scholarly journals SARS-CoV-2 Detection for Diagnosis Purposes in the Setting of a Molecular Biology Research Lab

2020 ◽  
Vol 3 (3) ◽  
pp. 59 ◽  
Author(s):  
Damien Coupeau ◽  
Nicolas Burton ◽  
Noémie Lejeune ◽  
Suzanne Loret ◽  
Astrid Petit ◽  
...  
Keyword(s):  
Molecular Biology ◽  
False Negative ◽  
Basic Research ◽  
Clinical Samples ◽  
Acid Extraction ◽  
Public Health Systems ◽  
Negative Results ◽  
False Negative Results ◽  
Biology Lab ◽  
Biology Research

The emergence of the SARS-CoV-2 virus and the exponential growth of COVID-19 cases have created a major crisis for public health systems. The critical identification of contagious asymptomatic carriers requires the isolation of viral nucleic acids, reverse transcription, and amplification by PCR. However, the shortage of specific proprietary reagents or the lack of automated platforms have seriously hampered diagnostic throughput in many countries. Here, we provide a procedure for SARS-CoV-2 detection for diagnostic purposes from clinical samples in the setting of a basic research molecular biology lab. The procedure details the necessary steps for daily analysis of up to 500 clinical samples with a team composed of 12 experienced researchers. The protocol has been designed to rely on widely available reagents and devices, to cope with heterogeneous clinical specimens, to guarantee nucleic acid extraction from very scarce biological material, and to minimize the rate of false-negative results.

scholarly journals Detection of LytA Genes in Streptococcus pneumoniae Isolated from sputum pneumonia patients

Biomedika ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 23-30
Author(s):  
Mustika Sari Hutabarat ◽  
Firdaus Hamid ◽  
Irawaty Djaharuddin ◽  
Alfian Zainuddin ◽  
Rossana Agus ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
False Negative ◽  
Pneumococcal Infection ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Biochemical Tests ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain

Streptococcus pneumoniae (pneumococcus) is a Gram-positive facultative anaerobic bacterium that is a major cause of morbidity and mortality worldwide. But the lack of reporting of disease by this bacterium in Indonesia, one of the causes is because the diagnosis of pneumococcal infection is often clinically not typical and conventional methods which are still the standard gold method often give false-negative results. So the purpose of this study was to evaluate the performance of culture and molecular diagnostic methods using the Polymerase Chain Reaction (PCR) technique in detecting Streptococcus pneumoniae in sputum clinical samples using the Autolysin (LytA) gene which is a virulence factor of this bacterium. 57 isolates from 60 samples were confirmed as Streptococcus sp through microscopic identification, culture, and biochemical tests. Then the sensitivity test with an optochin test of 9 (9%) compared the results descriptively with the PCR technique using the Autolysin A (LytA) gene which was obtained more sensitive by 15 (25%).

scholarly journals The Use of a Mimic to Detect Polymerase Chain Reaction– Inhibitory Factors in Feces Examined for the Presence of Lawsonia Intracellularis

2003 ◽  
Vol 15 (3) ◽  
pp. 268-273 ◽  
Author(s):  
Magdalena Jacobson ◽  
Stina Englund ◽  
András Ballagi-Pordány
Keyword(s):  
Polymerase Chain Reaction ◽  
False Negative ◽  
Clinical Samples ◽  
Lawsonia Intracellularis ◽  
Chain Reaction ◽  
Fecal Samples ◽  
Wild Boars ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain

Lawsonia intracellularis is an intracellular organism that causes proliferative enteritis in pigs. This bacterium is difficult to culture, and antemortem demonstration of the microbe is therefore often performed on fecal samples by polymerase chain reaction (PCR). Polymerase chain reaction is sensitive and specific, but inhibitory factors in feces might cause false-negative results. This article describes the construction and use of an internal standard, a mimic. The mimic is amplified by the same primers as those used for L. intracellularis DNA and thus could indicate false-negative results in clinical samples. The amplicon was clearly visible when as few as 10 mimic molecules were added per amplification reaction and when no inhibitors were present. When fecal samples were spiked with the mimic, the detection limit was 102 molecules per PCR. Sixty clinical samples, 20 from wild boars, 20 from growing pigs with diarrhea, and 20 from pigs without diarrhea, were prepared by a boiling procedure and subjected to PCR together with 103 mimic molecules. Nine samples were positive, of which 7 originated from pigs with diarrhea and 2 from pigs without diarrhea. In 14 samples from wild boars, in 8 samples from pigs without diarrhea, and in 3 samples from pigs with diarrhea, neither the mimic nor the target DNA was visible. This indicated the presence of inhibitors in these samples. It is concluded that the mimic can be used as an internal control in the diagnosis of L. intracellularis to indicate inhibition of PCR.

scholarly journals SARS-CoV-2 RNA detection using pooling of self-collected samples: Simple protocol may foster asymptomatic surveillance

2020 ◽  
Author(s):  
Giselle Ibette Lopez-Lopes ◽  
Rita de Cassia Compagnoli Carmona ◽  
Valéria Oliveira Silva ◽  
Cintia Mayumi Ahagon ◽  
Lincoln Spinazola do Prado ◽  
...  
Keyword(s):  
False Negative ◽  
Clinical Samples ◽  
Rna Detection ◽  
Negative Results ◽  
False Negative Results ◽  
Throat Wash ◽  
Asymptomatic Volunteers ◽  
Simple Protocol ◽  
Antigen Tests ◽  
Public Health Institute

1AbstractBackgroundSurveillance of COVID infection and isolation of infected individuals is one of the available tools to control the spread of SAR-CoV-2. Asymptomatic and pre symptomatic are responsible for substantial transmission. RNA or antigen tests are necessary to identify non-symptomatic individuals. We tested the feasibility of using samples pooling offering different collection alternatives (swab/throat wash/saliva) to volunteers of a public health institute.MethodsWe evaluated pool samples from frozen material from previously tested samples and a prospective collection from asymptomatic volunteers. Some collections were paired for comparison. Pools and some individual samples were extracted with QIAamp Viral RNA Mini Kit (Qiagen, USA) and/or Lucigen Quick Extract DNA extraction solution (BioSearch, USA) and submitted to rtPCR (Allplex, Seegene, Korea).ResultsA total of 240 samples from 130 new collections and 37 samples with known result were evaluated. Pool CT was generally higher than individual samples. Lucigen extraction showed higher CT, including false negative results for samples with high CT at Qiagen extraction. Paired Swab and TW samples showed comparable results. No volunteer from negative pools reported any symptom in the 2-3 days after collection.ConclusionsClinical samples pooling to detect SARS-CoV-2 RNA is feasible and an economical way to test for COVID-19, especially in surveillance strategies targeting more infectiousness, higher viremia individuals. The use of Lucigen reagents show lower sensibility that may lead to false negative results with lower viremia samples. Combining throat wash with saliva may provide and interesting self-collection alternative, but more comparative work is needed.

scholarly journals Impact of false negative results of RT-PCR test for SARS-CoV-2 in COVID-19 case count as applied to Mexico

2020 ◽  
Author(s):  
Isaac J. Núñez ◽  
Pablo F. Belaunzarán-Zamudio ◽  
Yanink Caro-Vega
Keyword(s):  
False Negative ◽  
Open Data ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Negative Results ◽  
Case Count ◽  
False Negative Results ◽  
True Number ◽  
High Prevalence ◽  
Polymerase Chain

Underestimation of the number of cases during the COVID-19 pandemic has been a constant concern worldwide. Case confirmation is based on identification of SARS-CoV-2 RNA using real time polymerase chain reaction (RT-PCR) in clinical samples. However, these tests have suboptimal sensitivity, especially during the early and late course of infection. Using open data, we estimated that among 1 343 730 people tested in Mexico since February 27th, there were 838 377 (95% CL 734 605 - 1 057 164) cases, compared with 604 376 considering only positive tests. ICU admissions and deaths were around 16% and 9% higher than reported. Thus, we show that accounting for the sensitivity of SARS-Cov-2 RT-PCR diagnostic tests is a simple way to improve estimations for the true number of COVID-19 cases in tested people, particularly in high-prevalence populations. This could aid to better inform public health measures and reopening policies.

scholarly journals Trueness evaluation and verification of inter-assay agreement of serum folate measuring systems

2020 ◽  
Vol 58 (10) ◽  
pp. 1697-1705
Author(s):  
Federica Braga ◽  
Erika Frusciante ◽  
Simona Ferraro ◽  
Mauro Panteghini
Keyword(s):  
False Negative ◽  
Serum Folate ◽  
Clinical Samples ◽  
Measuring Systems ◽  
Negative Results ◽  
False Negative Results ◽  
Performance Specifications ◽  
The Mean ◽  
Intercomparison Study ◽  
Is Value

AbstractBackgroundDefinitive data to establish if the use of the WHO International Standard (IS) 03/178 as a common calibrator of commercial measuring systems (MSs) has improved the harmonization of serum total folate (tFOL) measurements to a clinically suitable level are lacking. Here, we report the results of an intercomparison study aimed to verify if the current inter-assay variability is acceptable for clinical application of tFOL testing.MethodsAfter confirming their commutability, the IS 03/178 and National Institute for Standards and Technology SRM 3949 L1 were used for evaluating the correctness of traceability implementation by manufacturers and the MSs trueness, respectively. The inter-assay agreement was verified using 20 patient pools. The measurement uncertainty (U) of tFOL measurements on clinical samples was also estimated. An outcome-based model for defining desirable performance specifications for bias and imprecision for serum tFOL measurements was applied.ResultsThe majority of evaluated MSs overestimated the WHO IS value of +5% or more with the risk to produce an unacceptably high number of false-negative results in clinical practice. The mean inter-assay CV on all pools and on those with tFOL values >3.0 μg/L (n = 15) was 12.5% and 7.1%, respectively. In neither case the goal of 3.0% was fulfilled. The residual bias resulted in an excessive U of tFOL measurement on clinical samples.ConclusionsThe implementation of traceability of tFOL MSs to the WHO IS 03/178 is currently inadequate, resulting in an inter-assay variability that does not permit the use of a common threshold for detecting folate deficiency.

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 Soft Salt-Mannitol Agar–Cloxacillin Test: a Highly Specific Bedside Screening Test for Detection of Colonization with Methicillin-Resistant Staphylococcus aureus

1998 ◽  
Vol 36 (4) ◽  
pp. 986-989 ◽  
Author(s):  
Nuria Mir ◽  
Miguel Sánchez ◽  
Fernando Baquero ◽  
Blanca López ◽  
Celia Calderón ◽  
...  
Keyword(s):  
Intensive Care Unit ◽  
Staphylococcus Aureus ◽  
Intensive Care ◽  
Predictive Value ◽  
Color Change ◽  
False Negative ◽  
Clinical Samples ◽  
Methicillin Resistant ◽  
Negative Results ◽  
False Negative Results

The early detection of colonization with methicillin-resistantStaphylococcus aureus (MRSA) of patients in intensive-care units is an essential step in the strategy for preventing MRSA epidemics. In this study, tubes containing soft salt-mannitol agar with cloxacillin (6 μg/ml) (SSMAC) were prepared for inoculation of clinical samples at patients’ bedsides by personnel of an intensive-care unit. A total of 1,914 swabs from different sample sites of 81 patients were dipped into SSMAC tubes, and after 24 h of incubation (in an incubator located near the intensive-care unit), an evident color change was considered by the intensive-care-unit personnel to be an MRSA alarm. Sixty-three (3.3%) SSMAC tubes were considered positive for MRSA, 1,827 (95.4%) were considered negative, and 24 (1.2%) were considered intermediate. Compared with values for parallel conventional surveillance cultures for MRSA, excluding tubes with intermediate results, the SSMAC test had a sensitivity of 72.7%, a specificity of 99.2%, a positive predictive value of 76.2%, and a negative predictive value of 99.0%. When intermediate tubes were considered positive, the corresponding values were 75.3, 98.2, 63.2, and 99.0%, respectively. The sensitivity and specificity values of the test to identify MRSA-colonized patients were 89.4 and 100%, respectively. Oropharyngeal and naris specimens were the most reliable samples for MRSA detection. False-negative results were frequent in bronchial aspirates with low (<103 to 106CFU/ml) MRSA counts. False-positive results were mainly due to methicillin-resistant Staphylococcus haemolyticus. The SSMAC tube is a useful, rapid, and inexpensive tool for the early identification of MRSA-colonized patients and, consequently, for the implementation of measures to prevent the spread of MRSA.

Sandwich dot-immunogold filtration assay (DIGFA) for specific immunodiagnosis of active neuroangiostrongyliasis

Parasitology ◽  
2020 ◽  
pp. 1-6
Author(s):  
Praphathip Eamsobhana ◽  
Anchalee Tungtrongchitr ◽  
Hoi-Sen Yong ◽  
Anchana Prasartvit ◽  
Darawan Wanachiwanawin ◽  
...  
Keyword(s):  
False Negative ◽  
Clinical Samples ◽  
Serum Samples ◽  
Serological Tests ◽  
Past Infection ◽  
Negative Results ◽  
Resource Limited ◽  
False Negative Results ◽  
Circulating Antigens ◽  
Specific Antigens

Abstract Serological tests may yield false-negative results for specific antibodies detection before or at the early seroconversion phase. Tests that detect circulating antigens of Angiostrongylus cantonensis would therefore be of value in diagnosis to distinguish current or past infection. Here, a quick, easy to perform, portable and inexpensive diagnostic device for detection of 31-kDa A. cantonensis specific antigens had been developed. This sandwich dot-immunogold filtration assay (AcDIGFAAg), for detecting active angiostrongyliasis was produced using anti-A. cantonensis polyclonal antibody dotted on the nitrocellulose membrane as a capture agent and colloidal gold-labelled anti-31 kDa A. cantonensis antibody as a detection agent. A well-defined pink dot, indicating positivity, was seen readily by naked eye within 10–15 min. The AcDIGFAAg detected A. cantonensis-specific antigens in cerebrospinal fluid samples from 4 out of 10 serologically confirmed angiostrongyliasis cases and 2 out of 5 suspected cases with negative anti-A. cantonensis antibodies. Among the 19 patient sera with A. cantonensis infection, 2 showed positive reaction by AcDIGFAAg. No positive AcDIGFAAg reaction was observed in all the serum samples with other parasitic diseases, and the healthy controls. The present ‘AcDIGFAAg’ enables rapid qualitative detection of the specific 31-kDa antigens of A. cantonensis in clinical samples with potential for application even under resource-limited settings.

scholarly journals Target specific serologic analysis of COVID-19 convalescent plasma

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0249938
Author(s):  
Sachie Ikegami ◽  
Robert C. Benirschke ◽  
Hossein Fakhrai-Rad ◽  
Mohammad H. Motamedi ◽  
Rick Hockett ◽  
...  
Keyword(s):  
False Negative ◽  
High Specificity ◽  
Clinical Samples ◽  
Disease Symptoms ◽  
Clinical Sensitivity ◽  
Negative Results ◽  
False Negative Results ◽  
Assay Performance ◽  
Roche Diagnostics ◽  
Donor Characteristics

This study compared the performance of four serology assays for Coronavirus Disease 2019 (COVID-19) and investigated whether COVID-19 disease history correlates with assay performance. Samples were tested at Northshore using the Elecsys Anti-SARS-CoV-2 (Roche Diagnostics), Access SARS-CoV-2 IgG anti-RBD (Beckman Coulter), and LIAISON SARS-CoV-2 S1/S2 IgG (DiaSorin) as well as at Genalyte using Maverick Multi-Antigen Serology Panel. The study included one hundred clinical samples collected before December 2019 and ninety-seven samples collected from convalescent plasma donors originally diagnosed with COVID-19 by PCR. COVID-19 disease history was self-reported by the plasma donors. There was no difference in specificity between the assays tested. Clinical sensitivity of these four tests was 98% (Genalyte), 96% (Roche), 92% (DiaSorin), and 87% (Beckman). The only statistically significant differences in clinical sensitivity was between the Beckman assay and both Genalyte and Roche assays. Convalescent plasma donor characteristics and disease symptoms did not correlate with false negative results from the Beckman and DiaSorin assays. All four tests showed high specificity (100%) and varying sensitivities (89–98%). No correlations between disease history and serology results were observed. The Genalyte Multiplex assay showed as good or better sensitivity to three other previously validated assays with FDA Emergency Use Authorizations.

scholarly journals Multitarget PCR Assay for Direct Detection of Penicillinase-Producing Neisseria gonorrhoeae for Enhanced Surveillance of Gonococcal Antimicrobial Resistance

2015 ◽  
Vol 53 (8) ◽  
pp. 2706-2708 ◽  
Author(s):  
Cameron Buckley ◽  
Ella Trembizki ◽  
Robert W. Baird ◽  
Marcus Chen ◽  
Basil Donovan ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Direct Detection ◽  
False Negative ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
Negative Results ◽  
False Negative Results ◽  
Sequence Variations

A multitarget PCR was developed for the direct detection of penicillinase-producing Neisseria gonorrhoeae (PPNG). The assay was validated by testing 342 PPNG isolates and 415 clinical samples. The method is suitable for routine detection of PPNG strains. Its multitarget approach reduces the potential for false-negative results caused by sequence variations.

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