Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.

The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.

Metagenomic comparison of tracheal aspirate and mini-bronchial alveolar lavage for assessment of respiratory microbiota.

Accurate and informative microbiological testing is essential for guiding diagnosis and management of pneumonia in patients who are critically ill. Sampling of tracheal aspirate (TA) is less invasive compared with mini-bronchoalveolar lavage (mBAL) and is now recommended as a frontline diagnostic approach in patients who are mechanically ventilated, despite the historical belief that TA was suboptimal due to contamination from oral microbes. Advancements in metagenomic next-generation sequencing (mNGS) now permit assessment of airway microbiota without a need for culture and, as such, provide an opportunity to examine differences between mBAL and TA at a resolution previously unachievable. Here, we engaged shotgun mNGS to assess quantitatively the airway microbiome in matched mBAL and TA specimens from a prospective cohort of critically ill adults. We observed moderate differences between sample types across all subjects; however, we found significant compositional similarity in subjects with bacterial pneumonia, whose microbial communities were characterized by dominant pathogens. In contrast, in patients with noninfectious acute respiratory illnesses, significant differences were observed between sample types. Our findings suggest that TA sampling provides a similar assessment of airway microbiota as more invasive testing by mBAL in patients with pneumonia.