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.

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.

Antiviral Effect of Low Molecular Weight Chitooligosaccharide Against SARS-CoV-2 in Vitro

( Donghwan Jang ),( Dagyum Lee ),( Jihee Jung ),( Sungweon Ryoo ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-

Background We tried to assure the natural polysaccharide would be used as a disinfectant for Coronavirus disease 2019. Previous studies already reported that the chitosans are effective substances for viral diseases. Here, we showed that three different molecular weight chitosan-based substances (chitosan, chitooligosaccharide, and water soluble chitosan) have direct antiviral properties against SARS-CoV-2 in vitro using Vero cells. Methods The cytotoxicity of chitosan-based substances was measured by MTT assay. The antiviral effect was confirmed by quantitative viral RNA targeting the RdRp gene and plaque assay. The reduction of nucleocapsid protein expression in the virus-infected cells was confirmed by fluorescence microscopy. Results The chitosan-based substances have low cytotoxicity to Vero cells. Among the chitosan-based substances tested, chitooligosaccharide decreased the expression of SARS-CoV-2 nucleocapsid protein of SARS-CoV-2 infected cells and reduced SARS-CoV-2 virus infection in a concentration-dependent manner. Conclusions In conclusion, low molecular weight chitooligosaccharide can serve as an antiviral natural disinfectant for SARS-CoV-2 in vitro.

Comparative Analysis of KOGENE Mycobacterial Interspersed Repetitive Unit-variable Number of Tandem Repeat (MIRU-VNTR) Typing Kit and Its Application on Clinically Isolated Mycobacterium Tuberculosis

( 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.

Evaluation of the KOGENE Real-time PCR Kit for Identification on Beijing Strain of Mycobacterium Tuberculosis and Its Application on Clinical Isolates from the National Tuberculosis Hospital

( 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.

신에솔, 신재생에너지 종합평가 응용 솔루션 개발

김진영(Jin-Young Kim),김현구(Hyun-Goo Kim),강용혁(Yong-Heack Kang),김종현(Jonghyun Kim),이다겸(Dagyum Lee),신기열(Ki-Yeol Shin),전대성(Dae-Seong Jeoune),염기웅(Giwwong Yeom),이태형(Tae-Hyung Lee),오의석(Eui-Seok Oh) 한국신재생에너지학회 2019 한국신재생에너지학회 학술대회논문집 Vol.2019 No.5