Caught in a trap: DNA contamination in tsetse xenomonitoring can lead to over-estimates ofTrypanosoma bruceiinfection

Background: Tsetse flies (Glossina sp.) are vectors ofTrypanosoma bruceisubspecies that cause human African trypanosomiasis (HAT). Capturing and screening tsetse is critical for HAT surveillance. Classically, tsetse have been microscopically analysed to identify trypanosomes, but this is increasingly replaced with molecular xenomonitoring. Nonetheless, sensitiveT. brucei-detection assays, such as TBR-PCR, are vulnerable to DNA cross-contamination. This may occur at capture, when often multiple live tsetse are retained temporarily in the cage of a trap. This study set out to determine whether infected tsetse can contaminate naive tsetse withT. bruceiDNA via faeces when co-housed. Methodology/Principle Findings: Insectary-reared teneralG. morsitans morsitanswere fed an infectiousT. b. brucei-spiked bloodmeal. At 19 days post-infection, infected and naive tsetse were caged together in the following ratios: (T1) 9:3, (T2) 6:6 (T3) 1:11 and a control (C0) 0:12 in triplicate. Following 24-hour incubation, DNA was extracted from each fly and screened for parasite DNA presence using PCR and qPCR. All insectary-reared infected flies were positive forT. bruceiDNA using TBR-qPCR. However, naive tsetse also tested positive. Even at a ratio of 1 infected to 11 naive flies, 91% of naive tsetse gave positive TBR-qPCR results. Furthermore, the quantity ofT. bruceiDNA detected in naive tsetse was significantly correlated with cage infection ratio. With evidence of cross-contamination, field-caught tsetse from Tanzania were then assessed using the same screening protocol. End-point TBR-PCR predicted a sample population prevalence of 24.8%. Using qPCR and Cq cut-offs optimised on insectary-reared flies, we estimated that prevalence was 0.5% (95% confidence interval [0.36, 0.73]). Conclusions/Significance: Our results show that infected tsetse can contaminate naive flies withT. bruceiDNA when co-caged, and that the level of contamination can be extensive. Whilst simple PCR may overestimate infection prevalence, quantitative PCR offers a means of eliminating false positives.