Caught in a trap: DNA contamination in tsetse xenomonitoring can lead to over-estimates ofTrypanosoma bruceiinfection
Authors: Saldanha Isabel , Lea Rachel , Manangwa Oliver , Garrod Gala , Haines Lee R. , Acosta-Serrano Álvaro , Auty Harriet , Betson Martha , Lord Jennifer S. , Morrison Liam J. , Mramba Furaha , Torr Stephen J. , Cunningham Lucas J. 
Published: March 2024

AbstractBackgroundTsetse 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 naïve tsetse withT. bruceiDNA via faeces when co-housed.Methodology/Principle FindingsInsectary-reared teneralG. morsitans morsitanswere fed an infectiousT. 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 forT. bruceiDNA 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 ofT. bruceiDNA 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]).Conclusions/SignificanceOur results show that infected tsetse can contaminate naïve 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.Author SummaryTsetse flies (Glossina sp.) are vectors ofTrypanosoma bruceiparasites that cause human African trypanosomiasis, also known as sleeping sickness. As part of disease surveillance, tsetse can be captured in traps and checked for parasite presence. The molecular screening of disease vectors (such as mosquitoes, ticks and blackflies) for the presence of pathogen DNA has gained popularity in recent years. However, DNA contamination may occur at capture when live vectors are retained for a limited period in a trap cage. To explore this, we conducted experiments, initially with laboratory-reared tsetse and then field-caught tsetse from Tanzania. Our results show that infected tsetse can contaminate uninfected tsetse withT. bruceiDNA when retained together in a trap cage, and that the level of contamination can be extensive. Infected tsetse consistently shedT. bruceiDNA in their faeces, which in turn contaminates other tsetse. This can produce false-positive results, leading to inaccurate reporting of infection prevalence. These findings impact not only trypanosomiasis surveillance, but may also have ramifications for the xenomonitoring of other vector-borne neglected diseases. Future work should explore whether pathogen DNA contamination routes exist in other vector species and, if so, the methods to mitigate DNA contamination in entomological traps.