Bonhan Koo

An isothermal, label-free, and rapid one-step RNA amplification/detection assay for diagnosis of respiratory viral infections.

Abstract Recently, RNA viral infections caused by respiratory viruses, such as influenza, parainfluenza, respiratory syncytial virus, coronavirus, and Middle East respiratory syndrome-coronavirus (MERS-CoV), and Zika virus, are a major public health threats in the world. Although myriads of diagnostic methods based on RNA amplification have been developed in the last decades, they continue to lack speed, sensitivity, and specificity for clinical use. A rapid and accurate diagnostic method is needed for appropriate control, including isolation and treatment of the patients. Here, we report an isothermal, label-free, one-step RNA amplification and detection system, termed as iROAD, for the diagnosis of respiratory diseases. It couples a one-step isothermal RNA amplification method and a bio-optical sensor for simultaneous viral RNA amplification/detection in a label-free and real-time manner. The iROAD assay offers a one-step viral RNA amplification/detection example to rapid analysis (<20min). The detection limit of iROAD assay was found to be 10-times more sensitive than that of real-time reverse transcription-PCR method. We confirmed the clinical utility of the iROAD assay by detecting viral RNAs obtained from 63 human respiratory samples. We envision that the iROAD assay will be useful and potentially adaptable for better diagnosis of emerging infectious diseases including respiratory diseases.

Molecular epidemiology and environmental contamination during an outbreak of parainfluenza virus 3 in a haematology ward

Summary Background Although fomites or contaminated surfaces have been considered as transmission routes, the role of environmental contamination by human parainfluenza virus type 3 (hPIV-3) in healthcare settings is not established. Aim To describe an hPIV-3 nosocomial outbreak and the results of environmental sampling to elucidate the source of nosocomial transmission and the role of environmental contamination. Methods During an hPIV-3 outbreak between May and June 2016, environmental surfaces in contact with clustered patients were swabbed and respiratory specimens used from infected patients and epidemiologically unlinked controls. The epidemiologic relatedness of hPIV-3 strains was investigated by sequencing of the haemagglutinin–neuraminidase and fusion protein genes. Findings Of 19 hPIV-3-infected patients, eight were haematopoietic stem cell recipients and one was a healthcare worker. In addition, four had upper and 12 had lower respiratory tract infections. Of the 19 patients, six (32%) were community-onset infections (symptom onset within <7 days of hospitalization) and 13 (68%) were hospital-onset infections (≥7 days of hospitalization). Phylogenetic analysis identified two major clusters: five patients, and three patients plus one healthcare worker. Therefore, seven (37%) were classified as nosocomial transmissions. hPIV-3 was detected in 21 (43%) of 49 environmental swabs up to 12 days after negative respiratory polymerase chain reaction conversion. Conclusion At least one-third of a peak season nosocomial hPIV-3 outbreak originated from nosocomial transmission, with multiple importations of hPIV-3 from the community, providing experimental evidence for extensive environmental hPIV-3 contamination. Direct contact with the contaminated surfaces and fomites or indirect transmission from infected healthcare workers could be responsible for nosocomial transmission.

Rapid Diagnosis of Tick-Borne Illnesses by Use of One-Step Isothermal Nucleic Acid Amplification and Bio-Optical Sensor Detection

BACKGROUND Scrub typhus and severe fever with thrombocytopenia syndrome (SFTS) are the most common tick-borne illnesses in South Korea. Early differentiation of SFTS from scrub typhus in emergency departments is essential but difficult because of their overlapping epidemiology, shared risk factors, and similar clinical manifestations. METHODS We compared the diagnostic performance of one-step isothermal nucleic acid amplification with bio-optical sensor detection (iNAD) under isothermal conditions, which is rapid (20-30 min), with that of real-time PCR, in patients with a confirmed tick-borne illness. Fifteen patients with confirmed SFTS who provided a total of 15 initial blood samples and 5 follow-up blood samples, and 21 patients with confirmed scrub typhus, were evaluated. RESULTS The clinical sensitivity of iNAD (100%; 95% CI, 83-100) for SFTS was significantly higher than that of real-time PCR (75%; 95% CI, 51-91; P = 0.047), while its clinical specificity (86%; 95% CI, 65-97) was similar to that of real-time PCR (95%; 95% CI, 77-99; P = 0.61). The clinical sensitivity of iNAD for scrub typhus (100%; 95% CI, 81-100) was significantly higher than that of real-time PCR for scrub typhus (67%; 95% CI, 43-85; P = 0.009), while its clinical specificity (90%; 95% CI, 67-98) was similar to that of real-time PCR (95%; 95% CI, 73-100; P > 0.99). CONCLUSIONS iNAD is a valuable, rapid method of detecting SFTS virus and Orientia tsutsugamushi with high clinical sensitivity and specificity.

Use of Dimethyl Pimelimidate with Microfluidic System for Nucleic Acids Extraction without Electricity

The isolation of nucleic acids in the lab on a chip is crucial to achieve the maximal effectiveness of point-of-care testing for detection in clinical applications. Here, we report on the use of a simple and versatile single-channel microfluidic platform that combines dimethyl pimelimidate (DMP) for nucleic acids (both RNA and DNA) extraction without electricity using a thin-film system. The system is based on the adaption of DMP into nonchaotropic-based nucleic acids and the capture of reagents into a low-cost thin-film platform for use as a microfluidic total analysis system, which can be utilized for sample processing in clinical diagnostics. Moreover, we assessed the use of the DMP system for the extraction of nucleic acids from various samples, including mammalian cells, bacterial cells, and viruses from human disease, and we also confirmed that the quality and quantity of the nucleic acids extracted were sufficient to allow for the robust detection of biomarkers and/or pathogens in downstream analysis. Furthermore, this DMP system does not require any instruments and electricity, and has improved time efficiency, portability, and affordability. Thus, we believe that the DMP system may change the paradigm of sample processing in clinical diagnostics.

A single-tube approach for in vitro diagnostics using diatomaceous earth and optical sensor

Abstract Versatile, simple and efficient sample preparation is desirable for point-of-care testing of emerging diseases such as zoonoses, but current sample preparation assays are insensitive, labour-intensive and time-consuming and require multiple instruments. We developed a single-tube sample preparation approach involving direct pathogen enrichment and extraction from human specimens using diatomaceous earth (DE). Amine-modified DE was used to directly enrich a zoonotic pathogen, Brucella, in a large sample volume. Next, a complex of amine-modified DE and dimethyl suberimidate was used for nucleic acid extraction from the enriched pathogen. Using our single-tube approach, the pathogen can be enriched and extracted within 60min at a level of 1 colony formation unit (CFU) from a 1ml sample volume in the same tube. The performance of this approach is 10–100 times better than that of a commercial kit (102 to 103 CFU/ml) but does not require a large centrifuge. Finally, we combined the single-tube approach with a bio-optical sensor for rapid and accurate zoonotic pathogen detection in human urine samples. Using the combination system, Brucella in human urine can be efficiently enriched (~ 8-fold) and the detection limit is enhanced by up to 100 times (1CFU/ml bacteria in urine) compared with the commercial kit. This combined system is fast and highly sensitive and thus represents a promising approach for disease diagnosis in the clinical setting.

Simple and label-free pathogen enrichment via homobifunctional imidoesters using a microfluidic (SLIM) system for ultrasensitive pathogen detection in various clinical specimens

Abstract Diseases caused by pathogenic microorganisms including bacteria and viruses can cause serious medical issues including death and result in huge economic losses. Despite the myriad of recent advances in the rapid and accurate detection of pathogens, large volume clinical samples with a low concentration of pathogens continue to present challenges for diagnosis and surveillance. We here report a simple and label-free approach via homobifunctional imidoesters (HIs) with a microfluidic platform (SLIM) to efficiently enrich and extract pathogens at low concentrations from clinical samples. The SLIM system consists of an assembled double microfluidic chip for streamlining large volume processing and HIs for capturing pathogens and isolating nucleic acids by both electrostatic and covalent interaction without a chaotropic detergent or bulky instruments. The SLIM system significantly increases the enrichment and extraction rate of pathogens (up to 80% at 10 CFU (colony forming unit) in a 1 mL volume within 50 min). We demonstrated its clinical utility in large sample volumes from 46 clinical specimens including environmental swabs, saliva, and blood plasma. The SLIM system showed higher sensitivity with these samples and could detect pathogens that were below the threshold of detection with other methods. Finally, by combining our SLIM approach with an isothermal optical sensor, pathogens could be detected at a very high sensitivity in blood plasma samples within 80 min via enrichment, extraction and detection steps. Our SLIM system thus provides a simple, reliable, cost-effective and ultrasensitive pathogen diagnosis platform for use with large volume clinical samples and would thus have significant utility for various infectious diseases.

A microfluidic enrichment platform with a recombinase polymerase amplification sensor for pathogen diagnosis

Rapid and sensitive detection of low amounts of pathogen in large samples is needed for early diagnosis and treatment of patients and surveillance of pathogen. In this study, we report a microfluidic platform for detection of low pathogen levels in a large sample volume that couples an Magainin 1 based microfluidic platform for pathogen enrichment and a recombinase polymerase amplification (RPA) sensor for simultaneous pathogenic DNA amplification and detection in a label-free and real-time manner. Magainin 1 is used as a pathogen enrichment agent with a herringbone microfluidic chip. Using this enrichment platform, the detection limit was found to be 20 times more sensitive in 10 ml urine with Salmonella and 10 times more sensitive in 10 ml urine with Brucella than that of real-time PCR without the enrichment process. Furthermore, the combination system of the enrichment platform and an RPA sensor that based on an isothermal DNA amplification method with rapidity and sensitivity for detection can detect a pathogen at down to 50 CFU in 10 ml urine for Salmonella and 102 CFU in 10 ml urine for Brucella within 60 min. This system will be useful as it has the potential for better diagnosis of pathogens by increasing the capture efficiency of the pathogen in large samples, subsequently enhancing the detection limit of pathogenic DNA.

A rapid bio-optical sensor for diagnosing Q fever in clinical specimens

Recent zoonotic outbreaks, such as Zika, Middle East respiratory syndrome and Ebola, have highlighted the need for rapid and accurate diagnostic assays that can be used to aid pathogen control. Q fever is a zoonotic disease caused by the transmission of Coxiella burnetii that can cause serious illness in humans through aerosols and is considered a potential bioterrorism agent. However, the existing assays are not suitable for the detection of this pathogen due to its low levels in real samples. We here describe a rapid bio-optical sensor for the accurate detection of Q fever and validate its clinical utility. By combining a bio-optical sensor, that transduces the presence of the target DNA based on binding-induced changes in the refractive index on the waveguide surface in a label-free and real-time manner, with isothermal DNA amplification, this new diagnostic tool offers a rapid (<20 min), 1-step DNA amplification/detection method. We confirmed the clinical sensitivity (>90%) of the bio-optical sensor by detecting C. burnetii in 11 formalin-fixed, paraffin-embedded liver biopsy samples from acute Q fever hepatitis patients and in 16 blood plasma samples from patients in which Q fever is the cause of fever of unknown origin.

Arch-shaped multiple-target sensing for rapid diagnosis and identification of emerging infectious pathogens

Abstract Rapid identification of emerging infectious pathogens is crucial for preventing public health threats. Various pathogen detection techniques have been introduced; however, most techniques are time-consuming and lack multiple-target detection specificity. Although multiple-target detection techniques can distinguish emerging infectious pathogens from related pathogens, direct amplification methods have not been widely examined. Here, we present a novel arch-shaped multiple-target sensor capable of rapid pathogen identification using direct amplification in clinical samples. In this study, an arch-shaped amplification containing primer sequences was designed to rapidly amplify multiple targets. Further, the sensing platform allowed for sensitive and specific detection of human coronavirus, Middle East respiratory syndrome, Zika virus, and Ebola virus down to several copies. This platform also simultaneously distinguished between Middle East respiratory syndrome and human coronavirus in clinical specimens within 20 min. This arch-shaped multiple-target sensing assay can provide rapid, sensitive, and accurate diagnoses of emerging infectious diseases in clinical applications.

Large Instrument- and Detergent-Free Assay for Ultrasensitive Nucleic Acids Isolation via Binary Nanomaterial

Nucleic acid-based diagnostics are widely used for clinical applications due to their powerful recognition of biomolecule properties. Isolation and purification of nucleic acids such as DNA and RNA in the diagnostic system have been severely hampered in point-of-care testing because of low recovery yields, degradation of nucleic acids due to the use of chaotropic detergent and high temperature, and the requirement of large instruments such as centrifuges and thermal controllers. Here, we report a novel large instrument- and detergent-free assay via binary nanomaterial for ultrasensitive nucleic acid isolation and detection from cells (eukaryotic and prokaryotic). This binary nanomaterial couples a zinc oxide nanomultigonal shuttle (ZnO NMS) for cell membrane rupture without detergent and temperature control and diatomaceous earth with dimethyl suberimidate complex (DDS) for the capture and isolation of nucleic acids (NA) from cells. The ZnO NMS was synthesized to a size of 500 nm to permit efficient cell lysis at room temperature within 2 min using the biological, chemical, and physical properties of the nanomaterial. By combining the ZnO NMS with the DDS and proteinase K, the nucleic acid extraction could be completed in 15 min with high quantity and quality. For bacterial cells, DNA isolation with the binary nanomaterial yielded 100 times more DNA, than a commercial spin column based reference kit, as determined by the NanoDrop spectrophotometer. We believe that this binary nanomaterial will be a useful tool for rapid and sensitive nucleic acid isolation and detection without large instruments and detergent in the field of molecular diagnostics.