Evidence for SARS-CoV-2 Delta and Omicron co-infections and recombination (preprint)

Between November 2021 and February 2022, SARS-CoV-2 Delta and Omicron variants co-circulated in the United States, allowing for co-infections and possible recombination events. We sequenced 29,719 positive samples during this period and analyzed the presence and fraction of reads supporting mutations specific to either the Delta or Omicron variant. Our sequencing protocol uses hybridization capture and is thus less subject to artifacts observed in amplicon-based approaches that may lead to spurious signals for recombinants. We identified 20 co-infections, one of which displayed evidence of a low recombinant viral population. We also identified two independent cases of infection by a Delta-Omicron recombinant virus, where 100% of the viral RNA came from one clonal recombinant. In both cases, the 5'-end of the viral genome was from the Delta genome, and the 3'-end from Omicron, though the breakpoints were different. Delta-Omicron recombinant viruses were rare, and there is currently no evidence that the two Delta-Omicron recombinant viruses identified are more transmissible between hosts compared to the circulating Omicron lineages.

Sentinel Cards Provide Practical SARS-CoV-2 Monitoring in School Settings (preprint)

Accurate, high-resolution environmental monitoring of SARS-CoV-2 traces indoors through sentinel cards is a promising approach to help students safely return to in-person learning. Because SARS-CoV-2 RNA can persist for up to a week on several indoor surface types, there is a need for increased temporal resolution to determine whether consecutive surface positives arise from new infection events or continue to report past events. Cleaning sentinel cards after sampling would provide the needed resolution, but might interfere with assay performance. We tested the effect of three cleaning solutions (BZK wipes, wet wipes, RNase Away) at three different viral loads: "high" (4 x 104 GE/mL), "medium" (1 x 104 GE/mL), and "low" (2.5 x 103 GE/mL). RNAse Away, chosen as a positive control, was the most effective cleaning solution on all three viral loads. Wet wipes were found to be more effective than BZK wipes in the medium viral load condition. The low viral load condition was easily reset with all three cleaning solutions. These findings will enable temporal SARS-CoV-2 monitoring in indoor environments where transmission risk of the virus is high and the need to avoid individual-level sampling for privacy or compliance reasons exists.

Wastewater sequencing uncovers early, cryptic SARS-CoV-2 variant transmission (preprint)

As SARS-CoV-2 becomes an endemic pathogen, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing/sequencing capacity, which can also introduce biases. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here, we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We develop and deploy improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detect emerging variants of concern up to 14 days earlier in wastewater samples, and identify multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

SARS-CoV-2 Distribution in Residential Housing Suggests Contact Deposition and Correlates with Rothia (preprint)

Monitoring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on surfaces is emerging as an important tool for identifying past exposure to individuals shedding viral RNA. Our past work has demonstrated that SARS-CoV-2 reverse transcription-quantitative PCR (RT-qPCR) signals from surfaces can identify when infected individuals have touched surfaces such as Halloween candy, and when they have been present in hospital rooms or schools. However, the sensitivity and specificity of surface sampling as a method for detecting the presence of a SARS-CoV-2 positive individual, as well as guidance about where to sample, has not been established. To address these questions, and to test whether our past observations linking SARS-CoV-2 abundance to Rothia spp. in hospitals also hold in a residential setting, we performed detailed spatial sampling of three isolation housing units, assessing each sample for SARS-CoV-2 abundance by RT-qPCR, linking the results to 16S rRNA gene amplicon sequences to assess the bacterial community at each location and to the Cq value of the contemporaneous clinical test. Our results show that the highest SARS-CoV-2 load in this setting is on touched surfaces such as light switches and faucets, but detectable signal is present in many non-touched surfaces that may be more relevant in settings such as schools where mask wearing is enforced. As in past studies, the bacterial community predicts which samples are positive for SARS-CoV-2, with Rothia sp. showing a positive association. ImportanceSurface sampling for detecting SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is increasingly being used to locate infected individuals. We tested which indoor surfaces had high versus low viral loads by collecting 381 samples from three residential units where infected individuals resided, and interpreted the results in terms of whether SARS-CoV-2 was likely transmitted directly (e.g. touching a light switch) or indirectly (e.g. by droplets or aerosols settling). We found highest loads where the subject touched the surface directly, although enough virus was detected on indirectly contacted surfaces to make such locations useful for sampling (e.g. in schools, where students do not touch the light switches and also wear masks so they have no opportunity to touch their face and then the object). We also documented links between the bacteria present in a sample and the SARS-CoV-2 virus, consistent with earlier studies.

Comparison of heat-inactivated and infectious SARS-CoV-2 across indoor surface materials shows comparable RT-qPCR viral signal intensity and persistence (preprint)

Environmental monitoring in public spaces can be used to identify surfaces contaminated by persons with COVID-19 and inform appropriate infection mitigation responses. Research groups have reported detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) on surfaces days or weeks after the virus has been deposited, making it difficult to estimate when an infected individual may have shed virus onto a SARS-CoV-2 positive surface, which in turn complicates the process of establishing effective quarantine measures. In this study, we determined that reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection of viral RNA from heat-inactivated particles experiences minimal decay over seven days of monitoring on eight out of nine surfaces tested. The properties of the studied surfaces result in RT-qPCR signatures that can be segregated into two material categories, rough and smooth, where smooth surfaces have a lower limit of detection. RT-qPCR signal intensity (average quantification cycle (Cq) can be correlated to surface viral load using only one linear regression model per material category. The same experiment was performed with infectious viral particles on one surface from each category, with essentially identical results. The stability of RT-qPCR viral signal demonstrates the need to clean monitored surfaces after sampling to establish temporal resolution. Additionally, these findings can be used to minimize the number of materials and timepoints tested and allow for the use of heat-inactivated viral particles when optimizing environmental monitoring methods.

Automated, miniaturized, and scalable screening of healthcare workers, first responders, and students for SARS-CoV-2 in San Diego County (preprint)

Background: Successful containment strategies for SARS-CoV-2, the causative virus of the COVID-19 pandemic, have involved widespread population testing that identifies infections early and enables rapid contact tracing. In this study, we developed a rapid and inexpensive RT-qPCR testing pipeline for population-level SARS-CoV-2 detection, and used this pipeline to establish a clinical laboratory dedicated to COVID-19 testing at the University of California San Diego (UCSD) with a processing capacity of 6,000 samples per day and next-day result turnaround times. Methods and findings: Using this pipeline, we screened 6,786 healthcare workers and first responders, and 21,220 students, faculty, and staff from UCSD. Additionally, we screened 6,031 preschool-grade 12 students and staff from public and private schools across San Diego County that remained fully or partially open for in-person teaching during the pandemic. Between April 17, 2020 and February 5, 2021, participants provided 161,582 nasal swabs that were tested for the presence of SARS-CoV-2. Overall, 752 positive tests were obtained, yielding a test positivity rate of 0.47%. While the presence of symptoms was significantly correlated with higher viral load, most of the COVID-19 positive participants who participated in symptom surveys were asymptomatic at the time of testing. The positivity rate among preschool-grade 12 schools that remained open for in-person teaching was similar to the positivity rate at UCSD and lower than that of San Diego County, with the children in private schools being less likely to test positive than the adults at these schools. Conclusions: Most schools across the United States have been closed for in-person learning for much of the 2020-2021 school year, and their safe reopening is a national priority. However, as there are no vaccines against SARS-CoV-2 currently available to the majority of school-aged children, the traditional strategies of mandatory masking, physical distancing, and repeated viral testing of students and staff remain key components of risk mitigation in these settings. The data presented here suggest that the safety measures and repeated testing actions taken by participating healthcare and educational facilities were effective in preventing outbreaks, and that a similar combination of risk-mitigation strategies and repeated testing may be successfully adopted by other healthcare and educational systems.

Alternative swabs and storage for SARS-CoV-2 detection in a hospital environment (preprint)

Since its appearance in late 2019, COVID-19 has caused well over one million deaths worldwide. Large-scale testing and contact tracing remain critical for controlling viral spread. Complying with the US CDC and WHO protocols for sample collection requires a ready supply of inexpensive swabs and collection reagents. Unfortunately, CDC-approved clinical-grade sampling supplies are expensive, and additionally, current methods prevent further analysis of the microbiome due to the presence of antibiotics in viral transport media. Researchers sought out new testing supplies in a recent study comparing five consumer-grade swabs and one clinical-grade swab. They found that using 95% ethanol instead of viral transfer media reduced RNase activity, preserving samples for microbiome analysis, and extracting directly from the swab head instead of the surrounding liquid resulted in 2-4x higher RNA recovery than the clinical standard. The limit of detection was similar for samples collected with consumer-grade or research-grade plastic swabs compared to the CDC-approved synthetic swab, and microbiome analysis demonstrated that microbial communities were not impacted by swab type. These results suggest that improved SARS-CoV-2 detection may be possible using a less expensive technique that also preserves material for microbiome analysis.

Emergence of an early SARS-CoV-2 epidemic in the United States (preprint)

The emergence of the early COVID-19 epidemic in the United States (U.S.) went largely undetected, due to a lack of adequate testing and mitigation efforts. The city of New Orleans, Louisiana experienced one of the earliest and fastest accelerating outbreaks, coinciding with the annual Mardi Gras festival, which went ahead without precautions. To gain insight into the emergence of SARS-CoV-2 in the U.S. and how large, crowded events may have accelerated early transmission, we sequenced SARS-CoV-2 genomes during the first wave of the COVID-19 epidemic in Louisiana. We show that SARS-CoV-2 in Louisiana initially had limited sequence diversity compared to other U.S. states, and that one successful introduction of SARS-CoV-2 led to almost all of the early SARS-CoV-2 transmission in Louisiana. By analyzing mobility and genomic data, we show that SARS-CoV-2 was already present in New Orleans before Mardi Gras and that the festival dramatically accelerated transmission, eventually leading to secondary localized COVID-19 epidemics throughout the Southern U.S.. Our study provides an understanding of how superspreading during large-scale events played a key role during the early outbreak in the U.S. and can greatly accelerate COVID-19 epidemics on a local and regional scale.

Feasibility of using alternative swabs and storage solutions for paired SARS-CoV-2 detection and microbiome analysis in the hospital environment (preprint)

Background: Determining the role of fomites in the transmission of SARS-CoV-2 is essential in the hospital setting and will likely be important outside of medical facilities as governments around the world make plans to ease COVID-19 public health restrictions and attempt to safely reopen economies. Expanding COVID-19 testing to include environmental surfaces would ideally be performed with inexpensive swabs that could be transported safely without concern of being a source of new infections. However, CDC-approved clinical-grade sampling supplies and techniques using a synthetic swab are expensive, potentially expose laboratory workers to viable virus and prohibit analysis of the microbiome due to the presence of antibiotics in viral transport media (VTM). To this end, we performed a series of experiments comparing the diagnostic yield using five consumer-grade swabs (including plastic and wood shafts and various head materials including cotton, synthetic, and foam) and one clinical grade swab for inhibition to RNA. For three of these swabs, we evaluated performance to detect SARS-CoV-2 in twenty intensive care unit (ICU) hospital rooms of patients including COVID-19+ patients. All swabs were placed in 95% ethanol and further evaluated in terms of RNase activity. SARS-CoV-2 was measured both directly from the swab and from the swab eluent. Results: Compared to samples collected in VTM, 95% ethanol demonstrated significant inhibition properties against RNases. When extracting directly from the swab head as opposed to the eluent, RNA recovery was approximately 2-4x higher from all six swab types tested as compared to the clinical standard of testing the eluent from a CDC-approved synthetic (SYN) swab. The limit of detection (LoD) of SARSSARS-CoV-2 from floor samples collected using the consumer-grade plastic (CGp) or research-grade plastic The Microsetta Initiative (TMI) swabs was similar or better than the SYN swab, further suggesting that swab type does not impact RNA recovery as measured by the abundance of SARSSARS-CoV-2. The LoD for TMI was between 0-362.5 viral particles while SYN and CGp were both between 725-1450 particles. Lastly microbiome analyses (16S rRNA gene sequencing) of paired samples (nasal and floor from same patient-room) collected using different swab types in triplicate indicated that microbial communities were not impacted by swab type, but instead driven by the patient and sample type. Conclusions: Compared to using a clinical-grade synthetic swab, detection of SARS-CoV-2 from environmental samples collected from ICU rooms of patients with COVID was similar using consumer grade swabs, stored in 95% ethanol. The yield was best from the swab head rather than the eluent and the low level of RNase activity and lack of antibiotics in these samples makes it possible to perform concomitant microbiome analyses.

Microbial context predicts SARS-CoV-2 prevalence in patients and the hospital built environment (preprint)

Synergistic effects of bacteria on viral stability and transmission are widely documented but remain unclear in the context of SARS-CoV-2. We collected 972 samples from hospitalized patients with coronavirus disease 2019 (COVID-19), their health care providers, and hospital surfaces before, during, and after admission. We screened for SARS-CoV-2 using RT-qPCR, characterized microbial communities using 16S rRNA gene amplicon sequencing, and contextualized the massive microbial diversity in this dataset through meta-analysis of over 20,000 samples. Sixteen percent of surfaces from COVID-19 patient rooms were positive, with the highest prevalence in floor samples next to patient beds (39%) and directly outside their rooms (29%). Although bed rail samples increasingly resembled the patient microbiome over time, SARS-CoV-2 was detected less there (11%). Despite viral surface contamination in almost all patient rooms, no health care workers contracted the disease, suggesting that personal protective equipment was effective in preventing transmissions. SARS-CoV-2 positive samples had higher bacterial phylogenetic diversity across human and surface samples, and higher biomass in floor samples. 16S microbial community profiles allowed for high SARS-CoV-2 classifier accuracy in not only nares, but also forehead, stool, and floor samples. Across distinct microbial profiles, a single amplicon sequence variant from the genus Rothia was highly predictive of SARS-CoV-2 across sample types and had higher prevalence in positive surface and human samples, even compared to samples from patients in another intensive care unit prior to the COVID-19 pandemic. These results suggest that bacterial communities may contribute to viral prevalence both in the host and hospital environment. One Sentence SummaryMicrobial classifier highlights specific taxa predictive of SARS-CoV-2 prevalence across diverse microbial niches in a COVID-19 hospital unit.

High throughput wastewater SARS-CoV-2 detection enables forecasting of community infection dynamics in San Diego county (preprint)

Large-scale wastewater surveillance has the ability to greatly augment the tracking of infection dynamics especially in communities where the prevalence rates far exceed the testing capacity. However, current methods for viral detection in wastewater are severely lacking in terms of scaling up for high throughput. In the present study, we employed an automated magnetic-bead based concentration approach for viral detection in sewage that can effectively be scaled up for processing 24 samples in a single 40-minute run. The method compared favorably to conventionally used methods for viral wastewater concentrations with higher recovery efficiencies from input sample volumes as low as 10ml and can enable the processing of over 100 wastewater samples in a day. The sensitivity of the high-throughput protocol was shown to detect cases as low as 2 in a hospital building with a known COVID-19 caseload. Using the high throughput pipeline, samples from the influent stream of the primary wastewater treatment plant of San Diego county (serving 2.3 million residents) were processed for a period of 13 weeks. Wastewater estimates of SARS-CoV-2 viral genome copies in raw untreated wastewater correlated strongly with clinically reported cases by the county, and when used alongside past reported case numbers and temporal information in an autoregressive integrated moving average (ARIMA) model enabled prediction of new reported cases up to 3 weeks in advance. Taken together, the results show that the high-throughput surveillance could greatly ameliorate comprehensive community prevalence assessments by providing robust, rapid estimates. ImportanceWastewater monitoring has a lot of potential for revealing COVID-19 outbreaks before they happen because the virus is found in the wastewater before people have clinical symptoms. However, application of wastewater-based surveillance has been limited by long processing times specifically at the concentration step. Here we introduce a much faster method of processing the samples, and show that its robustness by demonstrating direct comparisons with existing methods and showing that we can predict cases in San Diego by a week with excellent accuracy, and three weeks with fair accuracy, using city sewage. The automated viral concentration method will greatly alleviate the major bottleneck in wastewater processing by reducing the turnaround time during epidemics.

A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities (preprint)

One goal among microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols. Accession numbersRaw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub (github.com/justinshaffer/Extraction_test_MagMAX). Methods summaryTo allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.

A Sensitive, Rapid, and Portable CasRx-based Diagnostic Assay for SARS-CoV2 (preprint)

Since its first emergence from China in late 2019, the SARS-CoV-2 virus has spread globally despite unprecedented containment efforts, resulting in a catastrophic worldwide pandemic. Successful identification and isolation of infected individuals can drastically curtail virus spread and limit outbreaks. However, during the early stages of global transmission, point-of-care diagnostics were largely unavailable and continue to remain difficult to procure, greatly inhibiting public health efforts to mitigate spread. Furthermore, the most prevalent testing kits rely on reagent- and time-intensive protocols to detect viral RNA, preventing rapid and cost-effective diagnosis. Therefore the development of an extensive toolkit for point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect COVID-19. Herein, we outline the development of a CRISPR-based nucleic acid molecular diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens (CasRx) to detect SARS-CoV-2, an approach we term SENSR (Sensitive Enzymatic Nucleic-acid Sequence Reporter). We demonstrate SENSR robustly detects SARS-CoV-2 sequences in both synthetic and patient-derived samples by lateral flow and fluorescence, thus expanding the available point-of-care diagnostics to combat current and future pandemics.

Bacterial modification of the host glycosaminoglycan heparan sulfate modulates SARS-CoV-2 infectivity (preprint)

The human microbiota has a close relationship with human disease and it remodels components of the glycocalyx including heparan sulfate (HS). Studies of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) spike protein receptor binding domain suggest that infection requires binding to HS and angiotensin converting enzyme 2 (ACE2) in a codependent manner. Here, we show that commensal host bacterial communities can modify HS and thereby modulate SARS-CoV-2 spike protein binding and that these communities change with host age and sex. Common human-associated commensal bacteria whose genomes encode HS-modifying enzymes were identified. The prevalence of these bacteria and the expression of key microbial glycosidases in bronchoalveolar lavage fluid (BALF) was lower in adult COVID-19 patients than in healthy controls. The presence of HS-modifying bacteria decreased with age in two large survey datasets, FINRISK 2002 and American Gut, revealing one possible mechanism for the observed increase in COVID-19 susceptibility with age. In vitro, bacterial glycosidases from unpurified culture media supernatants fully blocked SARS-CoV-2 spike binding to human H1299 protein lung adenocarcinoma cells. HS-modifying bacteria in human microbial communities may regulate viral adhesion, and loss of these commensals could predispose individuals to infection. Understanding the impact of shifts in microbial community composition and bacterial lyases on SARS-CoV-2 infection may lead to new therapeutics and diagnosis of susceptibility. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/238444v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@14ff1ecorg.highwire.dtl.DTLVardef@193d84corg.highwire.dtl.DTLVardef@15d6f9eorg.highwire.dtl.DTLVardef@14b16c6_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract. Diagram of hypothesis for bacterial mediation of SARS-CoV-2 infection through heparan sulfate (HS).It is well known that host microbes groom the mucosa where they reside. Recent investigations have shown that HS, a major component of mucosal layers, is necessary for SARS-CoV-2 infection. In this study we examine the impact of microbial modification of HS on viral attachment. C_FIG

KG-COVID-19: a framework to produce customized knowledge graphs for COVID-19 response (preprint)

Integrated, up-to-date data about SARS-CoV-2 and coronavirus disease 2019 (COVID-19) is crucial for the ongoing response to the COVID-19 pandemic by the biomedical research community. While rich biological knowledge exists for SARS-CoV-2 and related viruses (SARS-CoV, MERS-CoV), integrating this knowledge is difficult and time consuming, since much of it is in siloed databases or in textual format. Furthermore, the data required by the research community varies drastically for different tasks - the optimal data for a machine learning task, for example, is much different from the data used to populate a browsable user interface for clinicians. To address these challenges, we created KG-COVID-19, a flexible framework that ingests and integrates biomedical data to produce knowledge graphs (KGs) for COVID-19 response. This KG framework can also be applied to other problems in which siloed biomedical data must be quickly integrated for different research applications, including future pandemics. BIGGER PICTUREAn effective response to the COVID-19 pandemic relies on integration of many different types of data available about SARS-CoV-2 and related viruses. KG-COVID-19 is a framework for producing knowledge graphs that can be customized for downstream applications including machine learning tasks, hypothesis-based querying, and browsable user interface to enable researchers to explore COVID-19 data and discover relationships.

A Lymph Node Targeted Amphiphile Vaccine Induces Potent Cellular and Humoral Immunity to SARS-CoV-2 (preprint)

The SARS-CoV-2 pandemic has led to public health, economic, and social consequences that mandate urgent development of effective vaccines to contain or eradicate infection. To that end, we evaluated a novel amphiphile (AMP) vaccine adjuvant, AMP-CpG, composed of diacyl lipid-modified CpG, admixed with the SARS-CoV-2 Spike-2 receptor binding domain protein as a candidate vaccine (ELI-005) in mice. AMP immunogens are efficiently delivered to lymph nodes, where innate and adaptive immune responses are generated. Compared to alum, AMP immunization induced >25-fold higher antigen-specific T cells which produced multiple Th1 cytokines and trafficked into lung parenchyma and respiratory secretions. Antibody responses favored Th1 isotypes (IgG2bc, IgG3) and potently neutralized Spike-2-ACE2 receptor binding, with titers 265-fold higher than the natural immune response from convalescent COVID-19 patients; responses were maintained despite 10-fold dose-reduction in Spike antigen. Both cellular and humoral immune responses were preserved in aged mice. These advantages merit clinical translation to SARS-CoV-2 and other protein subunit vaccines.

SARS-CoV-2 Infection Among Symptom-Free Healthcare Workers (preprint)

Importance: Current evidence suggests that transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possible among symptom-free individuals but limited data are available on this topic in healthcare workers (HCW). The quality and acceptability of self-collected nasopharyngeal swabs (NPS) is unknown. Objective: To estimate the prevalence of SARS-CoV-2 infection and to assess the acceptability of self-collected NPS among HCW. Design: Cross-sectional convenience sample enrolled between April 20th and June 24th, 2020. We had >95% power to detect at least one positive test if the true underlying prevalence of SARS-CoV2 was > 1%. Setting: The metropolitan area surrounding Minneapolis and St. Paul, Minnesota. Participants: HCW free of self-reported upper respiratory symptoms were recruited. Exposures: Participants completed questionnaires regarding demographics, household characteristics, personal protective equipment (PPE) utilization and comorbidities. Outcomes: A participant self-collected nasopharyngeal swab (NPS) was obtained. SARS-CoV-2 infection was assessed via polymerase chain reaction. NPS discomfort was assessed on a scale of 1 (no discomfort) - 10 (extreme discomfort). NPS duration and depth into the nasopharynx, and willingness to perform future self-collections were assessed. Results: Among n=489 participants 80% were female and mean age+/-SD was 41+/-11. Participants reported being physicians (14%), nurse practitioners (8%), physicians assistants (4%), nurses (51%), medics (3%), or other which predominantly included laboratory technicians and administrative roles (22%). Exposure to a known/suspected COVID-19 case in the 14 days prior to enrollment was reported in 40% of participants. SARS-CoV-2 was not detected in any participant. The mean+/-SD discomfort level of the NPS was 4.5+/-2.0. 95% of participants reported that their self-swab was longer than or equal to the duration of patient swabs they had previously performed, and 89% reported the depth to be deeper than or equal to the depth of previous patient swabs. Over 95% of participants reported a willingness to repeat a self-collected NP swab in the future. Conclusions and Relevance: The point prevalence of SARS-CoV-2 infection was likely very low in symptom-free Minnesota healthcare workers from April 20th and June 24th, 2020. Self-collected NP swabs are well-tolerated and a viable alternative to provider-collected swabs to preserve PPE.

Feasibility of SARS-CoV-2 virus detection from consumer-grade cotton swabs (preprint)

BackgroundDetermining the role of fomites in the transmission of SARS-CoV-2 is essential in the hospital setting and will likely be important outside of medical facilities as governments around the world make plans to ease COVID-19 public health restrictions and attempt to safely reopen economies. Expanding COVID-19 testing to include environmental surfaces would ideally be performed with inexpensive swabs that could be transported safely without concern of being a source of new infections. However, CDC-approved clinical-grade sampling supplies and techniques using a polyester swab are expensive, potentially expose laboratory workers to viable virus and prohibit analysis of the microbiome due to the presence of antibiotics in viral transport media (VTM). To this end, we performed a series of experiments comparing the diagnostic yield using five consumer-grade swabs (including plastic and wood shafts and various head materials including cotton, polyester, and foam) and one clinical grade swab for inhibition to RNA. For three of these swabs, we evaluated performance to detect SARS-CoV-2 in twenty intensive care unit (ICU) hospital rooms of patients with 16 COVID-19+. All swabs were placed in 95% ethanol and further evaluated in terms of RNase activity. SARS-CoV-2 was measured both directly from the swab and from the swab eluent. ResultsCompared to samples collected in VTM, 95% ethanol demonstrated significant inhibition properties against RNases. When extracting directly from the swab head as opposed to the eluent, RNA recovery was approximately 2-4x higher from all six swab types tested as compared to the clinical standard of testing the eluent from a CDC-approved polyester swab. The limit of detection (LoD) of SARs-CoV-2 from floor samples collected using the CGp or TMI swabs was similar or better than the CDC standard, further suggesting that swab type does not impact RNA recovery as measured by SARs-CoV-2. The LoD for TMI was between 0-362.5 viral particles while PE and CGp were both between 725-1450 particles. Lastly microbiome analyses (16S rRNA) of paired samples (e.g., environment to host) collected using different swab types in triplicate indicated that microbial communities were not impacted by swab type but instead driven by the patient and sample type (floor or nasal). ConclusionsCompared to using a clinical-grade polyester swab, detection of SARS-CoV-2 from environmental samples collected from ICU rooms of patients with COVID was similar using consumer grade swabs, stored in 95% ethanol. The yield was best from the swab head rather than the eluent and the low level of RNase activity in these samples makes it possible to perform concomitant microbiome analysis.