In vivo study
Dabola prefecture (114,000 inhabitants) has an average altitude of 1,200 m and seasonal perennial malaria, with increased transmission between June and October. Plasmodium falciparum is the predominant species. Dabola reported 15,221 clinical malaria cases in 2003, accounting for 30% of the total outpatient consultations (MOH, 2003).
The efficacy study was based on the last WHO recommendations for the assessment and monitoring of antimalarial drug efficacy for area of high transmission . Children aged 6 to 59 months with measured fever (axillary temperature ≥ 37.5°C) were screened from outpatient lines. Children with a P. falciparum mono-infection and asexual parasitaemia between 2,000 and 200,000/μL were eligible for inclusion to the study. Exclusion criteria were (i) signs of severity or severe malaria according to WHO criteria, which included severe anaemia defined by haemoglobin < 5 g/dl , (ii) history of allergic reactions to the study drugs, (iii) presence of a concomitant febrile condition with the potential to confound study outcome (e.g. ARI, measles, severe diarrhoea, etc.) and (iv) severe malnutrition .
Treatment and follow-up
Study regimens consisted of either sulphadoxine-pyrimethamine 1.25-mg/Kg stat (Fansidar®, Roche, France); or amodiaquine 30 mg/Kg base divided into three daily doses of 10 mg/Kg (Camoquin®, Parke-Davis, Senegal); each combined with artesunate at a daily dose of 4 mg/Kg on days 0, 1 and 2 (Arsumax®, Guilin Pharmaceutical Works, China). Drugs were crushed and mixed with water and sugar, given in a spoon or a syringe to small children. All doses were directly observed and repeated if vomiting occurred within 30 minutes. Patients were randomly allocated (blocks of 20), without concealment, to receive either AS/SP or AS/AQ.
On day 0, blood samples were taken for haemoglobin measurement, parasitaemia including gametocytaemia and possible genotypic analysis to distinguish recrudescence from re-infection in the event of parasite recurrence. After treatment (days 0, 1, and 2), children were re-assessed clinically and parasitologically on days 3, 7, 14, 21, and 28. Children, who were parasitaemic but asymptomatic during follow-up were asked to return to the clinic every three days. Gametocyte carriage was re-measured at day 28, and a second blood sample for PCR genotyping was collected in case of symptomatic recurrent parasitaemia occurring after day 9 (recurrences on or before day 9 were assumed to be recrudescence, i.e. true failures), or at the end of follow-up in the presence of parasitaemia without symptoms (MSF/Epicentre guidelines). Rescue therapy (quinine hydrochloride 10 mg/Kg/8 hourly for seven days) was administered to treatment failures, orally or parenterally according to the patient's clinical condition.
Children were withdrawn from the study in case of (i) vomiting any study dose twice, (ii) withdrawal of consent, (iii) onset of a serious febrile illness, (iv) intake of any drug with antimalarial properties, (v) missing any treatment dose, (vi) mixed species parasitaemia or (vii) any protocol violation. Patients who missed follow up visits and did not come on the successive day despite tracing were considered lost to follow up. Patients were classified as early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) or adequate clinical and parasitological response (ACPR) as per WHO definitions .
Capillary blood was obtained by fingerprick. Thick and thin films, prepared on the same slide, were stained with 10% Giemsa (pH 7.2) for 15 minutes. Asexual parasitaemia was quantified against 200 to 500 leukocytes, assuming a white blood cell count of 8000/μL . Presence of gametocytes was recorded through this same method. All slides were re-read and any discordance (positivity, species or density for parasitaemia below 2,000 or above 2,000,000/μL) was resolved by a third reader. External quality control on a random sample of 92 slides was carried out by an independent and experienced laboratory technician in Geneva and showed only two negative/positive discordances (2.2%), which did not change the outcome classification. Haemoglobin was measured using the Lovibond technique (Assistant Co., Sondheim Rhon, Germany). Anaemia was defined as haemoglobin (Hb) < 11 g/dl .
Blood samples for PCR analysis were collected on Isocode® kits (Schleicher & Schuell, Ecquevilly, France). Kits were air dried and stored in cool and dark boxes with desiccants. Genotypic analysis was performed at the Epicentre laboratory at Mbarara University (Uganda) according to a published method considering the three P. falciparum gene loci merozoite surface protein-1 (msp-1), merozoite surface protein-2 (msp-2), and glutamate rich protein (GLURP) . Cases in which pre- and post-treatment genotypes were identical were considered as recrudescence, i.e. failures; cases in which pre- and post-treatment genotypes were different were considered as re-infections; mixed genotypes were classified as failures.
As no reliable estimates of antimalarial efficacy were available, all our calculations were based on an estimated 50% failure rate and a desired precision of 10%. Allowing for a type-1 error of 0.05 and considering a 15% drop out rate, 110 patients per study group were needed.
Data entry and analysis
Records were entered in Epidata 3.0 (The Epidata association, Odense Denmark) and analysed on SPSS® 11.0 for Windows (SPSS Inc. Chicago, Illinois) and EpiInfo 6.04b (CDC, Atlanta; 1996). Data entry errors were checked individually on each record. Appropriate statistical tests (Chi2, ANOVA, Kruskal-Wallis were used to compare the two groups. The main outcome was day-28 failure rate, defined as the number of true failures (recurrences on or before day 9 + PCR-confirmed recrudescence occurring after day 9) over the total number of patients analysed. Losses to follow-up and withdrawn patients, as well as PCR-confirmed new infections and indeterminate PCR results were excluded from the analysis according to the last WHO guideline for assessment and monitoring of antimalarial efficacy .
Lainé refugee camp (22,000 Liberian refugees and average altitude 470 m) borders Ivory Cost and Liberia. Malaria transmission is perennial. A total of 10,541 malaria confirmed cases were recorded in 2004 (Figure 1).
Patients from the outpatient clinic of the Lainé refugee camp with measured fever (axillary temperature ≥ 37.5°C) were screened for falciparum malaria using the Paracheck-Pf® rapid diagnostic test. After microscopic confirmation, samples from 181 consecutive patients were collected on Isocode® kits and transported to the London School of Hygiene and Tropical Medicine (LSHTM, London, United Kingdom). After DNA extraction, the PCR-Sequence Specific Oligonucleotide Probing (PCR-SSOP) method was used for molecular genotyping of point mutations in the dihydrofolate reductase (dhfr) and dyhydropteroate synthetase (dhps) genes [9, 10]. This method involves the PCR amplification of coding regions of dhfr and dhps genes, which are fixed onto membranes and probed with SSOP designed to detect each of the single base-pair changes which code for substitutions at codons 51 (N to I), 59 (C to R), and 108 (S to N, or T) of dhfr and codons 436 (S to A, or F), 437 (A to G), and 540 (K to E) of dhps. Since point mutations occur in a number of different configurations, each conferring differing degrees of resistance, data were to summarized in two ways. Firstly, by showing the allelic haplotypes (combination of mutations which are in the same gene), which were found among the parasites in Lainé. These can be determined easily in infections where there is a single genotype because the blood stage form of the parasite is haploid but are difficult or impossible to resolve in mixed infections [9, 10]. The frequency of allelic haplotypes in the parasite population was calculated after excluding mixed infections. The second way in which were summariszd the data, was to calculate the proportion of all infections (mixed and non-mixed), which were found to contain all three dhfr mutations plus the dhps 437 and 540 mutations. This particular genotype has been shown to be associated with SP treatment failure in a number of East African countries: Kenya, Malawi and Uganda but has only been reported rarely in West Africa [11–13].