Plasmodium Falciparum Dihydrofolate Reductase Research Articles

Mutational analysis of Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase genes in the interior division of Sabah, Malaysia

BackgroundThe sulphadoxine/pyrimethamine (SDX/PYR) combination had been chosen to treat uncomplicated falciparum malaria in Malaysia for more than 30 years. Non-silent mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes are responsible for the resistance to pyrimethamine and sulphadoxine, respectively. This study reports the mutational analysis of pfdhfr and pfdhps in single Plasmodium falciparum infection isolates from the interior division of Sabah, Malaysian Borneo.MethodsA total of 22 P. falciparum single infection isolates collected from two districts of the interior division of Sabah from February to November 2010 were recruited for the mutational study of pfdhfr and pfdhps. Both genes were amplified by nested PCR prior to DNA sequencing and mutational analysis.ResultsA total of three pfdhfr and four pfdhps alleles were identified. The most prevalent pfdhfr allele is ANRNL (86%) involving triple mutation at position 108(S to N), 59(C to R) and 164(I to L). In pfdhps, two novel alleles, SGTGA (73%) and AAKAA (5%) were identified. Alleles involving triple mutation in both pfdhfr (ANRNL) and pfdhps (SGTGA), which were absent in Sabah in a study conducted about 15 years ago, are now prevalent.ConclusionsHigh prevalence of mutations in SDX/PYR associated drug resistance genes are reported in this study. This mutational study of pfdhps and pfdhfr indicating that SDX/PYR should be discontinued in this region.

Plasmodium falciparumMutant Haplotype Infection during Pregnancy Associated with Reduced Birthweight, Tanzania

Intermittent preventive treatment during pregnancy with sulfadoxine–pyrimethamine (IPTp-SP) is a key strategy in the control of pregnancy-associated malaria. However, this strategy is compromised by widespread drug resistance from single-nucleotide polymorphisms in the Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthetase genes. During September 2008–October 2010, we monitored a cohort of 924 pregnant women in an area of Tanzania with declining malaria transmission. P. falciparum parasites were genotyped, and the effect of infecting haplotypes on birthweight was assessed. Of the genotyped parasites, 9.3%, 46.3%, and 44.4% had quadruple or less, quintuple, and sextuple mutated haplotypes, respectively. Mutant haplotypes were unrelated to SP doses. Compared with infections with the less-mutated haplotypes, infections with the sextuple haplotype mutation were associated with lower (359 g) birthweights. Continued use of the suboptimal IPTp-SP regimen should be reevaluated, and alternative strategies (e.g., intermittent screening and treatment or intermittent treatment with safe and effective alternative drugs) should be evaluated.

High prevalence of mutation in the Plasmodium falciparum dhfr and dhps genes in field isolates from Sabah, Northern Borneo

BackgroundSulphadoxine-pyrimethamine (SP) has been in use for the treatment of uncomplicated falciparum malaria in Malaysia since the 1970s and is still widely employed in spite of widespread clinical resistance. Resistance to SP is known to be mediated by mutations in the pfdhfr and pfdhps genes. The aim of the present study was to investigate the distribution of pfdhfr and pfdhps gene polymorphism in Plasmodium falciparum field isolates from Kalabakan, Sabah, in northern Borneo.MethodsA total number of 619 individuals were screened from 23 study sites of which 31 were positive for P. falciparum. Analysis of restriction fragment length polymorphisms (RFLP) was used to identify polymorphism in the pfdhfr and pfdhps genes at positions 16, 51, 59, 108, 164 and 437, 540, 581, respectively.ResultsAll samples had at least one mutation in each of the genes associated with drug resistance. The prevalence of pfdhfr 59arg, 164leu and 108asn were 100%, 80.65% and 58.06%, respectively. Pfdhps mutants 437gly and 581gly accounted for 100% and 74.19% respectively. In pfdhfr, the most common mutant genotypes were combination 59arg + 164leu (22.58%) and 59arg + 108asn + 164leu (51.61%). In pfdhps the most common genotype was 437gly + 581gly (74.19%). One individual (3.22%) harboured parasites with four pfdhfr (16 val + 59arg + 108asn + 164leu) and two pfdhps (437gly + 581gly) mutations. The highest quintuple pfdhfr/pfdhps (41.94%) was three pfdhfr (59arg + 108asn + 164gly) and two pfdhps (437gly + 581gly).ConclusionThe data suggest a high prevalence of genetic variations conferring resistance to SP which can predict treatment failure before becoming clinically evident. In areas like this, the use of SP may no longer be indicated.

Malaria treatment failure with novel mutation in the Plasmodium falciparum dihydrofolate reductase (pfdhfr) gene in Kolkata, West Bengal, India

The aim of this work was to define the cause of sulfadoxine/pyrimethamine (SP) treatment failure in Plasmodium falciparum infections in a malaria-endemic zone of India. Samples were collected from 176 patients in Kolkata from November 2008 to July 2009. In vitro susceptibility testing was performed on all isolates. Parasite DNA was extracted, and PCR and restriction fragment length polymorphism (RFLP) analysis of different codons of the dhfr gene (16, 51, 59, 108 and 164) and dhps gene (436, 437, 540, 581 and 613) were performed. Finally, sequencing of the products was performed to confirm the mutations. The in vivo treatment response to SP among the 176 patients was determined. A novel mutation of isoleucine was observed at codon 108 of the dhfr gene, which was highly correlated with in vitro SP resistance as well as early treatment failure. A double dhfr mutation (108I+51I) was observed in 77.3% of isolates, and triple mutation of the dhps gene was observed in 18.2% of isolates. In this endemic zone, SP treatment failure is due to a novel dhfr mutation (108I+51I) and any one of the dhps mutations (S436A, A437G, A581G or A613T/S). An increase in these mutations was highly correlated with SP resistance (P<0.0001).

Polymorphism of Plasmodium Falciparum Dihydrofolate Reductase and Dihydropteroate Synthase Genes among Pregnant Women with Falciparum Malaria in Banjar District, South Kalimantan Province, Indonesia

Pregnant women are highly vulnerable to malaria infection in its endemic areas, particularly infection by Plasmodium falciparum that can cause premature, low birth weight, severe anemia in pregnant women, and death. Sulfadoxine-pyrimethamine (SP) for Intermittent Preventive Treatment for pregnant (IPTp) is used for malaria control in pregnancy recommended by the World Health Organization that has already been implemented in Africa. The P. falciparum resistance to SP has been reported in several malarial endemic areas, and mutations in the genes of Plasmodium falciparum Dihydrofolate Reductase (Pfdhfr) and Dihydropteroate Synthase (Pfdhps) are shown to be associated with parasite resistance to SP treatment. Genetic analysis of Pfdhfr and Pfdhps genes in pregnant women infected with P. falciparum has not yet been examined in Indonesia. The cross-sectional study was conducted at two subdistricts, Sungai Pinang and Peramasan, in Banjar district of South Kalimantan Province, where 127 pregnant women were recruited from 2008 to April 2010. Two important mutations in Pfdhfr gene (amino acid positions at N51 and S108) and three in Pfdhps gene (A437, K540 and A581) were analyzed by nested PCR-RFLP method. All of the seven pregnant women samples infected with P. falciparum presented PfDHFR 108N and PfDHPS 437G mutations. One of the samples had the additional mutation at PfDHPS 540, in which Lys is substituted by Glu. These results suggested that P. falciparum might present only some resistance to SP at Sungai Pinang and Peramasan subdistricts, Banjar District, South Kalimantan province, Indonesia. Although there were limited number of samples, this study showed only few mutations of Pfdhfr and Pfdhps genes in P. falciparum at Banjar district, South Kalimantan Province, that suggests SP might be effective for IPTp in this area. Thus, further analysis of the other mutation sites in Pfdhfr and Pfdhps genes and in vivo efficacy study of SP with more sufficient sample numbers will be necessary to confirm this preliminarily result.Keywords: PfDHFR and PfDHPS genes, Pregnant women, Falciparum malaria

Malaria antifolate resistance with contrasting Plasmodium falciparum dihydrofolate reductase (DHFR) polymorphisms in humans and Anopheles mosquitoes.

Surveillance for drug-resistant parasites in human blood is a major effort in malaria control. Here we report contrasting antifolate resistance polymorphisms in Plasmodium falciparum when parasites in human blood were compared with parasites in Anopheles vector mosquitoes from sleeping huts in rural Zambia. DNA encoding P. falciparum dihydrofolate reductase (EC 1.5.1.3) was amplified by PCR with allele-specific restriction enzyme digestions. Markedly prevalent pyrimethamine-resistant mutants were evident in human P. falciparum infections--S108N (>90%), with N51I, C59R, and 108N+51I+59R triple mutants (30-80%). This resistance level may be from selection pressure due to decades of sulfadoxine/pyrimethamine use in the region. In contrast, cycloguanil-resistant mutants were detected in very low frequency in parasites from human blood samples-S108T (13%), with A16V and 108T+16V double mutants (∼4%). Surprisingly, pyrimethamine-resistant mutants were of very low prevalence (2-12%) in the midguts of Anopheles arabiensis vector mosquitoes, but cycloguanil-resistant mutants were highly prevalent-S108T (90%), with A16V and the 108T+16V double mutant (49-57%). Structural analysis of the dihydrofolate reductase by in silico modeling revealed a key difference in the enzyme within the NADPH binding pocket, predicting the S108N enzyme to have reduced stability but the S108T enzyme to have increased stability. We conclude that P. falciparum can bear highly host-specific drug-resistant polymorphisms, most likely reflecting different selective pressures found in humans and mosquitoes. Thus, it may be useful to sample both human and mosquito vector infections to accurately ascertain the epidemiological status of drug-resistant alleles.

Detection of high levels of mutations involved in anti-malarial drug resistance in Plasmodium falciparum and Plasmodium vivax at a rural hospital in southern Ethiopia

BackgroundIn Ethiopia, malaria is caused by Plasmodium falciparum and Plasmodium vivax, and anti-malarial drug resistance is the most pressing problem confronting control of the disease. Since co-infection by both species of parasite is common and sulphadoxine-pyrimethamine (SP) has been intensively used, resistance to these drugs has appeared in both P. falciparum and P. vivax populations. This study was conducted to assess the prevalence of anti-malarial drug resistance in P. falciparum and P. vivax isolates collected at a rural hospital in southern Ethiopia.MethodsA total of 1,147 patients with suspected malaria were studied in different months across the period 2007-2009. Plasmodium falciparum dhfr and dhps mutations and P. vivax dhfr polymorphisms associated with resistance to SP, as well as P. falciparum pfcrt and pfmdr1 mutations conferring chloroquine resistance, were assessed.ResultsPCR-based diagnosis showed that 125 of the 1147 patients had malaria. Of these, 52.8% and 37.6% of cases were due to P. falciparum and P. vivax respectively. A total of 10 cases (8%) showed co-infection by both species and two cases (1.6%) were infected by Plasmodium ovale. Pfdhfr triple mutation and pfdhfr/pfdhps quintuple mutation occurred in 90.8% (95% confidence interval [CI]: 82.2%-95.5%) and 82.9% (95% CI: 72.9%-89.7%) of P. falciparum isolates, respectively. Pfcrt T76 was observed in all cases and pfmdr1 Y86 and pfmdr1 Y1246 in 32.9% (95% CI: 23.4%-44.15%) and 17.1% (95% CI: 10.3-27.1%), respectively. The P. vivax dhfr core mutations, N117 and R58, were present in 98.2% (95% CI: 89.4-99.9%) and 91.2% (95% CI: 80.0-96.7%), respectively.ConclusionCurrent molecular data show an extraordinarily high frequency of drug-resistance mutations in both P. falciparum and P. vivax in southern Ethiopia. Urgent surveillance of the emergence and spread of resistance is thus called for. The level of resistance indicates the need for implementation of entire population access to the new first-line treatment with artemether-lumefantrine, accompanied by government monitoring to prevent the emergence of resistance to this treatment.

Molecular Epidemiology of Malaria in Cameroon. XXX. Sequence Analysis of Plasmodium falciparum ATPase 6, Dihydrofolate Reductase, and Dihydropteroate Synthase Resistance Markers in Clinical Isolates from Children Treated with an Artesunate-Sulfadoxine-Pyrimethamine Combination

Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes are reliable molecular markers for antifolate resistance. The P. falciparum ATPase 6 (pfatp6) gene has been proposed to be a potential marker for artemisinin resistance. In our previous clinical study, we showed that artesunate-sulfadoxine-pyrimethamine is highly effective against uncomplicated malaria in Yaoundé, Cameroon. In the present study, dhfr, dhps, and pfatp6 mutations in P. falciparum isolates obtained from children treated with artesunate-sulfadoxine-pyrimethamine were determined. All 61 isolates had wild-type Pfatp6 263, 623, and 769 alleles, and 11 (18%) had a single E431K substitution. Three additional mutations, E643Q, E432K, and E641Q, were detected. The results did not indicate any warning signal of serious concern (i.e., no parasites were seen with quintuple dhfr-dhps, DHFR Ile164Leu, or pfatp6 mutations), as confirmed by the high clinical efficacy of artesunate-sulfadoxine-pyrimethamine. Further studies are required to identify a molecular marker that reliably predicts artemisinin resistance.

Trypanosomal Dihydrofolate Reductase Reveals Natural Antifolate Resistance

Dihydrofolate reductase (DHFR) is a potential drug target for Trypanosoma brucei, a human parasite, which is the causative agent for African sleeping sickness. No drug is available against this target, since none of the classical antifolates such as pyrimethamine (PYR), cycloguanil, or trimethoprim are effective as selective inhibitors of T. brucei DHFR (TbDHFR). In order to design effective drugs that target TbDHFR, co-crystal structures with bound antifolates were studied. On comparison with malarial Plasmodium falciparum DHFR (PfDHFR), the co-crystal structures of wild-type TbDHFR reveal greater structural similarities to a mutant PfDHFR causing antifolate resistance than the wild-type enzyme. TbDHFR imposes steric hindrance for rigid inhibitors like PYR around Thr86, which is equivalent to Ser108Asn of the malarial enzymes. In addition, a missing residue on TbDHFR active-site loop together with the presence of Ile51 widens its active site even further than the structural effect of Asn51Ile, which is observed in PfDHFR structures. The structural similarities are paralleled by the similarly poor affinities of the trypanosomal enzyme for rigid inhibitors. Mutations of TbDHFR at Thr86 resulted in 10-fold enhancement or 7-fold reduction in the rigid inhibitors affinities for Thr86Ser or Thr86Asn, respectively. The co-crystal structure of TbDHFR with a flexible antifolate WR99210 suggests that its greater affinity result from its ability to avoid such Thr86 clash and occupy the widened binding space similarly to what is observed in the PfDHFR structures. Natural resistance to antifolates of TbDHFR can therefore be explained, and potential antifolate chemotherapy of trypanosomiasis should be possible taking this into account.

High prevalence of dhfr triple mutant and correlation with high rates of sulphadoxine-pyrimethamine treatment failures in vivo in Gabonese children

BackgroundDrug resistance contributes to the global malaria burden. Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) polymorphisms confer resistance to sulphadoxine-pyrimethamine (SP).MethodsThe study assessed the frequency of SP resistance-conferring polymorphisms in Plasmodium falciparum-positive samples from two clinical studies in Lambaréné. Their role on treatment responses and transmission potential was studied in an efficacy open-label clinical trial with a 28-day follow-up in 29 children under five with uncomplicated malaria.ResultsSP was well tolerated by all subjects in vivo. Three subjects were excluded from per-protocol analysis. PCR-corrected, 12/26 (46%) achieved an adequate clinical and parasitological response, 13/26 (50%) were late parasitological failures, while 1/26 (4%) had an early treatment failure, resulting in early trial discontinuation. Of 106 isolates, 98 (92%) carried the triple mutant dhfr haplotype. Three point mutations were found in dhps in a variety of haplotypic configurations. The 437G + 540E double mutant allele was found for the first time in Gabon.ConclusionsThere is a high prevalence of dhfr triple mutant with some dhps point mutations in Gabon, in line with treatment failures observed, and molecular markers of SP resistance should be closely monitored.Trial RegistrationClinicalTrials.gov: NCT00453856

Selection of drug resistant mutants from random library of Plasmodium falciparum dihydrofolate reductase in Plasmodium berghei model

BackgroundThe prevalence of drug resistance amongst the human malaria Plasmodium species has most commonly been associated with genomic mutation within the parasites. This phenomenon necessitates evolutionary predictive studies of possible resistance mutations, which may occur when a new drug is introduced. Therefore, identification of possible new Plasmodium falciparum dihydrofolate reductase (PfDHFR) mutants that confer resistance to antifolate drugs is essential in the process of antifolate anti-malarial drug development.MethodsA system to identify mutations in Pfdhfr gene that confer antifolate drug resistance using an animal Plasmodium parasite model was developed. By using error-prone PCR and Plasmodium transfection technologies, libraries of Pfdhfr mutant were generated and then episomally transfected to Plasmodium berghei parasites, from which pyrimethamine-resistant PfDHFR mutants were selected.ResultsThe principal mutation found from this experiment was S108N, coincident with the first pyrimethamine-resistance mutation isolated from the field. A transgenic P. berghei, in which endogenous Pbdhfr allele was replaced with the mutant PfdhfrS108N, was generated and confirmed to have normal growth rate comparing to parental non-transgenic parasite and also confer resistance to pyrimethamine.ConclusionThis study demonstrated the power of the transgenic P. berghei system to predict drug-resistant Pfdhfr mutations in an in vivo parasite/host setting. The system could be utilized for identification of possible novel drug-resistant mutants that could arise against new antifolate compounds and for prediction the evolution of resistance mutations.

Exploring QSAR, Pharmacophore Mapping and Docking Studies and Virtual Library Generation for Cycloguanil Derivatives as PfDHFR-TS Inhibitors

Resistance of available antimalarial drugs against Plasmodium species is one of the major problems of malaria control in the developing world. In the present study, we have performed QSAR, pharmacophore mapping and molecular docking studies of cycloguanil derivatives as Plasmodium falciparum dihydrofolate reductase thymidylate synthase (PfDHFR-TS) inhibitors to explore essential features required for the antimalarial activity and important interaction patterns between the enzyme and ligands for the design of new potent PfDHFR-TS inhibitors. The QSAR studies have been carried out using topological parameters along with thermodynamic and structural descriptors. Acceptable values of internal and external validation parameters for the developed QSAR models confirm acceptability of the models. Pharmacophore mapping revealed that two hydrogen bond donor (HBD) features and a hydrophobic feature (HYD) are important parameters for PfDHFR-TS inhibitory activity. The docking studies suggest that the PfDHFR-TS inhibitors interact with Asp54, Ile14, Ile164, ser108, Ser111, Tyr170, Met55, Ala16, Thr185, Leu46, Cys15, Phe58, Ile112, Trp48, Tyr57 and Leu119 amino acid residues. The QSAR, pharmacophore and docking studies inferred that i) branching of the substituents at R1 and R2 positions should be less (small alkyl chain substituents are favored); ii) the electronegativity of the molecules should be high but within some limit; iii) the size and volume of the molecules should be high; iv) molecules should be flexible enough; v) R configuration at C6 position of the triazine ring favors the inhibitory binding affinity; vi) the substituents of the phenyl ring at 3, 4 and 5 position of the phenyl ring should be small hydrophobic groups. Based on these studies, we have designed a library of cycloguanil derivatives with good in silico predicted PfDHFR-TS inhibitory activity.

High-Throughput Genotyping of Single Nucleotide Polymorphisms in the Plasmodium falciparum dhfr Gene by Asymmetric PCR and Melt-Curve Analysis

Mutations within the Plasmodium falciparum dihydrofolate reductase gene (Pfdhfr) contribute to resistance to antimalarials such as sulfadoxine-pyrimethamine (SP). Of particular importance are the single nucleotide polymorphisms (SNPs) within codons 51, 59, 108, and 164 in the Pfdhfr gene that are associated with SP treatment failure. Given that traditional genotyping methods are time-consuming and laborious, we developed an assay that provides the rapid, high-throughput analysis of parasite DNA isolated from clinical samples. This assay is based on asymmetric real-time PCR and melt-curve analysis (MCA) performed on the LightCycler platform. Unlabeled probes specific to each SNP are included in the reaction mixture and hybridize differentially to the mutant and wild-type sequences within the amplicon, generating distinct melting curves. Since the probe is present throughout PCR and MCA, the assay proceeds seamlessly with no further addition of reagents. This assay was validated for analytical sensitivity and specificity using plasmids, purified genomic DNA from reference strains, and parasite cultures. For all four SNPs, correct genotypes were identified with 100 copies of the template. The performance of the assay was evaluated with a blind panel of clinical isolates from travelers with low-level parasitemia. The concordance between our assay and DNA sequencing ranged from 84 to 100% depending on the SNP. We also directly compared our MCA assay to a published TaqMan real-time PCR assay and identified major issues with the specificity of the TaqMan probes. Our assay provides a number of technical improvements that facilitate the high-throughput screening of patient samples to identify SP-resistant malaria.

Differentiating Plasmodium falciparum alleles by transforming Cartesian X,Y data to polar coordinates

BackgroundDiagnosis of infectious diseases now benefits from advancing technology to perform multiplex analysis of a growing number of variables. These advances enable simultaneous surveillance of markers characterizing species and strain complexity, mutations associated with drug susceptibility, and antigen-based polymorphisms in relation to evaluation of vaccine effectiveness. We have recently developed assays detecting single nucleotide polymorphisms (SNPs) in the P. falciparum genome that take advantage of post-PCR ligation detection reaction and fluorescent microsphere labeling strategies. Data from these assays produce a spectrum of outcomes showing that infections result from single to multiple strains. Traditional methods for distinguishing true positive signal from background can cause false positive diagnoses leading to incorrect interpretation of outcomes associated with disease treatment.ResultsFollowing analysis of Plasmodium falciparum dihydrofolate reductase SNPs associated with resistance to a commonly used antimalarial drug, Fansidar (Sulfadoxine/pyrimethamine), and presumably neutral SNPs for parasite strain differentiation, we first evaluated our data after setting a background signal based on the mean plus three standard deviations for known negative control samples. Our analysis of single allelic controls suggested that background for the absent allele increased as the concentration of the target allele increased. To address this problem, we introduced a simple change of variables from customary (X,Y) (Cartesian) coordinates to planar polar coordinates (X = rcos(θ), Y = rsin(θ)). Classification of multidimensional fluorescence signals based on histograms of angular and radial data distributions proved more effective than classification based on Cartesian thresholds. Comparison with known diallelic dilution controls suggests that histogram-based classification is effective for major:minor allele concentration ratios as high as 10:1.ConclusionWe have observed that the diallelic SNP data resulting from analysis of P. falciparum mutations is more accurately diagnosed when a simple polar transform of the (X,Y) data into (r,θ) is used. The development of high through-put methods for genotyping P. falciparum SNPs and the refinement of analytical approaches for evaluating these molecular diagnostic results significantly advance the evaluation of parasite population diversity and antimalarial drug resistance.

Computer-aided molecular design of 1H-imidazole-2,4-diamine derivatives as potential inhibitors of Plasmodium falciparum DHFR enzyme

Design and discovery of new potential inhibitors of Plasmodium falciparum dihydrofolate reductase (PfDHFR), equally active against both the wild-type and mutant strains, is urgently needed. In this study, a computer-aided molecular design approach that involved ab initio molecular orbital and density functional theory calculations, along with molecular electrostatic potential analysis, and molecular docking studies was employed to design 15 1H-imidazole-2,4-diamine derivatives as potential inhibitors of PfDHFR enzyme. Visual inspection of the binding modes of the compounds demonstrated that they all interact, via H-bond interactions, with key amino acid residues (Asp54, Ileu/Leu164, Asn/Ser108 and Ile14) similar to those of WR99210 (3) in the active site of the enzymes used in the study. These interactions are known to be essential for enzyme inhibition. These compounds showed better or comparable binding affinities to that of the bound ligand (WR99210). In silico toxicity predictions, carried out using TOPKAT software, also indicated that the compounds are non-toxic.

Low infectivity of Plasmodium falciparum gametocytes to Anopheles gambiae following treatment with sulfadoxine–pyrimethamine in Mali

Sulfadoxine–pyrimethamine (SP) treatment increases the rate of gametocyte carriage and selects SP resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), raising concerns of increased malaria transmission and spread of drug resistance. In a setting in Mali where SP was highly efficacious, we measured the prevalence of DHFR and DHPS mutations in P. falciparum infections with microscopy-detected gametocytes following SP treatment, and used direct feeding to assess infectivity to Anopheles gambiae sensu lato. Children and young adults presenting with uncomplicated malaria were treated with SP or chloroquine and followed for 28 days. Gametocyte carriage peaked at 67% 1 week after treatment with a single dose of SP. Those post-SP gametocytes carried significantly more DHFR and DHPS mutations than pre-treatment asexual parasites from the same population. Only 0.5% of 1728 mosquitoes fed on SP-treated gametocyte carriers developed oocysts, while 11% of 198 mosquitoes fed on chloroquine-treated gametocyte carriers were positive for oocysts. This study shows that in an area of high SP efficacy, although SP treatment sharply increased gametocyte carriage, the infectiousness of these gametocytes to the vector may be very low. Accurate and robust methods for measuring infectivity are needed to guide malaria control interventions that affect transmission.

Plasmodium falciparum resistance to anti-malarial drugs in Papua New Guinea: evaluation of a community-based approach for the molecular monitoring of resistance

BackgroundMolecular monitoring of parasite resistance has become an important complementary tool in establishing rational anti-malarial drug policies. Community surveys provide a representative sample of the parasite population and can be carried out more rapidly than accrual of samples from clinical cases, but it is not known whether the frequencies of genetic resistance markers in clinical cases differ from those in the overall population, or whether such community surveys can provide good predictions of treatment failure rates.MethodsBetween 2003 and 2005, in vivo drug efficacy of amodiaquine or chloroquine plus sulphadoxine-pyrimethamine was determined at three sites in Papua New Guinea. The genetic drug resistance profile (i.e., 33 single nucleotide polymorphisms in Plasmodium falciparum crt, mdr1, dhfr, dhps, and ATPase6) was concurrently assessed in 639 community samples collected in the catchment areas of the respective health facilities by using a DNA microarray-based method. Mutant allele and haplotype frequencies were determined and their relationship with treatment failure rates at each site in each year was investigated.ResultsPCR-corrected in vivo treatment failure rates were between 12% and 28% and varied by site and year with variable longitudinal trends. In the community samples, the frequencies of mutations in pfcrt and pfmdr1 were high and did not show significant changes over time. Mutant allele frequencies in pfdhfr were moderate and those in pfdhps were low. No mutations were detected in pfATPase6. There was much more variation between sites than temporal, within-site, variation in allele and haplotype frequencies. This variation did not correlate well with treatment failure rates. Allele and haplotype frequencies were very similar in clinical and community samples from the same site.ConclusionsThe relationship between parasite genetics and in vivo treatment failure rate is not straightforward. The frequencies of genetic anti-malarial resistance markers appear to be very similar in community and clinical samples, but cannot be used to make precise predictions of clinical outcome. Thus, indicators based on molecular data have to be considered with caution and interpreted in the local context, especially with regard to prior drug usage and level of pre-existing immunity. Testing community samples for molecular drug resistance markers is a complementary tool that should help decision-making for the best treatment options and appropriate potential alternatives.

Binding modes of 2,4-diaminoquinazoline and 2,4-diaminopteridine analogs to P. falciparum dihydrofolate reductase enzyme: Molecular docking studies

A molecular docking study was carried out on 28 compounds belonging to 2,4-diaminoquinazoline and 2,4-diaminopteridine analogs using Glide, FlexX and GOLD programs and the X-ray crystallographic structures of the quadruple mutant (1J3K:pdb) and wild type (1J3I:pdb) Plasmodium falciparum dihydrofolate reductase enzyme. The experimental conformation the bound ligand WR99210 was precisely reproduced by the docking procedures as demonstrated by low (<2.00 Å) root-mean-square deviations. The results indicated that most of the compounds dock into the active sites of both the wild type and quadruple mutant P. falciparum dihydrofolate reductase enzymes. Visual inspection of the binding modes also demonstrated that most of the compounds could form H-bond interactions with the key amino acid residues (Asp54, Ile14 and Leu/Ile164) and with better docking scores than the bound compound (5). Their long side chains orient in the hydrophobic portion of the active site which is occupied by trichloro aryloxy side chain of WR99210 (5). Thus, avoid potential steric clashes with Asn108 (mutated from Ser108). Such a clash is known to be responsible for the resistance of the P. falciparum to pyrimethamine and cycloguanil.

A common feature-based 3D-pharmacophore model generation and virtual screening: identification of potential PfDHFR inhibitors

A four-feature 3D-pharmacophore model was built from a set of 24 compounds whose activities were reported against the V1/S strain of the Plasmodium falciparum dihydrofolate reductase (PfDHFR) enzyme. This is an enzyme harboring Asn51Ile + Cys59Arg + Ser108Asn + Ile164Leu mutations. The HipHop module of the Catalyst program was used to generate the model. Selection of the best model among the 10 hypotheses generated by HipHop was carried out based on rank and best-fit values or alignments of the training set compounds onto a particular hypothesis. The best model (hypo1) consisted of two H-bond donors, one hydrophobic aromatic, and one hydrophobic aliphatic features. Hypo1 was used as a query to virtually screen Maybridge2004 and NCI2000 databases. The hits obtained from the search were subsequently subjected to FlexX and Glide docking studies. Based on the binding scores and interactions in the active site of quadruple-mutant PfDHFR, a set of nine hits were identified as potential inhibitors.

Shape‐ and Chemical Feature‐Based 3D‐Pharmacophore Model Generation and Virtual Screening: Identification of Potential Leads for P. falciparum DHFR Enzyme Inhibition

Plasmodium falciparum dihydrofolate reductase (Pf DHFR) enzyme is one of the validated targets in the treatment of malaria using typical antifolates such as cycloguanil and pyrimethamine. However, point mutations at amino acid residues such as Ala16, Ile51, Cys59, Ser108 and Ile164 in the active site of the wild-type enzyme resulted in a widespread resistance of the parasite to these drugs. Thus, design and discovery of new potential Pf DHFR inhibitors, equally active against both the wild-type and mutant strains, is an urgent need. Catalyst software was used to generate a 3D pharmacophore query based on the bioactive conformation of WR99210 extracted from the X-ray crystal structure of quadruple mutant PfDHFR enzyme. Validation criteria based on the experimentally determined conformation of WR99210 and its key interactions with the protein were considered to identify hits from two chemical databases, namely, NCI2000 and Maybridge2004 using different virtual filters. Virtual screening based on FlexX, GOLD and Glide docking programs resulted in a total of 73 hits. The hits reported in this article showed good potential to be inhibitors of the above Pf DHFRs based on their (i) best-fit values (ii) binding scores (iii) binding modes and (iv) interactions with the key amino acid residues (Asp54, Ileu/Leu164, Asn/Ser108 and Ile14).