SARS-CoV-2 Viral Viability Culture and Sequencing from Immunocompromised Patients with Persistently Positive SARS-CoV-2 PCR Results

Background. Immunocompromised (IC) patients (pts) can have prolonged SARS-CoV-2 PCR positivity, even after resolution of COVID-19 symptoms. This study aimed to determine if viable virus could be detected in samples collected > 21 days after an initial positive (pos) SARS-CoV-2 PCR in IC pts. Methods. We obtained 20 remnant SARS-CoV-2 PCR pos nasopharyngeal swabs from IC pts (bone marrow or solid organ transplant, high dose steroids, immunosuppressive medications) with a pos repeat PCR within the previous 30 days. The repeat specimens were cultured on Vero-hACE2-TMPRSS2 cells and incubated for 96 hours to assess viral viability. Viable RNA and infectious virus in the cultured cells were measured by qPCR and infectious plaque assays. RNA sequencing was performed on a HiSeq platform (Illumina). Samples also underwent SARS-CoV-2 antigen (Ag) testing (BD Veritor). Clinical data were extracted from the electronic health record by chart review. Results. Pt characteristics are in Table 1. Viral cultures from the repeat specimen were negative (neg) for 18 pts and pos for 2 (Table 2). Pt 1 is a 60M treated with obinatuzumab 19 days prior to his first pos PCR test, with repeat specimen collected 21 days later (cycle threshold (Ct) not available). Pt 1 had a low viral titer (27 PFU/mL) & a D614G mutation on sequencing. Pt 2 is a 75M treated with rituximab 10 days prior to his first pos PCR test, with repeat specimen collected 23 days later (Ct 27.56/27.74). Pt 2 had a high viral titer (2e6 PFU/mL) and D614G, S98F, and S813I mutations. Demographics of Study Population (N=20) Characteristics of patients with a positive SARS-CoV-2 viral culture Conclusion. 90% of specimens collected > 21 days after an initial pos SARS-CoV-2 PCR did not have viable virus detected on their repeat specimen. The 2 pts with pos viral cultures had active hematologic malignancies treated with an anti-CD20 mAb at the time of COVID-19 diagnosis. One pt had a high concentration of active, viable virus. No known variants of concern were noted in this cohort, collected in Q2 2020, though prolonged replication is a risk for variant development. Further data are needed about risk factors for persistent viable viral shedding & methods to prevent transmission of viable virus from IC hosts.

High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro.

Influenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air-liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

Evaluation of hydroxychloroquine-based combination therapies for the treatment of COVID-19

Background: During the early COVID-19 pandemic a large number of investigational agents were utilized due to lack of therapeutic options. We evaluate the utility of commonly-used investigational agents combined with hydroxychloroquine (HCQ). Methods: This multicenter observational cohort study included patients admitted with COVID-19 between March - May 2020 in Detroit, Michigan who received at least 2 doses of HCQ. Our primary outcome was the change in Sequential Organ Failure Assessment (SOFA) score from presentation to day 5 of HCQ therapy with a secondary outcome of in-hospital mortality. Data collected included demographics, Charlson Comorbidity index (CCI), daily SOFA score, laboratory data and COVID-directed therapies. Multiple linear regressions were performed to control for potential confounders between different therapies and change in SOFA score. Results: Three hundred thirty-five patients receiving HCQ were included. Patients were 62 ± 14.8 years of age, male (54%) and African-American (82%) with a mean CCI of 1.7 ± 1.9. In our cohort, 32% were admitted to the intensive care unit and 35% expired. Therapies received by more than 20% of patients in addition to HCQ included azithromycin (80%), zinc (76%) and vitamin D (29%). In our unadjusted analysis, a significant improvement in SOFA score was observed with zinc (0.76) while no significant change was observed with azithromycin (-0.46) or vitamin D (0.05). However, there was no significant change in SOFA score after adjusting for confounders for azithromycin, zinc and vitamin D. No difference in mortality was observed between the groups. Conclusion: Overall, no benefit in end-organ damage or mortality was observed with the addition of azithromycin, zinc or vitamin D to HCQ. Further studies are needed to confirm this observation.