combination therapy in the future.