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COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses1-6. Although pathological innate immune activation is well-documented in severe disease1, the effect of autoantibodies on disease progression is less well-defined. Here we use a high-throughput autoantibody discovery technique known as rapid extracellular antigen profiling7 to screen a cohort of 194 individuals infected with SARS-CoV-2, comprising 172 patients with COVID-19 and 22 healthcare workers with mild disease or asymptomatic infection, for autoantibodies against 2,770 extracellular and secreted proteins (members of the exoproteome). We found that patients with COVID-19 exhibit marked increases in autoantibody reactivities as compared to uninfected individuals, and show a high prevalence of autoantibodies against immunomodulatory proteins (including cytokines, chemokines, complement components and cell-surface proteins). We established that these autoantibodies perturb immune function and impair virological control by inhibiting immunoreceptor signalling and by altering peripheral immune cell composition, and found that mouse surrogates of these autoantibodies increase disease severity in a mouse model of SARS-CoV-2 infection. Our analysis of autoantibodies against tissue-associated antigens revealed associations with specific clinical characteristics. Our findings suggest a pathological role for exoproteome-directed autoantibodies in COVID-19, with diverse effects on immune functionality and associations with clinical outcomes.

Saliva Specimens to Detect SARS-CoV-2 Infection In this letter, the investigators report that saliva specimens and nasopharyngeal swab specimens had similar sensitivity in the detection of SARS-CoV...

Solid organ transplant recipients are at increased risk of COVID-19-associated morbidity and mortality.We describe a nosocomial outbreak investigation on an immunocompromised inpatient unit.Patients positive for SARS-CoV-2 were identified. An epidemiologic investigation was assisted with whole genome sequencing of positive samples.Two patients were identified as potential index cases; one presented with diarrhea and was initially not isolated, and the other developed hypoxemia on hospital day 18 before testing positive. Following identification of a SARS-CoV-2 cluster, the unit was closed and all patients and staff received surveillance testing revealing eight additional positive patients and staff members. Whole genome sequencing confirmed an outbreak. Enhanced infection prevention practices mitigated further spread. Asymptomatic patients with COVID-19 were successfully treated with bamlanivimab.Preventing SARS-CoV-2 outbreaks in transplant units poses unique challenges as patients may have atypical presentations of COVID-19. Immunocompromised patients who test positive for SARS-CoV-2 while asymptomatic may benefit from monoclonal antibody therapy to prevent disease progression. All hospital staff members working with immunocompromised patients should be promptly encouraged to follow infection prevention behaviors and receive SARS-CoV-2 vaccination.SARS-CoV-2 outbreaks on immunocompromised units can be mitigated through prompt identification of cases and robust infection prevention practices.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continues to shape the coronavirus disease 2019 (Covid-19) pandemic. The detection and rapid spread of the SARS-CoV-2 ‘ Omicron’ variant (lineage B.1.1.529) in Botswana and South Africa became a global concern because it contained 15 mutations in the spike protein immunogenic receptor binding domain and was less neutralized by sera derived from vaccinees compared to the previously dominant Delta variant. To investigate if Omicron is more likely than Delta to cause infections in vaccinated persons, we analyzed 37,877 nasal swab PCR tests conducted from 12-26 December 2021 and calculated the test positivity rates for each variant by vaccination status. We found that the positivity rate among unvaccinated persons was higher for Delta (5.2%) than Omicron (4.5%). We found similar results in persons who received a single vaccine dose. Conversely, our results show that Omicron had higher positivity rates than Delta among those who received two doses within five months (Omicron = 4.7% vs. Delta = 2.6%), two doses more than five months ago (4.2% vs. 2.9%), and three vaccine doses (2.2% vs. 0.9%). Our estimates of Omicron positivity rates in persons receiving one or two vaccine doses were not significantly lower than unvaccinated persons but were 49.7% lower after three doses. In comparison, the reduction in Delta positivity rates from unvaccinated to 2 vaccine doses was 45.6-49.6% and to 3 vaccine doses was 83.2%. Despite the higher positivity rates for Omicron in vaccinated persons, we still found that 91.2% of the Omicron infections in our study occurred in persons who were eligible for 1 or more vaccine doses at the time of PCR testing. In conclusion, escape from vaccine-induced immunity likely contributed to the rapid rise in Omicron infections.

Background Infections with respiratory viruses [e.g. influenza and respiratory syncytial virus (RSV)] can increase the risk of severe pneumococcal infections. Likewise, pneumococcal coinfection is associated with poorer outcomes in viral respiratory infection. However, there are limited data describing the frequency of pneumococcus and SARS-CoV-2 coinfection and the role of coinfection in influencing COVID-19 severity. We, therefore, investigated the detection of pneumococcus in COVID-19 inpatients during the early pandemic period. Methods The study included patients aged 18 years and older, admitted to the Yale-New Haven Hospital who were symptomatic for respiratory infection and tested positive for SARS-CoV-2 during March–August 2020. Patients were tested for pneumococcus through culture-enrichment of saliva followed by RT-qPCR (to identify carriage) and serotype-specific urine antigen detection (UAD) assays (to identify presumed lower respiratory tract pneumococcal disease). Results Among 148 subjects, the median age was 65 years; 54.7% were male; 50.7% had an ICU stay; 64.9% received antibiotics; and 14.9% died while admitted. Pneumococcal carriage was detected in 3/96 (3.1%) individuals tested by saliva RT-qPCR. Additionally, pneumococcus was detected in 14/127 (11.0%) individuals tested by UAD, and more commonly in severe than moderate COVID-19 [OR: 2.20; 95% CI: (0.72, 7.48)]; however, the numbers were small with a high degree of uncertainty. None of the UAD-positive individuals died. Conclusions Pneumococcal lower respiratory tract infection (LRTI), as detected by positive UAD, occurred in patients hospitalized with COVID-19. Moreover, pneumococcal LRTI was more common in those with more serious COVID-19 outcomes. Future studies should assess how pneumococcus and SARS-CoV-2 interact to influence COVID-19 severity in hospitalized patients.

Early in the pandemic, a simple, open-source, RNA extraction-free RT-qPCR protocol for SARS-CoV-2 detection in saliva was developed and made widely available. This simplified approach (SalivaDirect) requires only sample treatment with proteinase K prior to PCR testing. However, feedback from clinical laboratories highlighted a need for a flexible workflow that can be seamlessly integrated into their current health and safety requirements for the receiving and handling of potentially infectious samples. To address these varying needs, we explored additional pre-PCR workflows. We built upon the original SalivaDirect workflow to include an initial incubation step (95°C for 30 minutes, 95°C for 5 minutes or 65°C for 15 minutes) with or without addition of proteinase K. The limit of detection for the workflows tested did not significantly differ from that of the original SalivaDirect workflow. When tested on de-identified saliva samples from confirmed COVID-19 individuals, these workflows also produced comparable virus detection and assay sensitivities, as determined by RT-qPCR analysis. Exclusion of proteinase K did not negatively affect the sensitivity of the assay. The addition of multiple heat pretreatment options to the SalivaDirect protocol increases the accessibility of this cost-effective SARS-CoV-2 test as it gives diagnostic laboratories the flexibility to implement the workflow which best suits their safety protocols.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant quickly rose to dominance in mid-2021, displacing other variants, including Alpha. Studies using data from the United Kingdom and India estimated that Delta was 40-80% more transmissible than Alpha, allowing Delta to become the globally dominant variant. However, it was unclear if the ostensible difference in relative transmissibility was due mostly to innate properties of Delta’s infectiousness or differences in the study populations. To investigate, we formed a partnership with SARS-CoV-2 genomic surveillance programs from all six New England US states. By comparing logistic growth rates, we found that Delta emerged 37-163% faster than Alpha in early 2021 (37% Massachusetts, 75% New Hampshire, 95% Maine, 98% Rhode Island, 151% Connecticut, and 163% Vermont). We next computed variant-specific effective reproductive numbers and estimated that Delta was 58-120% more transmissible than Alpha across New England (58% New Hampshire, 68% Massachusetts, 76% Connecticut, 85% Rhode Island, 98% Maine, and 120% Vermont). Finally, using RT-PCR data, we estimated that Delta infections generate on average ∼6 times more viral RNA copies per mL than Alpha infections. Overall, our evidence indicates that Delta’s enhanced transmissibility could be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on the underlying immunity and behavior of distinct populations.

Effectively monitoring the spread of SARS-CoV-2 variants is essential to efforts to counter the ongoing pandemic. Wastewater monitoring of SARS-CoV-2 RNA has proven an effective and efficient technique to approximate COVID-19 case rates in the population. Predicting variant abundances from wastewater, however, is technically challenging. Here we show that by sequencing SARS-CoV-2 RNA in wastewater and applying computational techniques initially used for RNA-Seq quantification, we can estimate the abundance of variants in wastewater samples. We show by sequencing samples from wastewater and clinical isolates in Connecticut U.S.A. between January and April 2021 that the temporal dynamics of variant strains broadly correspond. We further show that this technique can be used with other wastewater sequencing techniques by expanding to samples taken across the United States in a similar timeframe. We find high variability in signal among individual samples, and limited ability to detect the presence of variants with clinical frequencies

Emerging SARS-CoV-2 variants have shaped the second year of the COVID-19 pandemic and the public health discourse around effective control measures. Evaluating the public health threat posed by a new variant is essential for appropriately adapting response efforts when community transmission is detected. However, this assessment requires that a true comparison can be made between the new variant and its predecessors because factors other than the virus genotype may influence spread and transmission. In this study, we develop a framework that integrates genomic surveillance data to estimate the relative effective reproduction number (Rt) of co-circulating lineages. We use Connecticut, a state in the northeastern United States in which the SARS-CoV-2 variants B.1.1.7 and B.1.526 co-circulated in early 2021, as a case study for implementing this framework. We find that the Rt of B.1.1.7 was 6-10% larger than that of B.1.526 in Connecticut in the midst of a COVID-19 vaccination campaign. To assess the generalizability of this framework, we apply it to genomic surveillance data from New York City and observe the same trend. Finally, we use discrete phylogeography to demonstrate that while both variants were introduced into Connecticut at comparable frequencies, clades that resulted from introductions of B.1.1.7 were larger than those resulting from B.1.526 introductions. Our framework, which uses open-source methods requiring minimal computational resources, may be used to monitor near real-time variant dynamics in a myriad of settings.

With the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants that may increase transmissibility and/or cause escape from immune responses, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant, first detected in the United Kingdom, could be serendipitously detected by the Thermo Fisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69–70, would cause a “spike gene target failure” (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern (VOC) that lack spike Δ69–70, such as B.1.351 (also 501Y.V2), detected in South Africa, and P.1 (also 501Y.V3), recently detected in Brazil. We identified a deletion in the ORF1a gene (ORF1a Δ3675–3677) in all 3 variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675–3677 as the primary target and spike Δ69–70 to differentiate, we designed and validated an open-source PCR assay to detect SARS-CoV-2 VOC. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, and P.1., Surveillance for SARS-CoV-2 variants is very important, but sequencing is not always practical or affordable. This study presents a multiplex qPCR that is able to distinguish among different SARS-CoV-2 variants of concern that are currently circulating.

Most currently approved strategies for the collection of saliva for COVID-19 diagnostics require specialized tubes containing buffers promoted for the stabilization of SARS-CoV-2 RNA and virus inactivation. Yet many of these are expensive, in limited supply, and not necessarily validated specifically for viral RNA. While saliva is a promising sample type as it can be reliably self-collected for the sensitive detection of SARS-CoV-2, the expense and availability of these collection tubes are prohibitive to mass testing efforts. Therefore, we investigated the stability of SARS-CoV-2 RNA and infectious virus detection from saliva without supplementation. We tested RNA stability over extended periods of time (2-25 days) and at temperatures representing at-home storage and elevated temperatures which might be experienced when cold chain transport may be unavailable. We found SARS-CoV-2 RNA in saliva from infected individuals is stable at 4°C, room temperature (~19°C), and 30°C for prolonged periods and found limited evidence for viral replication in stored saliva samples. This work demonstrates that expensive saliva collection options involving RNA stabilization and virus inactivation buffers are not always needed, permitting the use of cheaper collection options. Affordable testing methods are urgently needed to meet current testing demands and for continued surveillance in reopening strategies.

SalivaDirect™ is an RNA-extraction free, dual-plexed RT-qPCR method for SARS-CoV-2 detection. It can be broadly implemented as it (1) does not require saliva collection tubes containing preservatives, (2) does not require specialized equipment for RNA extraction, and (3) is validated for use with products from multiple vendors. Thus, the simplicity and flexibility of SalivaDirect™ means that it is not as affected by supply chain bottlenecks as some other assays. Our method is RNA-extraction free which enables testing of low volume and minimally processed saliva in dual-plexed RT-qPCR for SARS-CoV-2 detection. Saliva will be treated with proteinase K followed by a heat inactivation step, and is then directly used as input in the dual-plexed RT-qPCR test. Our aim was not to design new primers and probes for RT-qPCR testing, but rather to use validated primer and probe sets (N1 and RP) developed by the US CDC. The human Ribonuclease P (RP) probe was modified with a different fluorophore so that the primer/probe set could be combined in a dualplex assay, reducing the number of tests to 1 assay with 2 sets. Version 2 includes: Optimized thermocycler conditions Locally validated alternative options for Proteinase K, RT-qPCR master mix, and thermocyclers Use of 8-strip tubes for sample processing step, due to contamination issues in 96-well plates. Version 3 has been updated to remove steps for sample self-collection. Version 4 has updated Ct thresholds for the ABI 7500 Fast Dx. Version 5 has an updated description for use, additional RP probe with ATTO647 fluorophore, and a detailed table with catalog numbers. Version 6 includes: Introducing multiple workflows for sample processing including heat Pre-treatment as an alternative to Proteinase K Updated list of RT-qPCR reagents, theromcyclers and protocol for 384 well format.

SARS-CoV-2 infection has so far affected over 42 million people worldwide, causing over 1.1 million deaths. With the large majority of SARS-CoV-2 infected individuals being asymptomatic, major concerns have been raised about possible long-term consequences of the infection. We developed an antigen capture assay to detect SARS-CoV-2 spike protein in urine samples from COVID-19 patients whose diagnosis was confirmed by PCR from nasopharyngeal swabs (NP-PCR+). The study used a collection of 233 urine samples from 132 participants from Yale New Haven Hospital and the Children’s Hospital of Philadelphia obtained during the pandemic (106 NP-PCR+ and 26 NP-PCR-) as well as a collection of 20 urine samples from 20 individuals collected before the pandemic. Our analysis identified 23 out of 91 (25%) NP-PCR+ adult participants with SARS-CoV-2 spike S1 protein in urine (Ur-S+). Interestingly, although all NP-PCR+ children were Ur-S-, 1 NP-PCR-child was found to be positive for spike protein in urine. Of the 23 Ur-S+ adults, only 1 individual showed detectable viral RNA in urine. Our analysis further showed that 24% and 21% of NP-PCR+ adults have high levels of albumin and cystatin C in urine, respectively. Among individuals with albuminuria (>0.3 mg/mg of creatinine) statistical correlation could be found between albumin and spike protein in urine. Together, our data showe that 1 of 4 of SARS-CoV-2 infected individuals develop renal abnormalities such as albuminuria. Awareness about the long-term impact of these findings is warranted.

While several clinical and immunological parameters correlate with disease severity and mortality in SARS-CoV-2 infection, work remains in identifying unifying correlates of coronavirus disease 2019 (COVID-19) that can be used to guide clinical practice. Here, we examine saliva and nasopharyngeal (NP) viral load over time and correlate them with patient demographics, and cellular and immune profiling. We found that saliva viral load was significantly higher in those with COVID-19 risk factors; that it correlated with increasing levels of disease severity and showed a superior ability over nasopharyngeal viral load as a predictor of mortality over time (AUC=0.90). A comprehensive analysis of immune factors and cell subsets revealed strong predictors of high and low saliva viral load, which were associated with increased disease severity or better overall outcomes, respectively. Saliva viral load was positively associated with many known COVID-19 inflammatory markers such as IL-6, IL-18, IL-10, and CXCL10, as well as type 1 immune response cytokines. Higher saliva viral loads strongly correlated with the progressive depletion of platelets, lymphocytes, and effector T cell subsets including circulating follicular CD4 T cells (cTfh). Anti-spike (S) and anti-receptor binding domain (RBD) IgG levels were negatively correlated with saliva viral load showing a strong temporal association that could help distinguish severity and mortality in COVID-19. Finally, patients with fatal COVID-19 exhibited higher viral loads, which correlated with the depletion of cTfh cells, and lower production of anti-RBD and anti-S IgG levels. Together these results demonstrated that viral load – as measured by saliva but not nasopharyngeal — is a dynamic unifying correlate of disease presentation, severity, and mortality over time.

With the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants that may increase transmissibility and/or cause escape from immune responses, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant, first detected in the United Kingdom, could be serendipitously detected by the Thermo Fisher TaqPath Coronavirus Disease 2019 (COVID-19) PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern (VOC) that lack spike Δ69-70, such as B.1.351 (also 501Y.V2), detected in South Africa, and P.1 (also 501Y.V3), recently detected in Brazil. We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all 3 variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open-source PCR assay to detect SARS-CoV-2 VOC. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, and P.1.

COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses1–8. While pathological innate immune activation is well documented in severe disease1, the impact of autoantibodies on disease progression is less defined. Here, we used a high-throughput autoantibody discovery technique called Rapid Extracellular Antigen Profiling (REAP) to screen a cohort of 194 SARS-CoV-2 infected COVID-19 patients and healthcare workers for autoantibodies against 2,770 extracellular and secreted proteins (the “exoproteome”). We found that COVID-19 patients exhibit dramatic increases in autoantibody reactivities compared to uninfected controls, with a high prevalence of autoantibodies against immunomodulatory proteins including cytokines, chemokines, complement components, and cell surface proteins. We established that these autoantibodies perturb immune function and impair virological control by inhibiting immunoreceptor signaling and by altering peripheral immune cell composition, and found that murine surrogates of these autoantibodies exacerbate disease severity in a mouse model of SARS-CoV-2 infection. Analysis of autoantibodies against tissue-associated antigens revealed associations with specific clinical characteristics and disease severity. In summary, these findings implicate a pathological role for exoproteome-directed autoantibodies in COVID-19 with diverse impacts on immune functionality and associations with clinical outcomes.

COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses

Background SARS-CoV-2 infections are characterized by viral proliferation and clearance phases and can be followed by low-level viral RNA shedding. The dynamics of viral RNA concentration, particularly in the early stages of infection, can inform clinical measures and interventions such as test-based screening. Methods We used prospective longitudinal RT-qPCR testing to measure the viral RNA trajectories for 68 individuals during the resumption of the 2019-20 National Basketball Association season. For 46 individuals with acute infections, we inferred the peak viral concentration and the duration of the viral proliferation and clearance phases. Findings On average, viral RNA concentrations peaked 2.7 days (95% credible interval [1.2, 3.8]) after first detection at a cycle threshold value of 22.4 [20.6, 24.1]. The viral clearance phase lasted longer for symptomatic individuals (10.5 days [6.5, 14.0]) than for asymptomatic individuals (6.7 days [3.2, 9.2]). A second test within 2 days after an initial positive PCR substantially improves certainty about a patient’s infection phase. The effective sensitivity of a test intended to identify infectious individuals declines substantially with test turnaround time. Interpretation SARS-CoV-2 viral concentrations peak rapidly regardless of symptoms. Sequential tests can help reveal a patient’s progress through infection stages. Frequent rapid-turnaround testing is needed to effectively screen individuals before they become infectious. Funding NWO Rubicon 019.181EN.004 (CBFV); clinical research agreement with the NBA and NBPA (NDG); Huffman Family Donor Advised Fund (NDG); Fast Grant funding from the Emergent Ventures at the Mercatus Center; George Mason University (NDG); the Morris-Singer Fund for the Center for Communicable Disease Dynamics at the Harvard T.H. Chan School of Public Health (YHG). Research in Context Evidence before this study SARS-CoV-2 viral dynamics affect clinical and public health measures, informing patient care, testing algorithms, contact tracing protocols, and clinical trial design. We searched Web of Science using the search terms “ALL = ((SARS-CoV-2 OR COVID-19) AND (viral OR RNA) AND (load OR concentration OR shedding) AND (dynamic* OR kinetic* OR trajector*))” which returned 83 references. Of these, 22 were not pertinent to within-host SARS-CoV-2 viral dynamics. The remaining 61 studies tracked SARS-CoV-2 viral trajectories in a variety of geographic locations and patient populations. Together, these studies report that viral titers normally peak at or before the onset of symptoms and that a long tail of intermittent positive tests can follow a period of acute infection. Plasma but not nasopharyngeal viral concentration is associated with increased disease severity. Most studies tracked hospitalized patients after the onset of symptoms. Two of the studies tracked pre-symptomatic and/or asymptomatic patients, but these were too sparsely sampled to clearly discern viral dynamics during the earliest stage of infection. Added value of this study We implemented prospective longitudinal real time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) testing for SARS-CoV-2 in a cohort of individuals during the resumption of the 2019-20 National Basketball Association season. This allowed us to explicitly measure viral titers during the full course of 46 acute infections. Consistent with other studies, we find that peak viral concentrations do not differ substantially between symptomatic and asymptomatic individuals but that symptomatic individuals take longer to clear the virus than asymptomatic individuals. For both symptomatic and asymptomatic individuals, viral titers normally peak within 3 days of the first positive test. This study is the first to describe the time course of viral concentrations during the earliest stage of infection when individuals are most likely to be infectious. Implications of all the available evidence Symptomatic and asymptomatic individuals follow similar SARS-CoV-2 viral trajectories. Due to the rapid progression from first possible detection to peak viral concentration, frequent rapid-turnaround testing is needed to screen individuals prior to them becoming infectious.

SalivaDirect™ is an RNA-extraction free, dual-plexed RT-qPCR method for SARS-CoV-2 detection. It can be broadly implemented as it (1) does not require saliva collection tubes containing preservatives, (2) does not require specialized equipment for RNA extraction, and (3) is validated for use with products from multiple vendors. Thus, the simplicity and flexibility of SalivaDirect™ means that it is not as affected by supply chain bottlenecks as some other assays. Our method is RNA-extraction free which enables testing of low volume and minimally processed saliva in dual-plexed RT-qPCR for SARS-CoV-2 detection. Saliva will be treated with proteinase K followed by a heat inactivation step, and is then directly used as input in the dual-plexed RT-qPCR test. Our aim was not to design new primers and probes for RT-qPCR testing, but rather to use validated primer and probe sets (N1 and RP) developed by the US CDC. The human Ribonuclease P (RP) probe was modified with a different fluorophore so that the primer/probe set could be combined in a dualplex assay, reducing the number of tests to 1 assay with 2 sets. Version 2 includes: Optimized thermocycler conditions Locally validated alternative options for Proteinase K, RT-qPCR master mix, and thermocyclers Use of 8-strip tubes for sample processing step, due to contamination issues in 96-well plates. Version 3 has been updated to remove steps for sample self-collection. Version 4 has updated Ct thresholds for the ABI 7500 Fast Dx. Version 5 has an updated description for use, additional RP probe with ATTO647 fluorophore, and a detailed table with catalog numbers.

Most currently approved strategies for the collection of saliva for COVID-19 diagnostics require specialized tubes containing buffers promoted for the stabilization of SARS-CoV-2 RNA and virus inactivation. Yet many of these are expensive, in limited supply, and not necessarily validated specifically for viral RNA. While saliva is a promising sample type as it can be reliably self-collected for the sensitive detection of SARS-CoV-2, the expense and availability of these collection tubes are prohibitive to mass testing efforts. Therefore, we investigated the stability of SARS-CoV-2 RNA and infectious virus detection from saliva without supplementation. We tested RNA stability over extended periods of time (2-25 days) and at temperatures representing at-home storage and elevated temperatures which might be experienced when cold chain transport may be unavailable. We found SARS-CoV-2 RNA in saliva from infected individuals is stable at 4°C, room temperature (∼19°C), and 30°C for prolonged periods and found limited evidence for viral replication in stored saliva samples. This work demonstrates that expensive saliva collection options involving RNA stabilization and virus inactivation buffers are not always needed, permitting the use of cheaper collection options. Affordable testing methods are urgently needed to meet current testing demands and for continued surveillance in reopening strategies.

The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse transcription–PCR (RT–qPCR) assays are being used by clinical, research and public health laboratories. However, it is currently unclear whether results from different tests are comparable. Our goal was to make independent evaluations of primer–probe sets used in four common SARS-CoV-2 diagnostic assays. From our comparisons of RT–qPCR analytical efficiency and sensitivity, we show that all primer–probe sets can be used to detect SARS-CoV-2 at 500 viral RNA copies per reaction. The exception for this is the RdRp-SARSr (Charite) confirmatory primer–probe set which has low sensitivity, probably due to a mismatch to circulating SARS-CoV-2 in the reverse primer. We did not find evidence for background amplification with pre-COVID-19 samples or recent SARS-CoV-2 evolution decreasing sensitivity. Our recommendation for SARS-CoV-2 diagnostic testing is to select an assay with high sensitivity and that is regionally used, to ease comparability between outcomes. This is a comparative analysis of the performance of the primer–probe sets from four open-source molecular diagnostic assays for SARS-CoV-2 recommended by the World Health Organization.

This protocol details recommendations for collecting and processing saliva for SARS-CoV-2 detection, as used in Wyllie et al. 2020, which details results of testing saliva collected from COVID-19+ inpatients and asymptomatic healthcare workers. COVID-19 inpatients. Saliva samples were self-collected by the patient. Upon waking, patients were asked to avoid food, water and brushing of teeth until the sample was collected. Patients were asked to repeatedly spit into a sterile 90 mL specimen collection cup until roughly a quarter full of liquid (excluding bubbles), before securely closing it. For patients unable to provide saliva (such as those on mechanical ventilation), clinical teams were advised that suction could be used to collect saliva into the cup (or a sputum trap container for improved containment and safety). Collected volumes ranged from 0.5 - 20 mL. All saliva samples were stored at room temperature and transported to the research lab at the Yale School of Public Health within 5 hours of collection, with RNA for SARS-CoV-2 detection extracted within 12 hours of collection. When possible, saliva samples were stored at +4°C, otherwise they were kept at room temperature. Asymptomatic healthcare workers. Asymptomatic healthcare workers were asked to collect ~10 mL of saliva into a sterile specimen collection cup. No specific instructions were given regarding food intake etc prior to collection. Samples were delivered to the research lab at the Yale School of Public Health within 6 hours of collection and stored for up to 6 hours at +4°C until aliquoting for RNA extraction. Samples collected on overnight shifts were stored at +4°C before delivery to the research lab.

The current quantitative reverse transcription PCR (RT-qPCR) assay recommended for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the United States requires analysis of 3 genomic targets per sample: 2 viral and 1 host. To simplify testing and reduce the volume of required reagents, we devised a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the Centers for Disease Control and Prevention, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the single assay adapted for research purposes. Low copy numbers (≥500 copies/reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current single diagnostics by saving reagents, costs, time, and labor., The current RT-qPCR assay recommended for SARS-CoV-2 testing in the United States requires analysis of three genomic targets per sample: two viral and one host. This study describes a multiplex PCR that saves both reagents and labor while accurately detecting SARS-CoV-2 RNA.

A growing body of evidence indicates sex differences in the clinical outcomes of coronavirus disease 2019 (COVID-19)1-4. However, whether immune responses against SARS-CoV-2 differ between sexes, and whether such differences explain male susceptibility to COVID-19, is currently unknown. In this study, we examined sex differences in viral loads, SARS-CoV-2-specific antibody titers, plasma cytokines, as well as blood cell phenotyping in COVID-19 patients. By focusing our analysis on patients with mild to moderate disease who had not received immunomodulatory medications, our results revealed that male patients had higher plasma levels of innate immune cytokines and chemokines including IL-8, IL-18, and CCL5, along with more robust induction of non-classical monocytes. In contrast, female patients mounted significantly more robust T cell activation than male patients during SARS-CoV-2 infection, which was sustained in old age. Importantly, we found that a poor T cell response negatively correlated with patients’ age and was predictive of worse disease outcome in male patients, but not in female patients. Conversely, higher innate immune cytokines in female patients associated with worse disease progression, but not in male patients. These findings reveal a possible explanation underlying observed sex biases in COVID-19, and provide important basis for the development of sex-based approach to the treatment and care of men and women with COVID-19.

The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assays are being used by clinical, research, and public health laboratories. However, it is currently unclear if results from different tests are comparable. Our goal was to evaluate the primer-probe sets used in four common diagnostic assays available on the World Health Organization (WHO) website. To facilitate this effort, we generated RNA transcripts to be used as assay standards and distributed them to other laboratories for internal validation. We then used these (1) RNA transcript standards, (2) full-length SARS-CoV-2 RNA, and (3) pre-COVID-19 nasopharyngeal swabs, and (4) clinical samples from COVID-19 patients to determine analytical efficiency and sensitivity of the qRT-PCR primer-probe sets. We show that all primer-probe sets can be used to detect SARS-CoV-2, but there are clear differences in the ability to differentiate between true negatives and positives with low amounts of virus. We found that several primer-probe sets cross-react with SARS-CoV-2-negative nasopharyngeal swabs. However, background cross-reactivity by the 2019-nCoV_N2 set issued by the US Centers for Disease Control and Prevention did not interfere with outcomes of the combined 9N19 and 9N29 assay when testing COVID-19 clinical samples. Our findings characterize the limitations of currently used primer-probe sets and can assist other laboratories in selecting appropriate assays for the detection of SARS-CoV-2.

Summary The novel coronavirus SARS-CoV-2 was first detected in the Pacific Northwest region of the United States in January 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the United States, we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated effects of federal travel restrictions. This study provides evidence of widespread sustained transmission of SARS-CoV-2 within the United States and highlights the critical need for local surveillance., Graphical Abstract, Highlights • Connecticut’s COVID-19 outbreak resulted from multiple domestic virus introductions • SARS-CoV-2 genomic data indicate that coast-to-coast spread occurred in the United States • Risk of introduction by domestic air travel exceeded international travel in March • Restrictions on international travel did not significantly alter risk estimates, Using genomics and air travel information, the spread of SARS-CoV-2 in the United States from coast to coast is shown to be more a consequence of domestic introductions than of international travel.

The protocol describes how to generate high-quality single-stranded RNA transcript standards (starting from a virus RNA stock) targeting the nsp10, RdRp, nsp14, envelope (E), and nucleocapsid (N) coding regions for use with China CDC, Hong Kong University (HKU), Corman et al. (Berlin), and US CDC SARS-CoV-2 primer and probe sets for qRT-PCR. (Sequences for transcripts generated, along with their corresponding assays, are provided under 'Guidelines.')

Cancer patients display immunomodulation related to malignancy and anti-cancer therapies, but how these factors impact COVID-19 remains unknown. To investigate immune responses in cancer patients with COVID-19, we undertook a prospective case-control study, enrolling hospitalized solid tumor patients with acute COVID-19, as well as age-, gender-, and comorbidity-matched COVID-19 patients without cancer as controls. Using biospecimens collected during hospitalization, we performed virologic measurements as well as in-depth immunophenotyping of cellular, antibody and cytokine responses. We enrolled 17 cancer patients (cases) admitted to Yale-New Haven Hospital between March 15 and June 30, 2020 with COVID-19, as well as 17 matched non-cancer patients (controls) admitted with COVID-19. No significant differences were observed between cases and controls based on patient characteristics (age, gender, race, co-morbidities, smoking history, days from symptom onset to COVID-19 diagnosis) or outcomes (COVID-19 severity, length of hospital stay, rate of intubation or mortality). The most common primary tumor sites were lung (4/17) and gastrointestinal (4/17); all cases had received cancer-directed therapy within 6 months of COVID-19 diagnosis, with 13/17 receiving treatment less than 1 month prior to hospitalization. Three of 17 cases had received immune checkpoint inhibitor therapies. Despite having similar SARS-CoV-2 viral RNA loads at the time of COVID-19 diagnosis when compared with controls, cancer cases had increased viral RNA abundance during hospitalization, suggesting slower clearance. Antibody responses against SARS-CoV-2 were preserved in cancer cases, with cases displaying similar levels of IgM and IgG antibodies directed against SARS-CoV-2 epitopes compared to controls. Cytokine profiling revealed higher plasma levels of CCL3, IL1A and CXCL12 in cancer cases compared to controls. Using flow cytometric immunophenotyping, we found that innate immune and non-T cell adaptive immune parameters were similar between cases and controls hospitalized with COVID-19. However, among cancer cases on conventional therapies, T cell lymphopenia was more profound, and these cases demonstrated higher levels of CD8+ exhausted (CD8+CD45RA−PD1+TIM3+), CD8+GranzymeB+ and CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ activated T cells when compared with controls; interestingly, these differences were not observed in patients who had received immune checkpoint inhibition. Thus, we found reduced viral RNA clearance and specific alterations in T cell and cytokine responses in cancer patients hospitalized with COVID-19 compared with matched controls with COVID-19. This dysregulated T cell response in cancer patients, which may reflect immune modulation due to chronic antigen stimulation as well as cancer therapies, may lead to altered virologic and clinical outcomes in this population. Citation Format: Michael Chiorazzi, Erin Silva, Kristina Brower, Patrick Wong, Carolina Lucas, Jon Klein, Feimei Liu, Maura Nakahata, Jun Zhao, Nur-Taz Rahman, Camila Odio, Santos Bermejo, Shelli F. Farhadian, Charles Dela Cruz, Arnau Casanovas-Massana, John Fournier, Catherine Muenker, Anne L. Wyllie, Chantal B.F. Vogels, Chaney C. Kalinich, Mary E. Petrone, Isabel M. Ott, Anne E. Watkins, Adam J. Moore, Tara Alpert, Yuval Kluger, Aaron Ring, Nathan D. Grubaugh, Akiko Iwasaki, Albert I. Ko, Roy S. Herbst. Cancer patients display diminished viral RNA clearance and altered T cell responses during SARS-CoV-2 infection [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr S03-03.

Background Scaling SARS-CoV-2 testing to meet demands of safe reopenings continues to be plagued by assay costs and supply chain shortages. In response, we developed SalivaDirect, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA). Methods We simplified our saliva-based diagnostic test by (1) not requiring collection tubes with preservatives, (2) replacing nucleic acid extraction with a simple enzymatic and heating step, and (3) testing specimens with a dualplex qRT-PCR assay. Moreover, we validated SalivaDirect with reagents and instruments from multiple vendors to minimize supply chain issues. Findings From our hospital cohort, we show a high positive agreement (94%) between saliva tested with SalivaDirect and nasopharyngeal swabs tested with a commercial qRT-PCR kit. In partnership with the National Basketball Association (NBA) and National Basketball Players Association (NBPA), we tested 3,779 saliva specimens from healthy individuals and detected low rates of invalid (0.3%) and false-positive (, Context and Significance Frequent testing is critical to limit SARS-CoV-2 transmission. In response to this need, we developed SalivaDirect, a sensitive, simplified, and flexible testing framework, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA). We tested saliva collected from a hospital cohort and showed a high positive agreement (94%) as compared to paired nasopharyngeal swabs tested with a commercial diagnostic kit. Then, we partnered with the National Basketball Association (NBA) to test a large cohort of mostly healthy individuals, and we detected low rates of invalid (0.3%) and false-positive (0.03%–0.05%) results. Our study shows that SalivaDirect can help to increase testing capacity by providing access to an affordable framework that is less prone to supply chain shortages., Graphical Abstract, Highlights SalivaDirect is a simplified saliva-based test for detection of SARS-CoV-2 The testing framework is flexible to minimize the risk of supply chain issues SalivaDirect is sensitive, with low rates of invalid and false-positive results Laboratories can be designated to use SalivaDirect to increase testing capacity, SalivaDirect is a sensitive saliva-based COVID-19 diagnostic test, which received Emergency Use Authorization from the U.S. FDA. With the ability to designate other laboratories, the flexible and simplified framework helps to increase the capacity of existing laboratory infrastructure for SARS-CoV-2 testing.

Genomic epidemiology can provide a unique, real-time understanding of SARS-CoV-2 transmission patterns. Yet the potential for genomic analyses to guide local policy and community-based behavioral decisions is limited because they are often oriented towards specially trained scientists and conducted on a national or global scale. Here, we propose a new paradigm: Phylogenetic analyses performed on a local level (municipal, county, or state), with results communicated in a clear, timely, and actionable manner to strengthen public health responses. We believe that presenting results rapidly, and tailored to a non-expert audience, can serve as a template for effective public health response to COVID-19 and other emerging viral diseases., This Perspective article proposes a new paradigm to present SARS-CoV-2 phylogenetic analyses performed on a local level (municipal, county, or state), with results communicated in a clear, timely, and actionable manner. The authors believe that presenting results rapidly, and tailored to a non-expert audience, can serve as a template for effective public health communication for COVID-19 and other emerging viral diseases.

The novel coronavirus SARS-CoV-2 was first detected in the Pacific Northwest region of the United States in January 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the United States, we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated effects of federal travel restrictions. This study provides evidence of widespread sustained transmission of SARS-CoV-2 within the United States and highlights the critical need for local surveillance.

Effective public health research and preparedness requires an accurate understanding of which virus species possess or are at risk of developing human transmissibility. Unfortunately, our ability to identify these viruses is limited by gaps in disease surveillance and an incomplete understanding of the process of viral adaptation. By fitting boosted regression trees to data on 224 human viruses and their associated traits, we developed a model that predicts the human transmission ability of zoonotic viruses with over 84% accuracy. This model identifies several viruses that may have an undocumented capacity for transmission between humans. Viral traits that predicted human transmissibility included infection of nonhuman primates, the absence of a lipid envelope, and detection in the human nervous system and respiratory tract. This predictive model can be used to prioritize high-risk viruses for future research and surveillance, and could inform an integrated early warning system for emerging infectious diseases.