Development of biomaterial composite hydrogel as a passive sampler with potential application in wastewater-based surveillance.

Nowadays, the need to track fast-spreading infectious diseases has raised due to the recent COVID-19 disease pandemic. As a response, Wastewater-based Surveillance (WBS) has emerged as an early detection and disease tracking method for large populations that enables a comprehensive overview of public health allowing for a faster response from public health sector to prevent large outbreaks. The process to achieve WBS requires a highly intensive sampling strategy with either expensive equipment or trained personnel to continuously sample. The sampling problem can be addressed by passive sampler development. Chitosan-based hydrogels are recognized for their capability to sample and remove various contaminants from wastewater, including metals, dyes, pharmaceuticals, among others. However, chitosan-based hydrogels unique characteristics, can be exploited to develop passive samplers of genetic material that can be a very valuable tool for WBS. This study aimed to develop a novel chitosan hydrogel formulation with enhanced characteristics suitable for use as a passive sampler of genetic material and its application to detect disease-causing pathogens present in wastewater. The study evaluates the effect of the concentration of different components on the formulation of a Chitosan composite hydrogel (Chitosan, Glutaraldehyde, Microcrystalline cellulose (MCC), and Polyethylene glycol (PEG)) on the hydrogel properties using a Box Hunter & Hunter experimental matrix. Hydrogels' weight, thickness, swelling ratio, microscopic morphology (SEM), FTIR assay, and zeta potential were characterized. The resulting hydrogel formulations were shown to be highly porous, positively charged (Zeta potential up to 35.80 ± 1.44 mV at pH 3) and with high water swelling capacity (up to 703.89 ± 15.00 %). Based on the results, a formulation from experimental design was selected and then evaluated its capacity to adsorb genetic material from a control spiked water with Influenza A virus synthetic vector. The adsorption capacity of the selected formulation was 4157.04 ± 64.74 Gene Copies/mL of Influenza A virus synthetic vector. The developed hydrogel showed potential to be used as passive sampler for pathogen detection in wastewater. However, deeper research can be conducted to improve adsorption, desorption and extraction techniques of genetic material from chitosan-hydrogel matrices.

Inter-institutional laboratory standardization for SARS-CoV-2 surveillance through wastewater-based epidemiology applied to Mexico City.

Objectives: Wastewater-based surveillance applied to SARS-CoV-2 viral load quantification for COVID-19 has become one of the most relevant complementary tools in epidemiologic prevention programs worldwide. However, this valuable decision-making tool still requires fine-tuning to produce comparable results between laboratories, especially when applied to the surveillance of megacities. Methods: Six laboratories across Mexico and one from the United States executed an interlaboratory study to set up a singular standardized protocol considering method cost, installed infrastructure, materials available, and supply availability for SARS-CoV-2 quantification from five Mexico City sampling sites across this megacity. Results: Comparable data from processing outcomes in the Mexican laboratories and in the external international laboratory serve as a validating data source. The Bland-Altman comparison showed consistency, with cycle threshold values within ±1.96 SD of SARS-CoV-2 genetic copies for the standard curve quantification, with a mismatch of two laboratories. In addition, MS2 bacteriophage recovery rates varied between 35% and 67% among all participating laboratories. Finally, the efficiency of viral genetic material recovered from all participating laboratories varied between 65% and 93% for the participating laboratories. Conclusion: This work lays the foundation for extensive and continuous wastewater-based surveillance application across independent Mexican laboratories in a time- and resource-effective manner.

Spread and persistence of antimicrobial resistance genes in wastewater from human and animal sources in São Paulo, Brazil.

The spread of antimicrobial resistance (AMR) through multiple reservoirs is a global concern. Wastewater is a critical AMR dissemination source, so this study aimed to assess the persistence of resistance genetic markers in wastewater using a culture-independent approach. Raw and treated wastewater samples (n = 121) from a wastewater treatment plant (WWTP), a human hospital, a veterinary hospital, and a pig farm were monthly collected and concentrated by filtration. DNA was extracted directly from filter membranes, and PCR was used in the qualitative search of 32 antimicrobial resistance genes (ARGs). Selected genes (blaCTX-M, blaKPC, qnrB, and mcr-1) were enumerated by quantitative real-time PCR (qPCR). Twenty-six ARGs were detected in the qualitative ARGs search, while quantitative data showed a low variation of the ARG's relative abundance (RA) throughout the months, especially at the human hospital and the WWTP. At the WWTP, despite significantly reducing the absolute number of gene copies/L after each treatment stage (p < 0.05), slight increases (p > 0.05) in the RAs of genes blaCTX-M, qnrB, and mcr-1 were observed in reused water (tertiary treatment) when compared with secondary effluent. Although the increase is not statistically significant, it is worth noting that there was some level of ARGs concentration after the disinfection process. No significant absolute or relative after-treatment quantification reductions were observed for any ARGs at the veterinary hospital or the pig farm. The spread of ARGs through sewage needs to be continuously addressed, because their release into natural environments may pose potential risks of exposure to resistant bacteria and impact local ecosystems.

Omicron and Delta variant prevalence detection and identification during the fourth COVID-19 wave in Mexico using wastewater-based epidemiology.

Objectives: To identify the SARS-CoV-2 variants Delta and Omicron during the fourth wave of the COVID-19 pandemic in Mexico using samples taken from 19 locations in 18 out of the 32 states. Methods: The genetic material concentration was done with PEG/NaCl precipitation, SARS-CoV-2 presence was confirmed by reverse transcriptase-quantitative polymerase chain reaction assay, the variant detection was carried out using a commercial mutation detection panel kit, and variant/mutation confirmation was done by amplicon sequencing of receptor-binding domain target region. The study used 41 samples. Results: The Delta variant was confirmed in two samples during August 2021 (Querétaro and CDMX) and in three samples during November 2021 (Aguascalientes, Ciudad Juárez campuses, and Nuevo Leon). In December 2021, another sample with the Delta variant was confirmed in Nuevo Leon. Between January to March 2022 only the presence of Omicron was confirmed, (variant BA.1). Additionally, in this period six samples were identified with the status "Variant Not Determined". Conclusion: To our knowledge, this study is one of the first to identify Omicron and Delta variants with polymerase chain reaction in Mexico and Latin America and its distribution across the country with 56% Mexican states making it a viable alternative for variant detection without conducting a large quantity of sequencing of clinical tests.

Passive Sampler Technology for Viral Detection in Wastewater-Based Surveillance: Current State and Nanomaterial Opportunities.

Although wastewater-based surveillance (WBS) is an efficient community-wide surveillance tool, its implementation for pathogen surveillance remains limited by ineffective sample treatment procedures, as the complex composition of wastewater often interferes with biomarker recovery. Moreover, current sampling protocols based on grab samples are susceptible to fluctuant biomarker concentrations and may increase operative costs, often rendering such systems inaccessible to communities in low-to-middle-income countries (LMICs). As a response, passive samplers have emerged as a way to make wastewater sampling more efficient and obtain more reliable, consistent data. Therefore, this study aims to review recent developments in passive sampling technologies to provide researchers with the tools to develop novel passive sampling strategies. Although promising advances in the development of nanostructured passive samplers have been reported, optimization remains a significant area of opportunity for researchers in the area, as methods for flexible, robust adsorption and recovery of viral genetic materials would greatly improve the efficacy of WBS systems while making them more accessible for communities worldwide.