Min-Yen Hsu

Paper-based ELISA to rapidly detect Escherichia coli.

Escherichia coli is a generic indicator of fecal contamination, and certain serotypes cause food- and water-borne illness such as O157:H7. In the clinic, detection of bacteriuria, which is often due to E. coli, is critical before certain surgical procedures or in cases of nosocomial infection to prevent further adverse events such as postoperative infection or sepsis. In low- and middle-income countries, where insufficient equipment and facilities preclude modern methods of detection, a simple, low-cost diagnostic device to detect E. coli in water and in the clinic will have significant impact. We have developed a simple paper-based colorimetric platform to detect E. coli contamination in 5h. On this platform, the mean color intensity for samples with 10(5)cells/mL is 0.118±0.002 (n=4), and 0.0145±0.003 (P<0.01⁎⁎) for uncontaminated samples. This technique is less time-consuming, easier to perform, and less expensive than conventional methods. Thus, paper-based ELISA is an innovative point-of-care diagnostic tool to rapidly detect E. coli, and possibly other pathogens when customized as appropriate, especially in areas that lack advanced clinical equipment.

Cotton-based Diagnostic Devices

A good diagnostic procedure avoids wasting medical resources, is easy to use, resists contamination, and provides accurate information quickly to allow for rapid follow-up therapies. We developed a novel diagnostic procedure using a “cotton-based diagnostic device” capable of real-time detection, i.e., in vitro diagnostics (IVD), which avoids reagent contamination problems common to existing biomedical devices and achieves the abovementioned goals of economy, efficiency, ease of use, and speed. Our research reinforces the advantages of an easy-to-use, highly accurate diagnostic device created from an inexpensive and readily available U.S. FDA-approved material (i.e., cotton as flow channel and chromatography paper as reaction zone) that adopts a standard calibration curve method in a buffer system (i.e., nitrite, BSA, urobilinogen and uric acid assays) to accurately obtain semi-quantitative information and limit the cross-contamination common to multiple-use tools. Our system, which specifically targets urinalysis diagnostics and employs a multiple biomarker approach, requires no electricity, no professional training, and is exceptionally portable for use in remote or home settings. This could be particularly useful in less industrialized areas.

Paper-based devices for isolation and characterization of extracellular vesicles.

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 μl. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

Detection of aqueous VEGF concentrations before and after intravitreal injection of anti-VEGF antibody using low-volume sampling paper-based ELISA

Intraocular vascular endothelial growth factor (VEGF) levels play an important role in the pathogenesis of blindness-related diseases, such as age-related macular degeneration (AMD). Here, we aimed to develop a paper-based enzyme-linked immunosorbent assay (P-ELISA) to analyze the suppression of aqueous VEGF concentrations following intravitreal injection (IVI) of anti-VEGF antibody (bevacizumab or ranibizumab). A total of 25 eyes with wet AMD, one with myopic neovascularization, and one with polypoidal choroidal vasculopathy were enrolled in this study. The limit of detection using P-ELISA was 0.03 pg/mL. Forty-six consecutive samples of aqueous humor were acquired. From all samples, 66.67% (10/15) achieved complete VEGF suppression (below the detection limit) within 5 weeks of receiving IVI of anti-VEGF antibody. Only 13.33% of samples (2/15) achieved complete VEGF suppression 5 weeks after receiving treatment. In some patients, elevated VEGF was still detected 5 weeks after receipt of anti-VEGF antibody, and all samples (10/10) were found to have elevated VEGF levels 49 days after treatment. Thus, we suggest that monthly IVI of anti-VEGF antibody may be required to ensure durable VEGF inhibition. Ultrasensitive P-ELISA can detect elevated VEGF at an earlier time point and may facilitate decision-making regarding appropriate treatment strategies.

Pharmacokinetics and Safety of Intravitreal Caspofungin

ABSTRACT Caspofungin exhibits potent antifungal activities against Candida and Aspergillus species. The elimination rate and retinal toxicity of caspofungin were determined in this study to assess its pharmacokinetics and safety in the treatment of fungal endophthalmitis. Intravitreal injections of 50 μg/0.1 ml of caspofungin were administered to rabbits. Levels of caspofungin in the vitreous and aqueous humors were determined using high-performance liquid chromatography (HPLC) at selected time intervals (10 min and 1, 2, 4, 8, 16, 24, and 48 h), and the half-lives were calculated. Eyes were intravitreally injected with caspofungin to obtain concentrations of 10 μg/ml, 50 μg/ml, 100 μg/ml, and 200 μg/ml. Electroretinograms were recorded 4 weeks after injections, and the injected eyes were examined histologically. The concentrations of intravitreal caspofungin at various time points exhibited an exponential decay with a half-life of 6.28 h. The mean vitreous concentration was 6.06 ± 1.76 μg/ml 1 h after intravitreal injection, and this declined to 0.47 ± 0.15 μg/ml at 24 h. The mean aqueous concentration showed undetectable levels at all time points. There were no statistical differences in scotopic a-wave and b-wave responses between control eyes and caspofungin-injected eyes. No focal necrosis or other abnormality in retinal histology was observed. Intravitreal caspofungin injection may be considered to be an alternative treatment for fungal endophthalmitis based on its antifungal activity, lower retinal toxicity, and lower elimination rate in the vitreous. More clinical data are needed to determine its potential role as primary therapy for fungal endophthalmitis.

Paper-based CRP Monitoring Devices

Here, we discuss the development of a paper-based diagnostic device that is inexpensive, portable, easy-to-use, robust, and capable of running simultaneous tests to monitor a relevant inflammatory protein for clinical diagnoses i.e. C-reactive protein (CRP). In this study, we first attempted to make a paper-based diagnostic device via the wax printing method, a process that was used in previous studies. This device has two distinct advantages: 1) reduced manufacturing and assay costs and operation duration via using wax printing method to define hydrophobic boundaries (for fluidic devices or general POC devices); and, 2) the hydrophilicity of filter paper, which is used to purify and chromatographically correct interference caused by whole blood components with a tiny amount of blood sample (only 5 μL). Diagnosis was based on serum stain length retained inside the paper channels of our device. This is a balanced function between surface tension and chromatographic force following immune reactions (CRP assays) with a paper-embedded biomarker.

Pterygium is related to a decrease in corneal endothelial cell density.

Purpose: The aim of this study was to investigate the relationship between pterygium and a decrease in the corneal endothelial cell density (ECD) in patients with unilateral primary pterygium. Methods: In this retrospective cross-sectional study, 90 consecutive patients with unilateral primary pterygium were enrolled from January 2010 to June 2012. Corneal ECD was measured in both eyes, and the fellow eyes were considered as controls. The relationship between the percentage of pterygium to cornea and a decrease in the ECD was analyzed. An increase in astigmatism in eyes with pterygium was evaluated for association with decreased ECD using the Pearson correlation test. Results: The percentage of pterygium to cornea ranged from 3.5% to 65.2%, with a median of 12.35%. The difference in the corneal ECD between eyes with pterygium and control eyes ranged from +9.6% to −37.7%, with a median of −9.75%. The results of the Pearson correlation statistical test showed a strong logarithmic correlation between a decrease in the corneal ECD and the percentage of pterygium to cornea (R = 0.688, P < 0.001). An increase in astigmatism was correlated with a decrease in the ECD in eyes with pterygium. Conclusions: Pterygium is related to a decrease in corneal ECD. Surgical intervention should be considered in patients with extensive pterygium involvement in the cornea or a significant increase in astigmatism.

Analysis of the Interleukin-6 (-174) Locus Polymorphism and Serum IL-6 Levels with the Severity of Normal Tension Glaucoma

Purpose: In normal tension glaucoma (NTG), factors other than elevated intraocular pressure are likely to have a role in the pathogenesis of optic neuropathy. Recent studies of glaucoma or retinal ganglion cells (RGCs) reveal that the cytokine interleukin-6 (IL-6) is linked to the pathogenesis of glaucoma and may regulate RGC survival or death. The IL-6 (-174) G allele has also been shown to increase the IL-6 protein. We hypothesized that the IL-6 (-174) polymorphism may be a predisposing genetic factor affecting the severity of glaucoma. The aim of the present study was to evaluate the IL-6 polymorphism and serum IL-6 levels as a potential risk factor related to the severity of NTG. Methods: A total of 256 subjects with NTG in the Chinese population were enrolled. The patients were genotyped for the IL-6 (-174) C/G polymorphism. Genomic DNA was amplified by a polymerase chain reaction, followed by the enzymatic restriction fragment length polymorphism technique. Serum IL-6 levels were measured by ELISA. Patient age at diagnosis, cup/disc (C/D) ratio, rim area (RA), retinal nerve fiber layer (RNFL) thickness, and visual field (VF) were analyzed. The associations between genotypes of IL-6 (-174) C/G and the clinical parameters were calculated using a logistic regression. Results: The IL-6 (-174) GC genotype in NTG patients was significantly associated with a smaller C/D ratio (p = 0.04), larger RA (p = 0.04), and thicker RNFL (p = 0.05) compared with IL-6 (-174) GG patients. The allele frequency of IL-6 (-174) C was significantly higher in the NTG patients at an early-moderate stage than at an advanced stage according to the C/D ratio (OR 0.55; 95% CI 0.31-0.99). Pattern standard deviation of VF was borderline lower in IL-6 (-174) GC patients (p = 0.06), and serum IL-6 levels were borderline higher in advanced stages than in early-moderate stages (7.66 ± 3.22 vs. 4.46 ± 3.83 pg/mL; p = 0.06). Conclusion: The IL-6 (-174) GC genotype is associated with a smaller C/D ratio, larger RA, and thicker RNFL compared with IL-6 (-174) GG in NTG patients. We found that the IL-6 (-174) G/C polymorphism and serum IL-6 levels may be associated with the severity of NTG.

Risk of developing open-angle glaucoma in patients with carotid artery stenosis: A nationwide cohort study

Whether carotid artery stenosis (CAS) is an independent risk factor for open-angle glaucoma remains unclear. In this study, we investigated the association between CAS and the development of open-angle glaucoma in the Taiwanese population-based cohort derived from a longitudinal database containing claims data from the Taiwan National Health Insurance (NHI) program; this study enrolled 2093 patients with CAS and 8372 patients without CAS matched by age and sex from 1999 to 2010. Diagnoses of open-angle glaucoma (OAG) were identified during a follow-up period lasting through December 31, 2013. A Cox proportional hazards model was applied to evaluate the hazard ratio (HR) for OAG in the CAS cohort compared with the matched cohort. We found that the HR for open-angle glaucoma in the CAS cohort compared with the matched cohort. The adjusted HR for OAG in the CAS cohort was 1.50 (95% confidence interval, 1.11–2.02, P = .008). The Kaplan-Meier analysis revealed that the CAS cohort had a higher cumulative incidence of OAG than did the matched cohort during the follow-up period (log-rank test, P < .001). We concluded that CAS is a significantly independent risk factor for the development of OAG. Our finding is clinically important for the aging population, which has an increasing prevalence of CAS.

Paper-Based Microfluidic Platforms for Understanding the Role of Exosomes in the Pathogenesis of Major Blindness-Threatening Diseases

Emerging roles of exosomes in the pathogenesis of major blindness-threatening diseases, such as age-related macular degeneration, glaucoma, and corneal dystrophy, were discovered by aqueous humor analysis. A new diagnostic method using cellulose-based devices and microfluidic chip techniques for the isolation of exosomes from aqueous humor is less cumbersome and saves time. This method will enable more investigations for aqueous humor analysis in the future.