Kyu Jam Hwang

Prion Diseases as Transmissible Zoonotic Diseases

n Abstractn n Prion diseases, also called transmissible spongiform encephalopathies (TSEs), lead to neurological dysfunction in animals and are fatal. Infectious prion proteins are causative agents of many mammalian TSEs, including scrapie (in sheep), chronic wasting disease (in deer and elk), bovine spongiform encephalopathy (BSE; in cattle), and Creutzfeldt–Jakob disease (CJD; in humans). BSE, better known as mad cow disease, is among the many recently discovered zoonotic diseases. BSE cases were first reported in the United Kingdom in 1986. Variant CJD (vCJD) is a disease that was first detected in 1996, which affects humans and is linked to the BSE epidemic in cattle. vCJD is presumed to be caused by consumption of contaminated meat and other food products derived from affected cattle. The BSE epidemic peaked in 1992 and decreased thereafter; this decline is continuing sharply owing to intensive surveillance and screening programs in the Western world. However, there are still new outbreaks and/or progression of prion diseases, including atypical BSE, and iatrogenic CJD and vCJD via organ transplantation and blood transfusion. This paper summarizes studies on prions, particularly on prion molecular mechanisms, BSE, vCJD, and diagnostic procedures. Risk perception and communication policies of the European Union for the prevention of prion diseases are also addressed to provide recommendations for appropriate government policies in Korea.n n

Discovery of Novel Anti-prion Compounds Using In Silico and In Vitro Approaches

Prion diseases are associated with the conformational conversion of the physiological form of cellular prion protein (PrPC) to the pathogenic form, PrPSc. Compounds that inhibit this process by blocking conversion to the PrPSc could provide useful anti-prion therapies. However, no suitable drugs have been identified to date. To identify novel anti-prion compounds, we developed a combined structure- and ligand-based virtual screening system in silico. Virtual screening of a 700,000-compound database, followed by cluster analysis, identified 37 compounds with strong interactions with essential hotspot PrP residues identified in a previous study of PrPC interaction with a known anti-prion compound (GN8). These compounds were tested in vitro using a multimer detection system, cell-based assays, and surface plasmon resonance. Some compounds effectively reduced PrPSc levels and one of these compounds also showed a high binding affinity for PrPC. These results provide a promising starting point for the development of anti-prion compounds.

Epidemiological Characteristics of Serologically Confirmed Q Fever Cases in South Korea, 2006–2011

Objectives: Q fever has been reported worldwide; however, there was almost no official report of Q fever in Korea. In this study, we describe the current status of human Q fever occurrence in Korea. Methods: Demographic data of Q fever patients were collected from the National Notifiable Diseases Surveillance System from 2006 to 2011. Case investigation reports from regional public health departments were used for additional information, like risk factors and clinical manifestation, of the patients since 2008. Results: There were 65 serologically confirmed cases during the study period. The annual notification rate of Q fever was 0.22 cases per million persons. The majority of cases were men (87.7%), adults (98.5%), and urban inhabitants (67.7%). Relevant exposures to risk factors were identified in 45.7% of patients. The most common symptoms of acute Q fever were fever (89.3%), myalgia (67.9%) and asthenia (53.6%). Two cases with endocarditis were identified in chronic Q fever. Conclusion: This study suggests that Q fever has a low endemicity in Korea. However, management and research at national level is required for prevention of a future epidemic.

Autochthonous Lyme Borreliosis in Humans and Ticks in Korea

Objective: This study aimed at finding epidemiological and clinical features of autochthonous Lyme borreliosis in humans through epidemiological investigations and identifying its vectors and pathogens through analysis of ticks. Method: Epidemiological investigations, including review of the retrospective medical records and patient interviews, were conducted in two cases that occurred in 2012. To identify the vectors and pathogens, ticks were collected between September 23 and October 6, 2012 from the area where the tick bite in the first patient occurred. The ticks were classified, and polymerase chain reaction (PCR) tests and cultures were performed. Results: The first patient, a 46-year-old female, visited a forest in Gangwon province, which was 900 m above sea level, where the tick bite occurred. Two weeks after the tick bite, erythema migrans (12 × 6 cm2 in size) appeared on the site of tick bite, along with fever, chill, fatigue, myalgia, and arthralgia on shoulders, knees, and hips. The second patient, a 44-year-old male, visited a mountain in Gangwon province, which was 1200 m above sea level, where a tick bite occurred. One month after the tick bite, erythema migrans appeared at the site of the tick bite, along with fatigue, myalgia, and arthralgia on the right shoulder and temporomandibular joint. Indirect fluorescent antibody testing and Western blotting were carried out in these two cases for diagnosis, and positive findings were obtained. As a result, Lyme borreliosis could be confirmed. To estimate the pathogens and vectors, the ticks were collected. A total of 122 ticks were collected and only two species, Haemaphysalis japonica and Haemaphysalis flava, were identified. PCR and culture were performed on ticks. However, Borrelia burgdo rferi sensu lato was not isolated from any collected ticks. Conclusions: This study is significant to confirm Lyme borreliosis officially at first by the national surveillance system, although identification of the mites and pathogens failed.

Alternative application of Tau protein in Creutzfeldt-Jakob disease diagnosis: Improvement for weakly positive 14-3-3 protein in the laboratory.

The 14-3-3 protein has been used as a biomarker for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). However, weakly positive 14-3-3 leads to false positive results and an incorrect diagnosis. We attempted to use quantitative data for tau protein to provide an accurate diagnosis based on weak 14-3-3 protein. Sixty-two patients with sCJD, including pathologically confirmed, clinically definite, and probable cases, and 89 non-CJD patients were investigated based on a Korean population. Among them, 20 sCJD and 14 non-CJD showed weakly positive 14-3-3. The total tau (t-tau) and phosphorylated tau (p-tau) protein levels were measured by ELISA, and the p-tau to t-tau ratio (p/t ratio) was calculated. The combined use of the 14-3-3 protein assay, t-tau levels, and p/t ratio improved the specificity of diagnosis compared with the use of the 14-3-3 protein assay alone (47% for 14-3-3 alone; 85.94% for 14-3-3 combined with t-tau; 90.62% for 14-3-3 combined with the p/t ratio). In addition, 18 of 20 sCJD and 12 of 14 non-CJD who were weakly positive for 14-3-3 were positive for the p/t ratio and negative for the p/t ratio, respectively. When used in combination with the 14-3-3 protein, the tau protein is useful as a biomarker for the precise diagnosis of sCJD.

Biological network inferences for a protection mechanism against familial Creutzfeldt-Jakob disease with E200K pathogenic mutation

BackgroundHuman prion diseases are caused by abnormal accumulation of misfolded prion protein in the brain tissue. Inherited prion diseases, including familial Creutzfeldt-Jakob disease (fCJD), are associated with mutations of the prion protein gene (PRNP). The glutamate (E)-to-lysine (K) substitution at codon 200 (E200K) in PRNP is the most common pathogenic mutation causing fCJD, but the E200K pathogenic mutation alone is regarded insufficient to cause prion diseases; thus, additional unidentified factors are proposed to explain the penetrance of E200K-dependent fCJD. Here, exome differences and biological network analysis between fCJD patients with E200K and healthy individuals, including a non-CJD individual with E200K, were analysed to gain new insights into possible mechanisms for CJD in individuals carrying E200K.MethodsExome sequencing of the three CJD patients with E200K and 11 of the family of one patient (case1) were performed using the Illumina HiSeq 2000. The exome sequences of 24 Healthy Koreans were used as control. The bioinformatic analysis of the exome sequences was performed using the CLC Genomics Workbench v5.5. Sanger sequencing for variants validation was processed using a BigDye Terminator Cycle Sequencing Kit and an ABI 3730xl automated sequencer. Biological networks were created using Cytoscape (v2.8.3 and v3.0.2) and Pathway Studio 9.0 software.ResultsNineteen sites were only observed in healthy individuals. Four proteins (NRXN2, KLKB1, KARS, and LAMA3) that harbour rarely observed single-nucleotide variants showed biological interactions that are associated with prion diseases and/or prion protein in our biological network analysis.ConclusionThrough this study, we confirmed that individuals can have a CJD-free life, even if they carry a pathogenic E200K mutation. Our research provides a possible mechanism that involves a candidate protective factor; this could be exploited to prevent fCJD onset in individuals carrying E200K.

Dermabacter jinjuensis sp. nov., a novel species of the genus Dermabacter isolated from a clinical specimen.

A Gram-stain-positive, catalase-positive, facultatively anaerobic, non-motile, coryneform bacterium, designated strain 32T, was isolated from a closed pus sample from a patient having finger necrosis in Korea. Strain 32T was considered as representing a novel species according to its initial identification by matrix-assisted laser desorption/ionization-time-of-flight MS. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 32T belonged to the genus Dermabacter and was closely related to Dermabacter hominis DSM 7083T (=ATCC 49369T) (98.34u2009% similarity). Optimal growth was observed at 30-40u2009°C and pH 7. Growth occurred in the presence of 0-6u2009% (w/v) NaCl. Menaquinones MK-8, MK-7 and MK-9 were the major respiratory quinones. The major polar lipids were phosphatidylethanolamine, phosphatidylcholine, glycolipid and two unknown lipids. The major cellular fatty acids were anteiso-C17u2009:u20090, anteiso-C15u2009:u20090, iso-C16u2009:u20090 and iso-C15u2009:u20090. The DNA G+C content of strain 32T was 62.58 mol%, and the mean level of DNA-DNA relatedness between strain 32T and D. hominis ATCC 49369T was 49±1.6u2009%. Based on the phenotypic and genotypic characteristics, strain 32T is confirmed to represent a novel species of the genus Dermabacter, for which the name Dermabacter jinjuensis sp. nov. is proposed. The type strain is 32T (=NCCP 16133T=DSM 101003T).

Taxonomic Identification of Bacillus Species Using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry

Background: In this study, we compared various methods of taxonomic identification of Bacillus strains: biochemical methods, 16S rRNA gene sequencing, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). We also developed a pathogenisolate resource database, thus increasing the identification rate when using MALDI-TOF MS. Methods: Thirty Bacillus strains were obtained from the NCCP (National Culture Collection for Pathogens) and were identified using the VITEK 2 system (bioMérieux, France), API kit (bioMérieux, France), 16S rRNA gene sequencing, and MALDI-TOF MS. The pathogenicity of Bacillus cereus was confirmed through the identification of virulent genes using a multiplex PCR, and both protein extraction for protein profiling in MALDI-TOF MS and repetitive-sequence fingerprinting were performed. Results: The identification rates at the species level were 40%, 80%, and 76.3% for the VITEK 2 system (bioMérieux), 16S rRNA gene sequencing, and MALDITOF MS, respectively. When the major spectrumprofiling dendrogram was compared with the phylogenetic tree, which was constructed based on the 16S rRNA gene sequences and rep-PCR fingerprinting, the classifications were confirmed to be effective. Conclusion: Identification of Bacillus strains using MALDI-TOF MS was more effective than that using the VITEK 2 system (bioMérieux), but was similar to that using 16S rRNA gene sequencing. Continual addition to a proteome-based database can result in increased identification rates for MALDI-TOF MS. (Ann Clin Microbiol 2016;19:110-120)

The first case of hand infection caused by Dermabacter jinjuensis in a symmetrical peripheral gangrene patient

Introduction Strains of the genus Dermabacter is a recently established species, recognized as relatively rare opportunistic human pathogen, and is infrequently isolated from clinical specimens, including blood cultures, abscesses, wounds, bone, eye, and skin. Presentation of case We present a 78-year old female with chronic symmetrical peripheral gangrene and hand infection. The patient underwent surgical debridement with amputation on gangrene with infection of both fingers. At 2 weeks postoperatively, pus discharge was newly observed and the patient underwent reoperation. In the subsequent reinfection, unknown organism has been repeatedly identified, may be the most likely causative agent. On the basis of phenotypic and genotypic distinctness and DNA–DNA hybridization results, new strain should be placed in the genus Dermabacter as representing a novel species, for which the name Dermabacter jinjuensis sp. nov. is proposed. Discussion We judged the novel species as the causative bacteria. Because of, a novel species called D. jinjuensis was repeatedly identified more than common bacteria. It can be considered as a postoperative nosocomial infection or opportunistic infection. It is not clear how the infection of D. jinjensis occurred. Conclusion This is the first reported case of a human D. jinjuensis infection. We were able to treat patients without any complications by operative treatment and administering appropriate antimicrobial agents according to antibiotics susceptibility test.

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.