Pfcrt Mutations Research Articles

Characterization of Plasmodium falciparum genes associated with drug resistance in Hodh Elgharbi, a malaria hotspot near Malian\u2013Mauritanian border

BackgroundA malaria hotspot in the southeastern region of Mauritania, near the Malian border, may hamper malaria control strategies. The objectives were to estimate the prevalence of genetic polymorphisms associated with drug resistance in Plasmodium falciparum isolates and establish baseline data.MethodsThe study was conducted in two malaria-endemic areas in Hodh Elgharbi, situated in the Malian–Mauritanian border area. Blood samples were collected from symptomatic patients. Single nucleotide polymorphisms in Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps were genotyped using PCR-restriction fragment length polymorphism, DNA sequencing and primer extension. The Pfmdr1 gene copy number was determined by real-time PCR.ResultsOf 280 P. falciparum-infected patients, 193 (68.9%) carried the Pfcrt 76T mutant allele. The Pfmdr1 86Y and 184F mutations were found in 61 (23.1%) of 264 isolates and 167 (67.6%) of 247 samples that were successfully genotyped, respectively. Pfmdr1 mutant alleles 1034C, 1042D and 1246Y were rarely observed. Of 102 P. falciparum isolates analysed, ten (9.8%) had more than one copy of Pfmdr1 gene. The prevalence of isolates harbouring at least triple mutant Pfdhfr 51I, 59R, 108 N/T was 42% (112/268), of which 42 (37.5%) had an additional Pfdhps 437G mutation. The Pfdhps 540E mutation was observed in four isolates (1.5%), including three associated with Pfdhfr triple mutant. Only two quintuple mutants (Pfdhfr-51I-59R-108N Pfdhps-437G-540E) were observed.ConclusionsThe observed mutations in Pfdhfr, Pfdhps, Pfmdr1, and Pfcrt may jeopardize the future of seasonal malaria chemoprevention based on amodiaquine-sulfadoxine-pyrimethamine, intermittent preventive treatment for pregnant women using sulfadoxine-pyrimethamine, and treatment with artesunate-amodiaquine. Complementary studies should be carried out to document the distribution, origin and circulation of P. falciparum populations in this region and more widely in the country to assess the risk of the spread of resistance.

Reemergence of chloroquine-sensitive pfcrt K76 Plasmodium falciparum genotype in southeastern Cameroon

BackgroundChloroquine had been used extensively during the last five decades in Cameroon. Its decreasing clinical effectiveness, supported by high proportions of clinical isolates carrying the mutant pfcrt haplotype (CVIET), led the health authorities to resort to amodiaquine monotherapy in 2002 and artemisinin-based combination therapy (ACT) in 2004 (artesunate–amodiaquine, with artemether–lumefantrine as an alternative since 2006) as the first-line treatment of uncomplicated malaria. The aim of the present study was to investigate whether the withdrawal of chloroquine was associated with a reduction in pfcrt mutant parasite population and reemergence of chloroquine-sensitive parasites in southeastern Cameroon between 2003 and 2012.MethodsThe frequency of pfcrt haplotypes at positions 72–76 in Plasmodium falciparum isolates collected from individuals in 2003 and 2012 in southeastern Cameroon was determined by sequence specific oligonucleotide probes-enzyme linked immunosorbent assay (SSOP-ELISA).ResultsThe proportions of parasites carrying the mutant haplotype CVIET and the wild-type CVMNK were 53.0 and 28.0% in 2003, respectively. The proportion of the mutant haplotype in samples collected 9 years later decreased to 25.3% whereas the proportion of parasites carrying the wild-type CVMNK haplotype was 53.7%.ConclusionsEven though the proportion of chloroquine-sensitive parasites seems to be increasing in southeastern Cameroon, a reintroduction of chloroquine cannot be recommended at present in Cameroon. The current national anti-malarial drug policy should be implemented and reinforced to combat drug-resistant malaria.

Prevalence of Multiple Drug-resistant Plasmodium falciparum Malaria Cases in Northeast India

Two numbers of Plasmodium falciparum field isolates from Gossingpara, Runikhata area in Chirang district of Assam had shown multiple mutations in Pfcrt-dhfr-dhps gene (up to seven mutations: One mutation in Pfcrt gene, three mutations in Pfdhfr gene and three mutations in Pfdhps gene). Similarly, two cases in Bat camp, Miao area under Changlang district of Arunachal Pradesh had shown a total of eight mutations, of which one mutation in Pfcrt gene, three mutations in Pfdhfr gene, three mutations in Pfdhps gene and one mutation in PfATPase6gene. One case in 3 Miles, Miao area of Changlang district has shown mutations in Pfcrt (one mutation), Pfdhfr (four mutations) and Pfdhps (three mutations) gene. These results indicated that there is an existence of multiple mutant P. falciparum malaria cases in northeastern region of India.

Drug-Resistant Polymorphisms and Copy Numbers in Plasmodium falciparum, Mozambique, 2015.

One of the fundamental steps toward malaria control is the use of antimalarial drugs. The success of antimalarial treatment can be affected by the presence of drug-resistant populations of Plasmodium falciparum. To assess resistance, we used molecular methods to examine 351 P. falciparum isolates collected from 4 sentinel sites in Mozambique for K13, pfmdr1, pfcrt, and pfdhps polymorphisms and for plasmepsin2 (pfpm2) and pfmdr1 copy numbers. We found multiple copies of pfpm2 in 1.1% of isolates. All isolates carried K13 wild-type alleles (3D7-like), except 4 novel polymorphisms (Leu619Leu, Phe656Ile, Val666Val, Gly690Gly). Prevalence of isolates with pfcrt mutant (K76T) allele was low (2.3%). Prevalence of isolates with pfdhps mutant alleles (A437G and K540E) was >80%, indicating persistence of sulfadoxine/pyrimethamine resistance; however, markers of artemisinin were absent, and markers of piperaquine resistance were low. Piperaquine resistance isolates may spread in Mozambique as dihydroartemisinin/piperaquine drug pressure increases.

Evolution of Fitness Cost-Neutral Mutant PfCRT Conferring P. falciparum 4-Aminoquinoline Drug Resistance Is Accompanied by Altered Parasite Metabolism and Digestive Vacuole Physiology.

Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pressures on the subcontinent have recurrently produced several allelic variants of parasite drug resistance genes, including the P. falciparum chloroquine resistance transporter (pfcrt). Despite significant reductions in the deployment of the 4-aminoquinoline drug chloroquine (CQ), which selected for the mutant pfcrt alleles that halted CQ efficacy decades ago, the parasite pfcrt locus is continuously evolving. This is highlighted by the presence of a highly mutated allele, Cam734 pfcrt, which has acquired the singular ability to confer parasite CQ resistance without an associated fitness cost. Here, we used pfcrt-specific zinc-finger nucleases to genetically dissect this allele in the pathogenic setting of asexual blood-stage infection. Comparative analysis of drug resistance and growth profiles of recombinant parasites that express Cam734 or variants thereof, Dd2 (the most common Southeast Asian variant), or wild-type pfcrt, revealed previously unknown roles for PfCRT mutations in modulating parasite susceptibility to multiple antimalarial agents. These results were generated in the GC03 strain, used in multiple earlier pfcrt studies, and might differ in natural isolates harboring this allele. Results presented herein show that Cam734-mediated CQ resistance is dependent on the rare A144F mutation that has not been observed beyond Southeast Asia, and reveal distinct impacts of this and other Cam734-specific mutations on CQ resistance and parasite growth rates. Biochemical assays revealed a broad impact of mutant PfCRT isoforms on parasite metabolism, including nucleoside triphosphate levels, hemoglobin catabolism and disposition of heme, as well as digestive vacuole volume and pH. Results from our study provide new insights into the complex molecular basis and physiological impact of PfCRT-mediated antimalarial drug resistance, and inform ongoing efforts to characterize novel pfcrt alleles that can undermine the efficacy of first-line antimalarial drug regimens.

Prevalence of crt and mdr-1 mutations in Plasmodium falciparum isolates from Grande Comore island after withdrawal of chloroquine.

BackgroundIn Comoros, the widespread of chloroquine (CQ)-resistant Plasmodium falciparum populations was a major obstacle to malaria control, which led to the official withdrawal of CQ in 2004. Continuous monitoring of CQ-resistant markers of the P. falciparum CQ resistant transporter (pfcrt) and the P. falciparum multiple drug resistance 1 (pfmdr-1) is necessary inder to obtain first-hand information on CQ susceptibility of parasite populations in the field. The objective of this study is to assess the prevalence and evolution of CQ-resistance in the P. falciparum populations on the Comoros’ Grande Comore island after withdrawal of CQ.MethodsA total of 207 P. falciparum clinical isolates were collected from the island, including 118 samples from 2006 to 2007 and 89 samples from 2013 to 2014. Nucleotide substitutions in the pfcrt and pfmdr-1 genes linked to CQ response in parasite isolates were assessed using nested PCR and DNA sequencing.ResultsFrom the pfcrt gene segment sequenced, we detected C72S, M74I, N75E, and K76T substitutions in the parasite isolates collected from both 2006–2007 to 2013–2014 periods. Significant decline of pfcrt resistant alleles at C72S (42.6 to 6.9 %), M74I (39.1 to 14.9 %), N75E (63.5 to 18.3 %), and K76T (72.2 to 19.5 %) from 2006–2007 to 2013–2014 were observed, and the frequency of pfcrt wild type allele was significantly increased from 19.1 % in 2006–2007 to 75.8 % in 2013–2014. Sequence analysis of pfmdr-1 also detected point mutations at codons N86Y, Y184F, and D1246Y, but not S1034C and N1042D, in the isolates collected from both examined periods. An increasing trend in the prevalence of the pfmdr-1 wild type allele (NYD, 4.3 % in 2006–2007; and 28.7 % in 2013–2014), and a decreasing trend for pfmdr-1 N86Y mutation (87.0 % in 2006–2007; and 40.2 % in 2013–2014) were observed in our samples.ConclusionsThe present data indicate that the prevalence and patterns of mutant pfcrt and pfmdr-1 dramatically decreased in the Grande Comore isolates from 2006 to 2014, suggesting that the CQ-sensitive P. falciparum strains have returned after the withdrawal of CQ. The data also suggests that the parasites with wild type pfcrt/pfdmr-1 genes may have growth and/or transmission advantages over the mutant parasites. The information obtained from this study will be useful for developing and updating anti-malarial treatment policy in Grande Comore island.

Molecular markers associated with resistance to commonly used antimalarial drugs among Plasmodium falciparum isolates from a malaria-endemic area in Taiz governorate—Yemen during the transmission season

Since 2005, artesunate (AS) plus sulfadoxine/pyrimethamine (SP) combination has been adopted as the first-line treatment for uncomplicated malaria in Yemen in response to the high level of Plasmodium falciparum resistance to chloroquine (CQ). Therefore, the aim of the present study was to determine the frequency distribution of molecular markers associated with resistance to CQ and AS plus SP combination among P. falciparum isolates from a malaria-endemic area in Taiz governorate, Yemen. Fifty P. falciparum isolates were collected during a cross-sectional study in Mawza district, Taiz, in the period from October 2013 to April 2014. The isolates were investigated for drug resistance-associated molecular markers in five genes, including P. falciparum CQ resistance transporter (pfcrt) 76T and P. falciparum multidrug resistance 1 (pfmdr1) 86Y as markers of resistance to CQ, mutations in the Kelch 13 (K13) propeller domain for resistance to AS, and P. falciparum dihydrofolate reductase (pfdhfr) and P. falciparum dihydropteroate synthase (pfdhps) genes for resistance to SP. Nested polymerase chain reaction was used to amplify target genes in DNA extracts of the isolates followed by restriction fragment length polymorphism for detecting 76T and 86Y mutations in pfcrt and pfmdr1, respectively, and by DNA sequencing for detecting mutations in K13, pfdhfr and pfdhps. All the investigated isolates from Mawza district were harboring the pfcrt 76T mutant and the pfmdr1 N86 wild-type alleles. The pfdhfr 51I/108N double mutant allele was found in 2.2% (1/45) of the isolates; however, no mutations were detected at codons 436, 437, 540, 581 and 613 of pfdhps. All P. falciparum isolates that were successfully sequenced (n=47) showed the K13 Y493, R539, I543 and C580 wild-type alleles. In conclusion, the pfcrt 76T mutant allele is fixed in the study area about six years after the official withdrawal of CQ, possibly indicating its over-the-counter availability and continued use as a self-medication in the study area. However, the almost predominant wild-type alleles of the genes associated with resistance to AS and SP among P. falciparum isolates in the present study indicates the sustained efficacy of the currently adopted first-line treatment of AS plus SP in the study area.

Examining Plasmodium falciparum and P. vivax clearance subsequent to antimalarial drug treatment in the Myanmar-China border area based on quantitative real-time polymerase chain reaction.

BackgroundRecent emergence of artemisinin-resistant P. falciparum has posed a serious hindrance to the elimination of malaria in the Greater Mekong Subregion. Parasite clearance time, a measure of change in peripheral parasitaemia in a sequence of samples taken after treatment, can be used to reflect the susceptibility of parasites or the efficiency of antimalarials. The association of genetic polymorphisms and artemisinin resistance has been documented. This study aims to examine clearance time of P. falciparum and P. vivax parasitemia as well as putative gene mutations associated with residual or recurred parasitemia in Myanmar.MethodsA total of 63 P. falciparum and 130 P. vivax samples collected from two internally-displaced populations and one surrounding village were examined for parasitemia changes. At least four samples were taken from each patient, at the first day of diagnosis up to 3 months following the initial treatment. The amount of parasite gene copy number was estimated using quantitative real-time PCR based on a species-specific region of the 18S rRNA gene. For samples that showed residual or recurred parasitemia after treatment, microsatellites were used to identify the ‘post-treatment’ parasite genotype and compared such with the ‘pre-treatment’ genotype. Mutations in genes pfcrt, pfmdr1, pfatp6, pfmrp1 and pfK13 that are potentially associated with ACT resistance were examined to identify if mutation is a factor for residual or persistent parasitemia.ResultsOver 30 % of the P. falciprium infections showed delayed clearance of parasitemia after 2–3 days of treatment and 9.5 % showed recurred parasitemia. Mutations in codon 876 of the pfmrp1 corroborated significance association with slow clearance time. However, no association was observed in the variation in pfmdr1 gene copy number as well as mutations of various codonsinpfatp6, pfcrt, and pfK13 with clearance time. For P. vivax, over 95 % of the infections indicated cleared parasitemia at days 2–3 of treatment. Four samples were found to be re-infected with new parasite strains based on microsatellite genotypes after initial treatment.ConclusionThe appearance of P.falciparum infected samples showing delayed clearance or recurred parasitemia after treatment raises concerns on current treatment and ACT drug resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1482-6) contains supplementary material, which is available to authorized users.

Mutational prevalence of chloroquine resistance transporter gene among Plasmodium falciparum field isolates in Assam and Arunachal Pradesh, India

Objective: The present study aims to find out the mutational prevalence of Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene in Assam and Arunachal Pradesh, India. Methods: To fulfil the objective of the study, a total of 54 P. falciparum malaria positive samples were attempted for genotyping of Pfcrt gene using polymerase chain reaction (PCR) and direct DNA sequencing method. Results: The K76T mutation was observed in 77.78% cases followed by M74I (61.11%), N75E (61.11%) and C72S (16.67%). Triple mutant allele M74I+N75E+K76T was found in 61.11% P. falciparum field isolates. Double mutant allele C72S+K76T was seen among 16.67% samples. Mutation studies have shown existence of three different haplotypes of which two were associated with chloroquine resistance. The haplotype CVIET was found preponderance and circulated in all four districts. The other haplotype SVMNT was observed only in Karbi Anglong district of Assam. Maximum haplotype diversity was also recorded from Karbi Anglong district of Assam. Conclusion: Our study has confirmed high prevalence of mutant Pfcrt genotypes associated with chloroquine resistance in Assam and Arunachal Pradesh, India.

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.

Combinatorial Genetic Modeling of pfcrt-Mediated Drug Resistance Evolution in Plasmodium falciparum.

The emergence of drug resistance continuously threatens global control of infectious diseases, including malaria caused by the protozoan parasite Plasmodium falciparum. A critical parasite determinant is the P. falciparum chloroquine resistance transporter (PfCRT), the primary mediator of chloroquine (CQ) resistance (CQR), and a pleiotropic modulator of susceptibility to several first-line artemisinin-based combination therapy partner drugs. Aside from the validated CQR molecular marker K76T, P. falciparum parasites have acquired at least three additional pfcrt mutations, whose contributions to resistance and fitness have been heretofore unclear. Focusing on the quadruple-mutant Ecuadorian PfCRT haplotype Ecu1110 (K76T/A220S/N326D/I356L), we genetically modified the pfcrt locus of isogenic, asexual blood stage P. falciparum parasites using zinc-finger nucleases, producing all possible combinations of intermediate pfcrt alleles. Our analysis included the related quintuple-mutant PfCRT haplotype 7G8 (Ecu1110 + C72S) that is widespread throughout South America and the Western Pacific. Drug susceptibilities and in vitro growth profiles of our combinatorial pfcrt-modified parasites were used to simulate the mutational trajectories accessible to parasites as they evolved CQR. Our results uncover unique contributions to parasite drug resistance and growth for mutations beyond K76T and predict critical roles for the CQ metabolite monodesethyl-CQ and the related quinoline-type drug amodiaquine in driving mutant pfcrt evolution. Modeling outputs further highlight the influence of parasite proliferation rates alongside gains in drug resistance in dictating successful trajectories. Our findings suggest that P. falciparum parasites have navigated constrained pfcrt adaptive landscapes by means of probabilistically rare mutational bursts that led to the infrequent emergence of pfcrt alleles in the field.

Polymorphisms in K13, pfcrt, pfmdr1, pfdhfr, and pfdhps in parasites isolated from symptomatic malaria patients in Burkina Faso.

Background: The emergence of resistance to artemisinin derivatives in western Cambodia is threatening to revert the recent advances made toward global malaria control and elimination. Known resistance-mediating polymorphisms in the K13, pfcrt, pfmdr1, pfdhfr, and pfdhps genes are of greatest importance for monitoring the spread of antimalarial drug resistance. Methods: Samples for the present study were collected from 244 patients with uncomplicated malaria in health centers of Bobo-Dioulasso, Burkina Faso. Blood sample was collected on filter paper before the subject received any treatment. The parasite DNA was then extracted and amplified by Polymerase Chain Reaction (PCR) to evaluate the prevalence of polymorphism of pfcrtK76T, pfmdr1 (N86Y, Y184F), and pfdhps (A437G, K540E). The K13 gene polymorphism was analyzed by nested PCR followed by sequencing. Results: The overall results showed 2.26% (5/221) of K13 synonymous mutant alleles (two C469C, one Y493Y, one G496G, and one V589V), 24.78%, 19.58%, 68.75%, 60.9%, 53.7%, 63.8%, and 64.28%, respectively, for mutant pfcrt 76T, pfmdr1-86Y, pfmdr1-184F, pfdhfr51I, pfdhfr59R, pfdhfr108N, and pfdhps 437G. We did not report any mutation at codon 540 of pfdhps. Conclusion: These results provide baseline prevalence of known drug resistance polymorphisms and suggest that artemisinin combination therapies may retain good efficacy in the treatment of uncomplicated malaria in Burkina Faso.

Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities.

Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.

Molecular mutation profile of Pfcrt and Pfmdr1 in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea

BackgroundAntimalarial drug resistance is a primary public health problem. Haplotypes of pfcrt and pfmdr1 gene have been implicated to be molecular markers of chloroquine (CQ) resistance. This study aims to explore mutation distribution of Pfcrt and Pfmdr1 in Bioko Island, Equatorial Guinea (EG). MethodsBlood samples were collected from different districts of Bioko. The single nucleotide polymorphisms in Pfcrt (codons 72 to 76) and Pfmdr1 (codons 86, 130, 184, 1034, 1042, 1109 and 1246) were assessed by nested PCR with DNA sequencing and haplotype prevalences were also determined. ResultsAnalysis of Pfcrt and Pfmdr1 mutations was successful in 151 and 157 samples respectively out of the 172 samples taken for this study. The mutations of Pfcrt and Pfmdr1 were found in 98.67% and 89.81% isolates, respectively. The Pfcrt 74–76, Pfmdr1 86, and Pfmdr1 184 were 92.05%, 50.32%, and 87.26% found mostly of mutation type, respectively. Three haplotypes coding 72–76 of Pfcrt were found including CVMNK, CVIET, and CVM/I N/E K/T, which accounted for 1.33%, 92.05%, and 6.62%, respectively. No mutation in Pfmdr1-N1 codon at 130 and Pfmdr1-N2 (S1034C, N1042D, V1109I, and D1246Y) was detected. The types coding 86 and 184 in Pfmdr1 were found including NY, YY, NF, YF, NY/F and YY/F, which accounted for 10.19%, 2.55%, 33.76%, 45.22%, 5.73% and 2.55%, respectively. ConclusionHigh prevalence of Pfcrt CVIET and Pfmdr1 86Y, 184F double mutations confirm high-level CQ resistance (CQR) and might suggest reduced susceptibility of Plasmodium falciparum isolates to AQ in Bioko, EG. It establishes fundamental data for detection of P. falciparum CQR with molecular markers and will promotes the surveillance level of drug resistance in Bioko, EG.

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.

Survey of chloroquine-resistant mutations in the Plasmodium falciparum pfcrt and pfmdr-1 genes in Hadhramout, Yemen

Malaria is still a major public health problem in Yemen. More than 95% of the malaria cases are due to Plasmodium ‎falciparum‎. Recently in Yemen, the antimalarial treatment policy was changed from chloroquine (CQ) to artemisinin combination therapy (ACTs). However, CQ is still available and prescribed in the Yemeni market. The persistence of CQ resistance will be prolonged if the shift to ACT and the simultaneous withdrawal of CQ are not rigorously implemented. The aim of the current survey is to detect chloroquine-resistant mutations in P. falciparum chloroquine-resistance transporter (pfcrt) and P. falciparum multi-drug resistance-1 (pfmdr1) genes. These data will be important for future monitoring and assessment of antimalarial drug policy in Yemen. Blood specimens were collected from 735 individuals from different districts of the Hadhramout province, Yemen by house-to-house visit. Mutation-specific nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (PCR–RFLP) methods were used to investigate the mutations in the pfmdr1(codons 86 and 1246) and pfcrt (codons 76, 271, 326, 356 and 371) genes. The overall prevalence of pfcrt mutations at codons 76, 271, 326 and 371 were 50.4%, 58.7%, 54.3% and 44.9%, respectively. All isolates had wild-type pfcrt 356 allele. The majority of pfmdr1 86 alleles (83.3%) and all pfmdr1 1246 alleles were wild type. There was no association between pfcrt mutations and symptomatology, gender and age groups. In conclusion, point mutations in codons 76, 271, 326 and 371 of pfcrt of P. falciparum are high suggesting a sustained high CQ resistance even after 4 years of shifting to ACTs. These findings warrant complete withdrawal of CQ use from the Yemeni market for P. falciparum and careful usage of CQ for treating Plasmodium vivax.

Persistence of chloroquine-resistant haplotypes of Plasmodium falciparum in children with uncomplicated Malaria in Lagos, Nigeria, four years after change of chloroquine as first-line antimalarial medicine.

BackgroundIn Nigeria, despite the change in National malaria drug policy to artemisinin combination therapy (ACT) in 2005 due to widespread chloroquine resistance, chloroquine (CQ) is still widely used in the treatment of malaria because it is cheap, affordable and accessible. The use of ACT for the management of uncomplicated malaria is currently being promoted. The employment of genetic markers to track circulating chloroquine-resistant parasites are useful in elucidating likely poor efficacy of chloroquine, especially in settings where it is not recommended for the treatment of uncomplicated falciparum malaria. This study determined the prevalence of pfcrt haplotypes and point mutations in pfmdr1 genes four years after the change in antimalarial treatment policy from CQ to the ACTs in Lagos, a commercial city in South-West, Nigeria.MethodsThis was a cross sectional study on uncomplicated malaria in children less than 12 years that presented with fever and other symptoms suggestive of malaria. Parasite DNA was extracted from 119 patients out of 251 children who were positive for Plasmodium falciparum by microscopy and amplified. The occurrence of haplotypes was investigated in pfcrt gene using probe-based qPCR and single nucleotide polymorphisms in pfmdr1 gene using nested PCR.ResultsOne hundred and nine (109) of the 119 children with P falciparum infection (91.6%) harbourd parasites with the mutant pfcrt haplotype (CVIET). Out of this, 4.2% comprised a mixture of genotypes encoding CVMNK and CVIET, while 4.2% had the wild type (CVMNK). Furthermore, the frequency of point mutations in pfmdr1 was 62.2% and 69.0% for codons Y86 and F184 respectively. There were no mutations at codons 1034, 1042 and 1246 of the Pfmdr1 genes.ConclusionThe high frequency of the CQ-resistant haplotypes (CVIET) and mutations in Pfmdr1 associated with CQ resistance in P. falciparum among these children suggest that CQ-resistant parasites are still in circulation. Continuous use of chloroquine may continue to increase the level of mutations in pfcrt and pfmdr1genes. There is need to strengthen current case management efforts at promoting ACT use as well as urgently restricting access to chloroquine by the National drug regulatory agency, National Agency for Food Drug Administration and Control (NAFDAC).Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2069472010142303

Molecular epidemiology of drug-resistant Plasmodium falciparum in Benguela province, Angola.

BackgroundThe malaria situation has been worsening in Angola, partly due to armed conflict until the recent past and drug-resistant Plasmodium falciparum. Malaria transmission is heterogeneous within the country, and data on drug-resistant malaria in different parts of the country are incomplete. The aim of the present study was to evaluate resistance to 4-aminoquinolines and antifolate drugs in P. falciparum isolates collected in Benguela province, central Angola, using molecular markers.MethodsFingerprick capillary blood was collected from asymptomatic children aged less than 15 years old during a household survey in and around Balombo town in 2010–2011. Samples were screened for P. falciparum by nested PCR. Molecular markers (P. falciparum dihydrofolate reductase [pfdhfr], P. falciparum dihydropteroate synthase [pfdhps], P. falciparum chloroquine resistance transporter [pfcrt], and P. falciparum multidrug-resistance gene 1 [pfmdr1]) were sequenced to determine the key codons associated with drug resistance.ResultsA total of 60 blood samples were positive for P. falciparum. Most isolates with successful PCR amplification had mutant pfdhfr alleles, with either double mutant AICNI (69%) or triple mutant AIRNI (21%) haplotypes. A16V, S108T, and I164L substitutions were not found. Many of the isolates were carriers of either SGKAA (60%) or AGKAA (27%) pfdhps haplotype. K540E substitution was absent. There were only two pfcrt haplotypes: wild-type CVMNK (11%) and mutant CVIET (89%). Wild-type pfmdr1 NYSND haplotype was found in 19% of the isolates, whereas single mutant pfmdr1 YYSND and NFSND haplotypes occurred in 48% and 11%, respectively. Double mutant pfmdr1 haplotypes (YFSND and YYSNY) occurred rarely.ConclusionsThe results suggest that the high prevalence of mutant pfcrt CVIET haplotype is in agreement with low clinical efficacy of chloroquine observed in earlier studies and that the double pfdhfr mutant AICNI and single pfdhps mutant SGKAA are currently the predominant haplotypes associated with antifolate resistance in Benguela province. The hallmark of clinical resistance observed in East Africa, i.e. triple pfdhfr mutant haplotype (AIRNI) and double pfdhps mutant haplotype (SGEAA), was absent. These molecular findings need to be further evaluated in parallel with clinical studies, in particular with the efficacy of intermittent preventive treatment using sulphadoxine-pyrimethamine in pregnant women and artesunate-amodiaquine for uncomplicated malaria.

Identification of chloroquine resistance Pfcrt-K76T and determination of Pfmdr1-N86Y copy number by SYBR Green I qPCR

ABSTRACT Objective To identify prevalence of chloroquine resistance point mutation at ( Pfcrt , K76T) and ( Pfmdr1 , N86Y) copy number variation. Methods SYBR Green I based real time PCR was used. One hundred and thirty-three samples were analyzed for ( Pfcrt , K76T) and ( Pfmdr1 , N86Y) copy number from dried blood spot. Parasite DNA was extracted using high pure DNA preparation kit. The amplification of DNA was done by using AccuPower 2× GreenStar™ q PCR Master mix. For quantification purpose a new primer pair was designed for 178 base pair template from complete genome sequence of Plasmodium falciparum strain 3D7 at NCBI . Absolute quantification method was used to determine the Pfmdr1 -N86Y copy number variations. Standard curve was built from strain 3D7 g DNA since it has single copy of Pfmdr1 per haploid genome. The known positive controls with single and multi-copy number of Pfmdr1 gene were included in each experiment. The copy number ratio of the samples to the standard calibrator was made to obtain the fold difference among the samples with respect to copy number variation. Results Out of 133 samples 73 (54.89%) were confirmed as mutant ( Pfcrt, 76T) and the remaining 60 (45.11%) were genotyped as wild type ( Pfcrt, K76). The ( Pfmdr1 , N86Y) copy number variation was determined for 133 clinical samples. Out of these samples 61 (45.86%) had single copy and the remaining 72 (54.14%) had multi-copy numbers higher than 1.5 copies per genome. Thirty-four (25.56%) multi-copies were between 1.5 and 2.5 copies per genome while 38 (28.57%) were more than 2.5 copies per genome. The minimum and maximum copies per genome were 0.474 and 4.741, respectively. Conclusions The study showed high prevalence level and fixation of Pfcrt , 76T mutation after chloroquine withdrawal. The prevalence of Pfmdr1 copy number variant suggested that the presence of modulating factor for emergence of Plasmodium falciparum strains with higher copy numbers. However, the prevalence level was not statistically significant.

Assessment of molecular markers for anti-malarial drug resistance after the introduction and scale-up of malaria control interventions in western Kenya.

BackgroundAlthough it is well known that drug pressure selects for drug-resistant parasites, the role of transmission reduction by insecticide-treated bed nets (ITNs) on drug resistance remains unclear. In this study, the drug resistance profile of current and previous first-line anti-malarials in Kenya was assessed within the context of drug policy change and scale-up of ITNs. National first-line treatment changed from chloroquine (CQ) to sulphadoxine-pyrimethamine (SP) in 1998 and to artemether-lumefantrine (AL) in 2004. ITN use was scaled-up in the Asembo, Gem and Karemo areas of western Kenya in 1997, 1999 and 2006, respectively.MethodsSmear-positive samples (N = 253) collected from a 2007 cross-sectional survey among children in Asembo, Gem and Karemo were genotyped for mutations in pfcrt and pfmdr1 (CQ), dhfr and dhps (SP), and at pfmdr-N86 and the gene copy number in pfmdr1 (lumefantrine). Results were compared among the three geographic areas in 2007 and to retrospective molecular data from children in Asembo in 2001.ResultsIn 2007, 69 and 85% of samples harboured the pfmdr1-86Y mutation and dhfr/dhps quintuple mutant, respectively, with no significant differences by study area. However, the prevalence of the pfcrt-76T mutation differed significantly among areas (p <0.02), between 76 and 94%, with the highest prevalence in Asembo. Several 2007 samples carried mutations at dhfr-164L, dhps-436A, or dhps-613T. From 2001 to 2007, there were significant increases in the pfcrt-76T mutation from 82 to 94% (p <0.03), dhfr/dhps quintuple mutant from 62 to 82% (p <0.03), and an increase in the septuple CQ and SP combined mutant haplotype, K76Y86I51R59N108G437E540, from 28 to 39%. The prevalence of the pfmdr1-86Y mutation remained unchanged. All samples were single copy for pfmdr1.ConclusionsMolecular markers associated with lumefantrine resistance were not detected in 2007. More recent samples will be needed to detect any selective effects by AL. The prevalence of CQ and SP resistance markers increased from 2001 to 2007 in the absence of changes in transmission intensity. In 2007, only the prevalence of pfcrt-76T mutation differed among study areas of varying transmission intensity. Resistant parasites were most likely selected by sustained drug pressure from the continued use of CQ, SP, and mechanistically similar drugs, such as amodiaquine and cotrimoxazole. There was no clear evidence that differences in transmission intensity, as a result of ITN scale-up, influenced the prevalence of drug resistance molecular markers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0588-4) contains supplementary material, which is available to authorized users.