Efficient wastewater sample filtration improves the detection of SARS-CoV-2 variants: An extensive analysis based on sequencing parameters.

During the SARS-CoV-2 pandemic, many countries established wastewater (WW) surveillance to objectively monitor the level of infection within the population. As new variants continue to emerge, it has become clear that WW surveillance is an essential tool for the early detection of variants. The EU Commission published a recommendation suggesting an approach to establish surveillance of SARS-CoV-2 and its variants in WW, besides specifying the methodology for WW concentration and RNA extraction. Therefore, different groups have approached the issue with different strategies, mainly focusing on WW concentration methods, but only a few groups highlighted the importance of prefiltering WW samples and/or purification of RNA samples. Aiming to obtain high-quality sequencing data allowing variants detection, we compared four experimental conditions generated from the treatment of: i) WW samples by WW filtration and ii) the extracted RNA by DNase treatment, purification and concentration of the extracted RNA. To evaluate the best condition, the results were assessed by focusing on several sequencing parameters, as the outcome of SARS-CoV-2 sequencing from WW is crucial for variant detection. Overall, the best sequencing result was obtained by filtering the WW sample. Moreover, the present study provides an overview of some sequencing parameters to consider when optimizing a method for monitoring SARS-CoV-2 variants from WW samples, which can also be applied to any sample preparation methodology.

Wastewater-based SARS-CoV-2 environmental monitoring for Piedmont, Italy.

The experience gained over the last hundred years clearly indicates that two groups of viruses represent the main risk for the development of highly transmissible epidemics and pandemics in the human species: influenza viruses and coronaviruses (CoV). Although the search for viruses with pandemic potential in the environment may have an important predictive and monitoring role, it is still based on empirical methodologies, mostly resulting from the clinic and not fully validated for environmental matrices. As far as the SARS-CoV-2 pandemic, currently underway, is concerned, environmental monitoring activities aiming at checking the presence of SARS-CoV-2 in wastewater can be extremely useful to predict and check the diffusion of the disease. For this reason, the present study aims at evaluating the SARS-CoV-2 diffusion by means of a wastewater-based environmental monitoring developed in Piedmont, N-W Italy, during the second and third pandemic waves. Wastewater sampling strategies, sampling points sample pre-treatments and analytical methods, data processing and standardization, have been developed and discussed to give representative and reliable results. The following outcomes has been highlighted by the present study: i) a strong correlation between SARS-CoV-2 concentration in untreated wastewater and epidemic evolution in the considered areas can be observed as well as a predictive potential that could provide decision-makers with indications to implement effective policies, to mitigate the effects of the ongoing pandemic and to prepare response plans for future pandemics that could certainly arise in the decades to come; ii) moreover, the data at disposal from our monitoring campaign (almost 500 samples analysed in 11 months) confirm that SARS-CoV-2 concentrations in wastewater are strongly variable and site-specific across the region: the highest SARS-CoV-2 concentration values have been found in sewer networks serving the most populated areas of the region; iii) normalization of viral concentrations in wastewater through Pepper Mild Mottle Virus (a specific faecal marker) has been carried out and commented; iv) the study highlights the potential of wastewater treatment plants to degrade the genetic material referable to SARS-CoV-2 as well. In conclusion, the preliminary data reported in the present paper, although they need to be complemented by further studies considering also other geographical regions, are very promising.

SARS-CoV-2 airborne transmission: A validated sampling and analytical method.

The airborne transmission path for SARS-CoV-2 is of primary scientific and health-related interest as it could actually involve management, accessibility, use and functionality of many activities, including hospitals), schools, workplaces, factories, transport, sport venues and outdoor environment. It is necessary to develop a sampling and analytical method for virus-laden bioaerosol that could be considered reliable and validated according to ISO/IEC 17025 requirements. The present paper defines sample pretreatments aiming at recover SARS-CoV-2 from glass-fiber and PTFE filters employed by low and high-volume air samplers. Recovery test results focused on the sample concentration step carried out by means of ultracentrifugation are reported as well. Human coronavirus strain OC43 (a surrogate ß-coronavirus with the same SARS-CoV-2 particle structure) was used to validate each step of the recovery tests. We found that the elution efficiency of coronavirus OC43 from glass-fiber and quartz filters could be strongly enhanced by using an elution buffer containing up to 40% of fetal calf serum. Moreover, the recovery from PTFE filters is much higher and easier than from glass-fiber filters: for glass-fiber filters a 3 h-shaking phase, followed by a 30 s-vortexing step, are necessary to elute viral infective particles; for PTFE, 60 min-shaking is enough. The effect of suction time on filters could be resumed as follows: sampling durations up to 20 min at a flow rate of 500 L/min do not affect recovery efficiencies from 10 cm glass-fiber filters, whereas the recovery efficiency of infectious virions from 4.7 cm PTFE filters decreases of a factor 2 after 3 h of sampling at a flow rate of 20 L/min. The recovery efficiency of ultracentrifugation turns out to be around 57%. The effect of storage temperature of filters immersed in a transport medium on coronavirus infectivity is assessed as well. Based on the sampling techniques and the analytical methods developed as described in the present study, many field tests were carried out reporting virus concentrations up to 50 genomic copies per cubic meter of air in domestic environment with poor ventilation condition, whereas in hospital wards the detectable concentrations of SARS-CoV-2 were generally lower than 10 genomic copies per cubic meter of air.