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To explore the potential modes of Severe Acute Respiratory Coronavirus-2 (SARS-CoV-2) transmission, we collected 535 diverse clinical and environmental samples from 75 infected hospitalized and community patients. Infectious SARS-CoV-2 with quantitative burdens varying from 5 plaque-forming units/mL (PFU/mL) up to 1.0 × 106 PFU/mL was detected in 151/459 (33%) of the specimens assayed and up to 1.3 × 106 PFU/mL on fomites with confirmation by plaque morphology, PCR, immunohistochemistry, and/or sequencing. Infectious virus in clinical and associated environmental samples correlated with time since symptom onset with no detection after 7–8 days in immunocompetent hosts and with N-gene based Ct values ≤ 25 significantly predictive of yielding plaques in culture. SARS-CoV-2 isolated from patient respiratory tract samples caused illness in a hamster model with a minimum infectious dose of ≤ 14 PFU. Together, our findings offer compelling evidence that large respiratory droplet and contact (direct and indirect i.e., fomites) are important modes of SARS-CoV-2 transmission.

SARS-CoV-2 antigen tests used at the point-of-care, such as the Abbott Panbio, have great potential to help combat the COVID-19 pandemic. The Panbio is Health Canada approved for the detection of SARS-CoV-2 in symptomatic individuals within the first 7 days of COVID-19 symptom onset(s). Symptomatic adults recently diagnosed with COVID-19 in the community were recruited into the study. Paired nasopharyngeal (NP), throat, and saliva swabs were collected, with one paired swab tested immediately with the Panbio, and the other transported in universal transport media and tested using real-time reverse-transcriptase polymerase chain reaction (RT-PCR). We also prospectively evaluated results from assessment centers within the community. For those individuals, an NP swab was collected for Panbio testing and paired with RT-PCR results from parallel NP or throat swabs. One hundred and forty-five individuals were included in the study. Collection of throat and saliva was stopped early due to poorer performance (throat sensitivity 57.7%, n=61, and saliva sensitivity 2.6%, n=41). NP swab sensitivity was 87.7% [n=145, 95% confidence interval (CI) 81.0-92.7%]. There were 1641 symptomatic individuals tested by Panbio in assessment centers with 268/1641 (16.3%) positive for SARS-CoV-2. There were 37 false negatives and 2 false positives, corresponding to a sensitivity and specificity of 86.1% [95% CI 81.3-90.0%] and 99.9% [95% CI 99.5-100.0%], respectively. The Panbio test reliably detects most cases of SARS-CoV-2 from adults in the community setting presenting within 7 days of symptom onset using nasopharyngeal swabs. Throat and saliva swabs are not reliable specimens for the Panbio.

Background: The recent emergence and rapid global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demonstrates the urgent need for laboratory-developed assays for clinical diagnosis and public health interventions in the absence of commercial assays. Methods: We outline the progression of reverse-transcriptase polymerase chain reaction (RT-PCR) assays that were developed and validated at the Alberta Precision Laboratories, Public Health Laboratory, Alberta, Canada, to respond to this pandemic. Initially, testing was performed using SARS-CoV-2–specific and pan-coronavirus gel-based assays that were soon superseded by real-time RT-PCR assays targeting the envelope and RNA-dependent RNA polymerase genes to accommodate the high anticipated volumes of samples. Throughput was further enhanced by multiplexing the different targets together with the co-detection of an internal extraction control. Results: These assays are comparable in sensitivity and specificity to the assays recommended by the World Health Organization and the US Centers for Disease Control and Prevention. Conclusions: The availability of real-time RT-PCR assays early in the pandemic was essential to provide valuable time to local health authorities to contain transmission and prepare for appropriate response strategies.

Few studies have assessed for infectious SARS-CoV-2 in multiple types of clinical and environmental samples. In almost 500 samples from 75 hospitalized and community cases, we detected infectious virus with quantitative burdens varying from 5.0 plaque-forming units/mL (PFU/mL) up to 1.0×106 PFU/mL in clinical specimens and up to 1.3×106 PFU/mL on fomites including facial tissues, nasal prongs, call bells/cell phones, dentures, and sputum deposits with confirmation by plaque morphology, PCR, immunohistochemistry, and sequencing. Expectorated sputum samples had the highest percentage of positive samples and virus titers (71%, 2.9×102 to 5.2×105 PFU/mL), followed by saliva (58%, 10 to 4.6×104 PFU/mL), and cough samples without sputum (19%, 5 to 1.9×103 PFU/mL). We also detected infectious SARS-CoV-2 from patients’ hands (28%, 60 to 2.3×102 PFU/mL) but no infectious virus was found in continuous speech samples despite finding high levels of infectious virus in the associated nasopharynx, throat, or saliva specimens. We demonstrated infectious virus stability in clinical samples, including those dried for prolonged periods of time. Infectious virus correlated with time since symptom onset with no detection after 7-8 days in immunocompetent hosts and with N-gene based Ct values ≤ 25 significantly predictive of yielding plaques in culture. One PFU was associated with ∼105 copies of N gene RNA across a diversity of samples and times from symptom onset. Clinical salivary isolates caused illness in a hamster model with a minimum infectious dose of ≤14 PFU/mL. Our findings of high quantitative burdens of infectious virus, stability even with drying, and a very low minimal infectious dose suggest multiple modes of transmission are exploited by SARS-CoV-2, including direct contact, large respiratory droplet, and fomite transmission and in the context of a high binding avidity to human cellular receptors, offer an explanation of the high contagiousness of this virus.Research in ContextEvidence before this studyWe searched the literature for articles that reported on the presence of infectious SARS-CoV-2 in patients’ samples from clinical and environmental sources. We found several key primary studies and systematic reviews providing valuable background on the carriage of infectious virus and the correlation with cycle threshold (Ct) and/or RNA copies/mL on PCR testing. Clinical correlations with respect to underlying clinical conditions and details on the onset of illness were not commonly reported with respect to the timing of obtaining specimens for culture. Few studies carefully assessed the presence of infectious virus in cough samples, sputum, nasal secretions, hands, and common high touch surfaces. A few published works were found on factors which may be associated with shedding of infectious virus.Added value of this studyWe assessed the presence of infectious virus shedding in almost 500 specimens from 75 patients with COVID-19 in both the hospital and community setting. High titers of infectious virus were detected in multiple clinical and environmental samples. The longest duration of recovery of infectious virus in a fomite sample was from a dried facial tissue found at a patient’s bedside table, used at least 9 hours earlier. Cough specimens revealed infectious virus in 28% of specimens with infectious virus titers as high as 5.2×105 PFU/mL. Hand samples contained infectious virus with titers ranging from 55 to 2.3×102 PFU/mL. Infectious viral loads correlated with N-gene based Ct values and showed that Ct values ≤ 25 were predictive of yielding plaques in culture. These experiments also showed that infectious virus is most often recovered during a 7 to 8-day period following illness onset in immunocompetent persons, and during that time the ratio of RNA/PFU in these clinical specimens varies relatively little, with a ratio ∼160,000:1. Infectious virus may be recovered for weeks to several months in immunosuppressed persons. We also showed that virus recovered from saliva specimens, representing a commonly encountered fomite sample, caused infection in the Syrian hamster model, hence demonstrating the infectiousness of the virus sourced from this type of specimen. A challenge dose as low as 14 PFU/mL yielded infection in this model.Implications of all the available evidenceWe have shown that SARS-CoV-2 is relatively easy to culture when obtained early in the course of illness and there are high levels of cultivatable SARS-CoV-2 in multiple types of clinical specimens and common fomites, including high-touch surfaces and demonstrated their infectiousness in a mammalian host. Our results demonstrate the presence of high quantitative burdens of SARS-CoV-2 in sputum, saliva, and droplets from coughing, which would lend support to large respiratory droplet transmission, hands which would support direct contact transmission, and fomites which would promote indirect contact transmission. We were unable to detect any infectious virus in continuous speech samples which suggests that brief conversations, without coughing or sneezing, pose little risk of transmitting SARS-CoV-2. Our findings provide an explanation for the high contagiousness of this virus and support current public health measures and infection prevention and control guidelines including physical distancing, hand hygiene, masking, and cleaning and disinfection.