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5. Streamlining whole genome sequencing for clinical diagnostics with ONT technology.

Author

Wu, Han-Chieh, Chiu, Yueh-Tzu, Wu, I-Ching, Liou, Ci-Hong, Cheng, Hung-Wei, Kuo, Shu-Chen, Lauderdale, Tsai-Ling, Sytwu, Huey-Kang, Liao, Yu-Chieh, and Chen, Feng-Jui

Subjects
*WHOLE genome sequencing, *NEISSERIA gonorrhoeae, *LIFE sciences, *GRAM-negative bacteria, *DRUG resistance in microorganisms
Abstract

Recent advances in whole-genome sequencing (WGS) have increased the accessibility of this tool, offering substantial potential for pathogen surveillance, outbreak response, and diagnostics. However, the routine clinical adoption of WGS is hindered by factors such as high costs, technical complexity, and the requirement for bioinformatics expertise for data analysis. To address these challenges, we propose RapidONT, a workflow designed for cost-effective and accessible WGS-based pathogen analysis. RapidONT employs a mechanical shearing–based DNA extraction protocol, followed by library construction by using a multiplexing Oxford nanopore technologies (ONT) rapid barcoding kit. Flye software is used for de novo assembly without manual intervention, followed by basic assembly polishing using Medaka and Homopolish. The polished assemblies are then analyzed using the user-friendly web-based platform Pathogenwatch, which facilitates species identification, molecular typing, and antimicrobial resistance (AMR) prediction, all while requiring minimal bioinformatics expertise. The efficacy of RapidONT was evaluated using nine clinically relevant pathogens, encompassing a total of 90 gram-positive and gram-negative bacterial strains. The workflow demonstrated high accuracy in critical tasks such as multilocus sequence typing (MLST) and AMR identification, using only ONT R9.4.1 flowcell data. Notably, limitations were observed with Salmonella spp. and Neisseria gonorrhoeae. Furthermore, RapidONT enabled the generation of genomic information for 48 bacterial isolates by using a single flow cell, significantly reducing sequencing costs. This approach eliminates the need for extensive experimentation in obtaining crucial genomic information. This workflow facilitates broader WGS implementation in clinical pathogen analysis and diagnostics. [ABSTRACT FROM AUTHOR]

Published
2025
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6. Clonal expansion of Tn1546-like transposon-carrying vancomycin-resistant Enterococcus faecium,a nationwide study in Taiwan, 2004-2018

Author

Huang, Ying-Chi, Chen, Feng-Jui, Huang, I-Wen, Wu, Han-Chieh, Kuo, Shu-Chen, Huang, Tzu-Wen, and Lauderdale, Tsai-Ling

Abstract

•E. faeciumaccounted for increasing ratios of enterococcal infections in Taiwan.•Vancomycin resistance in E. faeciumincreased from 3% in 2004 to 56.9% in 2018.•Transfer of Tn1546variants between E. faeciumisolates contributed to this increase.•The predominant clones of vancomycin-resistant E. faeciumbelong to ST17 and ST78.•Spread of ST17 and ST78 also contributed to the increased vancomycin resistance.

Published
2024
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13. Increased Expression of Programmed Death Ligand 1 in Hepatocellular Carcinoma of Patients with Hepatitis B Virus Pre-S2 Mutant

Author

Teng, Chiao-Fang, Li, Tsai-Chung, Wang, Ting, Wu, Tzu-Hua, Wang, John, Wu, Han-Chieh, Shyu, Woei-Cherng, Su, Ih-Jen, and Jeng, Long-Bin

Subjects
pre-S2 mutant, programmed death 1, programmed death ligand 1, hepatocellular carcinoma, hepatitis B virus, Journal of Hepatocellular Carcinoma, digestive system diseases, Original Research
Abstract

Chiao-Fang Teng,1– 3 Tsai-Chung Li,4,5 Ting Wang,2 Tzu-Hua Wu,2 John Wang,6 Han-Chieh Wu,7 Woei-Cherng Shyu,1,8– 10 Ih-Jen Su,11 Long-Bin Jeng2 1Graduate Institute of Biomedical Sciences, China Medical University, Taichung, Taiwan; 2Organ Transplantation Center, China Medical University Hospital, Taichung, Taiwan; 3Research Center for Cancer Biology, China Medical University, Taichung, Taiwan; 4Department of Public Health, College of Public Health, China Medical University, Taichung, Taiwan; 5Department of Healthcare Administration, College of Medical and Health Science, Asia University, Taichung, Taiwan; 6Department of Pathology, China Medical University Hospital, Taichung, Taiwan; 7National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan, Taiwan; 8Department of Occupational Therapy, Asia University, Taichung, Taiwan; 9Department of Neurology, China Medical University Hospital, Taichung, Taiwan; 10Translational Medicine Research Center, China Medical University Hospital, Taichung, Taiwan; 11Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, TaiwanCorrespondence: Chiao-Fang TengGraduate Institute of Biomedical Sciences, China Medical University, No. 91, Hsueh-Shih Road, Northern Dist., Taichung City 404, TaiwanTel + 886-4-2205-2121Fax + 886-4-2202-9083Email chiaofangteng@gmail.comLong-Bin JengOrgan Transplantation Center, China Medical University Hospital, No. 2, Yude Road, Northern Dist., Taichung City 404, TaiwanTel + 886-4-2205-2121Fax + 886-4-2202-9083Email longbin.cmuh@gmail.comPurpose: Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), a leading cause of cancer-related death worldwide. The HCC patients who harbor HBV pre-S2 mutant, an oncoprotein that plays key roles in HCC development, have been closely associated with a worse prognosis after curative surgical resection, suggesting an urgent need for alternative therapeutic options to improve their survival. In this study, we aimed to evaluate the expression profiles of programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1), two of the most well-studied immune checkpoint molecules that promote tumor immune evasion, in tumor of the pre-S2 mutant-positive/high HCC patients.Methods: We classified 40 HBV-related HCC patients into the pre-S2-positive/high and -negative/low groups by a next-generation sequencing-based approach. The fluorescent immunohistochemistry staining was performed to detect the expression of PD-1 and PD-L1 in HCC tissues of patients.Results: We showed that patients with either deletion spanning pre-S2 gene segment or high percentage of pre-S2 plus pre-S1+pre-S2 deletion (the pre-S2 mutant-positive/high group) exhibited a significantly higher density of PD-L1-positive cells in HCC tissues than those without. Moreover, the percentage of pre-S2 plus pre-S1+pre-S2 deletion displayed a high positive correlation with the density of PD-L1-positive cells in HCC tissues.Conclusion: The increased expression of PD-L1 in tumor tissues of the pre-S2 mutant-positive HCC patients suggest that pre-S2 mutant may play a potential role in dysregulation of tumor immune microenvironment in the progression of HBV-related HCC, implicating for the development of future therapeutic strategies.Keywords: hepatitis B virus, hepatocellular carcinoma, pre-S2 mutant, programmed death 1, programmed death ligand 1

Published
2020

14. Furin and TMPRSS2 Resistant Spike Induces Robust Humoral and Cellular Immunity Against SARS-CoV-2 Lethal Infection

Author

Lin, Jhe-Jhih, primary, Tien, Chih-Feng, additional, Kuo, Yi-Ping, additional, Lin, En-Ju, additional, Tsai, Wei-Hsiang, additional, Chen, Ming-Yu, additional, Tsai, Pei-Ju, additional, Su, Yu-Wen, additional, Pathak, Nikhil, additional, Yang, Jinn-Moon, additional, Yu, Chia-Yi, additional, Chuang, Zih-Shiuan, additional, Wu, Han-Chieh, additional, Tsai, Wan-Ting, additional, Dai, Shih-Syong, additional, Liao, Hung-Chun, additional, Chai, Kit Man, additional, Su, Yu-Siang, additional, Chuang, Tsung-Hsien, additional, Liu, Shih-Jen, additional, Chen, Hsin-Wei, additional, Dou, Horng-Yunn, additional, Chen, Feng-Jui, additional, Chen, Chiung-Tong, additional, Liao, Chin-Len, additional, and Yu, Guann-Yi, additional

Published
2022
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31. A pre-S gene chip to detect pre-S deletions in hepatitis B virus large surface antigen as a predictive marker for hepatoma risk in chronic hepatitis B virus carriers

Author

Hsieh Hui-Chuan, Chang Tsung-Chuan, Young Kung-Chia, Yao Wei-Jen, Hsieh Yi-Hsuan, Wu Han-Chieh, Su Ih-Jen, Shen Fan-Ching, Tsai Han-Ni, and Huang Wenya

Subjects
Medicine
Abstract

Abstract Background Chronic hepatitis B virus (HBV) infection is an important cause of hepatocellular carcinoma (HCC) worldwide. The pre-S1 and -S2 mutant large HBV surface antigen (LHBS), in which the pre-S1 and -S2 regions of the LHBS gene are partially deleted, are highly associated with HBV-related HCC. Methods The pre-S region of the LHBS gene in two hundred and one HBV-positive serum samples was PCR-amplified and sequenced. A pre-S oligonucleotide gene chip was developed to efficiently detect pre-S deletions in chronic HBV carriers. Twenty serum samples from chronic HBV carriers were analyzed using the chip. Results The pre-S deletion rates were relatively low (7%) in the sera of patients with acute HBV infection. They gradually increased in periods of persistent HBV infection: pre-S mutation rates were 37% in chronic HBV carriers, and as high as 60% in HCC patients. The Pre-S Gene Chip offers a highly sensitive and specific method for pre-S deletion detection and is less expensive and more efficient (turnaround time 3 days) than DNA sequencing analysis. Conclusion The pre-S1/2 mutants may emerge during the long-term persistence of the HBV genome in carriers and facilitate HCC development. Combined detection of pre-S mutations, other markers of HBV replication, and viral titers, offers a reliable predictive method for HCC risks in chronic HBV carriers.

Published
2009
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32. Susceptibility of Elizabethkingia spp. to commonly tested and novel antibiotics and concordance between broth microdilution and automated testing methods.

Author

Kuo, Shu-Chen, Tan, Mei-Chen, Huang, Wei-Cheng, Wu, Han-Chieh, Chen, Feng-Jui, Liao, Yu-Chieh, Wang, Hui-Ying, Shiau, Yih-Ru, and Lauderdale, Tsai-Ling

Abstract

Objectives: We aimed to determine susceptibilities of Elizabethkingia spp. to 25 commonly tested and 8 novel antibiotics, and to compare the performance of different susceptibility testing methods.Methods: Clinical isolates of Elizabethkingia spp., Chryseobacterium spp. and Flavobacterium spp. collected during 2002-18 (n = 210) in a nationwide surveillance programme in Taiwan were speciated by 16S rRNA sequencing. MICs were determined by broth microdilution. The broth microdilution results of 18 common antibiotics were compared with those obtained by the VITEK 2 automated system.Results: Among the Elizabethkingia spp. identified (n = 108), Elizabethkingia anophelis was the most prevalent (n = 90), followed by Elizabethkingia meningoseptica (n = 7) and Elizabethkingia miricola cluster [E. miricola (n = 6), Elizabethkingia bruuniana (n = 3) and Elizabethkingia ursingii (n = 2)]. Most isolates were recovered from respiratory or blood specimens from hospitalized, elderly patients. PFGE showed two major and several minor E. anophelis clones. All isolates were resistant to nearly all the tested β-lactams. Doxycycline, minocycline and trimethoprim/sulfamethoxazole inhibited >90% of Elizabethkingia spp. Rifampin inhibited E. meningoseptica (100%) and E. anophelis (81.1%). Fluoroquinolones and tigecycline were active against E. meningoseptica and E. miricola cluster isolates. Novel antibiotics, including imipenem/relebactam, meropenem/vaborbactam, ceftazidime/avibactam, cefepime/zidebactam, delafloxacin, eravacycline and omadacycline were ineffective but lascufloxacin inhibited half of Elizabethkingia spp. The very major discrepancy rates of VITEK 2 were >1.5% for ciprofloxacin, moxifloxacin and vancomycin. Major discrepancy rates were >3% for amikacin, tigecycline, piperacillin/tazobactam and trimethoprim/sulfamethoxazole.Conclusions: MDR, absence of standard interpretation criteria and poor intermethod concordance necessitate working guidelines to facilitate future research of emerging Elizabethkingia spp. [ABSTRACT FROM AUTHOR]

Published
2021
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35. HER2 testing in breast cancer by quantitative PCR: technical and clinical considerations.

Author

Yu, Rong, primary, Chen, Wen-Chung, additional, Chiu, Kung Y., additional, Lu, Sunny, additional, Liu, Yuan-Chil, additional, Ko, Hsiao-Yun, additional, Wu, Han-Chieh, additional, Hsieh, Wen-Chaun, additional, Lee, Zi-jia, additional, Zhang, Peter, additional, Shi, Lili, additional, Liu, Ming, additional, Chang, Kung-Chao, additional, Su, Ih-Jen, additional, Su, Peggy, additional, and Yeh, Yu-Ming, additional

Published
2015
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41. Milrinone Therapy for Enterovirus 71-Induced Pulmonary Edema and/or Neurogenic Shock in Children

Author

Chi, Chia-Yu, primary, Khanh, Truong Huu, additional, Thoa, Le Phan Kim, additional, Tseng, Fan-Chen, additional, Wang, Shih-Min, additional, Thinh, Le Quoc, additional, Lin, Chia-Chun, additional, Wu, Han-Chieh, additional, Wang, Jen-Ren, additional, Hung, Nguyen Thanh, additional, Thuong, Tang Chi, additional, Chang, Chung-Ming, additional, Su, Ih-Jen, additional, and Liu, Ching-Chuan, additional

Published
2013
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42. Histone deacetylase inhibitor suberoylanilide hydroxamic acid suppresses the pro-oncogenic effects induced by hepatitis B virus pre-S 2 mutant oncoprotein and represents a potential chemopreventive agent in high-risk chronic HBV patients

Author

Hsieh, Yi-Hsuan, primary, Su, Ih-Jen, additional, Yen, Chia-Jui, additional, Tsai, Ting-Fen, additional, Tsai, Hung-Wen, additional, Tsai, Han-Ni, additional, Huang, Yu-Jun, additional, Chen, Yen-Yu, additional, Ai, Yu-Lin, additional, Kao, Lin-Yuan, additional, Hsieh, Wen-Chuan, additional, Wu, Han-Chieh, additional, and Huang, Wenya, additional

Published
2012
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