Laboratory evaluation of molecular xenomonitoring using mosquito 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 the completion of field-based pilot studies aimed at assessing the efficacy of E/F screening.

Field evaluation of DNA detection of human filarial and malaria parasites using mosquito excreta/feces

“…Interestingly, we also detected Mansonella DNA in the excreta of female mosquitoes collected using gravid traps. Based on the experimental evidence previously collected [38], the reliable detection of parasite signal during E/F surveillance from non-vector mosquitoes is likely limited to the 72 hours following blood-feeding.…”

“…We extracted the DNA from mosquito E/F in the 1.5 ml collection tubes using the QIAamp DNA Micro Kit (QIAGEN, Germantown, Maryland) following a modified version of the manufacturer's protocol as described elsewhere [38]. To test the possibility of detecting the presence of W. bancrofti or P. falciparum developing or infective stage, mosquito carcasses were separated into two separate aliquots, heads and thoraces (H&T) and abdomens (Abd), which were then pooled in groups from up to five mosquitoes.…”

“…A sample was considered positive either if it allowed for amplification in both the initial reaction replicates or if it was positive in at least one replicate reaction upon retesting. As described elsewhere [38], highly sensitive digital PCR using the QuantStudio 3D Digital PCR instrument was used to retest E/F samples that were inconsistently positive upon initial testing, given the very small residual volumes available for retesting from these samples and the potential to improve detection when very low concentrations of target are present. Due to the prohibitive cost of digital PCR, qPCR was instead used to retest inconsistently positive blood and mosquito carcasses samples, as DNA extraction from these sample types produces considerably greater sample volumes.…”

Field evaluation of DNA amplification of human filarial and malaria parasites using mosquito excreta/feces

“…A sample was considered positive either if it allowed for amplification in both the initial reaction replicates or it was positive in at least one replicate reaction upon retesting. As described elsewhere [38], highly sensitive digital PCR using the QuantStudio 3D Digital PCR instrument was used to retest E/F samples that were inconsistently positive upon initial testing, given the very small residual volumes available for retesting from these samples and the potential to improve detection when very low concentrations of target are present. Due to the prohibitive cost of digital PCR, qPCR was instead used to retest inconsistently positive blood and mosquito carcasses samples, as DNA extraction from these sample types produces considerably greater sample volumes.…”