A single test approach for accurate and sensitive detection and taxonomic characterization of Trypanosomes by comprehensive analysis of ITS1 amplicons

The World Health Organization has targeted stopping the transmission of Human African Trypanosomiasis by 2030. To achieve this, better tools are urgently required to identify and monitor Trypanosome infections in human, animals, and tsetse fly vectors. This study presents a single test approach for detection and identification of Trypanosomes and their comprehensive characterization at species and sub-group level. Our method uses newly designed ITS1 PCR primers (a widely used method for detection of African Trypanosomes, amplifying the ITS1 region of ribosomal RNA genes) coupled to Illumina sequencing of the amplicon. The protocol is based on the widely used Illumina’s 16s bacterial metagenomic analysis procedure that makes use of multiplex PCR and dual indexing. We analyzed wild tsetse flies collected from Zambia and Zimbabwe. Our results show that the traditional method for Trypanosome species detection based on band size comparisons on a gel is unable to distinguish between T. vivax and T. godfreyi accurately. Additionally, this approach shows increased sensitivity of detection at species level. Through phylogenetic analysis, we identified Trypanosomes at species and sub-group level without the need for any additional tests. Our results show T. congolense Kilifi sub-group is more closely related to T. simiae than to other T. congolense sub-groups. This agrees with previous studies using satellite DNA and 18s RNA analysis. While current classification does not list any sub-groups for T. vivax and T. godfreyi, we observed distinct subgroups for these species. Interestingly, sequences matching T. congolense Tsavo (now classified as T. simiae Tsavo) clusters distinctly from the rest of the T. simiae Tsavo sequences suggesting that the Nannomonas group is more divergent than currently thought thus the need for a better classification criteria. This approach has the potential for refining classification of Trypanosomes and provide detailed molecular epidemiology information useful for surveillance and transmission control efforts.