Fig 1.
A flow diagram illustrating the protocol development and trial process.
Image created using BioRender https://BioRender.com/0s83hti [accessed 23/04/25].
Table 1.
Primers and probes used in the study.
Fig 2.
A photo showing a pyramidal trap in-situ (bottom) on the bank of the river Oluffe (trap G3-MCA).
This shows the typical This shows the typical environment, with mixed-used agricultural land and river access point (for bathing, retrieving water and livestock drinking) on the left, and thick brush vegetation surrounding the riverbank on the right.
Fig 3.
A flow diagram depicting steps in the trialled tsetse xenomonitoring protocol. Image created using BioRender https://BioRender.com/mpwx208 [accessed 23/04/25].
Fig 4.
Photographs demonstrating different stages of the training workshop.
Image (a) shows a lecture being given on PCR; (b) shows training being given in DNA extraction; (c) shows technician Victor operating the Mic qPCR machine; (d) shows the Arua team after the first week of the training workshop.
Fig 5.
A figure displaying the mean Cq value results of optimisation experiments for targets TBR(A), TCF (B), TVX (C) and UGWigg (D) in the Multi-Tryp multiplex probe-based qPCR assay.
Annealing temperature optimisation (left) and primer and probe concentration optimisation (centre) experiments were carried out using 10 pg/µL T. brucei AnTat 1.1 DNA, 100 fg/µL T. congolense Forest ANR4 DNA, 10 pg/µL T. vivax DNA or a 1 in 10 dilution of composite G. f. fuscipes (containing W. glossinidia) DNA in triplicate. A five-fold log dilution series (right) of Trypanosoma sp. DNA spiked into composite G. f. fuscipes DNA was carried out using optimised wet (blue) and dry (red) Multi-Tryp air-dryable probe mix (ADPM) in triplicate. A box and whisker plot (bottom right) shows Cq value results of UGWigg-qPCR target across all spiked composite G. f. fuscipes samples (n = 15) screened as part of the dilution series. Error bars represent min and max values.
Fig 6.
A figure displaying the Cq value results of a validation experiment for Multi-Tryp multiplex assay targets(a) TBR (Trypanosoma brucei s-l), (b) TCF (T. congolense Forest), (c) TVX (T. vivax) and (d) UGWigg (W. glossinidia).
For the experiment, 5 µL of 100 pg/µL (T. b. brucei (Tbb) AnTat 1.1, T. b. gambiense (Tbg) ELIANE, T. vivax (Tvx) Y486), or 1 pg/µL (T. congolense Forest (Tcf) ANR4) and 5 µL of composite West Nile G. f. fuscipes (containing W. glossinidia (UGWigg)) DNA was spiked into the dissected tissues of insectary-reared G. m. morsitans (3 male (M) and 3 female (F) per assay target and negative extraction control (NEC)). DNA extraction was carried out following the MagnaExtract DNA extraction protocol. All samples were then screened using the optimised dry-format Multi-Tryp qPCR assay.
Table 2.
Multi-Tryp qPCR results (positive) from 284 tsetse across the four assay targets (Wigglesworthia, T. b. brucei, T. congolense Forest and T. vivax) against tsetse sex and blood-fed status.
Fig 7.
Box-and-whisker plots displaying total Cq value results of Multi-Tryp qPCR screening of wild-caught G. f. fuscipes (n=280) across assay targets TBR (Trypanosoma brucei s-l), TCF (T. congolense Forest), TVX (T. vivax) and UGWigg (W. glossinidia).
Error bars represent minimum and maximum values, the central horizontal bar represents and median and the central cross (+) within the box represents the mean. Plotted symbols (TBR, yellow circle; TCF, orange square; TVX, brown upward triangle; UGWigg, purple downward triangle) represented individual sample Cq values.