Real-Time Quaking-Induced Conversion (RT-QuIC) is a highly sensitive laboratory test that detects minute amounts of specific abnormal proteins. It serves as a diagnostic tool to aid in the diagnosis of certain neurodegenerative conditions.
Understanding Prion Diseases
Prion diseases are a group of rare, progressive, and invariably fatal neurodegenerative disorders that affect both humans and animals. The causative agents are misfolded proteins called prions, which are proteinaceous infectious particles.
The normal form of the prion protein (PrPC) is found on the surface of many cells, particularly in the brain, though its exact function is not fully understood. In prion diseases, this normal protein undergoes a structural change, transforming into an abnormal, misfolded form (PrPSc). This misfolded PrPSc then acts as a template, inducing other normal PrPC molecules to also misfold and aggregate, leading to their accumulation in the brain. This abnormal buildup of proteins results in neuronal damage, spongiform changes, and the eventual loss of neurons.
Symptoms of human prion diseases, such as Creutzfeldt-Jakob disease (CJD), often include rapidly progressive dementia, memory loss, personality changes, and problems with coordination and balance. The rarity of these diseases, coupled with their varied and often non-specific symptoms that can mimic other neurological conditions, makes accurate diagnosis challenging. Historically, a definitive diagnosis of prion disease typically required neuropathological examination of brain tissue obtained post-mortem.
How RT-QuIC Works
The RT-QuIC assay leverages the unique ability of misfolded prion proteins to induce normal prion proteins to also misfold and aggregate. The test begins by adding a small amount of a patient’s sample, suspected of containing misfolded prions (PrPSc), to a reaction mixture. This mixture contains an excess of recombinant, normally folded prion protein (rPrPsen), which acts as a substrate. An amyloid-sensitive fluorescent dye, such as thioflavin T (ThT), is also included in the mixture.
The “quaking” aspect of the test refers to the intermittent shaking or agitation applied to the samples in a multiwell plate. If misfolded prions (PrPSc) are present in the patient’s sample, they act as “seeds,” prompting the recombinant normal prion proteins to misfold and aggregate into amyloid fibrils. The shaking helps break apart these newly formed aggregates, creating more surfaces for the misfolding process to continue and amplify the signal. As these amyloid fibrils form, the thioflavin T dye binds to them, causing an increase in fluorescence. This increase in fluorescence is monitored in real-time by a plate reader, providing a quantifiable signal that indicates the presence of misfolded prions.
The Importance of RT-QuIC for Diagnosis
RT-QuIC represents a significant advancement in the diagnosis of prion diseases, particularly Creutzfeldt-Jakob disease (CJD). Before its development, a definitive diagnosis often relied on brain biopsies or post-mortem examination, procedures that carry risks or are not feasible for living patients. Its ability to detect PrPSc in accessible biological specimens has improved early and accurate diagnosis.
The assay demonstrates high sensitivity, detecting minute amounts of misfolded prions, and high specificity, accurately identifying prions without false positives. For instance, studies have shown RT-QuIC to have a diagnostic sensitivity ranging from approximately 80% to over 95% and a specificity of nearly 100% for sporadic CJD when using cerebrospinal fluid (CSF) samples. This high level of accuracy helps differentiate prion diseases from other neurological conditions that present with similar symptoms, which is a common diagnostic challenge. The improved speed of testing, with results often available within hours to a few days, also contributes to better patient management and surveillance efforts.
Collecting Samples for RT-QuIC Testing
RT-QuIC testing can be performed on several types of biological samples, making it a versatile diagnostic tool. Cerebrospinal fluid (CSF) is a commonly used sample, typically collected via a lumbar puncture, also known as a spinal tap. CSF is chosen because it directly bathes the brain and spinal cord, making it a likely place to find prion proteins in neurodegenerative conditions.
Nasal brushings, specifically from the olfactory epithelium, have also shown promising results for RT-QuIC testing. This less invasive collection method involves gently brushing the nasal cavity to obtain cells where prions may accumulate. Some studies suggest that nasal brushings can yield stronger and faster RT-QuIC responses compared to CSF, with high sensitivity and specificity. Additionally, skin biopsies have been explored as a sample source for RT-QuIC, offering another accessible option for detecting misfolded proteins.