Optimization of RT-QuIC for detection of seeding activity in preclinical blood samples from prion-infected sheep.

Recent large-scale surveys of appendix samples estimate that as many as 1 in 2000 people in the UK have abnormal accumulation of disease-associated PrP, suggesting that they may be subclinically infected with variant Creutzfeldt-Jakob disease (vCJD) [1]. If these individuals are infectious, they pose a potential risk to others through blood and organ donation, or contamination of surgical instruments. Indeed, several cases of vCJD have been attributed to receiving blood products from individuals who donated blood during the preclinical stage of the disease [2]. Thus, a robust diagnostic test for vCJD is urgently required to detect these ‘silent’ (preclinical/subclinical) infections, in order to protect the UK blood supply.

Efforts to develop such a test are complicated by the extremely low concentrations of PrPSc present in readily accessible biological samples, such as blood, and this value is likely to be even lower in preclinical samples. Recently, several platforms have been developed which may be sensitive enough to overcome this challenge. One such technique, an ultrasensitive in vitro prion amplification assay, termed ‘real-time quaking induced conversion’ (RT-QuIC), has already proven to be a highly effective tool in the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD), when applied to samples of cerebrospinal fluid. [3] However, attempts to develop RT-QuIC into a blood-based diagnostic test for vCJD have been less successful.

Here we present a novel version of the RT-QuIC reaction, using a previously untested recombinant prion protein (recPrP) substrate. Due to a paucity of suitable human samples (which are necessary to optimize the platform and evaluate its performance in detecting preclinical infection), we have optimized our assay using an animal model of vCJD infection – sheep that have been experimentally infected with BSE. We show that our version of the RT-QuIC assay gives positive amplification results when applied to whole blood samples spiked with BSE-infected sheep brain homogenate. Positive samples are amplified within 20–40 h, with a high degree of analytical sensitivity, equivalent to a 10−6 dilution of BSE-infected brain homogenate. We then assess the performance of the assay on ‘endogenously’ infected whole blood samples, collected from BSE-infected sheep at regular time points, from the time of infection to the onset of clinical signs. If this version of the RT-QuIC reaction demonstrates high levels of sensitivity and specificity in identifying blood samples from pre-clinically infected individuals, it could then be adapted for use in humans.