SARS-CoV-2 RNA Detection in Wastewater and Its Effective Correlation with Clinical Data during the Outbreak of COVID-19 in Salamanca.

Wastewater treatment plants (WWTPs) are the final stage of the anthropogenic water cycle where a wide range of chemical and biological markers of human activity can be found. In COVID-19 disease contexts, wastewater surveillance has been used to infer community trends based on viral abundance and SARS-CoV-2 RNA variant composition, which has served to anticipate and establish appropriate protocols to prevent potential viral outbreaks. Numerous studies worldwide have provided reliable and robust tools to detect and quantify SARS-CoV-2 RNA in wastewater, although due to the high dilution and degradation rate of the viral RNA in such samples, the detection limit of the pathogen has been a bottleneck for the proposed protocols so far. The current work provides a comprehensive and systematic study of the different parameters that may affect the detection of SARS-CoV-2 RNA in wastewater and hinder its quantification. The results obtained using synthetic viral RNA as a template allow us to consider that 10 genome copies per µL is the minimum RNA concentration that provides reliable and consistent values for the quantification of SARS-CoV-2 RNA. RT-qPCR analysis of wastewater samples collected at the WWTP in Salamanca (western Spain) and at six pumping stations in the city showed that below this threshold, positive results must be confirmed by sequencing to identify the specific viral sequence. This allowed us to find correlations between the SARS-CoV-2 RNA levels found in wastewater and the COVID-19 clinical data reported by health authorities. The close match between environmental and clinical data from the Salamanca case study has been confirmed by similar experimental approaches in four other cities in the same region. The present methodological approach reinforces the usefulness of wastewater-based epidemiology (WBE) studies in the face of future pandemic outbreaks.

Influence of physical and chemical properties of HTSXT-FTIR samples on the quality of prediction models developed to determine absolute concentrations of total proteins, carbohydrates and triglycerides: a preliminary study on the determination of their absolute concentrations in fresh microalgal biomass.

Absolute concentrations of total macromolecules (triglycerides, proteins and carbohydrates) in microorganisms can be rapidly measured by FTIR spectroscopy, but caution is needed to avoid non-specific experimental bias. Here, we assess the limits within which this approach can be used on model solutions of macromolecules of interest. We used the Bruker HTSXT-FTIR system. Our results show that the solid deposits obtained after the sampling procedure present physical and chemical properties that influence the quality of the absolute concentration prediction models (univariate and multivariate). The accuracy of the models was degraded by a factor of 2 or 3 outside the recommended concentration interval of 0.5-35 µg spot(-1). Change occurred notably in the sample hydrogen bond network, which could, however, be controlled using an internal probe (pseudohalide anion). We also demonstrate that for aqueous solutions, accurate prediction of total carbohydrate quantities (in glucose equivalent) could not be made unless a constant amount of protein was added to the model solution (BSA). The results of the prediction model for more complex solutions, here with two components: glucose and BSA, were very encouraging, suggesting that this FTIR approach could be used as a rapid quantification method for mixtures of molecules of interest, provided the limits of use of the HTSXT-FTIR method are precisely known and respected. This last finding opens the way to direct quantification of total molecules of interest in more complex matrices.

Green improved processes to extract bioactive phenolic compounds from brown macroalgae using Sargassum muticum as model.

A comparative study between "alternative" extraction processes such as centrifugal partition extraction (CPE), supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE) and classical solid/liquid used in the laboratory are currently focusing on the efficiency (selectivity and productivity) to obtain bioactive phenolic compounds from the phaeophyte Sargassum muticum model. The choice of the best process was based on several measurements: (i) the total phenolic content measured by the colorimetric Folin-Ciocalteu assay, (ii) radical scavenger and antioxidant activities assessed by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay, and the ß-carotene bleaching method and finally (iii) the method productivity. Irrespective of the solvent used in the processes, alternative methods are always sharply more effective than classical ones. With the exception of SFE which does not allow extracting the totality of the active phenolic compounds, two of the other extraction methods were particularly promising. First, CPE afforded the most important yields in concentrated phenolic compounds (PC) (22.90±0.65% DW) also displaying the best activities (0.52±0.02 and 0.58±0.19 mg/mL for IC50 and AAC700, respectively). Secondly, PLE using an EtOH:water mixture 75:25 (v/v) allowed a good PC extraction (10.18±0.25% DW) with huge efficiency. Despite a lesser activity of the extracts (0.77±0.01 and 1.59±0.15 mg/mL for IC50 and AAC700, respectively) PLE is a green process and potentially complies European norms requirements for the prospective valorization of phenolic compounds from S. muticum in Brittany.