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

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.

COVID-19 neutralizing antibodies predict disease severity and survival (preprint)

COVID-19 exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, and high anti-RBD antibody levels. While anti-RBD IgG levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting protection from reinfection by this strain. However, SARS-CoV-2 sera was unable to cross-neutralize a highly-homologous pre-emergent bat coronavirus, WIV1-CoV, that has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics.

Cerebral Microvascular Injury in Severe COVID-19 (preprint)

IMPORTANCE: 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. OBJECTIVE: To determine the incidence, distribution, and clinical and histopathologic correlates of microvascular lesions in patients with severe COVID-19. DESIGN: Observational, retrospective cohort study: March to May 2020. SETTING: Single academic medical center. PARTICIPANTS: Consecutive patients (16) admitted to the intensive care unit with severe COVID-19, undergoing brain MRI for evaluation of coma or focal neurologic deficits. EXPOSURES: Not applicable. MAIN OUTCOME AND MEASURES: Hypointense 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. RESULTS: Eleven 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 RELEVANCE: SWI 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.

Dynamics and significance of the antibody response to SARS-CoV-2 infection (preprint)

BACKGROUND Characterizing the humoral immune response to SARS-CoV-2 and developing accurate serologic assays are needed for diagnostic purposes and estimating population-level seroprevalence. METHODS We measured the kinetics of early antibody responses to the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2 in a cohort of 259 symptomatic North American patients infected with SARS-CoV-2 (up to 75 days after symptom onset) compared to antibody levels in 1548 individuals whose blood samples were obtained prior to the pandemic. RESULTS Between 14-28 days from onset of symptoms, IgG, IgA, or IgM antibody responses to RBD were all accurate in identifying recently infected individuals, with 100% specificity and a sensitivity of 97%, 91%, and 81% respectively. Although the estimated median time to becoming seropositive was similar across isotypes, IgA and IgM antibodies against RBD were short-lived with most individuals estimated to become seronegative again by 51 and 47 days after symptom onset, respectively. IgG antibodies against RBD lasted longer and persisted through 75 days post-symptoms. IgG antibodies to SARS-CoV-2 RBD were highly correlated with neutralizing antibodies targeting the S protein. No cross-reactivity of the SARS-CoV-2 RBD-targeted antibodies was observed with several known circulating coronaviruses, HKU1, OC 229 E, OC43, and NL63. CONCLUSIONS Among symptomatic SARS-CoV-2 cases, RBD-targeted antibodies can be indicative of previous and recent infection. IgG antibodies are correlated with neutralizing antibodies and are possibly a correlate of protective immunity.

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

Introduction: The 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. Methods: A 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. Results: Clinical 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. Conclusion: PCR 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 (preprint)

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.