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 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 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 Lumipulse G SARS-CoV-2 Ag assay evaluation using clinical samples from different testing groups

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Giulia Menchinelli ◽  
Licia Bordi ◽  
Flora Marzia Liotti ◽  
Ivana Palucci ◽  
Maria Rosaria Capobianchi ◽  
...  
Keyword(s):  
Positive Result ◽  
Limit Of Detection ◽  
False Negative ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Repeated Testing ◽  
Rt Pcr ◽  
Subgenomic Rna ◽  
Negative Results ◽  
Percent Agreement

Abstract Objectives Compared to RT-PCR, lower performance of antigen detection assays, including the Lumipulse G SARS-CoV-2 Ag assay, may depend on specific testing scenarios. Methods We tested 594 nasopharyngeal swab samples from individuals with COVID-19 (RT-PCR cycle threshold [Ct] values ≤ 40) or non-COVID-19 (Ct values > 40) diagnoses. RT-PCR positive samples were assigned to diagnostic, screening, or monitoring groups of testing. Results With a limit of detection of 1.2 × 104 SARS-CoV-2 RNA copies/ml, Lumipulse showed positive percent agreement (PPA) of 79.9% (155/194) and negative percent agreement of 99.3% (397/400), whereas PPAs were 100% for samples with Ct values of <18 or 18–<25 and 92.5% for samples with Ct values of 25–<30. By three groups, Lumipulse showed PPA of 87.0% (60/69), 81.1% (43/53), or 72.2% (52/72), respectively, whereas PPA was 100% for samples with Ct values of <18 or 18–<25, and was 94.4, 80.0, or 100% for samples with Ct values of 25–<30, respectively. Additional testing of RT-PCR positive samples for SARS-CoV-2 subgenomic RNA showed that, by three groups, PPA was 63.8% (44/69), 62.3% (33/53), or 33.3% (24/72), respectively. PPAs dropped to 55.6, 20.0, or 41.7% for samples with Ct values of 25–<30, respectively. All 101 samples with a subgenomic RNA positive result had a Lumipulse assay’s antigen positive result, whereas only 54 (58.1%) of remaining 93 samples had a Lumipulse assay’s antigen positive result. Conclusions Lumipulse assay was highly sensitive in samples with low RT-PCR Ct values, implying repeated testing to reduce consequences of false-negative results.

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.

scholarly journals Association of COVID-19 RT-qPCR test false-negative rate with patient age, sex and time since diagnosis

2020 ◽  
Author(s):  
Matan Levine-Tiefenbrun ◽  
Idan Yelin ◽  
Hedva Uriel ◽  
Jacob Kuint ◽  
Licita Schreiber ◽  
...  
Keyword(s):  
False Negative ◽  
False Negative Rate ◽  
Disease Spread ◽  
Positive Test ◽  
N Gene ◽  
P Value ◽  
True Positive ◽  
Younger Patients ◽  
Viral Loads ◽  
Negative Rate

AbstractBackgroundRoutine testing for SARS-CoV-2 in the community is essential for guiding key epidemiological decisions from the quarantine of individual patients to enrolling regional and national preventive measures. Yet, the primary testing tool, the RT-qPCR based testing, is notoriously known for its low sensitivity, i.e. high risk of missed detection of carriers. Quantifying the false-negative rate (FNR) of the RT-qPCR test at the community settings and its dependence on patient demographic and disease progression is therefore key in designing and refining strategies for disease spread prevention.MethodsAnalyzing 843,917 test results of 521,696 patients, we identified false-negative (FN) and true-positive (TP) results as negative and positive results preceded by a COVID-19 diagnosis and followed by a later positive test. Regression analyses were used to determine associations of false-negative results with time of sampling after diagnosis, patient demographics and viral loads based on RT-qPCR Ct values of the next positive tests.FindingsThe overall FNR was 22.8%, which is consistent with previous studies. Yet, this rate was much lower at the first 5 days following diagnosis (10.7%) and only increased in later dates. Furthermore, the FNR was strongly associated with demographics, with odds ratio of 1.74 (95% CI: 1.58-1.90) for women over men and 1.36 (95% CI: 1.34-1.39) for 10 years younger patients. Finally, FNR was associated with viral loads (p-value 0.0005), with a difference of 1.50 (95% CI: 0.70-2.30) between the average Ct of the N gene in a positive test following a false-negative compared to a positive test following a true-positive.InterpretationOur results show that in the first few days following diagnosis, when results are critical for quarantine decisions, RT-qPCR testing is more reliable than previously reported. Yet the reliability of the test result is reduced in later days as well as for women and younger patients, where the viral loads are typically lower.FundingThis research was supported by the ISRAEL SCIENCE FOUNDATION (grant No. 3633/19) within the KillCorona – Curbing Coronavirus Research Program.

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 Effect of Sample Aliquot Size on the Limit of Detection and Reproducibility of Clinical Assays

2007 ◽  
Vol 53 (11) ◽  
pp. 1962-1965 ◽  
Author(s):  
Guorong Chen ◽  
Lori Kobayashi ◽  
Irina Nazarenko
Keyword(s):  
Limit Of Detection ◽  
Probability Model ◽  
False Negative ◽  
Success Probability ◽  
False Negative Rate ◽  
Sample Volume ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Minimum Number ◽  
The Impact

Abstract Background: Nucleic acid amplification technologies significantly improved the limit of detection (LOD) for diagnostic assays. The ability of these assays to amplify fewer than 10 target copies of DNA or RNA imposes new requirements on the preparation of clinical samples. We report a statistical method to determine how large of an aliquot is necessary to reproducibly provide a detectable number of cells. Methods: We determined the success probability (p) based on aliquot size and sample volume. The binomial distribution, based on p and the concentration of cells in sample, was used to calculate the probability of getting no target objects in an aliquot and to determine the minimum number of objects per aliquot necessary to generate a reproducible clinical assay. Results: The described method was applied to find a minimum aliquot volume required for a set LOD, false-negative rate (FNR), and %CV. For example, to keep FNR &lt;0.01% for 0.5%, 1% and 2% aliquots (minimum 2000, 1000, and 500 cells per sample) are required. Comparison between experimental and predicted FNR demonstrated good correlation for the small volume aliquots and/or low concentration of target. When 4 μL of 200 copies/mL of plasmid is amplified, predicted and experimental FNRs are 47.2% and 44.9%. Conclusion: This probability model is a useful tool to predict the impact of aliquot volume on the LOD and reproducibility of clinical assays. Even for samples for which pathogens are homogeneously distributed, it is theoretically impossible to collect a single pathogen consistently if the concentration of pathogen is below a certain limit.

scholarly journals Diagnostic utility of GeneXpert MTB/RIF assay versus conventional methods for diagnosis of pulmonary and extra-pulmonary tuberculosis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Asmaa Mohammed Elbrolosy ◽  
Rana H. El Helbawy ◽  
Osama M. Mansour ◽  
Reda Abdel Latif
Keyword(s):  
Risk Factors ◽  
False Negative ◽  
False Negative Rate ◽  
Multivariate Regression Analysis ◽  
Rifampicin Resistance ◽  
Clinical Samples ◽  
Smear Microscopy ◽  
Negative Rate ◽  
Extra Pulmonary ◽  
Conventional Methods

Abstract Background Tuberculosis (T.B) is one of the major infectious diseases in the developing countries. The diagnosis of extrapulmonary T.B (EPTB) remains problematic and emergence of resistant strains poses a significant threat. Improved diagnosis of tuberculosis is a global priority for proper control. The study aimed to assess the diagnostic accuracy of GeneXpert MTB/RIF assay for diagnosis of pulmonary TB (PTB) and EPTB and to evaluate the performance of GeneXpert system for demonstrating rifampicin resistance among the studied patients. Methods A total of 582 clinical samples (449 pulmonary; 430 sputum and 19 bronchoalveolar lavage (BAL) and 133 extra-pulmonary origins; 26 pleural fluid, 62 CSF, 19 ascetic fluid, 12 pus and 14 urine) were collected from patients under clinical and radiological assessment of either PTB or EPTB who were admitted to Menoufia Chest Hospital over a period of three years. Clinical samples were processed and investigated for detection of Mycobacterium tuberculosis (MTB) by both Xpert assay and the conventional methods including Ziehl-Neelsen (ZN)/acid-fast bacillus (AFB) smear microscopy and Lowenstein-Jensen (LJ) culture. Patients′ demographic, clinical characteristics and risk factors for acquiring rifampicin resistance were analyzed. Results The sensitivity, specificity, false- negative rate and total accuracy of AFB smear microscopy respectively were 72.1 %, 81.3 %, 27.9 and 78.8 % for PTB. However for EPTB, they were 63.2 %, 70.5 %, 36.8 and 68.4 % respectively in relation to LJ culture as the gold standard. GeneXpert MTB/RIF revealed better performance for PTB than EPTB. For PTB, it showed 90.2 % sensitivity, 86.9 % specificity, and 9.8 % false- negative rate. For EPTB, the assay showed a sensitivity of 81.6 %, specificity of 78.9 % and false- negative rate of 18.5 %. Multivariate regression analysis showed that presence of EPTB and contacts with known TB cases were independent risk factors for developing rifampicin resistance. Conclusions GeneXpert MTB/RIF assay is a rapid and highly sensitive technique for diagnosis of PTB or EPTB. Its simplicity and accuracy make this new method a very impressive tool for diagnosis of MTB and rifampicin resistance.

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