scholarly journals Inhibition of Polymerase Chain Reaction: Pathogen-Specific Controls are better than Human Gene Amplification

2019 ◽  
Author(s):  
Guillaume Roux ◽  
Christophe Ravel ◽  
Emmanuelle Varlet-Marie ◽  
Rachel Jendrowiak ◽  
Patrick Bastien ◽  
...  
Keyword(s):  
Human Gene ◽  
False Negative ◽  
Routine Practice ◽  
Pcr Inhibition ◽  
Specific Sequence ◽  
Negative Results ◽  
Albumin Gene ◽  
False Negative Results ◽  
Pcr Inhibitor ◽  
Pcr Assays

AbstractPCR inhibition is frequent in medical microbiology routine practice and may lead to false-negative results; however there is no consensus on how to detect it. Pathogen-specific and human gene amplifications are widely used to detect PCR inhibition. We aimed at comparing the value of PCR inhibitor detection using these two methods. We analysed Cp shifts (ΔCp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific sequence used in two laboratory-developed microbiological qPCR assays. 3152 samples including various matrixes were included. Pathogen-specific amplification and albumin qPCR identified 62/3152 samples (2.0 %), and 409/3152 (13.0%) samples, respectively, as inhibited. Only 16 samples were detected using both methods. In addition, the use of the Youden’s index failed to determine adequate Cp thresholds for albumin qPCR, even when we distinguished among the different sample matrixes. qPCR targeting the albumin gene therefore appears not adequate to identify the presence of PCR inhibitors in microbiological PCR assays. Our data may be extrapolated to other heterologous targets and should discourage their use to assess the presence of PCR inhibition in microbiological PCR assays.

scholarly journals False-Negative Nasopharyngeal Swab RT-PCR Assays in Typical COVID-19: Role of Ultra-low-dose Chest CT and Bronchoscopy in Diagnosis

2020 ◽  
Author(s):  
Marco Marando ◽  
Adriana Tamburello ◽  
Pietro Gianella
Keyword(s):  
Low Dose ◽  
False Negative ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Oral Swab ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain ◽  
Pcr Assays

On 11 March 2020, the WHO declared COVID-19 a pandemic and global health emergency. We describe the clinical features and role of ultra-low-dose chest computed tomography (CT) and bronchoscopy in the diagnosis of coronavirus disease (COVID-19). In our patient, who was highly suggestive clinically and radiologically for COVID-19, we had two false-negative results for nasopharyngeal and oral swab reverse-transcriptase polymerase chain reaction (RT-PCR) assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Eventually, we confirmed the diagnosis using bronchoscopy and bronchoalveolar lavage (BAL).

scholarly journals Assessment of Commercial DNA Cleanup Kits for Elimination of Real-Time PCR Inhibitors in the Detection of Cyclospora cayetanensis in Cilantro

2020 ◽  
Vol 83 (11) ◽  
pp. 1863-1870
Author(s):  
ANGELA ASSURIAN ◽  
HELEN MURPHY ◽  
ALICIA SHIPLEY ◽  
HEDIYE NESE CINAR ◽  
ALEXANDRE DA SILVA ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Threshold Values ◽  
Cyclospora Cayetanensis ◽  
Cycle Threshold ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Inhibitor ◽  
Zymo Research

ABSTRACT Inhibited reactions have occasionally been observed when cilantro samples were processed for the detection of Cyclospora cayetanensis using quantitative real-time PCR (qPCR). Partial or total inhibition of PCR reactions, including qPCR, can occur, leading to decreased sensitivity or false-negative results. If inhibition occurs, this implies the need for additional purification or cleanup treatments of the extracted DNA to remove inhibitors prior to molecular detection. Our objective was to evaluate the performance of five commercial DNA cleanup kits (QIAquick purification kit from Qiagen [kit 1], OneStep PCR inhibitor removal by Zymo Research [kit 2], NucleoSpin genomic DNA cleanup XS from Macherey-Nagel [kit 3], DNA IQ system by Promega [kit 4], and DNeasy PowerPlant pro kit from Qiagen [5]) to minimize qPCR inhibition using the U.S. Food and Drug Administration–validated Bacteriological Analytical Manual (BAM) Chapter 19b method for detection of C. cayetanensis in cilantro samples containing soil. Each of the five commercial DNA cleanup kits evaluated was able to reduce the qPCR internal amplification control cycle threshold values to those considered to be normal for noninhibited samples, allowing unambiguous interpretation of results in cilantro samples seeded at both a high oocyst level (200 oocysts) and a low oocyst level (10 oocysts). Of the five kits compared, kits 1, 2, and 3 did not show significant differences in the detection of C. cayetanensis, while significantly higher cycle threshold values, indicating lower recovery of the target DNA, were observed from kits 4 and/or 5 in samples seeded with 200 and 10 oocysts (P < 0.05). This comparative study provides recommendations on the use of commercial cleanup kits which could be implemented when inhibition is observed in the detection of C. cayetanensis in cilantro samples using the BAM Chapter 19b method. HIGHLIGHTS

scholarly journals An Algorithm to Optimize Pain Detection and Management in Older Patients in Routine Practice

2020 ◽  
Vol 4 (4) ◽  
Author(s):  
Sylvie Bonin-Guillaume ◽  
◽  
Patrice Rat ◽  
Keyword(s):  
Older Patients ◽  
Health Workers ◽  
False Negative ◽  
Medical Personnel ◽  
Routine Clinical Practice ◽  
Routine Practice ◽  
Negative Results ◽  
False Negative Results ◽  
Pain Scales ◽  
Pain Detection

Acute or persistent pain is a common occurrence and is often undertreated in older patients, especially those with an inability to communicate verbally (ICV). Regular comprehensive pain assessment, including self-rating and/or behavior scales, is critical but difficult to implement in routine clinical practice. The choice of the most appropriate scale for each patient is not easy, even for trained and skilled medical personnel. Indeed, the use of scales for short pain-behavior exposes to pain under-detection due to false-negative results, whereas extensive behavior scales are time-consuming, require pre-rating knowledge of the patient, and can also yield false-positive scores because of an overlap between pain-associated behaviors and those of other non-painful conditions (i.e., dementia, delirium, or depression). We describe the process used to devise an at-a-glance algorithm targeting medical personnel. This algorithm combines short and long pain scales specifically validated for geriatric populations with ICV or without. This algorithm has been tested by health workers in several settings. The final version of this algorithm reliably detected nociceptive acute or chronic pain in older patients and can easily be applied to older patients in routine practice in any setting. This algorithm ensured a rapid and easy-to-use comprehensive assessment of pain in older patients to determine the need for analgesic administration.

scholarly journals Development and application of a canine endogenous internal positive control for use in real-time PCR assays

2018 ◽  
Vol 30 (5) ◽  
pp. 789-792 ◽  
Author(s):  
Joseph J. Modarelli ◽  
Pamela J. Ferro ◽  
Maria D. Esteve-Gasent
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Positive Control ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Negative Results ◽  
False Negative Results ◽  
Internal Positive Control ◽  
Pcr Assays

Real-time PCR (rtPCR) tests have become a method of choice in many diagnostic settings, both animal and human. A concern remains, however, regarding rtPCR assay inhibition during nucleic acid extraction and/or rtPCR reaction process that may result in false-negative results. The use of an internal positive control, either endogenous or exogenous, to mitigate this issue has become more commonplace. We identified and standardized an endogenous internal positive control that can be utilized in rtPCR assays targeting canine-specific pathogens in either a singleplex or multiplex format. The target chosen for the endogenous internal positive control (EIPC-K9) was a highly conserved region in canine mitochondrial DNA. Samples from 240 dogs and 11 other species were screened with EIPC-K9; all canine samples were detected, and no cross-amplification with other species tested was observed. Additionally, no inhibition was noted when comparing singleplex to multiplex rtPCR formats.

scholarly journals Exogenous Controls Increase Negative Call Veracity in Multiplexed, Quantitative PCR Assays for Phakopsora pachyrhizi

Plant Disease ◽  
2011 ◽  
Vol 95 (3) ◽  
pp. 343-352 ◽  
Author(s):  
James S. Haudenshield ◽  
Glen L. Hartman
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Arbitrary Sequence ◽  
Phakopsora Pachyrhizi ◽  
Synthetic Dna ◽  
Negative Results ◽  
Detection And Identification ◽  
False Negative Results ◽  
Pcr Assays ◽  
Rust Pathogens

Quantitative polymerase chain reaction (Q-PCR) utilizing specific primer sequences and a fluorogenic, 5′-exonuclease linear hydrolysis probe is well established as a detection and identification method for Phakopsora pachyrhizi and P. meibomiae, two rust pathogens of soybean. Because of the extreme sensitivity of Q-PCR, the DNA of single urediniospores of these fungi can be detected from total DNA extracts of environmental samples. However, some DNA preparations unpredictably contain PCR inhibitors that increase the frequency of false negatives indistinguishable from true negatives. Three synthetic DNA molecules of arbitrary sequence were constructed as multiplexed internal controls (ICs) to cull false-negative results by producing a positive signal to validate the PCR process within each individual reaction. The first two, PpaIC and PmeIC, are a single-stranded oligonucleotide flanked by sequences complementary to the primers of either the P. pachyrhizi or P. meibomiae primary assay but hybridizing to a unique fluorogenic probe; the third contains unique primer- and probe-binding sequences, and was prepared as a cloned DNA fragment in a linearized plasmid. These ICs neither qualitatively nor quantitatively affected their primary assays. PpaIC and PmeIC were shown to successfully identify false-negative reactions resulting from endogenous or exogenous inhibitors, and can be readily adapted to function in a variety of diagnostic Q-PCR assays; the plasmid was found to successfully validate true negatives in similar Q-PCR assays for other soybean pathogens, as well as to function as a tracer molecule during DNA extraction and recovery.

scholarly journals Mutations in Animal SARS-CoV-2 Induce Mismatches with the Diagnostic PCR Assays

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 371
Author(s):  
Ahmed Elaswad ◽  
Mohamed Fawzy
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Animal Species ◽  
False Negative ◽  
Bioinformatic Analysis ◽  
Diagnostic Pcr ◽  
Animal Host ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Assays

Recently, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was detected in several animal species. After transmission to animals, the virus accumulates mutations in its genome as adaptation to the new animal host progresses. Therefore, we investigated whether these mutations result in mismatches with the diagnostic PCR assays and suggested proper modifications to the oligo sequences accordingly. A comprehensive bioinformatic analysis was conducted using 28 diagnostic PCR assays and 793 publicly available SARS-CoV-2 genomes isolated from animals. Sixteen out of the investigated 28 PCR assays displayed at least one mismatch with their targets at the 0.5% threshold. Mismatches were detected in seven, two, two, and six assays targeting the ORF1ab, spike, envelope, and nucleocapsid genes, respectively. Several of these mismatches, such as the deletions and mismatches at the 3’ end of the primer or probe, are expected to negatively affect the diagnostic PCR assays resulting in false-negative results. The modifications to the oligo sequences should result in stronger template binding by the oligos, better sensitivity of the assays, and higher confidence in the result. It is necessary to monitor the targets of diagnostic PCR assays for any future mutations that may occur as the virus continues to evolve in animals.

Critical Evaluation of Impedance Phlebography for the Diagnosis of Deep Vein Thrombosis

1974 ◽  
Vol 31 (02) ◽  
pp. 273-278
Author(s):  
Kenneth K Wu ◽  
John C Hoak ◽  
Robert W Barnes ◽  
Stuart L Frankel
Keyword(s):  
Deep Vein Thrombosis ◽  
False Positive ◽  
False Negative ◽  
Critical Evaluation ◽  
Original Method ◽  
Vein Thrombosis ◽  
Negative Results ◽  
False Negative Results ◽  
Deep Vein ◽  
Positive Results

SummaryIn order to evaluate its daily variability and reliability, impedance phlebography was performed daily or on alternate days on 61 patients with deep vein thrombosis, of whom 47 also had 125I-fibrinogen uptake tests and 22 had radiographic venography. The results showed that impedance phlebography was highly variable and poorly reliable. False positive results were noted in 8 limbs (18%) and false negative results in 3 limbs (7%). Despite its being simple, rapid and noninvasive, its clinical usefulness is doubtful when performed according to the original method.

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