Use of a SARS-CoV-2 Strand-specific Assay to evaluate for Prolonged Viral Replication among Hospitalized Patients. Tertiary Care Center, California 2020- 2022

Background. Determining if a patient with SARS-CoV-2 remains infectious is an infection control challenge in healthcare settings;specially, among critically ill or profoundly immunosuppressed patients. We use an assay that detects minus-strand RNA as a surrogate for actively replicating SARS-CoV-2. We report positive strand-specific assays in relationship to time since admission and describe patients with a detectable strand-specific assay >20 days since admission. Methods. We use a 2-step rRT-PCR specific to the minus strand of the SARS-CoV-2 envelope gene. The strand-specific assay is used to evaluate for infectivity in asymptomatic patients with a positive admission screening or pre-procedural test or if ongoing replication is suspected (critical illness or profound immunosuppression). We retrieved strand-specific test results for patients hospitalized at Stanford Healthcare during August 2020-March 2022. We describe clinical characteristics for patients with a detectable minus strand-specific test >20 days since admission. Results. A total of 774 strand-specific tests were collected from 624 hospitalized patients. A total of 523 patients had only one test (84%) and 101 (16%) had >=2 tests. The test positivity rate varied by time since admission: 19% in tests performed 0-5 days, 28% in 6-10 days, 22% in 11-20 days, and 41% in those >20 days since admission. Among 35 patients tested >20 days since admission, 13 (37%) had >=1 detectable minus strand-specific test. Most were male (n=8, 62%) and mean age was 59. Of 13 patients with a detectable assay, seven (54%) had prolonged viral replication with persistent symptoms and detectable minus strand assays for >20 days from symptom onset. Of these seven patients, four had a transplant (3 lung, 1 liver), 1 ovarian cancer, 1 CAR-T cell therapy, and 1 ESRD without immunosuppression. The remaining eight patients with a detectable assay >20 days since admission had illness onset while hospitalized. Conclusion. Among hospitalized patients with SARS-CoV-2 infection, we found a varying positivity rate according to the timing of testing, possibly reflecting different indications for the test. The strand-specific assay may help assess for infectiousness in profoundly immunocompromised patients.

Duration of Seroprevalence among Individuals Previously Infected with SARS-CoV-2 and their Household Contacts

Background. Serological tests directed against SARS-CoV-2 can provide information about the timing of infection and immunity against the virus. However, the kinetics of the host immune response to SARS-CoV-2 remain poorly understood. We established a household transmission study to analyze the serological responses within households, to determine longitudinal immune responses to infection. Methods. From April 2020 to April 2022, we prospectively enrolled 76 households with at least one RT-PCR confirmed case of COVID-19. Participants were asked to provide blood samples at three time points: at baseline within 2 weeks of the index's diagnosis of COVID-19, and at one- and three-months post-enrollment. Samples were tested for the presence of IgG antibodies against SARS-CoV-2 spike protein via an FDA EUA approved ELISA. Demographics, medical history, and symptomatology were also collected. Results. To date, we have analyzed 238 serologic samples from 135 participants, including 82 baseline samples, 89 one-month samples, and 67 three-month samples. At baseline, 67.8% (n=40/59) of all confirmed cases tested positive for SARS-CoV-2 antibodies, which increased to 86.4% (n=57/66) at the one month, and 85.1% at three months (n=40/47). Of those confirmed infected participants that failed to seroconvert at baseline, almost all reported symptoms (n=14/19, 73.7%) and did not have chronic medical conditions (n=17/19, 89.5%). Of the 19, 3 failed to seroconvert by their third visit. All individuals who were fully vaccinated at the time of each visit tested positive for antibodies at baseline (n=26), one-month (n=27), and three-months (n=20). Of those who were not fully vaccinated, 56 (41.1%) were positive for antibodies at baseline, 62 (59.7%) were positive at one -month, and 47 (63.8%) at three-months. Differences in seropositivity rates between pediatric and adult participants, as well as between index cases and household contacts, at each visit were also identified (Table 1). Conclusion. Identifying differences in seroprevalence in various demographic groups can provide insight into longitudinal immune responses post-infection. Future analyses on seropositivity among previously infected individuals who received therapeutics may be of interest.

Use of a SARS-CoV-2 Strand-specific Assay to detect Relapse or Ongoing Infection at a Tertiary Care Center, California 2020-2022

Background. Determining if a patient with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains infectious can be challenging in patients with severe disease or those profoundly immunosuppressed. We use an assay that detects minus-strand RNA as a surrogate for actively replicating SARS-CoV-2. Here, we describe a cohort of patients who had strand-assay reversion: a detectable minus strandspecific assay after having had an undetectable value which may signify relapse of infection or reinfection. Methods. We used a 2-step rRT-PCR specific to the minus strand of the SARS-CoV-2 envelope gene. The strand-specific assay is used to evaluate for infectivity in asymptomatic patients with a positive screening admission or pre-procedural test or in cases when ongoing infection was suspected (critical illness or profound immunosuppression). We collected minus strand-specific assays performed at Stanford Healthcare during August 2020-April 2022. We describe basic demographics and clinical characteristics for patients who reverted from undetectable to detectable using the minus strand-specific assay. Results. A total of 2,505 strand-specific tests were collected from August 2020-April 2022 from 2,064 patients. A total of 292 (14%) had two or more strandspecific tests. Of them, 19 (7%) had an undetected minus strand-specific assay followed by a subsequent detectable value. Of them, seven (37%) had a minus strandspecific CT value of < 33.Most were male (n=4), median age was 54 (range: 8-62). All were profoundly immunosuppressed: Four had hematologic malignancies and three were post transplantation (kidney, lung, bone marrow). Median time from onset of symptoms or first positive test to reversion was 41 days (range:27-159). Median time from undetectable to detectable minus strand specific test was 26 days (range:4-34). All cases were considered relapses or ongoing infection rather than a new infection. Conclusion. Among patients with SARS-CoV-2 infection and consecutive strand-specific testing, a small proportion reverted from negative to positive. Most were profoundly immunosuppressed. The strand-specific assay can be an important tool in the evaluation of suspected cases of relapse or reinfection.

SARS-CoV-2 RNA and N Antigen Quantification via Wastewater at the Campus Level, Building Cluster Level, and Individual-Building Level

Monitoring wastewater for SARS-CoV-2 from populations smaller than those served by wastewater treatment plants may help identify small spatial areas (subsewersheds) where COVID-19 infections are present. We sampled wastewater from three nested locations with different sized populations within the same sewer network at a university campus and quantified SARS-CoV-2 RNA using reverse transcriptase droplet digital polymerase chain reaction (PCR). SARS-CoV-2 RNA concentrations and/or concentrations normalized by PMMoV were positively associated with laboratory-confirmed COVID-19 cases for both the sewershed level and the subsewershed level. We also used an antigen-based assay to detect the nucleocapsid (N) antigen from SARS-CoV-2 in wastewater samples at the sewershed level. The N antigen was regularly detected at the sewershed level, but the results were not associated with either laboratory-confirmed COVID-19 cases or SARS-CoV-2 RNA concentrations. The results of this study indicate that wastewater monitoring based on quantification of SARS-CoV-2 RNA using PCR-based methods is associated with COVID-19 cases at multiple geographic scales within the subsewershed level and can serve to aid the public health response.

A microfluidic approach to rapid crispr-based detection of sars-cov-2 RNA

We will present a microfluidic assay to detect SARS-CoV-2 RNA from raw nasopharyngeal swab samples in <30 min. Our method combines isotachophoresis (ITP) and state-of-the-art CRISPR Cas12-based nucleic acid detection. We show, for the first time, that electric field gradients in ITP can be used to pre-concentrate Cas12-guide RNA complex, target nucleic acids, and reporter molecules by ~1,000-fold into a ~100 pL volume on-chip. This pre-concentration accelerates target-activated CRISPR enzymatic activity on reporter molecules. We combine our ITP-CRISPR method with ITP-based nucleic acid extraction and isothermal amplification to detect Covid-19 from both contrived and clinical patient samples. © 2020 CBMS-0001