John E. Hyde

Pyrimethamine–sulfadoxine resistance in Plasmodium falciparum: what next?

Chemotherapy remains the only practicable tool to control falciparum malaria in sub-Saharan Africa, where >90% of the worlds burden of malaria mortality and morbidity occurs. Resistance is rapidly eroding the efficacy of chloroquine, and the combination pyrimethamine-sulfadoxine is the most commonly chosen alternative. Resistant populations of Plasmodium falciparum were selected extremely rapidly in Southeast Asia and South America. If this happens in sub-Saharan Africa, it will be a public health disaster because no inexpensive alternative is currently available. This article reviews the molecular mechanisms of this resistance and discusses how to extend the therapeutic life of antifolate drugs.

Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilization.

Sulfadoxine/pyrimethamine (Fansidar) is widely used in Africa for treating chloroquine‐resistant falciparum malaria. To clarify how parasite resistance to this combination arises, various lines of Plasmodium falciparum were used to investigate the role of naturally occurring mutations in the target enzyme, dihydropteroate synthetase (DHPS), in the parasite response to sulfadoxine inhibition. An improved drug assay was employed to identify a clear correlation between sulfadoxine‐resistance levels and the number of DHPS mutations. Moreover, tight linkage was observed between DHPS mutations and high‐level resistance in the 16 progeny of a genetic cross between sulfadoxine‐sensitive (HB3) and sulfadoxine‐resistant (Dd2) parents. However, we also demonstrate a profound influence of exogenous folate on IC50 values, which, under physiological conditions, may have a major role in determining resistance levels. Importantly, this phenotype does not segregate with dhps genotypes in the cross, but shows complete linkage to the two alleles of the dihydrofolate reductase (dhfr) gene inherited from the parental lines. However, in unrelated lines, this folate effect correlates less well with DHFR sequence, indicating that the gene responsible may be closely linked to dhfr, rather than dhfr itself. These results have major implications for the acquisition of Fansidar resistance by malaria parasites.

Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine‐resistant malaria

We have exploited the recently developed ability to trans‐ fect the malaria parasite Plasmodium falciparum to investigate the role of polymorphisms in the enzyme dihydropteroate synthase (DHPS), identified in sulfadoxine‐resistant field isolates. By using a truncated form of the dhps gene, specific mutations were introduced into the endogenous gene by allelic replacement such that they were under the control of the endogenous promoter. Using this approach a series of mutant dhps alleles that mirror P.falciparum variants found in field isolates were found to confer different levels of sulfadoxine resistance. This analysis shows that alteration of Ala437 to Gly (A437G) confers on the parasite a 5‐fold increase in sulfadoxine resistance and addition of further mutations increases the level of resistance to 24‐fold above that seen for the transfectant expressing the wild‐type dhps allele. This indicates that resistance to high levels of sulfadoxine in P.falciparum has arisen by an accumulation of mutations and that Gly437 is a key residue, consistent with its occurrence in most dhps alleles from resistant isolates. These studies provide proof that the mechanism of resistance to sulfadoxine in P.falciparum involves mutations in the dhps gene and determines the relative contribution of these mutations to this phenotype.

Resistance to antifolates in Plasmodium falciparum monitored by sequence analysis of dihydropteroate synthetase and dihydrofolate reductase alleles in a large number of field samples of diverse origins

Resistance of Plasmodium falciparum to antifolate chemotherapy is a significant problem where combinations such as Fansidar (pyrimethamine-sulfadoxine; PYR-SDX) are used in the treatment of chloroquine-resistant malaria. Antifolate resistance has been associated with variant sequences of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS), the targets of PYR and SDX respectively. However, while the nature and distribution of mutations in the dhfr gene are well established, this is not yet the case for dhps. We have thus examined by DNA sequence analysis 141 field samples from several geographical regions with differing Fansidar usage (West and East Africa, the Middle East and Viet Nam) to establish a database of the frequency and repertoire of dhps mutations, which were found in 60% of the samples. We have also simultaneously determined from all samples their dhfr sequences, to better understand the relationship of both types of mutation to Fansidar resistance. Whilst the distribution of mutations was quite different across the regions surveyed, it broadly mirrored our understanding of relative Fansidar usage. In samples taken from individual patients before and after drug treatment, we found an association between the more highly mutated forms of dhps and/or dhfr and parasites that were not cleared by antifolate therapy. We also report a novel mutation in a Pakistani sample at position 16 of DHFR (A16S) that is combined with the familiar C59R mutation, but is wild-type at position 108. This is the first observation in a field sample of a mutant dhfr allele where the 108 codon is unchanged.

Prevalence of Toxoplasma gondii in commercial meat products as monitored by polymerase chain reaction – food for thought?

DNA was extracted from 71 meat samples obtained from UK retail outlets. All of these DNA preparations gave the expected polymerase chain reaction products when amplified with primers specific for the species from which the meat originated. A second polymerase chain reaction analysis, using primers specific for the Toxoplasma gondii SAG2 locus, revealed the presence of this parasite in 27 of the meat samples. Restriction analysis and DNA sequencing showed that 21 of the contaminated meats contained parasites genotyped as type I at the SAG2 locus, whilst six of the samples contained parasites of both types I and II. Toxoplasma- positive samples were subjected to further polymerase chain reaction analysis to determine whether any carried an allele of the dihydropteroate synthase gene that has recently been shown to be causally associated with sulfonamide resistance in T. gondii. In all cases, this analysis confirmed that parasites were present in the samples and, additionally, revealed that none of them carried the drug-resistant form of dihydropteroate synthase. These results suggest that a significant proportion of meats commercially available in the UK are contaminated with T. gondii. Although none of the parasites detected in this study carried the sulfonamide-resistance mutation, a simplified procedure for monitoring this situation merits development.

The establishment of genomic DNA libraries for the human malaria parasite Plasmodium falciparum and identification of individual clones by hybridisation

The DNA of Plasmodium falciparum has been purified and fragmented with the restriction endonucleases EcoRI and HindIII. The fragments have been incorporated in vitro into derivatives of bacteriophage lambda to make libraries in which most of the parasite DNA is represented. By Southern hybridisation we have been able to recover from these libraries specific clones containing (a) repetitive DNA sequences, (b) rRNA gene(s) and (c) sequences homologous to an actin gene probe. Parasite DNA from two independent sources differs markedly in the pattern of its repetitive DNA visualised by hybridisation to our repetitive clone. By contrast, the rRNA genes of the two isolates prove to be carried on identically sized fragments.

Drug-resistant malaria - an insight

Despite intensive research extending back to the 1930s, when the first synthetic antimalarial drugs made their appearance, the repertoire of clinically licensed formulations remains very limited. Moreover, widespread and increasing resistance to these drugs contributes enormously to the difficulties in controlling malaria, posing considerable intellectual, technical and humanitarian challenges. A detailed understanding of the molecular mechanisms underlying resistance to these agents is emerging that should permit new drugs to be rationally developed and older ones to be engineered to regain their efficacy. This review summarizes recent progress in analysing the causes of resistance to the major antimalarial drugs and its spread.

Mechanisms of resistance of Plasmodium falciparum to antimalarial drugs.

Chemotherapy and chemoprophylaxis are the principal means of combating malaria parasite infections in the human host. In the last 75 years, since the introduction of synthetic antimalarials, only a small number of compounds have been found suitable for clinical usage, and this limited armoury is now greatly compromised by the spread of drug-resistant parasite strains. Our current knowledge of the molecular mechanisms underlying resistance in the lethal species Plasmodium falciparum is reviewed here.

The dihydrofolate reductase-thymidylate synthetase gene in the drug resistance of malaria parasites

Resistance to antifolate drugs such as pyrimethamine is widespread among malaria parasites of the most pathogenic species Plasmodium falciparum. These drugs inhibit the dihydrofolate reductase activity of the dihydrofolate reductase-thymidylate synthetase (DHFR-TS) bifunctional enzyme. This review examines work done to characterize the enzyme, the cloning of plasmodial DHFR-TS genes, chromosomal mapping studies of these genes by pulsed-field gel electrophoresis, and the structural insights into the mechanism of drug resistance that have been gained by comparing genes from drug-sensitive parasites with those from drug-resistant strains that have arisen in the field or after experimental induction.

Protocols in molecular parasitology

Culturing and Biological Cloning of T. brucei. Culturing and Biological Cloning of T. cruzi. In Vitro Cultivation and Biological Cloning of Leishmania. Simple In Vitro Cultivation of the Malaria Parasite P. falciparum (Erythrocytic Stages) Suitable for Large-Scale Preparations. Synchronization and Cloning of Malaria Parasites. The Culture and Prparation of Gametocytes of Plasmodium falciparum for Immunochemical, Molecular, and Mosquito Infectivity Studies. The Culture of S. mansoni and Production of Life Cycle Stages. Preparation of DNA and RNA from T. brucei. Isolation of RNA and DNA from T. cruzi. Isolation of DNA and RNA from Leishmania. The Extraction and Purification of DNA and RNA from In Vitro Cultures of the Malaria Parasite P. falciparum. Extraction and Purificaiton of Nucleic Acids from Schistosomes. Application of DNA and Whole Organisms to Filter Supports for DNA Probe Analysis: Dot, Slot, and Touch Blotting. The Development and Use of Repetitive Sequences as DNA Probes for Parasite Detection and Species Identification. Analysis of the DNA of Parasitic Protozoa by Flow Cytometry. PCR for Low-Level Detection of Malaria Parasites in Blood. PCR Methods for Identification of Point Mutations and Gene Rearrangements. Antigenic Typing of Field Isolates of Plasmodium by DNA Techniques. Ribosomal RNA Probes for Detection and Identification of Species. Rapid Sequencing of Parasite rRNA. Antibody Screening of Expression Libraries. Antibody Select Procedure for Characterization of Expression Clones. Isolation of Parasite Genes Using Synthetic Oligonucleotides. Chromosome Mapping Methods for Parasitic Protozoa. Transfection of Leishmania and Trypanosoma brucei by Electroporation. Two-Dimensional Polyacrylamide Gel Electrophoresis. Isoenzyme Electrophoresis for Parasiste Characterization. The Surface Labeling of Schistosomes. Monoclonal Antibody Affinity Chromatography. Immunoblotting and Enzyme Linked Immunosorbent Assay. Immunofluorescence of Parasites. Immunoelectron Microscopic Localization of Antigens in Malaria Parasites. Preparation of Blotted Membrane for Protein Microsequencing. Identification of T-Cell Epitopes. Index.