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

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 qRT-PCR 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 rapidly 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 qRT-PCR 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. RNA was extracted from samples deriving from infected animals (n=2), an uninfected animal, an in vitro trypanosome cell pellet and subjected to small RNAseq.

人类与动物非洲锥虫病（分别简称HAT与AAT）在撒哈拉以南非洲大部分地区仍是严重的公共卫生与经济问题。有效控制AAT并实现HAT的根除，需要可在现场使用的低成本、高灵敏度与高特异性的诊断检测手段。血液或血清中的小RNA（small RNA）因其稳定性、可及性以及成熟的检测技术，成为极具吸引力的疾病生物标志物。通过RNA测序（RNAseq），我们鉴定出一种锥虫特异性小RNA，在感染牛的血清中呈高表达水平。该小RNA源自肽信号识别颗粒的非编码7SL RNA，且在感染牛血清中的检出水平显著高于寄生虫体内，提示其存在主动加工与分泌过程。我们利用定制化实时定量逆转录PCR（qRT-PCR）检测方法，实现了感染牛血清中该小RNA的有效检出。值得注意的是，该RNA可在血液显微镜检测到寄生虫血症（parasitaemia）之前被检出，且在显微镜无法检出寄生虫血症的感染缓解期也可被检测到。但在使用锥虫杀虫药（trypanocides）治疗后，RNA水平会快速下降，表明该标志物可精准预测活动性感染。尽管该小RNA序列在不同锥虫物种间保守，但序列内的核苷酸差异可用于开发高特异性检测方法，以区分布氏锥虫（Trypanosoma brucei）、刚果锥虫（Trypanosoma congolense）与vivax锥虫（Trypanosoma vivax）感染。最后，我们通过一步法qRT-PCR检测方法，无需样本预处理即可直接从血清中有效检出该小RNA。本研究鉴定出一种物种特异性锥虫小RNA，可在活动性寄生虫感染牛的血清中高浓度检出，为开发低成本、无创且高效的锥虫病诊断检测方法奠定了基础。我们从2只感染动物、1只未感染动物以及1份体外锥虫细胞沉淀样本中提取RNA，并进行小RNA测序（small RNAseq）。