Prevalence of olfactory and taste dysfunction in COVID-19 patients: a community care facility study.

PURPOSE: We aim to study the prevalence of olfactory and taste dysfunction (OTD) in subjects residing in a Community Care Facility (CCF), a center unique to Singapore that is dedicated to isolate foreign workers with COVID-19 infection who have mild disease with minimal or no symptoms. METHODS: This is a cross-sectional study analyzing data prospectively collected from COVID-19-positive subjects who were admitted into a single-center Singapore EXPO CCF from 1st May 2020 to 1st July 2020. The following variables were collected: age, gender, ethnicity, anosmia, ageusia and acute respiratory infection (ARI) symptoms. Symptoms of anosmia and ageusia were self-declared via a mandatory questionnaire administered on admission. RESULTS: A total of 1983 subjects were included. The overall prevalence of anosmia and ageusia is 3.0% and 2.6%, respectively. 58% of anosmic subjects have co-existent ageusia and 72.6% of anosmic subjects have no concurrent sinonasal symptoms. OTD is less likely to present in subjects who are asymptomatic for ARI, compared to those symptomatic for ARI (anosmia: 2.0% versus 4.4% p = 0.002; ageusia: 1.6% versus 4.2% p < 0.001). There is a difference in the prevalence of OTD between the different ethnic groups (Indian, Chinese, Bangladeshi and Others), with Chinese and Bangladeshi reporting a higher prevalence (p < 0.043) CONCLUSION: The true prevalence of OTD in COVID-19-positive subjects may be low with aggressive screening of all subjects, including those asymptomatic for ARI.

Prediction of overall survival following colorectal cancer surgery in elderly patients.

BACKGROUND: With advanced age and chronic illness, the life expectancy of a patient with colorectal cancer (CRC) becomes less dependent on the malignant disease and more on their pre-morbid condition. Justifying major surgery for these elderly patients can be challenging. An accurate tool demonstrating post-operative survival probability would be useful for surgeons and their patients. AIM: To integrate clinically significant prognostic factors relevant to elective colorectal surgery in the elderly into a validated pre-operative scoring system. METHODS: In this retrospective cohort study, patients aged 70 and above who underwent surgery for CRC at Singapore General Hospital between 1 January 2005 and 31 December 2012 were identified from a prospectively maintained database. Patients with evidence of metastatic disease, and those who underwent emergency surgery or had surgery for benign colorectal conditions were excluded from the analysis. The primary outcome was overall 3-year overall survival (OS) following surgery. A multivariate model predicting survival was derived and validated against an equivalent external surgical cohort from Kyungpook National University Chilgok Hospital, South Korea. Statistical analyses were performed using Stata/MP Version 15.1. RESULTS: A total of 1267 patients were identified for analysis. The median post-operative length of stay was 8 [interquartile range (IQR) 6-12] d and median follow-up duration was 47 (IQR 19-75) mo. Median OS was 78 (IQR 65-85) mo. Following multivariate analysis, the factors significant for predicting overall mortality were serum albumin < 35 g/dL, serum carcinoembryonic antigen ≥ 20 µg/L, T stage 3 or 4, moderate tumor cell differentiation or worse, mucinous histology, rectal tumors, and pre-existing chronic obstructive lung disease. Advanced age alone was not found to be significant. The Korean cohort consisted of 910 patients. The Singapore cohort exhibited a poorer OS, likely due to a higher proportion of advanced cancers. Despite the clinicopathologic differences, there was successful validation of the model following recalibration. An interactive online calculator was designed to facilitate post-operative survival prediction, available at http://bit.ly/sgh_crc. The main limitation of the study was selection bias, as patients who had undergone surgery would have tended to be physiologically fitter. CONCLUSION: This novel scoring system generates an individualized survival probability following colorectal resection and can assist in the decision-making process. Validation with an external population strengthens the generalizability of this model.

Breast reconstruction rate and profile in a Singapore patient population: a National University Hospital experience.

INTRODUCTION: Breast reconstruction is an integral part of breast cancer management with the aim of restoring a breast to its natural form. There is increasing awareness among women that it is a safe procedure and its benefits extend beyond aesthetics. Our aim was to establish the rate of breast reconstruction and provide an overview of the patients who underwent breast reconstruction at National University Hospital (NUH), Singapore. METHODS: We evaluated factors that impact a patient's decision to proceed with breast reconstruction, such as ethnicity, age, time and type of implant. We retrospectively reviewed the medical records of women who had breast cancer and underwent breast surgery at NUH between 2001 and 2010. RESULTS: The breast reconstruction rate in this study was 24.3%. There were 241 patients who underwent breast reconstruction surgeries (including delayed and immediate procedures) among 993 patients for whom mastectomies were done for breast cancer. Chinese patients were the largest ethnic group who underwent breast reconstruction after mastectomy (74.3%). Within a single ethnic patient group, Malay women had the largest proportion of women undergoing breast reconstruction (60.0%). The youngest woman in whom cancer was detected in our study was aged 20 years. Malay women showed the greatest preference for autologous tissue breast reconstruction (92.3%). The median age at cancer diagnosis of our cohort was 46 years. CONCLUSION: We noted increases in the age of patients undergoing breast reconstruction and the proportion of breast reconstruction cases over the ten-year study period.

Evaluation of fluorescent compound interference in 4 fluorescence polarization assays: 2 kinases, 1 protease, and 1 phosphatase.

With the increasing use of fluorescence-based assays in high-throughput screening (HTS), the possibility of interference by fluorescent compounds needs to be considered. To investigate compound interference, a well-defined sample set of biologically active compounds, LOPAC, was evaluated using 4 fluorescein-based fluorescence polarization (FP) assays. Two kinase assays, a protease assay, and a phosphatase assay were studied. Fluorescent compound interference and light scattering were observed in both mixture- and single-compound testing under certain circumstances. In the kinase assays, which used low levels (1-3 nM) of fluorophore, an increase in total fluorescence, an abnormal decrease in mP readings, and negative inhibition values were attributed to compound fluorescence. Light scattering was observed by an increase in total fluorescence and minimal reduction in mP, leading to false positives. The protease and phosphatase assays, which used a higher concentration of fluorophore (20-1200 nM) than the kinase assays, showed minimal interference from fluorescent compounds, demonstrating that an increase in the concentration of the fluorophore minimized potential fluorescent compound interference. The data also suggests that mixtures containing fluorescent compounds can result in either false negatives that can mask a potential "hit" or false positives, depending on the assay format. Cy dyes (e.g., Cy3B and Cy5 ) excite and emit further into the red region than fluorescein and, when used in place of fluorescein in kinase 1, eliminate fluorescence interference and light scattering by LOPAC compounds. This work demonstrates that fluorescent compound and light scattering interferences can be overcome by increasing the fluorophore concentration in an assay or by using longer wavelength dyes.

HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications.

HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions.Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays.Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.

Development of a HTRF kinase assay for determination of Syk activity.

Regulation of protein phosphorylation is a primary cellular signaling mechanism. Many cellular responses to internal and external events are mitigated by protein kinase signaling cascades. Dysfunction of protein kinase activity has been linked to a variety of human pathologies, in the areas of cancer, inflammation, metabolism, cell cycle, apoptosis, as well as cardiovascular, neurodegenerative and autoimmune diseases. As such, there is an important need for protein kinase activity detection methodologies for researchers engaged in Drug Discovery. A number of different technologies have been employed for the measurement of protein kinase activity, including radioactive methods, luminescent methods, and fluorescent methods. More recently, Homogeneous Time Resolved Fluorescence technology (HTRF), based on the principle of time-resolved fluorescent resonance energy transfer (TR-FRET), has been developed and applied for the measurement of protein kinase activity in vitro. This technology note describes the development of an HTRF assay for detection of Syk enzyme activity in a format consistent with the requirements of High-Throughput Screening (HTS) campaigns currently used in drug discovery.