Identification of Novel Biomarkers for Early Diagnosis of Atherosclerosis Using High-Resolution Metabolomics.

Atherosclerosis (AS) is a metabolic disorder and the pre-stage of several cardiovascular diseases, including myocardial infarction, stroke, and angina pectoris. Early detection of AS can provide the opportunity for effective management and better clinical results, along with the prevention of further progression of the disease. In the current study, an untargeted and targeted metabolomic approach was used to identify possible metabolic signatures that have altered levels in AS patients. A total of 200 serum samples from individuals with AS and normal were analyzed via liquid chromatography-high-resolution mass spectrometry. Univariate and multivariate analysis approaches were used to identify differential metabolites. A group of metabolites associated with bile acids, amino acids, steroid hormones, and purine metabolism were identified that are capable of distinguishing AS-risk sera from normal. Further, the targeted metabolomics approach confirmed that six metabolites, namely taurocholic acid, cholic acid, cortisol, hypoxanthine, trimethylamine N-oxide (TMAO), and isoleucine, were found to be significantly upregulated, while the concentrations of glycoursodeoxycholic acid, glycocholic acid, testosterone, leucine, methionine, phenylalanine, tyrosine, and valine were found to be significantly downregulated in the AS-risk sera. The receiver operating characteristic curves of three metabolites, including cortisol, hypoxanthine, and isoleucine, showed high sensitivity and specificity. Taken together, these findings suggest cortisol, hypoxanthine, and isoleucine as novel biomarkers for the early and non-invasive detection of AS. Thus, this study provides new insights for further investigations into the prevention and management of AS.

Continuous On-Chip Cell Washing Using Viscoelastic Microfluidics.

Medium exchange of particles/cells to a clean buffer with a low background is essential for biological, chemical, and clinical research, which has been conventionally conducted using centrifugation. However, owing to critical limitations, such as possible cell loss and physical stimulation of cells, microfluidic techniques have been adopted for medium exchange. This study demonstrates a continuous on-chip washing process in a co-flow system using viscoelastic and Newtonian fluids. The co-flow system was constructed by adding a small amount of biocompatible polymer (xanthan gum, XG) to a sample containing particles or cells and introducing Newtonian fluids as sheath flows. Polymer concentration-dependent and particle size-dependent lateral migration of particles in the co-flow system were examined, and then the optimal concentration and the critical particle size for medium exchange were determined at the fixed total flow rate of 100 µL/min. For clinical applications, the continuous on-chip washing of white blood cells (WBCs) in lysed blood samples was demonstrated, and the washing performance was evaluated using a scanning spectrophotometer.

Separation and Washing of Candida Cells from White Blood Cells Using Viscoelastic Microfluidics.

An early and accurate diagnosis of Candida albicans is critical for the rapid antifungal treatment of candidemia, a mortal bloodstream infection. This study demonstrates viscoelastic microfluidic techniques for continuous separation, concentration, and subsequent washing of Candida cells in the blood. The total sample preparation system contains two-step microfluidic devices: a closed-loop separation and concentration device and a co-flow cell-washing device. To determine the flow conditions of the closed-loop device, such as the flow rate factor, a mixture of 4 and 13 µm particles was used. Candida cells were successfully separated from the white blood cells (WBCs) and concentrated by 74.6-fold in the sample reservoir of the closed-loop system at 800 µL/min with a flow rate factor of 3.3. In addition, the collected Candida cells were washed with washing buffer (deionized water) in the microchannels with an aspect ratio of 2 at a total flow rate of 100 µL/min. Finally, Candida cells at extremely low concentrations (Ct > 35) became detectable after the removal of WBCs, the additional buffer solution in the closed-loop system (Ct = 30.3 ± 1.3), and further removal of blood lysate and washing (Ct = 23.3 ± 1.6).

Effects of Combinations of Toxin Binders with or without Natural Components on Broiler Breeders Exposed to Ochratoxin A.

The objective of this study was to investigate the effects of toxin binders on broiler breeders fed an ochratoxin A (OTA)-contaminated diet. A total of 60 45-week-old female Arbor Acres broiler breeder birds with an initial body weight of 3.65 ± 0.35 kg were randomly divided into 6 treatment groups, with 10 replicates per group and 1 bird per replicate. The trial was conducted for 9 weeks (including 1 week of adaptation). Feed additive 1 (FA1) was composed of clay minerals (85% bentonite and 12% clinoptilolite) with 3% charcoal. FA2 was composed of clay minerals (66.1% aluminosilicates) with natural components (0.8% artichoke and rosemary plant extracts), 7% yeast extract, 0.5% beta-glucans, and 25.6% carriers. The dietary treatment groups were as follows: (1) birds fed an OTA-free basal diet (Negative Control; NC); (2) lipopolysaccharide (LPS)-challenged birds fed a diet including OTA (4 mg/kg) (Positive Control, PC); (3) the PC with 0.05% FA1 (Treatment 1, T1); (4) the PC with 0.10% FA1 (Treatment 2, T2); (5) the PC with 0.10% FA2 (Treatment 3, T3); and (6) the PC with 0.20% FA2 (Treatment 4, T4). The LPS challenge (an intramuscular injection of 1 mg E. coli O55:B5 LPS per kg of body weight) was performed on the first day of the experiment. The results of this experiment show that the PC treatment negatively affected (p < 0.05) egg production, hatchability, Haugh unit, bone mineralization, relative organ weight (abdominal fat, liver), the levels of glutamic oxaloacetic transaminase (GOT), high-density lipoprotein (HDL), and total cholesterol in the blood, and OTA accumulation in the liver compared with the NC. However, supplementation with toxin binders mitigated (p < 0.05) the negative effects of the OTA. Specifically, supplementation with 0.10% FA1 and 0.10% FA2 increased (p < 0.05) eggshell strength by week 4, and the Haugh unit and bone mineralization (phosphorous) by week 8, while decreasing (p < 0.05) the relative weight of the liver and the levels of GOT and HDL in the blood. Supplementation with 0.10% FA2 led to greater improvements in various parameters, including laying performance and bone mineralization, than the other treatments. In conclusion, toxin binders with or without natural components can be effective tools in the mitigation of OTA-induced problems due to their synergistic effects.

Sinus augmentation with poly(ε)caprolactone-ß tricalcium phosphate scaffolds, mesenchymal stem cells and platelet rich plasma for one-stage dental implantation in minipigs.

PURPOSE: This study evaluated the efficacy of a tube-shaped poly(ε) caprolactone - ß tricalcium phosphate (PCL-TCP) scaffold with the incorporation of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) and platelet-rich plasma (PRP) for bone regeneration in the procedure of single-stage sinus augmentation and dental implantation in minipigs. METHODS: Implants were placed in the bilateral sides of the maxillary sinuses of 5 minipigs and allocated to a PCL-TCP+hUCMSCs+PRP group (n=5), a PCL-TCP+PRP group (n=5), and a PCL-TCP-only group (n=6). After 12 weeks, bone regeneration was evaluated with soft X-rays, micro-computed tomography, fluorescence microscopy, and histomorphometric analysis. RESULTS: Four implants failed (2 each in the PCL-TCP+hUCMSCs+PRP and PCL-TCP+hUCMSC groups). An analysis of the grayscale levels and bone-implant contact ratio showed significantly higher mean values in the PCL-TCP+hUCMSCs+PRP than in the PCL-TCP group (P=0.045 and P=0.016, respectively). In fluoromicroscopic images, new bone formation around the outer surfaces of the scaffolds was observed in the PCL-TCP+hUCMSCs+PRP group, suggesting a tenting effect of the specially designed scaffolds. Bone regeneration at the scaffold-implant interfaces was observed in all 3 groups. CONCLUSIONS: Using a tube-shaped, honeycombed PCL-TCP scaffold with hUCMSCs and PRP may serve to enhance bone formation and dental implants' osseointegration in the procedure of simultaneous sinus lifting and dental implantation.

Effect of black soldier fly larvae as substitutes for fishmeal in broiler diet.

This study investigated the effect of processed forms (defatted or hydrolyzed) of black soldier fly larvae (Hermetia illucens L., BSFL) as a protein substitute on broilers. Experiment 1 was a feeding experiment, and Experiment 2 was a metabolism experiment. In Experiment 1, a total of 120 day-old Arbor Acres broilers (initial body weight 39.52 ± 0.24 g) were used for 28 days. There were 8 replicate pens, and 5 broilers were assigned to each pen. In Experiment 2, a total of 36 day-old broilers (initial body weight 39.49 ± 0.21 g) were used for the metabolism trial. There were 2 broilers in a metabolism cage and six replicate cages per treatment. The dietary treatments were as follows: a basal diet (CON), a basal diet without fishmeal and substitute with defatted BSFL (T1), a basal diet without fishmeal and a substitute with hydrolyzed BSFL (T2). In Experiment 1, during the entire experimental period, the T2 group significantly increased (p < 0.05) body weight gain and feed intake compared to the CON and T1 groups. The feed conversion ratio showed a lower tendency (p = 0.057) in the T2 group than in the CON and T1 groups. At 2 weeks, the CON and T2 groups were significantly higher (p < 0.05) crude protein (CP) digestibility than the T1 group. At 4 weeks, the total protein level significantly increased (p < 0.05) in the CON and T2 groups compared to the T1 group. In Experiment 2, the CP digestibility significantly increased (p < 0.05) in the T2 group compared to the CON and T1 group at weeks 2 and 4. At week 4 amino acid digestibility, the T2 group significantly increased (p < 0.05) lysine, methionine, tryptophan, and glycine digestibility compared to the T1 group. There was no difference in fecal microbiota among the treatment groups. In conclusion, feeding hydrolyzed BSFL as a fishmeal substitute in broiler diets improved growth performance, CP digestibility, and specific amino acid digestibility. Therefore, it is considered that hydrolyzed BSFL in broiler diets can be sufficiently used as a new protein source.

A Continuous Microfluidic Concentrator for High-Sensitivity Detection of Bacteria in Water Sources.

Water contamination is a critical issue that threatens global public health. To enable the rapid and precise monitoring of pathogen contamination in drinking water, a concentration technique for bacterial cells is required to address the limitations of current detection methods, including the culture method and polymerase chain reaction. Here we present a viscoelastic microfluidic device for the continuous concentration of bacterial cells. To validate the device performance for cell concentration, the flow characteristics of 2-µm particles were estimated in viscoelastic fluids at different concentrations and flow rates. Based on the particle flow distributions, the flow rate factor, which is defined as the ratio of the inlet flow rate to the outlet flow rate at the center outlet, was optimized to achieve highly concentrated bacterial cells by removal of the additional suspending medium. The flow characteristics of 0.5-, 0.7-, and 1.0-µm-diameter particles were evaluated to consider the effect of a wide spectrum of bacterial size distribution. Finally, the concentration factor of bacterial cells, Staphylococcus aureus, suspended in a 2000-ppm polyethylene oxide solution was found to be 20.6-fold at a flow rate of 20 µL/min and a flow rate factor of 40.

Developing a multiplex loop-mediated isothermal amplification assay (LAMP) to determine severe fever with thrombocytopenia syndrome (SFTS) and scrub typhus.

Severe fever with thrombocytopenia syndrome (SFTS) and scrub typhus are endemic zoonotic diseases that pose significant public health threats in East Asia. As these two diseases share common clinical features, as well as overlapping disease regions, it is difficult to differentiate between SFTS and scrub typhus. A multiplex reverse-transcription loop­mediated isothermal amplification (RT-LAMP) assay was developed to detect large segments and GroES genes for SFTS virus (SFTSV) and Orientia tsutsugamushi (OT). The performance of the RT-LAMP assay was compared and evaluated with those of commercial PowerChek™ SFTSV real-time PCR and LiliF™ TSUTSU nested PCR for 23 SFTS and 12 scrub typhus clinical samples, respectively. The multiplex SFTSV/OT/Internal control (IC) RT-LAMP assay showed comparable sensitivity (91.3%) with that of commercial PowerChek™ SFTSV Real-time PCR (95.6%) and higher sensitivity (91.6%) than that of LiliF™ TSUTSU nested PCR (75%). In addition, the multiplex SFTSV/OT RT-LAMP assay showed 100% specificity and no cross-reactivity for blood from uninfected healthy patients and samples from patients infected with other fever viruses. Thus, the multiplex SFTSV/OT/IC RT-LAMP assay could serve as a useful point-of-care molecular diagnostic test for SFTS and scrub typhus.

High-Throughput Cell Concentration Using A Piezoelectric Pump in Closed-Loop Viscoelastic Microfluidics.

Cell concentration is a critical process in biological assays and clinical diagnostics for the pre-treatment of extremely rare disease-related cells. The conventional technique for sample preconcentration and centrifugation has the limitations of a batch process requiring expensive and large equipment. Therefore, a high-throughput continuous cell concentration technique needs to be developed. However, in single-pass operation, the required concentration ratio is hard to achieve. In this study, we propose a closed-loop continuous cell concentration system using a viscoelastic non-Newtonian fluid. For miniaturized and integrated systems, two piezoelectric pumps were adopted. The pumping capability generated by a piezoelectric pump in a microfluidic channel was evaluated depending on the applied voltage, frequency, sample viscosity, and channel length. The concentration performance of the device was evaluated using 13 µm particles and white blood cells (WBCs) with different channel lengths and voltages. In the closed-loop system, the focused cells collected at the center outlet were sent back to the inlet, while the buffer solution was removed to the side outlets. Finally, to expand the clinical applicability of our closed-loop system, WBCs in lysed blood samples with 70% hematocrit and prostate cancer cells in urine samples were used. Using the closed-loop system, WBCs were concentrated by ~63.4 ± 0.8-fold within 20 min to a final volume of 160 µL using 10 mL of lysed blood sample with 70% hematocrit (~3 cP). In addition, prostate cancer cells in 10 mL urine samples were concentrated by ~64.1-fold within ~11 min due to low viscosity (~1 cP).

Mesenchymal stem cells and platelet-rich plasma-impregnated polycaprolactone-ß tricalcium phosphate bio-scaffold enhanced bone regeneration around dental implants.

BACKGROUND: Finding a material that supports bone regeneration is the concern for many investigators. We supposed that a composite scaffold of poly(ε) caprolactone and ß-tricalcium phosphate (PCL-TCP) would entail desirable characteristics of biocompatibility, bioresorbability, rigidity, and osteoconductivity for a proper guided bone regeneration. Furthermore, the incorporation of mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) would boost the bone regeneration. We conducted this study to evaluate the bone regeneration capacity of PCL-TCP scaffold that is loaded with MSCs and PRP. MATERIALS AND METHODS: Five miniature pigs received 6 implants in 6 created-mandibular bony defects in the right and left lower premolar areas. The bony defects were managed according to the following three groups: the PCL-TCP scaffold loaded with MSCs and PRP (MSCs+PRP+PCL-TCP) group (n = 10), PCL-TCP scaffold loaded with PRP (PRP+PCL-TCP) group (n = 10), and PCL-TCP scaffold group (n = 10). After 12 weeks, the bone regeneration was assessed using fluorochrome bone labeling, µCT bone morphogenic analysis, and histomorphometric analysis. RESULTS: All of the three groups supported the bone regeneration around the dental implants. However, the PCL-TCP scaffold loaded with MSCs and PRP (MSCs+PRP+PCL-TCP) group showed non-significant higher bone surface, bone specific surface, and bone surface density than the other two groups as revealed by the µCT bone morphogenic analysis. Histologically, the same group revealed higher bone-implant contact ratio (BIC) (p = 0.017) and new bone height formation (NBH, mm) (p = 0.0097) with statistically significant difference compared to the PCL-TCP scaffold group. CONCLUSIONS: PCL-TCP scaffold is compatible for bone regeneration in bone defects surrounding dental implants. Moreover, the incorporation of MSCs and PRP optimized the bone regeneration process with respect to the rate of scaffold replacement, the height of the regenerated bone, and implant stability.

An Automated Microscopic Malaria Parasite Detection System Using Digital Image Analysis.

Rapid diagnosis and parasitemia measurement is crucial for management of malaria. Microscopic examination of peripheral blood (PB) smears is the gold standard for malaria detection. However, this method is labor-intensive. Here, we aimed to develop a completely automated microscopic system for malaria detection and parasitemia measurement. The automated system comprises a microscope, plastic chip, fluorescent dye, and an image analysis program. Analytical performance was evaluated regarding linearity, precision, and limit of detection and was compared with that of conventional microscopic PB smear examination and flow cytometry. The automated microscopic malaria parasite detection system showed a high degree of linearity for Plasmodium falciparum culture (R2 = 0.958, p = 0.005) and Plasmodium vivax infected samples (R2 = 0.931, p = 0.008). Precision was defined as the %CV of the assay results at each level of parasitemia and the %CV value for our system was lower than that for microscopic examination for all densities of parasitemia. The limit of detection analysis showed 95% probability for parasite detection was 0.00066112%, and a high correlation was observed among all three methods. The sensitivity and specificity of the system was both 100% (n = 21/21) and 100% (n = 50/50), respectively, and the system correctly identified all P. vivax and P. falciparum samples. The automated microscopic malaria parasite detection system offers several advantages over conventional microscopy for rapid diagnosis and parasite density monitoring of malaria.

Clinical Usefulness of LabChip Real-time PCR using Lab-On-a-Chip Technology for Diagnosing Malaria.

As malaria remains a major health problem worldwide, various diagnostic tests have been developed, including microscopy-based and rapid diagnostic tests. LabChip real-time PCR (LRP) is a small and portable device used to diagnose malaria using lab-on-a-chip technology. This study aimed to evaluate the diagnostic performance of LRP for detecting malaria parasites. Two hundred thirteen patients and 150 healthy individuals were enrolled from May 2009 to October 2015. A diagnostic detectability of LRP for malaria parasites was compared to that of conventional RT-PCR. Sensitivity of LRP for Plasmodium vivax, P. falciparum, P. malariae, and P. ovale was 95.5%, 96.0%, 100%, and 100%, respectively. Specificity of LRP for P. vivax, P. falciparum, P. malariae, and P. ovale was 100%, 99.3%, 100%, and 100%, respectively. Cohen's Kappa coefficients between LRP and CFX96 for detecting P. vivax, P. falciparum, P. malariae, and P. ovale were 0.96, 0.98, 1.00, and 1.00, respectively. Significant difference was not observed between the results of LRP and conventional RT-PCR and microscopic examination. A time required to amplify DNAs using LRP and conventional RT-PCR was 27 min and 86 min, respectively. LRP amplified DNAs 2 times more fast than conventional RT-PCR due to the faster heat transfer. Therefore, LRP could be employed as a useful tool for detecting malaria parasites in clinical laboratories.

Performance Evaluation of Alere i Influenza A&B in Detecting Influenza Viruses A and B.

OBJECTIVE: Alere i Influenza A&B is an isothermal nucleic acid amplification-based integrated system used for detecting and differentiating between influenza virus A and influenza virus B. We evaluated the clinical performances of Alere i Influenza A&B compared to that of real-time PCR, multiplex real-time PCR, and two rapid influenza diagnostic kits. METHODS: Nasopharyngeal aspiration specimens (n=315) from patients with signs of acute respiratory infection were collected between 2015 and 2016. Samples were tested using real-time PCR, the multiplex RT-PCR Anyplex II RV16 Detection kit, Alere i Influenza A&B, BD Veritor™ System Flu A+B, and the Sofia Influenza A+B Fluorescence Immunoassay. Positive influenza specimens detected by the Anyplex II RV16 Detection kit were tested by real-time PCR. RESULTS: Compared to that of multiplex RT-PCR (influenza A, n=88; influenza B, n=82; influenza-negative, n=145), the sensitivities of Alere i, Sofia, and Veritor for influenza A were 97.7%, 72.7%, and 71.6%, respectively, whereas for influenza B, the sensitivities were 96.3%, 80.4%, and 75.6%, respectively. The specificity of Alere i, Sofia, and Veritor was 100.0%. CONCLUSIONS: The clinical performance of Alere i Influenza A&B is satisfactory, with the advantage of a significantly shorter test time than other molecular assays. It is suitable for point-of-care testing and rapid influenza diagnostic tests because of its high sensitivity and specificity.

Development of a multiplex Loop-Mediated Isothermal Amplification (LAMP) assay for on-site diagnosis of SARS CoV-2.

A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).

Development and evaluation of a multiplex loop-mediated isothermal amplification (LAMP) assay for differentiation of Mycobacterium tuberculosis and non-tuberculosis mycobacterium in clinical samples.

INTRODUCTION: The rapid and accurate diagnosis of tuberculosis (TB) is important to reduce morbidity and mortality rates and risk of transmission. Therefore, molecular detection methods such as a real-time PCR-based assay for Mycobacterium tuberculosis (MTB) have been commonly used for diagnosis of TB. Loop-mediated isothermal amplification (LAMP) assay was believed to be a simple, quick, and cost-effective isothermal nucleic acid amplification diagnostic test for infectious diseases. In this study, we designed an in-house multiplex LAMP assay for the differential detection of MTB and non-tuberculosis mycobacterium (NTM), and evaluated the assay using clinical samples. MATERIAL AND METHODS: For the multiplex LAMP assay, two sets of specific primers were designed: the first one was specific for IS6110 genes of MTB, and the second one was universal for rpoB genes of mycobacterium species including NTM. MTB was confirmed with a positive reaction with both primer sets, and NTM was identified with a positive reaction by only the second primer set without a MTB-specific reaction. Total 333 clinical samples were analyzed to evaluate the multiplex LAMP assay. Clinical samples were composed of 195 positive samples (72 MTB and 123NTM) and 138 negative samples. All samples were confirmed positivity or negativity by real-time PCR for MTB and NTM. Analytical sensitivity and specificity were evaluated for the multiplex LAMP assay in comparison with acid fast bacilli staining and the culture method. RESULTS: Of 123 NTM samples, 121 were identified as NTM and 72/72 MTB were identified as MTB by the multiplex LAMP assay. False negative reactions were seen only in two NTM positive samples with co-infection of Candida spp. All 138 negative samples were identified as negative for MTB and NTM. Analytical sensitivity of the multiplex LAMP assay was 100% (72/72) for MTB, and 98.4% (121/123) for NTM. And the specificity of assay was 100% (138/138) for all. CONCLUSIONS: Our newly designed multiplex LAMP assay for MTB and NTM showed relatively good sensitivity in comparison with previously published data to detect isolated MTB. This multiplex LAMP assay is expected to become a useful tool for detecting and differentiating MTB from NTM rapidly at an acceptable sensitivity.

Development of a multiplex isothermal amplification molecular diagnosis method for on-site diagnosis of influenza.

Influenza, which is an acute respiratory disease caused by the influenza virus, represents a worldwide public health and economic problem owing to the significant morbidity and mortality caused by its seasonal epidemics and pandemics. Sensitive and convenient methodologies for the detection of influenza viruses are important for clinical care and infection control as well as epidemiological investigations. Here, we developed a multiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) with quencher/fluorescence oligonucleotides connected by a 5' backward loop (LF or LB) primer for the detection of two subtypes of influenza viruses: Influenza A (A/H1 and A/H3) and influenza B. The detection limits of the multiplex RT-LAMP assay were 103 copies and 102 copies of RNA for influenza A and influenza B, respectively. The sensitivities of the multiplex influenza A/B/IC RT-LAMP assay were 94.62% and 97.50% for influenza A and influenza B clinical samples, respectively. The specificities of the multiplex influenza A/B/IC RT-LAMP assay were 100% for influenza A, influenza B, and healthy clinical samples. In addition, the multiplex influenza A/B/IC RT-LAMP assay had no cross-reactivity with other respiratory viruses.

Acoustic mixing in a dome-shaped chamber-based SAW (DC-SAW) device.

The use of an open droplet system for surface acoustic wave (SAW)-based applications has been limited by droplet instability at high input power. This study introduces a dome-shaped chamber-based SAW (DC-SAW) device for the first time, which can be fabricated simply using a single adhesive tape and a drop of ultraviolet-curable material without soft lithography processes. The dome-shaped chamber device with a contact angle of 68° enables the maximizing of the effect of SAW transmitted at a refraction angle of roughly 22°, negating the droplet instability. The DC-SAW device was applied to acoustic mixing to estimate its capability. Acoustic mixing of two different fluids (i.e., deionized water and fluorescent particle suspension) was demonstrated in the dome-shaped chamber device. Moreover, the effect of flow rate and applied voltage on mixing performance was estimated. With the decreasing flow rate and increasing applied voltage, mixing performance was enhanced. At an applied voltage of 20 V, mixing indices were higher than 0.9 at a total flow rate of 300 µl min-1.

Sheathless Shape-Based Separation of Candida Albicans Using a Viscoelastic Non-Newtonian Fluid.

Rapid and accurate identification of Candida albicans from among other candida species is critical for cost-effective treatment and antifungal drug assays. Shape is a critical biomarker indicating cell type, cell cycle, and environmental conditions; however, most microfluidic techniques have been focused only on size-based particle/cell manipulation. This study demonstrates a sheathless shape-based separation of particles/cells using a viscoelastic non-Newtonian fluid. The size of C. albicans was measured at 37 °C depending on the incubation time (0 h, 1 h, and 2 h). The effects of flow rates on the flow patterns of candida cells with different shapes were examined. Finally, 2-h-incubated candida cells with germ tube formations (≥26 µm) were separated from spherical candida cells and shorter candida cells with a separation efficiency of 80.9% and a purity of 91.2% at 50 µL/min.

Performance evaluation of three rapid antigen tests for the diagnosis of group A Streptococci.

OBJECTIVE: To compare the diagnostic performance of three rapid antigen detection tests (RADTs) for group A Streptococcus (GAS). DESIGN: A hospital-based, cross-sectional, retrospective study. SETTING: A comparative study of rapid diagnostic tests for GAS using clinical specimens in a single institute. PARTICIPANTS: 225 children in the outpatient clinics ofKorea University Guro Hospitalwith suspicious symptoms were subjected to throat swab sampling. A dual-swab applicator was used. Samples were stored at below -70°C in a 10 mL transport tube containing 1 mL liquid Stuart's transport medium. OUTCOME MEASURES: All tests were performed in the laboratory by trained clinical laboratory scientists. Sensitivity, specificity, accuracy and kappa index of three RADTs were compared with the reference PCR test and culture results. RESULTS: Of the 225 patients suspected of having GAS, 67 and 90 were positive for GAS in the culture and PCR tests, respectively. Compared with the reference culture, the sensitivity for GAS was 92.5% (CI 83.4 to 97.5), 71.6% (CI 59.3 to 81.9) and 74.63% (CI 62.5 to 84.4) for careUS Strep A Plus, SD Bioline and BD Veritor, respectively, and the specificity was 97.0% (CI 93.1 to 99.0), 94.6% (CI 90.1 to 97.5) and 92.9% (CI 87.8 to 96.2) for careUS Strep A Plus, SD Bioline and BD Veritor, respectively. Compared with the reference GAS real-time PCR, the sensitivity was 73.3% (CI 62.9 to 82.1), 63.3% (CI 52.5 to 73.2) and 67.8% (CI 57.1 to 77.2) for careUS Strep A Plus, SD Bioline and BD Veritor, respectively, and the specificity was 99.3% (CI 95.9 to 99.9), 100.0% (CI 97.3 to 100.0) and 99.3% (CI 95.9 to 99.9) for careUS Strep A Plus, SD Bioline and BD Veritor, respectively. CONCLUSIONS: The careUS Strep A Plus is a useful test that showed highly comparable results with those of the culture test and superior performances among the three RADTs. The use of RADTs should be encouraged to provide acceptable and fast results using simple equipment.

Lamb wave-based molecular diagnosis using DNA hydrogel formation by rolling circle amplification (RCA) process.

Recent developments in microfluidics enable the lab-on-a-chip-based molecular diagnosis. Rapid and accurate diagnosis of infectious diseases is critical for preventing the transmission of the disease. Here, we characterize a Lamb wave-based device using various parameters including the contact angle and viscosity of the sample droplet, the applied voltage, and the temperature increase. Additionally, we demonstrate the functionality of the Lamb wave-based device in clinical application. Optimal temperature for rolling circle amplification (RCA) process is 30 °C, and it was achieved by Lamb wave generation at 17 V. Gene amplification due to RCA process could be detected by viscosity increase due to DNA hydrogel formation in a sample droplet, which induced the acoustic streaming velocity of suspended particles to be decreased. In our Lamb wave-based device, isothermal amplification of target nucleic acids could be successfully detected within 30 min using 10 µL of sessile droplet, and was validated by comparing that of commercial real-time fluorescence analysis. Our device enables simple and low-cost molecular diagnosis, which can be applied to resource-limited clinical settings.