Wastewater Surveillance for Identifying SARS-CoV-2 Infections in Long-Term Care Facilities, Kentucky, USA, 2021-2022.

Persons living in long-term care facilities (LTCFs) were disproportionately affected by COVID-19. We used wastewater surveillance to detect SARS-CoV-2 infection in this setting by collecting and testing 24-hour composite wastewater samples 2-4 times weekly at 6 LTCFs in Kentucky, USA, during March 2021-February 2022. The LTCFs routinely tested staff and symptomatic and exposed residents for SARS-CoV-2 using rapid antigen tests. Of 780 wastewater samples analyzed, 22% (n = 173) had detectable SARS-CoV-2 RNA. The LTCFs reported 161 positive (of 16,905) SARS-CoV-2 clinical tests. The wastewater SARS-CoV-2 signal showed variable correlation with clinical test data; we observed the strongest correlations in the LTCFs with the most positive clinical tests (n = 45 and n = 58). Wastewater surveillance was 48% sensitive and 80% specific in identifying SARS-CoV-2 infections found on clinical testing, which was limited by frequency, coverage, and rapid antigen test performance.

Simple SARS-CoV-2 concentration methods for wastewater surveillance in low resource settings.

Wastewater-based epidemiology (WBE) measures pathogens in wastewater to monitor infectious disease prevalence in communities. Due to the high dilution of pathogens in sewage, a concentration method is often required to achieve reliable biomarker signals. However, most of the current concentration methods rely on expensive equipment and labor-intensive processes, which limits the application of WBE in low-resource settings. Here, we compared the performance of four inexpensive and simple concentration methods to detect SARS-CoV-2 in wastewater samples: Solid Fraction, Porcine Gastric Mucin-conjugated Magnetic Beads, Calcium Flocculation-Citrate Dissolution (CFCD), and Nanotrap® Magnetic Beads (NMBs). The NMBs and CFCD methods yielded the highest concentration performance for SARS-CoV-2 (∼16-fold concentration and âˆ¼ 41 % recovery) and require <45 min processing time. CFCD has a relatively low consumable cost (<$2 per four sample replicates). All methods can be performed with basic laboratory equipment and minimal electricity usage which enables further application of WBE in remote areas and low resource settings.

Improving wastewater-based epidemiology performance through streamlined automation.

Wastewater-based epidemiology (WBE) has enabled us to describe Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections in populations. However, implementation of wastewater monitoring of SARS-CoV-2 is limited due to the need for expert staff, expensive equipment, and prolonged processing times. As WBE increases in scope (beyond SARS-CoV-2) and scale (beyond developed regions), there is a need to make WBE processes simpler, cheaper, and faster. We developed an automated workflow based on a simplified method termed exclusion-based sample preparation (ESP). Our automated workflow takes 40 min from raw wastewater to purified RNA, which is several times faster than conventional WBE methods. The total assay cost per sample/replicate is $6.50 which includes consumables and reagents for concentration, extraction, and RT-qPCR quantification. The assay complexity is reduced significantly, as extraction and concentration steps are integrated and automated. The high recovery efficiency of the automated assay (84.5 ± 25.4%) yielded an improved Limit of Detection (LoDAutomated=40 copies/mL) compared to the manual process (LoDManual=206 copies/mL), increasing analytical sensitivity. We validated the performance of the automated workflow by comparing it with the manual method using wastewater samples from several locations. The results from the two methods correlated strongly (r = 0.953), while the automated method was shown to be more precise. In 83% of the samples, the automated method showed lower variation between replicates, which is likely due to higher technical errors in the manual process e.g., pipetting. Our automated wastewater workflow can support the expansion of WBE in the fight against Coronavirus Disease of 2019 (COVID-19) and other epidemics.

Stabilization of SARS-CoV-2 RNA in wastewater via rapid RNA extraction.

Wastewater-based Epidemiology (WBE) has contributed to surveillance of SARS-CoV-2 in communities across the world. Both symptomatic and asymptomatic patients with COVID-19 can shed the virus through the gastrointestinal tract, enabling the quantification of the virus in stool and ultimately in wastewater (WW). Unfortunately, instability of SARS-CoV-2 RNA in wastewater limits the utility of WBE programs, particularly in remote/rural regions where reliable cold storage and/or rapid shipping may be unavailable. This study examined whether rapid SARS-CoV-2 RNA extraction on the day of sample collection could minimize degradation. Importantly, the extraction technology used in these experiments, termed exclusion-based sample preparation (ESP), is lightweight, portable, and electricity-free, making it suitable for implementation in remote settings. We demonstrated that immediate RNA extraction followed by ambient storage significantly increased the RNA half-life compared to raw wastewater samples stored at both 4 °C or ambient temperature. Given that RNA degradation negatively impacts both the sensitivity and precision of WBE measurements, efforts must be made to mitigate degradation in order to maximize the potential impact of WBE on public health.

Development and Validation of a Simplified Method for Analysis of SARS-CoV-2 RNA in University Dormitories.

Over the course of the COVID-19 pandemic, wastewater surveillance has become a useful tool for describing SARS-CoV-2 prevalence in populations of varying size, from individual facilities (e.g., university residence halls, nursing homes, prisons) to entire municipalities. Wastewater analysis for SARS-CoV-2 RNA requires specialized equipment, expensive consumables, and expert staff, limiting its feasibility and scalability. Further, the extremely labile nature of viral RNA complicates sample transportation, especially in regions with limited access to reliable cold chains. Here, we present a new method for wastewater analysis, termed exclusion-based sample preparation (ESP), that substantially simplifies workflow (at least 70% decrease in time; 40% decrease in consumable usage compared with traditional techniques) by targeting the labor-intensive processing steps of RNA purification and concentration. To optimize and validate this method, we analyzed wastewater samples from residence halls at the University of Kentucky, of which 34% (44/129) contained detectible SARS-CoV-2 RNA. Although concurrent clinical testing was not comprehensive, student infections were identified in the 7 days following a positive wastewater detection in 68% of samples. This pilot study among university residence halls validated the performance and utility of the ESP method, laying the foundation for future studies in regions of the world where wastewater testing is not currently feasible.

Exploring American and Italian consumer preferences for Californian and Italian red wines.

BACKGROUND: To increase the market share of Californian wines in other countries, wine preferences need to be explored in potential markets. This work studied the preferences of American and Italian consumers for red wines produced in California and Italy, focusing on wines made from the same varieties in each location. Descriptive analysis and consumer preference tests were performed. RESULTS: Americans scored each of the Californian wines significantly higher in preference than the Italian wines. In contrast, the Italian consumer preference scores for many Italian and Californian wines overlapped. By external preference mapping of the American consumer segments, the ideal flavour of one cluster was closest to the Californian Zinfandel, Merlot and Syrah, which had the 'most balanced' flavour profiles. Another cluster of Italians also preferred the Californian wines. In addition, one Italian cluster was driven by a dislike of the leather, band-aid and medicinal aromas of the Italian Merlot and Refosco. CONCLUSION: The results provided information that can contribute to wine marketing research necessary for successfully exporting Californian red wines to Italy and vice versa.

Polyphenols: factors influencing their sensory properties and their effects on food and beverage preferences.

Bitterness and astringency are found in a variety of foods, including nuts, fruits, chocolate, tea, wine, and soymilk. In fruits and beverages, the taste of bitterness and the tactile sensation of astringency are elicited primarily by flavanol polymers (proanthocyanidins or condensed tannins). Variations in proanthocyanidin composition, such as polymer size, extent of galloylation, and formation of derivatives, affect both bitterness and astringency. In beverages, other factors also influence these sensations, including the pH and the levels of ethanol, sweetness, and viscosity. Similarly, foods eaten with beverages can influence astringency. For example, eating dark chocolate increases the astringency of red wine more than does milk chocolate. Individuals perceive astringency differently because of variations in salivary flow rates, and preferences for and acceptance of a product may vary tremendously among individuals; decreasing bitterness and/or astringency may not increase preference. Factors influencing bitterness, astringency, and individual preference decisions are discussed.

Trilostane therapy for treatment of pituitary-dependent hyperadrenocorticism in 5 cats.

Hyperadrenocorticism is a rare syndrome in cats. Current medical therapy is unsatisfactory and prognosis for long-term survival with surgical treatment is guarded. We report on 5 cats treated with the 3-beta hydroxysteroid dehydrogenase inhibitor trilostane. Diagnosis was confirmed in all cats by endocrine testing. Three cats had concurrent diabetes mellitus. Trilostane reduced clinical signs and improved endocrine test results in all cats, but insulin requirements did not change and all continued to have some signs of hypercortisolemia. Two died or were euthanized after 16 and 140 days, whereas 3 were still alive 6, 11, and 20 months after the start of trilostane therapy. Trilostane ameliorates clinical signs of feline hyperadrenocorticism, but more research is needed before it can be recommended for treatment.

Characterization of odor-active compounds in Californian chardonnay wines using GC-olfactometry and GC-mass spectrometry.

Nineteen commercial Californian Chardonnay wines were analyzed by gas chromatography-mass spectrometry (GC-MS). Freon extracts of wines were separated by silica gel chromatography into three fractions. Volatiles were quantified by GC analysis of each fraction using internal standards added to the wine prior to Freon extraction. Twelve of the 19 wines were evaluated by GC-Olfactometry (GC-O). Of the 81 compounds shown to be odor-active (OA) by GC/O, 74 were quantified and 61 were tentatively identified, all of which had been previously reported in grapes or wines. Overall concentrations of compounds with floral or oak-related aromas were higher in wines shown by descriptive analysis to be high in intensity of either floral or oak notes, respectively. The relationship between sensory intensity ratings from a previous descriptive analysis of the wines and 74 OA compounds was modeled by partial least-squares regression (PLS) analysis. This PLS model only explained 17% of the variation in the OA variables, whereas a PLS using a subset of 16 OA peaks explained 64 and 47% of variance in the sensory and GC data, respectively. Fruity wines high in peach, citrus, and floral terms were separated from those high in oak-related sensory attributes (oak, vanilla, caramel, spice, and butter). In both PLS models, the fruity and floral terms were associated with isoamyl acetate, 2-phenylethyl acetate, linalool and two unknowns exhibiting minty and bandaid-caramel odors; the oaky attributes were associated with vanillin, oak-lactones, 4-ethyl guaiacol, gamma-nonalactone, 2-acetyl furan, eugenol, 2-methoxy phenol, and two unknowns with plastic and smoky odors.