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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 <jats:italic>T<\/jats:italic>. <jats:italic>brucei<\/jats:italic>-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 na\u00efve tsetse with <jats:italic>T<\/jats:italic>. <jats:italic>brucei<\/jats:italic> DNA via faeces when co-housed.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methodology\/Principle findings<\/jats:title>\n<jats:p>Insectary-reared teneral <jats:italic>G<\/jats:italic>. <jats:italic>morsitans morsitans<\/jats:italic> were fed an infectious <jats:italic>T<\/jats:italic>. <jats:italic>b<\/jats:italic>. <jats:italic>brucei<\/jats:italic>-spiked bloodmeal. At 19 days post-infection, infected and na\u00efve 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 <jats:italic>T<\/jats:italic>. <jats:italic>brucei<\/jats:italic> DNA using TBR-qPCR. However, na\u00efve tsetse also tested positive. Even at a ratio of 1 infected to 11 na\u00efve flies, 91% of na\u00efve tsetse gave positive TBR-qPCR results. Furthermore, the quantity of <jats:italic>T<\/jats:italic>. <jats:italic>brucei<\/jats:italic> DNA detected in na\u00efve 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]).<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Conclusions\/Significance<\/jats:title>\n<jats:p>Our results show that infected tsetse can contaminate na\u00efve flies with <jats:italic>T<\/jats:italic>. <jats:italic>brucei<\/jats:italic> 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.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pntd.0012095","type":"journal-article","created":{"date-parts":[[2024,8,12]],"date-time":"2024-08-12T17:36:23Z","timestamp":1723484183000},"page":"e0012095","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":3,"title":["Caught in a trap: DNA contamination in tsetse xenomonitoring can lead to over-estimates of Trypanosoma brucei infection"],"prefix":"10.1371","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4141-8625","authenticated-orcid":true,"given":"Isabel","family":"Saldanha","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rachel","family":"Lea","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Oliver","family":"Manangwa","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gala","family":"Garrod","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lee R.","family":"Haines","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"\u00c1lvaro","family":"Acosta-Serrano","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Harriet","family":"Auty","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Martha","family":"Betson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jennifer S.","family":"Lord","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Liam J.","family":"Morrison","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Furaha","family":"Mramba","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stephen J.","family":"Torr","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lucas J.","family":"Cunningham","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2024,8,12]]},"reference":[{"key":"pntd.0012095.ref001","first-page":"1","volume-title":"Tsetse Biology and Ecology: Their Role in the Epidemiology and Control of Trypanosomosis.","author":"SGA Leak","year":"1999","edition":"2"},{"key":"pntd.0012095.ref002","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1017\/S0007485300028352","article-title":"The Trypanosome Infections of Tsetse-flies in Northern Nigeria and a new Method of Estimation","volume":"14","author":"Lloyd Ll","year":"1924","journal-title":"Bull Entomol Res"},{"key":"pntd.0012095.ref003","first-page":"5","article-title":"Trypanosomiasis \u201crisk\u201d or \u201cchallenge\u201d: a review.","volume":"42","author":"DJ Rogers","year":"1985","journal-title":"Acta Trop"},{"key":"pntd.0012095.ref004","first-page":"63","article-title":"Some factors concerned in trypanosome challenge.","author":"IM Smith","year":"1958","journal-title":"Proceedings of the 7th Meeting of the International Scientific Committee on Trypanosomiasis Research. 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