This study was approved by the ethics committee of Xuanwu Hospital, Capital Medical University. Written informed consent was provided by all participants and/or their legal next of kin
Participants were referred to the Department of Neurology, Xuanwu Hospital, Capital Medical University from 2010 to April 2019. Although there is no clinical consensus at present, we defined RPD as a cognitive disorder that progressed from the onset of the first symptoms to dementia within 2 years [20, 24]. RPD patients that underwent the CSF 14-3-3 test and were followed up for 2 years or until death were included in this study. Accordingly, 317 patients were enrolled and the flow chart is shown in Figure 1.
The patients were divided into CJD and non-prion RPD groups, and the latter included neurodegenerative diseases, autoimmune encephalitis, infectious diseases, central nervous system (CNS) neoplasms or paraneoplastic diseases, cerebrovascular disease, inherited metabolic diseases, hypoxic-ischemic encephalopathy and unknown etiologies. Patients suspected of CJD were diagnosed according to the updated clinical diagnostic criteria for CJD published in 2009 , and validated by the current WHO criteria . Neurodegenerative diseases (e.g., Alzheimer’s disease, frontotemporal lobar degeneration and dementia with Lewy bodies) were diagnosed according to the relevant criteria [27-30]. Autoimmune encephalitis was diagnosed as per the criteria established by Graus et al . The National Institute of Neurological and Communicative Disorders and the Stroke Association criteria were used to define vascular dementia . Due to the extremely low rate of brain biopsy or post-mortem examinations in China, all patients were followed up for at least 2 years by telephone or face-to-face interviews to improve diagnostic accuracy.
Clinical data, including epidemiological information, clinical symptoms and signs, auxiliary examination and prognosis, were collected for all enrolled patients. The clinical symptoms and signs included psychiatric anomalies (anxiety, depression, apathy, autism, excitement, disinhibition, abnormal emotion or behavior, change in personality), visual disturbances (deteriorated or blurred vision, restricted visual field, and disturbed perception of structures and colors), visual hallucinations, myoclonus, epilepsy, pyramidal signs (spastic increase of muscle tone and the pyramidal reflexed of Babinski, Oppenheimer and Chaddock), extrapyramidal signs (rigidity, tremor, hypokinesia, dystonia and involuntary movement), disturbed cerebellar movements (ataxia, dysmetria, disdiadochokinesia and nystagmus), sleep disorders, weakness or sensory disturbance, speech disorders (aphasia and dysarthria), akinetic mutism, and decorticate rigidity.
MRI scans were performed on a 3.0 Tesla MRI system (Siemens Magnetom Trio Tim MRI system, Germany), and T1-weighted, T2-weighted, fluid-attenuated inversion recovery (FLAIR), diffusion-weighted image (DWI) and apparent diffusion coefficient (ADC) images were obtained. The typical MRI features for CJD were high DWI or FLAIR signals in the caudate/putamen or at least in two cortical regions (temporal, parietal or occipital). A 32-channel digital EEG system (DAVINCI-SAM, Micromed, Mogliano Veneto, Italy) was used to record EEG, and periodic sharp wave complexes (PSWCs) were recognized as the typical EEG feature.
The survival time was defined as the time from disease onset to death. Onset to lumbar puncture (LP) was defined as the time from beginning of the first symptom to lumbar puncture (LP) for the 14-3-3 test. The duration between LP to disease onset was divided by the total duration of disease, and the patients were accordingly divided into the first (< 0.33), second (0.33-0.66) and third (>0.66) stages.
Detection of 14-3-3 protein in CSF
CSF samples were obtained in our hospital and transferred to the Chinese Center for Disease Control and Prevention (CDC) for the 14-3-3 test. The samples were divided into 50𝜇l aliquots and stored at -80℃. CSF 14-3-3 protein was detected by Western blotting. Briefly, 20𝜇l CSF samples were diluted in 5X loading buffer and separated by 12% SDS-PAGE. The protein bands were transferred to nitrocellulose (NC) membranes (Whatman, USA) using the semi-90 dry method in transferring buffer. After blocking, the membranes were incubated for 2 hours at room temperature with anti-14-3-3 polyclonal antibody (1:1000; Santa Cruz Biological, Santa Cruz, USA). The membranes were then incubated with goat anti-rabbit HRP-conjugated secondary antibody and the positive bands were detected using an enhanced chemiluminescence kit (Amersham-Pharmacia Biotech, USA) .
Continuous variables with normal distribution are presented as the mean ± standard deviation (SD), and those not conforming to normal distribution as median and interquartile range (IQR). Categorical data are presented as the frequency (percentage). 𝛸2 test or Fisher exact test was used to compare categorical variables including the incidence of symptoms, signs and paraclinical test results. Mann-Whitney U test was used to compare continuous variables including age and Barthel Index. The Kaplan-Meier method was used to evaluate the association between onset to LP and the CSF 14-3-3 test. A two-sided p value<0.05 was considered statistically significant. All statistical analyses were performed using SPSS, version 25.