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

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 <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.

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

Processes ◽

10.3390/pr8111425 ◽

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.

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

10.1101/2020.08.28.20183970 ◽

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.

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Two Stage Hierarchical Group Testing Strategy to Increase SARS-CoV-2 Testing Capacity at an Institution of Higher Education: A Retrospective Analysis

10.1101/2021.04.14.21255494 ◽

2021 ◽

Author(s):

Troy J Ganz ◽

Markus Leslloyd Waithe-Alleyne ◽

Deirdre Slate ◽

Rachel Donner ◽

Kevin Hines ◽

...

Keyword(s):

Higher Education ◽

Limit Of Detection ◽

False Negative ◽

Rna Extraction ◽

Group Testing ◽

False Negative Rate ◽

Diagnostic Methods ◽

Nucleic Acid Amplification ◽

Configuration Model ◽

Analytical Sensitivities

Population testing for severe acute respiratory syndrome 2 (SAR-CoV-2) is necessary owing to the possibility of viral transmission from asymptomatic cases, yet scarcity of reagents and equipment has added to the cost prohibitive implementation of screening campaigns at institutions of higher education. The high analytical sensitivities of leading nucleic acid amplification diagnostic methods allow for group testing to increase testing capacity. A feasibility study was performed using an optimized testing configuration model for pooling three, five, and ten samples. Following the standard RNA extraction and purification workflow for quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) method using Thermo Fisher TaqPath COVID-19 multiplex primers and probes for the ORF1ab, N, and S genes, matrix and dilution effects were assessed using pooled negative samples as the diluent. Probit analysis produced a limit of detection of 16075 (ORF1ab), 1308 (N), and 1180182 (S) genomic copy equivalents per milliliter. Trials comparing neat to 1:5 dilution for 34 weak-to-strongly positive samples demonstrated average threshold cycle (CT) shifts of 2.31+/-1.16 (ORF1ab), 2.23+/-1.12 (N), and 2.79+/-1.40 (S). Notwithstanding observed S gene dropouts, the false negative rate was unaffected. As the ratio of asymptomatic positive to symptomatic positive SARS-CoV-2 infected individuals was approximately 4:1 and the average prevalence was 0.16% since we started testing in August 2020, pooled testing was identified as a viable, cost-effective option for monitoring the Northeastern University community.

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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

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa466 ◽

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.

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Abstract 1122‐000034: Systematic CT‐Perfusion acquisition in All Suspected Stroke Patients Increases Vascular Occlusion Detection and Thrombectomy Rates

Stroke: Vascular and Interventional Neurology ◽

10.1161/svin.01.suppl_1.000034 ◽

2021 ◽

Vol 1 (S1) ◽

Author(s):

Marta Olive‐Gadea ◽

Manuel Requena ◽

Facundo Diaz ◽

Alvaro Garcia‐Tornel ◽

Marta Rubiera ◽

...

Keyword(s):

Ischemic Stroke ◽

Acute Ischemic Stroke ◽

Clinical Symptoms ◽

Ct Perfusion ◽

False Negative ◽

False Negative Rate ◽

Vascular Occlusion ◽

Stroke Patients ◽

Imaging Protocols ◽

The Impact

Introduction : In acute ischemic stroke patients, current guidelines recommend noninvasive vascular imaging to identify intracranial vessel occlusions (VO) that may benefit from endovascular treatment (EVT). However, VO can be missed in CT angiography (CTA) readings. We aim to evaluate the impact of consistently including CT perfusion (CTP) in admission stroke imaging protocols on VO diagnosis and EVT rates. Methods : We included patients with a suspected acute ischemic stroke that underwent urgent non‐contrast CT, CTA and CTP from April to October 2020. Hypoperfusion areas defined by Tmax>6s delay (RAPID software), congruent with the clinical symptoms and a vascular territory, were considered due to a VO (CTP‐VO). Cases in which mechanical thrombectomy was performed were defined as therapeutically relevant VO (EVT‐VO). For patients that received EVT, site of VO according to digital subtraction angiography was recorded. Two experienced neuroradiologists blinded to CTP but not to clinical symptoms, retrospectively evaluated NCCT and CTA to identify intracranial VO (CTA‐VO). We analyzed CTA‐VO sensitivity and specificity at detecting CTP‐VO and EVT‐VO respecitvely. We performed a logistic regression to test the association of Tmax>6s volumes with CTA‐VO identification and indication of EVT. Results : Of the 338 patients included in the analysis, 157 (46.5%) presented a CTP‐VO, (median Tmax>6s: 73 [29‐127] ml). CTA‐VO was identified in 83 (24.5%) of the cases. Overall CTA‐VO sensitivity for the detection of CTP‐VO was 50.3% and specificity was 97.8%. Higher hypoperfusion volume was associated with an increased CTA‐VO detection, with an odds ratio of 1.03 (95% confidence interval 1.02‐1.04) (figure). DSA was indicated in 107 patients; in 4 of them no EVT was attempted due to recanalization or a too distal VO in the first angiographic run. EVT was performed in 103 patients (30.5%. Tmax>6s: 102 [63‐160] ml), representing 65.6% of all CTP‐VO. Overall CTA‐VO sensitivity for the detection of EVT‐VO was 69.9%. The CTA‐VO sensitivity for detecting patients with indication of EVT according to clinical guidelines was as follows: 91.7% for ICA occlusions and 84.4% for M1‐MCA occlusions. For all other occlusion sites that received EVT, the CTA‐VO sensitivity was 36.1%. The overall specificity was 95.3%. Among patients who received EVT, CTA‐VO was not detected in 31 cases, resulting in a false negative rate of 30.1%. False negative CTA‐VO cases had lower Tmax>6s volumes (69[46‐99.5] vs 126[84‐169.5]ml, p<0.001) and lower NIHSS (13[8.5‐16] vs 17[14‐21], p<0.001). Conclusions : Systematically including CTP perfusion in the acute stroke admission imaging protocols may increase the diagnosis of VO and rate of EVT.

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Evaluation of Rapid Molecular Detection Assays for Salmonella in Challenging Food Matrices at Low Inoculation Levels and Using Difficult-to-Detect Strains

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-098 ◽

2015 ◽

Vol 78 (9) ◽

pp. 1632-1641 ◽

Cited By ~ 7

Author(s):

GINA RYAN ◽

SHERRY ROOF ◽

LAURIE POST ◽

MARTIN WIEDMANN

Keyword(s):

Foodborne Pathogens ◽

Validation Studies ◽

Limit Of Detection ◽

False Negative ◽

Nucleic Acid Amplification ◽

Raw Material ◽

Dark Chocolate ◽

Pet Food ◽

End User ◽

Significant Difference

Assays for detection of foodborne pathogens are generally initially evaluated for performance in validation studies carried out according to guidelines provided by validation schemes (e.g., AOAC International or the International Organization for Standardization). End users often perform additional validation studies to evaluate the performance of assays in specific matrices (e.g., specific foods or raw material streams of interest) and with specific pathogen strains. However, these types of end-user validations are typically not well defined. This study was conducted to evaluate a secondary end user validation of four AOAC-validated commercial rapid detection assays (an isothermal nucleic acid amplification, an immunoassay, and two PCR-based assays) for their ability to detect Salmonella in two challenging matrices (dry pet food and dark chocolate). Inclusivity was evaluated with 68 diverse Salmonella strains at low population levels representing the limit of detection (LOD) for each assay. One assay detected all strains at the LOD, two assays detected multiple strains only at 10 times the LOD, and the fourth assay failed to detect two strains (Salmonella bongori and S. enterica subsp. houtenae) even at 1,000 times the LOD; this assay was not further evaluated. The three remaining assays were subsequently evaluated for their ability to detect five selected Salmonella strains in food samples contaminated at fractional levels. Unpaired comparisons revealed no significant difference between the results for each given assay and the results obtained with the reference assay. However, analysis of paired culture-confirmed results revealed assay false-negative rates of 4 to 26% for dry pet food and 12 to 16% for dark chocolate. Overall, our data indicate that rapid assays may have high false-negative rates when performance is evaluated under challenging conditions, including low-moisture matrices, strains that are difficult to detect, injured cells, and low inoculum levels.

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Performance evaluation of cobas HBV real-time PCR assay on Roche cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2017-1133 ◽

2018 ◽

Vol 56 (7) ◽

pp. 1133-1139 ◽

Cited By ~ 7

Author(s):

Hanah Kim ◽

Mina Hur ◽

Eunsin Bae ◽

Kyung-A Lee ◽

Woo-In Lee

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Nucleic Acid Amplification ◽

Coefficient Of Determination ◽

Clinical Samples ◽

Pcr Assay ◽

Cobas Taqman ◽

Limit Of Quantitation ◽

Two Systems

Abstract Background: Hepatitis B virus (HBV) nucleic acid amplification testing (NAAT) is important for the diagnosis and management of HBV infection. We evaluated the analytical performance of the cobas HBV NAAT (Roche Diagnostics GmbH, Mannheim, Germany) on the cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test (CAP/CTM HBV). Methods: Precision was evaluated using three levels of cobas HBV/HCV/HIV-1 Control Kit, and linearity was evaluated across the anticipated measuring range (10.0–1.0×109 IU/mL) at seven levels using clinical samples. Detection capability, including limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ), was verified using the 4th WHO International Standard for HBV DNA for NAT (NIBSC code: 10/266). Correlation between the two systems was compared using 205 clinical samples (102 sera and 103 EDTA plasma). Results: Repeatability and total imprecision (coefficient of variation) ranged from 0.5% to 3.8% and from 0.5% to 3.5%, respectively. Linearity (coefficient of determination, R2) was 0.999. LOB, LOD and LOQ were all acceptable within the observed proportion rate (85%). Correlation was very high between the two systems in both serum and plasma samples (correlation coefficient [r]=0.995). Conclusions: The new cobas HBV real-time PCR assay on the cobas 4800 System showed reliable analytical performances.

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Secondary Complete Cytoreduction in Recurrent Ovarian Cancer: Benefit of Optimal Patient Selection Using Scoring System

International Journal of Gynecological Cancer ◽

10.1097/igc.0000000000000051 ◽

2014 ◽

Vol 24 (2) ◽

pp. 238-246 ◽

Cited By ~ 15

Author(s):

Enora Laas ◽

Mathieu Luyckx ◽

Marjolein De Cuypere ◽

Frederic Selle ◽

Emile Daraï ◽

...

Keyword(s):

Ovarian Cancer ◽

Patient Selection ◽

Positive Impact ◽

False Negative ◽

False Negative Rate ◽

Recurrent Ovarian Cancer ◽

Complete Resection ◽

Predictive Values ◽

Independent Population ◽

The Impact

ObjectiveComplete tumor cytoreduction seems to be beneficial for patients with recurrent epithelial ovarian cancer (REOC). The challenge is to identify patients eligible for such surgery. Several scores based on simple clinical parameters have attempted to predict resectability and help in patient selection for surgery in REOC.The aims of this study were to assess the performance of these models in an independent population and to evaluate the impact of complete resection.Materials and MethodsA total of 194 patients with REOC between January 2000 and December 2010 were included in 2 French centers. Two scores were used: the AGO DESKTOP OVAR trial score and a score from Tian et al.The performance (sensitivity, specificity, and predictive values) of these scores was evaluated in our population. Survival curves were constructed to evaluate the survival impact of surgery on recurrence.ResultsPositive predictive values for complete resection were 80.6% and 74.0% for the DESKTOP trial score and the Tian score, respectively. The false-negative rate was high for both models (65.4% and 71.4%, respectively). We found a significantly higher survival in the patients with complete resection (59.4 vs 17.9 months,P< 0.01) even after adjustment for the confounding variables (hazard ratio [HR], 2.53; 95% confidence interval, 1.01–6.3;P= 0.04).ConclusionsIn REOC, surgery seems to have a positive impact on survival, if complete surgery can be achieved. However, factors predicting complete resection are not yet clearly defined. Recurrence-free interval and initial resection seem to be the most relevant factors. Laparoscopic evaluation could help to clarify the indications for surgery.

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

Applied and Environmental Microbiology ◽

10.1128/aem.07403-11 ◽

2011 ◽

Vol 78 (3) ◽

pp. 846-854 ◽

Cited By ~ 21

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.

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