Contreras Garcia Maria, Walshe Emily, Steketee Pieter C, Paxton Edith, Lopez-Vidal Javier, Pearce Michael C, Matthews Keith R, Ezzahra-Akki Fatima, Evans Alec, Fairlie-Clark Karen, Matthews Jacqueline B, Grey Finn, Morrison Liam J
Roslin Institute, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom.
Roslin Technologies Limited, Roslin Innovation Centre, University of Edinburgh, Edinburgh, United Kingdom.
Front Vet Sci. 2022 Apr 5;9:868912. doi: 10.3389/fvets.2022.868912. eCollection 2022.
Animal trypanosomiasis (AT) is a significant livestock disease, affecting millions of animals across Sub-Saharan Africa, Central and South America, and Asia, and is caused by the protozoan parasites , with the largest economic impact in cattle. There is over-reliance on presumptive chemotherapy due to inadequate existing diagnostic tests, highlighting the need for improved AT diagnostics. A small RNA species, the 7SL sRNA, is excreted/secreted by trypanosomes in infected animals, and has been previously shown to reliably diagnose active infection. We sought to explore key properties of 7SL sRNA RT-qPCR assays; namely, assessing the potential for cross-reaction with the widespread and benign , directly comparing assay performance against currently available diagnostic methods, quantitatively assessing specificity and sensitivity, and assessing the rate of decay of 7SL sRNA post-treatment. Results showed that the 7SL sRNA RT-qPCR assays specific for performed better than microscopy and DNA PCR in detecting infection. The 7SL sRNA signal was undetectable or significantly reduced by 96-h post treatment; at 1 × curative dose there was no detectable signal in 5/5 cattle infected with , and in 3/5 cattle infected with , with the signal being reduced 14,630-fold in the remaining two cattle. Additionally, the assays did not cross-react with . Finally, by using a large panel of validated infected and uninfected samples, the species-specific assays are shown to be highly sensitive and specific by receiver operating characteristic (ROC) analysis, with 100% sensitivity (95% CI, 96.44-100%) and 100% specificity (95% CI, 96.53-100%), 96.73% (95% CI, 95.54-99.96%) and 99.19% specificity (95% CI, 92.58-99.60%), and 93.42% (95% CI, 85.51-97.16% %) and 82.43% specificity (95% CI, 72.23-89.44% %) for the and assays, respectively, under the conditions used. These findings indicate that the 7SL sRNA has many attributes that would be required for a potential diagnostic marker of AT: no cross-reaction with , high specificity and sensitivity, early infection detection, continued signal even in the absence of detectable parasitaemia in blood, and clear discrimination between infected and treated animals.
动物锥虫病（AT）是一种重要的家畜疾病，影响着撒哈拉以南非洲、中美洲、南美洲和亚洲的数百万头牲畜，由原生动物寄生虫引起，对牛的经济影响最大。由于现有诊断测试不足，人们过度依赖推定化疗，这突出了改进AT诊断方法的必要性。一种小RNA物种，即7SL sRNA，由感染动物体内的锥虫排出/分泌，此前已证明它能可靠地诊断活动性感染。我们试图探索7SL sRNA RT-qPCR检测的关键特性；即评估与广泛存在且无害的[物种名称未给出]发生交叉反应的可能性，将检测性能与现有诊断方法直接进行比较，定量评估特异性和敏感性，以及评估治疗后7SL sRNA的衰减率。结果表明，针对[物种名称未给出]的7SL sRNA RT-qPCR检测在检测感染方面比显微镜检查和DNA PCR表现更好。治疗96小时后，7SL sRNA信号检测不到或显著降低；在1倍治疗剂量下，5头感染[物种名称未给出]的牛中有5头检测不到信号，5头感染[物种名称未给出]的牛中有3头检测不到信号，其余2头牛的信号降低了14630倍。此外，这些检测未与[物种名称未给出]发生交叉反应。最后，通过使用大量经过验证的感染和未感染样本，经受试者工作特征（ROC）分析表明，物种特异性检测具有高度敏感性和特异性，在所用条件下，[具体检测项目未明确给出]检测的敏感性为100%（95%置信区间，96.44 - 100%），特异性为100%（95%置信区间，96.53 - 100%）；[具体检测项目未明确给出]检测的敏感性为96.73%（95%置信区间，95.54 - 99.96%），特异性为99.19%（95%置信区间，92.58 - 99.60%）；[具体检测项目未明确给出]检测的敏感性为93.42%（95%置信区间，85.51 - 97.16%），特异性为82.43%（95%置信区间，72.23 - 89.44%）。这些发现表明，7SL sRNA具有作为AT潜在诊断标志物所需的许多特性：不与[物种名称未给出]发生交叉反应、高特异性和敏感性、能早期检测感染、即使在血液中检测不到寄生虫血症时仍有持续信号，以及能清晰区分感染动物和治疗动物。
