Sensitivity of ID NOW and RT-PCR for detection of SARS-CoV-2 in an ambulatory population.
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ABSTRACT:
SUBMITTER: Tu YP
PROVIDER: S-EPMC8081522 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Fast, accurate, and reliable diagnostic tests are critical for controlling the spread of the coronavirus disease 2019 (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The current gold standard for testing is real-time PCR; however, during the current pandemic, supplies of testing kits and reagents have been limited. We report the validation of a rapid (30 minutes), user-friendly, and accurate microchip real-time PCR assay for detection of SARS-CoV-2 from nasopharyngeal swab RNA extracts. Microchips preloaded with COVID-19 primers and probes for the N gene accommodate 1.2-μL reaction volumes, lowering the required reagents by 10-fold compared with tube-based real-time PCR. We validated our assay using contrived reference samples and 21 clinical samples from patients in Canada, determining a limit of detection of 1 copy per reaction. The microchip real-time PCR provides a significantly lower resource alternative to the Centers for Disease Control and Prevention-approved real-time RT-PCR assays with comparable sensitivity, showing 100% positive and negative predictive agreement of clinical samples.
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Project description:Bivalve shellfish are readily contaminated by human pathogens present in waters impacted by municipal sewage, and the detection of SARS-CoV-2 in feces of infected patients and in wastewater has drawn attention to the possible presence of the virus in bivalves. The aim of this study was to collect data on SARS-CoV-2 prevalence in bivalve mollusks from harvesting areas of Campania region. A total of 179 samples were collected between September 2019 and April 2021 and were tested using droplet digital RT-PCR (dd RT-PCR) and real-time RT-PCR. Combining results obtained with different assays, SARS-CoV-2 presence was detected in 27/179 (15.1%) of samples. A median viral concentration of 1.1 × 102 and 1.4 × 102 g.c./g was obtained using either Orf1b nsp14 or RdRp/gene E, respectively. Positive results were unevenly distributed among harvesting areas and over time, positive samples being more frequent after January 2021. Partial sequencing of the spike region was achieved for five samples, one of which displaying mutations characteristic of the Alpha variant (lineage B.1.1.7). This study confirms that bivalve mollusks may bioaccumulate SARS-CoV-2 to detectable levels and that they may represent a valuable approach to track SARS-CoV-2 in water bodies and to monitor outbreak trends and viral diversity.
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Project description:The early detection and differential diagnosis of respiratory infections increase the chances for successful control of COVID-19 disease. The nucleic acid RT-PCR test is regarded as the current standard for molecular diagnosis. However, the maximal specificity confirmation target ORF1ab gene is considered to be less sensitive than other targets in clinical application. In addition, recent evidence indicated that the initial missed diagnosis of asymptomatic patients with SARS-CoV-2 and discharged patients with "re-examination positive" might be due to low viral load, and the ability of rapid mutation of SARS-CoV-2 also increases the rate of false-negative results. Moreover, the mixed sample nucleic acid detection is helpful in seeking out the early community transmission of SARS-CoV-2 rapidly, but the detection kit needs ultra-high detection sensitivity. Herein, the lowest detection concentration of different nucleic acid detection kits was evaluated and compared to provide direct evidence for the selection of kits for mixed sample detection or make recommendations for the selection of validation kit, which is of great significance for the prevention and control of the current epidemic and the discharge criteria of low viral load patients.
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Project description:Severe acute respiratory syndrome (SARS) is an acute newly emerged infectious respiratory illness. The etiologic agent of SARS was named 'SARS-associated coronavirus' (SARS-CoV) that can be detected with reverse transcription-polymerase chain reaction (RT-PCR) assays. In this study, 12 sets of nested primers covering the SARS-CoV genome have been screened and showed sufficient sensitivity to detect SARS-CoV in RNA isolated from virus cultured in Vero 6 cells. To optimize further the reaction condition of those nested primers sets, seven sets of nested primers have been chosen to compare their reverse transcribed efficiency with specific and random primers, which is useful to combine RT with the first round of PCR into a one-step RT-PCR. Based on the sensitivity and simplicity of results, the no. 73 primer set was chosen as the candidate primer set for clinical diagnoses. To specify the amplicon to minimize false positive results, a Taqman RT-nested PCR system of no. 73 nested primer set was developed. Through investigations on a test panel of whole blood obtained from 30 SARS patients and 9 control persons, the specificity and sensitivity of the Taqman RT-nested PCR system was found to be 100 and 83%, respectively, which suggests that the method is a promising one to diagnose SARS in early stages.
| S-EPMC7119531 | biostudies-literature
Project description:BackgroundThe outbreak of novel coronavirus disease 2019 (COVID-19) has become a public health emergency of international concern. Quantitative testing of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus is demanded in evaluating the efficacy of antiviral drugs and vaccines and RT-PCR can be widely deployed in the clinical assay of viral loads. Here, we developed a quantitative RT-PCR method for SARS-CoV-2 virus detection in this study.MethodsRT-PCR kits targeting E (envelope) gene, N (nucleocapsid) gene and RdRP (RNA-dependent RNA polymerase) gene of SARS-CoV-2 from Roche Diagnostics were evaluated and E gene kit was employed for quantitative detection of COVID-19 virus using Cobas Z480. Viral load was calculated according to the standard curve established by series dilution of an E-gene RNA standard provided by Tib-Molbiol (a division of Roche Diagnostics). Assay performance was evaluated.ResultsThe performance of the assay is acceptable with limit of detection (LOD) below 10E1 copies/μL and lower limit of quantification (LLOQ) as 10E2 copies/μL.ConclusionA quantitative detection of the COVID-19 virus based on RT-PCR was established.
| S-EPMC8677905 | biostudies-literature
Project description:BackgroundUnderstanding the false negative rates of SARS-CoV-2 RT-PCR testing is pivotal for the management of the COVID-19 pandemic and it has implications for patient management. Our aim was to determine the real-life clinical sensitivity of SARS-CoV-2 RT-PCR.MethodsThis population-based retrospective study was conducted in March-April 2020 in the Helsinki Capital Region, Finland. Adults who were clinically suspected of SARS-CoV-2 infection and underwent SARS-CoV-2 RT-PCR testing, with sufficient data in their medical records for grading of clinical suspicion were eligible. In addition to examining the first RT-PCR test of repeat-tested individuals, we also used high clinical suspicion for COVID-19 as the reference standard for calculating the sensitivity of SARS-CoV-2 RT-PCR.ResultsAll 1,194 inpatients (mean [SD] age, 63.2 [18.3] years; 45.2% women) admitted to COVID-19 cohort wards during the study period were included. The outpatient cohort of 1,814 individuals (mean [SD] age, 45.4 [17.2] years; 69.1% women) was sampled from epidemiological line lists by systematic quasi-random sampling. The sensitivity (95% CI) for laboratory confirmed cases (repeat-tested patients) was 85.7% (81.5-89.1%) inpatients; 95.5% (92.2-97.5%) outpatients, 89.9% (88.2-92.1%) all. When also patients that were graded as high suspicion but never tested positive were included in the denominator, the sensitivity (95% CI) was: 67.5% (62.9-71.9%) inpatients; 34.9% (31.4-38.5%) outpatients; 47.3% (44.4-50.3%) all.ConclusionsThe clinical sensitivity of SARS-CoV-2 RT-PCR testing was only moderate at best. The relatively high false negative rates of SARS-CoV-2 RT-PCR testing need to be accounted for in clinical decision making, epidemiological interpretations, and when using RT-PCR as a reference for other tests.
| S-EPMC8139477 | biostudies-literature
Project description:The COVID-19 pandemic requires sensitive detection of the SARS-CoV-2 virus from samples to ensure accurate detection of infected patients, an essential component of effective national track and trace programs. Due to the scaling challenges of large sample numbers, sample pooling is an attractive solution to reduce both extraction and amplification reagent costs, if high sensitivity can be maintained. We demonstrate that the Erba Molecular ErbaMDx SARS-CoV-2 RT-PCR Kit (EM kit) delivers high sensitivity, achieving analytical detection of 5 copies/reaction SARS-CoV-2 genomic RNA, and 200 copies/mL SARS-CoV-2 inactivated virus spiked into nasopharyngeal swab (NP) samples and extracted through workflow. Furthermore, the EM Kit demonstrates high sensitivity in both pooled (1 in 5) and non-pooled NP samples when compared to an FDA Emergency Use Authorization approved assay, following published FDA guidelines. These findings demonstrate that the EM Kit is suitable for sample pooling, with minimal impact on assay performance. As the COVID-19 pandemic progresses, high sensitivity assays such as the EM Kit will have an important role in ensuring high throughput and sensitive testing using pooled samples can be maintained, delivering the most cost-effective sample extraction and amplification option for national test and trace programs.
| S-EPMC8965539 | biostudies-literature
Project description:To identify animals susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection or to determine whether SARS-CoV-2 contaminated meat is from a SARS-CoV-2-infected animal, a convenient and safe method was developed for rapid detection of SARS-CoV-2 in a replicating or non-replicating status in samples using reverse transcriptase-polymerase chain reaction (RT-PCR). This strategy can also be applied to develop assays for the detection of other viruses, either replicating or not.
| S-EPMC7825825 | biostudies-literature
Project description:BackgroundThe SARS-CoV-2 pandemic has created an urgent and unprecedented need for rapid large-scale diagnostic testing to inform timely patient management. However, robust data are lacking on the relative performance of available rapid molecular tests across a full range of viral concentrations.ObjectiveThis study aimed to compare two recently-authorized rapid tests, Cepheid Xpert Xpress SARS-CoV-2 and Abbott ID Now SARS-CoV-2, to the Roche cobas SARS-CoV-2 assay for samples with low, medium, and high viral concentrations.Study designA total of 113 nasopharyngeal swabs from remnant patient samples were tested, including 88 positives spanning the full range of observed Ct values on the cobas assay.ResultsCompared to cobas, the overall positive agreement was 73.9% with ID Now and 98.9% with Xpert. Negative agreement was 100% and 92.0% for ID Now and Xpert, respectively. Both ID Now and Xpert showed 100% positive agreement for medium and high viral concentrations (Ct value <30). However, for Ct values >30, positive agreement was 34.3% for ID Now and 97.1% for Xpert.ConclusionsWhile Xpert showed high agreement with cobas across a wide range of viral concentrations, this study highlights an important limitation of ID Now for specimens collected in viral or universal transport media with low viral concentrations. Further studies are needed to evaluate the performance of ID Now for direct swabs.
| S-EPMC7217789 | biostudies-literature
Project description:Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) is the gold standard method for the diagnosis of COVID‑19 infection. Due to pre‑analytical and technical limitations, samples with low viral load are often misdiagnosed as false‑negative samples. Therefore, it is important to evaluate other strategies able to overcome the limits of RT‑qPCR. Blinded swab samples from two individuals diagnosed positive and negative for COVID‑19 were analyzed by droplet digital PCR (ddPCR) and RT‑qPCR in order to assess the sensitivity of both methods. Intercalation chemistries and a World Health Organization (WHO)/Center for Disease Control and Prevention (CDC)‑approved probe for the SARS‑CoV‑2 N gene were used. SYBR‑Green RT‑qPCR is not able to diagnose as positive samples with low viral load, while, TaqMan Probe RT‑qPCR gave positive signals at very late Ct values. On the contrary, ddPCR showed higher sensitivity rate compared to RT‑qPCR and both EvaGreen and probe ddPCR were able to recognize the sample with low viral load as positive even at 10‑fold diluted concentration. In conclusion, ddPCR shows higher sensitivity and specificity compared to RT‑qPCR for the diagnosis of COVID‑19 infection in false‑negative samples with low viral load. Therefore, ddPCR is strongly recommended in clinical practice for the diagnosis of COVID‑19 and the follow‑up of positive patients until complete remission.
| S-EPMC7388836 | biostudies-literature