Ali Alloueche

Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a randomised trial

BACKGROUND RTS,S/AS02 is a pre-erythrocytic malaria vaccine based on the circumsporozoite surface protein of Plasmodium falciparum fused to HBsAg, incorporating a new adjuvant (AS02). We did a randomised trial of the efficacy of RTS,S/AS02 against natural P. falciparum infection in semi-immune adult men in The Gambia. METHODS 306 men aged 18-45 years were randomly assigned three doses of either RTS,S/AS02 or rabies vaccine (control). Volunteers were given sulfadoxine/pyrimethamine 2 weeks before dose 3, and kept under surveillance throughout the malaria transmission season. Blood smears were collected once a week and whenever a volunteer developed symptoms compatible with malaria. The primary endpoint was time to first infection with P. falciparum. Analysis was per protocol. FINDINGS 250 men (131 in the RTS,S/AS02 group and 119 in the control group) received three doses of vaccine and were followed up for 15 weeks. RTS,S/AS02 was safe and well tolerated. P. falciparum infections occurred significantly earlier in the control group than the RTS,S/AS02 group (Wilcoxons test p=0.018). Vaccine efficacy, adjusted for confounders, was 34% (95% CI 8.0-53, p=0.014). Protection seemed to wane: estimated efficacy during the first 9 weeks of follow-up was 71% (46-85), but decreased to 0% (-52 to 34) in the last 6 weeks. Vaccination induced strong antibody responses to circumsporozoite protein and strong T-cell responses. Protection was not limited to the NF54 parasite genotype from which the vaccine was derived. 158 men received a fourth dose the next year and were followed up for 9 weeks; during this time, vaccine efficacy was 47% (4-71, p=0.037). INTERPRETATION RTS,S/AS02 is safe, immunogenic, and is the first pre-erythrocytic vaccine to show significant protection against natural P. falciparum infection.

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.

Comparison of chlorproguanil-dapsone with sulfadoxine-pyrimethamine for the treatment of uncomplicated falciparum malaria in young African children: double-blind randomised controlled trial

BACKGROUND Increasing resistance to sulfadoxine-pyrimethamine is leading to a decline in its effectiveness. We aimed to assess the safety profile of chlorproguanil-dapsone (CD), and to compare the safety and efficacy of this drug with that of sulfadoxine-pyrimethamine (SP) as treatment for uncomplicated falciparum malaria. METHODS We undertook a double-blind, randomised trial in 1850 consecutively recruited children with uncomplicated falciparum malaria, pooling data from five African countries. Analyses were based on all randomised patients with available data. FINDINGS CD was significantly more efficacious than SP (odds ratio 3.1 [95% CI 2.0-4.8]); 1313 patients (96%) given CD and 306 (89%) given SP achieved acceptable clinical and parasitological response by day 14. Adverse events were reported in 46% and 50% of patients randomised to CD and SP, respectively (treatment difference -4.4%, [95% CI -10.1 to 1.3]). Haemoglobin in the CD group was significantly lower than in the SP group at day 7, a difference of -4 g/L (95% CI -6 to -2). Mean day 14 haemoglobin (measured only for the small number of patients whose day 7 data caused concern) was 94 g/L (92-96) and 97 g/L (92-102) after CD and SP, respectively. Glucose-6-phosphate dehydrogenase deficient patients on CD had greater odds than those on SP of having a fall of 20 g/dL or more in haemoglobin when baseline temperature was high. Methaemoglobinaemia was seen in the CD group (n=320, mean 0.4% [95% CI 0.4-0.4]) before treatment, 4.2% (95% CI 3.8-4.6) (n=301) at day 3, and 0.6% (0.6-0.7) (n=300) at day 7). INTERPRETATION CD had greater efficacy than SP in Africa and was well tolerated. Haematological adverse effects were more common with CD than with SP and were reversible. CD is a useful alternative where SP is failing due to resistance.

Combination Therapy Counteracts the Enhanced Transmission of Drug-Resistant Malaria Parasites to Mosquitoes

ABSTRACT Malaria parasites carrying genes conferring resistance to antimalarials are thought to have a selective advantage which leads to higher rates of transmissibility from the drug-treated host. This is a likely mechanism for the increasing prevalence of parasites with resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine in sub-Saharan Africa. Combination therapy is the key strategy being implemented to reduce the impact of resistance, but its effect on the transmission of genetically resistant parasites from treated patients to mosquito vectors has not been measured directly. In a trial comparing CQ monotherapy to the combination CQ plus artesunate (AS) in Gambian children with uncomplicated falciparum malaria, we measured transmissibility by feeding Anopheles gambiae mosquitoes with blood from 43 gametocyte-positive patients through a membrane. In the CQ-treated group, gametocytes from patients carrying parasites with the CQ resistance-associated allele pfcrt-76T prior to treatment produced infected mosquitoes with 38 times higher Plasmodium falciparum oocyst burdens than mosquitoes fed on gametocytes from patients infected with sensitive parasites (P < 0.001). Gametocytes from parasites carrying the resistance-associated allele pfmdr1-86Y produced 14-fold higher oocyst burdens than gametocytes from patients infected with sensitive parasites (P = 0.011). However, parasites carrying either of these resistance-associated alleles pretreatment were not associated with higher mosquito oocyst burdens in the CQ-AS-treated group. Thus, combination therapy overcomes the transmission advantage enjoyed by drug-resistant parasites.

Chloroquine/Sulphadoxine-Pyrimethamine for Gambian Children with Malaria: Transmission to Mosquitoes of Multidrug-Resistant Plasmodium falciparum

Objectives: In the Gambia, chloroquine (CQ) plus sulphadoxine-pyrimethamine (SP) is the first-line antimalarial treatment. Plasmodium falciparum parasites carrying mutations associated with resistance to each of these drugs were present in 2001 but did not cause a significant loss of therapeutic efficacy among children receiving the combination CQ/SP. We measured their effect on parasite transmission to Anopheles gambiae mosquitoes. Design: We conducted a single-blind, randomised, controlled trial with follow-up over 28 d. Mosquito feeding experiments were carried out 7, 10, or 14 d after treatment. Setting: The study took place in the town of Farafenni and surrounding villages in the Gambia. Participants: Participants were 500 children aged 6 mo to 10 y with uncomplicated P. falciparum malaria. Interventions: Children were randomised to receive CQ, SP, or CQ/SP. Outcome Measures: Outcomes related to transmission were determined, including posttreatment gametocyte prevalence and density. Infectiousness was assessed by membrane-feeding A. gambiae mosquitoes with blood from 70 gametocyte-positive patients. Mutations at seven loci in four genes associated with drug resistance were measured pre- and posttreatment and in the midguts of infected mosquitoes. Results: After SP treatment, the infectiousness of gametocytes was delayed, compared to the other two treatment groups, despite comparable gametocyte densities. Among bloodmeal gametocytes and the midguts of infected mosquitoes, the presence of the four-locus multidrug-resistant haplotype TYRG (consisting of mutations pfcrt-76T, pfmdr1-86Y, pfdhfr-59R, and pfdhps-437G) was associated with significantly higher oocyst burdens after treatment with the combination CQ/SP. Conclusions: Parasites with a multidrug-resistant genotype had a substantial transmission advantage after CQ/SP treatment but did not have a significant impact on in vivo efficacy of this drug combination. Protocols that include measuring transmission endpoints as well as therapeutic outcomes may be a useful strategy when monitoring the evolution of drug resistance in malaria parasites in vivo.

High-throughput sequence typing of T-cell epitope polymorphisms in Plasmodium falciparum circumsporozoite protein

We report a method for typing polymorphisms at the T-cell epitopes within the Th2R and Th3R regions of the Plasmodium falciparum circumsporozoite protein (CSP). This method combines the use of PCR and sequence specific oligonucleotide probes (PCR-SSOP), and allows the identification of single nucleotide polymorphisms in these epitope regions. PCR-SSOP is a robust and a high-throughput sequence typing technique which has the same specificity and fidelity as direct sequencing. This method has been developed specifically for the assessment of the protective efficacy of RTS,S/SBAS2 vaccine against the 3D7 strain of P. falciparum (RTS,S/SBAS2 vaccine contains a part of the 3D7 CSP protein) in a phase IIb trial in Gambia which has been completed recently. PCR-SSOP could be used to determine the allelic frequencies of other parasite antigens and their geographical distribution.

A randomized safety and tolerability trial of artesunate plus sulfadoxine--pyrimethamine versus sulfadoxine-pyrimethamine alone for the treatment of uncomplicated malaria in Gambian children.

Artemisinin derivatives, such as artesunate, have a short half-life and very rapid anti-malarial activity. Theoretically, using such agents in conjunction with well-established anti-malarial drugs such as sulfadoxine-pyrimethamine may reduce the rate of drug resistance. Such a combination has not previously been used in Africa. We have conducted a pilot safety trial of artesunate (4 mg/kg for 3 days) given with a single dose of sulfadoxine-pyrimethamine (25 mg/kg sulfadoxine) compared to sulfadoxine-pyrimethamine alone among 40 Gambian children with uncomplicated malaria. Both regimens were safe and well tolerated and there were no adverse experiences attributed to the combination. The addition of artesunate resulted in a higher proportion of afebrile children and children with a negative blood film on Day 2, and a reduction in the proportion of gametocyte carriers, when compared to sulfadoxine-pyrimethamine alone.

Mutations in dhfr in Plasmodium falciparum Infections Selected by Chlorproguanil-Dapsone Treatment

Treatment with the novel antifolate drug combination chlorproguanil-dapsone effectively cleared asymptomatic Plasmodium falciparum infections in 246 (93.5%) of 263 children in the Usambara Mountains of Tanzania during the course of a 2-week follow-up. Samples from 71 recurrent infections, collected over a 9-week follow-up, showed selection for parasites with the triple mutant Ile(51)-Arg(59)-Asn(108) in dihydrofolate reductase. There was no selection for mutations in dihydropteroate synthetase, the target enzyme of dapsone. Search for complete identity in the highly polymorphic genes coding for merozoite surface proteins 1 and 2 in parasite samples collected before and after treatment indicated that the majority of recurrent parasitemias were new infections. These observations on selection in Tanzania and the lack of selection reported from a less endemic area suggest that the active metabolite of chlorproguanil, which has a short half-life in the blood, may persist in the liver, where it exerts selective pressure on growing preerythrocytic stages.

A randomised, double-blind, placebo-controlled trial of atovaquone-proguanil vs. sulphadoxine-pyrimethamine in the treatment of malarial anaemia in Zambian children.

Objective  To compare the efficacy of atovaquone–proguanil (AP) and sulphadoxine–pyrimethamine (SP) in the treatment of malarial anaemia in Zambian children.

Rare, highly pyrimethamine-resistant alleles of the Plasmodium falciparum dihydrofolate reductase gene from 5 African sites

In eastern and southern Africa, there has been a rapid increase in the prevalence of alleles with mutations in the Plasmodium falciparum dihydrofolate reductase gene (dhfr) associated with increased risk of clinical failure of sulfadoxine-pyrimethamine (S/P). Molecular methods for surveillance of these mutations are now widespread, but the usual analysis detects only the most prevalent allele in a polyclonal sample. We used a yeast-expression system to identify rare, highly pyrimethamine-resistant alleles of dhfr in isolates from 5 African countries--Kenya, Tanzania, Malawi, Gabon, and Nigeria. Only the isolates from Nigeria yielded significant numbers of novel resistant alleles, and only 1 of the alleles from any location showed a >3-fold increase in resistance to S/P or to chlorproguanil-dapsone. Overall, these results suggest that dhfr alleles that confer high levels of resistance to antifolates are rare, even in eastern and southern Africa, where pyrimethamine has been intensively used.