1) We have furthered the development of a test for Creutzfeldt-Jakob disease (CJD) using nasal brushings with Gianluigi Zanusso and other collaborators because definite diagnosis of sporadic CJD in living patients remains a challenge. In our previous study The RT-QuIC assays seeded with nasal brushings were positive in 30 of 31 patients with CJD but were negative in 43 of 43 patients without CJD, indicating a sensitivity of 97% and specificity of 100% for the detection of CJD. By comparison, testing of cerebrospinal fluid (CSF) samples from the same group of patients had a sensitivity of 77% and a specificity of 100%. Since our initial report, we have collected these two specimen types simultaneously from 12 additional patients, with a sensitivity of 100% from olfactory mucosa (12 of 12 samples) and 83% from cerebrospinal fluid (10 of 12 samples). Thus, our current overall diagnostic performance for RT-QuIC of nasal brushings is 42 positive out of 43 CJD patients (98%) diagnostic sensitivity and 0 positive out of 43 non-CJD patients (100%) specificity. 2) In collaborations with Italian, Canadian and US colleagues, we have also improved our CSF-based testing for CJD using RT-QuIC. Previous RT-QuIC testing of human CSF has required 2.5 to 5 days. Our improved RT-QuIC assay identifies positive CSF samples within 4 to 14 h with better analytical sensitivity. Moreover, analysis of 11 CJD patients demonstrated that while 7 were RT-QuIC positive using the previous conditions, 10 were positive using the new assay. In these and further analyses, a total of 46 of 48 CSF samples from sporadic CJD patients were positive, while all 39 non-CJD patients were negative, giving 95.8% diagnostic sensitivity and 100% specificity. This second-generation RT-QuIC assay markedly improves the speed and sensitivity of detecting prion seeds in CSF specimens from CJD patients. This should enhance prospects for rapid and accurate ante mortem CJD diagnosis. 3) Statutory surveillance of bovine spongiform encephalopathy (BSE) indicates that cattle are susceptible to both classical BSE (C-BSE) and atypical forms of BSE. Atypical forms of BSE appear to be sporadic and thus may never be eradicated. A major challenge for prion surveillance is the lack of sufficiently practical and sensitive tests for routine BSE detection and strain discrimination. As noted above, the RT-QuIC test was known to be highly specific and sensitive for the detection of multiple human and animal prion diseases but not BSE. Collaborating with the Cristina Casalone and others, we have now tested brain tissue from cattle affected by C-BSE and atypical L-type bovine spongiform encephalopathy (L-type BSE or L-BSE) with the RT-QuIC assay and found that both BSE forms can be detected and distinguished using particular rPrPSen substrates. Specifically, L-BSE was detected using multiple rPrPSen substrates, while C-BSE was much more selective. This substrate-based approach suggests a diagnostic strategy for specific, sensitive, and rapid detection and discrimination of at least some BSE forms. 4) Because prions propagate as multiple strains in a wide variety of mammalian species, the detection of all such strains by a single ultrasensitive assay such as RT-QuIC would facilitate prion disease diagnosis, surveillance and research. In a collaboration with Romolo Nonno, Wenquan Zou, Bernadino Ghetti and Pierluigi Gambetti, we have found that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested.