David C. Warhurst

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.

The chemotherapy of rodent malaria. XLIV: studies on the mode of action of CM 6606, an indolo (3,2-c) quinoline N-oxide

CM 6606 differs in its mode of action from chloroquine but studies on its activity against parasites resistant to other antimalarials suggest that it may have some features in common with aminoalcohols. Similarities in drug-induced pigment changes are especially striking. Only halofantrine shows a reduced activity, however, against parasites that are highly resistant to CM 6606, while such parasites are slightly hypersensitive to sulfadoxine and clindamycin. Evidence suggesting that CM 6606 may function through an active metabolite, possibly CM 6609 in which the N-oxide is reduced, is discussed.

Malarone treatment failure and in vitro confirmation of resistance of Plasmodium falciparum isolate from Lagos, Nigeria

We report the first in vitro and genetic confirmation of Malarone® (GlaxoSmithKline; atovaquone and proguanil hydrochloride) resistance in Plasmodium falciparum acquired in Africa. On presenting with malaria two weeks after returning from a 4-week visit to Lagos, Nigeria without prophylaxis, a male patient was given a standard 3-day treatment course of Malarone®. Twenty-eight days later the parasitaemia recrudesced. Parasites were cultured from the blood and the isolate (NGATV01) was shown to be resistant to atovaquone and the antifolate pyrimethamine. The cytochrome b gene of isolate NGATV01 showed a single mutation, Tyr268Asn which has not been seen previously.

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.

Modified Fixed-Ratio Isobologram Method for Studying In Vitro Interactions between Atovaquone and Proguanil or Dihydroartemisinin against Drug-Resistant Strains of Plasmodium falciparum

ABSTRACT A modified fixed-ratio isobologram method for studying the in vitro interactions between antiplasmodial drugs is described. This method was used to examine the interactions between atovaquone, proguanil, and dihydroartemisinin. The interaction between atovaquone and proguanil was synergistic against atovaquone-sensitive strains K1 and T996; however, there was a loss of synergy against atovaquone-resistant strain NGATV01 isolated after Malarone (the combination of atovaquone and proguanil) treatment failure. While the interaction between atovaquone and dihydroartemisinin was indifferent against isolate NGATV01, the interaction displayed indifference tending toward antagonism against the atovaquone-sensitive strains tested. The relevance of in vitro interactions to in vivo treatment is discussed.

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.

Use of microdilution to assess in vitro antiamoebic activities of Brucea javanica fruits, Simarouba amara stem, and a number of quassinoids.

A microdilution technique for the assessment of in vitro activity against Entamoeba histolytica was devised and validated with metronidazole. The test was used to detect the antiamoebic activities of plant extracts prepared from the traditional remedies Brucea javanica fruits and Simarouba amara stems. The activity was associated with quassinoid-containing fractions. The 50% inhibitory concentrations for some quassinoids against amoebae were determined by using the microdilution method. These concentrations ranged from 0.019 micrograms.ml-1 for bruceantin, the most active quassinoid, to greater than 5 micrograms.ml-1 for glaucarubol, the least active compound tested. These results are discussed with reference to the known activities of these compounds against Plasmodium falciparum. Overall, the activities of the quassinoids against both protozoa are similar. The microdilution technique will be useful in the search for novel antiamoebic drugs.

Primary amoebic meningoencephalitis.

The third case in the UK of primary amoebic meningoencephalitis is reported; it affected an 11-year-old girl. Six days before admission the girl had swum in a pool fed by hot spring water in which the causative agent Naegleria fowleri was found. Early treatment with amphotericin B would seem to offer the only hope of recovery in this almost uniformly fatal infection.

The effect of combinations of qinghaosu (artemisinin) with standard antimalarial drugs in the suppressive treatment of malaria in mice

Artemisinin is a novel antimalarial drug isolated in China from the wormwood plant Artemisia annua L. Studies with rodent malaria were carried out to detect antagonism and synergism with a variety of antimalarial drugs. Isobolograms of drug interaction were plotted at the ED90 level. With a normally susceptible strain of Plasmodium berghei, marked potentiative synergism was found with mefloquine, tetracycline and spiramycin. There was some synergism also with primaquine. Combinations of artemisinin with dapsone, sulfadiazine, sulfadoxine, pyrimethamine, pyrimethamine/sulfadoxine and cycloguanil showed antagonism. A high degree of potentiation was shown between artemisinin and primaquine with a primaquine-resistant strain, whilst the combination with mefloquine showed enhanced potentiation with a mefloquine-resistant strain. Combinations of artemisinin with mefloquine, primaquine, tetracycline or clindamycin showed marked potentiation with an artemisinin-resistant strain. The mechanisms underlying the drug interactions observed are discussed.

In vittro studies on the mode of action of quassinoids with activity against chloroquine-resistant Plasmodium falciparum

Using the incorporation of [3H]isoleucine or [3H]hypoxanthine into acid-insoluble products as indices of protein- and nucleic acid-synthetic activity, respectively, it was shown that seven plant-derived quassinoids with differing chemical substitutions all inhibited protein synthesis more rapidly than nucleic acid synthesis in human erythrocytes infected with Plasmodium falciparum, in vitro. Five quassinoids (ailanthinone, bruceantin, bruceine B, glaucarubinone and holacanthone) were effective within 30 min at doses 10 times their 48 hr in vitro IC50 values. Chaparrin and glaucarubol differed in that they did not inhibit protein synthesis during the time course of these experiments when applied at 10 times their in vitro IC50 values. When these compounds were used at 209 and 114 times their respective IC50 values, their observed effects were identical to those of the other quassinoids studied. The time (t50) at which nucleic acid synthesis was reduced to 50% of control was directly proportional to the t50 for protein synthesis, suggesting that failure of nucleic acid synthesis is a consequence of inhibition of protein synthesis. It is concluded that in the malaria parasite, as in eukaryote models, quassinoids are rapid and potent inhibitors of protein synthesis, and that this is most likely due to effects upon the ribosome, rather than upon nucleic acid metabolism.