Impact of repeated nasal sampling on detection and quantification of SARS-CoV-2.
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ABSTRACT:
SUBMITTER: Levy JM
PROVIDER: S-EPMC8295385 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In the context of an unprecedented shortage of nasopharyngeal swabs (NPS) or sample transport media during the coronavirus disease 2019 (COVID-19) crisis, alternative methods for sample collection are needed. To address this need, we validated a cell culture medium as a viral transport medium, and compared the analytical sensitivity of SARS-CoV-2 RT-PCR in nasal wash (NW), oropharyngeal swab (OPS), and NPS specimens. Both the clinical and analytical sensitivity were comparable in these three sample types. OPS and NW specimens may therefore represent suitable alternatives to NPS for SARS-CoV-2 detection.
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Project description:Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is the agent responsible for the coronavirus disease 2019 (COVID-19) global pandemic. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2003 SARS outbreak. Although numerous reagents were developed to study SARS-CoV infections, few have been applicable to evaluating SARS-CoV-2 infection and immunity. Current limitations in studying SARS-CoV-2 include few validated assays with fully replication-competent wild-type virus. We have developed protocols to propagate, quantify, and work with infectious SARS-CoV-2. Here, we describe: (1) virus stock generation, (2) RT-qPCR quantification of SARS-CoV-2 RNA; (3) detection of SARS-CoV-2 antigen by flow cytometry, (4) quantification of infectious SARS-CoV-2 by focus-forming and plaque assays; and (5) validated protocols for virus inactivation. Collectively, these methods can be adapted to a variety of experimental designs, which should accelerate our understanding of SARS-CoV-2 biology and the development of effective countermeasures against COVID-19.
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Project description:Detection of SARS-CoV-2 using RT–PCR and other advanced methods can achieve high accuracy. However, their application is limited in countries that lack sufficient resources to handle large-scale testing during the COVID-19 pandemic. Here, we describe a method to detect SARS-CoV-2 in nasal swabs using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and machine learning analysis. This approach uses equipment and expertise commonly found in clinical laboratories in developing countries. We obtained mass spectra from a total of 362 samples (211 SARS-CoV-2-positive and 151 negative by RT–PCR) without prior sample preparation from three different laboratories. We tested two feature selection methods and six machine learning approaches to identify the top performing analysis approaches and determine the accuracy of SARS-CoV-2 detection. The support vector machine model provided the highest accuracy (93.9%), with 7% false positives and 5% false negatives. Our results suggest that MALDI-MS and machine learning analysis can be used to reliably detect SARS-CoV-2 in nasal swab samples.
2020-09-11 | PXD021388 | Pride
Project description:Nasal microbiome quantification identifies bacterial features specific to different sampling methods
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Project description:Background: The gold standard for COVID-19 diagnosis relies on quantitative reverse-transcriptase polymerase-chain reaction (RT-qPCR) from nasopharyngeal swab (NPS) specimens, but NPSs present several limitations. The simplicity, low invasive and possibility of self-collection of saliva imposed these specimens as a relevant alternative for SARS-CoV-2 detection. However, the discrepancy of saliva test results compared to NPSs made of its use controversial. Here, we assessed Salivettes®, as a standardized saliva collection device, and compared SARS-CoV-2 positivity on paired NPS and saliva specimens. Methods: A total of 303 individuals randomly selected among those investigated for SARS-CoV-2 were enrolled, including 30 (9.9%) patients previously positively tested using NPS (follow-up group), 90 (29.7%) mildly symptomatic and 183 (60.4%) asymptomatic. Results: The RT-qPCR revealed a positive rate of 11.6% (n = 35) and 17.2% (n = 52) for NPSs and saliva samples, respectively. The sensitivity and specificity of saliva samples were 82.9% and 91.4%, respectively, using NPS as reference. The highest proportion of discordant results concerned the follow-up group (33.3%). Although the agreement exceeded 90.0% in the symptomatic and asymptomatic groups, 17 individuals were detected positive only in saliva samples, with consistent medical arguments. Conclusion Saliva collected with Salivette® was more sensitive for detecting symptomatic and pre-symptomatic infections.
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Project description:Saliva has significant advantages as a test medium for detection of SARS-CoV-2 infection in patients, such as ease of collection, minimal requirement of supplies and trained personnel, and safety. Comprehensive validation in a large cohort of prospectively collected specimens with unknown SARS-CoV-2 status should be performed to evaluate the potential and limitations of saliva-based testing. We developed a saliva-based testing pipeline for detection of SARS-CoV-2 nucleic acids using real-time reverse transcription PCR (RT-PCR) and droplet digital PCR (ddPCR) readouts, and measured samples from 137 outpatients tested at a curbside testing facility and 29 inpatients hospitalized for COVID-19. These measurements were compared to the nasal swab results for each patient performed by a certified microbiology laboratory. We found that our saliva testing positively detects 100% (RT-PCR) and 93.75% (ddPCR) of curbside patients that were identified as SARS-CoV-2 positive by the Emergency Use Authorization (EUA) certified nasal swab testing assay. Quantification of viral loads by ddPCR revealed an extremely wide range, with 1 million-fold difference between individual patients. Our results demonstrate for both community screening and hospital settings that saliva testing reliability is on par with that of the nasal swabs in detecting infected cases, and has potential for higher sensitivity when combined with ddPCR in detecting low-abundance viral loads that evade traditional testing methods.
| S-EPMC8203799 | biostudies-literature
Project description:Respiratory infections, like the current COVID-19 pandemic, target epithelial cells in the respiratory tract. Alveolar macrophages (AMs) are tissue-resident macrophages located within the lung. They play a key role in the early phases of an immune response to respiratory viruses. AMs are likely the first immune cells to encounter SARS-CoV-2 during an infection and their reaction to the virus will have a profound impact on the outcome of the infection. Interferons (IFNs) are antiviral cytokines and among the first cytokines produced upon viral infection. In this study, AMs from non-infectious donors are challenged with SARS-CoV-2. We demonstrate that challenged AMs are incapable of sensing SARS-CoV-2 and of producing an IFN response in contrast to other respiratory viruses, like influenza A virus and Sendai virus, which trigger a robust IFN response. The absence of IFN production in AMs upon challenge with SARS-CoV2 could explain the initial asymptotic phase observed during COVID-19 and argues against AMs being the sources of proinflammatory cytokines later during infection.
| S-SCDT-EMBOR-2020-51252V1 | biostudies-other
Project description:We examined the detection rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using reverse transcription-PCR (RT-PCR) of side-by-side saliva and oropharyngeal swab (OPS) samples from 639 symptomatic and asymptomatic subjects, of which 47 subjects were found to be positive for SARS-CoV-2 in the OPS or saliva sample or both. It was found that the detection rate (93.6% for both OPS and saliva) as well as the sensitivity and specificity were comparable between the two sampling methods in this cohort. The sensitivity was 0.932 (95% confidence interval [CI], 0.818 to 0.977) and the specificity was 0.995 (95% CI, 0.985 to 0.998), both for saliva when OPS sampling was used as the reference and for OPS when saliva was used as the reference. Furthermore, the Cohen's kappa value was 0.926 (95% CI, 0.868 to 0.985), indicating strong agreement between the two sampling methods. In addition, the viral RNA stability in pure saliva and saliva mixed with preservation buffers was examined following storage at room temperature and at 4°C. It was found that pure saliva kept the viral RNA stable for 9 days at both temperatures and that the type of preservation buffer can either enhance or reduce the stability of the RNA. We conclude that self-administered saliva sampling is an attractive alternative to oropharyngeal swabbing for SARS-CoV-2 detection, and it might be useful in large-scale testing. IMPORTANCE It is not inconceivable that we will witness recurring surges of COVID-19 before the pandemic finally recedes. It is therefore still relevant to look for feasible, simple, and flexible screening methods so that schools, workplaces, and communities in general can avoid lockdowns. In this work, we analyzed two different sampling methods: oropharyngeal swabs and saliva collection. Oropharyngeal swabs must be collected by trained health personnel at clinics, whereas self-assisted saliva collection can be performed at any given location. It was found that the two sampling methods were comparable. Saliva sampling is a simple method that allows easy mass testing using minimal resources from the existing health care system, and this method may therefore prove to be an effective tool for containing the COVID-19 pandemic.
| S-EPMC9604035 | biostudies-literature
Project description:IntroductionMost SARS-CoV-2 antigen-detecting rapid diagnostic tests require nasopharyngeal sampling, which is frequently perceived as uncomfortable and requires healthcare professionals, thus limiting scale-up. Nasal sampling could enable self-sampling and increase acceptability. The term nasal sampling is often not used uniformly and sampling protocols differ.MethodsThis manufacturer-independent, prospective diagnostic accuracy study, compared professional anterior nasal and nasal mid-turbinate sampling for a WHO-listed SARS-CoV-2 antigen-detecting rapid diagnostic test. The second group of participants collected a nasal mid-turbinate sample themselves and underwent a professional nasopharyngeal swab for comparison. The reference standard was real-time polymerase chain reaction (RT-PCR) using combined oro-/nasopharyngeal sampling. Individuals with high suspicion of SARS-CoV-2 infection were tested. Sensitivity, specificity, and percent agreement were calculated. Self-sampling was observed without intervention. Feasibility was evaluated by observer and participant questionnaires.ResultsAmong 132 symptomatic adults, both professional anterior nasal and nasal mid-turbinate sampling yielded a sensitivity of 86.1% (31/36 RT-PCR positives detected; 95%CI: 71.3-93.9) and a specificity of 100.0% (95%CI: 95.7-100). The positive percent agreement was 100% (95%CI: 89.0-100). Among 96 additional adults, self nasal mid-turbinate and professional nasopharyngeal sampling yielded an identical sensitivity of 91.2% (31/34; 95%CI 77.0-97.0). Specificity was 98.4% (95%CI: 91.4-99.9) with nasal mid-turbinate and 100.0% (95%CI: 94.2-100) with nasopharyngeal sampling. The positive percent agreement was 96.8% (95%CI: 83.8-99.8). Most participants (85.3%) considered self-sampling as easy to perform.ConclusionProfessional anterior nasal and nasal mid-turbinate sampling are of equivalent accuracy for an antigen-detecting rapid diagnostic test in ambulatory symptomatic adults. Participants were able to reliably perform nasal mid-turbinate sampling themselves, following written and illustrated instructions. Nasal self-sampling will facilitate scaling of SARS-CoV-2 antigen testing.
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