The Reduction of SARS-CoV-2 RNA Concentration in the Presence of Sewer Biofilms

Wastewater surveillance has been widely used to track the prevalence of SARS-CoV-2 in communities. Although some studies have investigated the decay of SARS-CoV-2 RNA in wastewater, understanding about its fate during wastewater transport in real sewers is still limited. This study aims to assess the impact of sewer biofilms on the dynamics of SARS-CoV-2 RNA concentration in naturally contaminated real wastewater (raw influent wastewater without extra SARS-CoV-2 virus/gene seeding) using a simulated laboratory-scale sewer system. The results indicated that, with the sewer biofilms, a 90% concentration reduction of the SARS-CoV-2 RNA was observed within 2 h both in wastewater of gravity (GS, gravity-driven sewers) and rising main (RM, pressurized sewers) sewer reactors. In contrast, the 90% reduction time was 8–26 h in control reactors without biofilms. The concentration reduction of SARS-CoV-2 RNA in wastewater was significantly more in the presence of sewer biofilms. In addition, an accumulation of c.a. 260 and 110 genome copies/cm2 of the SARS-CoV-2 E gene was observed in the sewer biofilm samples from RM and GS reactors within 12 h, respectively. These results confirmed that the in-sewer concentration reduction of SARS-CoV-2 RNA in wastewater was likely caused by the partition to sewer biofilms. The need to investigate the in-sewer dynamic of SARS-CoV-2 RNA, such as the variation of RNA concentration in influent wastewater caused by biofilm attachment and detachment, was highlighted by the significantly enhanced reduction rate of SARS-CoV-2 RNA in wastewater of sewer biofilm reactors and the accumulation of SARS-CoV-2 RNA in sewer biofilms. Further research should be conducted to investigate the in-sewer transportation of SARS-CoV-2 and their RNA and evaluate the role of sewer biofilms in leading to underestimates of COVID-19 prevalence in communities.

Apical-Out Human Airway Organoids Modeling SARS-CoV-2 Infection.

The respiratory epithelium, particularly the airway epithelium, is the primary infection site for respiratory pathogens. The apical surface of epithelial cells is constantly exposed to external stimuli including invading pathogens. Efforts have been made to establish organoid cultures to recapitulate the human respiratory tract. However, a robust and simple model with an easily accessible apical surface would benefit respiratory research. Here, we report the generation and characterization of apical-out airway organoids from the long-term expandable lung organoids that we previously established. The apical-out airway organoids morphologically and functionally recapitulated the human airway epithelium at a comparable level to the apical-in airway organoids. Moreover, apical-out airway organoids sustained productive and multicycle replication of SARS-CoV-2, and accurately recapitulated the higher infectivity and replicative fitness of the Omicron variants BA.5 and B.1.1.529 and an ancestral virus. In conclusion, we established a physiologically relevant and convenient apical-out airway organoid model for studying respiratory biology and diseases.

Human airway and nasal organoids reveal escalating replicative fitness of SARS-CoV-2 emerging variants.

The high transmissibility of SARS-CoV-2 Omicron subvariants was generally ascribed to immune escape. It remained unclear whether the emerging variants have gradually acquired replicative fitness in human respiratory epithelial cells. We sought to evaluate the replicative fitness of BA.5 and earlier variants in physiologically active respiratory organoids. BA.5 exhibited a dramatically increased replicative capacity and infectivity than B.1.1.529 and an ancestral strain wildtype (WT) in human nasal and airway organoids. BA.5 spike pseudovirus showed a significantly higher entry efficiency than that carrying WT or B.1.1.529 spike. Notably, we observed prominent syncytium formation in BA.5-infected nasal and airway organoids, albeit elusive in WT- and B.1.1.529-infected organoids. BA.5 spike-triggered syncytium formation was verified by lentiviral overexpression of spike in nasal organoids. Moreover, BA.5 replicated modestly in alveolar organoids, with a significantly lower titer than B.1.1.529 and WT. Collectively, the higher entry efficiency and fusogenic activity of BA.5 spike potentiated viral spread through syncytium formation in the human airway epithelium, leading to enhanced replicative fitness and immune evasion, whereas the attenuated replicative capacity of BA.5 in the alveolar organoids may account for its benign clinical manifestation.

Tracing COVID-19 Trails in Wastewater: A Systematic Review of SARS-CoV-2 Surveillance with Viral Variants

The emergence of new variants of SARS-CoV-2 associated with varying infectivity, pathogenicity, diagnosis, and effectiveness against treatments challenged the overall management of the COVID-19 pandemic. Wastewater surveillance (WWS), i.e., monitoring COVID-19 infections in communities through detecting viruses in wastewater, was applied to track the emergence and spread of SARS-CoV-2 variants globally. However, there is a lack of comprehensive understanding of the use and effectiveness of WWS for new SARS-CoV-2 variants. Here we systematically reviewed published articles reporting monitoring of different SARS-CoV-2 variants in wastewater by following the PRISMA guidelines and provided the current state of the art of this study area. A total of 80 WWS studies were found that reported different monitoring variants of SARS-CoV-2 until November 2022. Most of these studies (66 out of the total 80, 82.5%) were conducted in Europe and North America, i.e., resource-rich countries. There was a high variation in WWS sampling strategy around the world, with composite sampling (50/66 total studies, 76%) as the primary method in resource-rich countries. In contrast, grab sampling was more common (8/14 total studies, 57%) in resource-limited countries. Among detection methods, the reverse transcriptase polymerase chain reaction (RT-PCR)-based sequencing method and quantitative RT-PCR method were commonly used for monitoring SARS-CoV-2 variants in wastewater. Among different variants, the B1.1.7 (Alpha) variant that appeared earlier in the pandemic was the most reported (48/80 total studies), followed by B.1.617.2 (Delta), B.1.351 (Beta), P.1 (Gamma), and others in wastewater. All variants reported in WWS studies followed the same pattern as the clinical reporting within the same timeline, demonstrating that WWS tracked all variants in a timely way when the variants emerged. Thus, wastewater monitoring may be utilized to identify the presence or absence of SARS-CoV-2 and follow the development and transmission of existing and emerging variants. Routine wastewater monitoring is a powerful infectious disease surveillance tool when implemented globally.

Moving forward with COVID-19: Future research prospects of wastewater-based epidemiology methodologies and applications.

Wastewater-based epidemiology (WBE) has been demonstrated for its great potential in tracking of coronavirus disease 2019 (COVID-19) transmission among populations despite some inherent methodological limitations. These include non-optimized sampling approaches and analytical methods; stability of viruses in sewer systems; partitioning/retention in biofilms; and the singular and inaccurate back-calculation step to predict the number of infected individuals in the community. Future research is expected to (1) standardize best practices in wastewater sampling, analysis and data reporting protocols for the sensitive and reproducible detection of viruses in wastewater; (2) understand the in-sewer viral stability and partitioning under the impacts of dynamic wastewater flow, properties, chemicals, biofilms and sediments; and (3) achieve smart wastewater surveillance with artificial intelligence and big data models. Further specific research is essential in the monitoring of other viral pathogens with pandemic potential and subcatchment applications to maximize the benefits of WBE beyond COVID-19.

Correlation between SARS-CoV-2 RNA concentration in wastewater and COVID-19 cases in community: A systematic review and meta-analysis.

Wastewater-based epidemiology (WBE) has been considered as a promising approach for population-wide surveillance of coronavirus disease 2019 (COVID-19). Many studies have successfully quantified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentration in wastewater (CRNA). However, the correlation between the CRNA and the COVID-19 clinically confirmed cases in the corresponding wastewater catchments varies and the impacts of environmental and other factors remain unclear. A systematic review and meta-analysis were conducted to identify the correlation between CRNA and various types of clinically confirmed case numbers, including prevalence and incidence rates. The impacts of environmental factors, WBE sampling design, and epidemiological conditions on the correlation were assessed for the same datasets. The systematic review identified 133 correlation coefficients, ranging from -0.38 to 0.99. The correlation between CRNA and new cases (either daily new, weekly new, or future cases) was stronger than that of active cases and cumulative cases. These correlation coefficients were potentially affected by environmental and epidemiological conditions and WBE sampling design. Larger variations of air temperature and clinical testing coverage, and the increase of catchment size showed strong negative impacts on the correlation between CRNA and COVID-19 case numbers. Interestingly, the sampling technique had negligible impact although increasing the sampling frequency improved the correlation. These findings highlight the importance of viral shedding dynamics, in-sewer decay, WBE sampling design and clinical testing on the accurate back-estimation of COVID-19 case numbers through the WBE approach.

SARS-CoV-2 shedding sources in wastewater and implications for wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) approach for COVID-19 surveillance is largely based on the assumption of SARS-CoV-2 RNA shedding into sewers by infected individuals. Recent studies found that SARS-CoV-2 RNA concentration in wastewater (CRNA) could not be accounted by the fecal shedding alone. This study aimed to determine potential major shedding sources based on literature data of CRNA, along with the COVID-19 prevalence in the catchment area through a systematic literature review. Theoretical CRNA under a certain prevalence was estimated using Monte Carlo simulations, with eight scenarios accommodating feces alone, and both feces and sputum as shedding sources. With feces alone, none of the WBE data was in the confidence interval of theoretical CRNA estimated with the mean feces shedding magnitude and probability, and 63% of CRNA in WBE reports were higher than the maximum theoretical concentration. With both sputum and feces, 91% of the WBE data were below the simulated maximum CRNA in wastewater. The inclusion of sputum as a major shedding source led to more comparable theoretical CRNA to the literature WBE data. Sputum discharging behavior of patients also resulted in great fluctuations of CRNA under a certain prevalence. Thus, sputum is a potential critical shedding source for COVID-19 WBE surveillance.

Prevalence of Visual Impairment Among Students Before and During the COVID-19 Pandemic, Findings From 1,057,061 Individuals in Guangzhou, Southern China.

BACKGROUND: Visual impairment (VI) is a growing public health concern among students as a result of the COVID-19 pandemic. OBJECTIVE: This study investigated the change in VI before and during the pandemic among students. METHODS: Data on 547,864 and 497,371 students were obtained from the Guangzhou Survey on Students' Constitution and Health (GSSCH) collected in October 2019 and October 2020, respectively. VI was defined as the unaided distance visual acuity lower than 20/25 Snellen equivalent (LogMAR 0.10) in the worse eye. Change in VI based on age and sociodemographic variables were evaluated by chi-square test for trend as appropriate. Comparisons of different categorical variables were tested by contingency tables-based chi-square test. We have further analysis of the students who went through both of the 2019 and 2020 examinations for evaluating the VI incidence during the COVID-19 pandemic. RESULTS: More than 1,045,235 students were involved in our study, among whom 271,790 (54.65%) out of 497,371 students in Guangzhou suffered from VI during the COVID-19 compared with 293,001(53.48%) visually impaired students (total tested participants = 547,864) before the COVID-19 pandemic. During the pandemic, the overall prevalence of VI actually showed an increased age tendency and reached the highest level in the 17 [80.04%, 95%Confidence interval (CI):79.53 to 80.54%] and the 18 (79.64, 95%CI: 79.06 to 80.23%) age groups. Rapid growth was detected among students aged between 9 and 16 years old (raised by 46.21) while older students were more likely to get moderate and severe VI than younger ones. Students involved in more screen-based activities [(64.83%, 2019); (66.59%, 2020)] appeared to have a higher prevalence of VI than those involved in less [(49.17%, 2019); (49.26%, 2020)]. CONCLUSION: A rising trend of VI among students was detected before and during the COVID-19 pandemic. Moreover, the pandemic appeared to be associated with a rapid VI shift in younger and boy populations. Potential danger may arise when public health emergencies occur in the school, and more effort should be made to improve students' vision.

Enhanced decay of coronaviruses in sewers with domestic wastewater.

Recent outbreaks caused by coronaviruses and their supposed potential fecal-oral transmission highlight the need for understanding the survival of infectious coronavirus in domestic sewers. To date, the survivability and decay of coronaviruses were predominately studied using small volumes of wastewater (normally 5-30 mL) in vials (in-vial tests). However, real sewers are more complicated than bulk wastewater (wastewater matrix only), in particular the presence of sewer biofilms and different operational conditions. This study investigated the decay of infectious human coronavirus 229E (HCoV-229E) and feline infectious peritonitis virus (FIPV), two typical surrogate coronaviruses, in laboratory-scale reactors mimicking the gravity (GS, gravity-driven sewers) and rising main sewers (RM, pressurized sewers) with and without sewer biofilms. The in-sewer decay of both coronaviruses was greatly enhanced in comparison to those reported in bulk wastewater through in-vial tests. 99% of HCoV-229E and FIPV decayed within 2 h under either GS or RM conditions with biofilms, in contrast to 6-10 h without biofilms. There is limited difference in the decay of HCoV and FIPV in reactors operated as RM or GS, with the T90 and T99 difference of 7-10 min and 14-20 min, respectively. The decay of both coronaviruses in sewer biofilm reactors can be simulated by biphasic first-order kinetic models, with the first-order rate constant 2-4 times higher during the first phase than the second phase. The decay of infectious HCoV and FIPV was significantly faster in the reactors with sewer biofilms than in the reactors without biofilms, suggesting an enhanced decay of these surrogate viruses due to the presence of biofilms and related processes. The mechanism of biofilms in virus adsorption and potential inactivation remains unclear and requires future investigations. The results indicate that the survivability of infectious coronaviruses detected using bulk wastewater overestimated the infectivity risk of coronavirus during wastewater transportations in sewers or the downstream treatment.

Current status and correlates of screen time among children in Guangdong during the COVID-19 pandemic

Objective To investigate the current status of online classes, screen time and its influencing factors among primary school students in Guangdong during the 2019 novel coronavirus pandemic. Methods Using the convenience sampling method, a total of 5 266 pupils aged 6-12-years-old from Guangzhou, Zhanjiang, and Zhongshan participated in the online questionnaire. ANOVA or chi-square tests were performed to compare differences in online classes and screen time between grades, and multinomial Logistic regression was performed to analyze the correlates of recreational screen time. Results The prevalence of prolonged recreational screen time was 42.2% and 55.2% on weekdays and weekends, respectively. Recreational screen time increased by 40.31 min/d on weekdays and 33.07 min/d on weekends, compared to usual school semesters. The average duration of an online class was (26.07+or-9.62) min, which totaled (110.41+or-51.98)min per day. Sex, grade, being the only child, and parents' education levels were identified as the influencing factors of prolonged recreational screen time. Children who practiced moderate levels (weekdays: OR=1.27;weekends: OR=1.40;P < 0.05) or lower levels of physical activity (weekdays: OR=1.86;weekend: OR=1.84;P < 0.05) were at a higher risk of prolonged recreational screen time than those who practiced more vigorous physical activity. Children whose parents limited their screen time to a moderate (weekdays: OR=1.61, P < 0.05) or lower level (weekdays: OR=1.32, P < 0.05) had a higher risk of prolonged recreational screen time than those with a higher frequency. Children with parents' recreational screen time 2 h/d had a higher risk of prolonged recreational screen time than the reference group;children who exhibited moderate to vigorous levels of physical activity < 1 h/d (weekdays: OR=1.31, P < 0.05), and those used electronic devices for learning 1-2 h/d (weekdays: OR=2.65;weekend: OR=2.65;P < 0.05) or for 2 h/d (weekdays: OR=4.05, weekend: OR=5.24, P < 0.05) were at a higher risk of prolonged recreational screen time than the reference group. Conclusion During the COVID-19 pandemic, the level of screen time among children in Guangdong was high. Behavioral monitoring and targeted interventions are needed to promote children's health.

Emotional and behavioral problems and determinants among primary and middle school students aged 6 to 17-year-old in Guangdong Province during the COVID-19 pandemic

Objective: To gain a better understanding of the emotional and behavioral problems associated determinants of primary and middle school students from Guangdong Province during the COVID-19 pandemic, so as to provide a basis for developing targeted intervention strategies.

Analytical performance comparison of four SARS-CoV-2 RT-qPCR primer-probe sets for wastewater samples.

Current studies have confirmed the feasibility of SARS-CoV-2 RNA detection by RT-qPCR assays in wastewater samples as an effective surveillance tool of COVID-19 prevalence in a community. Analytical performance of various RT-qPCR assays has been compared against wastewater samples based on the positive ratio. However, there is no systematic comparison work has been conducted for both analytical sensitivity and quantitative reliability against wastewater, which are essential factors for WBE. In this study, the detection performance of four RT-qPCR primer-probe sets, including CCDC-N, CDC-N1, N-Sarbeco, and E-Sarbeco, was systematically evaluated with pure synthetized plasmids, spiked wastewater mocks and raw wastewater samples. In addition to confirm RT-qPCR results, Nanopore sequencing was employed to delineate at molecular level for the analytical sensitivity and reproducibility of those primer-probe sets. CCDC-N showed high sensitivity and the broadest linearity range for wastewater samples. It was thus recommended to be the most efficient tool in the quantitative analysis of SARS-CoV-2 in wastewater. CDC-N1 had the highest sensitivity for real wastewater and thus would be suitable for the screening of wastewater for the presence of SARS-CoV-2. When applying the primer-probe sets to wastewater samples collected from different Australian catchments, increased active clinical cases were observed with the augment of SARS-CoV-2 RNA quantified by RT-qPCR in wastewater in low prevalence communities.

Data-driven estimation of COVID-19 community prevalence through wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) has been regarded as a potential tool for the prevalence estimation of coronavirus disease 2019 (COVID-19) in the community. However, the application of the conventional back-estimation approach is currently limited due to the methodological challenges and various uncertainties. This study systematically performed meta-analysis for WBE datasets and investigated the use of data-driven models for the COVID-19 community prevalence in lieu of the conventional WBE back-estimation approach. Three different data-driven models, i.e. multiple linear regression (MLR), artificial neural network (ANN), and adaptive neuro fuzzy inference system (ANFIS) were applied to the multi-national WBE dataset. To evaluate the robustness of these models, predictions for sixteen scenarios with partial inputs were compared against the actual prevalence reports from clinical testing. The performance of models was further validated using unseen data (data sets not included for establishing the model) from different stages of the COVID-19 outbreak. Generally, ANN and ANFIS models showed better accuracy and robustness over MLR models. Air and wastewater temperature played a critical role in the prevalence estimation by data-driven models, especially MLR models. With unseen datasets, ANN model reasonably estimated the prevalence of COVID-19 (cumulative cases) at the initial phase and forecasted the upcoming new cases in 2-4 days at the post-peak phase of the COVID-19 outbreak. This study provided essential information about the feasibility and accuracy of data-driven estimation of COVID-19 prevalence through the WBE approach.

Uncertainties in estimating SARS-CoV-2 prevalence by wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) is a promising approach for estimating population-wide COVID-19 prevalence through detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. However, various methodological challenges associated with WBE would affect the accuracy of prevalence estimation. To date, the overall uncertainty of WBE and the impact of each step on the prevalence estimation are largely unknown. This study divided the WBE approach into five steps (i.e., virus shedding; in-sewer transportation; sampling and storage; analysis of SARS-CoV-2 RNA concentration in wastewater; back-estimation) and further summarized and quantified the uncertainties associated with each step through a systematic review. Although the shedding of SARS-CoV-2 RNA varied greatly between COVID-19 positive patients, with more than 10 infected persons in the catchment area, the uncertainty caused by the excretion rate became limited for the prevalence estimation. Using a high-frequency flow-proportional sampling and estimating the prevalence through actual water usage data significantly reduced the overall uncertainties to around 20-40% (relative standard deviation, RSD). And under such a scenario, the analytical uncertainty of SARS-CoV-2 RNA in wastewater was the dominant factor. This highlights the importance of using surrogate viruses as internal or external standards during the wastewater analysis, and the need for further improvement on analytical approaches to minimize the analytical uncertainty. This study supports the application of WBE as a complementary surveillance strategy for monitoring COVID-19 prevalence and provides methodological improvements and suggestions to enhance the reliability for future studies.