Figure 3. Recognition profiles of plasma IgG from 202 Gambian children. Erythrocytes harbouring P. falciparum clone 3D7a stage V gametocytes (left column) and asexual parasite stages (right) were tested with each of 194 plasma (rows). Plasma are arranged in increasing order of the proportion of gametocyte recognition events in the right upper quadrant of the flow cytometry dot-blot. Positive antibody recognition is scored as dark grey. Pale fill indicates that antibodies could not be detected above the level of controls (see text). doi:10.1371/journal.pone.0002280.g003 inside intact host erythrocytes, and no appreciable gamete release had occurred during these experiments. Fifty-three (26.4%) of the 200 plasma successfully tested recognised the surface of erythrocytes infected with asexual parasites. Eleven test sera recognized both GSA and the surface of asexual-infected erythrocytes, but there was no association between these two reactivities. The relationship between recogni- tion of sexual and asexual parasites is shown for each plasma in Figure 3 and Table 1. This suggests these two parasite life cycle stages place distinct antigen sets on the host erythrocyte surface. Further, anti-GSA antibodies were more common among older children (mean age difference 0.621 years, 95% C.I. 0.041– 1.283 years; P = 0.066), but this was not true of antibodies against the 3D7 asexual parasite surface (mean age difference 0.349 years, 95% C.I. 20.516–1.22 years; P = 0.787). Surprisingly, day 7, 10 or 14 plasma antibodies that recognised GSA were less common among children that had harboured Gametocyte Surface Antigens gametocytes prior to treatment, although this was not statistically significant (Table 1). Children with plasma antibodies that recognised GSA in the flow cytometry assay were no more likely to exhibit transmission-blocking activity in the DMFA than other children. However, transmission blocking activity was significantly more common among children with antibodies recognising asexual parasites (Table 1), suggesting this may be related to either the longevity of infection, or the parasite load, but we did not have sufficient data from the DMFA to distinguish these possibilities. Mild anaemia, which is an indicator of a malaria infection of long duration, was unrelated to the presence of antibodies recognising either GSA or the surface of asexual parasites (data not shown). To further test whether the development of anti-GSA and anti- asexual antibodies depended on duration of infection, we compared the prevalence of antibodies in plasma taken at day 7 (N = 176) and at days 10 or 14 (N = 21). GSA antibodies were found in 11 of 20 samples from day 10 or 14 that were tested (55.0%) and only among 55 of 171 samples taken at day 7 (32.2%; O.R. 2.58, 95% C.I. 0.907–7.45; P = 0.042). Antibodies recognising asexual parasites did not occur in any of the plasma samples taken at day 10 or 14, but were present in 31 of 174 samples taken at day 7 (17.8%). Thus antibody reactivities to GSA, but not the surface of 3D7 asexual parasites, were more common in plasma taken 10 days or more after treatment than they were in day 7 plasma. Antibodies to GSA and recognition of gamete antigens Pfs48/45 and Pfs 230 Gamete antigen ELISA assays were conducted for each of 65 plasma collected in 2000. The relative Pfs48/45 and Pfs230 recognition score of each was tested for association with transmis- sion-blocking immunity as measured by DMFA, and with the ability to recognise both GSA and the surface of asexual parasites. Results of these analyses are presented in Table 2. Plasma samples with antibodies that recognised gametocytes, and those that recognised asexual parasites exhibited significantly stronger recognition of Pfs230 (but not Pfs48/45) in ELISA. The same was true of plasma with demonstrable transmission-blocking activity. Antibodies to GSA and persisting carriage of gametocytes To explore the possibility that antibodies detected in the day 7 plasma of children carrying gametocytes may have affected gametocyte survival in those hosts, we examined subsequent gametocyte carriage in children with and without plasma antibodies to GSA. Children carrying anti-GSA plasma antibodies at the day blood was donated for DMFA were found to have a lower mean gametocyte density at day 28, but this was affected by the treatment received. Among children followed up to day 28 and receiving non-artemisinin treatments (CQ, SP or CQ/SP; N = 99), Table 1. Associations between recognition of asexual- and sexual-stage parasites in flow cytometry, carriage of gametocytes at presentation and transmission-blocking immunity on plasma from 200 treated Gambian children. Odds Ratio, (95% C.I.) Antibodies to Asexual stages Gametocyte carriage at presentation Transmission blocking immunity * Antibodies to Mature gametocytes Stage V Antibodies to Mature Asexual Stages 1.14, (0.454–2.73) P = 0.756 0.531, (0.225–1.18) P = 0.098 1.59, (0.398–5.89) P = 0.435 , 1.38, (0.513–3.44) P = 0.461 5.85, (1.28–27.4) P = 0.010 *Only data from feeding experiments with at least one positive mosquito are included (N = 71). doi:10.1371/journal.pone.0002280.t001 PLoS ONE | www.plosone.org 5 May 2008 | Volume 3 | Issue 5 | e2280