Trends in risk factors and symptoms associated with SARS-CoV-2 and Rhinovirus test positivity in King County, Washington: A Test-Negative Design Study of the Greater Seattle Coronavirus Assessment Network

ImportanceFew US studies have reexamined risk factors for SARS-CoV-2 positivity in the context of widespread vaccination and new variants or considered risk factors for co-circulating endemic viruses, such as rhinovirus. ObjectiveTo understand how risk factors and symptoms associated with SARS-CoV-2 test positivity changed over the course of the pandemic and to compare these to the factors associated with rhinovirus test positivity. DesignThis test-negative design study used multivariable logistic regression to assess associations between SARS-CoV-2 and rhinovirus test positivity and self-reported demographic and symptom variables over a 22-month period. SettingKing County, Washington, June 2020-April 2022 Participants23,278 symptomatic individuals of all ages enrolled in a cross-sectional community surveillance study. ExposuresSelf-reported data for 15 demographic and health behavior variables and 16 symptoms. Main Outcome(s) and Measure(s)RT-PCR confirmed SARS-CoV-2 or rhinovirus infection. ResultsClose contact with a SARS-CoV-2 case (adjusted odds ratio, aOR 4.3, 95% CI 3.7-5.0) and loss of smell/taste (aOR 3.7, 95% CI 3.0-4.5) were the variables most associated with SARS-CoV-2 test positivity, but both attenuated during the Omicron period. Contact with a vaccinated case (aOR 2.4, 95% CI 1.7-3.3) was associated with a lower odds of test positivity than contact with an unvaccinated case (aOR 4.4, 95% CI 2.7-7.3). Sore throat was associated with Omicron infection (aOR 2.3, 95% CI 1.6-3.2) but not Delta. Vaccine effectiveness for participants fully vaccinated with a booster dose was 43% (95% CI 11-63%) for Omicron and 92% (95% CI 61-100%) for Delta. Variables associated with rhinovirus test positivity included age <12 years (aOR 4.0, 95% CI 3.5-4.6) and reporting a runny or stuffy nose (aOR 4.6, 95% CI 4.1-5.2). Race, region, and household crowding were significantly associated with both SARS-CoV-2 and rhinovirus test positivity. Conclusions and RelevanceEstimated risk factors and symptoms associated with SARS-CoV-2 infection have changed over time. There was a shift in reported symptoms between the Delta and Omicron variants as well as reductions in the protection provided by vaccines. Racial and socioeconomic disparities persisted in the third year of SARS-CoV-2 circulation and were also present in rhinovirus infection, although the causal pathways remain unclear. Trends in testing behavior and availability may influence these results. Key Points QuestionWhat are the characteristics associated with SARS-CoV-2 and rhinovirus infection? FindingsIn this test-negative design study of 23,278 participants, reporting close contact with a SARS-CoV-2 case was the strongest risk factor associated with test positivity. Loss of smell and taste was associated with the Delta variant, but not the Omicron variant. Vaccination and prior infection provided greater protection against Delta infection than Omicron Infection. Young age was the strongest predictor of rhinovirus positivity. Sociodemographic disparities were present for both SARS-CoV-2 and rhinovirus. MeaningMonitoring factors associated with respiratory pathogen test positivity remains important to identify at-risk populations in the post-SARS-CoV-2 pandemic period.

Mapping the emergence of SARS-CoV-2 Omicron variants on a university campus

Novel variants continue to emerge in the SARS-CoV-2 pandemic. University testing programs may provide timely epidemiologic and genomic surveillance data to inform public health responses. We conducted testing from September 2021 to February 2022 in a university population under vaccination and indoor mask mandates. A total of 3,048 of 24,393 individuals tested positive for SARS-CoV-2 by RT-PCR; whole genome sequencing identified 209 Delta and 1,730 Omicron genomes of the 1,939 total sequenced. Compared to Delta, Omicron had a shorter median serial interval between genetically identical, symptomatic infections within households (2 versus 6 days, P=0.021). Omicron also demonstrated a greater peak reproductive number (2.4 versus 1.8) and a 1.07 (95% confidence interval: 0.58, 1.57; P<0.0001) higher mean cycle threshold value. Despite near universal vaccination and stringent mitigation measures, Omicron rapidly displaced the Delta variant to become the predominant viral strain and led to a surge in cases in a university population.

Inexpensive workflow for simultaneous monitoring of HIV viral load and detection of SARS-CoV-2 infection

BackgroundCOVID-19 pandemic interrupted routine care for individuals living with HIV, putting them at risk of becoming virologically unsuppressed and ill. Often they are at high risk for exposure to SARS-CoV-2 infection and severe disease once infected. For this population, it is urgent to closely monitor HIV plasma viral load (VL) and screen for SARS-COV-2 infection. MethodWe have developed a non-proprietary method to isolate RNA from plasma, nasal secretions (NS), or both. HIV, SARS-CoV-2, and human RP targets in extracted RNA are then RT-qPCR to estimate the VL and classify HIV/SARS-CoV-2 status (i.e., HIV as VL failure or suppressed; SARS-CoV-2 as positive, presumptive positive, negative, or indeterminate). We evaluated this workflow on 133 clinical specimens: 40 plasma specimens (30 HIV-seropositive), 67 NS specimens (31 SARS-CoV-2-positive), and 26 pooled plasma/NS specimens (26 HIV-positive with 10 SARS-CoV-2-positive), and compared the results obtained using the in-house extraction to those using a commercial extraction kit. ResultsIn-house extraction had a detection limit of 200-copies/mL for HIV and 100-copies/mL for SARS-CoV-2. In-house and commercial methods yielded positively correlated HIV VL (R2: 0.98 for contrived samples; 0.81 for seropositive plasma). SARS-CoV-2 detection had 100% concordant classifications in contrived samples, and in clinical NS extracted by in-house method, excluding indeterminate results, was 95% concordant (25 positives, 6 presumptive positives, and 31 negatives) to those using the commercial method. Analysis of pooled plasma/NS showed R2 of 0.91 (contrived samples) and 0.71 (clinical specimens) for HIV VL correlations obtained by both extraction methods, while SARS-CoV-2 detection showed 100% concordance in contrived and clinical specimens. InterpretationOur low-cost workflow for molecular testing of HIV and SARS-CoV-2 could serve as an alternative to current standard assays for laboratories in low-resource settings.

Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes

The increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multi-target redundancy and an internal amplification control. A convenient and cost-effective target specific fluorescence detection system allows amplifications to be grouped by signal using adaptable probes for pooled reporting of SARS-COV-2 target amplifications or differentiation of the Internal Amplification Control. Over the course of the pandemic, primer coverage of viral lineages by the three redundant sub-assays has varied from assay to assay as they have diverged from the Wuhan-Hu-1 isolate sequence, but aggregate coverage has remained high for all variant sequences analyzed, with a minimum of 97.4% (Variant of Interest: Eta). In three instances (Delta, Gamma, Eta), a high frequency mismatch with one of the three sub-assays was observed, but overall coverage remained high due to multi-target redundancy. When challenged with extracted human samples the multiplexed assay showed 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction, and 100% specificity. These results are further evidence that conventional laboratory methodologies can be leveraged at the point-of-care for robust performance and diagnostic stability over time.

Harmony COVID-19: a ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection

RNA amplification tests allow sensitive detection of SARS-CoV-2 infection, but their complexity and cost are prohibitive for expanding COVID-19 testing. We developed "Harmony COVID-19", a point-of-care test using inexpensive consumables, ready-to-use reagents, and a simple device that processes up to 4 samples simultaneously. Our lyophilized reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) can detect as little as 15 SARS-CoV-2 RNA copies per reaction, and it can report as early as 17 min for samples with high viral load (2 x 105 RNA copies per reaction). Analysis of RNA extracted from clinical nasal specimens (n = 101) showed 95% concordance with RT-PCR, including 100% specificity in specimens positive for other viruses and bacteria. Analysis of contrived samples in the nasal matrix showed detection of 92% or 100% in samples with [≥]20 or [≥]100 particles per reaction, respectively. Usability testing showed 95% accuracy by healthcare workers operating the test for the first time. ONE SENTENCE SUMMARYHarmony COVID-19: point-of-care SARS-CoV-2 RNA detection

SARS-CoV-2 Epidemiology on a Public University Campus in Washington State

BackgroundTesting programs have been utilized as part of SARS-CoV-2 mitigation strategies on university campuses, and it is not known which strategies successfully identify cases and contain outbreaks. ObjectiveEvaluation of a testing program to control SARS-CoV-2 transmission at a large university. DesignProspective longitudinal study using remote contactless enrollment, daily mobile symptom and exposure tracking, and self-swab sample collection. Individuals were tested if the participant was (1) exposed to a known case, developed new symptoms, or reported high-risk behavior, (2) a member of a group experiencing an outbreak, or (3) at baseline upon enrollment. SettingAn urban, public university during Autumn quarter of 2020 ParticipantsStudents, staff, and faculty. MeasurementsSARS-CoV-2 PCR testing was conducted, and viral genome sequencing was performed. ResultsWe enrolled 16,476 individuals, performed 29,783 SARS-CoV-2 tests, and detected 236 infections. Greek community affiliation was the strongest risk factor for testing positive. 75.0% of positive cases reported at least one of the following: symptoms (60.8%), exposure (34.7%), or high-risk behaviors (21.5%). 88.1% of viral genomes (52/59) sequenced from Greek-affiliated students were genetically identical to at least one other genome detected, indicative of rapid SARS-CoV-2 spread within this group, compared to 37.9% (11/29) of genomes from non-Greek students and employees. LimitationsObservational study. ConclusionIn a setting of limited resources during a pandemic, we prioritized testing of individuals with symptoms and high-risk exposure during outbreaks. Rapid spread of SARS- CoV-2 occurred within outbreaks without evidence of further spread to the surrounding community. A testing program focused on high-risk populations may be effective as part of a comprehensive university-wide mitigation strategy to control the SARS-CoV-2 pandemic.

Viral genome sequencing places White House COVID-19 outbreak into phylogenetic context

In October 2020, an outbreak of at least 50 COVID-19 cases was reported surrounding individuals employed at or visiting the White House. Here, we applied genomic epidemiology to investigate the origins of this outbreak. We enrolled two individuals with exposures linked to the White House COVID-19 outbreak into an IRB-approved research study and sequenced their SARS-CoV-2 infections. We find these viral sequences are identical to each other, but are distinct from over 190,000 publicly available SARS-CoV-2 genomes. These genomes fall as part of a lineage circulating in the USA since April or May 2020 and detected in Virginia and Michigan. Looking forwards, sequencing of additional community SARS-CoV-2 infections collected in the USA prior to October 2020 may shed further light on its geographic ancestry. In sequencing of SARS-CoV-2 infections collected after October 2020, it may be possible to identify infections that likely descend from the White House COVID-19 outbreak.

Viral genomes reveal patterns of the SARS-CoV-2 outbreak in Washington State

The rapid spread of SARS-CoV-2 has gravely impacted societies around the world. Outbreaks in different parts of the globe are shaped by repeated introductions of new lineages and subsequent local transmission of those lineages. Here, we sequenced 3940 SARS-CoV-2 viral genomes from Washington State to characterize how the spread of SARS-CoV-2 in Washington State (USA) was shaped by differences in timing of mitigation strategies across counties, as well as by repeated introductions of viral lineages into the state. Additionally, we show that the increase in frequency of a potentially more transmissible viral variant (614G) over time can potentially be explained by regional mobility differences and multiple introductions of 614G, but not the other variant (614D) into the state. At an individual level, we see evidence of higher viral loads in patients infected with the 614G variant. However, using clinical records data, we do not find any evidence that the 614G variant impacts clinical severity or patient outcomes. Overall, this suggests that at least to date, the behavior of individuals has been more important in shaping the course of the pandemic than changes in the virus. One Sentence SummaryLocal outbreak dynamics of SARS-CoV-2 in Washington State (USA) were driven by regionally different mitigation measures and repeated introductions of unique viral variants with different viral loads.

Cryptic transmission of SARS-CoV-2 in Washington State

Following its emergence in Wuhan, China, in late November or early December 2019, the SARS-CoV-2 virus has rapidly spread throughout the world. Genome sequencing of SARS-CoV-2 strains allows for the reconstruction of transmission history connecting these infections. Here, we analyze 346 SARS-CoV-2 genomes from samples collected between 20 February and 15 March 2020 from infected patients in Washington State, USA. We found that the large majority of SARS-CoV-2 infections sampled during this time frame appeared to have derived from a single introduction event into the state in late January or early February 2020 and subsequent local spread, indicating cryptic spread of COVID-19 before active community surveillance was implemented. We estimate a common ancestor of this outbreak clade as occurring between 18 January and 9 February 2020. From genomic data, we estimate an exponential doubling between 2.4 and 5.1 days. These results highlight the need for large-scale community surveillance for SARS-CoV-2 and the power of pathogen genomics to inform epidemiological understanding.

Preliminary support for a 'dry swab, extraction free' protocol for SARS-CoV-2 testing via RT-qPCR

Structured AbstractO_ST_ABSBackgroundC_ST_ABSThe urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse transcription PCR (RT-qPCR) (1). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce (2). To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction. MethodsWe evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral activation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance. ResultsAfter optimization, SwabExpress has a low limit of detection at 2-4 molecules/uL, 100% sensitivity, and 99.4% specificity when compared side-by-side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time. ConclusionSwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.