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

Abstract

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.

Integrated xenosurveillance of Loa loa, Wuchereria bancrofti, Mansonella perstans and Plasmodium falciparum using mosquito carcasses and faeces: A pilot study in Cameroon

“…In addition, positive E/F samples were not associated with positive post-E/F mosquito carcasses. Taken together, these findings support the theory that, in incompetent vectors, ingested parasites undergo a rapid and complete expulsion in E/F, after which the parasite is no longer detectable in the carcass [ 18 ]. The reduced detection in mosquito carcasses processed 36 hours after collection highlights the critical importance to xenosurveillance activities of an appropriate sampling strategy and timely processing of samples.…”

“…Molecular processing of E/F samples was conducted at Smith College, Northampton, MA, USA, while processing of mosquito and fly carcasses was conducted at the Liverpool School of Tropical Medicine, UK. DNA was extracted from E/F samples using the methods previously described by Pilotte et al [ 18 ]. Briefly, a standard paper punch was used to take three punches from each collection card, and sample was eluted/recovered from these punches using the GenSolve DNA Recovery Kit (GenTegra).…”

“…Recent studies have also demonstrated the potential of screening mosquito excreta/feces (E/F) for the genetic material of pathogens, providing a nondestructive sampling approach with improved scope for pooling [13,14]. Xenosurveillance of mosquitoes and their E/F has shown strong potential for the community-level detection of multiple pathogens including blood-borne viruses [15,16], malaria parasites [17,18] and the filarial pathogens Wuchereria bancrofti, Brugia malayi, and Mansonella perstans [17,18]. As L. loa microfilariae have been detected in night blood of infected individuals, L. loa parasite DNA may also be taken up in sufficient quantities by night-biting mosquitoes to be detected through xenosurveillance methods [19,20].…”

A microfluidic platform for highly parallel bite by bite profiling of mosquito-borne pathogen transmission

“…In addition to requiring sample preparation of mosquitoes, these methods do not represent actual biting events thus providing limited information about the transmission dynamics such as the infectious viral load of a bite. More recently, alternative modes of sample collection exploiting e.g., sugar feeding or excreta have been proposed [15][16][17][18][19] . Although practical, these methods do not simulate biting events and typically cannot differentiate between pathogens in saliva versus the mosquito gut and as a consequence provide limited data regarding pathogen transmission.…”

Vectorchip: Microfluidic platform for highly parallel bite by bite profiling of mosquito-borne pathogen transmission

“…More recently, alternative modes of sample collection exploiting e.g. sugar feeding or excreta have been proposed [13][14][15][16][17], but similarly do not simulate biting events, cannot differentiate between pathogens in saliva vs gut, provide population level (i.e. pooled) 2 information with limited scope of improvement in resolution, and cannot quantify live viruses.…”