open access

https://hdl.handle.net/2115/73885

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

英語

2019-02-25

PLOS

PLoS neglected tropical diseases

13(2)

e0006842

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. Author summary Tsetse flies are central actors in the transmission of Trypanosomes to vertebrate hosts. Therefore, detection of Trypanosomes in the tsetse flies is important for understanding the epidemiology of African trypanosomiasis as a component of new control or surveillance strategies. We have developed a method that combines multiplex PCR and next-generation sequencing for the detection of different Trypanosome species and subspecies. Similar to the widely used bacterial metagenomic analysis protocol, this method uses a modular, two-step PCR process followed by sequencing of all amplicons in a single run, making sequencing of amplicons more efficient and cost-effective when dealing with large sample sizes. As part of this approach, we designed novel Internal Transcribed Spacer 1 primers optimized for short read sequencing and have slightly better sensitivity than conventional primers. Taxonomic identification of amplicons is based on BLAST searches against the constantly updated NCBI's nt database. Our approach is more accurate than traditional gel-based analyses which are prone to misidentification of species. It is also able to discriminate between subspecies of T. congolense, T. simiae, T. vivax, and T. godfreyi species. This method has the potential to provide new insights into the epidemiology of different Trypanosome genotypes and the discovery of new ones.

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Creative Commons Attribution 4.0 International

1935-2727

1935-2735