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RISS 인기검색어
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- 저자 : ( Yoohyun Hwang ) (검색결과 13 건)
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( 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.
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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.
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( Taeuk Kang ),( Yoohyun Hwang ),( Jiyeon Kim ),( Jihee Jung ),( Dagyum Lee ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-
Purpose Tuberculosis (TB) remains as public health burden in Korea. Beijing strain of Mycobacterium tuberculosis (M. tuberculosis) is assumed to be associated with hypervirulence and multi-drug resistance hence detection of such strain helps rapid detection of drug resistance. Thus, identification of Beijing strain is highly significant, in epidemiological and clinical aspects. Here, we present evaluation of the Real-time PCR kit for Beijing strain of Mycobacterium tuberculosis diagnostic® (KOGENE, Korea) (KOGENE RT-PCR) Method The 200 DNA samples, where 100 were clinical M. tuberculosis from Masan National Tuberculosis Hospital and other 100 were non-M. tuberculosis, were used. Strains of 100 M. tuberculosis were identified using MIRU-VNTR typing kit® (GenoScreen, France) and MIRU-VNTRplus database (URL: https://www.miru-vntrplus.org/MIRU/index.faces). Based on MIRU-VNTR result, the 84 out of 100 samples are identified as Beijing strain and remaining 16 samples are identified as non-Beijing strains. The KOGENE RT-PCR was conducted according to the manufacturer’s manual. Amplification of VIC fluorescence indicates M. tuberculosis-positive and amplification of both VIC and FAM fluorescences indicates Beijing strain of M. tuberculosis-positive. Result Of 200 samples, only 100 M. tuberculosis samples were amplified for VIC, showing 100% for both sensitivity and specificity with 95% Confidence of Interval (CI) value of 96.38-100%. Out of 100 M. tuberculosis, 84 Beijing strain of M. tuberculosis were positive for FAM, indicating 100% for both sensitivity and specificity with 95% CI values of 95.70-100% and 96.87-100% respectively (Table 1). Conclusion The clinical implication of Beijing strain is widely known. Here, investigated RT-PCR has shown capability in Beijing strain identification. Application of this tool could contribute in M. tuberculosis identification and prediction of possible drug resistance during pre- and initial treatment. Also, several outbreaks have been occurred by M. tuberculosis with Beijing strain. Hence, early identification of such strain is considered as highly useful in epidemiological TB investigation.
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( Taeuk Kang ),( Yoohyun Hwang ),( Jiyeon Kim ),( Jihee Jung ),( Dagyum Lee ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-
Purpose Tuberculosis (TB) remains as a serious public health burden in Korea due to ease transmission. Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) is preferred for epidemiological TB investigation. However, the MIRU-VNTR has issues that MIRU-VNTR kit by GenoScreen (GeMIRU) is hard to be procured in Korea hence the difficulty lies epidemiological TB investigation/research. Here, we have comparatively analyzed the MIRU-VNTR kit by Kogene (KoMIRU). Method The 200 DNA samples, where 100 samples were clinical M. tuberculosis DNA samples from Masan National Tuberculosis Hospital and other 100 samples were non-M. tuberculosis, were used. Then, 200 samples were typed using KoMIRU and GeMIRU and compared for comparative analysis. KoMIRU and GeMIRU were done using the protocols provided by each manufacturer’s manual. Lineage strains were identified using MIRU-VNTRplus database (https://www.miru-vntrplus.org/MIRU/ index.faces) Result Of 200 samples, all clinical M. tuberculosis samples were amplified while non-M. tuberculosis samples were not, showing 100% for both sensitivity and specificity with 95% Confidence of Interval value of 96.38 - 100.00% (Table 1). KoMIRU typing kit has shown successful differentiation of M. tuberculosis from non-M. tuberculosis species. Also, no discrepancies observed on determined lineage strains between KoMIRU and GeMIRU. Out of 100, 84 were identified as Beijing strains and remainings were identified as NEW-1 (n=8), Uganda (n=6), EAI (n=6), Turkey (n=2), and Harleem (n=1) (Figure 1). Interestingly, among those non-Beijing strains except EAI, they are geographically distant lineage strains, Uganda, Turkey, and Harleem, and relatively unique strain, NEW-1. Conclusion The effectiveness of preventive TB intervention using GeMIRU has been globally proved. In this study, KoMIRU has shown comparable capability as GeMIRU and localization of MIRU-VNTR by Kogene is expected to allow efficient TB investigation in Korea. Also, previous researches had suggested association between lineage strains and drug resistance hence, this can be applied to initial TB treatment.
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Biobank for Multidrug-resistant Tuberculosis Research: Importance of Sequential Samples
( Jiyeon Kim ),( Yoohyun Hwang ),( Jihye Kang ),( Junghee Lee ),( Seungkyu Park ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-
Purpose Since 2013, Masan National Tuberculosis Hospital (MNTH) has collected standardized specimens from its tuberculosis patients, which include a large number of multidrug-resistant strains. The repository collects matched participants and their bacilli samples, compiling sequential samples from the beginning of treatment. The repository aims to provide resources for in-depth international research. Method The MNTH biobank emphasizes on collecting and maintaining specimens from various types of disease-progress in its TB patients. Sputum, serum, urine, and bacterial strains are collected from each patient sequentially, the intervals depending on the condition of each patient. Strains are collected and stored under standardized protocols and stringent biosafety guidelines. Result The majority of specimens collected at MNTH for the past ten years are mostly made up of sputum and blood serum, with 13,196 and 10,905 samples respectively. A total of 1,354 distributions for the internal research institute and 1,022 distributions to external organizations were made as of 2021. The Results and related information are stored in the biobank database, helping form the groundwork in monitoring inpatients’ future treatment outcomes as well as providing future research resources. Analysis of sequential samples has shown a difference in the antibiotic and molecular profiles of the same patient. Currently, the MNTH biobank database stores the Results in the patterns of resistance of TB strains as pDST, molecular diagnosis, and WGS data. Conclusion MNTH presents this unique biobank and its protocols in the hopes that it will be taken as an example of a biorepository operation that may model the future management of TB biobanks. We always welcome infectious sample contributions and distribution requests and hope that our institute’s specimens and our database will allow research projects and clinical trials to become more accessible to all types of pharmaceuticals and medical technology development in our pursuit of the eradication of TB.
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( 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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( Jiyeon Kim ),( Taeyoon Kim ),( Yoohyun Hwang ),( Sengkyu Park ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-
Purpose While several automated AFB staining processes have been developed, the reading of slides still depends on a subjective, human aspect for an unambiguous judgement. Here we validate the scanning analysis system that combines automated microscopy with deep-learning based image analysis after auramine-staining method. This study evaluated the scanning analysis system microscopy by comparing it with manual microscopy, culture Results, and molecular diagnostic Results. We assessed the scanning system’s potential to read slides and summarize diagnosis-relevant image for a final judgement by a human aspect in order to assist with the visual workload. Method This study used 200 sputum samples that were stored in the Masan National Tuberculosis Hospital (MNTH) biobank. The main purpose of the study was to perform a routine check on the sputum of the biobank as a form of quality control. Auramine staining and microscopy For manual microscopy, auramine-stained slides (Auto Strainer Fluorescence(AT-2000F, Dagatron) were visually checked at a 400x magnification (40× objective and 10× ocular, Olympus) and the number of acid-fast bacteria was manually counted or estimated. The slides were classified according to the MNTH guideline, version 2020. Slide scanning Slides were scanned using a Metafer Slide-Scanning System from MetaSystems Hard & Software GmbH, Altlussheim. To meet the guideline requirement of 300 fields of view in 40x magnification with 23 mm ocular, an area-equivalent of 215 camera fields with a 20x magnification (Plan-Neofluar objective, Zeiss) and a CC4c color camera (MetaSystems) was scanned. Result From the 200 auramine-stained slides used for clinical validation, 129 came from culture positive specimens and 189 Real-time PCR MTB positive and 9 were detected M. intracellulare. Conclusion we can show that using an automated scanning and analysis system for auramine-stained slides to assist diagnosis can improve sensitivity of detection and reduce manual slide-handling time.
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( 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.
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