Antifolate screening using yeast expressing Plasmodium vivax dihydrofolate reductase and in vitro drug susceptibility assay for Plasmodium falciparum

The presence of homologous point mutations in the dhfr gene in Plasmodium vivax and Plasmodium falciparum is associated with resistance to antifolate drugs. The spread of antifolate resistance encouraged research for novel antifolate drugs active against both wild-type and dhfr-mutant strains of malaria parasites. Because P. vivax cannot be easily maintained in culture, we transformed a Saccharomyces cerevisiae DHFR-deleted mutant to express wild-type P. vivax dhfr gene and its mutant forms. Twenty-five dicyclic and tricyclic 2,4-diaminopyrimidine derivatives were screened. Six quinazoline compounds showed selective inhibition of yeast transformants expressing P. vivax dhfr genes. The 50% inhibitory concentration (IC(50)) of these six compounds was determined against field isolates of P. falciparum. Our results suggest that a close relationship between the yeast assay based on expression of P. vivax dhfr genes and the in vitro test using P. falciparum parasites in culture is a promising initial step for drug screening.     

Full-length extension of HLA allele sequences by HLA allele-specific hemizygous Sanger sequencing (SSBT)

The gold standard for typing at the allele level of the highly polymorphic Human Leucocyte Antigen (HLA) gene system is sequence based typing. Since sequencing strategies have mainly focused on identification of the peptide binding groove, full-length sequence information is lacking for >90% of the HLA alleles. One of the goals of the 17th IHIWS workshop is to establish full-length sequences for as many HLA alleles as possible. In our component “Extension of HLA sequences by full-length HLA allele-specific hemizygous Sanger sequencing” we have used full-length hemizygous Sanger Sequence Based Typing to achieve this goal. We selected samples of which full length sequences were not available in the IPD-IMGT/HLA database. In total we have generated the full-length sequences of 48 HLA-A, 45 -B and 31 -C alleles. For HLA-A extended alleles, 39/48 showed no intron differences compared to the first allele of the corresponding allele group, for HLA-B this was 26/45 and for HLA-C 20/31. Comparing the intron sequences to other alleles of the same allele group revealed that in 5/48 HLA-A, 16/45 HLA-B and 8/31 HLA-C alleles the intron sequence was identical to another allele of the same allele group. In the remaining 10 cases, the sequence either showed polymorphism at a conserved nucleotide or was the result of a gene conversion event. Elucidation of the full-length sequence gives insight in the polymorphic content of the alleles and facilitates the identification of its evolutionary origin.     

Mechanisms and dynamics of drug resistance in Plasmodium falciparum

The emergence of chloroquine resistance has been associated with a dramatic increase in malaria mortality in some human populations from endemic regions. Plasmodium falciparum drug resistant malaria originates from chromosomal mutations. Analysis using molecular, genetic and biochemical approaches has shown that 1) impaired intake of chloroquine by the parasite vacuole is a common characteristic of resistant strains, the chloroquine-resistance mechanism regulates the access of chloroquine to hematin, this phenotype correlates with Pfmdr1 and Pfcg2 gene mutations; 2) one to four point mutations of dihydrofolate reductase, the enzyme target of antifolinics (pyrimethamine and proguanil), give moderate to high levels of resistance to these drugs but there is a fitness cost to resistance; 3) the mechanism of resistance to sulfonamides and sulfones involves mutations of dihydropteroate synthase, their enzyme target; 4) treatment with sulphadoxine-pyrimethamine (SP) selected for the variants Ile(51), Arg(59) and Asn(108) of DHFR and for the variants Ser(436), Gly(437), and Glu(540) of DHPS; 5)clones that were resistant to some traditional antimalarial agents acquired resistance to new ones at high frequency (accelerated resistance to multiple drugs--ARMD). Amino-alcohol (quinine, mefloquine, halofantrine) mechanisms of resistance are still unclear. Population genetic studies have confirmed that selfing is more frequent in Plasmodium falciparum where the transmission rate is lower in some regions such as Papua-New Guinea, whereas isolates from individuals on the Thai-Burmese border, an area of hypoendemic transmission, revealed a higher number of genotypes per infected person. It has been suggested that intense intra-host competition between co-infecting clones, low numbers of genes required to encode resistance, and high drug usage all encourage the emergence of drug resistance. On the other hand, the greater effective recombination in high transmission areas may breakdown multiple drug resistance when it is coded for by two unlinked loci. Epidemiological studies have established that the frequency of chloroquine resistant mutants varies among parasite isolate populations while resistance to antifolinics is highly prevalent in most malarial endemic countries (more than 92% of Kenyan field isolates have undergone at least one point mutation). Established and strong drug pressure as well as low antiparasitic immunity probably explains the multidrug-resistance encountered in forests of Southeast Asia and South America. In Africa, frequent genetic recombinations in Plasmodium originate from a high level of malaria transmission, and falciparum chloroquine-resistant prevalence seems to stabilise at an equal level as chloroquine-sensitive malaria. Clinical studies demonstrated that control of clinical symptoms is better when chloroquine is used with sulphadoxine-pyrimethamine (SP) than when SP is used alone, and the cure rate also tends to be higher with the triple combination regimen.     

Point mutations in the dihydrofolate reductase-thymidylate synthase gene as the molecular basis for pyrimethamine resistance in Plasmodium falciparum

The dihydrofolate reductase-thymidylate synthase (DHFR-TS) bifunctional complex from pyrimethamine-sensitive (3D7) and drug-resistant (HB3 and 7G8) clones from Plasmodium falciparum was purified to homogeneity. A modified sequence of purification steps with a 10-formylfolate affinity column at its center, allows the isolation of the enzyme complex with a 10-fold higher yield than previously reported, irrespective of the pyrimethamine resistance of the parasites. Titration of the homogenous DHFR-TS complex with the inhibitor revealed a 500-fold lower affinity of the enzyme from clone 7G8 for the drug than found with the enzyme from clone 3D7. Direct comparison of the homogenous enzyme preparations on SDS-PAGE revealed no difference in the molecular mass of the DHFR-TS from the 3 clones, nor could a reproducible difference be detected in the peptide patterns obtained after digesting the DHFR-TS complex with various proteases. The amplification of segments from the DHFR-TS coding region of the 3 clones and 7 isolates of P. falciparum by polymerase chain reaction resulted in fragments of the predicted length without any size heterogeneity. The DNA sequence of the DHFR coding region from FCR-3, 3D7, HB3 and 7G8 differs in a total of 4 nucleotides. One point mutation changes amino acid residue 108 from threonine (FCR-3) or serine (3D7) to asparagine (HB3 and 7G8). The presence of asparagine-108 appears to be the molecular basis of pyrimethamine resistance of HB3 and 7G8. The degree of resistance is associated with a point mutation affecting the codon for amino acid 51 in 7G8.     

Mutagenesis of dihydrofolate reductase from Plasmodium falciparum: analysis in Saccharomyces cerevisiae of triple mutant alleles resistant to pyrimethamine or WR99210

Inhibitors of dihydrofolate reductase (DHFR) have been a mainstay of chemotherapy of falciparum malaria for >50 years. Unfortunately, point mutations in DHFR are the major cause of resistance to drugs of this class and mutations have rapidly diminished the clinical effectiveness of these drugs. We designed a simple yeast-based system to produce and analyze point mutations in the Plasmodium falciparum DHFR domain of the DHFR-thymidylate synthase gene that confers resistance to pyrimethamine (PM), the major antifolate currently used in malaria treatment, or to WR99210, an experimental antifolate. We used PCR mutagenesis, screened >1000 DHFR alleles that encoded functional enzymes and studied approximately 100 that were more resistant than a naturally occurring resistant allele (N51I and S108N). The IC(50) values for both drugs were determined for a subset of 44 alleles that carried only a single new mutation. Mutations that increased resistance to PM 10-100 fold (to >10(-4) M) were identified in three regions of the DHFR domain - around amino acids 50, 188 and 213. In contrast, mutations that caused WR-resistance were far less common and only conferred approximately 10-fold resistance (to approximately 10(-7) M). Even more interesting, only the mutations at 188 increased resistance to WR and mutations in the 213 and other regions either had no effect or actually increased sensitivity to WR. This collateral hypersensitivity raises the possibility that opposing selection for resistance/sensitivity to PM and WR might be used to slow selection of populations of P. falciparum resistant to antifolate treatment.     

Multiplex PCR and oligonucleotide microarray for detection of single-nucleotide polymorphisms associated with Plasmodium falciparum drug resistance

Drug resistance in Plasmodium falciparum is a serious public health threat in the countries where this organism is endemic. Since resistance has been associated with specific single-nucleotide polymorphisms (SNPs) in parasite genes, molecular markers are becoming useful surrogates for monitoring the emergence and dispersion of drug resistance. In this study, a multiplex PCR (mPCR) and oligonucleotide microarray method was developed for the detection of these SNPs in genes encoding chloroquine resistance transporter (Pfcrt), multidrug resistance 1 (Pfmdr1), dihydrofolate reductase (Pfdhfr), dihydropteroate synthetase (Pfdhps), and ATPase 6 (PfATPase6) of P. falciparum. The results show that DNA microarray technology, combined with mPCR, is a promising and time-saving tool that supports conventional detection methods, allowing sensitive, accurate, simultaneous analysis of the SNPs associated with drug resistance in P. falciparum.     

Genetic variations of the dihydrofolate reductase gene of Plasmodium vivax in Mandalay Division, Myanmar

Dihydrofolate reductase (DHFR; EC1.5.1.3) is a known target enzyme for antifolate agents, which are used as alternative chemotherapeutics for chloroquine-resistant malaria. Mutations in the dhfr gene of Plasmodium vivax are thought to be associated with resistance to the antifolate drugs. In this study, we have analyzed genetic variations in the dhfr genes of clinical isolates of P. vivax (n=21) in Myanmar, to monitor antifolate resistance in this country. Sequence variations within the entire dhfr gene were highly restricted to codons from 57 to 117, and the GGDN tandem repeat region. Double (S58R and S117N/T) or quadruple mutations (F57L/I, S58R, T61M, and S117N/T), which may be closely related to the drug resistance, were recognized in most of the isolates (20/21 cases). Our results suggest that antifolate-resistant P. vivax is becoming widespread in Myanmar, as it also is in the neighboring countries in Southeast Asia. It appears that the drug resistance situation may be worsening in the country.Byoung-Kuk Na and Hyeong-Woo Lee have contributed equally to the work.     

Functional expression of the dihydrofolate reductase and thymidylate synthetase activities of the human malaria parasite Plasmodium falciparum in Escherichia coli

We have developed a recombinant system that directs the functional expression from Escherichia coli of both dihydrofolate reductase-thymidylate synthetase (DHFR-TS) and the isolated DHFR domain from Plasmodium falciparum. Both products are inhibitory to a number of E. coli cell lines to the extent that cell growth ceases immediately upon induction. This dramatic inhibition is not seen in strain AB1899, in which amounts of plasmodial protein of up to 100 times the basal E. coli TS level can be accumulated. However, as well as the full-length DHFR-TS molecule, smaller proteins carrying an intact TS substrate-binding site are produced. These represent ca. 60-75% of the total plasmodial protein expressed and are observed in every E. coli strain examined. We show that they are not derived by degradation of the parent DHFR-TS molecule, but can be correlated with the sizes of proteins expected to be produced if erroneous initiation of translation were occurring at 3 internal methionine residues.     

Analysis of hepatitis B virus genotyping and drug resistance gene mutations based on massively parallel sequencing

Drug resistance to nucleoside analogs is a serious problem worldwide. Both drug resistance gene mutation detection and HBV genotyping are helpful for guiding clinical treatment. Total HBV DNA from 395 patients who were treated with single or multiple drugs including Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir and Emtricitabine were sequenced using the HiSeq 2000 sequencing system and validated using the 3730 sequencing system. In addition, a mixed sample of HBV plasmid DNA was used to determine the cutoff value for HiSeq-sequencing, and 52 of the 395 samples were sequenced three times to evaluate the repeatability and stability of this technology. Of the 395 samples sequenced using both HiSeq and 3730 sequencing, the results from 346 were consistent, and the results from 49 were inconsistent. Among the 49 inconsistent results, 13 samples were detected as drug-resistance-positive using HiSeq but negative using 3730, and the other 36 samples showed a higher number of drug-resistance-positive gene mutations using HiSeq 2000 than using 3730. Gene mutations had an apparent frequency of 1% as assessed by the plasmid testing. Therefore, a 1% cutoff value was adopted. Furthermore, the experiment was repeated three times, and the same results were obtained in 49/52 samples using the HiSeq sequencing system. HiSeq sequencing can be used to analyze HBV gene mutations with high sensitivity, high fidelity, high throughput and automation and is a potential method for hepatitis B virus gene mutation detection and genotyping.     

Pyrosequencing, a high-throughput method for detecting single nucleotide polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum

A pyrosequencing protocol was developed as a rapid and reliable method to identify the mutations of the dhfr and dhps genes of Plasmodium falciparum that are associated with antifolate resistance. The accuracy and specificity of this method were tested using six laboratory-cultured P. falciparum isolates harboring known single nucleotide polymorphisms (SNPs) in the genes dhfr (codons 50, 51, 59, 108, and 164) and dhps (codons 436, 437, 540, 581, and 613). The lowest threshold for detection of all the SNPs tested by pyrosequencing was the equivalent of two to four parasite genomes. Also, this method was highly specific for P. falciparum, as it did not amplify any DNA products from the other species of human malaria parasites. We also mixed wild-type and mutant-type parasite DNAs in various proportions to determine how pyrosequencing, restriction fragment length polymorphism (RFLP), and direct conventional sequencing (for dhfr) compared with each other in detecting different SNPs in the mixture. In general, pyrosequencing and RFLP showed comparable sensitivities in detecting most of the SNPs in dhfr except for the 164L mutation, which required at least twice the amount of DNA for pyroseqencing as for RFLP. For detecting SNPs in dhps, pyrosequencing was slightly more sensitive than RFLP and direct sequencing. Overall, pyrosequencing was faster and less expensive than either RFLP or direct sequencing. Thus, pyrosequencing is a practical alternative method that can be used in a high-throughput format for molecular surveillance of antimalarial-drug resistance.     

Structure of the knob protein (KP) gene of Plasmodium falciparum

We have determined the nucleotide sequence of the gene encoding the knob protein (KP) of Plasmodium falciparum (FCR-3/Gambia). The gene is interrupted by an intron which contains 34 imperfect tandemly repeated ATTTT sequences. The first exon encodes 33 amino acids with a hydrophobic core typical of signal peptides. The second exon has an open translational reading frame for 597 amino acids. The deduced protein sequence indicates that KP has multiple structural domains; unlike the N-terminal histidine-rich domain which we described previously, the C-terminal half is rich in lysine residues. Consistent with the apparent association of KP with the cytoplasmic surface of the host erythrocyte membrane, the protein is highly charged and hydrophilic.     

Mutational analysis of Plasmodium falciparum dihydrofolate reductase: the role of aspartate 54 and phenylalanine 223 on catalytic activity and antifolate binding

The catalytic activity and ability to confer resistance to antifolates of Plasmodium falciparum dihydrofolate reductase (pfDHFR) through single and double mutations at Asp-54 and Phe-223 were investigated. A single Asp54Glu (D54E) mutation in the pfDHFR domain greatly decreased the catalytic activity of the enzyme and affected both the K(m) values for the substrate dihydrofolate and the K(i) values for pyrimethamine, cycloguanil and WR99210. The Phe223Ser (F223S) single mutant had unperturbed kinetics but had very poor affinity with the first two antifolates. The ability to confer high resistance to the antifolates of F223S enzyme was, however, abolished in the D54E+F223S double mutant enzyme. When D54E mutation was present together with the A16V+S108T double mutation, the effects on the K(m) values for the substrate dihydrofolate and the binding affinity of antifolates were much more pronounced. The severely impaired kinetics and poor activity observed in A16V+S108T+D54E enzyme could, however, be restored when F223S was introduced, while the binding affinities to the antifolates remained poor. The experimental findings can be explained with a model for substrate and inhibitor binding. Our data not only indicate the importance of Asp-54 of pfDHFR in catalysis and inhibitor binding, but also provide evidence that infer the potentially crucial function of the C-terminal portion of pfDHFR domain.     

Use of DNA amplification (PCR) and direct DNA sequencing in the characterization of C4 alleles

A procedure for detailed characterization of individual C4 alleles has been developed. DNA containing the two polymorphic clusters of C4 was amplified in the polymerase chain reaction (PCR). Direct DNA sequencing of amplified DNA was then performed by a modification of previously described techniques. The results were confirmed by M13 sequencing.
Single C4A3 and C4B1 allele sequences were in accordance with previous reports. An individual typed C4A3B1 revealed double bands in the autoradiogram in the positions corresponding to the polymorphic nucleotides. We did not find the reported thymine in position 3641 specific for the C4A4 allele in an individual typed C4A4B2.     

Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species-specific real-time PCR assays

There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the "gold standard" in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.     

Plasmodium falciparum drug resistance in the Congo. Evaluation of surveys carried out from 1985 to 1989

Surveys on drug sensitivity of Plasmodium falciparum carried out between 1985 and 1989 included 7-day in vitro tests and in vivo tests. 485 in vivo tests were carried out in eight surveys conducted in Brazzaville and in several inland regions. The subjects were congolese children aged between 3 months and 15 years old. They were recruited in hospital, mother-child clinics or at school. The drugs studied were chloroquine, amodiaquine and the sulfadoxine-pyrimethamine combination. 182 strains were tested in vitro in two surveys (December 1985 and January 1987); amino-4-quinolines, quinine and mefloquine were studied. Although resistance to amino-4-quinolines is a recent occurrence, by 1985 it had spread widely in the indigenous population in the Centre and South of the country. Resistance has since increased gradually, especially for chloroquine which undergoes specific surveillance. The situation is less serious in the North, a less densely populated region which is still enclosed. In an in vivo comparative study with chloroquine conducted in Brazzaville in November 1986, amodiaquine was found to be only slightly more effective at a similar dosage. At that time, certain isolated observations already seem to imply that the sulfadoxine-pyrimethamine combination was also affected by resistance. This was not corroborated in an in vivo study carried out in 1989 on 40 children presenting with a malarial attack. Although the sensitivity to quinine may probably be decreased. This drug cannot yet be considered as being truly affected by resistance. The activity of mefloquine, the use of which is still limited, was satisfactory in 1987 in two different regions of the country.