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        Comparing Two Mycobacterium tuberculosis Genomes from Chinese Immigrants with Native Genomes Using Mauve Alignments

        ( Sungweon Ryoo ),( Jeongsoo Lee ),( Jee Youn Oh ),( Byeong Ki Kim ),( Young Kim ),( Je Hyeong Kim ),( Chol Shin ),( Seung Heon Lee ) 대한결핵 및 호흡기학회 2018 Tuberculosis and Respiratory Diseases Vol.81 No.3

        Background: The number of immigrants with tuberculosis (TB) increases each year in South Korea. Determining the transmission dynamics based on whole genome sequencing (WGS) to cluster the strains has been challenging. Methods: WGS, annotation refinement, and orthology assignment for the GenBank accession number acquisition were performed on two clinical isolates from Chinese immigrants. In addition, the genomes of the two isolates were compared with the genomes of Mycobacterium tuberculosis isolates, from two native Korean and five native Chinese individuals using a phylogenetic topology tree based on the Multiple Alignment of Conserved Genomic Sequence with Rearrangements (Mauve) package. Results: The newly assigned accession numbers for two clinical isolates were CP020381.2 (a Korean-Chinese from Yanbian Province) and CP022014.1 (a Chinese from Shandong Province), respectively. Mauve alignment classified all nine TB isolates into a discriminative collinear set with matched regions. The phylogenetic analysis revealed a rooted phylogenetic tree grouping the nine strains into two lineages: strains from Chinese individuals and strains from Korean individuals. Conclusion: Phylogenetic trees based on the Mauve alignments were supposed to be useful in revealing the dynamics of TB transmission from immigrants in South Korea, which can provide valuable information for scaling up the TB screening policy for immigrants.

      • SCOPUSKCI등재

        Comparing Two Mycobacterium tuberculosis Genomes from Chinese Immigrants with Native Genomes Using Mauve Alignments

        Ryoo, Sungweon,Lee, Jeongsoo,Oh, Jee Youn,Kim, Byeong Ki,Kim, Young,Kim, Je Hyeong,Shin, Chol,Lee, Seung Heon The Korean Academy of Tuberculosis and Respiratory 2018 Tuberculosis and Respiratory Diseases Vol.81 No.3

        Background: The number of immigrants with tuberculosis (TB) increases each year in South Korea. Determining the transmission dynamics based on whole genome sequencing (WGS) to cluster the strains has been challenging. Methods: WGS, annotation refinement, and orthology assignment for the GenBank accession number acquisition were performed on two clinical isolates from Chinese immigrants. In addition, the genomes of the two isolates were compared with the genomes of Mycobacterium tuberculosis isolates, from two native Korean and five native Chinese individuals using a phylogenetic topology tree based on the Multiple Alignment of Conserved Genomic Sequence with Rearrangements (Mauve) package. Results: The newly assigned accession numbers for two clinical isolates were CP020381.2 (a Korean-Chinese from Yanbian Province) and CP022014.1 (a Chinese from Shandong Province), respectively. Mauve alignment classified all nine TB isolates into a discriminative collinear set with matched regions. The phylogenetic analysis revealed a rooted phylogenetic tree grouping the nine strains into two lineages: strains from Chinese individuals and strains from Korean individuals. Conclusion: Phylogenetic trees based on the Mauve alignments were supposed to be useful in revealing the dynamics of TB transmission from immigrants in South Korea, which can provide valuable information for scaling up the TB screening policy for immigrants.

      • Characteristic Exploration of Sequential Mycobacterium Tuberculosis Strains Isolated from Patients with Linezolid Treatment

        ( Yoohyun Hwang ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-

        Background Linezolid (LZD) is an important antibiotic for the treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB), but mutations related to linezolid resistance remain inadequately characterized. In this study we investigated the resistance-related mutations of clinical isolates from MDR and XDR TB patients who failed linezolid treatment, using wholegenome sequencing. Methods We used the Sensititre™ plate to determine the phenotypic drug susceptibility and minimum inhibitory concentration (MIC) of first- and second-line anti-TB drugs, while using resazurin microtiter assay (REMA) for the MIC of linezolid. A total of 25 sequential isolates, previously collected from four MDR/XDR patients who failed LZD treatment, were used in this study. All isolates had whole genome sequencing performed with the Illumina® platform. Results All four TB patients had a form of multidrug-resistant TB, which is defined as a form of TB resistant to at least rifampicin and isoniazid, the first-line anti-TB drugs. Each patients’ isolates collected within three months of beginning LZD treatment were susceptible to LZD, but the isolates from the corresponding patient collected after three months had higher MIC for LZD and developed mutations in LZD resistance linked genes (Table 1). Whole genome sequencing of all strains collected from each patient revealed mutations in the LZD resistance linked genes rplC, rrl, and Rv0678 (Table 2). Conclusions After a given period of time, all four patients acquired resistance to LZD. However, the LZD MIC was below 32ug/ml, a concentration observed in a previous study. The rplC gene affects the ribosomal protein L3, the rrl gene affects the 23S rRNA, and Rv0678 is the regulator for the MmpS5-MmpL5 efflux pump. The combination of mutations in these genes caused the high-level linezolid resistance in each serial clinical isolate.

      • SCOPUSKCI등재
      • SCOPUSKCI등재

        Evaluation of the Selected 12-locus MIRU for Genotyping Beijing Family Mycobacterium Tuberculosis in Korea

        Kang, Heeyoon,Ryoo, Sungweon,Park, Youngkil,Lew, Woojin The Korean Academy of Tuberculosis and Respiratory 2009 Tuberculosis and Respiratory Diseases Vol.67 No.6

        Background: Mycobacterial Interspersed Repetitive Units (MIRUs) that are located mainly in intergenic regions dispersed throughout the Mycobacterium tuberculosis genome. The selected MIRU loci, which were composed of a 12-locus set, demonstrated a high power for discrimination of Mycobacterium tuberculosis isolates collected from Kangwon province of Korea. To evaluate its ability to discriminate the M. tuberculosis strains, 45 clinical isolates were genotyped using the methods IS6110 RFLP and MIRU. Methods: All the samples were collected during the period from January 2007 to December 2007 from TB patients, who were residents and registered to a public health center of Kangwon Province in Korea. A total of 45 DNAs were extracted from clinical isolated mycobacterial strains and genotyped using IS6110 RFLP, the MIRU method. Results: We compared the 12-MIRU with IS6110 RFLP in the 45 samples, the 12-locus version offered less discriminatory power (Hunter-Gaston discriminatory index [HGDI]: 0.959 vs 0.998; 57.78% of clustered cases vs 8.89%). Conclusion: This 12-locus MIRU can be useful when additional combinations of other loci for genotyping M. tuberculosis in Korea where the Beijing family strains are dominant.

      • Current Status on Training and Education on Public and Healthcare Workers Towards Tuberculosis Elimination: Case in Korea

        ( Taeuk Kang ),( Seungkyu Park ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-

        Background Tuberculosis still remains as intermediate-burden in Korea (Figure 1). For tuberculosis elimination, tuberculosis training/ education play pivotal role. Researches have suggested that provision of appropriate training/education could improve clinical outcome and prevent TB transmission. Despite significant role of TB training/education, TB training/education were poorly investigated. To assess current TB training/education program, identifying current status of TB training/education is inevitable. Hence, in this study, we reviewed articles and programs related to TB training/education in Korea and analyzed. Method A comprehensive review of literature on electronic databases and websites of TB-training providing organizations from year of 1995 to 2021 were undertaken. Result Several organizations from governmental/public sectors such as Korea Disease Control and Prevention Agency (KDCA), Korea National Tuberculosis Association (KNTA), Masan National Tuberculosis Hospital (MNTH), Korea Human Resource Development Institute for Health and Welfare (KOHI), Korea Nurses Association (KNA) were involved in provision of TB training/education. The KDCA conducts activities related to TB control and prevention at national, provincial, and local levels while KNTA, as commissioned by KDCA, provides TB trainings for healthcare workers with collaboration with KNA. In case of MNTH, this medical institute produced 14 TB medical specialists since 1993 and actively engaged in TB education at community level that 67,426 people were educated for TB prevention. Lastly, KOHI institutes provides several TB training/education programs for healthcare workers, medical technologists, and public (Table 1). Conclusion Although sufficient number of TB training/education are being provided. Its quality has never been assessed hence, there is difficulty lies in improvement of TB training/education due to lacking evaluation. Also, due to changing landscape of TB, training/education topics should reflect upcoming demands and be diversified such as inclusion of latent TB, drug-resistant TB and/or HIV/TB. The MNTH, with demonstrated history, is able to meet current demands from TB training/education.

      • The Usefulness of Determining the MIC of Newly Developed Anti-TB Drugs with the MBEC Biofilm Assay

        ( Yoohyun Hwang ),( Dagyum Lee ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-

        Background Drug-resistance is one of the ongoing topics in the treatment of bacterial infections; in the case of tuberculosis (TB), effective treatment is inhibited by the presence of the atypical mycobacterial cell wall and biofilm, which prevents antibiotic penetration into the cell. Additionally, the biofilm layer increases the mycobacterial antibiotic tolerance and delays the host immune response, which encourages drug-resistance. Methods We used the MBEC Assay Biofilm Inoculator (Innovotech) with Mycobacterium abscessus (M. abs) in this study. 96 pegs set on the lid of the assay was placed on a 96 well plate with an OD600 0.01 of M. abs, then incubated at rpm 80 for 3 days at 37℃ to form biofilm on the pegs. The pegs were then transferred to a new 96 well plate with various drug concentrations and incubated again under same conditions. Then 0.25% resazurin was added to measure minimum inhibitory concentration (MIC). Results Several antibiotics effective when used against the cells of M. abs in the resazurin microtiter assay (REMA) were ineffective in the treatment of M. abs cells with biofilm. In the presence of biofilm, the MIC50 for antibiotics were up to almost 3 times the MIC50 concentrations of aerobic M. abs growth conditions without biofilm. Conclusions Biofilm formation of mycobacteria is one of the complications behind the treatment of mycobacterium infections, including Mycobacterium tuberculosis, one of the leading infectious respiratory diseases. The presence of biofilm skews Results of MIC researched in vitro to the point of ineffectiveness in vivo. More prolific use of the MBEC biofilm assay to determine the effectiveness of newly developed drugs are needed before introducing them to most in vivo experiments.

      • Clomiphene Citrate Shows Effective and Sustained Antimicrobial Activity against Mycobacterium Abscessus

        ( Da-gyum Lee ),( Sungweon Ryoo ),( Yoohyun Hwang ),( Eun-jin Park ),( Jung-hyun Kim ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-

        Background Mycobacterium abscessus (M. abscessus) causes chronic pulmonary infections and is the most difficult Nontuberculous Mycobacteria (NTM) to treat due to its resistance to current antimicrobial drugs, with a treatment success rate of 45.6 %. Thus, novel treatment drugs are needed, of which we identified the drug Clomiphene Citrate (CC), which treats infertility in women, to exhibit inhibitory activity against M. abscessus. Methods To assess the potential of CC as a treatment for M. abscessus pulmonary diseases, we measured its efficacy in vitro and established the intracellular activity of CC against M. abscessus in human macrophages. Results CC significantly inhibited the growth of not only wild-type M. abscessus strains, but also clinical isolate strains and clarithromycin (CLR)-resistant strains of M. abscessus. CC’s drug-efficacy did not have a significant cytotoxicity in the infected macrophages. Furthermore, CC worked in anaerobic non-replicating conditions as well as in the presence of biofilm. Conclusions The Results of this in vitro study on M. abscessus activity suggest that CC is a potential new drug for the treatment of M. abscessus infections.

      • Operation Report of Biosafety Level 3 Laboratory at National Tuberculosis Hospital

        ( Donghwan Jang ),( Jiyeon Kim ),( Seungkyu Park ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-

        Background Since its opening in 1941, Masan National Tuberculosis Hospital (MNTH) has specialized in treating and managing refractory Tuberculosis (TB) patients. MNTH is operating TB specimen biobank. The risk of laboratory-acquired infection is increased due to the main procedures of diagnosis and research on TB and pandemic COVID-19. In addition, experiments to be performed strictly at BL3 facilities are growing. Methods During the first installation and operation permit in May 2017 to December 2020 of the MNTH BL3 laboratory, we analyzed the log records statistically indicate the annual BL3 usage hours, the subtotal time required for each task, and the number of daily entrances by the purpose of routine diagnosis and research. Results Total time for the routine diagnostic is decreasing from 188 to 53 hours per year. On the other hand, the time required for clinical research increased from 873 to 1,112 hours per year. The number of daily entrances decreased from 0.87 to 0.34 in routine diagnosis and increased from 0.83 to 1.76 in clinical research work. Conclusions Due to the aspect of MNTH, where the number of TB patients is decreasing every year, routine diagnostic tests performed in BL3 are rapidly decreasing. Meanwhile, in clinical research, the demand for not only TB-related research but also research related to COVID-19 is quickly increasing. MNTH Clinical Research Center is conducting five related studies, including joint research on mRNA COVID-19 vaccine development. Properly utilizing the well-established BL3 facilities is expected to present exemplary cases used in research projects such as the development of vaccines and therapeutics for COVID-19 pandemic.

      • Comparison of the STANDARD E TB-Feron ELISA and QuantiFERON-TB Gold Plus: The Advantageous Use of Whole Recombinant Protein Antigen for Latent TB Diagnosis

        ( Dagyum Lee ),( Jihye Kang ),( Yoohyun Hwang ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-

        Purpose The laboratory diagnosis of latent tuberculosis (LTB) is often performed using interferon-gamma(γ) release assays (IGRA). We compared two enzyme-linked immunosorbent assay (ELISA)-based IGRA, the Standard E TB-Feron ELISA (STFE; SD Biosensor, Gyeonggi-do, Republic of Korea) and the QuantiFERON-TB Gold PLUS assay (QFT-GP; Qiagen, Hilden, Germany), in 155 participants. Method All participants were classified into four groups and screened by both assays per the manufacturer’s protocols: group A who were not infected nor were in contact with TB patients, group B who were at an intermediate risk of TB infection, group C who were at a high risk of TB infection, and group D who were confirmed and completing TB treatment. Result The study conducted two statistical analysis. First, the Results of STFE were compared to QTF-GP, using the latter as the gold standard. STFE had a sensitivity and specificity of 98 % and 86 %, respectively. Next, the positivity and negativity concordance tests were calculated with a confidence interval of 95 %, using the characteristics of each group to differentiate healthy subjects and subjects with TB infection. The total concordance of healthy subjects was 80.00 % for QFT-GP and 75.80 % for STFE, while the total concordance of TB infected subjects was 58.00 % for QFT-GP and 82.00 % for STFE. Conclusion The IGRA test STFE had an improved clinical performance in detecting LTB and TB infected subjects, making it an acceptable alternative to QFT-GP.

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