Determinant Of Chloroquine Resistance Research Articles

Dissecting the loci of low-level quinine resistance in malaria parasites.

Quinine (QN) remains effective against Plasmodium falciparum, but its decreasing efficacy is documented from different continents. Multiple genes are likely to contribute to the evolution of QN resistance. To locate genes contributing to QN response variation, we have searched a P. falciparum genetic cross for quantitative trait loci (QTL). Results identify additive QTL in segments of chromosomes (Chrs) 13, 7 and 5, and pairwise effects from two additional loci of Chrs 9 and 6 that interact, respectively, with the QTL of Chrs 13 and 7. The mapped segments of Chrs 7 and 5 contain pfcrt, the determinant of chloroquine resistance (CQR), and pfmdr1, a gene known to affect QN responses. Association of pfcrt with a QTL of QN resistance supports anecdotal evidence for an evolutionary relationship between CQR and reduced QN sensitivity. The Chr 13 segment contains several candidate genes, one of which (pfnhe-1) encodes a putative Na(+)/H(+) exchanger. A repeat polymorphism in pfnhe-1 shows significant association with low QN response in a collection of P. falciparum strains from Asia, Africa and Central and South America. Dissection of the genes and modifiers involved in QN response will require experimental strategies that can evaluate multiple genes from different chromosomes in combination.

Chloroquine resistance in Plasmodium chabaudi: are chloroquine-resistance transporter (crt) and multi-drug resistance (mdr1) orthologues involved?

We have identified in the rodent malaria parasite Plasmodium chabaudi orthologues of two Plasmodium falciparum genes, pfcrt and pfmdr1 which have been implicated as determinants of chloroquine resistance in the latter species. The sequences of the P. chabaudi genes, denoted, respectively, pccg10 and pcmdr1, were first determined in the chloroquine-sensitive clone AS, and found to be highly similar to those of P. falciparum. For pccg10, there was a nucleotide sequence identity of 68.6% and amino acid sequence identity of 75.1% within the predicted coding region. For pcmdr1, the sequence identities were 75.0% (nucleotide) and 78.1% (amino acid). The sequences of the genes were then determined in three P. chabaudi clones selected from clone AS which possessed three different levels of resistance to chloroquine. The sequences of both genes in all mutants were found to be identical to those of the sensitive AS from which they had been derived. Polymorphic sites were found in both genes between the AS clones and a genetically unrelated sensitive clone AJ. Analysis of genetic crosses between AJ and resistant AS clones showed no linkage between inherited parental alleles of pccrt and pcmdr1 and drug responses of the cloned progeny. This showed that neither of these genes, nor genes closely linked to them, were determinants of the chloroquine resistance in the P. chabaudi mutants.

Plasmodium falciparum gametocytaemia in Nigerian children: Peripheral immature gametocytaemia as an indicator of a poor response to chloroquine treatment, and its relationship to molecular determinants of chloroquine resistance.

A group of 161 children who presented with acute, symptomatic, uncomplicated, Plasmodium falciparum malaria in an endemic area of Nigeria was investigated. The aims of the study were to determine the clinical characteristics and responses to oral chloroquine (CQ) therapy of children who were gametocytaemic on presentation and those who were not [including those who were found to have developed peripheral immature gametocytaemias (PIG) when checked 72 h after commencing treatment], and to follow the development of PIG 72-336 h after the start of treatment. The 40 consecutive children who did have peripheral gametocytaemia on presentation and the 40 who did not were similar in their clinical characteristics and responses to oral CQ therapy. Nine of the 40 children who did not initially have gametocytaemias but who subsequently developed PIG (stages I-III) 72-336 h after commencing CQ treatment failed the treatment. In order to evaluate the presence of PIG as an indicator of response to CQ, the smears of blood from 81 children--66 classified as resistant to CQ (60, five and one considered RI, RII, RIII, respectively) and 15 who, though considered to have sensitive responses to CQ, cleared their peripheral parasitaemias > or =72 h after commencing CQ therapy--were examined for PIG. Most (42) of the 66 children with CQ-resistant (CQ-R) infections but none of the 15 with sensitive responses had PIG. Among the 66 children with CQ-R infections, the clinical features of those with PIG were generally similar to those without PIG, although those with PIG were more likely to have hepato-splenomegaly and less likely to have hepatomegaly alone. In the children with CQ-R infections, plasma concentrations of CQ on days 7 and 14 were generally above the level necessary to clear sensitive infections in the study area. The results of molecular analyses of isolates from children with both CQ-R infections and PIG revealed that all 14 checked for mutations in pfcrt had the T76 mutation associated with CQ resistance, and that four of the five also checked for mutations in pfmdr1 had the Y86 mutation associated with CQ resistance. The detection of PIG 72 h after the commencement of CQ treatment may be used as a microscopical indicator of a poor response to CQ in children from this endemic area.

A Chemical Approach Towards Understanding the Mechanism and Reversal of Drug Resistance in Plasmodium falciparum: Is it Viable?

Genetic and biochemical approaches to studies of drug resistance mechanisms in Plasmodium falciparum have raised controversies and contradictions over the past several years. A different and novel chemical approach to this important problem is desirable at this point in time. Recently, the molecular basis of drug resistance in P. falciparum has been associated with mutations in the resistance genes, Chloroquine Resistance Transporter (PfCRT) and the P-glycoprotein homologue (Pgh1). Although not the determinant of chloroquine resistance in P. falciparum, mutations in Pgh1 have important implications for resistance to other antimalarial drugs. Because it is mutations in the aforementioned resistance genes rather than overexpression that has been associated with drug resistance in malaria, studies on mechanisms of drug resistance and its reversal by chemosensitisers should benefit from a chemical approach. Target-oriented organic synthesis of chemosensitisers against proteins implicated in drug resistance in malaria should shed light on mechanism of drug resistance and its reversal in this area. The effect of structurally diverse chemosensitisers should be examined on several putative resistance genes in P. falciparum to deal with antimalarial drug resistance in the broadest sense. Therefore, generating random mutations of these resistance proteins and subsequent screening in search of a specific phenotype followed by a search for mutations and/or chemosensitisers that affect a specific drug resistance pathway might be a viable strategy. This diversity-oriented organic synthesis approach should offer the means to simultaneously identify resistance proteins that can serve as targets for therapeutic intervention (therapeutic target validation) and chemosensitisers that modulate the functions of these proteins (chemical target validation).

Allelic modifications of the cg2 and cg1 genes do not alter the chloroquine response of drug-resistant Plasmodium falciparum

The determinant of chloroquine resistance (CQR) in a Plasmodium falciparum cross was previously mapped by linkage analysis to a 36 kb segment of chromosome 7. Candidate genes within this segment have been previously shown to include two genes, cg2 and cg1, that have complex polymorphisms linked to the CQR phenotype. Using DNA transfection and allelic exchange, we have replaced these polymorphisms in CQR parasites with cg2 and cg1 sequences from chloroquine sensitive parasites. Drug assays of the allelically-modified lines show no change in the degree of CQR, providing evidence against the hypothesis that these polymorphisms are important to the CQR phenotype. Similarly, no change was found in the degree to which verapamil or other chloroquine sensitizers reverse CQR in the transformants. These results and the high though not complete degree of association of CQR with cg2 and cg1 polymorphisms in field isolates suggest involvement of another nearby gene in the P. falciparum CQR mechanism.