Parasite specific 7SL-derived small RNA is an effective target for diagnosis of active trypanosomiasis infection

Abstract

Human and animal African trypanosomiasis (HAT & AAT, respectively) remain a significant health and economic issue across much of sub-Saharan Africa. Effective control of AAT and potential eradication of HAT requires affordable, sensitive and specific diagnostic tests that can be used in the field. Small RNAs in the blood or serum are attractive disease biomarkers due to their stability, accessibility and available technologies for detection. Using RNAseq, we have identified a trypanosome specific small RNA to be present at high levels in the serum of infected cattle. The small RNA is derived from the non-coding 7SL RNA of the peptide signal recognition particle and is detected in the serum of infected cattle at significantly higher levels than in the parasite, suggesting active processing and secretion. We show effective detection of the small RNA in the serum of infected cattle using a custom RT-qPCR assay. Strikingly, the RNA can be detected before microscopy detection of parasitaemia in the blood, and it can also be detected during remission periods of infection when no parasitaemia is detectable by microscopy. However, RNA levels drop following treatment with trypanocides, demonstrating accurate prediction of active infection. While the small RNA sequence is conserved between different species of trypanosome, nucleotide differences within the sequence allow generation of highly specific assays that can distinguish between infections with Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax. Finally, we demonstrate effective detection of the small RNA directly from serum, without the need for pre-processing, with a single step RT-qPCR assay. Our findings identify a species-specific trypanosome small RNA that can be detected at high levels in the serum of cattle with active parasite infections. This provides the basis for the development of a cheap, non-invasive and highly effective diagnostic test for trypanosomiasis. African trypanosomes cause significant disease in humans and animals across sub-Saharan Africa. For both human and animal infections diagnostics that can accurately identify an active infection are lacking–this is particularly the case in animal disease where most diagnosis is based upon clinical signs, which is not a specific or sensitive means of detecting infection. There is therefore a significant unmet need for a pathogen marker of active infection that accurately indicates whether an animal or human is currently infected. Through analysing the blood of cattle infected with trypanosomes, we identified a short sequence of RNA that was present at very high levels. This small RNA derives from the trypanosome genome, and we could identify its presence in the genome of all three species that are responsible for human and animal disease. We were able to design species-specific tests, and showed that in samples from infected animals the assays were more sensitive than the traditional microscope-based detection, importantly the signal disappeared relatively quickly after successful treatment, and when treatment failed, the assay was able to accurately identify when infection persisted. We also demonstrated that the causative agent of human trypanosomiasis secretes the marker at similar levels to that seen in the animal-infective trypanosomes. Therefore, we have discovered a marker of trypanosome infection that is present at high levels in the blood of infected animals, disappears quickly upon successful treatment, but is effective at detecting instances of unsuccessful treatment and persistent infection. This represents a potentially powerful diagnostic tool for human and animal trypanosomiasis.