SARS-CoV-2 Detection in Different Respiratory Sites: A Systematic Review and Meta-Analysis

BackgroundThe accurate detection of SARS-CoV-2 through respiratory sampling is critical for the prevention of further transmission and the timely initiation of treatment for COVID-19. There is a diverse range of SARS-CoV-2 detection rates in reported studies, with uncertainty as to the optimal sampling strategy for COVID-19 diagnosis and monitoring. MethodsWe performed a systematic review and meta-analysis of studies comparing respiratory sampling strategies for the detection of SARS-CoV-2 RNA. The inclusion criteria were studies that assessed at least two respiratory sampling sites (oropharyngeal swab, nasopharyngeal swab, and sputum) in participants with COVID-19. The percentage positive tests were compared between sampling modalities by constructing a Z-test assuming independence and using the standard errors obtained from the random effects meta-analysis. FindingsFrom 1039 total studies, we identified 11 studies that met our inclusion criteria, with SARS-CoV-2 testing results from a total of 3442 respiratory tract specimens. Compared to nasopharyngeal swab sampling, sputum testing resulted in significantly higher rates of SARS-CoV-2 RNA detection while oropharyngeal swab testing had lower rates of viral RNA detection. Earlier sampling after symptom onset was associated with improved detection rates, but the differences in SARS-CoV-2 RNA detection by sampling method was consistent regardless of the duration of symptoms. InterpretationThe results support sputum sampling as a primary method of COVID-19 diagnosis and monitoring, and highlight the importance of early testing after symptom onset to increase the rates of COVID-19 diagnosis. FundingThis study was funded in part by the NIH grants U01AI106701 and by the Harvard University for AIDS Research (NIAID 5P30AI060354).

A Rapid SARS-CoV-2 Variant Detection by Molecular-Clamping Based RT-qPCR

We applied XNA-based Molecular Clamping Technology to develop a multiplex qPCR assay for rapid and accurate detection of SARS-CoV-2 mutations. A total of 278 previously tested SARS-COV-2 positive samples originating primarily from San Francisco Bay Area were tested, including 139 Samples collected in middle January and 139 samples collected at the end of February 2021, respectively. The SARS-CoV-2 Spike-gene D614G mutation was detected from 58 samples (41.7%) collected in January 2021 and, 78 samples (56.1%) collected in February. Notably, while there were no N501Y mutation detected in samples from January, seven of the February samples were tested positive for the N501Y and D614G mutations. The results suggest a relatively recent and speedy spreading of the UK variant (B.1.1.7) in Northern California. This new Molecular Clamping technology-based multiplex RT-qPCR assay is highly sensitive and specific and can help speed up large scale testing for SARS-CoV-2 variants.

Saliva as a testing specimen with or without pooling for SARS-CoV-2 detection by multiplex RT-PCR test

BackgroundSensitive and high throughput molecular detection assays are essential during the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vast majority of the SARS-CoV-2 molecular assays use nasopharyngeal swab (NPS) or oropharyngeal swab (OPS) specimens collected from suspected individuals. However, using NPS or OPS as specimens has apparent drawbacks, e.g. the collection procedures for NPS or OPS specimens can be uncomfortable to some people and may cause sneezing and coughing which in turn generate droplets and/or aerosol particles that are of risk to healthcare workers, requiring heavy use of personal protective equipment. There have been recent studies indicating that self-collected saliva specimens can be used for molecular detection of SARS-CoV-2 and provides more comfort and ease of use for the patient. Here we report the performance of QuantiVirus SARS-CoV-2 multiplex test using saliva as the testing specimens with or without pooling. MethodsDevelopment and validation studies were conducted following FDA-EUA and molecular assay validation guidelines. Using SeraCare Accuplex SARS-CoV-2 reference panel, the limit of detection (LOD) and clinical evaluation studies were performed with the QuantiVirus SARS-CoV-2 multiplex test. For clinical evaluation, 85 known positive and 90 known negative clinical NPS samples were tested. Additionally, twenty paired NPS and saliva samples collected from recovering COVID-19 patients were tested and the results were further compared to that of the Abbott m2000 SARS-CoV-2 PCR assay. Results of community collected 389 saliva samples for COVID-19 screening by QuantiVirus SARS-CoV-2 multiplex test were also obtained and analyzed. Moreover, saliva pooling with 6 and 12 samples together were also evaluated. ResultsThe LOD for the QuantiVirus SARS-CoV-2 multiplex test was confirmed to be 100-200 copies/mL. The clinical evaluation using contrived saliva samples indicated that the positive percentage agreement (PPA) of the QuantiVirus SARS-CoV-2 multiplex test is 100% at 1xLOD, 1.5xLOD and 2.5xLOD. No cross-reactivity was observed for the QuantiVirus SARS-CoV-2 multiplex test with common respiratory pathogens. Testing of clinical samples showed a positive percentage agreement (PPA) of 100% (95% CI: 94.6% to 100%) and a negative percentage agreement (NPA) of 98.9% (95% CI: 93.1% to 99.9%). QuantiVirus SARS CoV-2 multiplex test had 80% concordance rate and no significant difference (p=0.13) in paired saliva and NPS specimens by Wilcoxon matched pairs signed rank test. Positive test rate was 1.79% for 389 saliva specimens collected from the communities for COVID-19 screening. Preliminary data showed that saliva sample pooling up to 6 samples for SARS-CoV-2 detection is feasible (sensitivity 94.8% and specificity 100%). ConclusionThe studies demonstrated that the QuantiVirus SARS-CoV-2 multiplex test has a LOD of 200 copies/mL in contrived saliva samples. The clinical performance of saliva-based testing is comparable to that of NPS-based testing. Pooling of saliva specimens for SARS-CoV-2 detection is feasible. Saliva based and high-throughput QuantiVirusSARS-CoV-2 multiplex test offers a highly desirable test during the ongoing COVID-19 pandemic.

Hospital-Based Donor Recruitment and Pre-Donation Serologic Testing for COVID-19 Convalescent Plasma

BackgroundMajor blood centers perform serologic testing on potential COVID-19 convalescent plasma donors retrospectively after blood donation. A hospital-based recruitment program for COVID-19 convalescent plasma (CCP) donors may be an efficient way to prospectively identify potential donors. Study Design and MethodsPatients who recovered from known or suspected COVID-19 were identified and recruited through medical record searches and public appeals. Participants were screened with a modified donor history questionnaire (DHQ), and if eligible, were consented and tested for SARS-CoV-2 antibodies (IgG and IgM). Participants who were positive for SARS-CoV-2 IgG were referred to a local blood center for convalescent plasma collection. ResultsOf 179 individuals screened, 128 completed serologic testing and 89 were referred for convalescent plasma donation to a local blood center (49.7% of those screened). IgG antibodies to SARS-CoV-2 were detected in 23/51 (45.1%) of participants with suspected COVID-19 and in 66/77 (85.7%) of participants with self-reported PCR-confirmed COVID-19. Testing was performed at a median of 38 days since last symptoms. Participant age positively correlated with anti-SARS-CoV-2 IgG and IgM levels. Time since last symptoms did not correlate with IgG or IgM levels. A wide range of SARS-CoV-2 IgG levels were observed. ConclusionA hospital based CCP donor recruitment program can prospectively identify potential CCP donors. Variability in SARS-CoV-2 IgG levels has implications for selection of CCP units for transfusion.

Point-of-care testing for COVID-19 using SHERLOCK diagnostics

The recent outbreak of the novel coronavirus SARS-CoV-2, which causes COVID-19, can be diagnosed using RT-qPCR, but inadequate access to reagents and equipment has slowed disease detection and impeded efforts to mitigate viral spread. Alternative approaches based on combinations of isothermal amplification and CRISPR-mediated detection, such as the SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) technique, offer reduced dependence on RT-qPCR equipment, but previously reported methods required multiple fluid handling steps, complicating their deployment outside clinical labs. Here we developed a simple test chemistry called STOP (SHERLOCK Testing in One Pot) for detecting SARS-CoV-2 in one hour that is suitable for point-of-care use. This simplified test, STOPCovid, provides sensitivity comparable to RT-qPCR-based SARS-CoV-2 tests and has a limit of detection of 100 copies of viral genome input in saliva or nasopharyngeal swabs per reaction. Using lateral flow readout, the test returns result in 70 minutes, and using fluorescence readout, the test returns result in 40 minutes. Moreover, we validated STOPCovid using nasopharyngeal swabs from COVID-19 patients and were able to correctly diagnose 12 positive and 5 negative patients out of 3 replicates. We envision that implementation of STOPCovid will significantly aid "test-trace-isolate" efforts, especially in low-resource settings, which will be critical for long-term public health safety and effective reopening of the society.

SARS-CoV-2 Viral Load is Associated with Increased Disease Severity and Mortality

The relationship between SARS-CoV-2 viral load and risk of disease progression remains largely undefined in coronavirus disease 2019 (COVID-19). We quantified SARS-CoV-2 viral load from participants with a diverse range of COVID-19 severity, including those requiring hospitalization, outpatients with mild disease, and individuals with resolved infection. SARS-CoV-2 plasma RNA was detected in 27% of hospitalized participants and 13% of outpatients diagnosed with COVID-19. Amongst the participants hospitalized with COVID-19, higher prevalence of detectable SARS-CoV-2 plasma viral load was associated with worse respiratory disease severity, lower absolute lymphocyte counts, and increased markers of inflammation, including C-reactive protein and IL-6. SARS-CoV-2 viral loads, especially plasma viremia, were associated with increased risk of mortality. SARS-CoV-2 viral load may aid in the risk stratification of patients with COVID-19 and its role in disease pathogenesis should be further explored.