Diagnostic and prognostic value of human prion detection in cerebrospinal fluid

Abstract

Objective: Several prion amplification systems have been proposed for detection of prions in cerebrospinal fluid (CSF), most recently, the measurements of prion seeding activity with second-generation real-time quaking-induced conversion (RT-QuIC). The objective of this study was to investigate the diagnostic performance of the RT-QuIC prion test in the broad phenotypic spectrum of prion diseases.

Methods: We performed CSF RT-QuIC testing in 2,141 patients who had rapidly progressive neurological disorders, determined diagnostic sensitivity and specificity in 272 cases that were autopsied, and evaluated the impact of mutations and polymorphisms in the PRNP gene, and type 1 or type 2 human prions on diagnostic performance.

Results: The 98.5% diagnostic specificity and 92% sensitivity of CSF RT-QuIC in a blinded retrospective analysis matched the 100% specificity and 95% sensitivity of a blind prospective study. The CSF RT-QuIC differentiated 94% of cases of sporadic Creutzfeldt-Jakob disease (sCJD) MM1 from the sCJD MM2 phenotype, and 80% of sCJD VV2 from sCJD VV1. The mixed prion type 1-2 and cases heterozygous for codon 129 generated intermediate CSF RT-QuIC patterns, whereas genetic prion diseases revealed distinct profiles for each PRNP gene mutation.

Interpretation: The diagnostic performance of the improved CSF RT-QuIC is superior to surrogate marker tests for prion diseases such as 14-3-3 and tau proteins, and together with PRNP gene sequencing the test allows the major prion subtypes to be differentiated in vivo. This differentiation facilitates prediction of the clinicopathological phenotype and duration of the disease-two important considerations for envisioned therapeutic interventions. ANN NEUROL 2017;81:79-92.

Figure 1

Analytical sensitivity and seeding response of second-generation CSF RT-QuIC to major human prions. (A) Serial dilution of brain homogenate of a typical sCJD MM1 case monitored in real time by thioflavin T fluorescence in second-generation RT-QuIC. The numbers above RT-QuIC curves denote the dilution and number of positive wells in four wells of the assay. (B) End point RT-QuIC sensitivity determined by serial dilutions of brain homogenate from three cases of sCJD MM1 and sCJD VV2 and monitored with second-generation RT-QuIC (red circles and green triangles). (C) Cumulative plot of second-generation CSF RT-QuIC data obtained in sCJD MM1 (n = 90) and in sCJD MM2 (n = 10). (D) Cumulative plot of CSF RT-QuIC data obtained in sCJD VV1 (n = 7; outlier with no remaining CSF for retesting has been eliminated) and in sCJD VV2 (n = 25). (E) Cumulative plot of CSF RT-QuIC data obtained in sCJD MV1 (n = 15) and in sCJD MV2 (n = 9). (F) Cumulative plot of CSF RT-QuIC data obtained in sCJD MM1–2 (n = 6), sCJD MV1–2 (n = 8), and in sCJD VV1–2 (n = 1). The curves are average ± S.D. of Thioflavin T florescence intensity at a given time point.

Figure 2

Effect of blood contamination on second generation CSF RT QuIC. Human blood was diluted into positive CSF samples received from patients with variable sCJD subtypes: sCJD VV2 (n = 3), sCJD MM1 (n = 3), sCJD MV1 (n = 2), MV1–2 (n = 1), gCJD with the E200K mutation (n = 1), and the second generation CSF RT QuIC was performed as described. Red blood cells (RBC) were counted before disruption by freezing and thawing, followed by in-plate absorbance reading for hemoglobin at 540nm. The curves are average ± S.D. of Thioflavin T florescence intensity in CSF RT QuIC (red circles), and hemoglobin absorbance (purple squares) at a given RBC count.

Figure 3

Differentiation of major human prion subtypes based on seeding potency in second-generation CSF RT-QuIC and relationship with the disease progression rate. (A) Individual data distribution of maximum CSF second-generation RT-QuIC fluorescence obtained in sCJD MM1 (n = 90) and sCJD MM2 (n = 10). (B) Cumulative survival curves of sCJD cases plotted in Figure 3A. (C) Cumulative plot of CSF RT-QuIC data obtained in sCJD VV1 (n = 7; outlier with no remaining CSF for retesting is not plotted) and in sCJD VV2 (n = 25). (D) Cumulative survival curves of sCJD cases plotted in Figure 3C (E) Cumulative plot of CSF RT-QuIC data obtained in genetic prion disease (E200K, n = 12; V210I, n = 2), GSS (n = 9), and FFI (n = 6) (F) Cumulative survival curves of the genetic prion cases plotted in Figure 3E.