Study area and samples
Blood samples positive with P. falciparum were collected from patients and asymptomatic individuals during two separate studies from 2005 to 2007, initiated by the IPTi consortium  and carried out at the Medical Research Unit of the Albert Schweitzer Hospital in Lambaréné, Gabon. In this study area of 30,000 inhabitants, malaria transmission is perennial, with little seasonal variation [20, 21].
One study was an IPTi trial (ClinicalTrials.gov identifier: NCT00167843) reported elsewhere [21, 22] and included into a meta-analysis of IPTi-SP trials across Africa .
The second study as reported here was an in vivo therapeutic efficacy trial of SP in children aged 6-59 months with uncomplicated falciparum malaria (ClinicalTrials.gov identifier: NCT00453856). Both studies were approved by the ethics committee of the International Foundation of the Albert Schweitzer Hospital.
Both studies included children from Lambaréné and its vicinity. Informed consent was obtained from parents or legal representative for each subject prior to enrolment. Children with acute febrile disease were physically examined; a thick blood film was screened for malaria parasites and a finger-prick blood sample for filter paper blood spotting (FTA Classic Card, Whatman Inc., Sanford, ME, USA) was obtained. The filter paper blood spots were air dried and stored at 4°C in individual plastic bags with desiccant and genotyping analysis later performed at the London School of Hygiene and Tropical Medicine (London, UK).
SP treatment in vivo study-specific procedures
The target sample size of 139 children was determined from a therapeutic efficacy between 70 and 95%, with a precision of 7.5% allowing for a 20% drop-out. The study end points were adequate clinical and parasitological response, late clinical failure, late parasitological failure and early treatment failure as defined by the World Health Organization . These were calculated in the per-protocol population, while safety and tolerance were evaluated in the intention-to-treat population, all children who received single-dose SP.
Study physicians examined all eligible children at enrolment, recording blood pressure, pulse and axillary temperature. Laboratory data recorded were parasite density of asexual and sexual forms of malaria, haemoglobin, haematocrit, white blood cell count, thrombocyte count (ABX Pentra 60®, ABX Diagnostics, Montpellier, France), creatinine and alanine- aminotransferase (ABX Mira Plus®, ABX Diagnostics).
A single dose of SP (25 mg/kg and 1.25 mg/kg; Maneesh Pharmaceuticals PVT Ltd, Govandi Mumbai, India) was crushed and mixed with glucose solution and administered orally by a study clinician. A patient was withdrawn from the trial if a re-dose was vomited and the outcome was early treatment failure, late clinical failure or late parasitological failure according to WHO definitions . These patients were subsequently treated with oral artemether-lumefantrine (COARTEM®, Novartis Pharma Ltd Beijing for Novartis Pharma AG, Basle Switzerland) or hospitalized, if oral treatment was not tolerated. Scheduled follow up visits of all study subjects were on days 1, 2, 3, 7, 14, 21 and 28 after oral administration of SP (day 0).
DNA extraction and PCR amplification of dhfr and dhps genes
DNA was extracted from bloodspots dried on filter papers by soaking overnight in 1 mL of 0.5% saponin-1x phosphate buffered saline. The segment was then washed twice in 1 mL of PBS and boiled for 8 min in 100 μL PCR quality water with 50 μL 20% Chelex suspension (pH 9.5).
Dhfr and dhps were PCR amplified using a nested PCR. The outer and nested dhfr/dhps PCR conditions, including primer sequences and reaction parameters, were as previously described . The nested PCR products were confirmed by electrophoresis on a 1% agarose gel along with a set of controls.
Molecular genotyping of point mutations using SSOP
Point mutations at codons 51, 59, 108 and 164 of the dhfr gene and codons 436, 437, 540, 581, and 613 of the dhps gene were genotyped using the Sequence Specific Oligonucleotide Probes (SSOP), a dot-blot methodology previously described by Pearce and colleagues . The probed blots were visualized through alkaline phosphatase-catalyzed breakdown of the fluorogenic substrate (ECF) (GE Healthcare, Buckinghamshire, UK) and the chemifluorescent signal scanned on a TYPHOON Trio® Phosphoimager (GE Healthcare, Buckinghamshire, UK).
The stringency and specificity of the hybridization process was confirmed by inspection of a series of four controls of known single genotype variant sequence. All blots with non-specifically bound probes were stripped and re-probed. A sequence variant was considered to be present in the PCR product when the intensity of signal was higher than that of the background. The presence, absence, and relative abundance of hybridization signal were recorded for every probe at each locus. Blood samples were categorized as having a single, a majority plus a minority, or a mixture of sequences at every locus. A sample was considered to have a single haplotype when only one sequence variant was found at each locus. Majority and mixed genotype infections were differentiated according to the relative intensity of signal.
Molecular treatment outcome measures
Each P. falciparum infection was characterized on the basis of the MSP-2 polymorphism . Allele-specific PCR to amplify FC27 or IC1/3D7 fragments was performed on paired pre- and post-treatment bloodspot samples. 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. Parasite clearance time was defined as the time from starting SP treatment until parasites were undetectable in two consecutive peripheral blood films at least 24 hrs apart. An experienced laboratory technician measured asexual parasitaemia per μL according to the Lambaréné method . The presence of single or multiple dhfr or dhps mutations from samples collected prior to treatment with SP were examined for their association with patients' treatment outcome. Each isolate was coded based on the presence or absence of a resistance associated allele. For example, infections with mixed wild-type/mutant alleles were treated as mutant.
Transmission potential was evaluated by measuring gametocytaemia on enrolment and at scheduled visits following the same technique used for asexual parasitaemia.
The study analysis consisted on calculating the proportion of malaria infections with mutations of interest present in samples from the IPTi plus those present at the time of enrolment in the SP in vivo study and estimated 95 percent binomial confidence intervals for prevalence of mutations in the study area.
For the SP treatment in vivo study, data were entered into an electronic database and validated by complete manual review. Statistical analysis was performed using Stata (Stat Corp., College Station, TX, USA) statistical software. Fever clearance time was calculated as the time from the start of treatment to the first of two consecutive axillary temperature measurements that recorded below 37.5°C and parasite elimination time as the time from the start of treatment to that of two consecutive negative blood smears.