At each survey, a single blood sample was obtained by finger prick (approximately 0·3 mL) for thick and thin blood films, filter paper blood collection (Whatman 3, Maidstone, UK), Haemoglobin test (HemoCue photometer) and for a Rapid Diagnostic Tests (RDT; Orchid Biomedical Systems, Goa, India) for malaria.

Filter papers were air-dried and stored in plastic bags with silica desiccant (silica gel type III; Sigma, Dorset, UK) and stored at −20°C. Plasma was see more diluted 1 : 1 in 0·1% sodium azide in PBS (reaching a final concentration of 0·05%). Individuals were followed up for 6 months by passive case detection with those who experienced a clinical malaria attack (temperature >37·5°C with parasites at any density) treated according to national treatment guidelines. Parasites were detected using three methods; microscopy, RDT and PCR. For microscopy, 100 fields of a Giemsa stained thick blood film were examined during the surveys, and at

all occasions, when a clinical malaria episode was suspected, RDTs (RDT; Orchid Biomedical Systems) were used for immediate detection of infection in the field. For PCR, DNA was extracted from filter paper samples using the QIAamp DNA mini kit (QIAGEN, Hilden, Germany), parasite detection carried out by nested-PCR amplification of the small subunit ribosomal RNA (rRNA) gene [16]. Immunoglobulin G (IgG) antibodies Quizartinib in vivo were assayed by ELISA, as described previously [14, 17]. Recombinant P. falciparum apical membrane antigen (AMA-1 FVO, provided by Takafumi Tusboi, Ehime PJ34 HCl University, Japan), merozoite surface protein 119 (MSP-119 Wellcome allele,

provided by Patrick Corran, London School of Hygiene & Tropical Medicine with permission of Tony Holder), merozoite surface protein 2 (MSP-2, Dd2 allele provided by David Cavanagh, Institute of Immunology and Infection Research, Edinburgh, UK), circumsporozoite protein (CSP; NANP16 peptide, provided by Patrick Corran, London School of Hygiene & Tropical Medicine) and Anopheles gambiae salivary antigen (gSG6 provided by Bruno Arcà, Sapienza University, Rome, Italy) were coated onto ELISA plates overnight at 4°C at a concentration of 1.25 ug/mL for AMA1, 5 μg/mL for gSG6 and 0.5 μg/mL for all the other antigens. Plates were washed using PBS plus 0·05% Tween 20 (PBS/T) and blocked with 1% (w/v) skimmed milk powder (Marvel, UK) in PBS/T. Serum samples were added in duplicate to each plate at a serum dilution of 1 : 400 for CSP, 1 : 2000 for AMA-1, 1 : 1000 for MSP-2 and MSP-119, and 1 : 100 for gSG6 in 1% bovine serum albumin (BSA) in PBS/T. A positive control of pooled hyperimmune serum collected from adults resident in a malaria endemic area was included in duplicates on each plate in a 4-fold serial dilution from 1 : 50 to 1/51 200 (6 concentrations in total) to allow standardization of day-to-day and plate-to-plate variation.