Underestimation of SARS-CoV-2 in wastewater due to single or double mutations in the N1 qPCR probe binding region (preprint)

Wastewater surveillance using RT-qPCR has now been widely adopted to track circulating levels of SARS-CoV-2 virus in many sewer sheds. The CDC qPCR assays targeting two regions (N1 and N2) within the N gene are commonly used, but a discrepancy between the two biomarkers has been noticed by many groups using this method since late 2021. The reason is presumed to be due to mutations in regions targeted by the qPCR probe. In this study, we systematically investigated and unequivocally confirmed that the underlying reason for this discrepancy was mutations in the N1 probe target, and that a single mutation could cause a significant drop in signal. We first confirmed the proportion of related mutations in wastewater samples (Jan 2021-Dec 2022) using nested PCR and LC-MS. Based on relative proportion of N1 alleles, we separated the wastewater data into four time periods corresponding to different variant waves: Period I (Alpha and Delta waves with 0 mutation), Period II (BA.1/BA.2 wave with a single mutation found in all Omicron strains), Period III (BA. 5.2* wave with two mutations), and Period IV (BQ.1* wave with two mutations). Significantly lower N1 copies relative to N2 copies in samples from Periods II-IV compared to those from Period I was observed in wastewater. To further pinpoint the extent to which each mutation impacted N1 quantification, we compared the qPCR response among different synthetic oligomers with corresponding mutations. This study highlighted the impact of even just one or two mutations on qPCR-based wastewater surveillance of SARS-CoV-2.

Ocular manifestations of COVID-19 infection: A retrospective study (preprint)

Introduction: Corona Virus Disease 2019 (COVID-19) swept the world rapidly to become the world’s first major public health problem. SARS-CoV-2’s receptors are expressed in multiple tissues throughout the body and can cause multiple organ damage when engaged by the virus. We collected and classified the clinical ocular manifestations of individuals infected with the SARS-CoV-2 during the pandemic and analyzed them to gain an understanding of the ocular manifestations of COVID-19. Methods: We conducted a retrospective study to collect the demographic characteristics, the SARS-CoV-2 infection signs and symptoms, and specifically, the ocular manifestations of the individuals with COVID-19. We used SPSS for all statistical analyses, to calculate the incidence of COVID-19 ocular manifestations and to analyze the possible associations between those and sex, age, and clinical manifestations of COVID-19. Results: Of the 452 records reviewed, 134 didn’t pass any tests to confirm COVID-19, so this group did not participate in the analysis. Of the remaining 318 reviewed records, 147 (46.2%, n=318) belonged to men and 171 (53.8%, n=318) to women. The most common systemic symptoms were cough, expectoration, chills, fever, and fatigue. In addition, we found women to be more likely than men to experience cough, expectoration, nasal congestion, runny nose, sore throat, hoarseness, low back pain, taste changes, and arthralgia. The overall incidence ofCOVID-19 eye manifestations was 26.7% (n=318) and the most common symptoms were eyeball pain, decreased vision , eye itching or foreign body sensation, and photophobia or tearing. Women were more likely than men to experience eyeball pain and orbital pain. We found sex and the prolonged screen time usage to be associated with COVID-19 ocular manifestations, whereas age, BMI, history of eye disease, basic diseases, types of COVID-19, vaccination status, and vaccination time were not associated with the presence of ocular manifestations. Conclusion: COVID-19 ocular manifestations are common, and the main ones include eyeball pain and decreased vision. The variables associated with eye discomfort are sex and the  prolonged screen time usage. Other variables such as age, BMI, underlying diseases, and type of COVID-19 were not associated with the occurrence of ocular symptoms in COVID-19.

Changes in telepractice use and perspectives among speech and language therapists in Singapore through the COVID-19 pandemic.

BACKGROUND: Much has been written about the changes in use and perspectives of telepractice among speech and language therapists (SLTs) during the global COVID-19 pandemic. However, no long-term study has been done to examine whether there is a permanent shift in attitudes towards telepractice as the world adjusts to new norms and endemic COVID-19. AIMS: To compare the speech telepractice use and perspectives of SLTs at two time points of the pandemic: during and after the height of the pandemic. METHODS & PROCEDURES: Two online surveys were distributed a year apart among SLTs in Singapore. The first survey was disseminated during an initial lockdown period in 2020 and the second survey was done in 2021 when Singapore was starting to reduce strict quarantine and safe-distancing regulations. These surveys were distributed via communication channels of the local speech therapy association. A total of 115 and 71 responses to the survey were collected in 2020 and 2021, respectively. Responses were captured and analysed using descriptive statistics and statistical analysis while qualitative content analysis was used to derive key themes from open-ended questions. OUTCOMES & RESULTS: Telepractice use across all age groups and client types peaked at the height of the pandemic. Even as lockdown measures were eased, telepractice utilization was still higher than what it was before the pandemic. Dysphagia management was the only area where SLTs reduced their use of telepractice during the stabilization phase. After more experience with telepractice during the height of the pandemic, SLTs acknowledged the benefit of being able to use a wide range of media through telepractice and were less worried about not having the resources or knowledge to set up telepractice. SLTs also reported increased confidence in providing telepractice, which was reflected in their willingness to continue to provide telepractice even after the pandemic ends. CONCLUSIONS & IMPLICATIONS: The increase in use of telepractice during this pandemic is likely to be sustained as a majority of respondents believed they would continue to provide this mode of service delivery even after the pandemic ends. However, clinicians will still have to assess for client suitability, as clients with more complex medical or behavioural issues may still require in-person therapy. Additionally, dysphagia management via telepractice will still be limited given that swallow presentations may be more variable. Lastly, although generic resources are helpful for clinicians, the long-term sustainability of telepractice can be boosted by the sharing of resources that are suitable for the local context. WHAT THIS PAPER ADDS: What is already known on this subject Current studies have examined telepractice use and perspectives of SLTs before and during the COVID-19 pandemic. Despite the evidence for the efficacy of telepractice, uptake was low due to perceived lack of training and resources. Although more clinicians provided telepractice during the pandemic, many still doubted its efficacy over in-person therapy and most studies concluded that longer term studies are required to ascertain if SLTs will continuing providing telepractice after the pandemic. What this paper adds to existing knowledge This study shows that there is a definitive shift in perspectives in favour of telepractice after the pandemic, as clinicians continued to provide telepractice across most service areas even without lockdown restrictions. The previous challenges of insufficient resources, knowledge and privacy concerns were reduced with more experience in providing telepractice. Although client suitability remained a major barrier, more clinicians saw benefits of easy access to therapy and range of resources used as benefits that they could harness from telepractice. What are the potential or actual clinical implications of this work? A majority of respondents stated that they are more confident in providing telepractice and would want to continue providing this service delivery mode after the pandemic, so more training and resources suitable for the local contexts can be provided by national associations to sustain this. More research and resources can be gathered to make telepractice more efficacious for dysphagia management and clients who may be deemed unsuitable for telepractice.

Metabolomic and Proteomic Profiling of Porcine Intestinal Epithelial Cells Infected with Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) infection results in severe epidemic diarrhea and the death of suckling pigs. Although new knowledge about the pathogenesis of PEDV has been improved, alterations in metabolic processes and the functional regulators involved in PEDV infection with host cells remain largely unknow. To identify cellular metabolites and proteins related to PEDV pathogenesis, we synergistically investigated the metabolome and proteome profiles of PEDV-infected porcine intestinal epithelial cells by liquid chromatography tandem mass spectrometry and isobaric tags for relative and absolute quantification techniques. We identified 522 differential metabolites in positive and negative ion modes and 295 differentially expressed proteins after PEDV infection. Pathways of cysteine and methionine metabolism, glycine, serine and threonine metabolism, and mineral absorption were significantly enriched by differential metabolites and differentially expressed proteins. The betaine-homocysteine S-methyltransferase (BHMT) was indicated as a potential regulator involved in these metabolic processes. We then knocked down the BHMT gene and observed that down-expression of BHMT obviously decreased copy numbers of PEDV and virus titers (p < 0.01). Our findings provide new insights into the metabolic and proteomic profiles in PEDV-infected host cells and contribute to our further understanding of PEDV pathogenesis.

SARS-CoV-2 productively infects human brain microvascular endothelial cells.

BACKGROUND: The emergence of the novel, pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global health emergency. SARS-CoV-2 is highly contagious and has a high mortality rate in severe patients. However, there is very limited information on the effect of SARS-CoV-2 infection on the integrity of the blood-brain barrier (BBB). METHODS: RNA-sequencing profiling was performed to analyze the transcriptomic changes in human brain microvascular endothelial cells (hBMECs) after SARS-CoV-2 infection. Bioinformatic tools were used for differential analysis. Immunofluorescence, real-time quantitative PCR, and Western blotting analysis were used to explore biological phenotypes. RESULTS: A total of 927 differentially expressed genes were identified, 610 of which were significantly upregulated while the remaining 317 were downregulated. We verified the significant induction of cytokines, chemokines, and adhesion molecules in hBMECs by SARS-CoV-2, suggesting an activation of the vascular endothelium in brain. Moreover, we demonstrated that SARS-CoV-2 infection could increase the BBB permeability, by downregulating as well as remodeling the intercellular tight junction proteins. CONCLUSIONS: Our findings demonstrated that SARS-CoV-2 infection can cause BBB dysfunction, providing novel insights into the understanding of SARS-CoV-2 neuropathogenesis. Moreover, this finding shall constitute a new approach for future prevention and treatment of SARS-CoV-2-induced CNS infection.

Wastewater to clinical case (WC) ratio of COVID-19 identifies insufficient clinical testing, onset of new variants of concern and population immunity in urban communities (preprint)

Clinical testing has been the cornerstone of public health monitoring and infection control efforts in communities throughout the COVID-19 pandemic. With the extant and anticipated reduction of clinical testing as the disease moves into an endemic state, SARS-CoV-2 wastewater surveillance (WWS) is likely to have greater value as an important diagnostic tool to inform public health. As the widespread adoption of WWS is relatively new at the scale employed for COVID-19, interpretation of data, including the relationship to clinical cases, has yet to be standardized. An in-depth analysis of the metrics derived from WWS is required for public health units/agencies to interpret and utilize WWS-acquired data effectively and efficiently. In this study, the SARS-CoV-2 wastewater signal to clinical cases (WC) ratio was investigated across seven different cities in Canada over periods ranging from 8 to 21 months. Significant increases in the WC ratio occurred when clinical testing eligibility was modified to appointment-only testing, identifying a period of insufficient clinical testing in these communities. The WC ratio decreased significantly during the emergence of the Alpha variant of concern (VOC) in a relatively non-immunized communitys wastewater (40-60% allelic proportion), while a more muted decrease in the WC ratio signaled the emergence of the Delta VOC in a relatively well-immunized communitys wastewater (40-60% allelic proportion). Finally, a rapid and significant decrease in the WC ratio signaled the emergence of the Omicron VOC, likely because of the variants greater effectiveness at evading immunity, leading to a significant number of new reported clinical cases, even when vaccine-induced community immunity was high. The WC ratio, used as an additional monitoring metric, complements clinical case counts and wastewater signals as individual metrics in its ability to identify important epidemiological occurrences, adding value to WWS as a diagnostic technology during the COVID-19 pandemic and likely for future pandemics.

Community Surveillance of Omicron in Ontario: Wastewater-based Epidemiology Comes of Age. (preprint)

Wastewater-based surveillance of SARS-CoV-2 RNA has been implemented at building, neighbourhood, and city levels throughout the world. Implementation strategies and analysis methods differ, but they all aim to provide rapid and reliable information about community COVID-19 health states. A viable and sustainable SARS-CoV-2 surveillance network must not only provide reliable and timely information about COVID-19 trends, but also provide for scalability as well as accurate detection of known or unknown emerging variants. Emergence of the SARS-CoV-2 variant of concern Omicron in late Fall 2021 presented an excellent opportunity to benchmark individual and aggregated data outputs of the Ontario Wastewater Surveillance Initiative in Canada; this public health-integrated surveillance network monitors wastewaters from over 10 million people across major population centres of the province. We demonstrate that this coordinated approach provides excellent situational awareness, comparing favourably with traditional clinical surveillance measures. Thus, aggregated datasets compiled from multiple wastewater-based surveillance nodes can provide sufficient sensitivity (i.e., early indication of increasing and decreasing incidence of SARS-CoV-2) and specificity (i.e., allele frequency estimation of emerging variants) with which to make informed public health decisions at regional- and state-levels.

Quantitative Trend Analysis of SARS-CoV-2 RNA in Municipal Wastewater Exemplified with Sewershed-Specific COVID-19 Clinical Case Counts (preprint)

We present and demonstrate a quantitative statistical linear trend analysis ( QTA ) approach to analyze and interpret SARS-CoV-2 RNA wastewater surveillance results concurrently with clinical case data. This demonstration is based on the work completed under the Ontario (Canada) Wastewater Surveillance Initiative (WSI) by two laboratories in four large sewersheds within the Toronto Public Health (TPH) jurisdiction. The sewersheds were sampled over a 9-month period and data were uploaded to the Ontario Wastewater Surveillance Data and Visualization Hub ( Ontario Dashboard ) along with clinical case counts, both on a sewershed-specific basis. The data from the last 5-months, representing a range of high and low cases, was used for this demonstration. The QTA was conducted on a sewershed specific approach using the recommendations for public health interpretation and use of wastewater surveillance data by the United States Centers for Disease Control and Prevention (US CDC). The interpretation of the QTA results was based on the integration of both clinical and wastewater virus signals using an integration matrix in an interim draft guide by the Public Health Agency of Canada (PHAC). The key steps in the QTA consisted of (i) the calculation of Pepper Mild Mottle Virus (PMMoV), flow and flow-PMMoV-normalized virus loads; (ii) computation of the linear trends including interval estimation to identify the key inflection points using a segmented linear regression method and (iii) integrated interpretations based on consideration of both the cases and wastewater signals, as well as end user actionability. This approach is considered a complementary tool to commonly used qualitative analyses of SARS-CoV-2 RNA in wastewater and is intended to directly support public health decisions using a systematic quantitative approach.

What can influence a county’s resilience and susceptibility to coronavirus in Nebraska?

This study sought to classify and assign the 93 counties in Nebraska into distinctive spatial-temporal clusters based on their trends in the number of COVID-19 confirmed cases and deaths. A secondary goal of this study was to assess the potential role of preexisting sociodemographic, environmental, and health conditions in determining the county’s resilience and susceptibility to 2019-nCov. Spatial-temporal cluster analysis yielded three distinct confirmed cases and death clusters. Results from spatial-temporal cluster and regression analyses suggested that COVID-19 and HIV and obesity prevention should be integrated. Emergency response planning and mitigation should focus on counties characterized by noticeable increases in the number of COVID-19 confirmed cases and deaths and/or counties with relatively high population and ethnic minorities. [ABSTRACT FROM AUTHOR] Copyright of Sociological Spectrum is the property of Routledge and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Stressors and coping strategies of migrant workers diagnosed with COVID-19 in Singapore: a qualitative study.

INTRODUCTION: The health, psychological and socioeconomic vulnerabilities of low-wage migrant workers have been magnified in the COVID-19 pandemic, especially in high-income receiving countries such as Singapore. We aimed to understand migrant worker concerns and coping strategies during the COVID-19 pandemic to address these during the crisis and inform on comprehensive support needed after the crisis. METHODS: In-depth semi-structured interviews were carried out with migrant workers diagnosed with COVID-19. The participants were recruited from a COVID-19 mass quarantine facility in Singapore through a purposive sampling approach. Interviews were transcribed verbatim and thematic analysis performed to derive themes in their collective experience during the crisis. RESULTS: Three theme categories were derived from 27 interviews: migrant worker concerns during COVID-19, coping during COVID-19 and priorities after COVID-19. Major stressors in the crisis included the inability to continue providing for their families when work is disrupted, their susceptibility to infection in crowded dormitories, the shock of receiving the COVID-19 diagnosis while asymptomatic, as well as the isolating conditions of the quarantine environment. The workers coped by keeping in contact with their families, accessing healthcare, keeping updated with the news and continuing to practise their faith and religion. They looked forward to a return to normalcy after the crisis with keeping healthy and having access to healthcare as new priorities. CONCLUSION: We identified coping strategies employed by the workers in quarantine, many of which were made possible through the considered design of care and service delivery in mass quarantine facilities in Singapore. These can be adopted in the set-up of other mass quarantine facilities around the world to support the health and mental well-being of those quarantined. Our findings highlight the importance of targeted policy intervention for migrant workers, in areas such as housing and working environments, equitable access to healthcare, and social protection during and after this crisis.

Association of Cardiovascular Manifestations with In-hospital Outcomes in Patients with COVID-19: A Hospital Staff Data (preprint)

Background: The outbreaks of coronavirus disease 2019 (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain a huge threat to the public health worldwide. Clinical data is limited up to now regarding the risk factors in favor of severe conversion of non-severe case with COVID-19. Aims: This study analyzed a hospital staff data to figure out general clinical features of COVID-19 in terms of the association of cardiovascular manifestations (CVMs) with in-hospital outcomes of COVID-19 cases. Methods: Retrospective, single-center case series of 41 consecutive hospitalized health staff with confirmed COVID-19 were collected at the Central Hospital of Wuhan in Wuhan, China, from January 15 to January 24, 2020. Epidemiological, demographic, clinical, laboratory, radiological, treatment data and in-hospital adverse events were collected and analyzed. Final date of follow-up was March 3, 2020. A comparative study was applied between cases with CVMs and those without CVMs. Results: Of all, clinicians and clinical nurses accounted for 80.5%, while 87.8% of all had history of patient contact. The population was presented with a mean age of 39.1 +- 9.2 and less comorbidities than community population. The three most frequent symptoms of COVID-19 cases analyzed were fever (82.9%), myalgia or fatigue (80.5%) and cough (63.4%). While, the three most frequent initial symptoms were myalgia or fatigue (80.5%), fever (73.2%) and cough (41.5%). There were 95.1% cases featured as non-severe course of disease according to the official standard in China. Patients with CVMs and those without CVMs accounted for 58.5% and 41.5%, respectively. Compared with cases without CVMs, patients with CVMs were presented with lower baseline lymphocyte count (0.99 +- 0.43 and 1.55 +- 0.61, P<0.001), more who had at least once positive nucleic acid detection of throat swab during admission (50.0% and 11.8%, P=0.011), and more received oxygen support (79.2% and 23.5%, P<0.001). The rate of in-hospital adverse events was significantly higher in patients with CVMs group (75.0% and 23.5%, P=0.001). Multivariable logistic regression model indicated that, coexisting with CVMs in COVID-19 patients was not independently associated with in-hospital adverse events. Conclusions: Most of hospital staff with COVID-19 had history of patient contact, featured non-severe course of disease. Cases with CVMs suffered from more in-hospital adverse events than those without CVMs. But concomitant CVMs were not independently associated with in-hospital adverse events in COVID-19 patients.