[20] suggests adhesion of sexual stages has some characteristics in common with, and others that differ from, asexual parasite adhesion. Further, the evidence that asexual and sexual stage parasites sequester in different tissues [20,21] suggests that distinct antigens exist on the surface of gametocyte-infected erythrocytes (gametocyte surface antigens, GSA). Such adhesins could conceiv- ably be either parasite-encoded molecules, altered host membrane components, or both. We reasoned that such antigens may elicit specific immune responses, independent of responses to asexual parasites, which may be capable of suppressing or killing gametocytes. No studies to date have demonstrated the presence of GSA. If the surface of gametocyte-infected erythrocytes do elicit immune responses, then a comparison of these to responses elicited by asexual parasites and to transmission-blocking antibod- ies would be of great interest. We present the results of experiments in which plasma collected from 200 Gambian children carrying microscopically confirmed gametocytes 7 to 14 days after treatment for uncomplicated P. falciparum malaria, were presented with live, cultured P. falciparum gametocytes. The degree of recognition of each test plasma was measured by flow cytometry. We demonstrate for the first time the existence of antibodies which specifically recognise the surface of gametocyte-infected erythrocytes, and explore associations with recognition of asexual parasites and gamete antigens Pfs 48/45 and Pfs 230. Methods and Materials Patients and plasma samples During the months of October to December 2000, 2001 & 2002, children under 10 years presenting as outpatients to Farafenni hospital, The Gambia, were recruited into clinical trials to study the effects of antimalarial treatment on P. falciparum transmission (N = 536, 500 and 497 in 2000, 2001 and 2002 respectively; refs 22–25). All participants or their parents/ guardians gave informed consent to participation in these studies as previously described. Children were treated with chloroquine (CQ), sulphadoxine-pyrimethamine (SP), CQ/SP in combination, CQ combined with artesunate (CQ/AS) or artemether-lumefan- trine (AL). Seventy, 72 and 27 children in each year respectively were identified as gametocyte carriers 7 days after treatment and donated venous blood for membrane-feeding of Anopheles mosqui- toes as previously described [23–26]. In 2002, an additional 35 patients, randomly selected from among those without gameto- cytes 7 days after treatment, donated blood for membrane-feeding of mosquitoes, giving a total of 204 experiments. Each blood sample was collected in the anti-coagulant citrate phosphate dextrose, and the plasma separated from cells by centrifugation. 200 plasma samples were available for the current analysis. Specific additional permission for the use of these plasma samples in the experiments described was obtained from the Joint Gambia Government/MRC Ethics Committee. Negative controls for flow cytometry experiments were seven individual sera from UK resident adults who had never been to a malaria endemic area, obtained from whole blood by removal of coagulated cellular material. Each of these donors provided written, informed consent under a protocol approved by the LSHTM Ethics Committee. A pool of these seven negative sera, and three other independent pools of sera from the same source of non-malaria exposed European donors were also used. The serum of an immune Dutch adult whose transmission blocking immunity had been previously assessed as high level using the standardised membrane feeding assay [27] was kindly provided by Teun Gametocyte Surface Antigens Bousema, St Radboud University Medical Centre, Nijmegen. This sample was collected more than 10 years prior to the study from an individual who gave verbal, but not written, informed consent to St Radboud UMC for his blood sample to be used for malaria research. Definition of transmission blocking from direct membrane-feeding assay (DMFA) In the DMFA, blood samples were centrifuged, plasma removed and the cells washed as described previously [23,26]. Cells, including gametocyte-infected erythrocytes, were then resus- pended either back in the autologous plasma (AP) sample itself or in control serum (CS) from European donors. This serum had been tested and shown to be permissive for gametocyte survival in in vitro cultures. The two parallel preparations of gametocytes were each presented to a cage of approximately 50 adult mosquitoes. Plasma from the Gambian children donating blood for DMFA experiments was classified as having transmission-blocking activity if either of the following two tests were satisfied: 1) 2) transmission occurred among mosquitoes fed with gameto- cytes in both AP and CS, and the ratio of arithmetic mean oocyst density was significantly higher in the CS cage than in the AP cage [23,24]. transmission occurred only among mosquitoes fed on gametocytes presented in CS, and i. ii. iii. the proportion of infected mosquitoes in the CS-fed cage was significantly greater than zero by Fisher’s exact test, or The proportion of infected mosquitoes in the CS-fed cage was not significantly greater than 0, but at least one mosquito was heavily infected (. = 4 oocysts) in CS and at least 13 uninfected mosquitoes from the AP-fed cage were dissected, or The proportion of infected mosquitoes in the CS-fed cage was not significantly greater than 0, but at least two mosquitoes were infected in CS and at least 13 uninfected mosquitoes from the AP-fed cage were dissected. Transmission-blocking immunity could not be evaluated in those DMFA experiments in which there were no infected mosquitoes in either cage. A heavy infection was defined as at least 4 oocysts (median of 536 positives among 5000 CS-fed mosquitoes 1998–2002). Thirteen mosquitoes is used as the cut-off because it encompasses the majority of our experiments. The prevalence of infection among 5000 AP-fed insects was 6.5%, and so on average 0.85 out of 13 AP-fed mosquitoes were infected. Parasite Culture and Purification Asexual blood stage parasites were cultured in human type O negative blood and RPMI 1640 (supplemented with 5.96 g l21 HEPES, 2 gL21 sodium bicarbonate, 50 mg l21 hypoxanthine, 3.96 g l21 glucose and 10% blood type AB serum) and incubated at 37uC in 3% CO2/1% O2/96% N2 according to established protocols [28]. Magnet-activated cell sorting (MACS; Miltenyi BioTec, Bergisch Gladbach, Germany) was used to synchronise asexual parasite cultures as follows: at ,10% mixed stage parasitaemia, the culture was washed three times in PBS supplemented with 2% bovine serum albumin (BSA). The culture was applied to the MACS apparatus using a CS Column mounted with a 21G flow resistor, and washed three times with PBS. Flow- through and washes were subsequently re-applied to the column once. Erythrocytes containing parasites in late developmental PLoS ONE | www.plosone.org 2 May 2008 | Volume 3 | Issue 5 | e2280