Cas10 based 7SL-sRNA diagnostic for the detection of active trypanosomiasis

Abstract

Animal Trypanosomosis (AT) is a significant disease affecting cattle across sub-Saharan Africa, Latin America, and Asia, posing a major threat to economic productivity and animal welfare. The absence of reliable diagnostic tests has led to an over-reliance on widespread pre-emptive drug treatments, which not only compromise animal health but also heighten the risk of drug resistance. The chronic nature of AT, characterized by cyclical low or undetectable parasite levels, and the necessity for field-applicable tests that can distinguish between active infection and prior exposure, present considerable challenges in developing effective diagnostics. In previous work, we identified a parasite-specific small RNA, 7SL-sRNA, which is detectable in the serum of infected cattle, even during the chronic stages of infection. However, existing methods for detecting sRNA require specialized equipment, making them unsuitable for field use. In this study, we have developed a lateral flow diagnostic test utilizing Cas10 technology for the detection of 7SL-sRNA. This assay demonstrates high sensitivity and specificity, effectively identifying active infections in cattle, including during chronic phases (with positive signals observed up to the 63 days post-infection end point). This is the first instance of Cas10 being used for small RNA detection, paving the way for a cost-effective, user-friendly, and field-deployable diagnostic test for AT, while establishing Cas10 technology for the detection of small RNAs in general. Author summaryAnimal Trypanosomosis (AT) is a parasitic disease that affects livestock, causing major economic losses and harm to animal health. Cheap effective diagnostics that can be easily used in the field would have a major impact on the disease burden by enabling better treatment practices. However, parasites causing the disease - trypanosomes - are often undetectable via conventional methods, especially during chronic disease stages. More accurate diagnostics are not suitable for use in the field and are therefore unavailable to farmers. This has led to the overuse of drug treatments that can harm animals and lead to the emergence of drug resistance. We previously discovered a trypanosome-specific small RNA, called 7SL-sRNA, that can be detected in the blood of infected cattle, even during the chronic stages of AT when parasites are not visible via microscopy. However, detecting this RNA has required complex equipment, limiting its use in field settings. Building on this work, we have now developed a field applicable test for trypanosomosis that uses Cas10 technology to detect 7SL-sRNA via a simple lateral flow assay. This test is easy to use and accurately identifies active infections, including during the chronic phase of the disease. This is the first time Cas10 technology has been used to detect small RNAs, offering a promising, cost-effective tool for diagnosing AT and potentially other neglected parasitic diseases.