https://scholarworks.korea.ac.kr/kumedicine/handle/2020.sw.kumedicine/528 2026-04-05T16:20:10Z https://scholarworks.korea.ac.kr/kumedicine/handle/2021.sw.kumedicine/79014 Title: Development of a LAMP assay using hybridization-based TaqMan-style probes (HyTaq) for simultaneous detection of wild-type and macrolide-resistant Mycoplasma pneumoniae Authors: Lee, Eunji; Jung, Woo Sang; Lim, Chae Seung; Jang, Woong Sik Abstract: Mycoplasma pneumoniae is a major cause of community-acquired pneumonia and increasingly exhibits macrolide resistance due to the A2063G and A2064G mutations in the 23S rRNA gene. We developed a loop-mediated isothermal amplification (LAMP) assay with hybridization-based TaqMan-style probes (HyTaq) to simultaneously detect M. pneumoniae wild-type strains and macrolide resistance mutations (A2063G and A2064G) within a single reaction (hereafter referred to as the MP-R HyTaq-LAMP assay). Analytical performance was evaluated using plasmid standards, and clinical validation was conducted with 224 nasopharyngeal swab samples. The MP-R HyTaq-LAMP assay detected wild-type strains and the A2063G and A2064G mutations with a limit of detection of 1 x 10(3) copies/mu L. In clinical samples, the assay demonstrated 95.79% sensitivity for A2063G and 100% sensitivity for wild-type strains, along with 100% specificity against 103 non-infection samples. Additionally, no cross-reactivity was observed with 16 other common respiratory pathogens. The MP-R HyTaq-LAMP assay provides rapid, multiplex detection of M. pneumoniae and associated macrolide resistance mutations without thermal cycling, demonstrating its strong potential as a point-of-care diagnostic tool.IMPORTANCEMacrolide-resistant Mycoplasma pneumoniae is a significant cause of treatment failure in community-acquired respiratory infections, particularly in children and adolescents. Early and accurate detection of resistance-associated mutations, such as A2063G and A2064G, is essential for guiding effective antibiotic therapy. In this study, we developed an MP-R HyTaq-based loop-mediated isothermal amplification assay capable of simultaneously detecting and discriminating wild-type strains and macrolide resistance mutations (A2063G and A2064G) in a single-tube, isothermal reaction. Its high specificity, rapid turnaround time, and minimal equipment requirements make this assay suitable for point-of-care testing in diverse clinical settings. 2026-01-01T00:00:00Z https://scholarworks.korea.ac.kr/kumedicine/handle/2021.sw.kumedicine/78982 Title: Residual WBC Count Using Image-Based Immunofluorescence Cell Counter with Microfluidic Concentration Chip Authors: Lim, C.; Jang, W. Abstract: Background/Case Studies: Accurate measurement of residual white blood cells (WBCs) is essential for quality control in leukoreduced blood components. Conventional methods such as the Nageotte chamber and flow cytometry are reliable but labor-intensive, time-consuming, and costly. These drawbacks limit their use in high-throughput or resource-limited settings. We evaluated a novel system combining the Microscanner C3—an image-based immunofluorescence cell counter—with a microfluidic concentration chip to enhance detection sensitivity and workflow efficiency. Study Design/Methods: Microfluidic Chip Design: The microfluidic concentration chip was fabricated using soft lithography with PDMS material and SU-8 photoresist molds. The main channel (50 μm wide, 25 μm high) and an expansion zone (700 μm wide) were designed to optimize leukocyte flow and enrichment. To reduce non-specific protein binding, channel surfaces were treated with Triton X-100. A hyaluronic acid-based viscoelastic solution was used to maintain stable flow characteristics. Red cell concentrates were serially diluted using leukoreduced RBCs and normal saline to prepare WBC concentrations of 200, 50, 25, 7.8, 1.9, and 0.5 WBCs/μL. The samples were stained with PI lysis solution (Biozentech, Korea) and analyzed using the Microscanner C3 equipped with the microfluidic concentration chip. The performance of the system was evaluated for repeatability, linearity, limit of detection (LOD), and precision. Results were compared to the Nageotte chamber method and flow cytometry (Navios EX, Beckman Coulter; LeucoCOUNT, BD Biosciences). Results/Findings: Residual WBC counting with the Microscanner C3 and microfluidic chip was completed within 5 min. The chip efficiently concentrated WBCs from the diluted samples. Linearity was maintained across a range of 0.5–200 WBCs/μL (R² > 0.92 vs. flow cytometry), and correlation with Nageotte counting was R² > 0.94. The coefficient of variation ranged from 9.4% to 18.7% (25–200 WBCs/μL). The system's detection limit reached 0.5 WBCs/μL using saline-diluted samples. Conclusions: The Microscanner C3 integrated with a microfluidic concentration chip enables rapid, reproducible, and cost-effective residual WBC quantification. This platform is well-suited for quality control in blood component manufacturing, particularly in settings with limited access to advanced cytometry systems. 2025-10-26T00:00:00Z https://scholarworks.korea.ac.kr/kumedicine/handle/2021.sw.kumedicine/78053 Title: Rapid and Multiplex Diagnosis of Malaria Using Chelex-100 Extraction and LAMP-MS Assay Authors: Lim, Min Sup; Choe, Young Lan; Jang, Woong Sik Abstract: Malaria remains a major public health threat, especially in tropical and subtropical regions. Accurate and rapid diagnosis is essential for effective disease management and control, yet conventional malaria diagnostics, including blood smear microscopy using Giemsa staining, PCR, and rapid diagnostic tests (RDTs), are limited by the need for trained personnel, reliance on laboratory infrastructure, and reduced sensitivity at low parasite densities, respectively. This protocol details an innovative, rapid, and economical diagnostic platform combining a simplified Chelex-100 resin-based nucleic acid extraction method with a multiplex loop-mediated isothermal amplification microscanner (LAMP-MS) assay. The malaria (Pan), and an internal control (IC) within 40 min, from DNA extraction to result interpretation. It demonstrates sensitivity and specificity comparable to traditional PCR-based diagnostics, making it a practical and scalable solution for use in resource-constrained environments. 2025-07-01T00:00:00Z https://scholarworks.korea.ac.kr/kumedicine/handle/2021.sw.kumedicine/76434 Title: Association of delayed asthma diagnosis with asthma exacerbations in children Authors: Wi, Chung-Il; Ryu, Euijung; King, Katherine S.; Kwon, Jung Hyun; Bublitz, Joshua T.; Park, Miguel; Chiarella, Sergio E.; Greenwood, Jason D.; Pongdee, Thanai; Myers, Lynnea; Nordlund, Björn; Sohn, Sunghwan; Sagheb, Elham; Kshatriya, Bhavani Singh Agnikula; Watson, Dave; Liu, Hongfang; Sheares, Beverley J.; Davis, Carla M.; Schulz, Wade; Juhn, Young J. Abstract: Background: There is a significant delay between symptom onset and diagnosis of childhood asthma, but the impact of this delay on asthma outcomes has not been well understood. Objectives: We sought to study the association of delayed diagnosis of asthma with asthma exacerbations (AEs) in children. Methods: Using the Mayo Clinic birth cohort, we identified children with a diagnosis of asthma from electronic health records. We defined onset date as the date when subjects first met predetermined asthma criteria ascertained by an electronic health records–based natural language processing algorithm. Delay in diagnosis (DD) was defined as first diagnosis >30 days from onset date (vs timely diagnosis [TD] within 30 days). The primary outcome was AE after the index date (for DD: first diagnosis date vs for TD: clinic visit at similar delay from diagnosis as matched DD counterpart). A Cox proportional hazard model was used to test the association between delayed diagnosis status and risk of AE, adjusting for sociodemographics, care quality, and asthma severity. Results: Among 537 matched pairs of DD and TD (median age at index date: 4.1 years), a total of 344 and 253 children in DD and TD, respectively, had ≥1 AE during median follow-up period of 9.3 years. Children in the DD group had a significantly increased risk of AE compared to TD (adjusted hazard ratio: 1.53; 95% CI: 1.28, 1.80; P < .001). Conclusions: DD of asthma in children is associated with an increased risk of AE compared to TD. TD of asthma should be an important priority in childhood asthma management. © 2025 The Author(s) 2025-05-01T00:00:00Z