Real‐time quaking‐induced conversion assay using a small‐scale substrate production workflow for the diagnosis of Creutzfeldt‐Jakob disease

Abstract

AbstractBackgroundThe lack of a dedicated surveillance program for prion disease, particularly in low‐ and middle‐income countries (LMICs), has hindered the global effort to address this public health threat. Although CSF Real‐time quaking‐induced conversion (RT‐QuIC) is considered the most reliable test for sporadic Creutzfeldt‐Jakob disease (sCJD), its availability in LMICs is limited due to its cost and technical difficulty in generating the recombinant prion protein substrate (recPrP). This study aimed to evaluate the performance of RT‐QuIC with recPrP produced in‐house through a small‐scale method – i.e. the application of reusable pre‐packed chromatography columns and subsequent dialysis.MethodCSF specimens from patients suspected of having prion disease were consecutively collected and stored between October 2015 and November 2022. Those with available CSF for RT‐QuIC analysis were included in this study. Electronic medical record data were reviewed to clinically classified participants as probable sCJD or non‐sCJD. CSF RT‐QuIC was performed using in‐house recPrP and 2nd generation (improved‐QuIC) protocol (Orrú et al., 2015). Its specificity and sensitivity for diagnosing probable sCJD were reported, along with details of other clinical data and investigations.ResultAmong 38 eligible participants, with a median (interquartile range) age of 64 (56.5‐70) years and 15 (39.5%) female, 12 had probable sCJD and the remaining 26 unequivocally suffered from non‐prion disorders. MRI and EEG were suggestive of sCJD in 100% (12/12) and 50% (6/12) of sCJD participants, respectively. PRNP gene sequencing was performed in all sCJD and 7 non‐sCJD participants, all of whom had MM genotype without mutation. RT‐QuIC was positive in 11/12 sCJD participants (sensitivity 0.92, 95% confidence interval (CI) 0.65‐0.99) and negative in all non‐sCJD participants (specificity 1.00, 95%CI 0.87‐1.00). CSF tau/p‐tau ratio showed sensitivity and specificity of 0.67‐1.0 and 0.85‐1.0, respectively.ConclusionRT‐QuIC using recPrP generated through a small‐scale workflow demonstrated great performance in detecting sCJD. Given its performance results along with its low cost, this technique could feasibly be implemented in LMICs and potentially be the first step towards establishing local prion disease surveillance programs that will strengthen the global efforts.