Development and Implementation of a Simple and Rapid Extraction-Free Saliva SARS-CoV-2 RT-LAMP Workflow for Workplace Surveillance (preprint)

Effective management of the COVID-19 pandemic requires widespread and frequent testing of the population for SARS-CoV-2 infection. Saliva has emerged as an attractive alternative to nasopharyngeal samples for surveillance testing as it does not require specialized personnel or materials for its collection and can be easily provided by the patient. We have developed a simple, fast, and sensitive saliva-based testing workflow that requires minimal sample treatment and equipment. After sample inactivation, RNA is quickly released and stabilized in an optimized buffer, followed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and detection of positive samples using a colorimetric and/or fluorescent readout. The workflow was optimized using 1,670 negative samples collected from 172 different individuals over the course of 6 months. Each sample was spiked with 50 copies/L of inactivated SARS-CoV-2 virus to monitor the efficiency of viral detection. Using pre-defined clinical samples, the test was determined to be 100% specific and 97% sensitive, with a limit of detection comparable to commercially available RT-qPCR-based diagnostics. The method was successfully implemented in a CLIA laboratory setting for workplace surveillance and reporting. From April 2021-February 2022, more than 30,000 self-collected samples from 755 individuals were tested and 85 employees tested positive mainly during December and January, consistent with high infections rates in Massachusetts and nationwide. The rapid identification and isolation of infected individuals with trace viral loads before symptom onset minimized viral spread in the workplace.

The Omicron variant mutation at position 28,311 in the SARS-CoV-2 N gene does not perturb CDC N1 target detection (preprint)

The emergence of new SARS-CoV-2 variants necessitates the reevaluation of current COVID-19 tests to ensure continued accuracy and reliability. The new SARS-CoV-2 variant, Omicron, is heavily mutated, with over 50 mutations within its RNA genome. Any of these mutations could adversely affect the ability of diagnostic assays to detect the virus in patient samples, potentially leading to inconclusive or false negative results. In fact, the U.S. Food and Drug Administration (FDA) has identified over two dozen diagnostic tests that contain a gene target that is expected to have significantly reduced sensitivity due to a mutation in the SAS-CoV-2 Omicron variant1. Additionally, one of the U.S. Centers for Disease Control and Prevention (CDC) Emergency Use Authorization (EUA) targets for COVID-19 tests, 2019-nCoV_N1, overlaps an Omicron mutation within the sequence targeted by the fluorescent probe. This target from the CDC has been used in many other EUA assays. Using in vitro transcribed (IVT) N gene RNA representing the wild-type (GenBank/GISAID ID MN908947.3) and Omicron variant (BA.1, GISAID ID EPI_ISL_6752027), we evaluated the performance of two different amplification protocols, both of which include the CDC 2019-nCoV_N1 primer-probe set. Both assays were able to detect the mutant N1 sequence as efficiently as the wild-type sequence. Consequently, these data suggest that diagnostic assays that use the 2019-nCoV-N1 primer-probe set are unlikely to be impacted by currently circulating Omicron lineage viruses.

Host, Viral, and Environmental Transcriptome Profiles of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (preprint)

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused thousands of deaths worldwide, including >18,000 in New York City (NYC) alone. The sudden emergence of this pandemic has highlighted a pressing clinical need for rapid, scalable diagnostics that can detect infection, interrogate strain evolution, and identify novel patient biomarkers. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs, plus a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, bacterial, and viral profiling. We applied both technologies across 857 SARS-CoV-2 clinical specimens and 86 NYC subway samples, providing a broad molecular portrait of the COVID-19 NYC outbreak. Our results define new features of SARS-CoV-2 evolution, nominate a novel, NYC-enriched viral subclade, reveal specific host responses in interferon, ACE, hematological, and olfaction pathways, and examine risks associated with use of ACE inhibitors and angiotensin receptor blockers. Together, these findings have immediate applications to SARS-CoV-2 diagnostics, public health, and new therapeutic targets.