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

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.

Rapid Detection of Salmonella spp. in Foods by Combination of a New Selective Enrichment and a Sandwich ELISA Using Two Monoclonal Antibodies against Dulcitol 1-Phosphate Dehydrogenase

1996 ◽  
Vol 59 (11) ◽  
pp. 1158-1163 ◽  
Author(s):  
HUAIZE TIAN ◽  
TAKAHISA MIYAMOTO ◽  
TAKASHI OKABE ◽  
YOICHIRO KURAMITSU ◽  
KEN-ICHI HONJOH ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Rapid Detection ◽  
Enrichment Culture ◽  
Sandwich Elisa ◽  
False Negative ◽  
Detection Sensitivity ◽  
Elisa Method ◽  
Salmonella Spp ◽  
Selective Enrichment ◽  
Raw Foods

A rapid-detection method was developed for food-borne dulcitol-positive Salmonella spp. in foods that involves a new preenrichment and selective enrichment system and a sandwich ELISA using two monoclonal antibodies against dulcitol 1-phosphate dehydrogenase. Preenrichment and selective enrichment were in Enterobacteriaceae enrichment mannitol (EEM) broth at 42°C for 6 h and in a new dulcitol-magnesium chloride-pyridinesulfonic acid brilliant green-novobiocin (DMPBN) medium at 42°C for 27 h, respectively. The cells were collected from the selective enrichment culture and suspended in 0.1 ml of 1 N NaOH for 2 min. The solution was neutralized with 0.1 ml of 2 M Tris-HCl buffer (pH 7.5) and the mixture was used as a sample for ELISA. The detection sensitivity of the ELISA was 105 CFU of Salmonella spp. per ml of culture. Competing non-Salmonella organisms in raw food did not interfere with the detection of Salmonella cells even when present at 107: 1 (non-Salmonella: Salmonella ratio) in food. Nonmotile Salmonella gallinarum was detected by the ELISA. The minimum detectable number of initial inoculum of Salmonella typhimurium was 0.69 CFU/25 g of raw chicken after the preenrichment in EEM broth and the selective enrichment in DMPBN medium. The present ELISA method required a total analysis time of 36 h including the preenrichment and selective enrichment periods. The ELISA method was compared with a conventional cultural method for the detection of Salmonella cells in 130 samples of raw foods. Of the samples tested, 16 were Salmonella-positive and 114 samples were negative by both methods. False-positive and false-negative results were not encountered.

scholarly journals DEVELOPMENT AND VALIDATION OF ULTRAVIOLET-SPECTROPHOTOMETRIC METHOD FOR DETERMINATION OF FEBUXOSTAT FOR CONDUCTING IN-VITRO QUALITY CONTROL TESTS IN BULK AND PHARMACEUTICAL DOSAGE FORMS

2018 ◽  
Vol 11 (9) ◽  
pp. 115
Author(s):  
Jaspreet Kaur ◽  
Daljit Kaur ◽  
Sukhmeet Singh
Keyword(s):  
Spectrophotometric Method ◽  
Limit Of Detection ◽  
Pharmaceutical Formulations ◽  
Dosage Forms ◽  
Pharmaceutical Dosage Forms ◽  
Relative Standard ◽  
Limit Of Quantitation ◽  
Development And Validation

Objective: A simple, accurate, and selective ultraviolet-spectrophotometric method has been developed for the estimation of febuxostat in the bulk and pharmaceutical dosage forms.Method: The method was developed and validated according to International Conference on Harmonization (ICH Q2 R1) guidelines. The developed method was validated statistically with respect to linearity, range, precision, accuracy, ruggedness, limit of detection (LOD), limit of quantitation (LOQ), and recovery. Specificity of the method was demonstrated by applying different stressed conditions to drug samples such as acid hydrolysis, alkaline hydrolysis, oxidative, photolytic, and thermal degradation.Results: The study was conducted using phosphate buffer pH 6.8 and λmax was found to be 312 nm. Standard plot having a concentration range of 1–10 μg/ml showed a good linear relationship with R2=0.999. The LOD and LOQ were found to be 0.118 μg/ml and 0.595 μg/ml, respectively. Recovery and percentage relative standard deviations were found to be 100.157±0.332% and <2%, respectively.Conclusion: Proposed method was successfully applicable to the pharmaceutical formulations containing febuxostat. Thus, the developed method is found to be simple, sensitive, accurate, precise, reproducible, and economical for the determination of febuxostat in pharmaceutical dosage forms.

scholarly journals 4-hour immuno-chromatographic detection of intestinal carriage of carbapenemase-producing Enterobacteriaceae: a validation study

2021 ◽  
Author(s):  
Salah Gallah ◽  
Khanh Villageois-Tran ◽  
Alexandre Godmer ◽  
Guillaume Arlet ◽  
Martin Rottman ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Low Cost ◽  
False Negative ◽  
Negative Likelihood Ratio ◽  
Clinical Settings ◽  
Predictive Values ◽  
Rectal Swabs ◽  
Public Health Challenge ◽  
Chromatographic Detection

Background. The increasing incidence of Carbapenemase-Producing Gram-Negative Bacilli (C-PGNB) represents a major public health challenge. Rapid detection of the digestive colonization with C-PGNB is fundamental to control their spread. Aim. Validation of a rapid protocol for C-PGNB detection directly on rectal swabs. Methods. We developed a protocol combining enrichment by a rapid selective subculture of the rectal swab medium and realization of a Resist-4 OKNV K-SeT® test on the bacterial pellet so obtained. The limit of detection and performances of this protocol were validated in vitro on 52 C-PGNB strains spiked on calibrated sample suspension; and confirmed in clinical settings on 144 rectal swabs sampled from patients with C-PGNB digestive colonization (n=48) and controls (patients with ESBL colonization (n=48) and without carbapenemase/ESBL (n=48)). Results. The protocol detected with 100% sensitivity the presence of the 15 OXA-48-, 14 KPC-, 13 NDM- and 10 VIM-producing GNB from 103 CFU/mL. The limit of detection was 2.102 CFU/mL. Among the 48 C-PGNB containing rectal swabs of the validation cohort, 46 were accurately detected. False negative were observed for 1 NDM-producing Acinetobacter baumanii and 1 OXA-48-producing E. coli. The 96 control swabs were negative. Sensitivity and specificity for C-PGNB detection were 97.7% 95%CI(87.7-100) and 100% 95%CI(96.2-100). The negative likelihood ratio was 0.04 95%CI(0.01-0.16). Considering a C-PGNB digestive colonization prevalence between 0.01% and 0.1%, positive and negative predictive values were 100%. Conclusion. Our protocol is a rapid and low-cost method detecting accurately the digestive colonization with C-PE in 4 hours without any requirement for specific equipment.

scholarly journals Development and Validation of a Spectrophotometric Method for Quantification and Dissolution Studies of Glimepiride in Tablets

2012 ◽  
Vol 9 (2) ◽  
pp. 993-998
Author(s):  
Madhusudhanareddy Induri ◽  
Bhagavan Raju M. ◽  
Rajendra Prasad Y. ◽  
Pavankumar Reddy K.
Keyword(s):  
Quantitative Determination ◽  
Spectrophotometric Method ◽  
Analytical Method ◽  
Limit Of Detection ◽  
Pharmaceutical Formulations ◽  
Spectrophotometric Methods ◽  
Dissolution Studies ◽  
Limit Of Quantitation ◽  
Development And Validation

The objective of present study was to develop and validate an analytical method for quantitative determination and dissolution studies of glimepiride in tablets. The glimepiride shows absorption maxima at 225 nm and obeyed Beer's law in the range of 6.0 – 14.0 µg/mL. The limit of detection and limit of quantitation were 0.06, and 0.17 µg/mL respectively. Percentage recovery of glimepiride for the proposed method ranged from 99.32 to 100.98% indicating no interference of the tablet excipients. It was concluded that the proposed method is simple, easy to apply, economical and used as an alternative to the existing spectrophotometric and non-spectrophotometric methods for the routine analysis of glimepiride in pharmaceutical formulations andin vitrodissolution studies.

scholarly journals Development and validation of a lateral flow immunoassay for rapid detection of VanA-producing enterococci

2020 ◽  
Vol 76 (1) ◽  
pp. 146-151 ◽  
Author(s):  
Saoussen Oueslati ◽  
Hervé Volland ◽  
Vincent Cattoir ◽  
Sandrine Bernabeu ◽  
Delphine Girlich ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Culture Medium ◽  
Limit Of Detection ◽  
Culture Media ◽  
Lateral Flow ◽  
Lateral Flow Immunoassay ◽  
Clinical Microbiology Laboratory ◽  
Mueller Hinton ◽  
Reliable Detection ◽  
Development And Validation

Abstract Background VRE are nosocomial pathogens with an increasing incidence in recent decades. Rapid detection is crucial to reduce their spread and prevent infections and outbreaks. Objectives To evaluate a lateral flow immunoassay (LFIA) (called NG-Test VanA) for the rapid and reliable detection of VanA-producing VRE (VanA-VRE) from colonies and broth. Methods NG-Test VanA was validated on 135 well-characterized enterococcal isolates grown on Mueller–Hinton (MH) agar (including 40 VanA-VRE). Different agar plates and culture broths widely used in routine laboratories for culture of enterococci were tested. Results All 40 VanA-VRE clinical isolates were correctly detected in less than 15 min irrespective of the species expressing the VanA ligase and the medium used for bacterial growth. No cross-reaction was observed with any other clinically relevant ligases (VanB, C1, C2, D, E, G, L, M and N). Overall, the sensitivity and specificity of the assay were 100% for VanA-VRE grown on MH agar plates. NG-Test VanA accurately detects VanA-VRE irrespective of the culture medium (agar and broth). Band intensity was increased when using bacteria grown on vancomycin-containing culture media or on MH close to the vancomycin disc as a consequence of VanA induction. The limit of detection of the assay was 6.3 × 106 cfu per test with bacteria grown on MH plates and 4.9 × 105 cfu per test with bacteria grown on ChromID® VRE plates. Conclusions NG-Test VanA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of VanA-VRE.

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.

DNA-Based Rapid Methods for the Detection of Foodborne Pathogens

1991 ◽  
Vol 54 (5) ◽  
pp. 387-401 ◽  
Author(s):  
MARK J. WOLCOTT
Keyword(s):  
Escherichia Coli ◽  
Yersinia Enterocolitica ◽  
Foodborne Pathogens ◽  
Food Microbiology ◽  
Detection Methods ◽  
Foodborne Illnesses ◽  
Salmonella Spp ◽  
Rapid Methods ◽  
Shigella Spp ◽  
Reliable Methods

The reports of foodborne illnesses are increasing and rapid, sensitive and reliable methods to detect and identify foodborne microorganisms are needed. This review encompasses a description of recent technologies upon which DNA detection methods are based. This review also provides current information on hybridization assays developed or under study for application in food microbiology. Literature pertaining to some of the most common foodborne microorganisms, including Salmonella spp., Shigella spp., Listeria spp., Escherichia coli, and Yersinia enterocolitica is emphasized.

scholarly journals Qualitative Variation among Commercial Immunoassays for Detection of Measles-Specific IgG

2020 ◽  
Vol 58 (6) ◽  
Author(s):  
Donald R. Latner ◽  
Sun B. Sowers ◽  
Kiana Anthony ◽  
Heather Colley ◽  
Christine Badeau ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Measles Virus ◽  
False Negative ◽  
Test Results ◽  
Serum Samples ◽  
Gold Standard Method ◽  
Negative Test ◽  
Specific Igg ◽  
False Negative Test

ABSTRACT Measurement of measles virus-specific IgG is used to assess presumptive evidence of immunity among immunocompetent individuals with uncertain immune or vaccination status. False-negative test results may lead to unnecessary quarantine and exclusion from activities such as employment, education, and travel or result in unnecessary revaccination. In contrast, false-positive results may fail to identify susceptible individuals and promote spread of disease by those who are exposed and unprotected. To better understand the performance characteristics of tests to detect measles IgG, we compared five widely used, commercially available measles IgG test platforms using a set of 223 well-characterized serum samples. Measles virus neutralizing antibodies were also measured by in vitro plaque reduction neutralization, the gold standard method, and compared to IgG test results. Discrepant results were observed for samples in the low-positive ranges of the most sensitive tests, but there was good agreement across platforms for IgG-negative sera and for samples with intermediate to high levels of IgG. False-negative test results occurred in approximately 11% of sera, which had low levels of neutralizing antibody.

Advances in Rapid Detection Methods for Foodborne Pathogens

2014 ◽  
Vol 24 (3) ◽  
pp. 297-312 ◽  
Author(s):  
Xihong Zhao ◽  
Chii-Wann Lin ◽  
Jun Wang ◽  
Deog Hwan Oh
Keyword(s):  
Foodborne Pathogens ◽  
Rapid Detection ◽  
Detection Methods
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