Will J R Stone1, Thomas S Churcher2, Wouter Graumans1, Geert-Jan van Gemert1, Martijn W Vos3, Kjerstin H W Lanke1, Marga G van de Vegte-Bolmer1, Rianne Siebelink-Stoter1, Koen J Dechering4, Ashley M Vaughan5, Nelly Camargo5, Stefan H I Kappe6, Robert W Sauerwein3, Teun Bousema7. 1. Department of Medical Microbiology, Radboud University Medical Center. 2. MRC Centre for Outbreak Analysis and Modelling, Department of Infectious Disease Epidemiology, Imperial College London. 3. Department of Medical Microbiology, Radboud University Medical Center TropIQ Health Sciences, Nijmegen, the Netherlands. 4. TropIQ Health Sciences, Nijmegen, the Netherlands. 5. Seattle Biomedical Research Institute. 6. Seattle Biomedical Research Institute Department of Global Health, University of Washington, Seattle. 7. Department of Medical Microbiology, Radboud University Medical Center Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, United Kingdom.
Abstract
BACKGROUND: The development of drugs and vaccines to reduce malaria transmission is an important part of eradication plans. The transmission-reducing activity (TRA) of these agents is currently determined in the standard membrane-feeding assay (SMFA), based on subjective microscopy-based readouts and with limitations in upscaling and throughput. METHODS: Using a Plasmodium falciparum strain expressing the firefly luciferase protein, we present a luminescence-based approach to SMFA evaluation that eliminates the requirement for mosquito dissections in favor of a simple approach in which whole mosquitoes are homogenized and examined directly for luciferase activity. RESULTS: Analysis of 6860 Anopheles stephensi mosquitoes across 68 experimental feeds shows that the luminescence assay was as sensitive as microscopy for infection detection. The mean luminescence intensity of individual and pooled mosquitoes accurately quantifies mean oocyst intensity and generates comparable TRA estimates. The luminescence assay presented here could increase SMFA throughput so that 10-30 experimental feeds could be evaluated in a single 96-well plate. CONCLUSIONS: This new method of assessing Plasmodium infection and transmission intensity could expedite the screening of novel drug compounds, vaccine candidates, and sera from malaria-exposed individuals for TRA. Luminescence-based estimates of oocyst intensity in individual mosquitoes should be interpreted with caution.
BACKGROUND: The development of drugs and vaccines to reduce malaria transmission is an important part of eradication plans. The transmission-reducing activity (TRA) of these agents is currently determined in the standard membrane-feeding assay (SMFA), based on subjective microscopy-based readouts and with limitations in upscaling and throughput. METHODS: Using a Plasmodium falciparum strain expressing the firefly luciferase protein, we present a luminescence-based approach to SMFA evaluation that eliminates the requirement for mosquito dissections in favor of a simple approach in which whole mosquitoes are homogenized and examined directly for luciferase activity. RESULTS: Analysis of 6860 Anopheles stephensi mosquitoes across 68 experimental feeds shows that the luminescence assay was as sensitive as microscopy for infection detection. The mean luminescence intensity of individual and pooled mosquitoes accurately quantifies mean oocyst intensity and generates comparable TRA estimates. The luminescence assay presented here could increase SMFA throughput so that 10-30 experimental feeds could be evaluated in a single 96-well plate. CONCLUSIONS: This new method of assessing Plasmodium infection and transmission intensity could expedite the screening of novel drug compounds, vaccine candidates, and sera from malaria-exposed individuals for TRA. Luminescence-based estimates of oocyst intensity in individual mosquitoes should be interpreted with caution.
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