Development of an ante-mortem & pre-symptomatic diagnostic test for human prion diseases using RT-QuIC & equic assays
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1 Development of an ante-mortem & pre-symptomatic diagnostic test for human prion diseases using RT-QuIC & equic assays Rocky Mountain Labs National Institute for Allergy & Infectious Diseases 12 th Annual CJD Foundation Family Conference Christina D. Orrú, Ph.D.
2 Human prion diseases INFECTIOUS: Kuru (cannibalism) iatrogenic CJD (e.g contaminated surgical instruments, prioninfected growth hormone injections) variant CJD (from BSE-infected cattle) FAMILIAL: prion protein mutations familial CJD Gerstmann-Sträussler-Scheinker syndrome (GSS) fatal familial insomnia (FFI) SPORADIC: no known prion protein mutations probably spontaneous disease 1 case per 2 million people annually worldwide accounts for 95% of human TSE
3 Diagnosis of Sporadic Creutzfeldt-Jakob Disease PSWs Diagnostic tools: EEG: periodic sharp waves complex CSF: Positive protein MRI: Hyperintensity in the basal ganglia and cortical regions PRNP: codon 129 polymorphism Lack of an Intravital Diagnostic test Definite Diagnosis is based on neuropathology & detection of pathological PrP in the brain CSF MRI PrP res protein Spongiosis PrP detection by immunoblot & immunocytochemistry
4 ThT fluorescence (sum of 8 replicate wells) Plate-based fluorescence detection of prion-seeded PrP amyloid (Real-time Quaking-Induced Conversion: RT-QuIC) dilution Sample + Normal PrP protein + Amyloid stain Scrapie brain 96-well plate Shaking fluorescence plate reader Normal brain Infectious PrP seed + Normal PrP PrP amyloid with fluorescent stain Extremely sensitive: up to 1 billion-fold amplification! Quantitative & Disease specific Much faster & cheaper than similarly sensitive tests Wilham, Orrú, Bessen et al, PLoS Pathogens, 2010
5 RT-QuIC analysis of human cerebrospinal fluid (CSF) o 85-89% overall sensitivity o % specificity much better than other CSF markers Mol Neurobiol; 2014 Characteristic CSF Prion Seeding Efficiency in Humans with Prion Diseases Maria Cramm & Matthias Schmitz & André Karch & Saima Zafar & Daniela Varges & Eva Mitrova & Bjoern Schroeder & Alex Raeber & Franziska Kuhn & Inga Zerr
6 Olfactory mucosa brushing procedure Orru`, Bongianni, Caughey & Zanusso et al., NEJM 2014
7 RT-QuIC analysis of olfactory mucosa samples from CJD & control patients Orru`, Bongianni, Caughey & Zanusso et al., NEJM 2014
8 RT-QuIC of olfactory mucosa from CJD & control patients: Brush vs. Swab
9 RT-QuIC of olfactory mucosa from CJD & control patients: Brush vs. Swab Brush 1 Swab 1 Swab 2 Patient Patient Patient Patient NA Patient NA Patient Patient Patient NA Patient NA Patient Patient Patient Patient Patient NA Patient Patient Patient Patient
10 % M a x im u m T h T F lu o re s c e n c e RT-QuIC of olfactory mucosa from CJD & control patients: Brush vs. Swab B ru s h 1 (1 8 ) S w a b 1 (1 8 ) S w a b 2 (1 3 ) T im e (h )
11 Faster RT-QuIC analysis of scjd olfactory mucosa samples Higher temperatures allow faster identification of scjd samples Orru`, Hughson, Groveman & Caughey, submitted 2014
12 In vivo analysis of Olfactory Mucosa & Cerebro Spinal Fluid from scjd patients and negative controls: preliminary update OM OM CSF CSF Tg66 mice ~400 days incubation In progress Raymond G. & Orru` C. & Caughey C., manuscript in preparation 2014
13 Faster & more sensitive RT-QuIC analysis of scjd CSF 42 C 55 C +
14 RT-QuIC detection of Genetic Human Prion Diseases Orru` C., Gambetti & Caughey, manuscript in preparation 2014
15 RT-QuIC detection of Genetic Human Prion Diseases Orru` C., Gambetti & Caughey, manuscript in preparation 2014
16 RT-QuIC analysis of olfactory mucosa samples from genetic CJD (E200K) & control patients
17 Conclusions o Succesfully tested additional pannel of scjd OM & CSF samples sensitivity 97% & specificity 100% o Comparable sensitivity from samples collected by Cyto-brushes or Flocked swab o Faster RT-QuIC detection of prion seeding activity in OM & CSF samples o Efficient detection of prion seeding activity in gcjd E200K OMs
18 NIAID, NIH Byron Caughey Bradley Groveman Andrew Hughson Gregory Raymond University of Verona Gianluigi Zanusso Matilde Bongianni Giovanni Tonoli Sergio Ferrari Michele Fiorini Salvatore Monaco Case Western Reserve University Pierluigi Gambetti Aaron Foutz Acknowledgements Istituto Superiore di Sanità Maurizio Pocchiari Franco Cardone Funding The CJD Foundation Intramural Research Program, National Institute for Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) Generous donations in memory of Steven Medeiros by his family and friends, of Donna Millard by her husband and friends, of Stephen Able by his wife. Generous donations to the NIAID Gift Fund from Mary Hilderman Smith, Zoё Smith Jaye, and Jenny Smith Unruh in memory of Jeffrey Smith
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