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

2021 ◽  
Author(s):  
Melissa Laverack ◽  
Rebecca L. Tallmadge ◽  
Roopa Venugopalan ◽  
Brittany Cronk ◽  
XiuLin Zhang ◽  
...  
Keyword(s):  
Clinical Evaluation ◽  
High Throughput ◽  
Predictive Value ◽  
Diagnostic Sensitivity ◽  
Analytical Sensitivity ◽  
Upper Respiratory Tract ◽  
Rt Pcr ◽  
Pcr Assay ◽  
P Gene ◽  
Upper Respiratory

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.

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

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.

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 Clinical evaluation of a fully automated, lab developed multiplex RT-PCR assay integrating dual-target SARS-CoV-2 and Influenza–A/B detection on a high-throughput platform

2020 ◽  
Author(s):  
Dominik Nörz ◽  
Armin Hoffmann ◽  
Martin Aepfelbacher ◽  
Susanne Pfefferle ◽  
Marc Lütgehetmann
Keyword(s):  
Clinical Evaluation ◽  
High Throughput ◽  
Influenza A ◽  
Influenza Season ◽  
Simultaneous Detection ◽  
Analytical Performance ◽  
Influenza B ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Dual Target

1AbstractBackgroundLaboratories worldwide face high demands for molecular testing during the SARS-CoV-2 pandemic that might be further aggravated with the upcoming influenza season in the northern hemisphere. Considering that symptoms of influenza are largely undistinguishable from COVID-19, both SARS-CoV-2 and the Influenza viruses require concurrent testing by RT-PCR in patients presenting with symptoms of respiratory tract infection. In this study, we adapted and evaluated a laboratory developed multiplex RT-PCR assay for simultaneous detection of SARS-CoV-2 (dual-target), Influenza-A and Influenza-B (SC2/InflA/InflB-UCT) on a fully automated high-throughput system (cobas6800).MethodsAnalytical performance was assessed by serial dilution of quantified reference material and cell culture stocks in transport medium, including pre-treatment for chemical inactivation. For clinical evaluation, residual portions of 164 predetermined patient samples containing SARS-CoV-2 (n=52), Influenza-A (n=43) or Influenza-B (n=19), as well as a set of negative samples was subjected to the novel multiplex assay.ResultsThe assay demonstrated analytical performance comparable to currently available commercial tests, with limits of detection of 94.9 cp/ml for SARS-CoV-2, 14.6 cp/ml for Influenza-A and 422.3 cp/ml for Influenza-B. Clinical evaluation showed excellent agreement with the comparator assays (sensitivity 98.1%, 97.7% and 100% for Sars-CoV-2, Influenza-A and -B respectively).ConclusionThe SC2/InflA/InflB-UCT allows for efficient high-throughput testing for all three pathogens and thus provides streamlined diagnostics while conserving resources during the Influenza-season.HighlightsSimultaneous detection of highly pathogenic respiratory viruses Influenza-A/B and SARS-CoV-2Including a dual-target assay for SARS-CoV-2 detectionFull automation on the cobas6800 high-throughput platform

scholarly journals 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

2020 ◽  
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.

scholarly journals Clinical evaluation of a fully automated, laboratory-developed multiplex RT-PCR assay integrating dual-target SARS-CoV-2 and influenza A/B detection on a high-throughput platform

2021 ◽  
Author(s):  
Dominik Nörz ◽  
Armin Hoffmann ◽  
Martin Aepfelbacher ◽  
Susanne Pfefferle ◽  
Marc Lütgehetmann
Keyword(s):  
Clinical Evaluation ◽  
High Throughput ◽  
Influenza A ◽  
Influenza Season ◽  
Influenza Viruses ◽  
Analytical Performance ◽  
Influenza B ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Dual Target

Introduction. Laboratories worldwide are facing high demand for molecular testing during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, which might be further aggravated by the upcoming influenza season in the northern hemisphere. Gap Statement. Given that the symptoms of influenza are largely indistinguishable from those of coronavirus disease 2019 (COVID-19), both SARS-CoV-2 and the influenza viruses require concurrent testing by RT-PCR in patients presenting with symptoms of respiratory tract infection. Aim. We adapted and evaluated a laboratory-developed multiplex RT-PCR assay for simultaneous detection of SARS-CoV-2 (dual target), influenza A and influenza B (SC2/InflA/InflB-UCT) on a fully automated high-throughput system (cobas6800). Methodology. Analytical performance was assessed by serial dilution of quantified reference material and cell culture stocks in transport medium, including pretreatment for chemical inactivation. For clinical evaluation, residual portions of 164 predetermined patient samples containing SARS-CoV-2 (n=52), influenza A (n=43) or influenza B (n=19), as well as a set of negative samples, were subjected to the novel multiplex assay. Results. The assay demonstrated comparable analytical performance to currently available commercial tests, with limits of detection of 94.9 cp ml−1 for SARS-CoV-2, 14.6 cp ml−1 for influenza A and 422.3 cp ml−1 for influenza B. Clinical evaluation showed excellent agreement with the comparator assays (sensitivity of 98.1, 97.7 and 100 % for Sars-CoV-2 and influenza A and B, respectively). Conclusion. The SC2/InflA/InflB-UCT allows for efficient high-throughput testing for all three pathogens and thus provides streamlined diagnostics while conserving resources during the influenza season.

scholarly journals The evaluation of maximum condyle-tragus distance can predict difficult airway management without exposing upper respiratory tract; a prospective observational study

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Hao Wu ◽  
Dandan Hu ◽  
Xu Chen ◽  
Xuebing Zhang ◽  
Min Xia ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Predictive Value ◽  
Prospective Observational Study ◽  
Difficult Laryngoscopy ◽  
Upper Respiratory Tract ◽  
Intubation Time ◽  
Clinical Trial Registry ◽  
Finger Width ◽  
Upper Respiratory ◽  
Interincisor Distance

Abstract Background Routine preoperative methods to assess airway such as the interincisor distance (IID), Mallampati classification, and upper lip bite test (ULBT) have a certain risk of upper respiratory tract exposure and virus spread. Condyle-tragus maximal distance(C-TMD) can be used to assess the airway, and does not require the patient to expose the upper respiratory tract, but its value in predicting difficult laryngoscopy compared to other indicators (Mallampati classification, IID, and ULBT) remains unknown. The purpose of this study was to observe the value of C-TMD to predict difficult laryngoscopy and the influence on intubation time and intubation attempts, and provide a new idea for preoperative airway assessment during epidemic. Methods Adult patients undergoing general anesthesia and tracheal intubation were enrolled. IID, Mallampati classification, ULBT, and C-TMD of each patient were evaluated before the initiation of anesthesia. The primary outcome was intubation time. The secondary outcomes were difficult laryngoscopy defined as the Cormack-Lehane Level > grade 2 and the number of intubation attempts. Results Three hundred four patients were successfully enrolled and completed the study, 39 patients were identified as difficult laryngoscopy. The intubation time was shorter with the C-TMD>1 finger group 46.8 ± 7.3 s, compared with the C-TMD<1 finger group 50.8 ± 8.6 s (p<0.01). First attempt success rate was higher in the C-TMD>1 finger group 98.9% than in the C-TMD<1 finger group 87.1% (P<0.01). The correlation between the C-TMD and Cormack-Lehane Level was 0.317 (Spearman correlation coefficient, P<0.001), and the area under the ROC curve was 0.699 (P<0.01). The C-TMD < 1 finger width was the most consistent with difficult laryngoscopy (κ = 0.485;95%CI:0.286–0.612) and its OR value was 10.09 (95%CI: 4.19–24.28), sensitivity was 0.469 (95%CI: 0.325–0.617), specificity was 0.929 (95%CI: 0.877–0.964), positive predictive value was 0.676 (95%CI: 0.484–0.745), negative predictive value was 0.847 (95%CI: 0.825–0.865). Conclusion Compared with the IID, Mallampati classification and ULBT, C-TMD has higher value in predicting difficult laryngoscopy and does not require the exposure of upper respiratory tract. Trial registration The study was registered on October 21, 2019 in the Chinese Clinical Trial Registry (ChiCTR1900026775).

scholarly journals Development and Evaluation of an iiPCR Assay for Salmonella and Shigella Detection on a Field-Deployable PCR System

2020 ◽  
Vol 2020 ◽  
pp. 1-5
Author(s):  
Tian Du ◽  
Ji-hong Lin ◽  
Jun-hua Zhao ◽  
Hai-bo Wang ◽  
Qiu-hua Mo
Keyword(s):  
Predictive Value ◽  
Large Scale ◽  
Limit Of Detection ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Preliminary Screening ◽  
Oral Transmission ◽  
Resource Limited ◽  
Clinical Accuracy ◽  
Insulated Isothermal Pcr

Background. Salmonella and Shigella are often associated with fecal-oral transmission and cause large-scale outbreaks in centralized catering units and, therefore, should be frequently and strictly monitored, especially among food handlers. However, no specific and sensitive on-site detection method is available until now. Methods. In this study, an insulated isothermal PCR assay for the detection of Salmonella and Shigella on a field-deployable PCR system was developed. Specificity, sensitivity, reproducibility, and clinical accuracy of the assay were characterized and evaluated. Results. The insulated isothermal PCR assay could be completed within 58 minutes with minimal pretreatment needed. The assay was specific and with good reproducibility. The limit of detection was 103 CFU/mL and 101 CFU/mL for Salmonella and Shigella, respectively, which was comparable to multiplex real-time PCR. Mock on-site clinical evaluation results showed that the analytical sensitivity and specificity of the insulated isothermal PCR assay were 100% and 96.6%, while the positive predictive value and negative predictive value were 94.1% and 100%, respectively. Conclusion. Based on our results, we believe that the assay developed herein could serve as an alternative method for preliminary screening and provide a valuable platform for the on-site detection of Salmonella and Shigella, especially in resource-limited and developing countries.

scholarly journals Evaluation of the PAX8/PPARG Translocation in Follicular Thyroid Cancer with a 4-Color Reverse-Transcription PCR Assay and Automated High-Resolution Fragment Analysis

2010 ◽  
Vol 56 (3) ◽  
pp. 391-398 ◽  
Author(s):  
Alicia Algeciras-Schimnich ◽  
Dragana Milosevic ◽  
Bryan McIver ◽  
Heather Flynn ◽  
Honey V Reddi ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
High Resolution ◽  
Reverse Transcription ◽  
Thyroid Tissue ◽  
Molecular Testing ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fragment Analysis ◽  
Reverse Transcription Pcr

Abstract Background: Molecular testing of thyroid malignancies, in combination with cytologic and histologic examination, is becoming increasingly attractive as a tool for refining traditional morphologic diagnosis. The molecular changes associated with follicular thyroid carcinoma (FTC) are point mutations in RAS oncogenes or the presence of PAX8/PPARG (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement. Methods: We developed and validated a clinical assay for the detection of PAX8/PPARG rearrangements that uses a 4-color reverse-transcription PCR (RT-PCR) assay and high-resolution fragment analysis. Results: The RT-PCR assay is applicable for detecting the various described fusion transcripts of PAX8/PPARG in formalin-fixed, paraffin-embedded thyroid tissue and in fine-needle aspirate biopsy washes from thyroid nodules. The analytical sensitivity of the assay is 1 abnormal cell in a background of 100–10 000 translocation-negative cells. A comparison of the RT-PCR assay with dual-fusion fluorescence in situ hybridization showed an overall concordance of 95%. With this assay, we obtained a prevalence for the PAX8/PPARG rearrangement in FTC of 62% (13 of 21 cases), compared with a 5% prevalence (3 of 55) for other follicular cell–derived neoplasms. Conclusions: The introduction of this assay into clinical practice could provide useful information for the diagnosis and possibly for the prognosis and treatment of thyroid cancer in the future.

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