Usefulness of electronic stimulation in restless legs syndrome: a pilot study.

PURPOSE: This study aims to investigate the effect of electronic stimulation (ES) as a non-pharmacological treatment in restless legs syndrome (RLS). METHODS: This is a randomized, single-blind study. A total of 46 patients were included, consisting of an active group and a sham group with 22 and 24 members, respectively. The stimulation was administered to bilateral lower legs using the tapping mode (3 Hz) on a handheld ES device, and symptom changes were measured in both groups. The effects of the stimuli were analyzed with repeated measures ANOVA. RESULTS: The symptom severity was significantly reduced in the active group, and showed significant interaction effects in the time * group (F = 4.441, p = 0.031). Although both the active and sham groups reported improved symptoms upon receiving longer periods of treatment, the effect of the ES was greater in the active group. CONCLUSIONS: ES treatment resulted in symptom improvement when using ideal levels of stimulation intensity. ES can be considered as a non-pharmacological treatment option for RLS.

Isolation of Coxiella burnetii in patients with nonspecific febrile illness in South Korea.

BACKGROUND: The number of human Q fever cases in South Korea has been rapidly increasing since 2015. We report the first isolation of Coxiella burnetii in Korea in two patients who initially presented with non-specific febrile illness and were finally diagnosed with acute Q fever in South Korea. CASE PRESENTATION: Two adult patients with fever had serologic tests against C. burnetii initially negative, and polymerase chain reaction against 16S rRNA using whole blood was also negative. After bacterial amplification of C. burnetii in immune-depressed mice, we isolated C. burnetii from patients with acute Q fever. The isolates KZQ2 and KZQ3 were confirmed by polymerase chain reaction, nucleotide sequence analysis, and morphologic observation using a transmission electron microscope. CONCLUSIONS: These results can help us understand the clinical and epidemiologic features of Q fever in South Korea.

Alterations in Salience Network Functional Connectivity in Individuals with Restless Legs Syndrome.

BACKGROUND AND PURPOSE: Restless legs syndrome (RLS) is a neurological disorder which is most commonly identified by an urge to move the legs. It often shows alterations in sensory processing which implies the salience network (SN) is experiencing changes. This study investigates the functional connectivity (FC) between the SN and other areas of the brain in RLS patients during the resting state period. METHODS: Thirty patients with drug naïve idiopathic RLS and 30 healthy age and gender matched controls were included in this study. Resting state fMRIs were performed in the morning during the asymptomatic period. The SN comparisons were conducted between the two groups. RESULTS: The RLS group showed a reduction in SN FC in the right pyramis, and an increase in SN FC in the bilateral orbitofrontal gyri and right postcentral gyrus. CONCLUSIONS: The results of this study give reason to believe that SN FC in RLS patients is altered during asymptomatic periods. This could have an influence on the processing of the saliency of information, particularly sensory information processing and inhibition mechanisms.

BMD42-2910, a Novel Benzoxazole Derivative, Shows a Potent Anti-prion Activity and Prolongs the Mean Survival in an Animal Model of Prion Disease.

Prion diseases are a group of neurodegenerative and fatal central nervous system disorders. The pathogenic mechanism involves the conversion of cellular prion protein (PrPC) to an altered scrapie isoform (PrPSc), which accumulates in amyloid deposits in the brain. However, no therapeutic drugs have demonstrated efficacy in clinical trials. We previously reported that BMD42-29, a synthetic compound discovered in silico, is a novel anti-prion compound that inhibits the conversion of PrPC to protease K (PK)-resistant PrPSc fragments (PrPres). In the present study, 14 derivatives of BMD42-29 were obtained from BMD42-29 by modifying in the side chain by in silico feedback, with the aim to determine whether they improve anti-prion activity. These derivatives were assessed in a PrPSc-infected cell model and some derivatives were further tested using real timequaking induced conversion (RT-QuIC). Among them, BMD42-2910 showed high anti-prion activity at low concentrations in vitro and also no toxic effects in a mouse model. Interestingly, abundant PrPres was reduced in brains of mice infected with prion strain when treated with BMD42- 2910, and the mice survived longer than control mice and even that treated with BMD42-29. Finally, high binding affinity was predicted in the virtual binding sites (Asn159, Gln 160, Lys194, and Glu196) when PrPC was combined with BMD-42-2910. Our findings showed that BMD42-2910 sufficiently reduces PrPres generation in vitro and in vivo and may be a promising novel anti-prion compound.

Resting-state connectivity and the effects of treatment in restless legs syndrome.

OBJECTIVES: Resting-state brain connectivity has been shown to differ for Restless Legs Syndrome (RLS) compared to healthy control (CON) groups. This study evaluates the degree these RLS-CON differences are changed by concurrent treatment. METHODS: Resting-state functional MRIs were obtained from 32 idiopathic RLS patients during the morning asymptomatic period and 16 age and gender-matched CON subjects. Of the 32 RLS patients, 16 were drug-naïve (DN-RLS), and 16 were regularly drug-treated using a dopamine agonist (DT-RLS). Various assessments of disease characteristics were also performed. The primary purpose was to assess the replicability of prior results and the effects of treatment on these differences between controls and untreated RLS patients. Resting-state connectivity was analyzed by a seed-based method using the bilateral ventral-posterolateral nuclei (VPLN) in the thalamus. RESULTS: In the DN-RLS group, compared to the CON group, three areas (the bilateral lingual gyri and right middle temporal gyrus) were replicated. The three replicated areas did not significantly differ for DT-RLS compared to DN-RLS. DT-RLS compared to DN-RLS had significantly higher thalamic connectivity for the left uvula, right tuber, left anterior insula, and right declive. CONCLUSIONS: Thalamic connectivity to the bilateral lingual gyri and right middle temporal gyrus is a replicable finding in DN-RLS that was not affected by dopamine agonist treatments. Other changes in thalamic connectivity were altered by dopamine agonist treatment. These treatment effects may be pertinent to the known treatment benefits of a dopamine agonist on RLS symptoms.

Sensitivity and specificity evaluation of multiple neurodegenerative proteins for Creutzfeldt-Jakob disease diagnosis using a deep-learning approach.

The diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) can only be confirmed by abnormal protease-resistant prion protein accumulation in post-mortem brain tissue. The relationships between sCJD and cerebrospinal fluid (CSF) proteins such as 14-3-3, tau, and α-synuclein (a-syn) have been investigated for their potential value in pre-mortem diagnosis. Recently, deep-learning (DL) methods have attracted attention in neurodegenerative disease research. We established DL-aided pre-mortem diagnostic methods for CJD using multiple CSF biomarkers to improve their discriminatory sensitivity and specificity. Enzyme-linked immunosorbent assays were performed on phospho-tau (p-tau), total-tau (t-tau), a-syn, and ß-amyloid (1-42), and western blot analysis was performed for 14-3-3 protein from CSF samples of 49 sCJD and 256 non-CJD Korean patients, respectively. The deep neural network structure comprised one input, five hidden, and one output layers, with 20, 40, 30, 20 and 12 hidden unit numbers per hidden layer, respectively. The best performing DL model demonstrated 90.38% accuracy, 83.33% sensitivity, and 92.5% specificity for the three-protein combination of t-tau, p-tau, and a-syn, and all other patients in a separate CSF set (n = 15) with other neuronal diseases were correctly predicted to not have CJD. Thus, DL-aided pre-mortem diagnosis may provide a suitable tool for discriminating CJD patients from non-CJD patients.

Case Report: Scrub Typhus and Q Fever Coinfection.

A 56-year-old female goat herder had scrub typhus that persisted after receiving doxycycline for 5 days. Her symptoms continued, prompting us to perform further examinations that revealed coinfection of Q fever and scrub typhus via molecular and serological testing. We also isolated Orientia tsutsugamushi using BALB/c mice and L929 cells.

Diagnosis and molecular characteristics of human infections caused by Anaplasma phagocytophilum in South Korea.

Human granulocytic anaplasmosis (HGA) is a tick borne infection caused by Anaplasma phagocytophilum. HGA cases in South Korea have been identified since the first report in 2014. In this study, we investigated the serological response in 594 clinical samples of patients with acute febrile illness and molecular characteristics of A. phagocytophilum clinical isolates obtained from HGA patients. In serological test for A. phagocytophilum, 7.91% (47/594 cases) were positive for IgG and Ig M and 13 of 47 cases showed seroconversion. In the detection rate of the 16S rRNA, msp2(p44), and ankA, genes were showed 3.68% (14/380 cases) for A. phagocytophilum-specific 16S rRNA gene. Phylogenetic analysis of three clinical isolates demonstrated high sequence similarity (98.58-100%) with A. phagocytophilum 16S rRNA sequences identified from public databases. Analysis of the msp2(p44) gene showed highly variable similarity rates (7.24-98.85%) even within isolated countries and host ranges. These results provide clues into the bacterial characterization of A. phagocytophilum originating from Korean patients, providing useful guidance for treatment and improving clinical outcomes.

High-Level Production of High-Purity Human and Murine Recombinant Prion Proteins Functionally Compatible to In Vitro Seeding Assay.

Recombinant (rec) prion protein (PrP) is an extremely useful resource for studying protein misfolding and subsequent protein aggregation events. Here, we report mass production of high-purity rec-polypeptide encoding the C-terminal globular domain of PrP; (90-230) for human and (89-231) for murine PrP. These proteins were expressed as His-tagged fusion proteins in E. coli cultured by a high cell-density aerobic fermentation method. RecPrPs recovered from inclusion bodies were slowly refolded under reducing conditions. Purification was performed by a sequence of metal-affinity, cation-exchange, and reverse-phase chromatography. The current procedure yielded several dozens of milligrams of recPrP per liter with ＞95% purity. The purified recPrPs predominantly adopted an α-helix-rich conformation and were functionally sufficient as substrates to measure the seeding activity of human and animal prions. Establishment of a procedure for high-level production of high-purity recPrP supports the advancement of in vitro investigations of PrP including diagnosis for prion diseases.

THERPA: A small molecule database related to prion protein regulation and prion diseases progression.

Prion diseases are fatal neurodegenerative disorders that affect humans and animals. Although various small molecules have been evaluated for application in the treatment of prion diseases, none have been shown to be efficacious. Expanding our knowledge of these molecules is important for understanding of the complex mechanisms of prion diseases. To improve access to the scattered information on small molecules related to prion diseases, we built a database of therapeutic molecules associated with prion diseases (THERPA, therpa.pythonanywhere.com). THERPA includes 119 small molecules and their 283 relationships with prion diseases. THERPA is an interactive visual database and useful for improving search efficiency which can help researchers identify intrinsic small molecules that can be used for developing therapeutics for prion diseases.

Diurnal variation of default mode network in patients with restless legs syndrome.

OBJECTIVE: Restless legs syndrome (RLS) patients compared to controls have been found to have abnormal patterns in the default mode network (DMN) in the morning when symptom threshold is the highest and symptoms are least likely to occur. If these morning abnormalities in DMN are pertinent to disease expression, then similar or further detectable differences may be expected on a nighttime assessment when RLS symptom threshold is at its lowest. The purpose of this study was to elucidate the potential neural mechanisms underlying the circadian aspect of RLS symptom expression by assessing diurnal changes in DMN. METHODS: Fifteen drug-naïve subjects with idiopathic RLS and 15 age- and gender-matched healthy subjects had fMRI scans in the morning and evening. The DMN patterns were compared both for differences between morning and evening and between RLS and controls. RESULTS: RLS patients compared to the healthy controls showed significant differences in morning and evening DMN. In particular, RLS patients showed consistent increased connectivity in the parietal lobule in both the morning and evening. In contrast, connectivity in the thalamus was increased in the morning and reduced in the evening. In addition, there were negative correlations between thalamic connectivity and the Korean versions of the international RLS scale and the quality-of-life subscore. CONCLUSIONS: The results indicated diurnal disturbances of the DMN in RLS subjects are consistent with both the circadian rhythm and severity of RLS. The circadian expression of RLS may relate to changes in arousal cortical-activation thresholds occurring with diurnal changes in the thalamic circuits of the DMN.

Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT.

BACKGROUND: Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. METHODOLOGY/PRINCIPAL FINDINGS: The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, p<0.001). CONCLUSIONS/SIGNIFICANCE: The mAb developed in this study shows high sensitivity and specificity, confirming its clinical utility with the RFFIT to measure the rabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV.

Clinical Isolation of Anaplasma phagocytophilum in South Korea.

We report the first isolation of Anaplasma phagocytophilum in South Korea. A 61-year-old woman presented with a 6-day history of fever, headache, and myalgia. Initial investigation showed neutropenia and thrombocytopenia. We diagnosed human granulocytic anaplasmosis by microscopic examination and serologic testing. The patient recovered fully without antibiotic therapy. The isolate was obtained from the patient's blood by cell culture and mouse inoculation. Its identity was confirmed by an immunofluorescence assay, sequencing of the 16S rRNA gene, msp2 (p44), and ankA genes, and staining and electron microscopy of morulae of A. phagocytophilum in cultured human promyelocytic leukemia HL-60 cells.

Case Report: Polymerase Chain Reaction Testing of Tick Bite Site Samples for the Diagnosis of Human Granulocytic Anaplasmosis.

Human granulocytic anaplasmosis (HGA) is a tick-borne infectious disease caused by Anaplasma phagocytophilum, an obligate intracellular bacterium. Until now, the utility of tick-bite site samples for HGA diagnosis has not been reported. Using a patient's buffy coat and tick-bite site crust samples, we performed polymerase chain reaction (PCR) testing using Ehrlichia- or Anaplasma-specific primers. PCR with buffy coat and crust samples obtained before doxycycline administration was positive. Six days after doxycycline administration, PCR with the buffy coat sample was negative but PCR with a crust tissue sample from the tick-bite site remained positive. This is the first case to suggest that crust tissue at the tick-bite site may be useful for early HGA diagnosis in patients who have already been treated with antibiotics such as doxycycline.

A Recombinant 47-kDa Outer Membrane Protein Induces an Immune Response against Orientia tsutsugamushi Strain Boryong.

We investigated the 47-kDa outer membrane protein (OMP), which is a periplasmic serine protease and an antigenic major surface protein of Orientia tsutsugamushi, as a vaccine candidate. We developed a conventional subunit vaccine expressing recombinant 47-kDa OMP (rec47) and a DNA vaccine (p47). In mouse immunization experiments, intranasal immunization with rec47 alone or with rec47 plus heat-labile enterotoxin B subunit from Escherichia coli or plus cholera toxin (CT) as adjuvants induced a higher amount of rec47-specific antibodies than intramuscular immunization with p47 alone or with p47 plus pBOOST2-samIRF7/3 (pB) as adjuvant. Moreover, the combination of rec47 and CT induced a strong cellular immune response to 47-kDa OMP, as demonstrated by a spleen cell proliferation assay, and also induced Th1- and Th2-type cytokine production, as demonstrated by a cytokine enzyme-linked immunosorbent assay. Intranasal immunization with rec47 plus CT was the most effective method for the induction of humoral and cell-mediated immune responses. Furthermore, relatively strong protection against homologous O. tsutsugamushi strain Boryong challenge was observed in mice immunized with rec47 plus CT. Therefore, 47-kDa OMP is an attractive candidate for developing a prophylactic vaccine against scrub typhus by O. tsutsugamushi infection.

Clinical and Genetic Features of Coxiella burnetii in a Patient with an Acute Febrile Illness in Korea.

Although Q fever is an important zoonotic infection with a worldwide distribution, no human isolates of Coxiella burnetii have been identified in Korea. For the first time, we identified the nucleotide sequence of C. burnetii from a 32-year-old man with an acute febrile illness in Korea. Diagnosis of acute Q fever was confirmed by seroconversion using indirect immunofluorescence antibody assays. Phylogenetic analysis demonstrated high sequence similarity (99.6%-100%) with C. burnetii 16S rRNA sequences identified from the reservoir. These results are the first genetic analysis of C. burnetii in a human case of Q fever in Korea.

Anti-Prion Screening for Acridine, Dextran, and Tannic Acid using Real Time-Quaking Induced Conversion: A Comparison with PrPSc-Infected Cell Screening.

Prion propagation is mediated by the structural alteration of normal prion protein (PrPC) to generate pathogenic prion protein (PrPSc). To date, compounds for the inhibition of prion propagation have mainly been screened using PrPSc-infected cells. Real time-quaking-induced conversion (RT-QuIC) is one alternative screening method. In this study, we assessed the propagation inhibition effects of known anti-prion compounds using RT-QuIC and compared the results with those from a PrPSc-infected cell assay. Compounds were applied to RT-QuIC reactions at 0 h or 22 h after prion propagation to determine whether they inhibited propagation or reduced amplified aggregates. RT-QuIC reactions in presence of acridine, dextran sulfate sodium (DSS), and tannic acid inhibited seeded aggregation with sporadic Creutzfeldt-Jakob disease at 0 h. After treatment at 22 h, amplified fluorescence was decreased in wells treated with either acridine or tannic acid. Compound activities were verified by western blot of RT-QuIC products and in a dye-independent conversion assay, the Multimer Detection System. Protease K-resistant PrPSc fragments (PrPres) were reduced by DSS and tannic acid in the PrPSc-infected cell assay. Importantly, these inhibitory effects were similar despite different treatment times (0 h versus 3 days). Consequentially, RT-QuIC enabled the more specific classification of compounds according to action (i.e., inhibition of prion propagation versus reduction of amplified aggregates). RT-QuIC addresses the limitations of cell-based screening methods and can be used to further aid our understanding of the mechanisms of action of anti-prion compounds.

Molecular detection of Rickettsia species in ticks collected from the southwestern provinces of the Republic of Korea.

BACKGROUND: Rickettsiae constitute a group of arthropod-borne, Gram-negative, obligate intracellular bacteria that are the causative agents of diseases ranging from mild to life threatening that impact on medical and veterinary health worldwide. METHODS: A total of 6,484 ticks were collected by tick drag from June-October 2013 in the southwestern provinces of the Republic of Korea (ROK) (Jeollanam, n = 3,995; Jeollabuk, n = 680; Chungcheongnam, n = 1,478; and Chungcheongbuk, n = 331). Ticks were sorted into 311 pools according to species, collection site, and stage of development. DNA preparations of tick pools were assayed for rickettsiae by 17 kDa antigen gene and ompA nested PCR (nPCR) assays and the resulting amplicons sequenced to determine the identity and prevalence of spotted fever group rickettsiae (SFGR). RESULTS: Haemaphysalis longicornis (4,471; 52 adults, 123 nymphs and 4,296 larvae) were the most commonly collected ticks, followed by Haemaphysalis flava (1,582; 28 adults, 263 nymphs and 1,291 larvae), and Ixodes nipponensis (431; 25 adults, 5 nymphs and 401 larvae). The minimum field infection rate/100 ticks (assuming 1 positive tick/pool) was 0.93% for the 17 kDa antigen gene and 0.82% for the ompA nPCR assays. The partial 17 kDa antigen and ompA gene sequences from positive pools of H. longicornis were similar to: Rickettsia sp. HI550 (99.4-100%), Rickettsia sp. FUJ98 (99.3-100%), Rickettsia sp. HIR/D91 (99.3-100%), and R. japonica (99.7%). One sequence of the partial 17 kDa antigen gene for H. flava was similar to Rickettsia sp. 17kd-005 (99.7%), while seven sequences of the 17 kDa antigen gene obtained from I. nipponensis ticks were similar to R. monacensis IrR/Munich (98.7-100%) and Rickettsia sp. IRS3 (98.9%). CONCLUSIONS: SFG rickettsiae were detected in three species of ixodid ticks collected in the southwestern provinces of the ROK during 2013. A number of rickettsiae have been recently reported from ticks in Korea, some of which were identified as medically important. Results from this study and previous reports demonstrate the need to conduct longitudinal investigations to identify tick-borne rickettsiae and better understand their geographical distributions and potential impact on medical and veterinary health, in addition to risk communication and development of rickettsial disease prevention strategies.

Impact of Vancomycin MIC on Treatment Outcomes in Invasive Staphylococcus aureus Infections.

There are conflicting data on the association of vancomycin MIC (VAN-MIC) with treatment outcomes in Staphylococcus aureus infections. We investigated the relationship between high VAN-MIC and 30-day mortality and identified the risk factors for mortality in a large cohort of patients with invasive S. aureus (ISA) infections, defined as the isolation of S. aureus from a normally sterile site. Over a 2-year period, 1,027 adult patients with ISA infections were enrolled in 10 hospitals, including 673 (66%) patients with methicillin-resistant S. aureus (MRSA) infections. There were 200 (19.5%) isolates with high VAN-MIC (≥1.5 mg/liter) by Etest and 87 (8.5%) by broth microdilution (BMD). The all-cause 30-day mortality rate was 27.4%. High VAN-MIC by either method was not associated with all-cause 30-day mortality, and this finding was consistent across MIC methodologies and methicillin susceptibilities. We conclude that high VAN-MIC is not associated with increased risk of all-cause 30-day mortality in ISA infections. Our data support the view that VAN-MIC alone is not sufficient evidence to change current clinical practice.

White matter hyperintensities on brain magnetic resonance imaging: comparison of early-onset and late-onset restless legs syndrome.

OBJECTIVE: Previous studies have suggested that early-onset RLS (EOR) and late-onset RLS (LOR) might have different etiopathophysiologies. Few previous studies have examined accumulation of cerebrovascular ischemic changes as a potential cause of LOR. METHODS: We recruited 39 RLS subjects (LOR: defined as age of RLS onset ≥45, n = 18 and EOR: age of onset <45; n = 21); and 39 healthy control subjects matched on age and sex. Structural magnetic resonance imaging (MRI) of the brain was performed for each subject, and images were graded for severity of periventricular white matter hyperintensities (PVH) and deep white matter hyperintensities (DWMH) independently by trained raters according to standardized methods. RESULTS: Interrater reliabilities were 0.861 (p <0.001) for PVH and 0.900 (p <0.001) for DWMH. LOR subjects had a significantly higher grade of DWMH than the EOR subjects (p = 0.043) and age- and sex-matched controls (p = 0.015). In contrast, there was no difference in DWMH severity rating between the EOR group and the EC group or in PVH severity between the LOR or EOR groups and their age-matched controls. CONCLUSION: Our findings suggest that the presence and severity of DWMH is associated with LOR, but not with EOR. Further examination of the contribution of cerebrovascular disease to the etiopathogenesis of LOR is warranted.