Caught in a trap: DNA contamination in tsetse xenomonitoring can lead to over-estimates of Trypanosoma brucei infection

Background
\nTsetse flies (Glossina sp.) are vectors of Trypanosoma brucei subspecies 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, sensitive T. 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 naïve tsetse with T. brucei DNA via faeces when co-housed.
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\nMethodology/Principle findings
\nInsectary-reared teneral G. morsitans morsitans were fed an infectious T. b. brucei-spiked bloodmeal. At 19 days post-infection, infected and naïve 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 for T. brucei DNA using TBR-qPCR. However, naïve tsetse also tested positive. Even at a ratio of 1 infected to 11 naïve flies, 91% of naïve tsetse gave positive TBR-qPCR results. Furthermore, the quantity of T. brucei DNA detected in naïve 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]).
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\nConclusions/Significance
\nOur results show that infected tsetse can contaminate naïve flies with T. brucei DNA 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.