Dennis E. Kyle

Malaria: progress, perils, and prospects for eradication

There are still approximately 500 million cases of malaria and 1 million deaths from malaria each year. Yet recently, malaria incidence has been dramatically reduced in some parts of Africa by increasing deployment of anti-mosquito measures and new artemisinin-containing treatments, prompting renewed calls for global eradication. However, treatment and mosquito control currently depend on too few compounds and thus are vulnerable to the emergence of compound-resistant parasites and mosquitoes. As discussed in this Review, new drugs, vaccines, and insecticides, as well as improved surveillance methods, are research priorities. Insights into parasite biology, human immunity, and vector behavior will guide efforts to translate parasite and mosquito genome sequences into novel interventions.

Evidence for the shikimate pathway in apicomplexan parasites

Parasites of the phylum Apicomplexa cause substantial morbidity, mortality and economic losses, and new medicines to treat them are needed urgently,. The shikimate pathway is an attractive target for herbicides and antimicrobial agents because it is essential in algae, higher plants, bacteria and fungi, but absent from mammals,. Here we present biochemical, genetic and chemotherapeutic evidence for the presence of enzymes of the shikimate pathway in apicomplexan parasites. In vitro growth of Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum was inhibited by the herbicide glyphosate, a well-characterized inhibitor of the shikimate pathway enzyme 5-enolpyruvyl shikimate 3-phosphate synthase. This effect on T. gondii and P. falciparum was reversed by treatment with p-aminobenzoate, which suggests that the shikimate pathway supplies folate precursors for their growth. Glyphosate in combination with pyrimethamine limited T. gondii infection in mice. Four shikimate pathway enzymes were detected in extracts of T. gondii and glyphosate inhibited 5-enolpyruvyl shikimate 3-phosphate synthase activity. Genes encoding chorismate synthase, the final shikimate pathway enzyme, were cloned from T. gondii and P. falciparum. This discovery of a functional shikimate pathway in apicomplexan parasites provides several targets for the development of new antiparasite agents.

Amplification of pfmdr1 associated with mefloquine and halofantrine resistance in Plasmodium falciparum from Thailand

Drug resistance in Plasmodium falciparum is an expanding problem in most endemic areas. Recent studies have suggested the potential involvement of genes in the MDR gene family in resistance to quinoline-containing compounds in P. falciparum. In this study a molecular analysis of pfmdr 1 in recent isolates from Thailand was done (1) to further examine the role of pfmdr 1 in drug-resistant isolates and (2) to examine the reported association of pfmdr 1 intragenic alleles and chloroquine resistance. Most of the isolates (10 of 11) were resistant to all compounds tested. Analysis of pfmdr 1 revealed an apparent association between increased gene copy number and increased level of expression of pfmdr 1 and decreased susceptibility to mefloquine and halofantrine. Sequence analysis of pfmdr 1 in these isolates revealed no association of intragenic alleles with chloroquine resistance.

Quinolone-3-Diarylethers: A New Class of Antimalarial Drug

ELQ-300, an investigational drug for treating and preventing malaria, shows potent transmission-blocking activity in rodent models of malaria. Taking the Bite Out of Malaria Malaria is spread from person to person by mosquitoes that inject 8 to 10 sporozoite forms of the parasite in a single bite. The sporozoites reproduce in the liver to produce 10,000 to 30,000 merozoites before the liver schizont ruptures and parasites flood into the bloodstream where the absolute parasite burden may increase to a thousand billion (1012) circulating parasites. Some of these parasites develop into gametocytes that may be ingested by another mosquito where they progress through ookinete, oocyst, and sporozoite stages to complete the cycle. Like quinine, most antimalarial drugs in use today target only the symptomatic blood stage. The efficacy of these drugs has been compromised by resistance, and so there is a pressing need for new drugs that target multiple stages of the parasite life cycle for use in malaria treatment and prevention. Clearly, it is advantageous to strike at the liver stage where parasite numbers are low, to diminish the likelihood of selecting for a resistant mutant and before the infection has a chance to weaken the defenses of the human host. In a new study, Nilsen and colleagues describe ELQ-300, a 4(1H)-quinolone-3-diarylether, which targets the liver and blood stages, including the forms that are crucial to disease transmission (gametocytes, zygotes, and ookinetes). In mouse models of malaria, a single oral dose of 0.03 mg/kg prevented sporozoite-induced infections, whereas four daily doses of 1 mg/kg achieved complete cures of patent infections. ELQ-300 is a preclinical candidate that may be coformulated with other antimalarials to prevent and treat malaria, with the potential to aid in eradication of the disease. The goal for developing new antimalarial drugs is to find a molecule that can target multiple stages of the parasite’s life cycle, thus impacting prevention, treatment, and transmission of the disease. The 4(1H)-quinolone-3-diarylethers are selective potent inhibitors of the parasite’s mitochondrial cytochrome bc1 complex. These compounds are highly active against the human malaria parasites Plasmodium falciparum and Plasmodium vivax. They target both the liver and blood stages of the parasite as well as the forms that are crucial for disease transmission, that is, the gametocytes, the zygote, the ookinete, and the oocyst. Selected as a preclinical candidate, ELQ-300 has good oral bioavailability at efficacious doses in mice, is metabolically stable, and is highly active in blocking transmission in rodent models of malaria. Given its predicted low dose in patients and its predicted long half-life, ELQ-300 has potential as a new drug for the treatment, prevention, and, ultimately, eradication of human malaria.

Genetic Diversity of Plasmodium falciparum Histidine-Rich Protein 2 (PfHRP2) and Its Effect on the Performance of PfHRP2-Based Rapid Diagnostic Tests

Rising costs of antimalarial agents are increasing the demand for accurate diagnosis of malaria. Rapid diagnostic tests (RDTs) offer great potential to improve the diagnosis of malaria, particularly in remote areas. Many RDTs are based on the detection of Plasmodium falciparum histidine-rich protein (PfHRP) 2, but reports from field tests have questioned their sensitivity and reliability. We hypothesize that the variability in the results of PfHRP2-based RDTs is related to the variability in the target antigen. We tested this hypothesis by examining the genetic diversity of PfHRP2, which includes numerous amino acid repeats, in 75 P. falciparum lines and isolates originating from 19 countries and testing a subset of parasites by use of 2 PfHRP2-based RDTs. We observed extensive diversity in PfHRP2 sequences, both within and between countries. Logistic regression analysis indicated that 2 types of repeats were predictive of RDT detection sensitivity (87.5% accuracy), with predictions suggesting that only 84% of P. falciparum parasites in the Asia-Pacific region are likely to be detected at densities < or = 250 parasites/microL. Our data also indicated that PfHRP3 may play a role in the performance of PfHRP2-based RDTs. These findings provide an alternative explanation for the variable sensitivity in field tests of malaria RDTs that is not due to the quality of the RDTs.

Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with pfcrt polymorphism in Papua New Guinea and South America

The mechanistic basis for chloroquine resistance (CQR) in Plasmodium falciparum recently has been linked to the polymorphic gene pfcrt. Alleles associated with CQR in natural parasite isolates harbor threonine (T), as opposed to lysine (K) at amino acid 76. P. falciparum CQR strains of African and Southeast Asian origin carry pfcrt alleles encoding an amino acid haplotype of CVIET (residues 72–76), whereas most South American CQR strains studied carry an allele encoding an SVMNT haplotype; chloroquine-sensitive strains from malarious regions around the world carry a CVMNK haplotype. Upon investigating the origin of pfcrt alleles in Papua New Guinean (PNG) P. falciparum we found either the chloroquine-sensitive-associated CVMNK or CQR-associated SVMNT haplotypes previously seen in Brazilian isolates. Remarkably we did not find the CVIET haplotype observed in CQR strains from Southeast Asian regions more proximal to PNG. Further we found a previously undescribed CQR phenotype to be associated with the SVMNT haplotype from PNG and South America. This CQR phenotype is significantly less responsive to verapamil chemosensitization compared with the effect associated with the CVIET haplotype. Consistent with this, we observed that verapamil treatment of P. falciparum isolates carrying pfcrt SVMNT is associated with an attenuated increase in digestive vacuole pH relative to CVIET pfcrt-carrying isolates. These data suggest a key role for pH-dependent changes in hematin receptor concentration in the P. falciparum CQR mechanism. Our findings also suggest that P. falciparum CQR has arisen through multiple evolutionary pathways associated with pfcrt K76T.

Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I

Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.

Randomised double-blind placebo-controlled trial of SPf66 malaria vaccine in children in northwestern Thailand

Summary Background Previous efficacy trials of SPf66 malaria vaccine have produced conflicting results in different populations. We report a randomised double-blind trial of the SPf66 vaccine conducted in Karen children aged 2–15 living in a malarious region of northwestern Thailand. Recombinant hepatitis B vaccine was used as a comparator. Methods The study had a power of 90% to detect an efficacy of 30%, defined as a reduction in the incidence of first cases of symptomatic falciparum malaria after three doses of vaccine. 1221 children received three immunisations and were eligible for the primary efficacy analysis. Intense active and passive case detection continued over 15 months of follow-up. Findings The SPf66 vaccine was well tolerated, although 26 children had mild or moderately severe local or systemic allergic reactions, compared with none in the comparator group. The vaccine was immunogenic; after three doses, 73% of recipients had seroconverted. There were no deaths due to malaria during the study. During the 15-month period of evaluation there were 379 first cases of symptomatic falciparum malaria (195 in the SPf66 recipients, 184 in the comparator group); an SPf66 efficacy of −9% (95% Cl -33 to 14, p=0·41). No significant differences between the two study groups in parasite density or any other measure of malaria-related morbidity were detected. Interpretation These findings are consistent with a recent study showing lack of efficacy of SPf66 among Gambian infants and differ from earlier positive reports from South America and evidence of borderline efficacy from Tanzania. We conclude that SPf66 does not protect against clinical falciparum malaria and that further efficacy trials are not warranted.

Artemisinin-Induced Dormancy in Plasmodium falciparum: Duration, Recovery Rates, and Implications in Treatment Failure

BACKGROUND Despite the remarkable activity of artemisinin and its derivatives, monotherapy with these agents has been associated with high rates of recrudescence. The temporary arrest of the growth of ring-stage parasites (dormancy) after exposure to artemisinin drugs provides a plausible explanation for this phenomenon. METHODS Ring-stage parasites of several Plasmodium falciparum lines were exposed to different doses of dihydroartemisinin (DHA) alone or in combination with mefloquine. For each regime, the proportion of recovering parasites was determined daily for 20 days. RESULTS Parasite development was abruptly arrested after a single exposure to DHA, with some parasites being dormant for up to 20 days. Approximately 50% of dormant parasites recovered to resume growth within the first 9 days. The overall proportion of parasites recovering was dose dependent, with recovery rates ranging from 0.044% to 1.313%. Repeated treatment with DHA or with DHA in combination with mefloquine led to a delay in recovery and an approximately 10-fold reduction in total recovery. Strains with different genetic backgrounds appeared to vary in their capacity to recover. CONCLUSIONS These results imply that artemisinin-induced arrest of growth occurs readily in laboratory-treated parasites and may be a key factor in P. falciparum malaria treatment failure.

Clinical features cannot predict a diagnosis of malaria or differentiate the infecting species in children living in an area of low transmission

The differentiation of malaria from other causes of fever in the absence of microscopy is notoriously difficult. Clinical predictors of malaria have been studied in an area of low and unstable transmission on the western border of Thailand. In 1527 children aged 2-15 years who were followed prospectively for 7 months, 82% (1254) had at least one febrile episode. Malaria caused 24% (301) of the first febrile episodes (Plasmodium falciparum 128, P. vivax 151, P. malariae 1, mixed infections with P. falciparum and P. vivax 21). Each malaria case was matched with the next child of similar age presenting to the dispensary with another cause of fever. Clinical symptoms or signs associated with a final diagnosis of malaria were: confirmed fever (> or = 38 degrees C) (odds ratio [OR] 1.6, 95% confidence interval [95% CI] 1.4-1.9), headache (OR 1.5, 95% CI 1.3-1.9), muscle and/or joint pain (OR 2.0, 95% CI 1.6-2.8), nausea (OR 1.7, 95% CI 1.4-2.3), clinical anaemia (OR 1.4, 95% CI 1.3-3.3), palpable spleen (OR 1.3, 95% CI 1.1-1.7), palpable liver (OR 1.4, 95% CI 1.1-2.1), absence of cough (OR 1.6, 95% CI 1.4-2.0), and absence of diarrhoea (OR 1.5, 95% CI 1.2-2.4). None of these signs alone or in combination proved a good predictor of malaria. The best diagnostic algorithms (history of fever and headache without cough, and history of fever with an oral temperature > or = 38 degrees C [sensitivity 51% for both, specificity 72 and 71%, respectively]) would result in prescription of antimalarial drugs in 28-29% of the non-malaria febrile episodes, and only 49% of the true malaria cases. Thus half of the potentially life-threatening P. falciparum infections would not be treated. Although multivariate analysis identified vomiting, confirmed fever, splenomegaly and hepatomegaly as independent risk factors for a diagnosis of falciparum malaria, use of these signs to differentiate falciparum from vivax malaria, and thus to determine antimalarial treatment, was insufficiently sensitive or specific. Malaria diagnosis should be confirmed by microscopical examination of a blood slide or the use of specific dipstick tests in areas of low transmission where highly drug-resistant P. falciparum coexists with P. vivax.