Assessment of virus concentration methods for detecting SARS-CoV-2 IN wastewater.

Wastewater-based epidemiology has been described as a valuable tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a community. However, there is no consensus on the best concentration method to allow reliable detection of SARS-CoV-2 in this matrix, considering different laboratory facilities. This study compares two viral concentration methods, ultracentrifugation (ULT) and skimmed-milk flocculation (SMF), for detecting SARS-CoV-2 in wastewater samples. The analytical sensitivity (limits of detection and quantification [LoD/LoQ]) of both methods was evaluated using a bovine respiratory syncytial virus (BRSV) as a surrogate. Three different approaches were conducted to establish LoD of each method based on the assays on the standard curve (ALoDsc), on the dilution of internal control (ALoDiC), and the processing steps (PLoD). For PLoD, ULT method had the lowest value (1.86 × 103 genome copy/microliter [GC/µL]) when compared to the SMF method (1.26 × 107 GC/µL). The LoQ determination showed a mean value of 1.55 × 105 GC/µL and 3.56 × 108 GC/µL to ULT and SMF, respectively. The detection of SARSCoV-2 in naturally contaminated wastewater revealed 100% (12/12) and 25% (3/12) of detection using ULT and SMF with quantification ranging from 5.2 to 7.2 log10 genome copy/liter (GC/L) and 5.06 to 5.46 log10 GC/L, respectively. The detection success rate of BRSV used as an internal control process was 100% (12/12) for ULT and 67% (8/12) for SMF, with an efficiency recovery rate ranging from 12 to 38% and 0.1 to 5%, respectively. Our data consolidates the importance of assessing the methods used; however, further analysis should be carried out to improve low-cost concentration methodologies, essential for use in low-income and developing countries.

The Matrix Effect in the RT-PCR Detection of SARS-CoV-2 Using Saliva without RNA Extraction.

The present work focuses on the detection of SARS-CoV-2 in saliva, contributing to understanding the inhibition effect of the matrix and its influence on the results. Detection of viral genes ORF1ab, N, and E was performed by RT-PCR using saliva directly in the reaction without RNA extraction. Different amounts of saliva were spiked with increasing amounts of viral RNA from COVID-19 patients and subjected to RT-PCR detection. In parallel, 64 saliva samples from confirmed COVID-19 patients were used in two different amounts directly in the RT-PCR reaction and their results compared. The presence of saliva in the RT-PCR always causes a positive shift of the Ct values, but a very high between-person variability of its magnitude was obtained, with increases ranging from 0.93 to 11.36. Viral targets are also affected differently depending on the initial number of viral particles. Due to inhibitors present in saliva, the duplication of sample volume causes only 48 to 61% of the expected Ct value decrease depending on the viral target gene. The use of saliva has advantages, but also limitations, due to potential inhibitors present in the matrix. However, the choice of the target and the right amount of sample may significantly influence the results.

Development of an Ultraviolet-C Irradiation Room in a Public Portuguese Hospital for Safe Re-Utilization of Personal Protective Respirators.

Almost two years have passed since COVID-19 was officially declared a pandemic by the World Health Organization. However, it still holds a tight grasp on the entire human population. Several variants of concern, one after another, have spread throughout the world. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant may become the fastest spreading virus in history. Therefore, it is more than evident that the use of personal protective equipment (PPE) will continue to play a pivotal role during the current pandemic. This work depicts an integrative approach attesting to the effectiveness of ultra-violet-C (UV-C) energy density for the sterilization of personal protective equipment, in particular FFP2 respirators used by the health care staff in intensive care units. It is increasingly clear that this approach should not be limited to health care units. Due to the record-breaking spreading rates of SARS-CoV-2, it is apparent that the use of PPE, in particular masks and respirators, will remain a critical tool to mitigate future pandemics. Therefore, similar UV-C disinfecting rooms should be considered for use within institutions and companies and even incorporated within household devices to avoid PPE shortages and, most importantly, to reduce environmental burdens.

A sociotechnical approach to vaccine manufacturer selection as part of a global immunization strategy against epidemics and pandemics.

These are unprecedented times while the world weathers the highly infectious respiratory pandemic caused by coronavirus disease-19 (COVID-19). Humanity has experienced other cataclysmic events, but something as novel as this pandemic cannot be easily described. A safe COVID-19 vaccine is often hailed as the only effective public health method to prevent the further spread of this virus. New vaccines' cost has increased even as policymakers struggle with limited resources and budget constraints. Thus, more decision-support tools are needed to facilitate the selection of vaccine manufacturers as part of a global immunization strategy against COVID-19 or other epidemics and pandemics. This study sought to address this issue by combining three well-established operational research methods (i.e., cognitive mapping, decision-making trial and evaluation laboratory, and the Choquet integral). Based on the insights provided by a panel of experts on vaccination and infectious diseases, a vaccine manufacturer selection mechanism was developed that incorporates the World Health Organization's guidelines. This approach facilitated the identification of multiple selection criteria regarding vaccine manufacturers, their allocation into six major clusters (i.e., soundness of scientific approach and technology used; speed of delivery; cost; liability and risk sharing; ability to supply sufficient quantities through production capacity development; and global solidarity), and subsequent analysis of the respective cause-and-effect relationships. The results of a real-life application of the proposed selection system were further consolidated by a member of Saint Francisco Xavier Hospital Infectious Diseases Unit in Lisbon, Portugal. The mechanism's advantages and limitations are also discussed.

Performance of saliva as a specimen to detect SARS-CoV-2.

Massive testing to detect SARS-CoV-2 is an imperious need in times of epidemic but also presents challenges in terms of its concretization. The use of saliva as an alternative to nasopharyngeal swabs (NPS) has advantages, being more friendly to the patient and not requiring trained health workers, so much needed in other functions. This study used a total of 452 dual samples (saliva and NPS) of patients suspected of having COVID-19 to compare results obtained for the different specimens when using RT-PCR of RNA extracted from NPS and saliva, as well as saliva directly without RNA extraction. SARS-CoV-2 was not detected in 13 saliva (direct) of the 80 positive NPS samples and in 16 saliva (RNA) of a total of 76 NPS positive samples. Sensitivity of detection of viral genes ORF1ab, E and N in saliva is affected differently and detection of these genes in saliva samples presents great variability when NPS samples present Ct-values above approximately 20, with sensitivities ranging from 76.3% to 86.3%. On average an increase in 7.3 Ct-values (average standard deviation of 4.78) is observed in saliva samples when compared to NPS. The use of this specimen should be carefully considered due to the false negative rate and the system used for detection may be also very relevant since the different viral genes are affected differently in terms of detection sensitivity using saliva.

Treatment of elderly patients with refractory/relapsed multiple myeloma: oral drugs adherence and the COVID-19 outbreak.

Once the treatment of refractory/relapsed multiple myeloma in the elderly is greatly influenced by the adherence of patients and family members, clinicians should be aware of patients' behavior and lifestyle, as it may influence the individual treatment plan for each patient. Furthermore, treatment with oral chemotherapy is of special value during the COVID-19 outbreak. Multidisciplinary healthcare involvement is crucial in the management of polypharmacy, adverse events and dose adjustment due to comorbidities and natural loss of renal function with age. Oral drugs simplify intake, reduce hospital visits, and improve autonomy and quality of life. However, although oral drugs have advantages, they also transfer control and responsibility from the healthcare professional to the patient, who must be able to understand and follow the directions given. Therefore, patient education and communication with healthcare professionals are critical for adherence.

Real-time physiological measurements of oxygen using a non-invasive self-referencing optical fiber microsensor.

Reactive molecular oxygen (O2) plays important roles in bioenergetics and metabolism and is implicated in biochemical pathways underlying angiogenesis, fertilization, wound healing and regeneration. Here we describe how to use the scanning micro-optrode technique (SMOT) to measure extracellular fluxes of dissolved O2. The self-referencing O2-specific micro-optrode (also termed micro-optode and optical fiber microsensor) is a tapered optical fiber with an O2-sensitive fluorophore coated onto the tip. The O2 concentration is quantified by fluorescence quenching of the fluorophore emission upon excitation with blue-green light. The micro-optrode presents high spatial and temporal resolutions with improved signal-to-noise ratio (in the picomole range). In this protocol, we provide step-by-step instructions for micro-optrode calibration, validation, example applications and data analysis. We describe how to use the technique for cells (Xenopus oocyte), tissues (Xenopus epithelium and rat cornea), organs (Xenopus gills and mouse skin) and appendages (Xenopus tail), and provide recommendations on how to adapt the approach to different model systems. The basic, user-friendly system presented here can be readily installed to reliably and accurately measure physiological O2 fluxes in a wide spectrum of biological models and physiological responses. The full protocol can be performed in ~4 h.