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Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here, we present the development and validation of a rapid COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K/Q/A, and N501Y mutations, at least one of which is found in nearly all major variants. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all targeted SNP mutations. An analysis pipeline for CRISPR-based SNP identification from 261 clinical samples yielded a SNP concordance of 97.3% and agreement of 98.9% (258 of 261) for SARS-CoV-2 lineage classification, using SARS-CoV-2 whole-genome sequencing and/or real-time RT-PCR as test comparators. We also showed that detection of the single E484A mutation was necessary and sufficient to accurately identify Omicron from other major circulating variants in patient samples. These findings demonstrate the utility of CRISPR-based DETECTR as a faster and simpler diagnostic method compared with sequencing for SARS-CoV-2 variant identification in clinical and public health laboratories.

Analytical sensitivity for SARS-CoV-2 detection is a key performance metric for the evaluation of viral detection assays. We determined analytical limits of detection for seven SARS-CoV-2 assays using serial dilutions of pooled patient material quantified with droplet digital PCR. Limits of detection ranged from ≤10 to 74 copies/ml for commercial high-throughput laboratory analyzers (Roche Cobas, Abbott m2000, and Hologic Panther Fusion) and 167 to 511 copies/ml for sample-to-answer (DiaSorin Simplexa, GenMark ePlex) and point-of-care instruments (Abbott ID NOW). The CDC assay yielded limits of detection ranging from 85 to 499 copies/ml, depending on the extraction method and thermocycler used. These results can help to inform the assay choice for testing approaches to manage the current COVID-19 outbreak.

INTRODUCTIONWith established applications of next-generation sequencing in inherited diseases and oncology, clinical laboratories are evaluating the use of metagenomics for identification of infectious agents directly from patient samples, to aid in the diagnosis of infections. Metagenomic next-generation sequencing for infectious diseases promises an unbiased approach to detection of microbes that does not depend on growth in culture or the targeting of specific pathogens. However, the issues of contamination, interpretation of results, selection of databases used for analysis, and prediction of antimicrobial susceptibilities from sequencing data remain challenges. In this Point-Counterpoint, Steve Miller and Charles Chiu discuss the pros of using direct metagenomic sequencing, while Kyle Rodino and Melissa Miller argue for the use of caution.

Rapidly growing mycobacteria are rarely found in central nervous system infections. We describe a case of polymicrobial infection in a brain abscess including two rapidly growing Mycobacterium species, M. immunogenum and M. llatzerense. The Mycobacterium isolates were distinguishable by molecular methods, and whole-genome sequencing showed