Endpoint Quaking-Induced Conversion: a Sensitive, Specific, and High-Throughput Method for Antemortem Diagnosis of Creutzfeldt-Jacob Disease

Abstract

The Prion Laboratory Section of the Public Health Agency of Canada supports heath care professionals dealing with patients suspected to have Creutzfeldt-Jakob disease (CJD) by testing cerebrospinal fluid (CSF) for protein markers of CJD. To better serve Canadian diagnostic requirements, a quaking-induced conversion (QuIC)-based assay has been added to the test panel. The QuIC tests exploit the ability of disease-associated prion protein, found in the CSF of a majority of CJD patients, to convert a recombinant prion protein (rPrP) into detectable amounts of a misfolded, aggregated form of rPrP. The rPrP aggregates interact with a specific dye, causing a measurable change in the dye's fluorescence emission spectrum. Optimal test and analysis parameters were empirically determined. Taking both practical and performance considerations into account, an endpoint QuIC (EP-QuIC) configuration was chosen. EP-QuIC uses a thermo-mixer to perform the shaking necessary to produce the quaking-induced conversions. Fluorescence readings are obtained from a microwell fluorescence reader only at the beginning and the end of EP-QuIC reactions. Samples for which the relative fluorescence unit ratio between the initial and final readings represent a ≥4 increase in signal intensity in at least two of the three replicates are classified as positive. A retrospective analysis of 91 CSF samples that included 45 confirmed cases of CJD and 46 non-CJD cases was used to estimate the performance characteristics of the EP-QuIC assay. The diagnostic sensitivity and specificity of the EP-QuIC test of this set of samples were 98 and 91%, respectively.

Chlamydia trachomatis-induced Fitz-Hugh–Curtis syndrome: a case report

“…However, Chlamydia culture is cumbersome, expensive and needs a lot of expertise affecting the assiduity of the results [20] and it therefore is cost-ineffective especially in resource-limited settings where individuals struggle to finance health through out of pocket payments. Chlamydia trachomatis (Enzyme Immuno Assay) with a specificity of 100% and a sensitivity range of 45–70% [21] is widely used in the diagnoses of Chlamydia [19, 22] in resource-limited settings, due to its relative cost-effectiveness. This method has its limitations as its high specificity makes it a good screening test but the low sensitivity hinders its relative diagnostic value.…”

An Evaluation of Rapidly Progressive Dementia Culminating in a Diagnosis of Creutzfeldt–Jakob Disease

“…EP-QuIC is an empirically validated assay that utilizes the intrinsic properties of disease-associated prion protein in patients' CSF to cause misfolding and aggregation of the recombinant prion protein. The protein aggregates interact with a dye resulting in detectable changes in its fluorescence pattern [ 17 ]. EP-QuIC is 80–90% sensitive and 99–100% specific for the diagnosis of prion disease [ 4 , 12 ].…”

The Predictive Value of Endpoint Quaking-Induced Conversion in Creutzfeldt-Jakob Disease

“…Canadian practitioners should be aware that although EP-QuIC is a highly useful clinical test, it has not yet been reported to have the near-perfect specificity of RT-QuIC. 7,8 Rectification of our false-positive result using the current modified EP-QuIC protocol is encouraging, but continued evaluation of test sensitivity, specificity, positive and negative predictive value is needed before it can be said with certainty that EP-QuIC may serve as a substitute for RT-QuIC in newly proposed diagnostic criteria for CJD. 4 Since no non-pathologic test for CJD can provide a definitive diagnosis, practitioners must consider all the available clinical and ancillary test information when evaluating suspected cases.…”

“…1,7 The Canadian National Microbiology Laboratory (NML) in Winnipeg, Manitoba now offers a similar test that instead uses Eppendorf ThermoMixer-aided shaking, called endpoint quaking-induced conversion (EP-QuIC). 8 In a retrospective case-control analysis comparing EP-QuIC to RT-QuIC, EP-QuIC was found to have a sensitivity and specificity of 98% and 91%, respectively, for the diagnosis of CJD, indicating a slightly lower specificity but higher sensitivity than RT-QuIC. 8 Importantly, while RT-QuIC has been shown to have a high negative predictive value (NPV) of 91% and excellent positive predictive value (PPV) of 100% in the diagnosis of CJD, a similar investigation into the predictive value of EP-QuIC has not been performed.…”

“…8 In a retrospective case-control analysis comparing EP-QuIC to RT-QuIC, EP-QuIC was found to have a sensitivity and specificity of 98% and 91%, respectively, for the diagnosis of CJD, indicating a slightly lower specificity but higher sensitivity than RT-QuIC. 8 Importantly, while RT-QuIC has been shown to have a high negative predictive value (NPV) of 91% and excellent positive predictive value (PPV) of 100% in the diagnosis of CJD, a similar investigation into the predictive value of EP-QuIC has not been performed. 9 In particular, evaluation of the PPV of EP-QuIC is needed, since its potentially slightly lower specificity compared to RT-QuIC may translate to false-positives when tested in a low-prevalence population.…”