scholarly journals SARS-CoV2 Testing: The Limit of Detection Matters

2020 ◽  
Author(s):  
Ramy Arnaout ◽  
Rose A. Lee ◽  
Ghee Rye Lee ◽  
Cody Callahan ◽  
Christina F. Yen ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Diagnostic Testing ◽  
False Negative ◽  
False Negative Rate ◽  
Fold Increase ◽  
Test Results ◽  
Rt Pcr ◽  
False Negatives ◽  
Healthcare Network ◽  
Negative Rate

AbstractResolving the COVID-19 pandemic requires diagnostic testing to determine which individuals are infected and which are not. The current gold standard is to perform RT-PCR on nasopharyngeal samples. Best-in-class assays demonstrate a limit of detection (LoD) of ~100 copies of viral RNA per milliliter of transport media. However, LoDs of currently approved assays vary over 10,000-fold. Assays with higher LoDs will miss more infected patients, resulting in more false negatives. However, the false-negative rate for a given LoD remains unknown. Here we address this question using over 27,500 test results for patients from across our healthcare network tested using the Abbott RealTime SARS-CoV-2 EUA. These results suggest that each 10-fold increase in LoD is expected to increase the false negative rate by 13%, missing an additional one in eight infected patients. The highest LoDs on the market will miss a majority of infected patients, with false negative rates as high as 70%. These results suggest that choice of assay has meaningful clinical and epidemiological consequences. The limit of detection matters.

scholarly journals Microfluidic Nano-Scale qPCR Enables Ultra-Sensitive and Quantitative Detection of SARS-CoV-2

Processes ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 1425
Author(s):  
Xin Xie ◽  
Tamara Gjorgjieva ◽  
Zaynoun Attieh ◽  
Mame Massar Dieng ◽  
Marc Arnoux ◽  
...  
Keyword(s):  
Viral Infections ◽  
Limit Of Detection ◽  
False Negative ◽  
False Negative Rate ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Viral Loads ◽  
Nano Scale ◽  
Negative Rate

A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. In this study, we implemented a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification, and nano-scale qPCR based on a commercially available microfluidic chip. Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of this microfluidic RT-qPCR by 1000-fold, enabling detection below 1 copy/µL. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible and quantitative detection of SARS-CoV-2 over five orders of magnitude (<1 to 106 viral copies/µL). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/µL) in 17 samples with negative clinical diagnosis, indicating a potential false-negative rate of 18.7% by clinical diagnostic procedures. In summary, this three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (<1 viral copy/µL) and has the potential to reduce the false-negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections.

scholarly journals Microfluidic nano-scale qPCR enables ultra-sensitive detection of SARS-CoV-2

2020 ◽  
Author(s):  
Xin Xie ◽  
Tamara Gjorgjieva ◽  
Zaynoun Attieh ◽  
Mame Massar Dieng ◽  
Marc Arnoux ◽  
...  
Keyword(s):  
Viral Infections ◽  
Limit Of Detection ◽  
False Negative ◽  
False Negative Rate ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Viral Loads ◽  
Nano Scale ◽  
Negative Rate

Background: A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used standard RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. Methods: We implement a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification and nano-scale qPCR based on the Fluidigm 192.24 microfluidic chip. We validate the method using both positive controls and nasopharyngeal swab samples. Results: Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of the Fluidigm method by 1,000-fold, enabling detection below 1 copy/μl. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible detection of SARS-CoV-2 over five orders of magnitude (< 1 to 106 viral copies/μl). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/μl) in 17 samples with negative clinical diagnosis, indicating a potential false negative rate of 18.7% by clinical diagnostic procedures. Conclusion: The three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (< 1 viral copy/μl) and has the potential to reduce the false negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections.

scholarly journals FAST: a Flexible, Accurate and Speedy Test for COVID-19

2020 ◽  
Author(s):  
Linjiajie Fang ◽  
Bing-Yi Jing ◽  
Shen Ling ◽  
Qing Yang
Keyword(s):  
Close Contact ◽  
False Negative ◽  
Group Testing ◽  
False Negative Rate ◽  
Rt Pcr ◽  
False Negatives ◽  
Improve Efficiency ◽  
Negative Rate ◽  
Pcr Test

AbstractAs the COVID-19 pandemic continues worldwide, there is an urgent need to detect infected patients as quickly and accurately as possible. Group testing proposed by Technion [1][2] could improve efficiency greatly. However, the false negative rate (FNR) would be doubled. Using USA as an example, group testing would have over 70,000 false negatives, compared to 35,000 false negatives by individual testing.In this paper, we propose a Flexible, Accurate and Speedy Test (FAST), which is faster and more accurate than any existing tests. FAST first forms small close contact subgroups, e.g. families and friends. It then pools subgroups to form larger groups before RT-PCR test is done. FAST needs a similar number of tests to Technion’s method, but sharply reduces the FNR to a negligible level. For example, FAST brings down the number of false negatives in USA to just 2000, and it is seven times faster than individual testing.

scholarly journals False-Negative Rate and Recovery Efficiency Performance of a Validated Sponge Wipe Sampling Method

2011 ◽  
Vol 78 (3) ◽  
pp. 846-854 ◽  
Author(s):  
Paula A. Krauter ◽  
Greg F. Piepel ◽  
Raymond Boucher ◽  
Matt Tezak ◽  
Brett G. Amidan ◽  
...  
Keyword(s):  
Surface Roughness ◽  
Stainless Steel ◽  
Limit Of Detection ◽  
False Negative ◽  
False Negative Rate ◽  
Recovery Efficiency ◽  
Surface Material ◽  
Content Type ◽  
Negative Rate ◽  
Efficiency Performance

ABSTRACTRecovery of spores from environmental surfaces varies due to sampling and analysis methods, spore size and characteristics, surface materials, and environmental conditions. Tests were performed to evaluate a new, validated sponge wipe method usingBacillus atrophaeusspores. Testing evaluated the effects of spore concentration and surface material on recovery efficiency (RE), false-negative rate (FNR), limit of detection (LOD), and their uncertainties. Ceramic tile and stainless steel had the highest mean RE values (48.9 and 48.1%, respectively). Faux leather, vinyl tile, and painted wood had mean RE values of 30.3, 25.6, and 25.5, respectively, while plastic had the lowest mean RE (9.8%). Results show roughly linear dependences of RE and FNR on surface roughness, with smoother surfaces resulting in higher mean REs and lower FNRs. REs were not influenced by the low spore concentrations tested (3.10 × 10−3to 1.86 CFU/cm2). Stainless steel had the lowest mean FNR (0.123), and plastic had the highest mean FNR (0.479). The LOD90(≥1 CFU detected 90% of the time) varied with surface material, from 0.015 CFU/cm2on stainless steel up to 0.039 on plastic. It may be possible to improve sampling results by considering surface roughness in selecting sampling locations and interpreting spore recovery data. Further, FNR values (calculated as a function of concentration and surface material) can be used presampling to calculate the numbers of samples for statistical sampling plans with desired performance and postsampling to calculate the confidence in characterization and clearance decisions.

Outcomes of Indeterminate Thyroid Nodules Managed Nonoperatively after Molecular Testing

2020 ◽  
Author(s):  
Catherine Y Zhu ◽  
Ines Donangelo ◽  
Deepashree Gupta ◽  
Dalena T Nguyen ◽  
Joana E Ochoa ◽  
...  
Keyword(s):  
Thyroid Nodules ◽  
False Negative ◽  
False Negative Rate ◽  
Molecular Testing ◽  
Test Results ◽  
Molecular Test ◽  
Negative Rate ◽  
Indeterminate Nodules

Abstract Context Molecular testing to refine the diagnosis of cytologically indeterminate thyroid nodules has become increasingly popular, but data on long-term durability of test results and the rate of delayed operation are limited. Objective Determine the delayed rate of surgical resection in indeterminate nodules with benign/negative molecular testing and the risk of false-negative molecular test results. Design Prospective follow-up of the Gene Expression Classifier vs Targeted Next-Generation Sequencing in the Management of Indeterminate Thyroid Nodules randomized controlled trial comparing the diagnostic test performance of Afirma Gene Expression Classifier and ThyroSeq v2. Setting University of California, Los Angeles. Participants Patients who underwent thyroid biopsy with indeterminate (Bethesda III/IV) cytology (April 2016 to July 2017). Intervention Ultrasound surveillance. Main Outcome Measure False-negative rate of molecular testing. Results Of 95 indeterminate nodules with negative/benign molecular test results, 12 nodules underwent immediate resection (11 benign nodules, 1 noninvasive follicular thyroid neoplasm nodule with papillary-like nuclear features). Nonoperative management was pursued for 83 (87.4%) nodules. The median surveillance was 26.7 months. Ten nodules were resected during surveillance and malignancy was identified in 4 nodules (overall false-negative rate of 5.8%). In the 4 malignant nodules that underwent delayed operation, surgery was prompted by sonographic changes during surveillance. Conclusions The majority of indeterminate nodules with negative molecular testing have a stable clinical course over 3 years of follow-up, but our finding of a 6% false-negative rate highlights the importance of continuing sonographic surveillance. Long-term studies are needed to determine the optimal length of follow-up.

High False-Negative Rate of HER2 Quantitative Reverse Transcription Polymerase Chain Reaction of the Oncotype DX Test: An Independent Quality Assurance Study

2011 ◽  
Vol 29 (32) ◽  
pp. 4279-4285 ◽  
Author(s):  
David J. Dabbs ◽  
Molly E. Klein ◽  
Syed K. Mohsin ◽  
Raymond R. Tubbs ◽  
Yongli Shuai ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quality Assurance ◽  
Reverse Transcription ◽  
False Negative ◽  
False Negative Rate ◽  
Oncotype Dx ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Negative Rate ◽  
Polymerase Chain

Purpose HER2 (ERBB2) status is an important prognostic and predictive marker in breast carcinoma. In recent years, Genomic Health (GHI), purveyors of the Oncotype DX test, has been separately reporting HER2 by reverse transcription polymerase chain reaction (RT-PCR) to oncologists. Because of the lack of independent evaluation, this quality assurance study was undertaken to define the concordance rate between immunohistochemistry (IHC)/fluorescent in situ hybridization (FISH) and GHI RT-PCR HER2 assay. Methods All patients at three participating laboratories (Magee-Womens Hospital [Pittsburgh, PA], Cleveland Clinic [Cleveland, OH], and Riverside Methodist Hospital [Columbus, OH]) with available HER2 RT-PCR results from GHI were included in this study. All IHC-positive and equivocal patient cases were further evaluated and classified by FISH at respective laboratories. Results Of the total 843 patient cases, 784 (93%) were classified as negative, 36 (4%) as positive, and 23 (3%) as equivocal at the three institutions using IHC/FISH. Of the 784 negative patient cases, 779 (99%) were also classified as negative by GHI RT-PCR assay. However, all 23 equivocal patient cases were reported as negative by GHI. Of the 36 positive cases, only 10 (28%; 95% CI, 14% to 45%) were reported as positive, 12 (33%) as equivocal, and 14 (39%) as negative. Conclusion There was an unacceptable false-negative rate for HER2 status with GHI HER2 assay in this independent study. This could create confusion in the decision-making process for targeted treatment and potentially lead to mismanagement of patients with breast cancer if only GHI HER2 information is used.

scholarly journals Variation in False Negative Rate of RT-PCR Based SARS-CoV-2 Tests by Time Since Exposure

2020 ◽  
Author(s):  
Lauren M Kucirka ◽  
Stephen A Lauer ◽  
Oliver Laeyendecker ◽  
Denali Boon ◽  
Justin Lessler
Keyword(s):  
Symptom Onset ◽  
Infected Individual ◽  
False Negative ◽  
False Negative Rate ◽  
Published Data ◽  
Rt Pcr ◽  
Considerable Uncertainty ◽  
Negative Rate ◽  
High Clinical Suspicion ◽  
Cubic Polynomial

ABSTRACTSARS-CoV-2 RT-PCR based tests are being used to “rule out” infection among high-risk individuals such as exposed inpatients and healthcare workers. It is critical to understand how the predictive value of the test varies with time from exposure and symptom onset in order to avoid being falsely reassured by negative tests. As such, the goal of our study was to estimate the false negative rate by day since infection. We used previously published data on RT-PCR sensitivity on samples derived from nasal swabs by day since symptom onset (n=633) and fit a cubic polynomial spline to calculate the false negative rate by day since exposure and symptom onset. Over the four days of infection prior to the typical time of symptom onset (day 5) the probability of a false negative test in an infected individual falls from 100% on day one (95% CI 69-100%) to 61% on day four (95% CI 18-98%), though there is considerable uncertainty in these numbers. On the day of symptom onset, the median false negative rate was 39% (95% CI 16-77%). This decreased to 26% (95% CI 18-34%) on day 8 (3 days after symptom onset), then began to rise again, from 27% (95% CI 20-34%) on day 9 to 61% (95% CI 54-67%) on day 21. Care must be taken when interpreting RT-PCR tests for SARS-CoV-2 infection, particularly if performed early in the course of infection, when using these results as a basis for removing precautions intended to prevent onward transmission. If there is high clinical suspicion, patients should not be ruled out on the basis of RT-PCR alone, and the clinical and epidemiologic situation should be carefully considered.

scholarly journals Does CT help in reducing RT-PCR false negative rate for COVID-19?

2021 ◽  
Vol 31 (5) ◽  
pp. 80
Author(s):  
RichaD Jain ◽  
Anirudh Kohli ◽  
Anagha Joshi ◽  
Ankur Shah ◽  
Abhishek Gorlawar ◽  
...  
Keyword(s):  
False Negative ◽  
False Negative Rate ◽  
Rt Pcr ◽  
Negative Rate
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