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

2015 ◽  
Vol 78 (9) ◽  
pp. 1632-1641 ◽  
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.

scholarly journals FTIR spectroscopy for estimation of efavirenz in raw material and tablet dosage form

2015 ◽  
Vol 4 (6) ◽  
pp. 390-395 ◽  
Author(s):  
Nareddy Preethi Reddy ◽  
Yenumula Padmavathi ◽  
Perika Mounika ◽  
Akari Anjali
Keyword(s):  
Liquid Phase ◽  
Direct Measurement ◽  
Limit Of Detection ◽  
Quantitative Estimation ◽  
Pharmaceutical Formulations ◽  
Raw Material ◽  
Coefficient Of Determination ◽  
Determination Limit ◽  
Pharmaceutical Dosage Forms ◽  
Significant Difference

A Fourier transform infrared (FTIR) spectrophotometric method was developed for rapid and direct measurement of efavirenz in pharmaceutical formulations. The method involves extraction of efavirenz from tablets with chloroform by sonication and the direct measurement of the absorbance in liquid phase using a reduced path length cell. In general, the spectrum was measured in transmission mode. The equipment was configured to collect a spectrum at 8 cm-1 resolution and 45 scans per sec .The spectra were collected between 4000 cm-1 and 450cm-1, the band obtained at 1750cm-1 (carbonyl group) showed intense, clear peak in the liquid phase for quantitation. The method was validated as per ICH guidelines. The method fulfilled most validation requirements in the linearity range 200-1000µg/mL. The coefficient of determination, limit of detection and quantification was found to be 0.993, 49.12?g/mL and 148.84?g/mL respectively. Results of developed FTIR method were compared with the results obtained with the existing UV method statistically by using t-test, which indicated that there is no significant difference between the methods at P=0.05. The proposed FTIR method reduces the solvent consumption and also eliminates the use of reagents. Thus the developed method offers a good alternative for the quantitative estimation of efavirenz in bulk and pharmaceutical dosage forms and also to quantify efavirenz when combined with other API in the same dosage form.International Current Pharmaceutical Journal, May 2015, 4(6): 390-395

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 <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.

Evaluation of invA Diversity among Salmonella Species Suggests Why Some Commercially Available Rapid Detection Kits May Fail To Detect Multiple Salmonella Subspecies and Species

2019 ◽  
Vol 82 (4) ◽  
pp. 710-717 ◽  
Author(s):  
ARIEL J. BUEHLER ◽  
MARTIN WIEDMANN ◽  
ZEINA KASSAIFY ◽  
RACHEL A. CHENG
Keyword(s):  
Foodborne Pathogens ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
False Negative ◽  
Detection Methods ◽  
Test Results ◽  
Salmonella Spp ◽  
Development And Validation ◽  
In Silico Analyses

ABSTRACTinvA is a common molecular target for Salmonella-specific detection methods and is recommended by the U.S. Food and Drug Administration Bacteriological Analytical Manual as a target for PCR confirmation of putative Salmonella isolates. Novel assays designed for the rapid detection of foodborne pathogens are often validated according to guidelines provided by validation schemes, such as the AOAC International or the International Organization for Standardization. However, these validation guidelines allow for flexibility in the validation study experimental design, which may inflate the assay's ability to detect foodborne pathogens, especially for foodborne pathogens such as Salmonella, exhibiting tremendous species diversity with >2,600 confirmed serovars. This study was conducted to (i) describe the sequence diversity of invA, across a diverse set of Salmonella serovars and (ii) evaluate the ability of two commercially available, AOAC International–validated rapid detection assays to detect a diverse collection of Salmonella spp. strains. In silico analyses identified 362 of 2,058 nucleotide sites that were variable among invA sequences from a diverse collection, representing 86 unique serovars spanning all species and subspecies. Not surprisingly, the majority of variable sites (308 of 2,058) occurred in non–Salmonella enterica subsp. enterica strains, including Salmonella bongori and the other S. enterica subspecies. In vitro testing showed that both rapid detection assays, examined here, failed to detect all Salmonella strains at 1 log above the limit of detection, with assay A failing to detect S. enterica subsp. salamae, and assay B failing to detect S. bongori. Both strains were eventually detected at 100,000 times the limit of detection. Taken together, our study highlights the need to include non–subsp. S. enterica strains in the development and validation of rapid detection methods to limit false-negative test results.HIGHLIGHTS

scholarly journals A CRISPR-Cas12a-based specific enhancer for more sensitive detection of SARS-CoV-2 infection

2020 ◽  
Author(s):  
Weiren Huang ◽  
Lei Yu ◽  
Donghua Wen ◽  
Dong Wei ◽  
Yangyang Sun ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
False Negative ◽  
Dna Recognition ◽  
Nucleic Acid Amplification ◽  
Grey Zone ◽  
Reverse Transcription Pcr ◽  
Target Dna ◽  
Highly Sensitive ◽  
Dna Reporter

AbstractHigh Ct-values falling in the grey zone are frequently encountered in SARS-CoV-2 detection by real-time reverse transcription PCR (rRT-PCR) and have brought urgent challenges in diagnosis of samples with low viral load. Based on the single-stranded DNA reporter trans-cleavage activity by Cas12a upon target DNA recognition, we create a Specific Enhancer for detection of PCR-amplified Nucleic Acids (SENA) to confirm SARS-CoV-2 detection through specifically targeting its rRT-PCR amplicons. SENA is highly sensitive, with its limit of detection being at least 2 copies/reaction lower than that of the corresponding rRT-PCR, and highly specific, which identifies both false-negative and false-positive cases in clinic applications. SENA provides effective confirmation for nucleic acid amplification-based molecular diagnosis, and may immediately eliminate the uncertainty problems of rRT-PCR in SARS-CoV-2 clinic detection.One Sentence SummaryCRISPR-Cas12a-based COVID-19 diagnosis.

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

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.

Clinical evaluation of a new thyroglobulin immunoradiometric assay in the follow-up of differentiated thyroid carcinoma

2003 ◽  
Vol 42 (02) ◽  
pp. 71-77 ◽  
Author(s):  
I. Schreivogel ◽  
C. Angerstein ◽  
U. Siefker ◽  
K. Lehmann ◽  
G. Altenvoerde ◽  
...  
Keyword(s):  
Thyroid Carcinoma ◽  
Residual Disease ◽  
False Negative ◽  
Differentiated Thyroid Carcinoma ◽  
Threshold Concentration ◽  
High Rate ◽  
Functional Assay ◽  
Hypothyroid Patient ◽  
Assay Sensitivity ◽  
Significant Difference

SummaryAim: Formal and clinical comparison of a new 3rd-gene-ration-Tg-IRMA (3-G-IRMA; Dynotest®Tg-plus) with a conventional Tg-IRMA (3-G-IRMA; SELco®Tg-assay) for patients with differentiated thyroid carcinoma. In addition we evaluated, if thyroglobulin (Tg) levels above a specific threshold concentration indicate the need for further investigations for residual disease. Patients, methods: Tg concentration of 105 sera of 93 consecutive patients with a differentiated thyroid cancer was determined with both assays and compared at different cut-off values (Dynotest®Tg-plus: 0.2, 1, 2 ng/ml; SELco®Tg-assay: 0.5, 1, 2 ng/ml) with the clinical results in respect to the corresponding TSH concentration. Results: Tg concentration did not show any significant difference (SELco®Tg-assay 0.5 ng/ml, Dynotest® Tg-plus 0.2 ng/ml). The Tg-values of both assays correlated with 97%. However, correlation of recovery in both assays was small (40%). The sensitivities and specificities of both assays at different cut-offs and TSH values did not reveal significant differences. In patients with TSH concentration >30 µU/ml the functional assay sensitivity was superior to arbitrary cut-offs in the decision to start further evaluations. Conclusions: In our study neither formal nor clinical significant differences between two Tg-assays were found. In a hypothyroid patient (TSH >30 µU/ml, Tg concentration exceeding the functional assay sensitivity) further investigations for residual disease are warranted. Higher thresholds are of limited value, due to a inacceptable high rate of false negative results.

Effects of Previous Surgery on the Detection of Sentinel Nodes in Women With Vulvar Cancer

2011 ◽  
Vol 21 (9) ◽  
pp. 1679-1683 ◽  
Author(s):  
Tessa A. Ennik ◽  
David G. Allen ◽  
Ruud L.M. Bekkers ◽  
Simon E. Hyde ◽  
Peter T. Grant
Keyword(s):  
Vulvar Cancer ◽  
False Negative ◽  
False Negative Rate ◽  
Blue Dye ◽  
Previous Surgery ◽  
Detection Rates ◽  
Reliable Technique ◽  
Appearance Time ◽  
Negative Rate ◽  
Significant Difference

BackgroundThere is a growing interest to apply the sentinel node (SN) procedure in the treatment of vulvar cancer. Previous vulvar surgery might disrupt lymphatic patterns and thereby decrease SN detection rates, lengthen scintigraphic appearance time (SAT), and increase SN false-negative rate. The aims of this study were to evaluate the SN detection rates at the Mercy Hospital for Women in Melbourne and to investigate whether previous vulvar surgery affects SN detection rates, SAT, and SN false-negative rate.MethodsData on all patients with vulvar cancer who underwent an SN procedure (blue dye, technetium, or combined technique) from November 2000 to July 2010 were retrospectively collected.ResultsSixty-five SN procedures were performed. Overall detection rate was 94% per person and 80% per groin. Detection rates in the group of patients who underwent previous excision of the primary tumor were not lower compared with the group without previous surgery or with just an incisional biopsy. There was no statistical significant difference in SAT between the previous excision group and the other patients. None of the patients with a false-negative SN had undergone previous excision.ConclusionsResults indicate that previous excision of a primary vulvar malignancy does not decrease SN detection rates or increase SN false-negative rate. Therefore, the SN procedure appears to be a reliable technique in patients who have previously undergone vulvar surgery. Previous excision did not significantly lengthen SAT, but the sample size in this subgroup analysis was small.

scholarly journals Digital Droplet PCR for SARS-CoV-2 Resolves Borderline Cases

2021 ◽  
Author(s):  
Jing Xu ◽  
Timothy Kirtek ◽  
Yan Xu ◽  
Hui Zheng ◽  
Huiyu Yao ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
False Negative ◽  
Accurate Method ◽  
Viral Envelope ◽  
Chain Reaction ◽  
Borderline Cases ◽  
Droplet Pcr ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Abstract Objectives The Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories) was the first droplet digital polymerase chain reaction (ddPCR) assay to receive Food and Drug Administration (FDA) Emergency Use Authorization approval, but it has not been evaluated clinically. We describe the performance of ddPCR—in particular, its ability to confirm weak-positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results. Methods We clinically validated the Bio-Rad Triplex Probe ddPCR Assay. The limit of detection was determined by using serial dilutions of SARS-CoV-2 RNA in an artificial viral envelope. The ddPCR assay was performed according to the manufacturer’s specifications on specimens confirmed to be positive (n = 48) or negative (n = 30) by an FDA-validated reverse transcription–polymerase chain reaction assay on the m2000 RealTime system (Abbott). Ten borderline positive cases were also evaluated. Results The limit of detection was 50 copies/mL (19 of 20 positive). Forty-seven specimens spanning a range of quantification cycles (2.9-25.9 cycle numbers) were positive by this assay (47 of 48; 97.9% positive precent agreement), and 30 negative samples were confirmed as negative (30 of 30; 100% negative percent agreement). Nine of 10 borderline cases were positive when tested in triplicate. Conclusions The ddPCR of SARS-CoV-2 is an accurate method, with superior sensitivity for viral RNA detection. It could provide definitive evaluation of borderline positive cases or suspected false-negative cases.

scholarly journals The underestimated issue of non-reproducible cardiac troponin I and T results: case series and systematic review of the literature

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Julien Favresse ◽  
Jean-Louis Bayart ◽  
Damien Gruson ◽  
Sergio Bernardini ◽  
Aldo Clerico ◽  
...  
Keyword(s):  
Systematic Review ◽  
Clinical Judgment ◽  
Troponin I ◽  
Troponin T ◽  
Case Reports ◽  
False Negative ◽  
Case Series ◽  
Elevated Alkaline Phosphatase ◽  
Negative Results ◽  
Significant Difference

Abstract Cardiac troponins (cTn) are the preferred biomarkers for the evaluation of myocardial injury and play a key role in the diagnosis of acute myocardial infarction (MI). Pre-analytical or analytical issues and interferences affecting troponin T and I assays are therefore of major concern given the risk of misdiagnosis. False positive troponin results have been related to various interferences including anti-troponin antibodies, heterophilic antibodies, or elevated alkaline phosphatase level. On the other hand, false negative results have been reported in the case of a large biotin intake. These interferences are characterized with erroneous but reproducible troponin results. Of interest, non-reproducible results have also been reported in the literature. In other words, if the sample is reanalyzed a second time, a significant difference in troponin results will be observed. These interferences have been named “fliers” or “outliers”. Compared to the biotin interference that received major attention in the literature, troponin outliers are also able to induce harmful clinical consequences for the patient. Moreover, the prevalence of outliers in recent studies was found to be higher (0.28–0.57%) compared to the biotin interference. The aim of this systematic review is to warn clinicians about these non-reproducible results that may alter their clinical judgment. Four case reports that occurred in the Clinique of Saint-Luc Bouge are presented to attest this point. Moreover, we aimed at identifying the nature of these non-reproducible troponin results, determining their occurrence, and describing the best way for their identification.

scholarly journals Nitric oxide (NO) in the Bohai Sea and the Yellow Sea

2019 ◽  
Vol 16 (22) ◽  
pp. 4485-4496 ◽  
Author(s):  
Ye Tian ◽  
Chao Xue ◽  
Chun-Ying Liu ◽  
Gui-Peng Yang ◽  
Pei-Feng Li ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Surface Layer ◽  
Yellow Sea ◽  
Bohai Sea ◽  
Limit Of Detection ◽  
Bottom Layer ◽  
The Yellow Sea ◽  
The Bohai Sea ◽  
Significant Difference ◽  
Average Flux

Abstract. Nitric oxide (NO) is a short-lived compound of the marine nitrogen cycle; however, our knowledge about its oceanic distribution and turnover is rudimentary. Here we present the measurements of dissolved NO in the surface and bottom layers at 75 stations in the Bohai Sea (BS) and the Yellow Sea (YS) in June 2011. Moreover, NO photoproduction rates were determined at 27 stations in both seas. The NO concentrations in the surface and bottom layers were highly variable and ranged from below the limit of detection (i.e., 32 pmol L−1) to 616 pmol L−1 in the surface layer and 482 pmol L−1 in the bottom layer. There was no significant difference (p>0.05) between the mean NO concentrations in the surface (186±108 pmol L−1) and bottom (174±123 pmol L−1) layers. A decreasing trend of NO in bottom-layer concentrations with salinity indicates a NO input by submarine groundwater discharge. NO in the surface layer was supersaturated at all stations during both day and night and therefore the BS and YS were a persistent source of NO to the atmosphere at the time of our measurements. The average flux was about 4.5×10-16 mol cm−2 s−1 and the flux showed significant positive relationship with the wind speed. The accumulation of NO during daytime was a result of photochemical production, and photoproduction rates were correlated to illuminance. The persistent nighttime NO supersaturation pointed to an unidentified NO dark production. NO sea-to-air flux densities were much lower than the NO photoproduction rates. Therefore, we conclude that the bulk of the NO produced in the mixed layer was rapidly consumed before its release to the atmosphere.

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