Accelerated Development of a COVID-19 Lateral Flow Test in an Academic Setting: Lessons Learned.

ConspectusThe COVID-19 pandemic further demonstrated the need for usable, reliable, and cost-effective point-of-care diagnostics that can be broadly deployed, ideally for self-testing at home. Antigen tests using more-detectable reporter labels (usually at the cost of reader complexity) achieve better diagnostic sensitivity, supporting the value of higher-analytical-sensitivity reporter technologies in lateral flow.We developed a new approach to simple, inexpensive lateral flow assays (LFAs) of great sensitivity, based on the glow stick peroxyoxalate chemistry widely used in emergency settings and in children's toys. At the peak of the COVID-19 pandemic, we had the opportunity to participate in the pandemic-driven NIH Rapid Acceleration of Diagnostics (RADx) initiative aiming to develop a deployable lateral flow diagnostic for SARS-CoV-2 nucleoprotein based on our novel glow stick-inspired light-emitting reporter technology. During this project, we screened more than 250 antibody pairs for analytical sensitivity and specificity directly in LFA format, using recombinant nucleoprotein and then gamma-irradiated virions spiked into negative nasal swab extracts. Membranes and other LFA materials and swabs and extraction reagent components also were screened and selected. Optimization of conjugate preparation and spraying as well as pretreatment/conditioning of the sample pad led to the final optimized LFA strip. Technology development also included optimization of excitation liquid enclosed in disposable droppers, design of a custom cartridge and smartphone-based reader, and app development, even a prototype reader usable with any mobile phone. Excellent preclinical performance was first demonstrated with contrived samples and then with leftover clinical samples. Moving beyond traditional academic focus areas, we were able to establish a quality management system (QMS), produce large numbers of customized LFA cassettes by contract injection molding, build in-house facilities to assemble and store thousands of complete tests for verification and validation and usability studies, and source kitting/packaging services and quality standard reagents and build partnerships for clinical translation, regulatory guidance, scale up, and market deployment. We were not able to bring this early stage technology to the point of commercialization within the limited time and resources available, but we did achieve strong proof-of-concept and advance translational aspects of the platform including initial high-performance LFAs, reading by the iPhone app using only a $2 plastic dark box with no lens, and convenient, usable excitation liquid packaging in droppers manufacturable in very large numbers.In this Account, we aim to provide a concise overview of our 18-month sprint toward the practical development of a deployable antigen lateral flow assay under pandemic conditions and the challenges and successes experienced by our team. We highlight what it takes to coach a technically savvy but commercially inexperienced academic team through the accelerated translation of an early stage technology into a useful product. Finally, we provide a guided tutorial and workflow to empower others interested in the rapid development of translatable LFAs.

Correction: Smartphone-read phage lateral flow assay for point-of-care detection of infection.

Correction for 'Smartphone-read phage lateral flow assay for point-of-care detection of infection' by Maede Chabi, et al., Analyst, 2023, 148, 839-848, https://doi.org/10.1039/D2AN01499H.

"Glow ELISA": sensitive immunoassay with minimal equipment and stable reagents.

Glow enzyme-linked immunosorbent assay (glow ELISA) uses inexpensive and shelf-stable glow stick reagents to chemically excite fluorescent reporters, obviating the need for excitation light sources, filters, and complex optics. It achieves excellent limits of detection while offering portability and equipment cost comparable to lateral flow immunoassays.

Oxidized Xanthan Gum Crosslinked NOCC: Hydrogel System and Their Biological Stability from Oxidation Levels of the Polymer.

Dynamic hydrogel systems from N,O-carboxymethyl chitosan (NOCC) are investigated in the past years, which has facilitated their widespread use in many biomedical engineering applications. However, the influence of the polymer's oxidation levels on the hydrogel biological properties is not fully investigated. In this study, chitosan is converted into NOCC and introduced to react spontaneously with oxidized xanthan gum (OXG) to form several injectable hydrogels with controlled degradability. Different oxidation levels of xanthan gum, as well as NOCC/OXG volume ratios, are trialed. The infrared spectroscopy spectra verify chemical modification on OXG and successful crosslinking. With increasing oxidation levels, more dialdehyde groups are introduced into the OXG, resulting in changes in physical properties including gelation, swelling, and self-healing efficiency. Under different volume ratios, the hydrogel shows a stable structure and rigidity with higher mechanical properties, and a slower degradation rate. The shear-thinning and self-healing properties of the hydrogels are confirmed. In vitro assays with L929 cells show the biocompatibility of all formulations although the use of a high amount of OXG15 and OXG25 limited the cell proliferation capacity. Findings in this study suggested a suitable amount of OXG at different oxidation levels in NOCC hydrogel systems for tissue engineering applications.

Smartphone-readable RPA-LFA for the high-sensitivity detection of Leishmania kDNA using nanophosphor reporters.

Early diagnosis of infectious diseases improves outcomes by enabling earlier delivery of effective treatment, and helps prevent further transmission by undiagnosed persons. We demonstrated a proof-of-concept assay combining isothermal amplification and lateral flow assay (LFA) for early diagnosis of cutaneous leishmaniasis, a vector-borne infectious disease that affects ca. 700,000 to 1.2 million people annually. Conventional molecular diagnostic techniques based on polymerase chain reaction (PCR) require complex apparatus for temperature cycling. Recombinase polymerase amplification (RPA) is an isothermal DNA amplification method that has shown promise for use in low-resource settings. Combined with lateral flow assay as the readout, RPA-LFA can be used as a point-of-care diagnostic tool with high sensitivity and specificity, but reagent costs can be problematic. In this work, we developed a highly-sensitive smartphone-based RPA-LFA for the detection of Leishmania panamensis DNA using blue-emitting [(Sr0.625Ba0.375)1.96Eu0.01Dy0.03]MgSi2O7 (SBMSO) persistent luminescent nanophosphors as LFA reporters. The greater detectability of nanophosphors allows the use of a reduced volume of RPA reagents, potentially reducing the cost of RPA-LFA. The limit of detection (LOD) of RPA with gold nanoparticle-based LFA readout is estimated at 1 parasite per reaction, but LOD can be 100-fold better, 0.01 parasites per reaction, for LFA based on SBMSO. This approach may be useful for sensitive and cost-effective point-of-care diagnosis and contribute to improved clinical and economic outcomes, especially in resource-limited settings.

Improving Visual Defect Detection and Localization in Industrial Thermal Images Using Autoencoders.

Reliable functionality in anomaly detection in thermal image datasets is crucial for defect detection of industrial products. Nevertheless, achieving reliable functionality is challenging, especially when datasets are image sequences captured during equipment runtime with a smooth transition from healthy to defective images. This causes contamination of healthy training data with defective samples. Anomaly detection methods based on autoencoders are susceptible to a slight violation of a clean training dataset and lead to challenging threshold determination for sample classification. This paper indicates that combining anomaly scores leads to better threshold determination that effectively separates healthy and defective data. Our research results show that our approach helps to overcome these challenges. The autoencoder models in our research are trained with healthy images optimizing two loss functions: mean squared error (MSE) and structural similarity index measure (SSIM). Anomaly score outputs are used for classification. Three anomaly scores are applied: MSE, SSIM, and kernel density estimation (KDE). The proposed method is trained and tested on the 32 × 32-sized thermal images, including one contaminated dataset. The model achieved the following average accuracies across the datasets: MSE, 95.33%; SSIM, 88.37%; and KDE, 92.81%. Using a combination of anomaly scores could assist in solving a low classification accuracy. The use of KDE improves performance when healthy training data are contaminated. The MSE+ and SSIM+ methods, as well as two parameters to control quantitative anomaly localization using SSIM, are introduced.

Development of a quantitative fluorescence lateral flow immunoassay (LFIA) prototype for point-of-need detection of anti-Müllerian hormone.

Objective: Anti-Müllerian Hormone (AMH) is a quantitative marker for ovarian reserve and is used to predict response during ovarian stimulation. Streamlining testing to the clinic or even to the physician's office would reduce inconvenience, turnaround time, patient stress and potentially also the total cost of testing, allowing for more frequent monitoring. In this paper, AMH is used as a model biomarker to describe the rational development and optimization of sensitive, quantitative, clinic-based rapid diagnostic tests. Design and Methods: We developed a one-step lateral-flow europium (III) chelate-based fluorescent immunoassay (LFIA) for the detection of AMH on a portable fluorescent reader, optimizing the capture/detection antibodies, running buffer, and reporter conjugates. Results: A panel of commercial calibrators was used to develop a standard curve to determine the analytical sensitivity (LOD = 0.41 ng/ml) and the analytical range (0.41-15.6 ng/ml) of the LFIA. Commercial controls were then tested to perform an initial evaluation of the prototype performance and showed a high degree of precision (Control I CV 2.18%; Control II CV 3.61%) and accuracy (Control I recovery 126%; Control II recovery 103%). Conclusions: This initial evaluation suggests that, in future clinical testing, the AMH LFIA will likely have the capability of distinguishing women with low ovarian reserve (<1 ng/ml AMH) from women with normal (1-4 ng/ml AMH) ovarian reserve. Furthermore, the LFIA demonstrated a wide linear range, indicating the assay's applicability to the detection of other health conditions such as PCOS, which requires AMH measurement at higher concentrations (>6 ng/ml).

Smartphone-read phage lateral flow assay for point-of-care detection of infection.

The COVID-19 pandemic has highlighted the urgent need for sensitive, affordable, and widely accessible testing at the point of care. Here we demonstrate a new, universal LFA platform technology using M13 phage conjugated with antibodies and HRP enzymes that offers high analytical sensitivity and excellent performance in a complex clinical matrix. We also report its complete integration into a sensitive chemiluminescence-based smartphone-readable lateral flow assay for the detection of SARS-CoV-2 nucleoprotein. We screened 84 anti-nucleoprotein monoclonal antibody pairs in phage LFA and identified an antibody pair that gave an LoD of 25 pg mL-1 nucleoprotein in nasal swab extract using a FluorChem gel documentation system and 100 pg mL-1 when the test was imaged and analyzed by an in-house-developed smartphone reader. The smartphone-read LFA signals for positive clinical samples tested (N = 15, with known Ct) were statistically different (p < 0.001) from signals for negative clinical samples (N = 11). The phage LFA technology combined with smartphone chemiluminescence imaging can enable the timely development of ultrasensitive, affordable point-of-care testing platforms for SARS-CoV-2 and beyond.

Expression and Characterization of Intein-Cyclized Trimer of Staphylococcus aureus Protein A Domain Z.

Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 ℃ increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of -33.0 kcal/mol and -32.7 kcal/mol, and -T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.

Protein A-Nanoluciferase fusion protein for generalized, sensitive detection of immunoglobulin G.

Detection and quantification of antibodies, especially immunoglobulin G (IgG), is a cornerstone of ELISAs, many diagnostics, and the development of antibody-based drugs. Current state-of-the-art immunoassay techniques for antibody detection require species-specific secondary antibodies and carefully-controlled bioconjugations. Poor conjugation efficiency degrades assay performance and increases the risk of clinical false positives due to non-specific binding. We developed a generic, highly-sensitive platform for IgG quantification by fusing the IgG-Fc binding Z domain of Staphylococcal Protein A with the ultrabright bioluminescence reporter Nanoluc-luciferase (Nluc). We demonstrated the application of this fusion protein in a sandwich IgG detection immunoassay using surface-bound antigens to capture target IgG and protein A-Nanoluc fusion as the detector. We optimized the platform's sensitivity by incorporating multiple repeats of the Z domain into the fusion protein constructs. Using rabbit and mouse anti-SARS-CoV-2 Nucleoprotein IgGs as model analytes, we performed ELISAs in two different formats, either with SARS-CoV-2 Nucleoprotein as the capture antigen or with polyclonal chicken IgY as the capture antibody. Using standard laboratory equipment, the platform enabled the quantitation of antibody analytes at concentrations as low as 10 pg/mL (67 fM).

Continuous monitoring of IgG using immobilized fluorescent reporters.

In the manufacture of therapeutic monoclonal antibodies, the clarified cell culture fluid (CCF) is typically loaded onto an initial protein A affinity capture column. Imperfect mass transfer and loading to maximum capacity can risk antibody breakthrough and loss of valuable product, but conservative underloading wastes expensive protein A resin. In addition, the effects of column fouling and ligand degradation require the frequent optimization of immunoglobulin G (IgG) loading to avoid wastage. Continuous real-time monitoring of IgG flowthrough is of great interest, therefore. We previously developed a fluorescence-based monitoring technology that allows batch mix-and-read mAb detection in the CCF. Here, we report the use of reporters immobilized on cyanogenbromide-activated Sepharose 4B resin for continuous detection of IgG in column breakthrough. The column effluent is continuously contacted with immobilized fluorescein-labeled Fc-binding ligands in a small monitoring column to produce an immediately-detectable change in fluorescence intensity. The technology allows rapid and reliable monitoring of IgG in a flowing stream of clarified CCF emerging from a protein A column, without prior sample preparation. We observed a significant change in fluorescence intensity at 0.5 g/L human IgG, sufficient to detect a 5% breakthrough of a 10 g/L load, within 18 s at a flow rate of 0.5 ml/min. The current small-scale technology is suitable for use in process development, but the chemistry should be readily adaptable to larger scale applications using fiber-optic sensors, and continuous IgG monitoring could be applicable in a variety of upstream and downstream process settings.

Glowstick-inspired smartphone-readable reporters for sensitive, multiplexed lateral flow immunoassays.

The COVID-19 pandemic has increased demand for point-of-care (POC) screening tests such as lateral flow assays (LFAs) and highlighted the need for sensitive and cost-effective POC diagnostic platforms. Here, we demonstrate an LFA platform using standard fluorescent nanoparticle reporters in which optical excitation is replaced by chemical excitation using the peroxyoxalate-based chemistry of inexpensive, shelf-stable glowsticks. The one-step chemi-excitation of fluorescent particles produces visible light readable by an unmodified smartphone, enhancing sensitivity while preserving simplicity and cost-effectiveness. Our Glow LFA detected the common model analyte human chorionic gonadotropin with a limit of detection (LoD) of 39 pg/mL-over ten times more sensitive than standard gold nanoparticles using the same antibodies. We also demonstrate its application to the detection of SARS-CoV-2 nucleoprotein at 100 pg/mL in nasal swab extract. Multiple fluorescent dyes can be chemi-excited by a single reagent, allowing for color multiplexing on a single LFA strip with a smartphone camera. The detection of three analytes on a single LFA test line was demonstrated using red, green, and blue fluorescent reporter particles, making glow LFA a promising platform for multiplexed detection.

A novel technology for home monitoring of lupus nephritis that tracks the pathogenic urine biomarker ALCAM.

Introduction: The gold standard for diagnosis of active lupus nephritis (ALN), a kidney biopsy, is invasive with attendant morbidity and cannot be serially repeated. Urinary ALCAM (uALCAM) has shown high diagnostic accuracy for renal pathology activity in ALN patients. Methods: Lateral flow assays (LFA) for assaying uALCAM were engineered using persistent luminescent nanoparticles, read by a smartphone. The stability and reproducibility of the assembled LFA strips and freeze-dried conjugated nanoparticles were verified, as was analyte specificity. Results: The LFA tests for both un-normalized uALCAM (AUC=0.93) and urine normalizer (HVEM)-normalized uALCAM (AUC=0.91) exhibited excellent accuracies in distinguishing ALN from healthy controls. The accuracies for distinguishing ALN from all other lupus patients were 0.86 and 0.74, respectively. Conclusion: Periodic monitoring of uALCAM using this easy-to-use LFA test by the patient at home could potentially accelerate early detection of renal involvement or disease flares in lupus patients, and hence reduce morbidity and mortality.

Flash Characterization of Smartphones Used in Point-of-Care Diagnostics.

Rapidly growing interest in smartphone cameras as the basis of point-of-need diagnostic and bioanalytical technologies increases the importance of quantitative characterization of phone optical performance under real-world operating conditions. In the context of our development of lateral-flow immunoassays based on phosphorescent nanoparticles, we have developed a suite of tools for characterizing the temporal and spectral profiles of smartphone torch and flash emissions, and their dependence on phone power state. In this work, these tools are described and documented to make them easily available to others, and demonstrated by application to characterization of Apple iPhone 5s, iPhone 6s, iPhone 8, iPhone XR, and Samsung Note8 flash performance as a function of time and wavelength, at a variety of power settings. Flash and torch intensity and duration vary with phone state and among phone models. Flash has high variability when the battery charge is below 10%, thus, smartphone-based Point-of-Care (POC) tests should only be performed at a battery level of at least 15%. Some output variations could substantially affect the results of assays that rely on the smartphone flash.

Isolation and Barcoding of Trace Pollen-free DNA for Authentication of Honey.

Adulteration and mislabeling of honey to mask its true origin have become a global concern. Pollen microscopy, the current gold standard for identifying honey's geographical and plant origins, is laborious, requires extensive training, and fails to identify filtered honey and honey spiked with pollen from a more favorable plant to disguise its origins. We successfully isolated pollen-free DNA from filtered honey using three types of adsorbents: (i) anti-dsDNA antibodies coupled to magnetic microspheres; (ii) anion-exchange adsorbent; and (iii) ceramic hydroxyapatite. The internal transcribed spacer 2 region of the captured pollen-free DNA was polymerase chain reaction-amplified and subjected to next-generation sequencing. Using an in-house bioinformatics pipeline, initial experiments showed that anion exchange had the greatest capacity to capture trace pollen-free DNA, and it was successfully applied to isolate DNA from five honey samples. Enrichment of trace pollen-free DNA from filtered honey samples opens a new approach for identifying the true origins of honey.

Microplastics in sewage sludge: Distribution, toxicity, identification methods, and engineered technologies.

Microplastic pollution is becoming a global challenge due to its long-term accumulation in the environment, causing adverse effects on human health and the ecosystem. Sludge discharged from wastewater treatment plants (WWTPs) plays a critical role as a carrier and primary source of environmental microplastic contamination. A significantly average microplastic variation between 1000 and 301,400 particles kg-1 has been reported in the sludge samples. In recent years, advanced technologies have been successfully applied to address this issue, including adsorption, advanced oxidation processes (AOPs), and membrane bioreactors (MBRs). Adsorption technologies are essential to utilizing novel adsorbents (e.g., biochar, graphene, zeolites) for effectively removing MPs. Especially, the removal efficiency of polymer microspheres from an aqueous solution by Mg/Zn modified magnetic biochars (Mg/Zn-MBC) was obtained at more than 95%. Also, advanced oxidation processes (AOPs) are widely applied to degrade microplastic contaminants, in which photocatalytic by semiconductors (e.g., TiO2 and ZnO) is a highly suitable approach to promote the degradation reactions owing to strongly hydroxyl radicals (OH*). Biological degradation-aided microorganisms (e.g., bacterial and fungal strains) have been reported to be suitable for removing microplastics. Yet, it was affected by biotic and abiotic factors of the environmental conditions (e.g., pH, light, temperature, moisture, bio-surfactants, microorganisms, enzymes) as well as their polymer characteristics, i.e., molecular weight, functional groups, and crystallinity. Notably, membrane bioreactors (MBRs) showed the highest efficiency in removing up to 99% microplastic particles and minimizing their contamination in sewage sludge. Further, MBRs illustrate the suitability for treating high-strength compounds, e.g., polymer debris and microplastic fibers from complex industrial wastewater. Finally, this study provided a comprehensive understanding of potential adverse risks, transportation pathways, and removal mechanisms of microplastic, which full-filled the knowledge gaps in this field.

Optimization of Oligomer Chitosan/Polyvinylpyrrolidone Coating for Enhancing Antibacterial, Hemostatic Effects and Biocompatibility of Nanofibrous Wound Dressing.

A synergistic multilayer membrane design is necessary to satisfy a multitude of requirements of an ideal wound dressing. In this study, trilayer dressings with asymmetric wettability, composed of electrospun polycaprolactone (PCL) base membranes coated with oligomer chitosan (COS) in various concentrations of polyvinylpyrrolidone (PVP), are fabricated for wound dressing application. The membranes are expected to synergize the hygroscopic, antibacterial, hemostatic, and biocompatible properties of PCL and COS. The wound dressing was coated by spraying the solution of 3% COS and 6% PVP on the PCL base membrane (PVP6-3) three times, which shows good interaction with biological subjects, including bacterial strains and blood components. PVP6-3 samples confirm the diameter of inhibition zones of 20.0 ± 2.5 and 17.9 ± 2.5 mm against Pseudomonas aeruginosa and Staphylococcus aureus, respectively. The membrane induces hemostasis with a blood clotting index of 74% after 5 min of contact. In the mice model, wounds treated with PVP6-3 closed 95% of the area after 10 days. Histological study determines the progression of skin regeneration with the construction of granulation tissue, new vascular systems, and hair follicles. Furthermore, the newly-growth skin shares structural resemblances to that of native tissue. This study suggests a simple approach to a multi-purpose wound dressing for clinical treatment.

Insulin Resistance in Gestational Diabetes Mellitus and Its Association With Anthropometric Fetal Indices.

Background: In pregnant women with gestational diabetes mellitus (GDM), insulin resistance (IR) increases the risk of developing manifest type 2 diabetes mellitus and is associated with complications in both mother and fetus. Objectives: This research aimed to evaluate the associations between IR evaluated by 3 indices (namely updated homeostasis model assessment model (HOMA2), QUICKI, and McAuley's index) and the diabetes risk factors and the fetal growth indices in Vietnamese women with GDM. Methods: A cross-sectional descriptive study was conducted on 370 women with GDM and 40 healthy pregnant women from January 2015 to May 2019. IR was calculated by HOMA2 (HOMA2-IR), QUICKI, and McAuley's index. Fetal anthropometric measurements were assessed via ultrasound which was performed and interpreted by ultrasound experts. Results: In the simple regression analysis, McAuley's index illustrated had statistically significant correlations to the highest number of risk factors of diabetes mellitus compared with HOMA2-IR and QUICKI indices. Moreover, McAuley's index correlated statistically significantly to the highest number of fetal ultrasound measurements factors such as including biparietal diameter (BPD) (r = -0.271, P < .001), head circumference (HC) (r = -0.225, P < .001), abdominal circumference (AC) (r = -0.214, P < .001), femur length (FL) (r = -0.231, P < .001), estimated fetal weight (EFW) (r = -0.239, P < .001) and fetal estimated age (r = -0.299, P < .001). In the multivariable analysis, the McAuley's index contributed the greatest to AC (Standardized B of -0.656, P < .001). Conclusion: The McAuley's index was significantly associated with a higher number of more risk factors for diabetes mellitus as well as fetal ultrasound sonography findings measurements than compared with HOMA2-IR and QUICKI indices.

Lower Plasma Albumin, Higher White Blood Cell Count and High-Sensitivity C-Reactive Protein are Associated with Femoral Artery Intima-Media Thickness Among Newly Diagnosed Patients with Type 2 Diabetes Mellitus.

Introduction: Low albumin levels, high levels of high-sensitivity C-reactive protein (hs-CRP), and high white blood cell count were risk factors for changes in arterial intima-media thickness (IMT). Femoral artery IMT damages were one of the common peripheral artery type 2 diabetes. This study was conducted to determine the association between femoral artery IMT and plasma albumin, hs-CRP levels, and white blood cell count in newly diagnosed patients with type 2 diabetes mellitus (nT2D). Materials and Methods: From January 2015 to May 2020, 306 patients with nT2D were recruited for this cross-sectional descriptive study at Vietnam's National Endocrinology Hospital. We measured IMT by Doppler ultrasound. Results: There was a statistically significant difference in albumin, hs-CRP levels, hs-CRP-to-albumin ratio, and white blood cell counts between three different IMT groups namely normal IMT, thick IMT, and atherosclerosis (p = 0.003, p = 0.001, p = 0.001 and p = 0.049, respectively). In the multivariate linear regression analysis, white blood cell count, and hs-CRP levels showed a significantly positive correlation to IMT (standardized B and p of 0.17, 0.015 and 0.163, 0.024, respectively), but albumin levels were a significantly negative correlation to IMT (standardized B = -0.151, p = 0.029). The multivariate logistic regression analysis showed that albumin (OR = 0.79, 95% CI 0.65-0.90, p = 0.018), hs-CRP (OR = 1.09, 95% CI 1.01-1.18, p = 0.026), and white blood cell count (OR = 1.36, 95% CI 1.03-1.81, p = 0.033) had correlation to atherosclerosis of femoral artery. Conclusion: Reduced plasma albumin, elevated hs-CRP, and white blood cell count associated with IMT increased the odds for atherosclerosis of femoral artery among nT2D.

Simple and Economical Process for Producing Amantadine Hydrochloride.

A simple and economical process for producing amantadine hydrochloride (1) on a 250 g scale, an antiviral and anti-Parkinson drug, has been developed. Several methods for the preparation of 1 through intermediate N-(1-adamantyl)-acetamide (4) in four or three steps were reported. These procedures started with adamantine (2) or 1-bromoadamantane (3), acetonitrile, and sulfuric acid by using the Ritter-type reaction to obtain N-(1-adamantyl)-acetamide, which was deacetylated to afford 1-amino-adamantane (5) and then the salt formed with anhydrous HCl gives 1 with the overall yield of 1 being 50-58%. In this article, a two-step procedure for the synthesis of 1 from 1-bromadamantane (3) and formamide via N-(1-adamantyl)-formamide (6) in two steps with an overall yield of 88% was reported. In this procedure, the preparation of 6 from 3 is a key step with a yield of 94%, followed by the hydrolysis of 6 with an aq. solution of HCl to give 1 in high yield (93%). The procedure was also carried out under optimal conditions established to reduce the use of toxic reagents or solvents and was carried out in one pot to make it more environmentally friendly. The procedure can be considered as more suitable for the large-scale production of 1. The structures of product 1 and intermediate 6 were confirmed by IR, MS, 1H NMR, 13C NMR.