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The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting. We further developed an mCARMEN panel to enable the identification of 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 2,088 patient specimens with near-perfect concordance to sequencing-based variant classification. Lastly, we implemented a combined Cas13 and Cas12 approach that enables quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples. The mCARMEN platform enables high-throughput surveillance of multiple viruses and variants simultaneously, enabling rapid detection of SARS-CoV-2 variants.

The global spread and continued evolution of SARS-CoV-2 has driven an unprecedented surge in viral genomic surveillance. Amplicon-based sequencing methods provide a sensitive, low-cost and rapid approach but suffer a high potential for contamination, which can undermine laboratory processes and results. This challenge will increase with the expanding global production of sequences across a variety of laboratories for epidemiological and clinical interpretation, as well as for genomic surveillance of emerging diseases in future outbreaks. We present SDSI + AmpSeq, an approach that uses 96 synthetic DNA spike-ins (SDSIs) to track samples and detect inter-sample contamination throughout the sequencing workflow. We apply SDSIs to the ARTIC Consortium's amplicon design, demonstrate their utility and efficiency in a real-time investigation of a suspected hospital cluster of SARS-CoV-2 cases and validate them across 6,676 diagnostic samples at multiple laboratories. We establish that SDSI + AmpSeq provides increased confidence in genomic data by detecting and correcting for relatively common, yet previously unobserved modes of error, including spillover and sample swaps, without impacting genome recovery.

The impact of coronavirus disease 2019 vaccination on viral characteristics of breakthrough infections is unknown. In this prospective cohort study, incidence of severe acute respiratory syndrome coronavirus 2 infection decreased following vaccination. Although asymptomatic positive tests were observed following vaccination, the higher cycle thresholds, repeat negative tests, and inability to culture virus raise questions about their clinical significance.

The rapid global spread and continued evolution of SARS-CoV-2 has highlighted an unprecedented need for viral genomic surveillance and clinical viral sequencing. Amplicon-based sequencing methods provide a sensitive, low-cost and rapid approach but suffer a high potential for contamination, which can undermine lab processes and results. This challenge will only increase with expanding global production of sequences by diverse research groups for epidemiological and clinical interpretation. We present an approach which uses synthetic DNA spike-ins (SDSIs) to track samples and detect inter-sample contamination through a sequencing workflow. Applying this approach to the ARTIC Consortium’s amplicon design, we define a series of best practices for Illumina-based sequencing and provide a detailed characterization of approaches to increase sensitivity for low-viral load samples incorporating the SDSIs. We demonstrate the utility and efficiency of the SDSI method amidst a real-time investigation of a suspected hospital cluster of SARS-CoV-2 cases.

Phylogenetics of superspreading One important characteristic of coronavirus epidemiology is the occurrence of superspreading events. These are marked by a disproportionate number of cases originating from often-times asymptomatic individuals. Using a rich sequence dataset from the early stages of the Boston outbreak, Lemieux et al. identified superspreading events in specific settings and analyzed them phylogenetically (see the Perspective by Alizon). Using ancestral trait inference, the authors identified several importation events, further investigated the context and contribution of particular superspreading events to the establishment of local and wider SARS-CoV-2 transmission, and used viral phylogenies to describe sustained transmission. Science , this issue p. eabe3261 ; see also p. 574

The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a sensitive and specific diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We identify the optimal conditions to allow RPA-based amplification and Cas13-based detection to occur in a single step, simplifying assay preparation and reducing run-time. We improve HUDSON to rapidly inactivate viruses in nasopharyngeal swabs and saliva in 10 min. SHINE’s results can be visualized with an in-tube fluorescent readout — reducing contamination risk as amplification reaction tubes remain sealed — and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-qPCR with a sample-to-answer time of 50 min. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities., The COVID-19 pandemic has highlighted the need for user-friendly diagnostic techniques. Here, the authors present SHINE, a streamlined and optimised Cas13-based method with accompanying smartphone app for visual diagnosis.

We evaluated the incidence, distribution, and histopathologic correlates of microvascular brain lesions in patients with severe COVID-19. Sixteen consecutive patients admitted to the intensive care unit with severe COVID-19 undergoing brain MRI for evaluation of coma or neurologic deficits were retrospectively identified. Eleven patients had punctate susceptibility-weighted imaging (SWI) lesions in the subcortical and deep white matter, eight patients had >10 SWI lesions, and four patients had lesions involving the corpus callosum. The distribution of SWI lesions was similar to that seen in patients with hypoxic respiratory failure, sepsis, and disseminated intravascular coagulation. Brain autopsy in one patient revealed that SWI lesions corresponded to widespread microvascular injury, characterized by perivascular and parenchymal petechial hemorrhages and microscopic ischemic lesions. Collectively, these radiologic and histopathologic findings add to growing evidence that patients with severe COVID-19 are at risk for multifocal microvascular hemorrhagic and ischemic lesions in the subcortical and deep white matter., National Institute of Neurological Disorders and Stroke (Grants R21NS109627, R21AG067562, RF1NS115268), NIH Director’s Office (Grant DP2HD101400), NIH National Institute of Mental Health (Grant K23MH115812), NIH National Institutes of Allergy and Infectious Diseases (Grant 2U19AI110818)

Developing and deploying new diagnostic tests is difficult, but the need to do so in response to a rapidly emerging pandemic such as COVID-19 is crucially important for an effective response. In the early stages of a pandemic, laboratories play a key role in helping health care providers and public health authorities detect active infection, a task most commonly achieved using nucleic acid-based assays. While the landscape of diagnostics is rapidly evolving, polymerase chain reaction (PCR) remains the gold-standard of nucleic acid-based diagnostic assays, in part due to its reliability, flexibility, and wide deployment. To address a critical local shortage of testing capacity persisting during the COVID-19 outbreak, our hospital set up a molecular based laboratory developed test (LDT) to accurately and safely diagnose SARS-CoV-2. We describe here the process of developing an emergency-use LDT, in the hope that our experience will be useful to other laboratories in future outbreaks and will help to lower barriers to fast and accurate diagnostic testing in crisis conditions.

SARS-CoV-2 has caused a severe, ongoing outbreak of COVID-19 in Massachusetts with 111,070 confirmed cases and 8,433 deaths as of August 1, 2020. To investigate the introduction, spread, and epidemiology of COVID-19 in the Boston area, we sequenced and analyzed 772 complete SARS-CoV-2 genomes from the region, including nearly all confirmed cases within the first week of the epidemic and hundreds of cases from major outbreaks at a conference, a nursing facility, and among homeless shelter guests and staff. The data reveal over 80 introductions into the Boston area, predominantly from elsewhere in the United States and Europe. We studied two superspreading events covered by the data, events that led to very different outcomes because of the timing and populations involved. One produced rapid spread in a vulnerable population but little onward transmission, while the other was a major contributor to sustained community transmission, including outbreaks in homeless populations, and was exported to several other domestic and international sites. The same two events differed significantly in the number of new mutations seen, raising the possibility that SARS-CoV-2 superspreading might encompass disparate transmission dynamics. Our results highlight the failure of measures to prevent importation into MA early in the outbreak, underscore the role of superspreading in amplifying an outbreak in a major urban area, and lay a foundation for contact tracing informed by genetic data.

Structured AbstractImportanceCerebral microvascular lesions are common in patients with severe COVID-19. Radiologic-pathologic correlation in one case suggests a combination of microvascular hemorrhagic and ischemic lesions that may reflect an underlying hypoxic mechanism of injury, which requires validation in larger studies.ObjectiveTo determine the incidence, distribution, and clinical and histopathologic correlates of microvascular lesions in patients with severe COVID-19.DesignObservational, retrospective cohort study: March to May 2020.SettingSingle academic medical center.ParticipantsConsecutive patients (n=16) admitted to the intensive care unit with severe COVID-19, undergoing brain MRI for evaluation of coma or focal neurologic deficits.ExposuresNot applicable.Main Outcome and MeasuresHypointense microvascular lesions identified by a prototype ultrafast high-resolution susceptibility-weighted imaging (SWI) MRI sequence, counted by two neuroradiologists and categorized by neuroanatomic location. Clinical and laboratory data (most recent measurements before brain MRI). Brain autopsy and cerebrospinal fluid PCR for SARS-CoV-2 in one patient who died from severe COVID-19.ResultsEleven of 16 patients (69%) had punctate and linear SWI lesions in the subcortical and deep white matter, and eight patients (50%) had >10 SWI lesions. In 4/16 patients (25%), lesions involved the corpus callosum. Brain autopsy in one patient revealed that SWI lesions corresponded to widespread microvascular injury, characterized by perivascular and parenchymal petechial hemorrhages and microscopic ischemic lesions.Conclusions and RelevanceSWI lesions are common in patients with neurological manifestations of severe COVID-19 (coma and focal neurologic deficits). The distribution of lesions is similar to that seen in patients with hypoxic respiratory failure, sepsis, and disseminated intravascular coagulation. Collectively, these radiologic and histopathologic findings suggest that patients with severe COVID-19 are at risk for multifocal microvascular hemorrhagic and ischemic lesions in the subcortical and deep white matter.Key Points SectionQuestionWhat is the prevalence and pathophysiology of cerebral microvascular injury in patients with severe COVID-19?FindingsIn this retrospective cohort study of 16 patients undergoing MRI for neurologic complications of severe COVID-19, microvascular lesions were observed in 11 patients and showed an anatomic distribution similar to that seen in patients with hypoxic respiratory failure and sepsis. In one patient who died, brain autopsy revealed widespread microvascular injury, including perivascular microhemorrhages and microscopic ischemic lesions.MeaningMicrovascular injury is common in patients with severe COVID-19. Radiologic-pathologic correlation, though limited to a single case, provides insights into possible mechanisms of injury.

The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a sensitive and specific diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We identify the optimal conditions to allow RPA-based amplification and Cas13-based detection to occur in a single step, simplifying assay preparation and reducing run-time. We improve HUDSON to rapidly inactivate viruses in nasopharyngeal swabs and saliva in 10 min. SHINE's results can be visualized with an in-tube fluorescent readout - reducing contamination risk as amplification reaction tubes remain sealed - and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-qPCR with a sample-to-answer time of 50 min. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities.

Objective Little is known about CSF profiles in patients with acute COVID-19 infection and neurological symptoms. Here, CSF was tested for SARS-CoV-2 RNA and inflammatory cytokines and chemokines and compared to controls and patients with known neurotropic pathogens. Methods CSF from twenty-seven consecutive patients with COVID-19 and neurological symptoms was assayed for SARS-CoV-2 RNA using quantitative reverse transcription PCR (RT-qPCR) and unbiased metagenomic sequencing. Assays for blood brain barrier (BBB) breakdown (CSF:serum albumin ratio (Q-Alb)), and proinflammatory cytokines and chemokines (IL-6, IL-8, IL-15, IL-16, monocyte chemoattractant protein −1 (MCP-1) and monocyte inhibitory protein – 1β (MIP-1β)) were performed in 23 patients and compared to CSF from patients with HIV-1 (16 virally suppressed, 5 unsuppressed), West Nile virus (WNV) (n = 4) and 16 healthy controls (HC). Results Median CSF cell count for COVID-19 patients was 1 white blood cell/μL; two patients were infected with a second pathogen (Neisseria, Cryptococcus neoformans). No CSF samples had detectable SARS-CoV-2 RNA by either detection method. In patients with COVID-19 only, CSF IL-6, IL-8, IL-15, and MIP-1β levels were higher than HC and suppressed HIV (corrected-p, Graphical abstract Unlabelled Image