Erythrocyte invasion profiles are associated with a common invasion ligand polymorphism in Senegalese isolates of Plasmodium falciparum.

Plasmodium falciparum parasites use multiple ligand-receptor interactions to invade human erythrocytes. Variant expression levels of members of the PfRh and PfEBA ligand families are associated with the use of different erythrocyte receptors, defining invasion pathways. Here we analyse a major polymorphism, a large sequence deletion in the PfRh2b ligand, and erythrocyte invasion profiles in uncultured Senegalese isolates. Parasites vary considerably in their use of sialic acid-containing and protease-sensitive erythrocyte receptors for invasion. The erythrocyte selectivity index was not related to invasion pathway usage, while parasite multiplication rate was associated with enhanced use of a trypsin-resistant invasion pathway. PfRh2b protein was expressed in all parasite isolates, although the PfRh2b deletion was present in a subset (approximately 68%). Parasites with the PfRh2b deletion were found to preferentially utilize protease-resistant pathways for erythrocyte invasion. Sialic acid-independent invasion is reduced in parasites with the PfRh2b deletion, but only in isolates derived from blood group O patients. Our results suggest a significant role for PfRh2b sequence polymorphism in discriminating between alternative erythrocyte receptors for invasion and as a possible determinant of virulence.

Rational use of drugs against Plasmodium falciparum.

Recent studies on resistance to blood schizontocides in Plasmodium falciparum give a rational basis for the use of artemisinins combined with arylaminoalcohols for the treatment of uncomplicated chloroquine-resistant malaria in Africa. In areas where such combinations are introduced, there is reason to believe that the continued use of chloroquine in the community will help protect the new drugs from resistance. In view of several laboratory studies, combinations of artemisinins with antifolates or chloroquine pose a risk of antagonistic interaction. This can be avoided by use of the artemisinin and the companion drug sequentially.

Linkage disequilibrium between two chromosomally distinct loci associated with increased resistance to chloroquine in Plasmodium falciparum.

Chloroquine-resistance in Plasmodium falciparum is associated with polymorphisms in a locus on or near the cg2 gene on chromosome 7, and in the pfmdr1 gene on chromosome 5. In this study we typed P. falciparum DNA from uncomplicated malaria cases in The Gambia in 1990, 1995 and 1996 for size polymorphism in the omega repeat of cg2, for sequence polymorphisms in pfmdr1 at codons 86 and 184, in dhfr at codon 108 and in the msp2 gene. Chloroquine sensitivity tests were conducted in vitro. A significant but incomplete association was found between the presence of the cg2 Dd2-like omega repeat size polymorphism and in vitro resistance, and between the tyr-86 allele of pfmdr1 and in vitro resistance. Furthermore there was strong linkage disequilibrium between the pfmdr1 asn-86 allele and the cg2 not Dd2-like omega repeat allele located on different chromosomes. In contrast, no linkage disequilibrium was found between these alleles and either the dhfr ser-108 allele or the msp2 IC sequence polymorphism. No significant linkage was measured between pfmdr1 asn-86 and phe-184 although these loci are separated only by 296 base pairs. Our results suggest that genetic elements linked to the cg2 and the pfmdr1 genes are important determinants of chloroquine resistance. It can be concluded that the observed linkage disequilibrium is maintained epistatically through selection by chloroquine.

Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr1 gene of Plasmodium falciparum.

The declining efficacy of chloroquine and pyrimethamine/sulphadoxine in the treatment of human malaria has led to the use of newer antimalarials such as mefloquine and artemisinin. Sequence polymorphisms in the pfmdr1 gene, the gene encoding the plasmodial homologue of mammalian multidrug resistance transporters, have previously been linked to resistance to chloroquine in some, but not all, studies. In this study, we have used a genetic cross between the strains HB3 and 3D7 to study inheritance of sensitivity to the structurally unrelated drugs mefloquine and artemisinin, and to several other antimalarials. We find a complete allelic association between the HB3-like pfmdr1 allele and increased sensitivity to these drugs in the progeny. Different pfmdr1 sequence polymorphisms in other unrelated lines were also associated with increased sensitivity to these drugs. Our results indicate that the pfmdr1 gene is an important determinant of susceptibility to antimalarials, which has major implications for the future development of resistance.

The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin.

Although chloroquine-resistance (CQR) in Plasmodium falciparum is increasing and resistance to other blood schizonticidal anti-malarials has been reported, the molecular basis remains unclear. In this study fresh field isolates were obtained from The Gambia, an area of emerging CQR and tested for sensitivity to the anti-malarial drugs mefloquine, halofantrine, artemisinin, dihydroartemisinin, chloroquine and quinine. Sequence polymorphisms in the pfmdr1 gene and size polymorphisms in the cg2 gene were assessed using PCR-based systems. A strong association was observed between the presence of the tyr-86 allele of pfmdr1 and increased sensitivity to mefloquine and halofantrine, as well as the structurally unrelated drugs artemisinin and dihydroartemisinin. A weaker association was found between the presence of tyr-86 and increased resistance to chloroquine and quinine. The cg2 Dd2-like omega repeat size polymorphism was associated with increased resistance to chloroquine and increased sensitivity to mefloquine and halofantrine. An intragenic association was also found between a polymorphism in the polyasparagine linker region of pfmdr1 and the tyr-86 allele, which may be due to genetic hitchhiking, indicative of recent selection by chloroquine. Our data support a hypothesis where the pfmdr1 gene confers a true multidrug resistance phenotype which is lost by mutation.

Efficacy of artesunate plus pyrimethamine-sulphadoxine for uncomplicated malaria in Gambian children: a double-blind, randomised, controlled trial.

BACKGROUND: Resistance to cheap effective antimalarial drugs, especially to pyrimethaminesulphadoxine (Fansidar), is likely to have a striking impact on childhood mortality in sub-Sharan Africa. The use of artesunate (artesunic acid) [corrected] in combination with pyrimethamine-sulphadoxine may delay or prevent resistance. We investigated the efficacy, safety, and tolerability of this combined treatment. METHODS: We did a double-blind, randomised, placebo-controlled trial in The Gambia. 600 children with acute uncomplicated Plasmodium falciparum malaria, aged 6 months to 10 years, at five health centres were randomly assigned pyrimethaminesulphadoxine (25 mg/500 mg) with placebo; pyrimethamine-sulphadoxine plus one dose of artesunate (4mg/kg bodyweight); or pyrimethamine-sulphadoxine plus one dose 4 mg/kg bodyweight artesunate daily for 3 days. Children were visited at home each day after the start of treatment until parasitaemia had cleared. FINDINGS: The combined treatment was well tolerated. No adverse reactions attributable to treatment were recorded. By day 1, only 178 (47%) of 381 children treated with artesunate were still parasitaemic, compared with 157 (81%) of 195 children in the pyrimethamine-sulphadoxine alone group (relative risk 1.7 [95% CI 1.5-2.0], p<0.001). Treatment-failure rates at day 14 were 3.1% in the pyrimethamine sulphadoxine alone group, and 3.7% in the one-dose artesunate group (risk difference -0.6% [-4.2 to 3.0]) and 1.6% in the three-dose group (1.5 [1.5-4.5], p=0.048). Symptoms resolved faster in children who received artesunate, but there was no additional benefit for three doses of artesunate over one dose. Children given artesunate were less likely to be gametocytaemic after treatment. INTERPRETATION: The combined treatment was safe, well tolerated, and effective. The addition of artesunate to malaria treatment regimens in Africa results in lower gametocyte rates and may lower transmission rates.

PIP: This double-blind, randomized, controlled study investigated the efficacy, safety and tolerability of artesunate plus pyrimethamine-sulphadoxine for uncomplicated malaria among Gambian children. Combined use of artesunate and pyrimethamine-sulphadoxine was hypothesized to delay or prevent resistance, which proved to be effective in reducing childhood mortality in sub-Saharan Africa. A total of 600 children with acute uncomplicated Plasmodium falciparum malaria, 6 months to 10 years old, were randomly administered pyrimethamine-sulphadoxine (25 mg/500 mg) with placebo, 4 mg/kg body weight pyrimethamine-sulphadoxine plus 1 dose of artesunate, or pyrimethamine-sulphadoxine plus 4 mg/kg body weight artesunate for 3 days. Results indicate that combined treatment was well tolerated. On day 1, 178 of 381 children treated with artesunate were still parasitemic compared with 157 of 195 children in the pyrimethamine-sulphadoxine group. On the other hand, failure rates on day 14 were 3.1% in the pyrimethamine-sulphadoxine group and 3.7% in the 1-dose artesunate group and 1.6% in the 3-dose group. Insignificant differences were found among children administered 1-dose and 3-dose artesunate, and were found less likely to be gametocytemic after treatment. In conclusion, this study confirms the safety and efficacy of a combined treatment, which eventually results in lower gametocyte rates and lower transmission rates.

Inoculum effect leads to overestimation of in vitro resistance for artemisinin derivatives and standard antimalarials: a Gambian field study.

Artemisinin (QHS) and its derivatives are new antimalarials which are effective against Plasmodium falciparum parasites resistant to chloroquine (CQ). As these drugs are introduced it is imperative that resistance is monitored. In this paper we demonstrate that the inoculum size used in in vitro testing influences the measured in vitro susceptibility to QHS and its derivative dihydroartemisinin (DHA) and to mefloquine (MEF) and CQ over the range of parasitaemias routinely used in testing with the WHO in vitro microtest. An increase in parasitaemia and/or haematocrit was accompanied by a decrease in the measured sensitivity of 2 laboratory lines. In the context of a field study testing in vitro susceptibility of parasite isolates from patients with uncomplicated malaria in Fajara, The Gambia we demonstrate that failure to control for inoculum size significantly overestimates the level of resistance to QHS and DHA as well as MEF, halofantrine (HAL) and quinine (QUIN). When controlling for the inoculum effect, cross-resistance was observed between QHS, MEF and HAL suggesting the presence of a multidrug resistance-like mechanism. These studies underline the importance of inoculum size in in vitro susceptibility testing.

The pfmdr1 gene is associated with a multidrug-resistant phenotype in Plasmodium falciparum from the western border of Thailand.

On the western border of Thailand, Plasmodium falciparum has become resistant to almost all antimalarial agents. The molecular basis of resistance in these parasite populations has not been well characterized. This study assessed genetic polymorphisms in the pfmdr1 gene in 54 parasites collected from the western border of Thailand to determine the relationship of pfmdr1 copy number and codon mutations with parasite sensitivities to mefloquine, chloroquine, halofantrine, quinine, and artesunate assessed in vitro. A point mutation at codon 86 (resulting in a change of Asn to Tyr) was associated with a significantly lower 50% inhibitory concentration (IC(50)) of mefloquine (median, 9 ng/ml versus 52.4 ng/ml; P = 0.003). Overall 35% of the isolates (19 of 54) had an increase in pfmdr1 copy number, and all 19 carried the wild-type allele at codon 86. Increased pfmdr1 copy number was associated with higher IC(50)s of mefloquine (P = 0.04) and artesunate (P = 0.005), independent of polymorphism at codon 86. The relationship between pfmdr1 and resistance to structurally distinct antimalarial agents confirms the presence of a true multidrug-resistant phenotype.

Plasmodium falciparum: selection of serine 108 of dihydrofolate reductase during treatment of uncomplicated malaria with co-trimoxazole in Ugandan children.

In vivo testing for resistance of Plasmodium falciparum to co-trimoxazole (trimethoprim/sulfamethoxazole) was performed in Uganda in 41 children with uncomplicated malaria, and blood samples were screened before and after treatment for polymorphisms in the antifolate target genes for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). Selection towards a specific genotype at some codons of the DHFR and DHPS genes was observed in samples collected after exposure to co-trimoxazole drug pressure. The alleles 51-isoleucine, 59-arginine, and 108-serine of DHFR were significantly associated with clinical resistance, as was allele 581-alanine of DHPS. Resistance against antifolate combinations probably requires resistance-related polymorphisms in both the DHFR and the DHPS genes. In addition, it appears that the trimethoprim-resistant DHFR genotype differs from that for pyrimethamine at residue 108.

Polymorphisms in the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) genes of Plasmodium falciparum and in vivo resistance to sulphadoxine/pyrimethamine in isolates from Tanzania.

The efficacy of sulphadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Africa is increasingly compromised by development of resistance. The occurrence of mutations associated with the active site sequence in the Plasmodium falciparum genes coding for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) is associated with in vitro resistance to pyrimethamine and sulphadoxine. This study investigates the occurrence of these mutations in infected blood samples taken from Tanzanian children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results show that alleles of DHPS (436-alanine, 437-alanine and 540-lysine) were significantly reduced in prevalence on day 7 after S/P treatment. In this area, a DHPS with 436-serine, 437-glycine and 540-glutamate appears to play a major role in resistance to S/P in vivo. Evidence for the influence of mutations in the DHFR gene in this investigation is not clear, probably because of the high prevalence of 'resistance-related' mutations at day 0 in the local parasite population. For apparently the same reason, it was not possible to show a statistical association between S/P resistance and the presence of particular polymorphisms in the DHFR and DHPS genes before treatment.

Plasmodium falciparum: detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestion.

With the spread of resistance to chloroquine, the combination of sulphadoxine and pyrimethamine is growing in importance for the treatment of infection with the malaria parasite Plasmodium falciparum. Mutations in the dhfr gene of P. falciparum have been associated with resistance to pyrimethamine. Recently, several polymorphisms have been identified in the P. falciparum dhps gene which may correlate with sulphadoxine-resistance. Simple and rapid tests have been developed to detect these polymorphisms, using PCR followed by restriction digestion. These tests can accurately identify all the polymorphisms described to date at codons 16, 51, 59, 108, and 164 in the dhfr gene and those at codons 436, 437, 540, 581, and 613 in the dhps gene. A nested system has been developed which allows the accurate detection of these polymorphisms in samples of fingerprick blood collected on glass fiber membranes and filter papers, some with very low parasitaemias.

In vivo selection for a specific genotype of dihydropteroate synthetase of Plasmodium falciparum by pyrimethamine-sulfadoxine but not chlorproguanil-dapsone treatment.

Plasmodium falciparum present in blood samples collected before and 3 weeks after treatment with either pyrimethamine-sulfadoxine or chlorproguanil-dapsone was analyzed for variants of the genes coding for the target enzymes of antifolate drugs, dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). Fragments of the genes were amplified by polymerase chain reactions, and variants were identified by specific restriction endonuclease digestion. Treatment with either drug combination selected for the variants Ile51, Arg59, and Asn108 of DHFR, which have been associated with in vitro resistance to pyrimethamine and cycloguanil. The genotype Ser436, Gly437, and Glu540 of DHPS was selected by pyrimethamine-sulfadoxine but not chlorproguanil-dapsone treatment, showing that a combination of these three variants is important for in vivo resistance to sulfadoxine in the area studied.

High prevalence of mutations in the dihydrofolate reductase gene of Plasmodium falciparum in isolates from Tanzania without evidence of an association to clinical sulfadoxine/pyrimethamine resistance.

Recently the efficacy of sulfadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Tanzania has been seriously compromised by the development of resistance. The occurrence of active site mutations in the Plasmodium falciparum gene sequence coding for dihydrofolate reductase (DHFR) is known to confer resistance to pyrimethamine. This study investigates the occurrence of these mutations in infected blood samples taken from Tanzanian children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results confirm the occurrence of one or more DHFR mutations in all the samples, but no relationship was found with the presence of parasites in the blood at day 7. The results suggest that alterations in the coding region for dihydropteroate synthetase (DHPS), the enzyme target for sulfadoxine, should be studied in order to predict resistance to the S/P combination. It has been proposed earlier that sulfadoxine could itself act on DHFR, because of a false dihydrofolate produced by drug metabolism through DHPS and dihydrofolate synthase. The results of this treatment study suggest that such a possibility is unlikely.

Polymorphism of the Pfmdr1 gene and chloroquine resistance in Plasmodium falciparum in The Gambia.

To assess the level of resistance to chloroquine (CQ) of Plasmodium falciparum in The Gambia in 1995-1996 we measured susceptibility in vivo by quantifying parasitaemia of children with mild malaria on days 4 and 8 after treatment. Pretreatment blood samples were used for susceptibility testing in vitro by the World Health Organization microtest and the prevalence of the tyrosine (tyr)86 allele of the Pfmdr1 gene was assessed by the polymerase chain reaction and restriction fragment length polymorphism analysis. Seven of 42 children (17%) treated with CQ remained parasitaemic on day 4 and required a change of antimalarial treatment. Susceptibility assays in vitro were performed on 50 P. falciparum isolates obtained from eligible children before treatment; 36 (72%) were resistant to CQ (> or = 1.6 mumol/L). The median minimum inhibitory concentration (MIC) of artemether was 3.38 nmol/L (range 0.42-13.51 nmol/L) and the median MIC of dihydroartemisinin was 0.88 nmol/L (range 0.22-14.04 nmol/L). Susceptibility in vitro to CQ and the Pfmdr1 genotype were determined for 31 fresh isolates. The allele was present in 12 of 22 isolates found to be resistant to CQ in vitro, but in none of the 9 isolates which were susceptible (Fisher's exact test, P = 0.005). All P. falciparum isolates with the tyr86 allele were CQ resistant in vitro, but since only half of all resistant isolates contained the allele, additional explanations for CQ resistance are required.

Evidence for selection for the tyrosine-86 allele of the pfmdr 1 gene of Plasmodium falciparum by chloroquine and amodiaquine.

The 4-aminoquinolines chloroquine (CQ) and amodiaquine (AM) were used to treat Gambian children with uncomplicated falciparum malaria in a randomized drug trial. Blood samples were taken immediately before treatment (day 0), and at day 7 and day 28 after treatment. Samples from those parasitologically positive at day 7 following treatment ('early positives') and those positive at day 28 but negative at day 7 ('late positives') have been studied by PCR followed by restriction enzyme digestion to determine the allelic status of the pfmdr 1 locus at the codon-86 position (asparagine or tyrosine), previously associated with resistance to CQ. A significantly higher prevalence of the tyr-86 allele was observed in samples taken immediately before treatment (day 0) in the early positives group when compared with the late positives group. This suggests the tyr-86 allele contributes to drug resistance in the early positives group. This association remained significant for both CQ and AM groups, implying a common genetic basis of resistance. Predominance of the allele at day 7 is consistent with a strong selection in the first week following treatment. In the late positives group, a significantly higher prevalence of the tyr-86 allele was observed in the samples at day 28 when compared with those at day 0, suggestive of selection during the period day 7 to day 28. Differences were observed in the extent of this selection in the CQ and AM groups. The samples were genotyped at 3 unlinked polymorphic loci. These analyses suggested that most parasites observed at day 7 were probably recrudescences whereas most of those at day 28 were reinfections.

Direct evidence that asparagine at position 108 of the Plasmodium falciparum dihydrofolate reductase is involved in resistance to antifolate drugs in Tanzania.

A nested polymerase chain reaction was used to amplify a fragment of the gene for dihydrofolate reductase of Plasmodium falciparum containing codon 108, where a point mutation, causing a serine to asparagine change, occurs in pyrimethamine resistant parasites. The presence of the mutation was detected by restriction enzyme digestion. Parasites in blood samples collected from asymptomatic children before, and 3 weeks after, treatment with pyrimethamine-sulfadoxine or chlorproguanil-dapsone were analysed. Parasites in the samples taken at 3 weeks carried only the asparagine mutant.