Effect of seasonal malaria chemoprevention plus azithromycin on Plasmodium falciparum transmission: gametocyte infectivity and mosquito fitness.

BACKGROUND: Seasonal malaria chemoprevention (SMC) consists of administration of sulfadoxine-pyrimethamine (SP) + amodiaquine (AQ) at monthly intervals to children during the malaria transmission period. Whether the addition of azithromycin (AZ) to SMC could potentiate the benefit of the intervention was tested through a double-blind, randomized, placebo-controlled trial. The effect of SMC and the addition of AZ, on malaria transmission and on the life history traits of Anopheles gambiae mosquitoes have been investigated. METHODS: The study included 438 children randomly selected from among participants in the SMC + AZ trial and 198 children from the same area who did not receive chemoprevention. For each participant in the SMC + AZ trial, blood was collected 14 to 21 days post treatment, examined for the presence of malaria sexual and asexual stages and provided as a blood meal to An. gambiae females using a direct membrane-feeding assay. RESULTS: The SMC treatment, with or without AZ, significantly reduced the prevalence of asexual Plasmodium falciparum (LRT X22 = 69, P < 0.0001) and the gametocyte prevalence (LRT X22 = 54, P < 0.0001). In addition, the proportion of infectious feeds (LRT X22 = 61, P < 0.0001) and the prevalence of oocysts among exposed mosquitoes (LRT X22 = 22.8, P < 0.001) was reduced when mosquitoes were fed on blood from treated children compared to untreated controls. The addition of AZ to SPAQ was associated with an increased proportion of infectious feeds (LRT X21 = 5.2, P = 0.02), suggesting a significant effect of AZ on gametocyte infectivity. There was a slight negative effect of SPAQ and SPAQ + AZ on mosquito survival compared to mosquitoes fed with blood from control children (LRTX22 = 330, P < 0.0001). CONCLUSION: This study demonstrates that SMC may contribute to a reduction in human to mosquito transmission of P. falciparum, and the reduced mosquito longevity observed for females fed on treated blood may increase the benefit of this intervention in control of malaria. The addition of AZ to SPAQ in SMC appeared to enhance the infectivity of gametocytes providing further evidence that this combination is not an appropriate intervention

Additional information on <i>P</i>. <i>falciparum</i> isolates.

MSP1-genotyping results for P. falciparum isolates (p1-p6) used in infections with An. gambiae (An. gam), An. coluzzii (An. col), and/or VK5-collected An. coluzzii (VK5-An. col) (See also Methods). (XLSX)</p

<i>P</i>. <i>falciparum</i> oocyst growth is negatively linked to egg development.

(A) In An. coluzzii controls (dsGFP-injected), egg numbers are negatively associated with oocyst size, but this association is lost following dsEcR treatment (GLMM, LRT). (B) In An. gambiae, egg numbers are negatively associated with oocyst size in both control and dsEcR conditions (GLMM, LRT), but (C) this association differentially varies across oocyst density in control and dsEcR females (3-way interaction, treatment*egg#*oocyst#, GLMM, LRT, X21 = 8.57, p = 0.003). Lines across egg numbers and oocyst size graphically represent the model-based analysis that was performed, which used nested individual oocyst measurements. Shading shows 95% confidence interval. N = sample size, or number of mosquitoes. Number of individual oocyst measurements including in analysis were: An. coluzzii controls = 926, An. coluzzii dsEcR = 1003, An. gambiae controls = 669, An. gambiae dsEcR = 760.</p

Additional infection data for colony mosquitoes and VK5-<i>An</i>. <i>coluzzii</i>.

(A) The majority of VK5-An. coluzzii females that were provided a parasite-infected (p1-p6) blood meal, failed to develop any eggs. (B) Oocyst prevalence (P) and intensity for individual infections with VK5-An. coluzzii. (C) Mean oocyst size per VK5-An. coluzzii female for each infection with a different parasite isolate (P#). Mean oocyst sizes are shown for simplicity, but all analyses were done with all individual oocyst measurements nested by mosquito. N = sample size. P# = parasite isolate. (TIF)</p

Gametocytemia and COI for <i>P</i>. <i>falciparum</i> field isolates.

Gametocytemia and COI for P. falciparum field isolates.</p

Full-factor GLMM Output for VK5 <i>An</i>. <i>coluzzii</i>.

Effect of egg development (eggs vs no eggs), oocyst number, and their interactions on oocyst size. In this model, egg development and oocyst number were considered as fixed effects whereas parasite isolate, mosquito generation, and mosquito ID were set as random effects. Significant effects are in bold. (XLSX)</p

Egg development for individual infections.

(A-B) The effects of EcR-silencing on egg development across individual infections for (A) An. coluzzii (unpaired t-test and Mann-Whitney) and (B) An. gambiae (unpaired t-test and Mann-Whitney) compared to controls (Cntrl). N = sample size. p# = parasite isolate. (TIF)</p

Full-factor GLMM Output for <i>An</i>. <i>coluzzii</i>.

Effect of treatment (dsGFP vs dsEcR), oocyst number, egg number, and their interactions on oocyst size. In this model, treatment, oocyst number and egg number were considered as fixed effects whereas parasite isolate and mosquito ID were set as random effects. Significant effects are in bold. (XLSX)</p

Full-factor GLMM Output for <i>An</i>. <i>gambiae</i>.

Effect of treatment (dsGFP vs dsEcR), oocyst number, egg number, and their interactions on oocyst size. In this model, treatment, oocyst number and egg number were considered as fixed effects whereas parasite isolate and mosquito ID were set as random effects. Significant effects are in bold. (XLSX)</p

Oocyst prevalence and intensity for individual infections.

(A-B) The effects of EcR-silencing on the prevalence (Fisher’s Exact) and intensity (unpaired t-test and Mann-Whitney) of oocysts across individual infections for (A) An. coluzzii and (B) An. gambiae, compared to dsGFP-injected (Cntrl) females. P = oocyst prevalence. N = sample size. p# = parasite isolate. (TIF)</p