Pfcrt K76T Mutation Research Articles

Analysis of <em>Pfmdr1</em> and <em>Pfcrt drug</em> resistant markers in <em>Plasmodium falciparum</em> following treatments of patients from Nyando and Mbita, Nyanza region with Artemisinin-based combination therapy

The multidrug resistance of Plasmodium falciparum to antimalarial drugs is a major public health concern in the global management of malaria, especially in developing countries. As of today, malaria treatment is primarily dependent on the sustained efficacy of artemisinin-based combination therapy (ACT). Recent reports indicating decline in efficacy of ACT and artesunate monotherapy in Southeast Asia are a major threat to global malaria control achievements. Initially, mutations in Pfmdr1 were associated with chloroquine and amodiaquine resistance only, but there are now emerging field study reports of Pfmdr1 mutation conferring tolerance to ACT. The current study used Restriction Fragment Length Polymorphism to evaluate the prevalence of genetic polymorphisms of Plasmodium falciparum multidrug resistance (Pfmdr1) and Plasmodium falciparum chloroquine-resistant transporter (Pfcrt) gene mutations in Mbita and Nyando field isolates. The findings report that the prevalence of Pfcrt K76T mutation in Plasmodium falciparum field isolates in Mbita declined significantly from 76.3% (95% CI 21.0-31.0) in 2008 to 51.9% (95% CI 19.0-25.0) in 2014, a reduction of 24.4% (χ2df =1 = 4.709, P = 0.0000). In Nyando, prevalence stood at 66.8% (95% CI 12.0-14.0) in 2014 down from 73.7% (95% CI 27.0-48.0), insignificant reduction of 6.9% (χ2df =1 = 17.699, P = 0.0913). Prevalence for Pfmdr1 in Mbita stood at 14.7% (95% CI 44.0-51.0) in 2014 down from 21.9% (95% CI 31.0-49.0) in 2008, a significant reduction of 4.3% (χ2df =1 = 9.011, P = 0.001). While in Nyando, field isolate samples recorded 16.2% (95% CI 22.0-31.0) in 2014, down from 30.6% (95% CI 12.0-27.0), insignificant reduction of 1.9% (χ2df =1 = 11.777, P = 0.0591). Our findings confirm a common trend reported by previous studies: that there has been a small decline in the prevalence of the Pfcrt mutation over a decade since the withdrawal of Chloroquine as the first line of treatment for uncomplicated cases of malaria in Kenya.

Prevalence of polymorphisms in marker genes associated with antimalarial drug resistance in Plasmodium falciparum following 10 years of artemisinin-based combination therapy implementation in urban Kolkata.

Resistance to antimalarial drugs is one of the major challenges for malaria elimination. In India, artemisinin combination therapy (artesunate-sulfadoxin pyrimethamine) was introduced in place of chloroquine (CQ) for the treatment of uncomplicated falciparum malaria in 2010. Periodical monitoring of polymorphisms in antimalarial drug resistance marker genes will be useful for assessing drug pressure, mapping and monitoring of drug resistance status; and will be helpful for searching alternative treatments. This study was conducted to study the polymorphisms in antimalarial drug resistance marker genes among clinical Plasmodium falciparum isolates collected from Kolkata after 10 years of artemisinin-based combination therapie (ACT) implementation. Blood samples were collected from P. falciparum mono-infected patients and polymorphisms in P. falciparum CQ resistance transporter (pfcrt), P. falciparum multidrug resistance (pfmdr-1), P. falciparum dihydrofolate reductase (pfdhfr), P. falciparum dihydropteroate synthetase (pfdhps), pfATPase6 and pfK-13 propeller genes were analysed by polymerase chain reaction and DNA sequencing. In pfcrt gene, C72S, and K76T mutation was recorded in 100% isolates and no mutations was detected in any of the targeted codons of pfmdr-1 gene. A double mutant pfcrt haplotype SVMNT and wildtype haplotype NYD in pfmdr-1 were prevalent in 100% of study isolates. Triple mutant pfdhfr-pfdhps haplotype ANRNI-SGKAA was recorded. No polymorphism in pfK13 gene was documented in any of the isolates. Observed wild codon N86 along with Y184 and D1246 of pfmdr-1 gene might be an indication of the reappearance of CQ sensitivity. The absence of quadruple and quintuple haplotypes in pfdhfr-pfdhps gene along with the wild haplotype of pfK13 is evidence of ACT effectivity. Hence, similar studies with large sample size are highly suggested for monitoring the drug resistance status of P. falciparum.

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.

Molecular detection of drug-resistant Plasmodium falciparum mutants in Assam.

The spread of drug-resistant Plasmodium falciparum (Pf) poses a serious threat to the control and elimination of malaria. The objective of this study was to detect the molecular biomarkers of antimalarial drug resistance in Pf in patients visiting a tertiary care hospital in Assam. Malaria was first detected in fever cases using microscopy and a rapid diagnostic test (RDT), and then confirmed using PCR. Pf chloroquine resistance transporter (Pfcrt), Pf multidrug resistance-1 (Pfmdr-1), and single-nucleotide polymorphisms linked to delayed parasite clearance after treatment with artemisinin MAL 10-688956 and MAL 13-1718319 and Kelch-13 propeller (PfK-13) genes were evaluated by PCR-restriction fragment length polymorphism (RFLP). Sixty nine cases of malaria were found among 300 cases of fever. Of these, 54 were positive for Pf, 47 of which were confirmed by PCR. Pfcrt-K76T mutation was seen in 96.6 per cent and Pfmdr1-N86Y mutation in 84.2 per cent of cases. Mutation was not detected in MAL10 and MAL13 genes. Sequence analysis of Kelch-13 gene showed the presence of a novel mutation at amino acid position 675. Statistically, no significant association was found between the molecular biomarkers and demographic profile, clinical presentation and outcome of the cases. Molecular surveillance is essential to assess the therapeutic efficacy of the drugs against circulating Pf isolates in Assam which are found to be highly resistant to CQ. The role of the new mutation found in the Kelch-13 gene in the development of artemisinin resistance in Assam needs to be thoroughly monitored in future research.

Biennial surveillance of Plasmodium falciparum anti-malarial drug resistance markers in Democratic Republic of Congo, 2017 and 2019

BackgroundBecause of the loss of chloroquine (CQ) effectiveness, the Democratic Republic of Congo (DRC)’s malaria treatment policy replaced CQ by sulfadoxine–pyrimethamine (SP) as first-line treatment of uncomplicated malaria in 2003, which in turn was replaced by artemisinin-based combination therapies (ACT) in 2005. The World Health Organization (WHO) recommends monitoring of anti-malarial drug resistance every 2 years. The study aimed to provide baseline data for biennial molecular surveillance of anti-malarial drug resistance by comparing data from a study conducted in 2019 to previously published data from a similar study conducted in 2017 in the DRC.MethodsFrom July to November 2019, a cross-sectional study was conducted in ten sites which were previously selected for a similar study conducted in 2017 across the DRC. P. falciparum malaria was diagnosed by a rapid diagnostic test (RDT) or by microscopy and dried blood samples (DBS) were taken from patients who had a positive test. Segments of interest in pfcrt and pfk13 genes were amplified by conventional PCR before sequencing.ResultsOut of 1087 enrolled patients, 906 (83.3%) were PCR-confirmed for P. falciparum. Like in the 2017-study, none of the mutations known to be associated with Artemisinine (ART) resistance in Southeast Asia was detected. However, non-synonymous (NS) mutations with unknown functions were observed among which, A578S was detected in both 2017 and 2019-studies. The overall prevalence of pfcrt-K76T mutation that confers CQ-resistance was 22.7% in 2019-study compared to 28.5% in 2017-study (p-value = 0.069), but there was high variability between sites in the two studies. Like in 2017-study, the pfcrt 72–76 SVMNT haplotype associated with resistance to amodiaquine was not detected.ConclusionThe study reported, within 2 years, the non-presence of molecular markers currently known to be associated with resistance to ART and to AQ in P. falciparum isolated in the DRC. However, the presence of polymorphisms with as-yet unknown functions was observed, requiring further characterization. Moreover, an overall decrease in the prevalence of CQ-resistance marker was observed in the DRC, but this prevalence remained highly variable from region to region.

Analysis of drug resistance marker genes of Plasmodium falciparum after implementation of artemisinin-based combination therapy in Pune district, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is a major problem in malaria control and elimination. In this study, samples from Pune district were characterized to determine prevalence of molecular markers of resistance to chloroquine (pfcrt codons C72S, M74I, N75E, K76T and pfmdr-1 N86Y, Y184F), pyrimethamine (pfdhfr C50R, N51I, C59R, S108N), sulfadoxine (pfdhps, S436A, A437G, K540E, A581G), and artemisinin (pfkelch13, C580Y, R539T). The pfcrt K76T mutation was found in 78% samples as CVMNT, SVMNT and CVIET haplotype. The pfmdr-1 N86Y and Y184F mutations were found in 54% of samples. The pfdhfr double mutation C59R + S108N was present in 67% of samples, while the pfdhfr triple mutation (N51I + C59R + S108N) was not detected. The pfdhps mutations A437G and K540E were found in 67% of samples. Single mutants of pfdhps were rare, with K540E detected in only 6 patient samples. Similarly, pfdhps A581G was found in 13 of the isolates. The molecular markers associated with artemisinin resistance (mutations in pfkelch13 C580Y, R539T) were not detected in any of the isolates. These results suggest an emerging problem with multidrug-resistant P. falciparum. Though the genotype conventionally associated with artemisinin resistance was not observed, chloroquine-resistant genotype has reached complete fixation in the population. Moreover, the prevalence of mutations in both pfdhfr and pfdhps, with the presence of the quadruple mutant, indicates that continued monitoring is required to assess whether sulfadoxine-pyrimethamine can be used efficiently as a partner drug for artemisinin for the treatment of P. falciparum.

Prevalence of Sickle Cell Anemia in Association of Plasmodium falciparum from Rajnandgaon, India

Sickle cell anemia is a blood disorder resulting from the inheritance of abnormal genes from parents. It is caused due to mutation in the β-globulin gene. Sickle cell anemia is widespread across the world and in Central India. The present study was undertaken to study the prevalence of the disorder in the Rajnandgaon district of Central India. A random sampling of 6088 people was done to test the sickle cell anemia problem by slide test method and a total of 249 (4.09%) people were found sickled positive. Further electrophoresis test was performed for all 249 of which 67 were found homozygous (HbSS) and 182 were found heterozygous (HbAS) positive. Besides the above analysis, chloroquine prophylaxis associated with a high prevalence of Plasmodium falciparum Pfcrt K76T mutation in people (n=26) with sickle cell anemia was also analyzed. The genotype of the subject was screened using the hemoglobin electrophoresis system and the P. falciparum Pfcrt genotyping was carried out using PCR-Restriction Fragment Length Polymorphism (RFLP). The prevalence rate of Pfcrt K76T mutant gene was proportionately found higher in the hemoglobin SS (n=40, m=32, r=0.67) genotype individuals than the hemoglobin AS (n=52, m= 27, r=0.519) and AA (n=182, m= 68, r= 0.37).

Surveillance of molecular markers of antimalarial drug resistance in Plasmodium falciparum and Plasmodium vivax in Federally Administered Tribal Area (FATA), Pakistan.

ABSTRACTThis molecular epidemiological study was designed to determine the antimalarial drug resistance pattern, and the genetic diversity of malaria isolates collected from a war-altered Federally Administered Tribal Area (FATA), in Pakistan. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai and Kurram agencies of FATA region between May 2017 and May 2018, and they underwent DNA extraction and amplification. The investigation of gene polymorphisms in drug resistance genes (dhfr, dhps, crt, and mdr1) of Plasmodium falciparum and Plasmodium vivax was carried out by pyrosequencing and Sanger sequencing, respectively. Out of 679 PCR-confirmed malaria samples, 523 (77%) were P. vivax, 121 (18%) P. falciparum, and 35 (5%) had mixed-species infections. All P. falciparum isolates had pfdhfr double mutants (C59R+S108N), while pfdhfr/pfdhps triple mutants (C59R+S108N+A437G) were detected in 11.5% of the samples. About 97.4% of P. falciparum isolates contained pfcrt K76T mutation, while pfmdr1 N86Y and Y184F mutations were present in 18.2% and 10.2% of the samples. P. vivax pvdhfr S58R mutation was present in 24.9% of isolates and the S117N mutation in 36.2%, while no mutation in the pvdhps gene was found. Pvmdr1 F1076L mutation was found in nearly all samples, as it was observed in 98.9% of isolates. No significant anti-folate and chloroquine resistance was observed in P. vivax; however, mutations associated with antifolate-resistance were found, and the chloroquine-resistant gene has been observed in 100% of P. falciparum isolates. Chloroquine and sulphadoxine-pyrimethamine resistance were found to be high in P. falciparum and low in P. vivax. Chloroquine could still be used for P. vivax infection but need to be tested in vivo, whereas a replacement of the artemisinin combination therapy for P. falciparum appears to be justified.

Surveillance of molecular markers of antimalarial drug resistance in Plasmodium falciparum and Plasmodium vivax in Federally Administered Tribal Area (FATA), Pakistan

This molecular epidemiological study was designed to determine the antimalarial drug resistance pattern, and the genetic diversity of malaria isolates collected from a war-altered Federally Administered Tribal Area (FATA), in Pakistan. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai and Kurram agencies of FATA region between May 2017 and May 2018, and they underwent DNA extraction and amplification. The investigation of gene polymorphisms in drug resistance genes (dhfr, dhps, crt, and mdr1) of Plasmodium falciparum and Plasmodium vivax was carried out by pyrosequencing and Sanger sequencing, respectively. Out of 679 PCR-confirmed malaria samples, 523 (77%) were P. vivax, 121 (18%) P. falciparum, and 35 (5%) had mixed-species infections. All P. falciparum isolates had pfdhfr double mutants (C59R+S108N), while pfdhfr/pfdhps triple mutants (C59R+S108N+A437G) were detected in 11.5% of the samples. About 97.4% of P. falciparum isolates contained pfcrt K76T mutation, while pfmdr1 N86Y and Y184F mutations were present in 18.2% and 10.2% of the samples. P. vivax pvdhfr S58R mutation was present in 24.9% of isolates and the S117N mutation in 36.2%, while no mutation in the pvdhps gene was found. Pvmdr1 F1076L mutation was found in nearly all samples, as it was observed in 98.9% of isolates. No significant anti-folate and chloroquine resistance was observed in P. vivax; however, mutations associated with antifolate-resistance were found, and the chloroquine-resistant gene has been observed in 100% of P. falciparum isolates. Chloroquine and sulphadoxine-pyrimethamine resistance were found to be high in P. falciparum and low in P. vivax. Chloroquine could still be used for P. vivax infection but need to be tested in vivo, whereas a replacement of the artemisinin combination therapy for P. falciparum appears to be justified.

Efficacy of artemether-lumefantrine for treating uncomplicated Plasmodium falciparum cases and molecular surveillance of drug resistance genes in Western Myanmar

BackgroundCurrently, artemisinin-based combination therapy (ACT) is the first-line anti-malarial treatment in malaria-endemic areas. However, resistance in Plasmodium falciparum to artemisinin-based combinations emerging in the Greater Mekong Sub-region is a major problem hindering malaria elimination. To continuously monitor the potential spread of ACT-resistant parasites, this study assessed the efficacy of artemether-lumefantrine (AL) for falciparum malaria in western Myanmar.MethodsNinety-five patients with malaria symptoms from Paletwa Township, Chin State, Myanmar were screened for P. falciparum infections in 2015. After excluding six patients with a parasite density below 100 or over 150,000/µL, 41 P. falciparum patients were treated with AL and followed for 28 days. Molecular markers associated with resistance to 4-amino-quinoline drugs (pfcrt and pfmdr1), antifolate drugs (pfdhps and pfdhfr) and artemisinin (pfk13) were genotyped to determine the prevalence of mutations associated with anti-malarial drug resistance.ResultsFor the 41 P. falciparum patients (27 children and 14 adults), the 28-day AL therapeutic efficacy was 100%, but five cases (12.2%) were parasite positive on day 3 by microscopy. For the pfk13 gene, the frequency of NN insert after the position 136 was 100% in the day-3 parasite-positive group as compared to 50.0% in the day-3 parasite-negative group, albeit the difference was not statistically significant (P = 0.113). The pfk13 K189T mutation (10.0%) was found in Myanmar for the first time. The pfcrt K76T and A220S mutations were all fixed in the parasite population. In pfmdr1, the Y184F mutation was present in 23.3% of the parasite population, and found in both day-3 parasite-positive and -negative parasites. The G968A mutation of pfmdr1 gene was first reported in Myanmar. Prevalence of all the mutations in pfdhfr and pfdhps genes assessed was over 70%, with the exception of the pfdhps A581G mutation, which was 3.3%.ConclusionsAL remained highly efficacious in western Myanmar. Pfk13 mutations associated with artemisinin resistance were not found. The high prevalence of mutations in pfcrt, pfdhfr and pfdhps suggests high-degree resistance to chloroquine and antifolate drugs. The pfmdr1 N86/184F/D1246 haplotype associated with selection by AL in Africa reached > 20% in this study. The detection of > 10% patients who were day-3 parasite-positive after AL treatment emphasizes the necessity of continuously monitoring ACT efficacy in western Myanmar.

Polymorphisms of pfcrt, pfmdr1, and K13-propeller genes in imported falciparum malaria isolates from Africa in Guizhou province, China

BackgroundImported falciparum malaria from Africa has become a key public health challenge in Guizhou Province since 2012. Understanding the polymorphisms of molecular markers of drug resistance can guide selection of antimalarial drugs for the treatment of malaria. This study was aimed to analyze the polymorphisms of pfcrt, pfmdr1, and K13-propeller among imported falciparum malaria cases in Guizhou Province, China.MethodFifty-five imported falciparum malaria cases in Guizhou Province during 2012–2016 were included in this study. Their demographic information and filter paper blood samples were collected. Genomic DNA of Plasmodium falciparum was extracted from the blood samples, and polymorphisms of pfcrt, pfmdr1, and K13-propeller were analyzed with nested PCR amplification followed by sequencing. Data were analyzed with the SPSS17.0 software.ResultsThe prevalence of pfcrt K76T, pfmdr1 N86Y, and pfmdr1 Y184F mutation was 56.6, 22.2, and 72.2%, respectively, in imported falciparum malaria cases in Guizhou Province. We detected two mutant haplotypes of pfcrt, IET and MNT, with IET being more commonly found (54.7%), and five mutant haplotypes of pfmdr1, of which NFD was the most frequent (53.7%). There were totally 10 combined haplotypes of pfcrt and pfmdr1, of which the haplotype IETNFD possessed a predominance of 28.8%. In addition, three nonsynonymous mutations (S459T, C469F, and V692L) and two synonymous mutations (R471R and V589V) were detected in K13-propeller, all having prevalence less than 6.0%. In particular, a candidate K13 resistance mutation, C469F, was identified for the first time from Democratic Republic of the Congo with the prevalence of 2.0%.ConclusionsThe high prevalence of IET haplotype of pfcrt and NFD haplotype of pfmdr1 suggests the presence of chloroquine, artemether/lumefantrine, and dihydroartemisinin/piperaquine resistance in these cases. Therefore cautions should be made to artemisinin therapy for P. falciparum in Africa. Continuous monitoring of anti-malarial drug efficacy in imported malaria cases is helpful for optimizing antimalarial drug therapy in Guizhou Province, China.

Genetic Variations Associated with Drug Resistance Markers in Asymptomatic Plasmodium falciparum Infections in Myanmar.

The emergence and spread of drug resistance is a problem hindering malaria elimination in Southeast Asia. In this study, genetic variations in drug resistance markers of Plasmodium falciparum were determined in parasites from asymptomatic populations located in three geographically dispersed townships of Myanmar by PCR and sequencing. Mutations in dihydrofolate reductase (pfdhfr), dihydropteroate synthase (pfdhps), chloroquine resistance transporter (pfcrt), multidrug resistance protein 1 (pfmdr1), multidrug resistance-associated protein 1 (pfmrp1), and Kelch protein 13 (k13) were present in 92.3%, 97.6%, 84.0%, 98.8%, and 68.3% of the parasites, respectively. The pfcrt K76T, pfmdr1 N86Y, pfmdr1 I185K, and pfmrp1 I876V mutations were present in 82.7%, 2.5%, 87.5%, and 59.8% isolates, respectively. The most prevalent haplotypes for pfdhfr, pfdhps, pfcrt and pfmdr1 were 51I/59R/108N/164L, 436A/437G/540E/581A, 74I/75E/76T/220S/271E/326N/356T/371I, and 86N/130E/184Y/185K/1225V, respectively. In addition, 57 isolates had three different point mutations (K191T, F446I, and P574L) and three types of N-terminal insertions (N, NN, NNN) in the k13 gene. In total, 43 distinct haplotypes potentially associated with multidrug resistance were identified. These findings demonstrate a high prevalence of multidrug-resistant P. falciparum in asymptomatic infections from diverse townships in Myanmar, emphasizing the importance of targeting asymptomatic infections to prevent the spread of drug-resistant P. falciparum.

Prevalence of Plasmodium falciparum Pfcrt and Pfmdr1 alleles in settings with different levels of Plasmodium vivax co-endemicity in Ethiopia

Plasmodium falciparum and P. vivax co-exist at different endemicity levels across Ethiopia. For over two decades Artemether-Lumefantrine (AL) is the first line treatment for uncomplicated P. falciparum, while chloroquine (CQ) is still used to treat P. vivax. It is currently unclear whether a shift from CQ to AL for P. falciparum treatment has implications for AL efficacy and results in a reversal of mutations in genes associated to CQ resistance, given the high co-endemicity of the two species and the continued availability of CQ for the treatment of P. vivax. This study thus assessed the prevalence of Pfcrt-K76T and Pfmdr1-N86Y point mutations in P. falciparum. 18S RNA gene based nested PCR confirmed P. falciparum samples (N = 183) collected through community and health facility targeted cross-sectional surveys from settings with varying P. vivax and P. falciparum endemicity were used. The proportion of Plasmodium infections that were P. vivax was 62.2% in Adama, 41.4% in Babile, 30.0% in Benishangul-Gumuz to 6.9% in Gambella. The Pfcrt-76T mutant haplotype was observed more from samples with higher endemicity of P. vivax as being 98.4% (61/62), 100% (31/31), 65.2% (15/23) and 41.5% (22/53) in samples from Adama, Babile, Benishangul-Gumuz and Gambella, respectively. However, a relatively higher proportion of Pfmdr1-N86 allele (77.3–100%) were maintained in all sites. The observed high level of the mutant Pfcrt-76T allele in P. vivax co-endemic sites might require that utilization of CQ needs to be re-evaluated in settings co-endemic for the two species. A country-wide assessment is recommended to clarify the implication of the observed level of variation in drug resistance markers on the efficacy of AL-based treatment against uncomplicated P. falciparum malaria.

Low rates of Plasmodium falciparum Pfcrt K76T mutation in three sentinel sites of malaria monitoring in Côte d'Ivoire.

Despite efforts to eliminate it, malaria remains a major public health concern, particularly in Côte d'Ivoire. Chloroquine (CQ) was one of the first drugs used for its treatment, but was officially withdrawn from the market in 2007 following reports of high levels of chloroquine resistance. The present study was carried out after the withdrawal of CQ and provides an update on the rates of CQ resistance in Côte d'Ivoire. Samples were collected between September 2013 and March 2014 in Abidjan and from January to May 2016 in Abengourou and San Pedro through cross-sectional studies. Parasitemia was assessed by microscopy, and single nucleotide polymorphism in the Pfcrt (codon 76) gene was analyzed by nested PCR and restriction fragment length polymorphism. A total of 343 samples were analyzed: 119, 106 and 118 were from Abidjan, Abengourou, and San Pedro, respectively. The sex ratio of patients was 0.92. The mean age of patients enrolled was 9.6 years (SD = 10.8). The geometric mean of parasite density was 21,337 parasites/μL (SD = 49,508; range, 2,000-200,000). Molecular analysis revealed 57 K76T mutants (16.6%): 33, 9, and 15 in Abidjan, Abengourou and in San Pedro, respectively. Most of these were found in patients aged ≤15 years (42/57) who had parasitemia greater than 10,000 parasites/μL (40/57). This is the first study conducted in Côte d'Ivoire reporting a decline in Pfcrt K76T mutation rate. Thus, our results indicate the importance of following up on the observed trend also at a national level.

Prevalence of Plasmodium falciparum Molecular Markers of Antimalarial Drug Resistance in a Residual Malaria Focus Area in Sabah, Malaysia.

Chloroquine (CQ) and fansidar (sulphadoxine-pyrimethamine, SP) were widely used for treatment of Plasmodium falciparum for several decades in Malaysia prior to the introduction of Artemisinin-based Combination Therapy (ACT) in 2008. Our previous study in Kalabakan, located in south-east coast of Sabah showed a high prevalence of resistance to CQ and SP, suggesting the use of the treatment may no longer be effective in the area. This study aimed to provide a baseline data of antimalarial drug resistant markers on P. falciparum isolates in Kota Marudu located in the north-east coast of Sabah. Mutations on genes associated with CQ (pfcrt and pfmdr1) and SP (pfdhps and pfdhfr) were assessed by PCR amplification and restriction fragment length polymorphism. Mutations on the kelch13 marker (K13) associated with artemisinin resistance were determined by DNA sequencing technique. The assessment of pfmdr1 copy number variation associated with mefloquine resistant was done by real-time PCR technique. A low prevalence (6.9%) was indicated for both pfcrt K76T and pfmdr1 N86Y mutations. All P. falciparum isolates harboured the pfdhps A437G mutation. Prevalence of pfdhfr gene mutations, S108N and I164L, were 100% and 10.3%, respectively. Combining the different resistant markers, only two isolates were conferred to have CQ and SP treatment failure markers as they contained mutant alleles of pfcrt and pfmdr1 together with quintuple pfdhps/pfdhfr mutation (combination of pfdhps A437G+A581G and pfdhfr C59R+S108N+I164L). All P. falciparum isolates carried single copy number of pfmdr1 and wild type K13 marker. This study has demonstrated a low prevalence of CQ and SP resistance alleles in the study area. Continuous monitoring of antimalarial drug efficacy is warranted and the findings provide information for policy makers in ensuring a proper malaria control.

Prevalence of anti-malarial resistance genes in Dakar, Senegal from 2013 to 2014.

BackgroundTo determine the impact of the introduction of artemisinin-based combination therapy (ACT) on parasite susceptibility, a molecular surveillance for antimalarial drug resistance was conducted on local isolates from the Hôpital Principal de Dakar between November 2013 and January 2014 and between August 2014 and December 2014.MethodsThe prevalence of genetic polymorphisms in antimalarial resistance genes (pfcrt, pfmdr1, pfdhfr and pfdhps) was evaluated in 103 isolates.ResultsThe chloroquine-resistant haplotypes CVIET and CVMET were identified in 31.4 and 3.9 % of the isolates, respectively. The frequency of the pfcrt K76T mutation was increased from 29.3 % in 2013–2014 to 43.2 % in 2014. The pfmdr1 N86Y and Y184F mutations were identified in 6.1 and 53.5 % of the isolates, respectively. The pfdhfr triple mutant (S108N, N51I and C59R) was detected in the majority of the isolates (82.3 %). The prevalence of quadruple mutants (pfdhfr S108N, N51I, C59R and pfdhps A437G) was 40.4 %. One isolate (1.1 %) harboured the pfdhps mutations A437G and K540E and the pfdhfr mutations S108N, N51I and C59R.ConclusionsDespite a decline in the prevalence of chloroquine resistance due to the official withdrawal of the drug and to the introduction of ACT, the spread of resistance to chloroquine has continued. Furthermore, susceptibility to amodiaquine may be decreased as a result of cross-resistance. The frequency of the pfmdr1 mutation N86Y declined while the Y184F mutation increased in prevalence, suggesting that selective pressure is acting on pfmdr1, leading to a high prevalence of mutations in these isolates and the lack of specific mutations. The 50.5 % prevalence of the pfmdr1 polymorphisms N86Y and Y184F suggests a decrease in lumefantrine susceptibility. Based on these results, intensive surveillance of ACT partner drugs must be conducted regularly in Senegal.

Synergism between Pfcrt and Pfmdr1 genes could account for the slow recovery of chloroquine sensitive Plasmodium falciparum strains in Ghana after chloroquine withdrawal.

Unlike other countries, the chloroquine resistant marker Pfcrt T76 mutant has remained fairly stable in Ghana several years after official disuse of chloroquine. Certain mutations in Pfmdr1 may potentiate Pfcrt T76, offering a possible explanation for this observation. To understand the phenomenon, the co-existence of mutations in Pfmdr1 with Pfcrt T76 in Ghanaian Plasmodium falciparum isolates was studied. The reported presence of parasites with reduced sensitivity to amodiaquine and quinine in the country was also studied. Blood samples collected from confirmed malaria patients presenting at health facilities in two distinct ecological zones were analyzed. The prevalence of Pfcrt K76T and the five point mutations in Pfmdr1 were determined using nested PCR followed by RFLP analysis. The association between genes was determined by chi square analysis, and synergism between the two genes was ascertained using the Jonckheere-Terptra (J-T) test followed by Monte Carlo simulation (MCS). Nearly fifty-four percent (53.7%) of the P. falciparum isolates examined had the Pfcrt T76 gene, out of which 18.3% had both K76 and T76 alleles. Mutations at codon 86, 184, 1034, 1042 and 1246 of the Pfmdr1 gene were detected in 36.0%, 87.9%, 71.0%, 91.6% and 8.4% of the isolates, respectively. The haplotypes of Pfmdr1 present were NFCDD (43.46%), YFCDD (27.57%), NFSDD (7.48%), NYSNY (5.14%) and YFSDD (4.67%). Pfcrt T76 was significantly associated with a double mutation at codon 86 and 184 of Pfmdr1 (YF; χ2=18.045, p=0.006). Associations were observed between Pfcrt K76T and Pfmdr1 triple mutation at codons 86, 184 and 1034 (NFC; χ2=13.770, p=0.032 and YFC; χ2=16.489, p=0.011). The J-T test showed significant synergism between Pfcrt 76 and Pfmdr1 polymorphisms (p<0.0001), which was confirmed by MCS at 99% CI. Synergism between Pfcrt and Pfmdr1 mutant genes could account for the slow recovery of chloroquine sensitive P. falciparum in Ghana. The same phenomenon could explain resistance to amodiaquine and quinine. The outcomes of this study also indicated a possible emergence of artemether-lumefantrine resistance in Ghana.

Chloroquine sensitivity: diminished prevalence of chloroquine-resistant gene marker pfcrt-76 13years after cessation of chloroquine use in Msambweni, Kenya.

BackgroundPlasmodium falciparum resistance to chloroquine (CQ) denied healthcare providers access to a cheap and effective anti-malarial drug. Resistance has been proven to be due to point mutations on the parasite’s pfcrt gene, particularly on codon 76, resulting in an amino acid change from lysine to threonine. This study sought to determine the prevalence of the pfcrt K76T mutation 13 years after CQ cessation in Msambweni, Kenya.MethodsFinger-prick whole blood was collected on 3MM Whatman® filter paper from 99 falciparum malaria patients. Parasite DNA was extracted via the Chelex method from individual blood spots and used as template in nested PCR amplification of pfcrt. Apo1 restriction enzyme was used to digest the amplified DNA to identify the samples as wild type or sensitive at codon 76. Prevalence figures of the mutant pfcrt 76T gene were calculated by dividing the number of samples bearing the mutant gene with the total number of samples multiplied by 100 %. Chi square tests were used to test the significance of the findings against previous prevalence figures.ResultsOut of 99 clinical samples collected in 2013, prevalence of the mutant pfcrt 76T gene stood at 41 %.ConclusionThe results indicate a significant [χ2 test, P ≤ 0.05 (2006 vs 2013)] reversal to sensitivity by the P. falciparum population in the study site compared to the situation reported in 2006 at the same study site. This could primarily be driven by diminished use of CQ in the study area in line with the official policy. Studies to establish prevalence of the pfcrt 76T gene could be expanded countrywide to establish the CQ sensitivity status and predict a date when CQ may be re-introduced as part of malaria chemotherapy.

Temporal changes in Plasmodium falciparum anti-malarial drug sensitivity in vitro and resistance-associated genetic mutations in isolates from Papua New Guinea

BackgroundIn northern Papua New Guinea (PNG), most Plasmodium falciparum isolates proved resistant to chloroquine (CQ) in vitro between 2005 and 2007, and there was near-fixation of pfcrt K76T, pfdhfr C59R/S108N and pfmdr1 N86Y. To determine whether the subsequent introduction of artemisinin combination therapy (ACT) and reduced CQ-sulphadoxine-pyrimethamine pressure had attenuated parasite drug susceptibility and resistance-associated mutations, these parameters were re-assessed between 2011 and 2013.MethodsA validated fluorescence-based assay was used to assess growth inhibition of 52 P. falciparum isolates from children in a clinical trial in Madang Province. Responses to CQ, lumefantrine, piperaquine, naphthoquine, pyronaridine, artesunate, dihydroartemisinin, artemether were assessed. Molecular resistance markers were detected using a multiplex PCR ligase detection reaction fluorescent microsphere assay.ResultsCQ resistance (in vitro concentration required for 50% parasite growth inhibition (IC50) >100 nM) was present in 19% of isolates. All piperaquine and naphthoquine IC50s were <100 nM and those for lumefantrine, pyronaridine and the artemisinin derivatives were in low nM ranges. Factor analysis of IC50s showed three groupings (lumefantrine; CQ, piperaquine, naphthoquine; pyronaridine, dihydroartemisinin, artemether, artesunate). Most isolates (96%) were monoclonal pfcrt K76T (SVMNT) mutants and most (86%) contained pfmdr1 N86Y (YYSND). No wild-type pfdhfr was found but most isolates contained wild-type (SAKAA) pfdhps. Compared with 2005–2007, the geometric mean (95% CI) CQ IC50 was lower (87 (71–107) vs 167 (141–197) nM) and there had been no change in the prevalence of pfcrt K76T or pfmdr1 mutations. There were fewer isolates of the pfdhps (SAKAA) wild-type (60 vs 100%) and pfdhfr mutations persisted.ConclusionsReflecting less drug pressure, in vitro CQ sensitivity appears to be improving in Madang Province despite continued near-fixation of pfcrt K76T and pfmdr1 mutations. Temporal changes in IC50s for other anti-malarial drugs were inconsistent but susceptibility was preserved. Retention or increases in pfdhfr and pfdhps mutations reflect continued use of sulphadoxine-pyrimethamine in the study area including through paediatric intermittent preventive treatment. The susceptibility of local isolates to lumefantrine may be unrelated to those of other ACT partner drugs.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12610000913077.

Dynamic of plasmodium falciparum chloroquine resistance transporter gene Pfcrt K76T mutation five years after withdrawal of chloroquine in Burkina Faso.

We investigated the evolution of Pfcrt K76T mutation five years after the withdrawal of chloroquine in Burkina Faso. A total of 675 clinical isolates collected from October 2010 to September 2012 were successfully genotyped. Single nucleotide polymorphism in Pfcrt (codon 76) gene was analyzed. The prevalence of resistant Pfcrt 76T allele was 20.55%. There was a progressive decrease of the proportion of mutant type pfcrt T76 from 2010 to 2012 (X2=5.508 p=0.0189). Our results suggest a progressive return of the wild type Pfcrt K76 in Burkina Faso but the prevalence of the mutants Pfcrt T76 still remains high.