Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples

Abstract The aim of this study was to identify and validate a sensitive, high-throughput and cost-effective SARS-CoV-2 RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a University surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The EZ-SARS-CoV-2 RT-PCR assay analytical sensitivity was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. Clinical performance of the EZ-SARS-CoV-2 assay was determined using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94% and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by Cohen’s kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity, excellent performance in AN sample matrix and in pooled upper respiratory samples support its use in a high-throughput surveillance testing program.

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Comparison of two commercial molecular tests and a laboratory-developed modification of the CDC 2019-nCOV RT-PCR assay for the qualitative detection of SARS-CoV-2 from upper respiratory tract specimens

10.1101/2020.05.02.20088740 ◽

2020 ◽

Cited By ~ 2

Author(s):

Nicholas M. Moore ◽

Haiying Li ◽

Debra Schejbal ◽

Jennifer Lindsley ◽

Mary K. Hayden

Keyword(s):

Respiratory Tract ◽

Medical Record ◽

Medical Record Review ◽

Nasopharyngeal Swab ◽

Upper Respiratory Tract ◽

Rt Pcr ◽

Pcr Assay ◽

Upper Respiratory ◽

Record Review ◽

Swab Specimen

ABSTRACTWe compared the ability of 2 commercial molecular amplification assays [RealTime SARS-CoV-2 on the m2000 (Abbott) and ID NOW™ COVID-19 (Abbott)] and a laboratory-developed test [modified CDC 2019-nCoV RT-PCR assay with RNA extraction by eMag® (bioMérieux) and amplification on QuantStudio™ 6 or ABI 7500 Real-Time PCR System (Life Technologies)] to detect SARS-CoV-2 RNA in upper respiratory tract specimens. Discrepant results were adjudicated by medical record review. 200 nasopharyngeal swab specimens in viral transport medium (VTM) were collected from symptomatic patients between March 27 and April 9, 2020. Results were concordant for 167 specimens (83.5% overall agreement), including 94 positive and 73 negative specimens. The RealTime SARS-CoV-2 assay on the m2000 yielded 33 additional positive results, 25 of which were also positive by the modified CDC assay but not by the ID NOW™ COVID-19 assay. In a follow-up evaluation, 97 patients for whom a dry nasal swab specimen yielded negative results by the ID NOW™ COVID-19 assay had a paired nasopharyngeal swab specimen collected in VTM and tested by the RealTime SARS-CoV-2 assay; SARS-CoV-2 RNA was detected in 13 (13.4%) of these specimens. Medical record review deemed all discrepant results to be true positives. The ID NOW™ COVID-19 test was the easiest to perform and provided a result in the shortest time: as soon as 5 minutes for positive and 13 minutes for negative result. The RealTime SARS-CoV-2 assay on the m2000 detected more cases of COVID-19 infection than the modified CDC assay or the ID NOW™ COVID-19 test.

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Clinical evaluation of the efficacy and safety of Bioaron C® in children with recurrent bacterial and viral infections of the upper respiratory tract

Planta Medica ◽

10.1055/s-2007-986816 ◽

2007 ◽

Vol 73 (09) ◽

Cited By ~ 1

Author(s):

A Pampura ◽

N Beuscher ◽

M Smirnova ◽

M Horoszkiewicz-Hassan ◽

K Schönknecht

Keyword(s):

Respiratory Tract ◽

Clinical Evaluation ◽

Viral Infections ◽

Upper Respiratory Tract ◽

Efficacy And Safety ◽

Upper Respiratory

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Is it enough for COVID-19 screening test? Limitation of swab test and general characteristics of mild symptom patients

Science Progress ◽

10.1177/00368504211026152 ◽

2021 ◽

Vol 104 (2) ◽

pp. 003685042110261

Author(s):

Sungwoo Choi ◽

Hyo Jeong Choi ◽

Ho Jung Kim

Keyword(s):

Screening Test ◽

Community Treatment ◽

Upper Respiratory Tract ◽

Test Results ◽

Rt Pcr ◽

Pcr Assay ◽

Positive Rate ◽

Upper Respiratory ◽

Polymerase Chain ◽

Discharge Criteria

The most common method for SARS-CoV-2 testing is throat or nasal swabbing by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. In South Korea, drive-through swab test is used for screening system and community treatment centers (CTCs), which admit and treat confirmed COVID-19 patients with mild symptoms, are being used. This retrospective study was conducted on patients admitted to a CTC on March 6, 2020. A total of 313 patients were admitted. The nasal and throat swabs were collected from the upper respiratory tract, and a sputum test was performed to obtain lower respiratory samples. The positive rate of the first set of test, sputum test was higher than that of the swab test ( p = 0.011). In the second set of test, 1 week after the first ones, the rate of positive swab tests was relatively high ( p = 0.026). In the first set of test, 66 of 152 (43.4%) patients showed 24-h consecutive negative swab test results, when the sputum test results were considered together, that number fell to 29 patients (19.1%) ( p < 0.001). Also, in the second set of test, 63 of 164 (38.4%) patients met the discharge criteria only when the swab test was considered; that number fell to 30 (18.3%) when the sputum test results were also considered ( p < 0.001). Using the swab test alone is insufficient for screening test and discharge decision. Patients who may have positive result in the sputum test can be missed.

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A randomized double blind placebo controlled clinical evaluation of extract of Andrographis paniculata (KalmCold™) in patients with uncomplicated upper respiratory tract infection

Phytomedicine ◽

10.1016/j.phymed.2009.12.001 ◽

2010 ◽

Vol 17 (3-4) ◽

pp. 178-185 ◽

Cited By ~ 47

Author(s):

R.C. Saxena ◽

R. Singh ◽

P. Kumar ◽

S.C. Yadav ◽

M.P.S. Negi ◽

...

Keyword(s):

Tract Infection ◽

Respiratory Tract ◽

Respiratory Tract Infection ◽

Clinical Evaluation ◽

Upper Respiratory Tract Infection ◽

Andrographis Paniculata ◽

Upper Respiratory Tract ◽

Double Blind ◽

Double Blind Placebo ◽

Upper Respiratory

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Paired nasopharyngeal and deep lung testing for SARS-CoV2 reveals a viral gradient in critically ill patients: a multi-centre study

10.1101/2020.07.19.20156869 ◽

2020 ◽

Author(s):

Islam Hamed ◽

Nesreen Shaban ◽

Marwan Nassar ◽

Sam Love ◽

Martin D Curran ◽

...

Keyword(s):

Viral Load ◽

Respiratory Tract ◽

Critically Ill ◽

Critically Ill Patients ◽

Lower Respiratory Tract ◽

Limit Of Detection ◽

Upper Respiratory Tract ◽

Rt Pcr ◽

Multi Centre Study ◽

Upper Respiratory

Introduction Samples for diagnostic tests for SARS-CoV-2 can be obtained from the upper (nasopharyngeal/oropharyngeal swabs) or lower respiratory tract (sputum or tracheal aspirate or broncho-alveolar lavage - BAL). Data from different testing sites indicates different rates of positivity. Reverse-transcriptase polymerase chain reaction (RT-PCR) allows for semi-quantitative estimates of viral load as time to crossing threshold (Ct) is inversely related to viral load. Objectives The objective of our study was to evaluate SARS-CoV2 RNA loads between paired nasopharyngeal (NP) and deep lung (endotracheal aspirate or BAL) samples from critically ill patients. Methods SARS-CoV-2 RT-PCR results were retrospectively reviewed for 51 critically ill patients from 5 intensive care units in 3 hospitals ; Addenbrookes Hospital Cambridge (3 units), Royal Papworth Cambridge (1 unit), and Royal Sunderland Hospital (1 unit). At the times when paired NP and deep lung samples were obtained, one patient had been on oxygen only, 6 patients on non-invasive ventilation, 18 patients on ECMO, and 26 patients mechanically ventilated. Results Results collected showed significant gradient between NP and deep lung viral loads. Median Ct value was 29 for NP samples and 24 for deep lung samples. Of 51 paired samples, 16 were negative (below limit of detection) on NP swabs but positive (above limit of detection) on deep lung sample, whilst 2 were negative on deep sample but positive on NP (both patients were on ECMO). Conclusions It has been suggested that whilst SARS-CoV1 tends to replicate in the lower respiratory tract, SARS-CoV2 replicates more vigorously in the upper respiratory tract. These data challenge that assumption. These data suggest that viral migration to, and proliferation in, the lower respiratory tract may be a key factor in the progression to critical illness and the development of severe acute respiratory syndrome (SARS). Factors which promote this migration should be examined for association with severe COVID-19. From a practical point of view, patients with suspected severe COVID-19 should have virological samples obtained from the lower respiratory tract where-ever possible, as upper respiratory samples have a significant negative rate.

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

IRAQI JOURNAL OF AGRICULTURAL SCIENCES ◽

10.36103/ijas.v51i2.982 ◽

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.

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