Falciparum Chloroquine Resistance Transporter Gene Research Articles

Designing hybrid CRISPR-Cas12 and LAMP detection systems for treatment-resistant Plasmodium falciparum with in silico method.

Genes associated with drug resistance of first line drugs for Plasmodium falciparum have been identified and characterized of which three genes most commonly associated with drug resistance are P. falciparum chloroquine resistance transporter gene (PfCRT), P. falciparum multidrug drug resistance gene 1 (PfMDR1), and P. falciparum Kelch protein K13 gene (PfKelch13). Polymorphism in these genes could be used as molecular markers for identifying drug resistant strains. Nucleic acid amplification test (NAAT) along with DNA sequencing is a powerful diagnostic tool that could identify these polymorphisms. However, current NAAT and DNA sequencing technologies require specific instruments which might limit its application in rural areas. More recently, a combination of isothermal amplification and CRISPR detection system showed promising results in detecting mutations at a nucleic acid level. Moreover, the Loop-mediated isothermal amplification (LAMP)-CRISPR systems offer robust and straightforward detection, enabling it to be deployed in rural and remote areas. The aim of this study was to develop a novel diagnostic method, based on LAMP of targeted genes, that would enable the identification of drug-resistant P. falciparum strains. The methods were centered on sequence analysis of P. falciparum genome, LAMP primers design, and CRISPR target prediction. Our designed primers are satisfactory for identifying polymorphism associated with drug resistant in PfCRT, PfMDR1, and PfKelch13. Overall, the developed system is promising to be used as a detection method for P. falciparum treatment-resistant strains. However, optimization and further validation the developed CRISPR-LAMP assay are needed to ensure its accuracy, reliability, and feasibility.

Rebound of multiple infections and prevalence of anti-malarial resistance associated markers following malaria upsurges in Dielmo village, Senegal, West Africa

BackgroundThanks to the scale up of malaria control interventions, the malaria burden in Senegal has decreased substantially to the point that the National Malaria Control Programme plans to achieve malaria elimination by 2030. To guide such efforts, measuring and monitoring parasite population evolution and anti-malarial drugs resistance is extremely important. Information on the prevalence of parasite mutations related to drug resistance can provide a first signal of emergence, introduction and selection that can help with refining drug interventions. The aim of this study was to analyse the prevalence of anti-malarial drug resistance-associated markers before and after the implementation of artemisinin-based combination therapy (ACT) from 2005 to 2014 in Dielmo, a model site for malaria intervention studies in Senegal.MethodsSamples from both malaria patients and Plasmodium falciparum asymptomatic carriers were analysed with high resolution melting (HRM) technique to genotype P. falciparum chloroquine resistance transporter (Pfcrt) gene haplotypes and multidrug-resistant protein 1 (Pfmdr1) gene at codons N86 and Y184.ResultsAmong the 539 samples analysed, 474, 486, and 511 were successfully genotyped for Pfmdr1 N86, Y184, and Pfcrt, respectively. The prevalence of drug resistance markers was high, particularly during the malaria upsurges. Following the scale-up in bed net distribution, only the mutant (86F-like) variant of Pfmdr1 86 was present while during the malaria upsurges the predominance of two types 86Y-86N (43%) and 86F-like (56%) were observed. Most infections (87%) carried the wild type Y-allele at Pfmdr1 184 during the period of nets scale-up while during the malaria upsurges only 16% of infections had wild type and 79% of infections had mixed (mutant/wild) type. The frequency of the mixed genotypes SVMNT-like_CVMNK and SVMNT-like_CVIET within Pfcrt gene was particularly low during bednet scale up. Their frequency increased significantly (P < 0.001) during the malaria upsurges.ConclusionThis data demonstrated the effect of multiple interventions on the dynamics of drug resistance-associated mutations in the main malaria parasite P. falciparum in an endemic village in Senegal. Monitoring drug resistance markers should be conducted periodically to detect threats of emergence or resurgence that could compromise the efficacy of anti-malarial drugs.

IFNγ, TNFα polymorphisms and IFNγ serum levels are associated with the clearance of drug-resistant P. falciparum in Malian children.

Host immunity has been suggested to clear drug-resistant parasites in malaria-endemic settings. However, the immunogenetic mechanisms involved in parasite clearance are poorly understood. Characterizing the host's immunity and genes involved in controlling the parasitic infection can inform the development of blood-stage malaria vaccines. This study investigates host regulatory cytokines and immunogenomic factors associated with the clearance of Plasmodium falciparum carrying a chloroquine resistance genotype. Biological samples from participants of previous drug efficacy trials conducted in two Malian localities were retrieved. The P. falciparum chloroquine resistance transporter (Pfcrt) gene was genotyped using parasite DNA. Children carrying parasites with the mutant allele (Pfcrt-76T) were classified based on their ability to clear their parasites. The levels of the different cytokines were measured in serum. The polymorphisms of specific human genes involved in malaria susceptibility were genotyped using human DNA. The prevalence of the Pfcrt-76T was significantly higher in Kolle than in Bandiagara (81.6% vs 38.6%, p<10-6). The prevalence of children who cleared their mutant parasites was significantly higher in Bandiagara than in Kolle (82.2% vs 67.4%, p<0.05). The genotyping of host genes revealed that IFN-γ -874T and TNF-α -308A alleles were positively associated with parasite clearance. Cytokine profiling revealed that IFN-γ level was positively associated with parasite clearance (p=0.04). This study highlights the role of host's immunity and immunogenetic factors to clear resistant parasites, suggesting further characterization of these polymorphisms may help to develop novel approaches to antiparasitic treatment strategies.

Monitoring antimalarial drug-resistance markers in Somalia.

The use of an effective antimalarial drug is the cornerstone of malaria control. However, the development and spread of resistant Plasmodium falciparum strains have placed the global eradication of malaria in serious jeopardy. Molecular marker analysis constitutes the hallmark of the monitoring of Plasmodium drug-resistance. This study included 96 P. falciparum PCR-positive samples from southern Somalia. The P. falciparum chloroquine resistance transporter gene had high frequencies of K76T, A220S, Q271E, N326S, and R371I point mutations. The N86Y and Y184F mutant alleles of the P. falciparum multidrug resistance 1 gene were present in 84.7 and 62.4% of the isolates, respectively. No mutation was found in the P. falciparum Kelch-13 gene. This study revealed that chloroquine resistance markers are present at high frequencies, while the parasite remains sensitive to artemisinin (ART). The continuous monitoring of ART-resistant markers and in vitro susceptibility testing are strongly recommended to track resistant strains in real time.

Molecular profile of Pfatpase-6 gene from Plasmodium falciparum isolates in Nigeria

Anti-malarial drug resistance is one of the biggest public health burdens confronting global malaria control programmes. The emergence of P. falciparum chloroquine resistance transporter and multi-drug resistance mutant genes had devastating effects on the therapeutic efficacy of chloroquine when it was the drug of choice for malaria treatment. The artemisinins have proven to be an excellent therapeutic alternative to fill the void in chemotherapeutic options left by resistance mechanisms. The sarco/endoplasmic reticulum Ca2+-ATPase ortholog of P. falciparum (PfATPase-6) has been suggested as one of the targets of the artemisinins. Consequently, Pfatpase-6 gene polymorphisms are being investigated as markers of artemisinin resistance elsewhere. The present study assessed the molecular profile of the current prevalence of four P. falciparum candidate artemisinin resistance biomarkers L263E, E431K, A623E, and S769N in the Pfatpase-6 gene in 113 samples of P. falciparum isolates collected from Ibadan, Oyo State, Nigeria between 2017 and 2018. The frequency of occurrence of E431K mutation was 17% from collected samples. No A623E, L263E and S769N were detected. The result suggests that resistance to artemisinin has either not yet been selected in Nigeria or other genes mutations might be responsible for such, if at all

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.

Investigation of &amp;lt;i&amp;gt;Plasmodium falciparum&amp;lt;/i&amp;gt; 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.

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 11years 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 as follows: 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.

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.

The Drug Resistance of Plasmodium falciparum and P. vivax in Iran: A Review Article.

Background:One of the main obstacles to malaria control in the world has been the emergence of resistance in Plasmodium falciparum to chloroquine and other anti-malarial drugs. This study aimed to review studies in Iran on resistance in P. falciparum and P. vivax to drugs, and to reveal the mechanisms and molecular markers of resistance of these two species.Methods:The databases of PubMed, Scopus, Google Scholar, Magiran, and reputable Iranian journals were searched to find published studies on the resistance in P. falciparum and P. vivax to antimalarial drugs in Iran.Results:There is a significant relationship between resistance to chloroquine in P. falciparum and the emergence of K76T mutation in the P. falciparum chloroquine-resistance transporter gene in Iran. Resistance to sulfadoxine-pyrimethamine (SP) in P. falciparum is also significantly associated with the development of mutations in the dihydrofolate reductase and dihydropteroate synthase genes. Resistance to chloroquine in P. vivax has not been reported in Iran and it is used as a first-line treatment for P. vivax malaria.Conclusion:P. falciparum has become resistant to chloroquine in different regions of Iran and is not currently used to treat malaria. Besides, cases have emerged of P. falciparum resistance to SP in different parts of southern Iran, and SP is not administered alone for treating P. falciparum.

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.

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.

Molecular Epidemiology of Plasmodium falciparum Chloroquine Resistance Transporter Genes among School Children in Kwara State, Southwestern Nigeria

Background: Plasmodium falciparum existence continues to develop resistance to conventional antimalaria drugs in malaria endemic areas. Plasmodia often prevent drugs from interacting with the target site, hence, developing resistance to antimalaria drugs. Mutations in the Plasmodium falciparum chloroquine resistance transporter (Pfcrt), are the major determinant of chloroquine resistance in human malaria parasite.&#x0D; Methodology: Malaria infection, Pfcrt and Pfmdr1 genes of isolates among school students within the age range of 11-22 years from four selected rural communities of Kwara state were studied. One hundred and eighty seven subjects (187) were selected for the study. Blood samples were collected by finger prick method for malaria screening. Nested PCR and restriction fragment length polymorphism (RFLP) were done to detect alleles of pfcrt at codon 76 and pfmdr1 at codon 86. DNA of isolates was appropriately extracted from the filter paper blots using the methanol fixation method. Logistic regression was performed on the binary observations obtained while linear regression was conducted on the fifty (50) subjects that tested positive to malaria.&#x0D; Results: Out of 187 subjects screened, 26.7% (50) were positive to P. falciparum. Highest malaria parasite count of 36.4% was recorded in 14-16 years age group while 20-22 years age group had the least malaria parasite count (15.4%). The result of the studied isolates indicated that out of 50 isolates analyzed for Pfcrt gene, wild type alleles accounted for 32% (16) while mutant alleles accounted for 68% (34). Alakuko Community accounted for the least number of T76 mutant alleles 10% (5) while Apado community recorded the highest number of T76 mutant gene 22% (11). For Pfmdr1 gene analysis at codon 86, isolates from Apado community showed the highest mutant type alleles (Y86) of 22% (11), while Igbonla community in Ifelodun local government had the least mutant alleles, 6% (3).&#x0D; Conclusion: The overall result revealed existence of mutant alleles in both the Pfcrt and Pfmdr1 genes which was higher than the wild type gene in both cases. The presence of chloroquine resistance genes among the studied population implies that alternative antimalaria drugs should be designed by pharmaceutical industry.

DNA recovery from archived RDTs for genetic characterization of Plasmodium falciparum in a routine setting in Lambar\xe9n\xe9, Gabon

BackgroundRapid diagnostic tests (RDTs) have been described as a source of genetic material to analyse malaria parasites in proof-of-concept studies. The increasing use of RDTs (e.g., in focal or mass screening and treatment campaigns) makes this approach particularly attractive for large-scale investigations of parasite populations. In this study, the complexity of Plasmodium falciparum infections, parasite load and chloroquine resistance transporter gene mutations were investigated in DNA samples extracted from positive RDTs, obtained in a routine setting and archived at ambient temperature.MethodsA total of 669 archived RDTs collected from malaria cases in urban, semi-urban and rural areas of central Gabon were used for P. falciparum DNA extraction. Performance of RDTs as a source of DNA for PCR was determined using: (i) amplification of a single copy merozoite surface protein 1 (msp1) gene followed by highly sensitive and automated capillary electrophoresis; (ii) genotyping of the pfcrt gene locus 72–76 using haplotype-specific-probe-based real-time PCR to characterize chloroquine resistance; and, (iii) real-time PCR targeting 18S genes to detect and quantify Plasmodium parasites.ResultsOut of the 669 archived RDTs, amplification of P. falciparum nucleic materials had a success rate of 97% for 18S real-time PCR, and 88% for the msp1 gene. The multiplicity of infections (MOI) of the whole population was 2.6 (95% CI 2.5–2.8). The highest number of alleles detected in one infection was 11. The MOI decreased with increasing age (β = − 0.0046, p = 0.02) and residence in Lambaréné was associated with smaller MOIs (p < 0.001). The overall prevalence of mutations associated with chloroquine resistance was 78.5% and was not associated with age. In Lambaréné, prevalence of chloroquine resistance was lower compared to rural Moyen-Ogooué (β = − 0.809, p-value = 0.011).ConclusionRDT is a reliable source of DNA for P. falciparum detection and genotyping assays. Furthermore, the increasing use of RDTs allows them to be an alternative source of DNA for large-scale genetic epidemiological studies. Parasite populations in the study area are highly diverse and prevalence of chloroquine-resistant P. falciparum remains high, especially in rural areas.

Prevalence of Mutant Alleles Responsible for Chloroquine Resistance among Plasmodium falciparum Isolates in North Central, Nigeria

Background: Although chloroquine (CQ) has been officially replaced with artemisinin combination therapy (ACT) as first line drug for the treatment of malaria in Nigeria since 2005, a lot of people still believe that chloroquine is more effective chiefly because of the decline in the sensitivity of Plasmodium falciparum to ACT. Thus resulting into unofficial use of CQ for self medication. This study was conducted in order to survey the current status of chloroquine resistant strains of pfcrt and pfmdr1 in view of possible re-introduction of chloroquine for malaria treatment.&#x0D; Methods: DNA was extracted from one hundred (100) microscopically confirmed Plasmodium falciparum positive blood samples spotted on 3 mm Whatman filter paper. The detection of mutations in Plasmodium falciparum chloroquine resistance transporter (Pfcrt) and Plasmodium falciparum multidrug resistance (Pfmdr1) genes was performed by nested polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP).&#x0D; Results: Results showed the presence of mutant alleles of Pfcrt and Pfmdr1 in 60% and 41% of the samples respectively. However, there was no significant correlation in the prevalence of mutant alleles (T76/Y86) in relation to gender (p = 0.59/ 0.08) and age (p=0.59/0.93) of participants respectively.&#x0D; Conclusion: The observed high prevalence of chloroquine resistance despite thirteen years of withdrawal calls for serious concern.

Multiple Novel Mutations in Plasmodium falciparum Chloroquine Resistance Transporter Gene during Implementation of Artemisinin Combination Therapy in Thailand.

Mutations in the chloroquine resistance transporter gene of Plasmodium falciparum (Pfcrt) are associated with drug susceptibility status of chloroquine and other antimalarials that interfere with heme detoxification process including artemisinin. We aim to investigate whether an increase in duration of artemisinin combination therapy (ACT) in Thailand could affect mutations in Pfcrt. The complete coding sequences of Pfcrt and dihydrofolate reductase (Pfdhfr), and size polymorphisms of the merozoite surface proteins-1 and 2 (Pfmsp-1 and Pfmsp-2) of 189 P. falciparum isolates collected during 1991 and 2016 were analyzed. In total, 12 novel amino acid substitutions and 13 novel PfCRT haplotypes were identified. The most prevalent haplotype belonged to the Dd2 sequence and no wild type was found. A significant positive correlation between the frequency of Pfcrt mutants and the year of sample collection was observed during nationwide ACT implementation (r = 0.780; P = 0.038). The number of haplotypes and nucleotide diversity of isolates collected during 3-day ACT (2009-2016) significantly outnumbered those collected before this treatment regimen. Positive Darwinian selection occurred in the transmembrane domains only among isolates collected during 3-day ACT but not among those collected before this period. No remarkable change was observed in the molecular indices for other loci analyzed when similar comparisons were performed. An increase in the duration of artesunate in combination therapy in Thailand could exert selective pressure on the Pfcrt locus, resulting in emergence of novel variants. The impact of these novel haplotypes on antimalarial susceptibilities requires further study.

Pfcrt genotypes and related microsatellite DNA polymorphisms on Plasmodium falciparum differed among populations in the Greater Mekong Subregion

Malaria morbidity and mortality have decreased gradually in the Greater Mekong Subregion (GMS). Presently, WHO sets a goal to eliminate malaria by 2030 in the GMS. However, drug-resistant malaria has been reported from several endemic areas. To achieve the goal of elimination, the status of the emergence and spread of drug resistance should be monitored. In this study, the genotype of the Plasmodium falciparum chloroquine (CQ) resistance transporter gene (pfcrt) and 6 microsatellite DNA loci flanking the gene were examined. P. falciparum isolates (n = 136) was collected from malaria patients in Thailand (n = 50, 2002–2005), Vietnam (n = 39, 2004), Laos (n = 15, 2007) and Cambodia (n = 32, 2009). Amino acid sequences at codons 72–76 on the gene were determined. All of the isolates from Thailand were CQ-resistant (CVIET), as were all of the isolates from Cambodia (CVIET, CVIDT). Thirteen of the 15 isolates (87%) from Laos were CQ-resistant (CVIET, CVIDT), whereas the other 2 (13%) were CQ-susceptible (CVMNK). In contrast, 27 of the 39 isolates (69%) from Vietnam were CQ-susceptible (CVMNK), whereas the other 12 (31%) were CQ-resistant (CVIET, CVIDT, CVMDT) or mixed (CVMNK/CVIDT). The mean of expected heterozygosity of the microsatellite loci was 0.444 in the Thai population, 0.482 in the Cambodian population, and 0.734 in the Vietnamese population. Genetic diversity in the Thai population was significantly lower than that in the Vietnamese population. These results suggested that chloroquine selective pressure on P. falciparum populations is heterogeneous in the GMS. Therefore, further examination to understand the mechanisms behind the emergence and spread of drug-resistant malaria are needed.

Variation in intronic microsatellites and exon 2 of the Plasmodium falciparum chloroquine resistance transporter gene during modification of artemisinin combination therapy in Thailand

The amino acid substitution at residue 76 of the food vacuolar transmembrane protein encoded by the chloroquine resistance transporter gene of Plasmodium falciparum (Pfcrt) is an important, albeit imperfect, determinant of chloroquine susceptibility status of the parasite. Other mutations in Pfcrt can modulate susceptibility of P. falciparum to other antimalarials capable of interfering with heme detoxification process, and may exert compensatory effect on parasite growth rate. To address whether nationwide implementation of artemisinin combination therapy (ACT) in Thailand could affect sequence variation in exon 2 and introns of Pfcrt, we analyzed 136 P. falciparum isolates collected during 1997 and 2016 from endemic areas bordering Myanmar, Cambodia and Malaysia. Results revealed 6 haplotypes in exon 2 of Pfcrt with 2 novel substitutions at c.243A > G (p.R81) and c.251A > T (p.N84I). Positive selection was observed at amino acid residues 75, 76 and 97. Four, 3, and 2 alleles of microsatellite (AT/TA) repeats occurred in introns 1, 2 and 4, respectively, resulting in 7 different 3-locus haplotypes. The number of haplotypes and haplotype diversity of exon 2, and introns 1, 2 and 4 were significantly greater among isolates collected during 2009 and 2016 than those collected during 1997 and 2008 when 3-day ACT and 2-day ACT regimens were implemented nationwide, respectively (p < 0.05). By contrast, the number of haplotypes and haplotype diversity of the merozoite surface proteins 1 and 2 of these parasite populations did not differ significantly between these periods. Therefore, the Pfcrt locus of P. falciparum in Thailand continues to evolve and could have been affected by selective pressure from modification of ACT regimen.

Cross sectional study to determine chloroquine resistance among Plasmodium falciparum clinical isolates from Khartoum, Sudan

Background: Malaria continues to present a global health threat; the World Health Organization (WHO) reported 214 million cases of malaria by the year 2015 with a death rate of 438000. Sudan is endemic to malaria with over 95% of malaria cases due to Plasmodium falciparum. Chloroquine is a well-established drug in the treatment of P. falciparum malaria although its use has declined since its introduction as the drug of choice in treatment of malaria in Sudan. The mechanism of resistance has been attributed to mutations in P. falciparum Chloroquine resistance transporter gene coding for a key food vacuole proteins. In current study we aimed at verifying the genetic cause of resistance to Chloroquine in field isolates of P. falciparum. Methods: Twenty P. falciparum cases were diagnosed from East Nile hospital in Khartoum and recruited in the investigation. Nested PCR was conducted to isolate mutation region in the PfCRT gene and the amplicons were sequenced using Sanger sequencing technique (Macrogen, Soule Korea). Results: 16/20 (80%) of the field isolates contained base pair mutation of codon 76 in the pfcrt gene thus being resistant to chloroquine treatment and only 4/20 (20%) did not contain such mutation. Conclusions: High treatment failures associated with Chloroquine treatment is evident of the high prevalence of mutant strains of P. falciparum field isolates thus suggesting the reduced relevance of Chloroquine as a treatment choice in the management of P. falciparum malaria.