A single test approach for accurate and sensitive detection and taxonomic characterization of Trypanosomes by comprehensive analysis of internal transcribed spacer 1 amplicons

Abstract

To improve our knowledge on the epidemiological status of African trypanosomiasis, better tools are required to monitor Trypanosome genotypes circulating in both mammalian hosts and tsetse fly vectors. This is important in determining the diversity of Trypanosomes and understanding how environmental factors and control efforts affect Trypanosome evolution. We present a single test approach for molecular detection of different Trypanosome species and subspecies using newly designed primers to amplify the Internal Transcribed Spacer 1 region of ribosomal RNA genes, coupled to Illumina sequencing of the amplicons. The protocol is based on Illumina's widely used 16s bacterial metagenomic analysis procedure that makes use of multiplex PCR and dual indexing. Results from analysis of wild tsetse flies collected from Zambia and Zimbabwe show that conventional methods for Trypanosome species detection based on band size comparisons on gels is not always able to accurately distinguish between T. vivax and T. godfreyi. Additionally, this approach shows increased sensitivity in the detection of Trypanosomes at species level with the exception of the Trypanozoon subgenus. We identified subspecies of T. congolense, T. simiae, T. vivax, and T. godfreyi without the need for additional tests. Results show T. congolense Kilifi subspecies is more closely related to T. simiae than to other T. congolense subspecies. This agrees with previous studies using satellite DNA and 18s RNA analysis. While current classification does not list any subspecies for T. godfreyi, we observed two distinct clusters for these species. Interestingly, sequences matching T. congolense Tsavo (now classified as T. simiae Tsavo) clusters distinctly from other T. simiae Tsavo sequences suggesting the Nannomonas group is more divergent than currently thought thus the need for better classification criteria. This method presents a simple but comprehensive way of identification of Trypanosome species and subspecies-specific using one PCR assay for molecular epidemiology of trypanosomes.

Fig 1. Map of Zambia and Zimbabwe showing areas of tsetse fly collection.

The map was sourced from Simarro P, et al, 2012 [40] and modified in Adobe Illustrator CC 2019 v23.0.1. Areas where tsetse flies were caught are marked in red stars: (A) Kafue in Zambia, (B) Rufunsa in Zambia and (C) Hurungwe in Zimbabwe.

Fig 2. Workflow for read analysis using AMPtk pipeline.

Fig 3. Representative replicates analysis results.

(A) Gel analysis of Rufunsa samples done in replicate showing matching bands per sample. (B) Amplicon sequence analysis of the same samples in showing number of reads detected per species in each sample.

Fig 4. Representative gel and sequence analysis results.

(A) Arrows showing bands are not visible after the 1st PCR become visible after 2nd PCR. (B) By gel analysis, amplicon bands of samples 5, 7 and 10 are indistinguishable by size and are deemed to be all T. godfreyi while sequencing reveals that the amplicon of sample 10 is, in fact, T. vivax. Positive controls comprise; Tbg (T. brucei gambiense), Tbr (T. brucei rhodesiense), Tb/Tc (an artificial mixture of equal amounts of T. brucei gambiense and T. congolense DNA).

Fig 5. Phylogenetic tree of unique ASVs generated from amplicon sequence data.

A Bodo caudatus ITS1 sequence was included as outgroup. Individual Trypanosome species and subspecies cluster into distinct clades. ASV are named after their respective blast best hit matches.

Fig 6. The distribution of Trypanosome species amongst infected tsetse flies.

(A1) Pie chart showing the prevalence of Trypanosome species in wild-caught tsetse flies collected from Kafue, Zambia. (A2) Bar graph showing the Trypanosome species infecting male and female flies from flies collected in Kafue, Zambia. (A3) Graphical representation showing the infection rates of male and female flies collected in Kafue, Zambia. (B) Pie chart showing the prevalence of Trypanosome species in wild-caught tsetse flies collected from Hurungwe, Zimbabwe. (C) Pie chart showing the prevalence of Trypanosome species in wild-caught tsetse flies collected from Rufunsa, Zambia. TBE = T. brucei/T. evansi, TV = T. vivax, TS = T. simiae, TG = T. godfreyi, and TC = T. congolense.