Early Laboratory Preparedness of the Korea Disease Control and Prevention Agency and Response to Unknown Pneumonia Outbreak from Wuhan, China, in January 2020.

We report the response process of the Laboratory Analysis Task Force (LATF) for Unknown Disease Outbreaks (UDOs) at the Korea Disease Control and Prevention Agency (KDCA) during January 2020 to coronavirus disease 2019 (COVID-19), which developed as a UDO in Korea. The advanced preparedness offered by the laboratory diagnostic algorithm for UDOs related to respiratory syndromes was critical for the rapid identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and enabled us to establish and expand the diagnostic capacity for COVID-19 on a national scale in a timely manner.

Genetic Characterization of Clostridium botulinum Isolated from the First Case of Infant Botulism in Korea.

Botulism is a neuroparalytic disease caused by a neurotoxin produced by Clostridium botulinum. This study aimed to genetically characterize C. botulinum strain isolated from the first case of infant botulism in Korea reported on June 17, 2019. We isolated C. botulinum strain CB-27 from a stool sample of the patient and analyzed the toxin types and toxin gene cluster compositions of the strain using a mouse bioassay, real-time PCR, and genome sequencing. Toxin gene cluster analysis showed that strain CB-27 possesses a C. botulinum neurotoxin type A harboring an unexpressed B gene. Although the nucleotide and amino acid sequences of toxin genes as well as the toxin gene cluster arrangements in strain CB-27 were identical to those of the known strain CDC_69094, the total nucleotide sequences of the toxin gene clusters of CB-27 differed from those of CDC_69094 by 0.47%, indicating genetic diversity of toxin gene clusters of CB-27 among other previously reported C. botulinum strains. To our knowledge, this is the first description of a C. botulinum strain with two separate toxin gene clusters in Korea.

2019 Tabletop Exercise for Laboratory Diagnosis and Analyses of Unknown Disease Outbreaks by the Korea Centers for Disease Control and Prevention.

OBJECTIVES: The Korea Centers for Disease Control and Prevention has published "A Guideline for Unknown Disease Outbreaks (UDO)." The aim of this report was to introduce tabletop exercises (TTX) to prepare for UDO in the future. METHODS: The UDO Laboratory Analyses Task Force in Korea Centers for Disease Control and Prevention in April 2018, assigned unknown diseases into 5 syndromes, designed an algorithm for diagnosis, and made a panel list for diagnosis by exclusion. Using the guidelines and laboratory analyses for UDO, TTX were introduced. RESULTS: Since September 9th, 2018, the UDO Laboratory Analyses Task Force has been preparing TTX based on a scenario of an outbreak caused by a novel coronavirus. In December 2019, through TTX, individual missions, epidemiological investigations, sample treatments, diagnosis by exclusions, and next generation sequencing analysis were discussed, and a novel coronavirus was identified as the causal pathogen. CONCLUSION: Guideline and laboratory analyses for UDO successfully applied in TTX. Conclusions drawn from TTX could be applied effectively in the analyses for the initial response to COVID-19, an ongoing epidemic of 2019 - 2020. Therefore, TTX should continuously be conducted for the response and preparation against UDO.

A waterborne outbreak of multiple diarrhoeagenic Escherichia coli infections associated with drinking water at a school camp.

BACKGROUND: In June 2015, a local public health laboratory was notified that students had developed gastroenteritis symptoms after attending a camp. An outbreak investigation was conducted to determine the extent and cause of the outbreak. METHOD: A retrospective cohort study was conducted to determine the correlations between the illness and specific exposures at the school camp. All attendees were interviewed with a standard questionnaire that addressed clinical symptoms, food consumption, and environmental exposures. Clinical specimens were cultured using standard microbiological methods for bacterial and viral pathogens. The genetic relationships of all isolates were determined using pulsed-field gel electrophoresis (PFGE). RESULTS: A total 188 patients with symptoms of diarrhoea, abdominal pain, and nausea were identified. The completed questionnaires suggested that the consumption of drinking water was likely to be linked to this outbreak. Using microbiological methods, enterohaemorrhagic Escherichia coli, enteropathogenic E. coli, and enteroaggregative E. coli were isolated, and the isolates from both patient stool and environmental water samples displayed indistinguishable XbaI-PFGE patterns. The water system in the camp used groundwater drawn from a private underground reservoir for cooking and drinking. The environmental investigation revealed some problems with the water supply system, such as the use of inappropriate filters in the water purifier and a defect in the pipeline between the reservoir and the chlorination device. CONCLUSIONS: This outbreak points to the importance of drinking water quality management in group facilities where underground water is used and emphasizes the need for periodic sanitation and inspection to prevent possible waterborne outbreaks.

Outbreak of CTX-M-15-Producing Enterotoxigenic Escherichia coli O159:H20 in the Republic of Korea in 2016.

We investigated an outbreak of enterotoxigenic Escherichia coli (ETEC) O159:H20 associated with the consumption of a tossed-noodle dish in a high school in 2016. Thirty-three ETEC strains isolated from clinical and food samples were genetically indistinguishable. The outbreak strains were resistant to third-generation cephalosporins and harbored a blaCTX-M-15 gene on a 97-kb self-transferable IncK plasmid. This is the first outbreak caused by CTX-M-15-producing ETEC strains.

Bidirectional cross-regulation between ErbB2 and ß-adrenergic signalling pathways.

AIMS: Despite the observation that ErbB2 regulates sensitivity of the heart to doxorubicin or ErbB2-targeted cancer therapies, mechanisms that regulate ErbB2 expression and activity have not been studied. Since isoproterenol up-regulates ErbB2 in kidney and salivary glands and ß2AR and ErbB2 complex in brain and heart, we hypothesized that ß-adrenergic receptors (AR) modulate ErbB2 signalling status. METHODS AND RESULTS: ErbB2 transfection of HEK293 cells up-regulates ß2AR, and ß2AR transfection of HEK293 up-regulates ErbB2. Interestingly, cardiomyocytes isolated from myocyte-specific ErbB2-overexpressing (ErbB2(tg)) mice have amplified response to selective ß2-agonist zinterol, and right ventricular trabeculae baseline force generation is markedly reduced with ß2-antagonist ICI-118 551. Consistently, receptor binding assays and western blotting demonstrate that ß2ARs levels are markedly increased in ErbB2(tg) myocardium and reduced by EGFR/ErbB2 inhibitor, lapatinib. Intriguingly, acute treatment of mice with ß1- and ß2-AR agonist isoproterenol resulted in myocardial ErbB2 increase, while inhibition with either ß1- or ß2-AR antagonist did not completely prevent isoproterenol-induced ErbB2 expression. Furthermore, inhibition of ErbB2 kinase predisposed mice hearts to injury from chronic isoproterenol treatment while significantly reducing isoproterenol-induced pAKT and pERK levels, suggesting ErbB2's role in transactivation in the heart. CONCLUSION: Our studies show that myocardial ErbB2 and ßAR signalling are linked in a feedback loop with ßAR activation leading to increased ErbB2 expression and activity, and increased ErbB2 activity regulating ß2AR expression. Most importantly, ErbB2 kinase activity is crucial for cardioprotection in the setting of ß-adrenergic stress, suggesting that this mechanism is important in the pathophysiology and treatment of cardiomyopathy induced by ErbB2-targeting antineoplastic drugs.

Tumorigenicity of hypoxic respiring cancer cells revealed by a hypoxia-cell cycle dual reporter.

Although aerobic glycolysis provides an advantage in the hypoxic tumor microenvironment, some cancer cells can also respire via oxidative phosphorylation. These respiring ("non-Warburg") cells were previously thought not to play a key role in tumorigenesis and thus fell from favor in the literature. We sought to determine whether subpopulations of hypoxic cancer cells have different metabolic phenotypes and gene-expression profiles that could influence tumorigenicity and therapeutic response, and we therefore developed a dual fluorescent protein reporter, HypoxCR, that detects hypoxic [hypoxia-inducible factor (HIF) active] and/or cycling cells. Using HEK293T cells as a model, we identified four distinct hypoxic cell populations by flow cytometry. The non-HIF/noncycling cell population expressed a unique set of genes involved in mitochondrial function. Relative to the other subpopulations, these hypoxic "non-Warburg" cells had highest oxygen consumption rates and mitochondrial capacity consistent with increased mitochondrial respiration. We found that these respiring cells were unexpectedly tumorigenic, suggesting that continued respiration under limiting oxygen conditions may be required for tumorigenicity.

Clinical and epidemiological features of hepatitis C virus infection in South Korea: a prospective, multicenter cohort study.

The epidemiological and clinical features of hepatitis C virus (HCV) infection in South Korea were examined in a prospective, multicenter cohort study that included 1,173 adult patients with positive results for anti-HCV antibody who completed a questionnaire survey on the risk factors for HCV infection from January 2007 to December 2011 at five university hospitals. The HCV cohort had a mean age of 55.4 years with 48.3% men, and diagnostic categories of acute hepatitis (n = 63, 5.3%), past infection (n = 37, 3.2%), chronic hepatitis (n = 777, 66.2%), cirrhosis of the liver (n = 179, 15.3%), and hepatocellular carcinoma (n = 117, 10.0%). The major HCV genotypes were genotype 1 (52.7%) and genotype 2 (45.3%). Liver biopsy was performed in 301 patients (25.7%), and 42.8% of the subjects received antiviral therapy against HCV. The behavioral risk factors possibly related to HCV infection were intravenous drug use (5%), needle stick injury (7%), blood transfusion before 1995 (19%), sexual relationship with more than three partners (28%), piercings (35%), tattoos (36%), surgery (43%), acupuncture (83%), diagnostic endoscopy (85%), and dental procedures (93%). Age, intravenous drug use, needle stick injury, transfusion before 1995, and tattoos were the independent risk factors of HCV infection.

Clinical and epidemiological characteristics of Korean patients with hepatitis C virus genotype 6.

BACKGROUND/AIMS: The distribution of hepatitis C virus (HCV) genotypes varies geographically. In Korea, genotypes 1 and 2 comprise more than 90% of HCV infections, while genotype 6 is very rare. This study compared the clinical and epidemiological characteristics of patients with genotype 6 HCV infection with those infected with HCV genotypes 1 and 2. METHODS: This was a prospective, multicenter HCV cohort study that enrolled 1,173 adult patients, of which 930 underwent HCV genotype analysis, and only 9 (1.0%) were found to be infected with genotype 6 HCV. The clinical and epidemiological parameters of the genotypes were compared. RESULTS: The patients with genotype 6 HCV had a mean age of 41.5 years, 77.8% were male, and they had no distinct laboratory features. A sustained virologic response (SVR) was observed in four (67%) of six patients who received antiviral therapy. Risk factors such as the presence of a tattoo (n=6, 66.7%), more than three sexual partners (n=3, 33.3%), and injection drug use (n=3, 33.3%) were more common among genotype 6 patients than among genotypes 1 or 2. CONCLUSIONS: The epidemiology and treatment response of patients infected with genotype 6 HCV differed significantly from those with genotypes 1 or 2, warranting continuous monitoring.

Risk factors for neurologic complications of hand, foot and mouth disease in the Republic of Korea, 2009.

In 2009, the first outbreak of hand, foot and mouth disease (HFMD) or herpangina (HP) caused by enterovirus 71 occurred in the Republic of Korea. This study inquired into risk factors associated with complications of HFMD or HP. A retrospective medical records review was conducted on HFMD or HP patients for whom etiologic viruses had been verified in 2009. One hundred sixty-eight patients were examined for this investigation. Eighty patients were without complications while 88 were accompanied by complications, and 2 had expired. Enterovirus 71 subgenotype C4a was the most prevalent in number with 67 cases (54.9%). In the univariate analysis, the disease patterns of HFMD rather than HP, fever longer than 4 days, peak body temperature over 39℃, vomiting, headache, neurologic signs, serum glucose over 100 mg/dL, and having an enterovirus 71 as a causative virus were significant risk factors of the complications. After multiple logistic analysis, headache (Odds ratio [OR], 10.75; P < 0.001) and neurologic signs (OR, 42.76; P < 0.001) were found to be the most significant factors. Early detection and proper management of patients with aforementioned risk factors would be necessary in order to attain a better clinical outcome.

Echovirus 30 induced neuronal cell death through TRIO-RhoA signaling activation.

BACKGROUND: Echovirus 30 (Echo30) is one of the most frequently identified human enteroviruses (EVs) causing aseptic meningitis and encephalitis. However the mechanism underlying the pathogenesis of Echo30 infection with significant clinical outcomes is not completely understood. The aim of this investigation is to illustrate molecular pathologic alteration in neuronal cells induced by Echo30 infection using clinical isolate from young patient with neurologic involvement. METHODOLOGY/PRINCIPAL FINDINGS: To characterize the neuronal cellular response to Echo30 infection, we performed a proteomic analysis based on two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF Mass Spectrophotometric (MS) analysis. We identified significant alteration of several protein expression levels in Echo30-infected SK-N-SH cells. Among these proteins, we focused on an outstanding up-regulation of Triple functional domain (TRIO) in Echo30-infected SK-N-SH cells. Generally, TRIO acts as a key component in the regulation of axon guidance and cell migration. In this study, we determined that TRIO plays a role in the novel pathways in Echo30 induced neuronal cell death. CONCLUSIONS/SIGNIFICANCE: Our finding shows that TRIO plays a critical role in neuronal cell death by Echo30 infection. Echo30 infection activates TRIO-guanine nucleotide exchange factor (GEF) domains (GEFD2) and RhoA signaling in turn. These results suggest that Echo30 infection induced neuronal cell death by activation of the TRIO-RhoA signaling. We expect the regulation of TRIO-RhoA signaling may represent a new therapeutic approach in treating aseptic meningitis and encephalitis induced by Echo30.

Enterovirus 71-associated hand, foot and mouth diseases with neurologic symptoms, a university hospital experience in Korea, 2009.

PURPOSE: Hand-foot-mouth disease (HFMD) is a common viral illness in children, which is usually mild and self-limiting. However, in recent epidemics of HFMD in Asia, enterovirus 71 (EV71) has been recognized as a causative agent with severe neurological symptoms with or without cardiopulmonary involvement. HFMD was epidemic in Korea in the spring of 2009. Severe cases with complications including death have been reported. The clinical characteristics in children with neurologic manifestations of EV71 were studied in Ewha Womans University Mokdong Hospital. METHODS: Examinations for EV71 were performed from the stools, respiratory secretion or CSF of children who presented neurologic symptoms associated with HFMD by realtime PCR. Clinical and radiologic data of the patients were collected and analyzed. RESULTS: EV71 was isolated from the stool of 16 patients but not from respiratory secretion or CSF. Among the 16 patients, meningitis (n=10) was the most common manifestation, followed by Guillain-Barré syndrome (n=3), meningoencephalitis (n=2), poliomyelitis-like paralytic disease (n=1), and myoclonus (n=1). Gene analysis showed that most of them were caused by EV71 subgenotype C4a, which was prevalent in China in 2008. CONCLUSION: Because EV71 causes severe complications and death in children, a surveillance system to predict upcoming outbreaks should be established and maintained and adequate public health measures are needed to control disease.

ApoE-epsilon 4-dependent association of the choline acetyltransferase gene polymorphisms (2384G>A and 1882G>A) with Alzheimer's disease.

BACKGROUND: One of the characteristic features of Alzheimer's disease (AD) is the degeneration of the cholinergic system. The gene encoding choline acetyltransferase (ChAT), a key enzyme in cholinergic function, is a candidate gene conferring risk for AD. But the genetic association of the enzyme with AD has been controversial. We analyzed 2 ChAT single nucleotide polymorphisms (SNPs), 2384G>A (rs3810950; Ala120Thr) and 1882G>A (rs1880676; Asp7Asn) and the ApoE polymorphisms in Korean population. METHODS: The samples from 316 AD patients and 264 age-matched healthy controls were analyzed. The differences in genotype frequencies were assessed. RESULTS: The 2 ChAT SNPs were almost completely linked with each other (r2=0.99, |D'|=1.0). No significant difference in the ChAT genotype distribution was observed between the patients and the controls. However, in non-ApoE-epsilon4 allele carriers, multiple logistic regression analysis showed that both the GA and the GA/AA genotypes were associated with AD (OR=1.639, 95% CI, 1.050-2.559, p=0.0297 for GA; OR=1.630, 95% CI, 1.049-2.532, p=0.0297 for GA/AA), suggesting a dominant effect of A allele. CONCLUSION: There is considerable effect of the ChAT polymorphisms on AD in Korean population and this effect is dependent on ApoE genotypes.

Cyclic AMP-dependent protein kinase A and CREB are involved in neuregulin-induced synapse-specific expression of acetylcholine receptor gene.

Neuregulin is reported to stimulate synapse-specific transcription of acetylcholine receptor (AChR) genes in the skeletal muscle fiber by multiple signaling pathways such as ERK, PI3K, and JNK. The co-localization of PKA mRNA with AChR and ErbBs, receptors for neuregulin, at the confined region of synapse implicates the putative role of PKA in neuregulin-induced AChR gene expression. In the present study, we found that mRNA and protein of a regulatory subunit of PKA (PKARIalpha) were concentrated at synaptic sites of the rat sternomastoid muscle fiber, while those of ERK and PI3K were uniformly distributed throughout the muscle fiber. Neuregulin (100 ng/ml) increased both PKA activity in the nucleus and AChRdelta subunit gene transcription in cultured Sol8 myotubes. These increases were significantly blocked by a specific PKA inhibitor H-89 (100 nM) and an adenylcyclase inhibitor SQ 22536 (200 microM) (72.5% and 60.1%, respectively). Furthermore, neuregulin phosphorylated CREB, a well-known down-stream transcription factor of PKA. While H-89 inhibited CREB phosphorylation, H-89 and PD098059 (50 microM), a specific MEK1/2 inhibitor, did not inhibit the phosphorylation of ERK and CREB, respectively, suggesting no cross-talk between PKA and ERK pathways. In conclusion, neuregulin increases AChRdelta subunit gene transcription, in part, by the activation of PKA/CREB, an alternative route to the previously reported ERK signaling pathway.