Self-Diagnosis of SARS-CoV-2 from Saliva Samples at Home: Isothermal Amplification Enabled by Do-It-Yourself Portable Incubators and Laminated Poly-ethyl Sulfonate Membranes.

COVID-19 made explicit the need for rethinking the way in which we conduct testing for epidemic emergencies. During the COVID-19 pandemic, the dependence on centralized lab facilities and resource-intensive methodologies (e.g., RT-qPCR methods) greatly limited the deployment of widespread testing efforts in many developed and underdeveloped countries. Here, we illustrate the development of a simple and portable diagnostic kit that enables self-diagnosis of COVID-19 at home from saliva samples. We describe the development of a do-it-yourself (DIY) incubator for Eppendorf tubes that can be used to conduct SARS-CoV-2 detection with competitive sensitivity and selectivity from saliva at home. In a proof-of-concept experiment, we assembled Eppendorf-tube incubators at our home shop, prepared a single-tube mix of reagents and LAMP primers in our lab, and deployed these COVID-19 detection kits using urban delivery systems (i.e., Rappifavor or Uber) to more than 15 different locations in Monterrey, México. This straightforward strategy enabled rapid and cost-effective at-home molecular diagnostics of SARS-CoV-2 from real saliva samples with a high sensitivity (100%) and high selectivity (87%).

3D-Printed Tumor-on-Chip for the Culture of Colorectal Cancer Microspheres: Mass Transport Characterization and Anti-Cancer Drug Assays.

Tumor-on-chips have become an effective resource in cancer research. However, their widespread use remains limited due to issues related to their practicality in fabrication and use. To address some of these limitations, we introduce a 3D-printed chip, which is large enough to host ~1 cm3 of tissue and fosters well-mixed conditions in the liquid niche, while still enabling the formation of the concentration profiles that occur in real tissues due to diffusive transport. We compared the mass transport performance in its rhomboidal culture chamber when empty, when filled with GelMA/alginate hydrogel microbeads, or when occupied with a monolithic piece of hydrogel with a central channel, allowing communication between the inlet and outlet. We show that our chip filled with hydrogel microspheres in the culture chamber promotes adequate mixing and enhanced distribution of culture media. In proof-of-concept pharmacological assays, we biofabricated hydrogel microspheres containing embedded Caco2 cells, which developed into microtumors. Microtumors cultured in the device developed throughout the 10-day culture showing >75% of viability. Microtumors subjected to 5-fluorouracil treatment displayed <20% cell survival and lower VEGF-A and E-cadherin expression than untreated controls. Overall, our tumor-on-chip device proved suitable for studying cancer biology and performing drug response assays.

Placing biofabrication into the context of human disease modeling.

The field of biofabrication has seen tremendous advances in the past decade. More recently, the emerging role of biofabrication in allowing faithful generation of models of human tissues in their healthy and diseased states has been demonstrated and has rapidly expanded. These biomimetic models are potentially widely applicable in a range of research and translational areas including but not limited to fundamental biology studies as well as screening of chemical compounds, such as therapeutic agents. The United States Food and Drug Administration Modernization Act 2.0, which now no longer requires animal tests before approving human drug trials, will likely further boost the field in the years to come. This Special Issue, with a collection of 11 excellent research articles, thus focuses on showcasing the latest developments of biofabrication towards human disease modeling, spanning from 3D (bo)printing to organ-on-a-chip as well as their integration.

Structural and biological engineering of 3D hydrogels for wound healing.

Chronic wounds have become one of the most important issues for healthcare systems and are a leading cause of death worldwide. Wound dressings are necessary to facilitate wound treatment. Engineering wound dressings may substantially reduce healing time, reduce the risk of recurrent infections, and reduce the disability and costs associated. In the path of engineering of an ideal wound dressing, hydrogels have played a leading role. Hydrogels are 3D hydrophilic polymeric structures that can provide a protective barrier, mimic the native extracellular matrix (ECM), and provide a humid environment. Due to their advantages, hydrogels (with different architectural, physical, mechanical, and biological properties) have been extensively explored as wound dressing platforms. Here we describe recent studies on hydrogels for wound healing applications with a strong focus on the interplay between the fabrication method used and the architectural, mechanical, and biological performance achieved. Moreover, we review different categories of additives which can enhance wound regeneration using 3D hydrogel dressings. Hydrogel engineering for wound healing applications promises the generation of smart solutions to solve this pressing problem, enabling key functionalities such as bacterial growth inhibition, enhanced re-epithelialization, vascularization, improved recovery of the tissue functionality, and overall, accelerated and effective wound healing.

Neuron(s)-on-a-chip: A review of the design and use of microfluidic systems for neural tissue culture.

Neuron-on-chip (NoC) systems-microfluidic devices in which neurons are cultured-have become a promising alternative to replace or minimize the use of animal models and have greatly facilitated in vitro research. Here, we review and discuss current developments in neuron-on-chip platforms, with a particular emphasis on existing biological models, culturing techniques, biomaterials, and topologies. We also discuss how the architecture, flow, and gradients affect neuronal growth, differentiation, and development. Finally, we discuss some of the most recent applications of NoCs in fundamental research (i.e., studies on the effects of electrical, mechanical/topological, or chemical stimuli) and in disease modeling.

Gelatin-methacryloyl hydrogels containing turnip mosaic virus for fabrication of nanostructured materials for tissue engineering.

Current tissue engineering techniques frequently rely on hydrogels to support cell growth, as these materials strongly mimic the extracellular matrix. However, hydrogels often need ad hoc customization to generate specific tissue constructs. One popular strategy for hydrogel functionalization is to add nanoparticles to them. Here, we present a plant viral nanoparticle the turnip mosaic virus (TuMV), as a promising additive for gelatin methacryloyl (GelMA) hydrogels for the engineering of mammalian tissues. TuMV is a flexuous, elongated, tubular protein nanoparticle (700-750 nm long and 12-15 nm wide) and is incapable of infecting mammalian cells. These flexuous nanoparticles spontaneously form entangled nanomeshes in aqueous environments, and we hypothesized that this nanomesh structure could serve as a nanoscaffold for cells. Human fibroblasts loaded into GelMA-TuMV hydrogels exhibited similar metabolic activity to that of cells loaded in pristine GelMA hydrogels. However, cells cultured in GelMA-TuMV formed clusters and assumed an elongated morphology in contrast to the homogeneous and confluent cultures seen on GelMA surfaces, suggesting that the nanoscaffold material per se did not favor cell adhesion. We also covalently conjugated TuMV particles with epidermal growth factor (EGF) using a straightforward reaction scheme based on a Staudinger reaction. BJ cells cultured on the functionalized scaffolds increased their confluency by approximately 30% compared to growth with unconjugated EGF. We also provide examples of the use of GelMA-TuMV hydrogels in different biofabrication scenarios, include casting, flow-based-manufacture of filaments, and bioprinting. We envision TuMV as a versatile nanobiomaterial that can be useful for tissue engineering.

One-Step Bioprinting of Multi-Channel Hydrogel Filaments Using Chaotic Advection: Fabrication of Pre-Vascularized Muscle-Like Tissues.

The biofabrication of living constructs containing hollow channels is critical for manufacturing thick tissues. However, current technologies are limited in their effectiveness in the fabrication of channels with diameters smaller than hundreds of micrometers. It is demonstrated that the co-extrusion of cell-laden hydrogels and sacrificial materials through printheads containing Kenics static mixing elements enables the continuous and one-step fabrication of thin hydrogel filaments (1 mm in diameter) containing dozens of hollow microchannels with widths as small as a single cell. Pre-vascularized skeletal muscle-like filaments are bioprinted by loading murine myoblasts (C2C12 cells) in gelatin methacryloyl - alginate hydrogels and using hydroxyethyl cellulose as a sacrificial material. Higher viability and metabolic activity are observed in filaments with hollow multi-channels than in solid constructs. The presence of hollow channels promotes the expression of Ki67 (a proliferation biomarker), mitigates the expression of hypoxia-inducible factor 1-alpha , and markedly enhances cell alignment (i.e., 82% of muscle myofibrils aligned (in ±10°) to the main direction of the microchannels after seven days of culture). The emergence of sarcomeric α-actin is verified through immunofluorescence and gene expression. Overall, this work presents an effective and practical tool for the fabrication of pre-vascularized engineered tissues.

nurP28, a New-to-Nature Zein-Derived Peptide, Enhances the Therapeutic Effect of Docetaxel in Breast Cancer Monolayers and Spheroids.

The development of novel cancer therapeutic strategies has garnered increasing interest in cancer research. Among the therapeutic choices, chemosensitizers have shown exciting prospects. Peptides are an attractive alternative among the molecules that may be used as chemosensitizers. We rationally designed a new-to-nature peptide, nurP28, derived from the 22-kDa α-zein protein sequence (entry Q00919_MAIZE). The resultant sequence of the nurP28 peptide after the addition of arginine residues was LALLALLRLRRRATTAFIIP, and we added acetyl and amide groups at the N- and C-terminus, respectively, for capping. We evaluated the cytotoxicity of the nurP28 peptide alone and in combination with docetaxel in fibroblast monolayers and breast cancer monolayers and spheroids. Our results indicated that nurP28 is not cytotoxic to human fibroblasts or cancer cells. Nevertheless, when combined with 1 µM docetaxel, 3 ng/mL nurP28 induced equivalent (in MCF7 monolayers) and higher (in MCF7 spheroids) cytotoxic effects than 10-fold higher doses of docetaxel alone. These findings suggest that nurP28 may act as a chemosensitizer in breast cancer treatment. This study describes the enhancing "anti-cancer" effects of nurP28 in breast cancer 2D and 3D cultures treated with docetaxel. Further studies should explore the mechanisms underlying these effects and assess the clinical potential of our findings using animal models.

Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies.

The use of organoids has become increasingly popular recently due to their self-organizing abilities, which facilitate developmental and disease modeling. Various methods have been described to create embryoid bodies (EBs) generated from embryonic or pluripotent stem cells but with varying levels of differentiation success and producing organoids of variable size. Commercial ultra-low attachment (ULA) V-bottom well plates are frequently used to generate EBs. These plates are relatively expensive and not as widely available as standard concave well plates. Here, we describe a cost-effective and low labor-intensive method that creates homogeneous EBs at high yield in standard V- and U-bottom well plates by applying an anti-adherence solution to reduce surface attachment, followed by centrifugation to enhance cellular aggregation. We also explore the effect of different seeding densities, in the range of 1 to 11 ×103 cells per well, for the fabrication of neuroepithelial EBs. Our results show that the use of V-bottom well plates briefly treated with anti-adherent solution (for 5 min at room temperature) consistently yields functional neural EBs in the range of seeding densities from 5 to 11×103 cells per well. A brief post-seeding centrifugation step further enhances EB establishment. EBs fabricated using centrifugation exhibited lower variability in their final size than their non-centrifuged counterparts, and centrifugation also improved EB yield. The span of conditions for reliable EB production is narrower in U-bottom wells than in V-bottom wells (i.e., seeding densities between 7×103 and 11×103 and using a centrifugation step). We show that EBs generated by the protocols introduced here successfully developed into neural organoids and expressed the relevant markers associated with their lineages. We anticipate that the cost-effective and easily implemented protocols presented here will greatly facilitate the generation of EBs, thereby further democratizing the worldwide ability to conduct organoid-based research.

Culture of cancer spheroids and evaluation of anti-cancer drugs in 3D-printed miniaturized continuous stirred tank reactors (mCSTRs).

Cancer continues to be a leading cause of mortality in modern societies; therefore, improved and more reliablein vitrocancer models are needed to expedite fundamental research and anti-cancer drug development. Here, we describe the use of a miniaturized continuous stirred tank reactor (mCSTR) to first fabricate and mature cancer spheroids (i.e. derived from MCF7 cells, DU145 cells, and a mix of MCF7 cells and fibroblasts), and then to conduct anti-cancer drug assays under continuous perfusion. This 3 ml mCSTR features an off-center agitation system that enables homogeneous chaotic laminar mixing at low speeds to support cell aggregation. We incubated cell suspensions for 3 d in ultra-low-attachment plates to allow formation of discoid cell aggregates (∼600µm in diameter). These cell aggregates were then transferred into mCSTRs and continuously fed with culture medium. We characterized the spheroid morphology and the expression of relevant tumor biomarkers at different maturation times for up to 4 weeks. The spheroids progressively increased in size during the first 5-6 d of culture to reach a steady diameter between 600 and 800µm. In proof-of-principle experiments, we demonstrated the use of this mCSTR in anti-cancer drug testing. Three drugs commonly used in breast cancer treatment (doxorubicin, docetaxel, and paclitaxel) were probed at different concentrations in MCF7-derived spheroids. In these experiments, we evaluated cell viability, glucose consumption, spheroid morphology, lactate dehydrogenase activity, and the expression of genes associated with drug resistance (ABCB1andABCC1) and anti-apoptosis (Bcl2). We envision the use of this agitated system as a tumor-on-a-chip platform to expedite efficacy and safety testing of novel anti-cancer drugs and possibly in personalized medicine applications.

Acquired insights from the long-term surveillance of SARS-CoV-2 RNA for COVID-19 monitoring: The case of Monterrey Metropolitan Area (Mexico).

Wastewater-based epidemiology offers a time- and cost-effective way to monitor SARS-CoV-2 spread in communities and therefore represents a complement to clinical testing. WBE applicability has been demonstrated in a number of cases over short-term periods as a method for tracking the prevalence of SARS-CoV-2 and an early-warning tool for predicting outbreaks in the population. This study reports SARS-CoV-2 viral loads from wastewater treatment plants (WWTPs) and hospitals over a 6-month period (June to December 2020). Results show that the overall range of viral load in positive tested samples was between 1.2 × 103 and 3.5 × 106 gene copies/l, unveiling that secondary-treated wastewaters mirrored the viral load of influents. The interpretation suggests that the viral titers found in three out of four WWTPs were associated to clinical COVID-19 surveillance indicators preceding 2-7 days the rise of reported clinical cases. The median wastewater detection rate of SARS-CoV-2 was one out of 14,300 reported new cases. Preliminary model estimates of prevalence ranged from 0.02 to 4.6% for the studied period. This comprehensive statistical and epidemiological analysis demonstrates that the applied wastewater-based approach to COVID-19 surveillance is in general consistent and feasible, although there is room for improvements.

Low-Cost Light-Based GelMA 3D Bioprinting via Retrofitting: Manufacturability Test and Cell Culture Assessment.

Light-based bioprinter manufacturing technology is still prohibitively expensive for organizations that rely on accessing three-dimensional biological constructs for research and tissue engineering endeavors. Currently, most of the bioprinting systems are based on commercial-grade-based systems or modified DIY (do it yourself) extrusion apparatuses. However, to date, few examples of the adoption of low-cost equipment have been found for light-based bioprinters. The requirement of large volumes of bioinks, their associated cost, and the lack of information regarding the parameter selection have undermined the adoption of this technology. This paper showcases the retrofitting and assessing of a low-cost Light-Based 3D printing system for tissue engineering. To evaluate the potential of a proposed design, a manufacturability test for different features, machine parameters, and Gelatin Methacryloyl (GelMA) concentrations for 7.5% and 10% was performed. Furthermore, a case study of a previously seeded hydrogel with C2C12 cells was successfully implemented as a proof of concept. On the manufacturability test, deviational errors were found between 0.7% to 13.3% for layer exposure times of 15 and 20 s. Live/Dead and Actin-Dapi fluorescence assays after 5 days of culture showed promising results in the cell viability, elongation, and alignment of 3D bioprinted structures. The retrofitting of low-cost equipment has the potential to enable researchers to create high-resolution structures and three-dimensional in vitro models.

Social Non-profit Bioentrepreneurship: Current Status and Future Impact on Global Health.

For-profit biotechnological and pharmaceutical companies have played an essential role in the research and development (R&D) of innovative medical products and drugs for many decades and embody a trillion-dollar industry. The past decades have been marked by an increase in growth of social non-profit biotechnology companies and organizations led by entrepreneurs committed to solve (global) health issues. In this review, we define the concept of social bioentrepreneurship and consider the potential impact of such ventures on global health. We analyse the current status of non-profit biotechnology and clarify the strategy, motivation, funding, and marketing techniques of these enterprises. We find that these non-profit ventures mainly focus on neglected and rare diseases by using different but also similar funding, marketing, and business strategy approaches to for-profit biotechnology enterprises. We also identify good leadership, multidisciplinary teams, and public awareness as key components to achieve long-term survival and higher success rates. Challenges faced by bioentrepreneurs include the lack of a clearly defined regulatory environment or governmental incentives to support their endeavors. Overall, with this qualitative data review and market analysis we draw a promising picture of social non-profit bioentrepreneurship and underscore its current and future impact on global health issues.

Portable and Label-Free Quantitative Loop-Mediated Isothermal Amplification (LF-qLamp) for Reliable COVID-19 Diagnostics in Three Minutes of Reaction Time: Arduino-Based Detection System Assisted by a pH Microelectrode.

Loop-mediated isothermal amplification (LAMP) has been recently studied as an alternative method for cost-effective diagnostics in the context of the current COVID-19 pandemic. Recent reports document that LAMP-based diagnostic methods have a comparable sensitivity and specificity to that of RT-qPCR. We report the use of a portable Arduino-based LAMP-based amplification system assisted by pH microelectrodes for the accurate and reliable diagnosis of SARS-CoV-2 during the first 3 min of the amplification reaction. We show that this simple system enables a straightforward discrimination between samples containing or not containing artificial SARS-CoV-2 genetic material in the range of 10 to 10,000 copies per 50 µL of reaction mix. We also spiked saliva samples with SARS-CoV-2 synthetic material and corroborated that the LAMP reaction can be successfully monitored in real time using microelectrodes in saliva samples as well. These results may have profound implications for the design of real-time and portable quantitative systems for the reliable detection of viral pathogens including SARS-CoV-2.

Fabrication of Multilayered Composite Nanofibers Using Continuous Chaotic Printing and Electrospinning: Chaotic Electrospinning.

Multi-material and multilayered micro- and nanostructures are prominently featured in nature and engineering and are recognized by their remarkable properties. Unfortunately, the fabrication of micro- and nanostructured materials through conventional processes is challenging and costly. Herein, we introduce a high-throughput, continuous, and versatile strategy for the fabrication of polymer fibers with complex multilayered nanostructures. Chaotic electrospinning (ChE) is based on the coupling of continuous chaotic printing (CCP) and electrospinning, which produces fibers with an internal multi-material microstructure. When a CCP printhead is used as an electrospinning nozzle, the diameter of the fibers is further scaled down by 3 orders of magnitude while preserving their internal structure. ChE enables the use of various polymer inks for the creation of nanofibers with a customizable number of internal nanolayers. Our results showcase the versatility and tunability of ChE to fabricate multilayered structures at the nanoscale at high throughput. We apply ChE to the synthesis of unique carbon textile electrodes composed of nanofibers with striations carved into their surface at regular intervals. These striated carbon electrodes with high surface areas exhibit 3- to 4-fold increases in specific capacitance compared to regular carbon nanofibers; ChE holds great promise for the cost-effective fabrication of electrodes for supercapacitors and other applications.

Three-Dimensional Printing Using a Maize Protein: Zein-Based Inks in Biomedical Applications.

The use of three-dimensional (3D) printing for biomedical applications has expanded exponentially in recent years. However, the current portfolio of 3D printable inks is still limited. For instance, only few protein matrices have been explored as printing/bioprinting materials. Here, we introduce the use of zein, the primary constitutive protein in maize seeds, as a 3D printable material. Zein-based inks were prepared by dissolving commercial zein powder in ethanol with or without polyethylene glycol (PEG400) as a plasticizer. The rheological characteristics of our materials, studied during 21 days of aging/maturation, showed an increase in the apparent viscosity as a function of time in all formulations. The addition of PEG400 decreased the apparent viscosity. Inks with and without PEG400 and at different maturation times were tested for printability in a BioX bioprinter. We optimized the 3D printing parameters for each ink formulation in terms of extrusion pressure and linear printing velocity. Higher fidelity structures were obtained with inks that had maturation times of 10 to 14 days. We present different proof-of-concept experiments to demonstrate the versatility of the engineered zein inks for diverse biomedical applications. These include printing of complex and/or free-standing 3D structures, tablets for controlled drug release, and scaffolds for cell culture.

Modeling vaccination strategies in an Excel spreadsheet: Increasing the rate of vaccination is more effective than increasing the vaccination coverage for containing COVID-19.

We have investigated the importance of the rate of vaccination to contain COVID-19 in urban areas. We used an extremely simple epidemiological model that is amenable to implementation in an Excel spreadsheet and includes the demographics of social distancing, efficacy of massive testing and quarantine, and coverage and rate of vaccination as the main parameters to model the progression of COVID-19 pandemics in densely populated urban areas. Our model predicts that effective containment of pandemic progression in densely populated cities would be more effectively achieved by vaccination campaigns that consider the fast distribution and application of vaccines (i.e., 50% coverage in 6 months) while social distancing measures are still in place. Our results suggest that the rate of vaccination is more important than the overall vaccination coverage for containing COVID-19. In addition, our modeling indicates that widespread testing and quarantining of infected subjects would greatly benefit the success of vaccination campaigns. We envision this simple model as a friendly, readily accessible, and cost-effective tool for assisting health officials and local governments in the rational design/planning of vaccination strategies.

Bioinks for 3D Bioprinting: A Scientometric Analysis of Two Decades of Progress.

This scientometric analysis of 393 original papers published from January 2000 to June 2019 describes the development and use of bioinks for 3D bioprinting. The main trends for bioink applications and the primary considerations guiding the selection and design of current bioink components (i.e., cell types, hydrogels, and additives) were reviewed. The cost, availability, practicality, and basic biological considerations (e.g., cytocompatibility and cell attachment) are the most popular parameters guiding bioink use and development. Today, extrusion bioprinting is the most widely used bioprinting technique. The most reported use of bioinks is the generic characterization of bioink formulations or bioprinting technologies (32%), followed by cartilage bioprinting applications (16%). Similarly, the cell-type choice is mostly generic, as cells are typically used as models to assess bioink formulations or new bioprinting methodologies rather than to fabricate specific tissues. The cell-binding motif arginine-glycine-aspartate is the most common bioink additive. Many articles reported the development of advanced functional bioinks for specific biomedical applications; however, most bioinks remain the basic compositions that meet the simple criteria: Manufacturability and essential biological performance. Alginate and gelatin methacryloyl are the most popular hydrogels that meet these criteria. Our analysis suggests that present-day bioinks still represent a stage of emergence of bioprinting technology.

High-Throughput and Continuous Chaotic Bioprinting of Spatially Controlled Bacterial Microcosms.

Microorganisms do not work alone but instead function as collaborative microsocieties. The spatial distribution of different bacterial strains (micro-biogeography) in a shared volumetric space and their degree of intimacy greatly influences their societal behavior. Current microbiological techniques are commonly focused on the culture of well-mixed bacterial communities and fail to reproduce the micro-biogeography of polybacterial societies. Here, we bioprinted fine-scale bacterial microcosms using chaotic flows induced by a printhead containing a static mixer. This straightforward approach (i.e., continuous chaotic bacterial bioprinting) enables the fabrication of hydrogel constructs with intercalated layers of bacterial strains. These multilayered constructs are used to analyze how the spatial distributions of bacteria affect their social behavior. For example, we show that bacteria within these biological microsystems engage in either cooperation or competition, depending on the degree of shared interface. The extent of inhibition in predator-prey scenarios (i.e., probiotic-pathogen bacteria) increases when bacteria are in greater intimacy. Furthermore, two Escherichia coli strains exhibit competitive behavior in well-mixed microenvironments, whereas stable coexistence prevails for longer times in spatially structured communities. We anticipate that chaotic bioprinting will contribute to the development of a greater complexity of polybacterial microsystems, tissue-microbiota models, and biomanufactured materials.

The presence of SARS-CoV-2 RNA in different freshwater environments in urban settings determined by RT-qPCR: Implications for water safety.

This study is the first focused on the presence of SARS-CoV-2 in different freshwater environments in an urban setting. Groundwater and surface water reservoirs for drinking water as well as water from receiving rivers of the Monterrey Metropolitan Area were sampled repeatedly during a SARS-CoV-2 peak phase between October 2020 and January 2021, and viral RNA was measured by quantitative reverse transcription polymerase chain reaction. Forty-four percent of the groundwater samples had detectable viral loads between 2.6 and 38.3 copies/ml. A significant correlation between viral load and sucralose concentration in groundwater reaffirmed the hypothesis of leaching and infiltrating effluent from surface and/or failing sewage pipes and emphasized the importance of water disinfection. Twelve percent of the surface water dam samples tested positive for viral RNA, with values varying between 3.3 and 3.8 copies/ml. Finally, 13% of the river samples were positive for viral RNA, with concentrations ranging from 2.5 to 7.0 copies/ml. Untreated wastewater samples taken in the same period showed viral loads of up to 3535 copies/ml, demonstrating a dilution effect and/or wastewater facilities efficiency of three orders of magnitude. Variations in the viral loads in the groundwater and surface water over time and at the submetropolitan level generally reflected the reported trends in infection cases for Monterrey. The viral loads in the freshwater environments of Monterrey represent a low risk for recreational activities according to a preliminary risk assessment model. However, this result should not be taken lightly due to uncertainty regarding data and model constraints and the possibility of situations where the infection risk may increase considerably.