A scalable assessment of Plasmodium falciparum transmission in the standard membrane-feeding assay, using transgenic parasites expressing green fluorescent protein-luciferase.
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.
Department of Medical Microbiology, Radboud University Medical Center.
Will J R
WJ
Stone
MRC Centre for Outbreak Analysis and Modelling, Department of Infectious Disease Epidemiology, Imperial College London.
Thomas S
TS
Churcher
Department of Medical Microbiology, Radboud University Medical Center.
Wouter
W
Graumans
Department of Medical Microbiology, Radboud University Medical Center.
Geert-Jan
GJ
van Gemert
Department of Medical Microbiology, Radboud University Medical Center TropIQ Health Sciences, Nijmegen, the Netherlands.
Martijn W
MW
Vos
Department of Medical Microbiology, Radboud University Medical Center.
Kjerstin H W
KH
Lanke
Department of Medical Microbiology, Radboud University Medical Center.
Marga G
MG
van de Vegte-Bolmer
Department of Medical Microbiology, Radboud University Medical Center.
Rianne
R
Siebelink-Stoter
TropIQ Health Sciences, Nijmegen, the Netherlands.
Koen J
KJ
Dechering
Seattle Biomedical Research Institute.
Ashley M
AM
Vaughan
Seattle Biomedical Research Institute.
Nelly
N
Camargo
Seattle Biomedical Research Institute Department of Global Health, University of Washington, Seattle.
Stefan H I
SH
Kappe
Department of Medical Microbiology, Radboud University Medical Center TropIQ Health Sciences, Nijmegen, the Netherlands.
Robert W
RW
Sauerwein
Department of Medical Microbiology, Radboud University Medical Center Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, United Kingdom.
Teun
T
Bousema
D000818:Animals; D000852:Anopheles / Q000469:parasitology*; D005260:Female; D049452:Green Fluorescent Proteins; D006801:Humans; D008156:Luciferases; D008163:Luminescent Measurements; D016778:Malaria, Falciparum / Q000635:transmission*; D008853:Microscopy; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000502:physiology; D010963:Plasmodium falciparum / Q000235:genetics / Q000502:physiology*
D049452:Green Fluorescent Proteins; D008156:Luciferases
Anopheles; P. falciparum; SMFA; infectivity; luminescence; malaria; mosquito feeding assay; mosquitoes; oocysts; transmission-reducing activity