SARS-CoV-2 and other respiratory pathogens are detected in continuous air samples from congregate settings.

Two years after the emergence of SARS-CoV-2, there is still a need for better ways to assess the risk of transmission in congregate spaces. We deployed active air samplers to monitor the presence of SARS-CoV-2 in real-world settings across communities in the Upper Midwestern states of Wisconsin and Minnesota. Over 29 weeks, we collected 527 air samples from 15 congregate settings and detected 106 SARS-CoV-2 positive samples, demonstrating SARS-CoV-2 can be detected in air collected from daily and weekly sampling intervals. We expanded the utility of air surveillance to test for 40 other respiratory pathogens. Surveillance data revealed differences in timing and location of SARS-CoV-2 and influenza A virus detection in the community. In addition, we obtained SARS-CoV-2 genome sequences from air samples to identify variant lineages. Collectively, this shows air surveillance is a scalable, cost-effective, and high throughput alternative to individual testing for detecting respiratory pathogens in congregate settings.

Oil Immersed Lossless Total Analysis System (OIL-TAS): Integrated RNA Extraction and Detection for SARS-CoV-2 Testing

The coronavirus disease 2019 (COVID-19) pandemic exposed difficulties in scaling current quantitative PCR (qPCR)-based diagnostic methodologies for large-scale infectious disease testing. Bottlenecks include the lengthy multi-step process of nucleic acid extraction followed by qPCR readouts, which require costly instrumentation and infrastructure, as well as reagent and plastic consumable shortages stemming from supply chain constraints. Here we report a novel Oil Immersed Lossless Total Analysis System (OIL-TAS), which integrates RNA extraction and detection onto a single device that is simple, rapid, cost effective, uses minimal supplies and requires reduced infrastructure to perform. We validated the performance of OIL-TAS using contrived samples containing inactivated SARS-CoV-2 viral particles, which show that the assay can reliably detect an input concentration of 10 copies/L and sporadically detect down to 1 copy/L. The OIL-TAS method can serve as a faster, cheaper, and easier-to-deploy alternative to current qPCR-based methods for infectious disease testing.

Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary patient samples

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed samples, albeit with approximately 100-fold lower sensitivity than qRT-PCR. We demonstrate that adding lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Overall, the limit of detection (LOD) of RT-LAMP using direct nasopharyngeal swab or saliva samples without RNA extraction is 1x105-1x106 copies/ml. This LOD is sufficient to detect samples from which infectious virus can be cultured. Therefore, samples that test positive in this assay contain levels of virus that are most likely to perpetuate transmission. Furthermore, purified RNA achieves a similar LOD to qRT-PCR. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 screening programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.

Initial evaluation of a mobile SARS-CoV-2 RT-LAMP testing strategy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control in the United States remains hampered, in part, by testing limitations. We evaluated a simple, outdoor, mobile, colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay workflow where self-collected saliva is tested for SARS-CoV-2 RNA. From July 16 to November 19, 2020, 4,704 surveillance samples were collected from volunteers and tested for SARS-CoV-2 at 5 sites. A total of 21 samples tested positive for SARS-CoV-2 by RT-LAMP; 12 were confirmed positive by subsequent quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, while 8 were negative for SARS-CoV-2 RNA, and 1 could not be confirmed because the donor did not consent to further molecular testing. We estimated the RT-LAMP assays false-negative rate from July 16 to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or less and testing for SARS-CoV-2 RNA by qRT-PCR. We observed a 98.8% concordance between the RT-LAMP and qRT-PCR assays, with only 5 of 421 RT-LAMP negative pools (2,493 samples) testing positive in the more sensitive qRT-PCR assay. Overall, we demonstrate a rapid testing method that can be implemented outside the traditional laboratory setting by individuals with basic molecular biology skills and can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.