scholarly journals Pooling Upper Respiratory Specimens for Rapid Mass Screening of COVID-19 by Real-Time RT-PCR

2020 ◽  
Vol 26 (10) ◽  
pp. 2469-2472
Author(s):  
So Yeon Kim ◽  
Jaehyeon Lee ◽  
Heungsup Sung ◽  
Hyukmin Lee ◽  
Myung Guk Han ◽  
...  
Keyword(s):  
Real Time ◽  
Mass Screening ◽  
Rt Pcr ◽  
Upper Respiratory ◽  
Rapid Mass ◽  
Respiratory Specimens

scholarly journals Pooling of Upper Respiratory Specimens Using a SARS-CoV-2 Real-time RT-PCR Assay Authorized for Emergency Use in Low-Prevalence Populations for High-Throughput Testing

2020 ◽  
Vol 7 (11) ◽  
Author(s):  
Gwynngelle A Borillo ◽  
Ron M Kagan ◽  
Russell E Baumann ◽  
Boris M Fainstein ◽  
Lamela Umaru ◽  
...  
Keyword(s):  
Real Time ◽  
False Negative ◽  
False Negative Rate ◽  
Nucleic Acid Amplification ◽  
Rt Pcr ◽  
Pooled Testing ◽  
Negative Results ◽  
Single Positive ◽  
Upper Respiratory ◽  
Respiratory Specimens

Abstract Background Nucleic acid amplification testing is a critical tool for addressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Specimen pooling can increase throughput and conserve testing resources but requires validation to ensure that reduced sensitivity does not increase the false-negative rate. We evaluated the performance of a real-time reverse transcription polymerase chain reaction (RT-PCR) test authorized by the US Food and Drug Administration (FDA) for emergency use for pooled testing of upper respiratory specimens. Methods Positive specimens were selected from 3 prevalence groups, 1%–3%, >3%–6%, and >6%–10%. Positive percent agreement (PPA) was assessed by pooling single-positive specimens with 3 negative specimens; performance was assessed using Passing-Bablok regression. Additionally, we assessed the distributions of RT-PCR cycle threshold (Ct) values for 3091 positive specimens. Results PPA was 100% for the 101 pooled specimens. There was a linear relationship between Ct values for pooled and single-tested specimens (r = 0.96–0.99; slope ≈ 1). The mean pooled Ct shifts at 40 cycles were 2.38 and 1.90, respectively, for the N1 and N3 targets. The median Cts for 3091 positive specimens were 25.9 (N1) and 24.7 (N3). The percentage of positive specimens with Cts between 40 and the shifted Ct was 1.42% (N1) and 0.0% (N3). Conclusions Pooled and individual testing of specimens positive for SARS-CoV-2 demonstrated 100% agreement, which demonstrates the viability of pooled specimens for SARS-COV-2 testing using a dual-target RT-PCR system. Pooled specimen testing can help increase testing capacity for SARS-CoV-2 with a low risk of false-negative results.

scholarly journals Comparison of real-time RT-PCR and RT-PCR/MALDI-TOF methods for SARS-CoV-2 detection in saliva

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S9-S9
Author(s):  
Matthew M Hernandez ◽  
Radhika Banu ◽  
Paras Shrestha ◽  
Armi Patel ◽  
Feng Chen ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
N Gene ◽  
Rt Pcr ◽  
Upper Respiratory ◽  
High Level ◽  
Respiratory Specimens ◽  
Pcr Test ◽  
Roche Cobas

Abstract Background The coronavirus disease 2019 pandemic has accelerated the need for rapid validation and implementation of assays for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in diagnostic specimens. Multiple molecular methods have received emergency use authorization by the U.S. Food and Drug Administration for detection of SARS-CoV-2 in upper respiratory specimens, with testing of nasopharyngeal (NP) specimens serving as the foundation for these assays. However, supply chain constraints and the need for improved ease and safety of collection have prompted consideration of other specimen types as alternatives to NP specimens for detection of SARS-CoV-2. Here, we compared two methods for SARS-CoV-2 detection in saliva: the Roche cobas® 6800 SARS-CoV-2 real-time RT-PCR Test (“Roche”), which tests for viral ORF1ab (target 1, T1) and envelope E genes (target 2, T2); and the Agena Biosciences MassARRAY® SARS-CoV-2 Panel/MassARRAY® System (“Agena”), which tests for targets in the ORF1ab gene (ORF1, Orf1ab) and nucleocapsid N gene (N1, N2, N3). Methods Sixty saliva specimens collected within 48 hours of SARS-CoV-2 detection in an upper respiratory (anterior nares or NP) specimen from the same individual were tested in both the Roche and Agena platforms. Each system was evaluated for overall detection results and agreement with results of matched upper respiratory specimens. In addition, we determined the limit of detection (LoD) for each system and its component targets using an in-house SARS-CoV-2 standard generated from pooled positive saliva specimens quantitated against a commercially available standard (ZeptoMetrix NATSARS(COV2)-ERC). Results Both platforms demonstrated a similarly high sensitivity (97%) and specificity (100%) when compared to matched patient upper respiratory specimens and had high agreement with one another (Cohen’s κ = 0.9321, p = 2.6x10-13). Overall, the LoD (copies/mL) for the Roche assay was four times lower than that of Agena for saliva specimens (390.6 v. 1562.5). Furthermore, we determined that the LoD differed among the target components of each assay. The experimental LoD was comparable across Roche targets, but probit analyses indicate T2 has greater sensitivity (LoD: 228.6), Of the five Agena targets, the N2 target had the lowest LoD (1562.5). Conclusions In sum, we demonstrate that saliva is an acceptable specimen for testing in both the Roche cobas® 6800 SARS-CoV-2 real-time RT-PCR Test and the Agena Biosciences MassARRAY® SARS-CoV-2 Panel/MassARRAY® System, and both provide sensitive and specific detection of SARS-CoV-2 in saliva specimens. Although there was a high level of agreement between platforms, the LoD was lower for the Roche compared to the Agena assay with T2 and N2 being the most sensitive targets on each platform, respectively. The addition of saliva as an acceptable specimen and understanding the sensitivity for testing on these platforms can further inform public health measures for screening and detection to combat the pandemic.

scholarly journals MULTIPLEX SYBR GREEN ASSAY FOR CORONAVIRUS DETECTION USING FAST REAL-TIME RT-PCR

2020 ◽  
Vol 51 (2) ◽  
pp. 556-564
Author(s):  
Salah & et al.
Keyword(s):  
Respiratory Tract ◽  
Real Time ◽  
Melting Curve ◽  
First Record ◽  
Sybr Green ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Efficient Detection ◽  
Upper Respiratory ◽  
Tract Infections

This study was aimed to provide a local database for detection of coronavirus (CoV) species in suspect individual with respiratory tract infections like influenza type A and a tuberculosis using multiplex Sybr green reverse transcriptase real-time PCR (rRT-PCR) technique. A total of 500 samples was collected from individuals suffering from upper and/or lower respiratory tract diseases for testing of 4 CoV species (229E, OC43, NL63, and HKU1). RNA extracted, amplified and subsequent the positive samples sequencing. The results showed melting curve analysis (Tm) of the specific amplicons (79.73±0.36) and 9% positive for CoVs  and some of them have other co-infection such as influenza virus 26.67%, and TB 11.11%. On the other hands, the CoVs were detected 4.62% in upper respiratory samples and 20.39% with lower respiratory samples. Sequencing results pointed out two isolates were CoV-NL63 and four isolates were CoV-229E, with first record accession number MN086823.1 and MN086824.1, respectively in GenBank. In conclusion, this rRT-PCR showed the rapid and efficient detection of CoVs with few copies number. This allows being used for the diagnosis of CoVs along with other respiratory viruses in a multiplex assay to reduce processing time. Subsequent applied nested RT-PCR to overcome the low viral load.

scholarly journals The Effect of Sample Site, Illness Duration, and the Presence of Pneumonia on the Detection of SARS-CoV-2 by Real-time Reverse Transcription PCR

2020 ◽  
Vol 7 (9) ◽  
Author(s):  
Stephanie Sutjipto ◽  
Pei Hua Lee ◽  
Jun Yang Tay ◽  
Shehara M Mendis ◽  
Mohammad Yazid Abdad ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Sampling Site ◽  
Upper Respiratory Tract ◽  
Clinical Sensitivity ◽  
Illness Onset ◽  
Reverse Transcription Pcr ◽  
Upper Respiratory ◽  
Polymerase Chain ◽  
Respiratory Specimens

Abstract Background The performance of real-time reverse transcription polymerase chain reaction (rRT-PCR) for SARS-CoV-2 varies with sampling site(s), illness stage, and infection site. Methods Unilateral nasopharyngeal, nasal midturbinate, throat swabs, and saliva were simultaneously sampled for SARS-CoV-2 rRT-PCR from suspected or confirmed cases of COVID-19. True positives were defined as patients with at least 1 SARS-CoV-2 detected by rRT-PCR from any site on the evaluation day or at any time point thereafter, until discharge. Diagnostic performance was assessed and extrapolated for site combinations. Results We evaluated 105 patients; 73 had active SARS-CoV-2 infection. Overall, nasopharyngeal specimens had the highest clinical sensitivity at 85%, followed by throat, 80%, midturbinate, 62%, and saliva, 38%–52%. Clinical sensitivity for nasopharyngeal, throat, midturbinate, and saliva was 95%, 88%, 72%, and 44%–56%, respectively, if taken ≤7 days from onset of illness, and 70%, 67%, 47%, 28%–44% if >7 days of illness. Comparing patients with upper respiratory tract infection (URTI) vs pneumonia, clinical sensitivity for nasopharyngeal, throat, midturbinate, and saliva was 92% vs 70%, 88% vs 61%, 70% vs 44%, 43%–54% vs 26%–45%, respectively. A combination of nasopharyngeal plus throat or midturbinate plus throat specimen afforded overall clinical sensitivities of 89%–92%; this rose to 96% for persons with URTI and 98% for persons ≤7 days from illness onset. Conclusions Nasopharyngeal specimens, followed by throat specimens, offer the highest clinical sensitivity for COVID-19 diagnosis in early illness. Clinical sensitivity improves and is similar when either midturbinate or nasopharyngeal specimens are combined with throat specimens. Upper respiratory specimens perform poorly if taken after the first week of illness or if there is pneumonia.

scholarly journals Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 test for bronchoalveolar lavage

2021 ◽  
Author(s):  
Tung Phan ◽  
Ashley Mays ◽  
Melissa McCullough ◽  
Alan Wells
Keyword(s):  
Bronchoalveolar Lavage ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Rapid Test ◽  
Rt Pcr ◽  
Qualitative Detection ◽  
Upper Respiratory ◽  
Prompt Diagnosis ◽  
Respiratory Specimens ◽  
Rapid Laboratory

Accurate and rapid laboratory tests are essential for the prompt diagnosis of COVID-19, which is important to patients and infection control. The Xpert Xpress SARS-CoV-2 test is a real-time RT-PCR intended for the qualitative detection of nucleic acid from SARS-CoV-2 in upper respiratory specimens. In this study, we assessed the analytical and clinical performance characteristics of this rapid test for SARS-CoV-2 in 60 bronchoalveolar lavage (BAL) specimens. BAL is a specimen type that is not authorized under EUA for the Xpert Xpress SARS-CoV-2 test. The limit of detection of the Xpert Xpress SARS-CoV-2 test was 500 copies/ml. The overall agreement of the Xpert Xpress SARS-CoV-2 test was 100%. The Xpert Xpress SARS-CoV-2 test is sensitive and specific to aid in diagnosis of COVID-19 using bronchoalveolar lavage.

scholarly journals Comparison of a singleplex real-time RT-PCR assay and multiplex respiratory viral panel assay for detection of influenza “A” in respiratory specimens

2010 ◽  
Vol 5 (2) ◽  
pp. 99-103 ◽  
Author(s):  
Kanti Pabbaraju ◽  
Sallene Wong ◽  
Bonita Lee ◽  
Raymond Tellier ◽  
Kevin Fonseca ◽  
...  
Keyword(s):  
Real Time ◽  
Influenza A ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Respiratory Specimens

scholarly journals Development of the one-step qualitative RT-PCR assay to detect SARS-CoV-2 Omicron (B.1.1.529) variant in respiratory specimens

2022 ◽  
Author(s):  
Tung Phan ◽  
Stephanie Boes ◽  
Melissa McCullough ◽  
Jamie Gribschaw ◽  
Alan Wells
Keyword(s):  
Limit Of Detection ◽  
Cross Reactivity ◽  
Upper Respiratory Tract ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Qualitative Detection ◽  
One Step ◽  
Upper Respiratory ◽  
The One ◽  
Respiratory Specimens

A new SARS-CoV-2 Omicron (B.1.1.529) Variant of Concern has been emerging worldwide. We are seeing an unprecedented surge in patients due to Omicron in this COVID-19 pandemic. A rapid and accurate molecular test that effectively differentiates Omicron from other SARS-CoV-2 variants would be important for both epidemiologic value and for directing variant-specific therapies such as monoclonal antibody infusions. In this study, we developed a real-time RT-PCR assay for the qualitative detection of Omicron from routine clinical specimens sampling the upper respiratory tract. The limit of detection of the SARS-CoV-2 Omicron variant RT-PCR assay was 2 copies/μl. Notably, the assay did not show any cross-reactivity with other SARS-CoV-2 variants including Delta (B.1.617.2). This SARS-CoV-2 Omicron variant RT-PCR laboratory-developed assay is sensitive and specific to detect Omicron in nasopharyngeal and nasal swab specimens.

scholarly journals SARS-CoV-2 virus culture from the upper respiratory tract: Correlation with viral load, subgenomic viral RNA and duration of illness.

2020 ◽  
Author(s):  
Ranawaka APM Perera ◽  
Eugene Tso ◽  
Owen TY Tsang ◽  
Dominic NC Tsang ◽  
Kitty Fung ◽  
...  
Keyword(s):  
Viral Load ◽  
Upper Respiratory Tract ◽  
Genomic Rna ◽  
Rt Pcr ◽  
Mild Disease ◽  
Virus Rna ◽  
Duration Of Illness ◽  
Virus Culture ◽  
Upper Respiratory ◽  
Respiratory Specimens

In 68 respiratory specimens from a cohort of 35 COVID-19 patients, 32 of them with mild disease, we found SARS coronavirus-2 virus culture and sub-genomic RNA was rarely detectable beyond 8 days after onset of illness although virus RNA by RT-PCR remained detectable for many weeks.

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