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

This study presents a single test approach for detection and identification of Trypanosomes and their comprehensive characterization at species and sub-group level. The method uses newly designed ITS1 PCR primers (a method used widely in the detection of African Trypanosomes, amplifying the ITS1 region that lies between 18s and 5.8s 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 shows the increased sensitivity of detection for all known species. Through phylogenetic analysis, we detected and identified Trypanosomes at species and sub-group level without the need for any additional tests. Our results show T. congolense Kilifi sub-group seems to be more closely related to T simiae than to other T. congolense subgroups. 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 the two species. Interestingly, sequences matching T. congolense Tsavo (now classified as T. simiae Tsavo) clustered distinctly from the rest of the T. simiae Tsavo sequences suggesting that the Nannomonas group is more divergent than currently thought and there is need for a better classification criteria. This approach has the potential for refining classification of Trypanosomes and provides detailed molecular epidemiology information useful for surveillance and transmission control efforts.