Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and SARS-CoV-2 variants

The COVID-19 pandemic has demonstrated a clear need for high-throughput, multiplexed, and sensitive assays for detecting SARS-CoV-2 and other respiratory viruses as well as their emerging variants. Here, we present microfluidic CARMEN (mCARMEN), a cost-effective virus and variant detection platform that combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel (RVP) and demonstrated its diagnostic-grade performance on 533 patient specimens in an academic setting and then 166 specimens in a clinical setting. We further developed a panel to distinguish 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 106 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 viral copies in samples. mCARMEN enables high-throughput surveillance of multiple viruses and variants simultaneously.

Phylogenetic analysis of SARS-CoV-2 in the Boston area highlights the role of recurrent importation and superspreading events

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.

Clinical Sensitivity and Interpretation of PCR and Serological COVID-19 Diagnostics for Patients Presenting to the Hospital

IntroductionThe diagnosis of COVID-19 requires integration of clinical and laboratory data. SARS-CoV-2 diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS-CoV-2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. MethodsA single-center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR-positive SARS-CoV-2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. To calculate daily clinical sensitivity by serology, we utilized 157 PCR- positive patients with a total of 197 specimens tested by enzyme-linked immunosorbent assay for IgM, IgG, and IgA anti-SARS-CoV-2 antibodies. ResultsClinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70-71% from days 9-11, and 30% at day 21. In contrast, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after day 7, >80% after day 12, and 100% by day 21. ConclusionPCR and serology are complimentary modalities that require time- dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.

Clinical assessment and validation of a rapid and sensitive SARS-CoV-2 test using reverse-transcription loop-mediated isothermal amplification

Amid the enduring COVID-19 pandemic, there is an urgent need for expanded access to rapid and sensitive SARS-CoV-2 testing worldwide. Here we present a simple clinical workflow that uses a sensitive and highly specific colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) to detect SARS-CoV-2 and takes forty minutes from sample collection to result. This test requires no specialized equipment and costs a few dollars per sample. Nasopharyngeal samples collected in saline were added either directly (unprocessed) to RT-LAMP reactions or first inactivated by a combined chemical and heat treatment step to inhibit RNases and lyse virions and human cells. The specimens were then amplified with two SARS-CoV-2-specific primer sets and an internal specimen control; the resulting color change was visually interpreted. While direct addition of unprocessed specimens to RT-LAMP reactions could reliably detect samples with abundant SARS-CoV-2, the assay sensitivity markedly increased after the addition of an inactivation step. In 62 clinical samples with a wide range of SARS-CoV-2 nucleic acid concentrations, the assay had 87.5% sensitivity and 100% specificity with a limit of detection at least 25 copies/L, making it an ideal test to rule in infection. To increase sensitivity, samples that tested negative for SARS-CoV-2 by direct sample addition could be reflexed to a purification step, to increase the effective per-reaction sample input volume. In 40 purified samples, the assay yielded a 90% sensitivity and 100% specificity, with a limit of detection comparable to commercially available real-time PCR-based diagnostics that have received Emergency Use Authorization (EUA) from the FDA. This test for SARS-CoV-2 can be performed in a range of settings for a fraction of the price of other available tests, with limited equipment, and without relying on over-burdened supply chains to increase overall testing capacity.