Genomic Analysis of Monkeypox Virus During the 2023 Epidemic in Korea.

We aimed to characterize the genomes of monkeypox virus isolates from the Far East, providing insights into viral transmission and evolution. Genomic analysis was conducted on 8 isolates obtained from patients with monkeypox virus disease in the Republic of Korea between May 2022 and early 2023. These isolates were classified into Clade IIb. Distinct lineages, including B.1.1, A.2.1, and B.1.3, were observed in 2022 and 2023 isolates, with only the B.1.3 lineage detected in six isolates of 2023. These genetic features were specific to Far East isolates (the Republic of Korea, Japan, and Taiwan), distinguishing them from the diverse lineages found in the Americas, Europe, Africa, and Oceania. In early 2023, the prevalence of the B.1.3 lineage of monkeypox virus identified in six patients with no overseas travel history is considered as an indicator of the potential initiation of local transmission in the Republic of Korea.

Prolonged viral shedding in an immunocompromised Korean patient infected with hMPXV, sub-lineage B.1.3, with acquired drug resistant mutations during tecovirimat treatment.

Following the worldwide surge in mpox (monkeypox) in 2022, cases have persisted in Asia, including South Korea, and sexual contact is presumed as the predominant mode of transmission, with a discernible surge in prevalence among immunocompromised patients. Drugs such as tecovirimat can result in drug-resistant mutations, presenting obstacles to treatment. This study aimed to ascertain the presence of tecovirimat-related resistant mutations through genomic analysis of the monkeypox virus isolated from a reported case involving prolonged viral shedding in South Korea. Here, tecovirimat-resistant mutations, previously identified in the B.1 clade, were observed in the B.1.3 clade, predominant in South Korea. These mutations exhibited diverse patterns across different samples from the same patient and reflected the varied distribution of viral subpopulations in different anatomical regions. The A290V and A288P mutant strains we isolated hold promise for elucidating these mechanisms, enabling a comprehensive analysis of viral pathogenesis, replication strategies, and host interactions. Our findings imply that acquired drug-resistant mutations, may present a challenge to individual patient treatment. Moreover, they have the potential to give rise to transmitted drug-resistant mutations, thereby imposing a burden on the public health system. Consequently, the meticulous genomic surveillance among immunocompromised patients, conducted in this research, assumes paramount importance.

Molecular Detection and Phylogenetic Analysis of Tick-Borne Encephalitis Virus from Ticks Collected from Cattle in Kyrgyzstan, 2023.

Ticks are important vectors of the tick-borne encephalitis virus (TBEV). In Kyrgyzstan, the livestock farming trade and nomadic lifestyle enable tick-borne diseases to be imported from neighboring countries, but there are few relevant studies. In this study, we collected 40 ticks from cattle in Kyrgyzstan. Molecular marker analysis identified the ticks as Ixodes persulcatus (97.5%; n = 39) and Haemaphysalis punctata (2.5%; n = 1). Real-time PCR screening revealed two ticks to be positive for TBEV, but only one tick was amplified using nested PCR targeting the TBEV envelope (E) and non-structure 5 (NS5) gene. The obtained sequences belonged to the TBEV Siberian subtype and phylogenetic tree analysis results confirmed that the virus was related to the Bosnia strain. We also performed next-generation sequencing, which confirmed the TBEV Siberian subtype. Continuous research and surveillance of TBEV in Kyrgyzstan are required to provide further information on tick-borne diseases.

Complete Genome Sequence of Monkeypox Virus Strain MPXV-ROK-P1-2022 Isolated from the First Monkeypox Patient in the Republic of Korea.

We report the complete genome sequence of the monkeypox virus strain MPXV-ROK-P1-2022, isolated from the first patient diagnosed with monkeypox in the Republic of Korea in June 2022. The virus was fully sequenced on the Illumina MiSeq instrument, and the phylogenetic tree showed that the strain belongs to lineage B.1.1.

Isolation and identification of monkeypox virus MPXV-ROK-P1-2022 from the first case in the Republic of Korea.

OBJECTIVES: Monkeypox outbreaks in nonendemic countries have been reported since early May 2022. The first case of monkeypox in the Republic of Korea was confirmed in a patient who traveled to Europe in June 2022, and an attempt was made to isolate and identify the monkeypox virus (MPXV) from the patient's specimens. METHODS: Clinical specimens from the patient were inoculated in Vero E6 cells. The isolated virus was identified as MPXV by the observation of cytopathic effects on Vero E6 cells, transmission electron microscopy, conventional polymerase chain reaction (PCR), and sequencing of PCR products. RESULTS: Cytopathic effects were observed in Vero E6 cells that were inoculated with skin lesion swab eluates. After multiple passages from the primary culture, orthopoxvirus morphology was observed using transmission electron microscopy. In addition, both MPXV-specific (F3L and ATI) and orthopoxvirus-specific genes (A39R, B2R, and HA) were confirmed by conventional PCR and Sanger sequencing. CONCLUSION: These results indicate the successful isolation and identification of MPXV from the first patient in the Republic of Korea. The isolated virus was named MPXV-ROK-P1-2022.

Characterization and validation of an alternative reference bacterium Korean Pharmacopoeia Staphylococcus aureus strain.

The National Culture Collection of Pathogens (NCCP) is a microbial resource bank in Korea that collects pathogen resources causing infectious disease in human and distributes them for research and education. The NCCP bank attempts to discover strains with various characteristics and specific purposes to provide diverse resources to researchers. Staphylococcus aureus American Type Culture Collection (ATCC) 6538P is used as a reference strain in the microbial assay for antibiotics in the Korean and in the United States Pharmacopoeias. We aimed to analyze domestically isolated microbial resources from the NCCP to replace the S. aureus reference strain. Staphylococcus aureus strains were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the VITEK-2 system and characterized by multilocus sequence typing, 16S rRNA sequencing, and antibiotic susceptibility testing. Several candidate strains had similar characteristics as the reference strain. Among them, the nucleotide sequence of the 16S rRNA region of NCCP 16830 was 100% identical to that of the reference strain; it was sensitive to six types of antibiotics and showed results most similar to the reference strain. A validity evaluation was conducted using the cylinder-plate method. NCCP 16830 presented valid results and had the same performance as ATCC 6538P; therefore, it was selected as an alternative candidate strain.

Draft Genome Sequence of Francisella tularensis subsp. holarctica Strain H0001, Isolated from a Tularemia Patient in the Republic of Korea.

Francisella tularensis is the etiological agent of the zoonosis tularemia. Here, we report the draft genome sequence of F. tularensis subsp. holarctica H0001, which was isolated from a tularemia patient in the Republic of Korea.

Evaluation of global evolutionary variations in the early stage of SARS-CoV-2 pandemic.

To understand the origin of variants and their evolutionary history in the early stage of the COVID-19 pandemic, time-scaled phylogenetic and gene variation analyses were performed. The mutation patterns and evolution characteristics were examined using the Bayesian Evolutionary Analysis Sampling Trees (BEAST) with 349 whole-genome sequences available by March 2020. The results revealed five phylogenetic clusters (Groups A-E), with 408 nucleotide variants. The mutations including the deletion of three nucleotides underwent various and complicated changes in the whole genome over time, while some frequency or transient mutations were also observed. Phylogenetic analysis demonstrated that SARS-CoV-2 originated from China and was transmitted to other Asian countries, followed by North America and Europe. This study could help to comprehensively understand the evolutionary characteristics of SARS-CoV-2 with a special emphasis on its global variation patterns.

Cohnella cholangitidis sp. nov., a novel species of the genus Cohnella isolated from a clinical specimen in Korea.

A Gram-positive, aerobic, rod-shaped bacterium, designated as strain 1605-214T, was isolated from the blood sample of a patient with cholangitis. Based on its 16S rRNA gene sequence, the strain 1605-214T belonged to the genus Cohnella and exhibited 97.9% sequence identity with Cohnella luojiensis DSM 24270T (GQ214052). DNA-DNA hybridization, digital DNA-DNA hybridization, and average nucleotide identity values between the two species were 23% ± 1.9, 21.1%, and 77.2%, respectively. The cellular fatty acids of strain 1605-214T were mainly comprised of anteiso-C15:0 (36.1%), iso-C16:0 (16.5%), and C16:0 (15.1%). The predominant quinone was menaquinone-7; predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and aminophospholipid-1. The cell wall peptidoglycan of strain 1605-214T contained meso-diaminopimelic acid. DNA G + C content of strain 1605-214T was 50.6 mol%. 5187 genes out of a total of 5413 (94.6%) were assigned putative functions using eggNOG v5.0. Based on genotypic characteristics and genomic sequence analysis results, strain 1605-214T was confirmed to represent a novel species of genus Cohnella, for which the name Cohnella cholangitidis sp. nov., was proposed.

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.

Complete Genome Sequences of Micrococcus luteus Strains NCCP 15687 and NCCP 16831, Isolated in South Korea.

In this study, the complete genome sequences of Micrococcus luteus strains NCCP 15687 and NCCP 16831 were determined and deposited in the National Culture Collection for Pathogens (NCCP) of South Korea. Genomic DNA was isolated from blood samples from patients infected with M. luteus.

CLUSTOM-CLOUD: In-Memory Data Grid-Based Software for Clustering 16S rRNA Sequence Data in the Cloud Environment.

High-throughput sequencing can produce hundreds of thousands of 16S rRNA sequence reads corresponding to different organisms present in the environmental samples. Typically, analysis of microbial diversity in bioinformatics starts from pre-processing followed by clustering 16S rRNA reads into relatively fewer operational taxonomic units (OTUs). The OTUs are reliable indicators of microbial diversity and greatly accelerate the downstream analysis time. However, existing hierarchical clustering algorithms that are generally more accurate than greedy heuristic algorithms struggle with large sequence datasets. To keep pace with the rapid rise in sequencing data, we present CLUSTOM-CLOUD, which is the first distributed sequence clustering program based on In-Memory Data Grid (IMDG) technology-a distributed data structure to store all data in the main memory of multiple computing nodes. The IMDG technology helps CLUSTOM-CLOUD to enhance both its capability of handling larger datasets and its computational scalability better than its ancestor, CLUSTOM, while maintaining high accuracy. Clustering speed of CLUSTOM-CLOUD was evaluated on published 16S rRNA human microbiome sequence datasets using the small laboratory cluster (10 nodes) and under the Amazon EC2 cloud-computing environments. Under the laboratory environment, it required only ~3 hours to process dataset of size 200 K reads regardless of the complexity of the human microbiome data. In turn, one million reads were processed in approximately 20, 14, and 11 hours when utilizing 20, 30, and 40 nodes on the Amazon EC2 cloud-computing environment. The running time evaluation indicates that CLUSTOM-CLOUD can handle much larger sequence datasets than CLUSTOM and is also a scalable distributed processing system. The comparative accuracy test using 16S rRNA pyrosequences of a mock community shows that CLUSTOM-CLOUD achieves higher accuracy than DOTUR, mothur, ESPRIT-Tree, UCLUST and Swarm. CLUSTOM-CLOUD is written in JAVA and is freely available at http://clustomcloud.kopri.re.kr.

Hippocampus-Sparing Whole-Brain Radiotherapy and Simultaneous Integrated Boost for Multiple Brain Metastases From Lung Adenocarcinoma: Early Response and Dosimetric Evaluation.

In this study, the volume response and treatment outcome after hippocampus-sparing whole-brain radiotherapy (HS-WBRT) with simultaneous integrated boost (SIB) using tomotherapy were evaluated. Patients with primary lung adenocarcinoma and multiple brain metastases who had a Karnofsky performance status ≥ 70 and exhibited well-controlled extracranial disease were treated. The prescribed dose was administered in 10 to 14 fractions as 25 to 28 Gy to whole-brain parenchyma, as 40 to 48 Gy to the gross metastatic lesion, and as 30 to 42 Gy to a 5-mm margin to the metastatic lesion. Double-dose gadolinium contrast-enhanced magnetic resonance imaging at 1-mm slice thickness was performed before treatment and at 1, 4, and 7 months post-treatment. The tumor volume reduction ratio was calculated for each follow-up. Between July 2011 and September 2012, 11 patients with 70 lesions were included in this analysis. The median number of lesions per patient was 4 (range, 2-15). The median initial tumor volume was 0.235 cm(3) (range, 0.020-10.140 cm(3)). The treatment plans were evaluated regarding conformation number (CN), target coverage (TC), and homogeneity index (HI). The median follow-up duration was 14 months (range, 3-25 months) and the 1-year intracranial control rate was 67%. The tumor volume reduction was most prominent during the first month with a median reduction rate of 0.717 (range, -0.190 to 1.000). Complete remission was seen in 22 (33%) lesions, and 45 (64%) lesions showed more than 65% reduction in tumor volume. The CN, TC, and HI values were comparable to that of previous studies, and the mean hippocampal dose was 13.65 Gy. No treatment breaks or ≥ G3 acute toxicities were observed during or after treatment. The HS-WBRT with SIB in patients with multiple brain metastases was effective and feasible for volume reduction and showed excellent intracranial control.

Successful salvage treatment of myxoid liposarcoma with multiple peritoneal seeding using helical tomotherapy-based intraperitoneal radiotherapy: a case report.

BACKGROUND: Myxoid liposarcoma is the most common soft-tissue sarcoma that metastasizes to the peritoneal cavity. Recently, an advanced intensity-modulated radiotherapy, known as helical tomotherapy, has been introduced to improve target coverage, while reducing normal tissue radiation. Here, we report a case of myxoid liposarcoma with multiple peritoneal seeding that was chemotherapy-refractory, but was successfully salvaged by helical tomotherapy-based intraperitoneal radiotherapy. CASE PRESENTATION: A 71-year-old East-Asian male was initially diagnosed with myxoid liposarcoma in his left thigh by excision. Six years later, the patient underwent a left pneumonectomy for metastatic myxoid liposarcoma in the left lung. Since then, the patient was treated with two segmental resections, and multiple lines of chemotherapy, for repeated recurrences in the peritoneal cavity. The patient underwent intraperitoneal radiotherapy followed by tumor boost radiotherapy, as salvage treatment for chemotherapy-resistant metastatic peritoneal myxoid liposarcoma. The prescribed dose was 24 Gy delivered in 15 fractions of 1.6 Gy over 3 weeks, followed by a 16 Gy boost dose administered in eight fractions of 2 Gy, to multifocal peritoneal lesions. A positron emission tomography scan obtained 8 weeks after completion of radiotherapy, showed a complete metabolic response of metastatic peritoneal lesions. Radiotherapy was well tolerated, without any side effects. In a computed tomography scan obtained 20 weeks after completion of radiotherapy, most of the peritoneal metastatic lesions had disappeared, except for two small residual nodules. CONCLUSION: This case suggests that low fraction-sized intraperitoneal radiotherapy (1.6 Gy administered once daily), followed by a focal boost using helical tomotherapy, is a feasible treatment without side effects. It produced an excellent tumor response, and durable intraperitoneal control for metastatic peritoneal myxoid liposarcoma.

Predictive factors of palliative radiotherapy response and survival in patients with spinal metastases from hepatocellular carcinoma.

BACKGROUND/AIMS: Spinal metastases often severely limit the quality of life by causing severe pain and neurological deficits. The purpose of this study was to evaluate the palliative effect of radiotherapy (RT) for spinal metastases from hepatocellular carcinoma (HCC) and to identify factors predictive of survival in HCC patients with spinal metastases who received RT. METHODS: A retrospective analysis was performed on 192 patients with spinal metastases from HCC who received RT. RESULTS: Of 192 patients with spinal metastases from HCC, an overall pain response to palliative RT occurred in 187 patients (97.4%), with a complete pain response (CR) in 41 patients (21.4%) and a partial response in 151 patients (78.6%). A higher biologically effective dose (BED) and more advanced RT techniques were identified as predictive factors for a CR. The 1- and 2-year overall survival (OS) rates were 18.1% and 6.3%, respectively, and the median survival time was 4.5 months. A long OS was associated with good performance status, controlled primary HCC, absence of extrahepatic metastases, and a higher BED. CONCLUSIONS: RT provided effective palliation for patients with painful spinal metastases from HCC. Our results provide information regarding pain control, survival outcomes, and predictive factors for the prognosis of HCC patients with spinal metastases treated with RT.

A prospective phase 2 multicenter study for the efficacy of radiation therapy following incomplete transarterial chemoembolization in unresectable hepatocellular carcinoma.

PURPOSE: The purpose of this study was to investigate the efficacy and toxicity of radiation therapy (RT) following incomplete transarterial chemoembolization (TACE) in unresectable hepatocellular carcinoma (HCC). METHODS AND MATERIALS: The study was designed as a prospective phase 2 multicenter trial. Patients with unresectable HCC, who had viable tumor after TACE of no more than 3 courses, were eligible. Three-dimensional conformal RT (3D-CRT) was added for HCC treatment with incomplete uptake of iodized oil, and the interval from TACE to RT was 4 to 6 weeks. The primary endpoint of this study was the tumor response after RT following incomplete TACE in unresectable HCC. Secondary endpoints were patterns of failure, progression-free survival (PFS), time to tumor progression (TTP), overall survival (OS) rates at 2 years, and treatment-associated toxicity. Survival was calculated from the start of RT. RESULTS: Between August 2008 and December 2010, 31 patients were enrolled. RT was delivered at a median dose of 54 Gy (range, 46-59.4 Gy) at 1.8 to 2 Gy per fraction. A best objective in-field response rate was achieved in 83.9% of patients, with complete response (CR) in 22.6% of patients and partial response in 61.3% of patients within 12 weeks post-RT. A best objective overall response rate was achieved in 64.5% of patients with CR in 19.4% of patients and PR in 45.1% of patients. The 2-year in-field PFS, PFS, TTP, and OS rates were 45.2%, 29.0%, 36.6%, and 61.3%, respectively. The Barcelona Clinic liver cancer stage was a significant independent prognostic factor for PFS (P=.023). Classic radiation-induced liver disease was not observed. There were no treatment-related deaths or hepatic failure. CONCLUSIONS: Early 3D-CRT following incomplete TACE is a safe and practical treatment option for patients with unresectable HCC.

Multimodality Management for Barcelona Clinic Liver Cancer Stage C Hepatocellular Carcinoma.

This review summarizes the contents of a workshop on multimodality management for Barcelona Clinic Liver Cancer (BCLC) stage C hepatocellular carcinoma (HCC) held on July 6, 2013, under the auspices of the 4th Asia-Pacific Primary Liver Cancer Expert Meeting Scientific Advisory Committee. BCLC stage C HCC represents a varied disease spectrum and, therefore, further stratification of BCLC stage C should be explored. Although sorafenib is currently the standard treatment for BCLC stage C HCC, the survival benefits are modest and new treatment strategies are still needed. Based on the opinions of Asian experts, there are numerous alternative options aside from sorafenib for the treatment of BCLC stage C HCC, including surgical resection, hepatic arterial infusion chemotherapy, transarterial chemoembolization, and external radiotherapy. Moreover, there are several studies on the multimodality management of BCLC stage C HCC, mainly in the form of retrospective studies and a few phase I and II trials. Multimodality management with combinations of various locoregional therapies or locoregional therapies with systemic targeted therapy using sorafenib needs to be actively investigated. The Asia-Pacific clinical practice guidelines on multimodality management for BCLC stage C HCC need recommendations based on the level of evidence, the strength of the data, and the strength of recommendations of previously reported systems.

Role of radiotherapy for pancreatobiliary neuroendocrine tumors.

PURPOSE: We investigated the role of radiotherapy (RT) for pancreatobiliary neuroendocrine tumors (PB-NETs). MATERIALS AND METHODS: We identified 9 patients with PB-NETs who received RT between January 2005 and March 2012. Of these 9 patients, 4 were diagnosed with NETs in the pancreas and 5 were diagnosed with NETs in the gallbladder. All patients received RT to the primary tumor or resection bed with a median total irradiation dose of 50.4 Gy, with or without chemotherapy. RESULTS: The tumor response rate and tumor control rate in the RT field were 60% and 100 %, respectively. All 4 patients who underwent surgery had no evidence of disease in the RT field. Of the 5 patients who received RT to the primary gross tumor, 1 had complete response, 2 had partial response, and 2 had stable disease in the RT field. The median time to progression was 11 months. Of the 9 patients, four patients had no progression, and 5 patients had progression of disease (locoregional, 2; distant, 2; locoregional/distant, 1). Of the 4 patients without progression, 3 were treated with RT in adjuvant or neoadjuvant setting, and one received RT to primary tumor. One patient experienced radiation-induced duodenitis at 3 months after concurrent chemoradiation without treatment-related mortality. CONCLUSION: RT can yield local control for advanced PB-NETs. RT should be considered an essential part of multimodality treatment in management of advanced PB-NETs.

Effect of troglitazone on radiation sensitivity in cervix cancer cells.

PURPOSE: Troglitazone (TRO) is a peroxisome proliferator-activated receptor γ (PPARγ) agonist. TRO has antiproliferative activity on many kinds of cancer cells via G1 arrest. TRO also increases Cu(2+)/Zn(2+)-superoxide dismutase (CuZnSOD) and catalase. Cell cycle, and SOD and catalase may affect on radiation sensitivity. We investigated the effect of TRO on radiation sensitivity in cancer cells in vitro. MATERIALS AND METHODS: Three human cervix cancer cell lines (HeLa, Me180, and SiHa) were used. The protein expressions of SOD and catalase, and catalase activities were measured at 2-10 µM of TRO for 24 hours. Cell cycle was evaluated with flow cytometry. Reactive oxygen species (ROS) was measured using 2',7'-dichlorofluorescin diacetate. Cell survival by radiation was measured with clonogenic assay. RESULTS: By 5 µM TRO for 24 hours, the mRNA, protein expression and activity of catalase were increased in all three cell lines. G0-G1 phase cells were increased in HeLa and Me180 by 5 µM TRO for 24 hours, but those were not increased in SiHa. By pretreatment with 5 µM TRO radiation sensitivity was increased in HeLa and Me180, but it was decreased in SiHa. In Me180, with 2 µM TRO which increased catalase but not increased G0-G1 cells, radiosensitization was not observed. ROS produced by radiation was decreased with TRO. CONCLUSION: TRO increases radiation sensitivity through G0-G1 arrest or decreases radiation sensitivity through catalase-mediated ROS scavenging according to TRO dose or cell types. The change of radiation sensitivity by combined with TRO is not dependent on the PPARγ expression level.