Interactions among 17 respiratory pathogens: a cross-sectional study using clinical and community surveillance data.

Background: Co-circulating respiratory pathogens can interfere with or promote each other, leading to important effects on disease epidemiology. Estimating the magnitude of pathogen-pathogen interactions from clinical specimens is challenging because sampling from symptomatic individuals can create biased estimates. Methods: We conducted an observational, cross-sectional study using samples collected by the Seattle Flu Study between 11 November 2018 and 20 August 2021. Samples that tested positive via RT-qPCR for at least one of 17 potential respiratory pathogens were included in this study. Semi-quantitative cycle threshold (Ct) values were used to measure pathogen load. Differences in pathogen load between monoinfected and coinfected samples were assessed using linear regression adjusting for age, season, and recruitment channel. Results: 21,686 samples were positive for at least one potential pathogen. Most prevalent were rhinovirus (33·5%), Streptococcus pneumoniae (SPn, 29·0%), SARS-CoV-2 (13.8%) and influenza A/H1N1 (9·6%). 140 potential pathogen pairs were included for analysis, and 56 (40%) pairs yielded significant Ct differences (p < 0.01) between monoinfected and co-infected samples. We observed no virus-virus pairs showing evidence of significant facilitating interactions, and found significant viral load decrease among 37 of 108 (34%) assessed pairs. Samples positive with SPn and a virus were consistently associated with increased SPn load. Conclusions: Viral load data can be used to overcome sampling bias in studies of pathogen-pathogen interactions. When applied to respiratory pathogens, we found evidence of viral-SPn facilitation and several examples of viral-viral interference. Multipathogen surveillance is a cost-efficient data collection approach, with added clinical and epidemiological informational value over single-pathogen testing, but requires careful analysis to mitigate selection bias.

SwabExpress: An End-to-End Protocol for Extraction-Free COVID-19 Testing.

BACKGROUND: The 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). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce. 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. METHODS: We 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 inactivation 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. RESULTS: After optimization, SwabExpress has a low limit of detection at 2-4 molecules/µL, 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. CONCLUSION: SwabExpress 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.

Comparison of Symptoms and RNA Levels in Children and Adults With SARS-CoV-2 Infection in the Community Setting.

Importance: The association between COVID-19 symptoms and SARS-CoV-2 viral levels in children living in the community is not well understood. Objective: To characterize symptoms of pediatric COVID-19 in the community and analyze the association between symptoms and SARS-CoV-2 RNA levels, as approximated by cycle threshold (Ct) values, in children and adults. Design, Setting, and Participants: This cross-sectional study used a respiratory virus surveillance platform in persons of all ages to detect community COVID-19 cases from March 23 to November 9, 2020. A population-based convenience sample of children younger than 18 years and adults in King County, Washington, who enrolled online for home self-collection of upper respiratory samples for SARS-CoV-2 testing were included. Exposures: Detection of SARS-CoV-2 RNA by reverse transcription-polymerase chain reaction (RT-PCR) from participant-collected samples. Main Outcomes and Measures: RT-PCR-confirmed SARS-CoV-2 infection, with Ct values stratified by age and symptoms. Results: Among 555 SARS-CoV-2-positive participants (mean [SD] age, 33.7 [20.1] years; 320 were female [57.7%]), 47 of 123 children (38.2%) were asymptomatic compared with 31 of 432 adults (7.2%). When symptomatic, fewer symptoms were reported in children compared with adults (mean [SD], 1.6 [2.0] vs 4.5 [3.1]). Symptomatic individuals had lower Ct values (which corresponded to higher viral RNA levels) than asymptomatic individuals (adjusted estimate for children, -3.0; 95% CI, -5.5 to -0.6; P = .02; adjusted estimate for adults, -2.9; 95% CI, -5.2 to -0.6; P = .01). The difference in mean Ct values was neither statistically significant between symptomatic children and symptomatic adults (adjusted estimate, -0.7; 95% CI, -2.2 to 0.9; P = .41) nor between asymptomatic children and asymptomatic adults (adjusted estimate, -0.6; 95% CI, -4.0 to 2.8; P = .74). Conclusions and Relevance: In this community-based cross-sectional study, SARS-CoV-2 RNA levels, as determined by Ct values, were significantly higher in symptomatic individuals than in asymptomatic individuals and no significant age-related differences were found. Further research is needed to understand the role of SARS-CoV-2 RNA levels and viral transmission.

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

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has gravely affected societies around the world. Outbreaks in different parts of the globe have been shaped by repeated introductions of new viral lineages and subsequent local transmission of those lineages. Here, we sequenced 3940 SARS-CoV-2 viral genomes from Washington State (USA) to characterize how the spread of SARS-CoV-2 in Washington State in early 2020 was shaped by differences in timing of mitigation strategies across counties and by repeated introductions of viral lineages into the state. In addition, 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 observed evidence of higher viral loads in patients infected with the 614G variant. However, using clinical records data, we did not find any evidence that the 614G variant affects clinical severity or patient outcomes. Overall, this suggests that with regard to D614G, the behavior of individuals has been more important in shaping the course of the pandemic in Washington State than this variant of the virus.

Effects of weather-related social distancing on city-scale transmission of respiratory viruses: a retrospective cohort study.

BACKGROUND: Unusually high snowfall in western Washington State in February 2019 led to widespread school and workplace closures. We assessed the impact of social distancing caused by this extreme weather event on the transmission of respiratory viruses. METHODS: Residual specimens from patients evaluated for acute respiratory illness at hospitals in the Seattle metropolitan area were screened for a panel of respiratory viruses. Transmission models were fit to each virus to estimate the magnitude reduction in transmission due to weather-related disruptions. Changes in contact rates and care-seeking were informed by data on local traffic volumes and hospital visits. RESULTS: Disruption in contact patterns reduced effective contact rates during the intervention period by 16 to 95%, and cumulative disease incidence through the remainder of the season by 3 to 9%. Incidence reductions were greatest for viruses that were peaking when the disruption occurred and least for viruses in an early epidemic phase. CONCLUSION: High-intensity, short-duration social distancing measures may substantially reduce total incidence in a respiratory virus epidemic if implemented near the epidemic peak. For SARS-CoV-2, this suggests that, even when SARS-CoV-2 spread is out of control, implementing short-term disruptions can prevent COVID-19 deaths.

Evaluating Specimen Quality and Results from a Community-Wide, Home-Based Respiratory Surveillance Study.

While influenza and other respiratory pathogens cause significant morbidity and mortality, the community-based burden of these infections remains incompletely understood. The development of novel methods to detect respiratory infections is essential for mitigating epidemics and developing pandemic-preparedness infrastructure. From October 2019 to March 2020, we conducted a home-based cross-sectional study in the greater Seattle, WA, area, utilizing electronic consent and data collection instruments. Participants received nasal swab collection kits via rapid delivery within 24 hours of self-reporting respiratory symptoms. Samples were returned to the laboratory and were screened for 26 respiratory pathogens and a housekeeping gene. Participant data were recorded via online survey at the time of sample collection and 1 week later. Of the 4,572 consented participants, 4,359 (95.3%) received a home swab kit and 3,648 (83.7%) returned a nasal specimen for respiratory pathogen screening. The 3,638 testable samples had a mean RNase P relative cycle threshold (Crt ) value of 19.0 (SD, 3.4), and 1,232 (33.9%) samples had positive results for one or more pathogens, including 645 (17.7%) influenza-positive specimens. Among the testable samples, the median time between shipment of the home swab kit and completion of laboratory testing was 8.0 days (interquartile range [IQR], 7.0 to 14.0). A single adverse event occurred and did not cause long-term effects or require medical attention. Home-based surveillance using online participant enrollment and specimen self-collection is a safe and feasible method for community-level monitoring of influenza and other respiratory pathogens, which can readily be adapted for use during pandemics.

SwabExpress: An end-to-end protocol for extraction-free COVID-19 testing.

BACKGROUND: The 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. METHODS: We 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. RESULTS: After 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. CONCLUSION: SwabExpress 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.

Comparable specimen collection from both ends of at-home mid-turbinate swabs.

Unsupervised upper respiratory specimen collection is a key factor in the ability to massively scale SARS-CoV-2 testing. But there is concern that unsupervised specimen collection may produce inferior samples. Across two studies that included unsupervised at-home mid-turbinate specimen collection, ~1% of participants used the wrong end of the swab. We found that molecular detection of respiratory pathogens and a human biomarker were comparable between specimens collected from the handle of the swab and those collected correctly. Older participants were more likely to use the swab backwards. Our results suggest that errors made during home-collection of nasal specimens do not preclude molecular detection of pathogens and specialized swabs may be an unnecessary luxury during a pandemic.

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.

The Seattle Flu Study: a multiarm community-based prospective study protocol for assessing influenza prevalence, transmission and genomic epidemiology.

INTRODUCTION: Influenza epidemics and pandemics cause significant morbidity and mortality. An effective response to a potential pandemic requires the infrastructure to rapidly detect, characterise, and potentially contain new and emerging influenza strains at both an individual and population level. The objective of this study is to use data gathered simultaneously from community and hospital sites to develop a model of how influenza enters and spreads in a population. METHODS AND ANALYSIS: Starting in the 2018-2019 season, we have been enrolling individuals with acute respiratory illness from community sites throughout the Seattle metropolitan area, including clinics, childcare facilities, Seattle-Tacoma International Airport, workplaces, college campuses and homeless shelters. At these sites, we collect clinical data and mid-nasal swabs from individuals with at least two acute respiratory symptoms. Additionally, we collect residual nasal swabs and data from individuals who seek care for respiratory symptoms at four regional hospitals. Samples are tested using a multiplex molecular assay, and influenza whole genome sequencing is performed for samples with influenza detected. Geospatial mapping and computational modelling platforms are in development to characterise the regional spread of influenza and other respiratory pathogens. ETHICS AND DISSEMINATION: The study was approved by the University of Washington's Institutional Review Board (STUDY00006181). Results will be disseminated through talks at conferences, peer-reviewed publications and on the study website (www.seattleflu.org).

Cryptic transmission of SARS-CoV-2 in Washington state.

After its emergence in Wuhan, China, in late November or early December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus rapidly spread globally. Genome sequencing of SARS-CoV-2 allows the reconstruction of its transmission history, although this is contingent on sampling. We analyzed 453 SARS-CoV-2 genomes collected between 20 February and 15 March 2020 from infected patients in Washington state in the United States. We find that most SARS-CoV-2 infections sampled during this time derive from a single introduction in late January or early February 2020, which subsequently spread locally before active community surveillance was implemented.

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. On March 11, 2020, the World Health Organization declared Coronavirus Disease 2019 (COVID-19) a pandemic. 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, strongly suggesting cryptic spread of COVID-19 during the months of January and February 2020, 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 introductions and spread and the power of pathogen genomics to inform epidemiological understanding.

Evidence for Limited Early Spread of COVID-19 Within the United States, January-February 2020.

From January 21 through February 23, 2020, public health agencies detected 14 U.S. cases of coronavirus disease 2019 (COVID-19), all related to travel from China (1,2). The first nontravel-related U.S. case was confirmed on February 26 in a California resident who had become ill on February 13 (3). Two days later, on February 28, a second nontravel-related case was confirmed in the state of Washington (4,5). Examination of four lines of evidence provides insight into the timing of introduction and early transmission of SARS-CoV-2, the virus that causes COVID-19, into the United States before the detection of these two cases. First, syndromic surveillance based on emergency department records from counties affected early by the pandemic did not show an increase in visits for COVID-19-like illness before February 28. Second, retrospective SARS-CoV-2 testing of approximately 11,000 respiratory specimens from several U.S. locations beginning January 1 identified no positive results before February 20. Third, analysis of viral RNA sequences from early cases suggested that a single lineage of virus imported directly or indirectly from China began circulating in the United States between January 18 and February 9, followed by several SARS-CoV-2 importations from Europe. Finally, the occurrence of three cases, one in a California resident who died on February 6, a second in another resident of the same county who died February 17, and a third in an unidentified passenger or crew member aboard a Pacific cruise ship that left San Francisco on February 11, confirms cryptic circulation of the virus by early February. These data indicate that sustained, community transmission had begun before detection of the first two nontravel-related U.S. cases, likely resulting from the importation of a single lineage of virus from China in late January or early February, followed by several importations from Europe. The widespread emergence of COVID-19 throughout the United States after February highlights the importance of robust public health systems to respond rapidly to emerging infectious threats.

Diagnostic accuracy of an app-guided, self-administered test for influenza among individuals presenting to general practice with influenza-like illness: study protocol.

INTRODUCTION: Diagnostic tests for influenza in Australia are currently only authorised for use in clinical settings. At-home diagnostic testing for influenza could reduce the need for patient contact with healthcare services, which potentially could contribute to symptomatic improvement and reduced spread of influenza. We aim to determine the accuracy of an app-guided nasal self-swab combined with a lateral flow immunoassay for influenza conducted by individuals with influenza-like illness (ILI). METHODS AND ANALYSIS: Adults (≥18 years) presenting with ILI will be recruited by general practitioners (GP) participating in Australian Sentinel Practices Research Network. Eligible participants will have a nasal swab obtained by their GP for verification of influenza A/B status using reverse transcription polymerase chain reaction (RT-PCR) test at an accredited laboratory. Participants will receive an influenza test kit and will download an app that collects self-reported symptoms and influenza risk factors, then instructs them in obtaining a low-nasal self-swab, running a QuickVue influenza A+B lateral flow immunoassay (Quidel Corporation) and interpreting the results. Participants will also interpret an enhanced image of the test strip in the app. The primary outcome will be the accuracy of participants' test interpretation compared with the laboratory RT-PCR reference standard. Secondary analyses will include accuracy of the enhanced test strip image, accuracy of an automatic test strip reader algorithm and validation of prediction rules for influenza based on self-reported symptoms. A post-test survey will be used to obtain participant feedback on self-test procedures. ETHICS AND DISSEMINATION: The study was approved by the Human Research and Ethic Committee (HREC) at the University of Adelaide (H-2019-116). Protocol details and any amendments will be reported to https://www.tga.gov.au/. Results will be published in the peer-reviewed literature, and shared with stakeholders in the primary care and diagnostics communities. TRIAL REGISTRATION NUMBER: Australia New Zealand Clinical Trial Registry (U1111-1237-0688).

Immunosequencing identifies signatures of cytomegalovirus exposure history and HLA-mediated effects on the T cell repertoire.

An individual's T cell repertoire dynamically encodes their pathogen exposure history. To determine whether pathogen exposure signatures can be identified by documenting public T cell receptors (TCRs), we profiled the T cell repertoire of 666 subjects with known cytomegalovirus (CMV) serostatus by immunosequencing. We developed a statistical classification framework that could diagnose CMV status from the resulting catalog of TCRß sequences with high specificity and sensitivity in both the original cohort and a validation cohort of 120 different subjects. We also confirmed that three of the identified CMV-associated TCRß molecules bind CMV in vitro, and, moreover, we used this approach to accurately predict the HLA-A and HLA-B alleles of most subjects in the first cohort. As all memory T cell responses are encoded in the common format of somatic TCR recombination, our approach could potentially be generalized to a wide variety of disease states, as well as other immunological phenotypes, as a highly parallelizable diagnostic strategy.

Annotation of pseudogenic gene segments by massively parallel sequencing of rearranged lymphocyte receptor loci.

BACKGROUND: The adaptive immune system generates a remarkable range of antigen-specific T-cell receptors (TCRs), allowing the recognition of a diverse set of antigens. Most of this diversity is encoded in the complementarity determining region 3 (CDR3) of the ß chain of the αß TCR, which is generated by somatic recombination of noncontiguous variable (V), diversity (D), and joining (J) gene segments. Deletion and non-templated insertion of nucleotides at the D-J and V-DJ junctions further increases diversity. Many of these gene segments are annotated as non-functional owing to defects in their primary sequence, the absence of motifs necessary for rearrangement, or chromosomal locations outside the TCR locus. METHODS: We sought to utilize a novel method, based on high-throughput sequencing of rearranged TCR genes in a large cohort of individuals, to evaluate the use of functional and non-functional alleles. We amplified and sequenced genomic DNA from the peripheral blood of 587 healthy volunteers using a multiplexed polymerase chain reaction assay that targets the variable region of the rearranged TCRß locus, and we determined the presence and the proportion of productive rearrangements for each TCRß V gene segment in each individual. We then used this information to annotate the functional status of TCRß V gene segments in this cohort. RESULTS: For most TCRß V gene segments, our method agrees with previously reported functional annotations. However, we identified novel non-functional alleles for several gene segments, some of which were used exclusively in our cohort to the detriment of reported functional alleles. We also saw that some gene segments reported to have both functional and non-functional alleles consistently behaved in our cohort as either functional or non-functional, suggesting that some reported alleles were not present in the population studied. CONCLUSIONS: In this proof-of-principle study, we used high-throughput sequencing of the TCRß locus of a large cohort of healthy volunteers to evaluate the use of functional and non-functional alleles of individual TCRß V gene segments. With some modifications, our method has the potential to be extended to gene segments in the α, γ, and δ TCR loci, as well as the genes encoding for B-cell receptor chains.

High-throughput pairing of T cell receptor α and ß sequences.

The T cell receptor (TCR) protein is a heterodimer composed of an α chain and a ß chain. TCR genes undergo somatic DNA rearrangements to generate the diversity of T cell binding specificities needed for effective immunity. Recently, high-throughput immunosequencing methods have been developed to profile the TCR α (TCRA) and TCR ß (TCRB) repertoires. However, these methods cannot determine which TCRA and TCRB chains combine to form a specific TCR, which is essential for many functional and therapeutic applications. We describe and validate a method called pairSEQ, which can leverage the diversity of TCR sequences to accurately pair hundreds of thousands of TCRA and TCRB sequences in a single experiment. Our TCR pairing method uses standard laboratory consumables and equipment without the need for single-cell technologies. We show that pairSEQ can be applied to T cells from both blood and solid tissues, such as tumors.