Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0024530 (malaria)
 44,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A mutation-specific polymerase chain reaction method was used to estimate the proportion of pyrimethamine-resistant parasites in 101 children reporting with malaria at the hospital in Ifakara, a town in southern Tanzania. The method is based on the observation that a point mutation (Asn-108) in the dihydroifolate reductase gene confers resistance to pyrimethamine. Twenty-eight percent of the examined 101 children had pyrimethamine-resistant parasites, 65% had pyrimethamine-sensitive parasites with the wild-type Ser-108 codon, and 9% had both alleles, suggesting a mixed infection. None of the 21 children with clinical malaria had pyrimethamine-resistant parasites. Currently, sulfadoxine-pyrimethamine is considered a potential first-line drug for malaria treatment in most African countries. We suggest that although sulfadoxine-pyrimethamine could still be effective against chloroquine-resistant malaria in this area, its judicious use is important so as to minimize the spread of resistance.
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PMID:Pyrimethamine-resistant Plasmodium falciparum parasites among Tanzanian children: a facility-based study using the polymerase chain reaction. 931 47

We have exploited the experimental accessibility of the protozoan parasite Toxoplasma gondii and its similarity to Plasmodium falciparum to investigate the influence of specific dihydrofolate reductase polymorphisms known from field isolates of drug-resistant malaria. By engineering appropriate recombinant shuttle vectors, it is feasible to examine mutations by transient or stable transformation of T. gondii parasites, in bacterial and yeast complementation assays, and through biochemical analysis of purified enzyme. A series of mutant alleles that mirror P. falciparum variants reveals that the key mutation Asn-108 (Asn-83 in T. gondii) probably confers resistance to pyrimethamine by affecting critical interactions in the ternary complex. Mutations such as Arg-59 (T. gondii 36) have limited effect in isolation, but in combination with other mutations they enhance the competitive ability of folate by increasing the speed of product turnover. Val-16 (T. gondii 10) confers low level resistance to cycloguanil but hypersensitivity to pyrimethamine. This mutation precludes Asn-108, probably because compression of the folate binding pocket introduced by this combination is incompatible with enzyme function. These studies permit detailed biochemical, kinetic, and structural analysis of drug resistance mutations and reconstruction of the probable phylogeny of antifolate resistance in malaria.
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PMID:A biochemical and genetic model for parasite resistance to antifolates. Toxoplasma gondii provides insights into pyrimethamine and cycloguanil resistance in Plasmodium falciparum. 945 69

A basis for the intrinsic resistance of some Plasmodium vivax isolates to pyrimethamine is suggested following the isolation of the bifunctional gene encoding dihydrofolate reductase-thymidylate synthase (DHFR-TS) of this human malaria parasite. Malaria parasites are dependent on this enzyme for folate biosynthesis. Specific inhibition of the DHFR domain of the enzyme by pyrimethamine blocks pyrimidine biosynthesis, leading to an inhibition of DNA replication. The gene was isolated by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotides designed to hybridize on the highly conserved regions of the sequence. The nucleotide sequence was completed by screening P. vivax genomic bank. Sequence analysis revealed an open reading frame (ORF) of 1872 nucleotides encoding a deduced protein of 623 amino acids (aa). Alignment with other malarial DHFR-TS genes showed that a 237-residue DHFR domain and a 286-residue TS domain were separated by a 100-aa linker region. Comparison with other malarial species showed low and essentially no isology in the DHFR and junctional domains, respectively, whereas an extensive isology was observed in the TS domain. The characteristic features of the P. vivax DHFR-TS gene sequence include an insertion of a short repetitive tandem array within the DHFR domain that is absent in another human malaria parasite, P. falciparum, and a GC-biased aa composition, giving rise to highly GC-rich DHFR (50.8%), junctional (58.7%), and TS (40.5%) domains, as compared with other malaria parasites. Analysis of the 5' noncoding region revealed the presence of a putative TATA box at 116 nucleotides upstream of the ATG start codon as well as a putative GC box at -636. Comparison of the DHFR sequences from pyrimethamine-sensitive and pyrimethamine-resistant P. vivax isolates revealed two residue changes: Ser Arg-58 and Ser Asn-117. These aa residues correspond to codons 59 and 108 in the P. falciparum DHFR active site in which similar aa substitutions (Cys Arg-59 and Ser Asn-108) are associated with pyrimethamine resistance. These findings may explain the intrinsic resistance of some P. vivax isolates to pyrimethamine.
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PMID:Analysis of the Plasmodium vivax dihydrofolate reductase-thymidylate synthase gene sequence. 957 57

The prevalence and severity of drug-resistant malaria is emerging rapidly in the Amazon basin of Brazil. In support of clinical trials using the new antimalarial drug combination of atovaquone and proguanil, we performed in vitro drug sensitivities, molecular characterization of parasite populations using the circumsporozoite protein, merozoite surface antigen-1 (MSA-1), and MSA-2 markers, and an analysis of the Plasmodium falciparum multidrug resistance (pfmdr1) gene sequence and copy number in 26 isolates of P. falciparum obtained in a gold-mining endemic area in Peixoto de Azevedo, Mato Grosso State. All 26 isolates were found to be resistant to chloroquine (50% inhibitory concentration [IC50] = 100-620 nM) and sensitive to mefloquine (IC50 < 23 nM) and halofantrine (IC50 < 6 nM). The isolates also show reduced susceptibility to quinine (IC50 = 48-280 nM). Sequence analysis of the pfmdr1 gene revealed Asn, Phe, Cys, Asp, and Tyr in positions 86, 184, 1034, 1042, and 1246, respectively. These point mutations were similar to that previously described in other Brazilian isolates. Southern blot analysis revealed no amplification of the pfmdr1 gene. These results suggest that three different mechanisms for drug resistance exist for chloroquine, mefloquine, and quinine.
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PMID:Characterization of Plasmodium falciparum isolated from the Amazon region of Brazil: evidence for quinine resistance. 959 53

The gene encoding dihydrofolate reductase-thymidylate synthase of the human malaria parasite, Plasmodium vivax, was isolated by polymerase chain reaction from genomic DNA and cloned. The sequences of the dihydrofolate reductase domain of 30 clinical isolates originating from various geographic areas were compared. Interstrain analysis revealed several genotypic variations, including short tandem repeat arrays which produced length polymorphism between different parasite isolates and point mutations in the putative dihydrofolate reductase active site cavity corresponding to those associated with pyrimethamine resistance in P. falciparum and rodent malaria parasites. Amino acid substitutions Ser-->Asn-117 and Ser-->Arg-58 were associated with decreased level of in vitro pyrimethamine sensitivity. These findings suggest that the P. vivax dihydrofolate reductase domain is characterized by polymorphism that has not been observed in P. falciparum and may explain the resistance of some P. vivax isolates to pyrimethamine. Nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDJB databases under the accession numbers X98123 (isolate ARI/Pakistan), AJ003050 (isolate CNC/Thailand), AJ003051 (isolate COU/unknown geographic origin), AJ003052 (isolate DUF/French Guiana), AJ003053 (isolate GRO/Madagascar), AJ003054 (isolate HRT/Comoros Islands), AJ003071 (isolate LFT/Cambodia), AJ003072 (isolate LGF/'India), AJ003073 (isolate MAN/Comoros Islands), AJ003074 (isolate MAT/Surinam), AJ003075 (isolate PHI/Djibouti), AJ003076 (isolate PIT/Madagascar), AJ003077 (isolate YTZ/Indonesia), AJ222630 (isolate Burma-1), AJ222631 (isolate Burma-151), AJ222632 (isolate Burma-5), AJ222633 (isolate Burma-6), AJ222634 (isolate Burma-98).
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PMID:Sequence variations in the Plasmodium vivax dihydrofolate reductase-thymidylate synthase gene and their relationship with pyrimethamine resistance. 965 31

Two new dihydrofolate reductase (DHFR) mutations were recently discovered in Plasmodium falciparum samples from an area of Bolivia with high rates of in vivo resistance to pyrimethamine-sulfadoxine: a Cys-->Arg point mutation in codon 50 and a five amino acid insertion after codon 30, termed the Bolivia repeat. We used a yeast expression system to screen these new DHFR mutants, as well as all of the other known DHFR mutant genotypes, against four antifolates: pyrimethamine, cycloguanil, chlorcycloguanil, and WR99210. The prodrug proguanil was also evaluated. The primary 108-Asn mutation, the known secondary mutations 51-Ile, 59-Arg and 164-Leu, as well as the 50-Arg mutation, all progressively enhanced pyrimethamine resistance in naturally observed combinations with one another, with the presence of 164-Leu most significantly increasing resistance. Cycloguanil and chlorcycloguanil resistance were most impacted by 164-Leu and the paired 16-Val/108-Thr. Proguanil had no effect on malaria DHFR. All DHFRs analyzed were sensitive to WR99210. The Bolivia repeat did not markedly affect drug sensitivity. We conclude that malaria DHFR can be reliably, rapidly and inexpensively analyzed in yeast for activity against a broad spectrum of antifolates. This system may be useful for initially characterizing newly discovered genotypes before proceeding to P. falciparum transfection; for large-scale geographic surveys of drug resistance; and for screening new antifolates or new antifolate combinations for their effectiveness against a large panel of DHFR mutants.
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PMID:Antifolate resistance due to new and known Plasmodium falciparum dihydrofolate reductase mutations expressed in yeast. 974 71

Merozoite surface antigen 1 (MSA1) is a promising candidate for vaccine development against malaria parasites. Here, we report the complete nucleotide sequence of the gene encoding the precursor to this major surface antigen of Plasmodium berghei strain ANKA using cDNA library screening and polymerase chain reaction techniques. A single open reading frame of 5,376 basepairs encoding a protein with a calculated molecular mass of 197 kD was defined. The protein contains a putative signal peptide of 19 amino acids, a membrane anchor sequence of 18 residues, and shows two epidermal growth factor-like domains rich in Cys residues at the C-terminus. There are four repeat sequences of oligopeptides in the molecule: tetrapeptide (Ser-Thr-Thr-Thr), tripeptide (Pro-Thr-Pro and Pro-Ala-Ala), and dipeptide (Ser-Gly). Furthermore, three nine-residue stretches of a motif (Ala-Ser-Asn-Pro-Gly-Ala-Ser-Ala-Ser) are located near each other. All of these repeat sequences are unexceptionally located in the variable regions when compared with other MSA1 molecules. The molecule displays 79% overall identity to the analogous antigen of P. yoelii yoelii strain YM, 70% to that of P. chabaudi chabaudi strain AS, and 38% to that of P. falciparum strain Wellcome.
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PMID:Cloning and characterization of the merozoite surface antigen 1 gene of Plasmodium berghei. 1040 33

Sulfadoxine/pyrimethamine (SP) is considered an alternative treatment for acute uncomplicated malaria caused by Plasmodium falciparum resistant to chloroquine. However, the appearance of resistance to this drug has been reported since its initial use in Colombia. Molecular analysis of the dihydrofolate reductase gene indicates a correlation between in vitro resistance to SP and the Asn-108 point mutation. Little is known about the association of this point mutation and in vivo resistance to SP. We used a mutation-specific polymerase chain reaction strategy to analyze the presence of the Asn-108 point mutation in 48 clinical samples with adequate clinical response (ACR), 2 early treatment failures (ETF), and 1 late treatment failure (LTF). The Asn-108 mutation was detected in 36 of the ACR samples and in all of the ETF and LTF samples. Eleven ACR samples amplified with the wild-type-specific primer and one amplified with the primer for the Thr-108 mutation described for resistance to cycloguanil. These results suggest that the Asn-108 marker may not be useful in predicting SP treatment failure.
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PMID:Lack of an association between the ASN-108 mutation in the dihydrofolate reductase gene and in vivo resistance to sulfadoxine/pyrimethamine in Plasmodium falciparum. 1046 74

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.
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PMID:[Mechanisms and dynamics of drug resistance in Plasmodium falciparum]. 1057 58

Pyrimethamine, in combination with sulfadoxine, is currently one of the major alternative drugs used for the treatment of chloroquine-resistant Plasmodium falciparum malaria infections in Africa. The mechanism of pyrimethamine resistance has been strongly associated with a single, key point mutation in the dihydrofolate reductase-thymidylate synthase gene, resulting in the substitution of the wild-type allele Ser-108 by either Asn-108 or Thr-108. The pyrimethamine-resistant phenotype and/or genotype were determined in 273 Cameroonian clinical isolates obtained in Yaounde by in vitro assays and polymerase chain reaction-restriction fragment length polymorphism over a 5-year period. The in vitro assays showed that 42% (18 of 43) and 63% (69 of 110) of the isolates obtained in 1994-1995 and 1997-1998, respectively, were resistant to pyrimethamine (50% inhibitory concentration [IC50] > 100 nM). The polymerase chain reaction showed that 43% (55 of 127) and 59% (50 of 85) of the isolates in 1994-1995 and 1997-1998, respectively, had the mutant Asn-108 allele. The pyrimethamine-resistant genotype (Asn-108) corresponded with the pyrimethamine-resistant phenotype (IC50 > or = 100 nM) in a large majority (> 95%) of the isolates. The results of our study suggest an increasing prevalence of pyrimethamine resistance in Yaounde. Our study further suggests that pyrimethamine resistance can be monitored by a technique that can be adopted by malaria research centers in Africa.
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PMID:Molecular epidemiology of malaria in Yaounde, Cameroon IV. Evolution of pyrimethamine resistance between 1994 and 1998. 1058 15


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