Laboratory evaluation of molecular xenomonitoring using mosquito and tsetse fly excreta/feces to amplify Plasmodium, Brugia, and Trypanosoma DNA

Background:   Results from an increasing number of studies suggest that mosquito excreta/feces (E/F) testing has considerable potential to serve as a supplement for traditional molecular xenomonitoring techniques. However, as the catalogue of possible use-cases for this methodology expands, and the list of amenable pathogens grows, a number of fundamental methods-based questions remain. Answering these questions is critical to maximizing the utility of this approach and to facilitating its successful implementation as an effective tool for molecular xenomonitoring. Methods: :  Utilizing E/F produced by mosquitoes or tsetse flies experimentally exposed to Brugia malayi , Plasmodium falciparum , or Trypanosoma brucei brucei , factors such as limits of detection, throughput of testing, adaptability to use with competent and incompetent vector species, and effects of additional blood feedings post parasite-exposure were evaluated.  Two platforms for the detection of pathogen signal (quantitative real-time PCR and digital PCR (dPCR)) were also compared, with strengths and weaknesses examined for each.       Results: :  Experimental results indicated that high throughput testing is possible when evaluating mosquito E/F for the presence of either B. malayi or P. falciparum from both competent and incompetent vector mosquito species.  Furthermore, following exposure to pathogen, providing mosquitoes with a second, uninfected bloodmeal did not expand the temporal window for E/F collection during which pathogen detection was possible.  However, this collection window did appear longer in E/F collected from tsetse flies following exposure to T. b. brucei .  Testing also suggested that dPCR may facilitate detection through its increased sensitivity.  Unfortunately, logistical obstacles will likely make the large-scale use of dPCR impractical for this purpose. Conclusions: :  By examining many E/F testing variables, expansion of this technology to a field-ready platform has become increasingly feasible.  However, translation of this methodology from the lab to the field will first require field-based pilot studies aimed at assessing the efficacy of E/F screening.