The Omicron variant mutation at position 28,311 in the SARS-CoV-2 N gene does not perturb CDC N1 target detection

The emergence of new SARS-CoV-2 variants necessitates the reevaluation of current COVID-19 tests to ensure continued accuracy and reliability. The new SARS-CoV-2 variant, Omicron, is heavily mutated, with over 50 mutations within its RNA genome. Any of these mutations could adversely affect the ability of diagnostic assays to detect the virus in patient samples, potentially leading to inconclusive or false negative results. In fact, the U.S. Food and Drug Administration (FDA) has identified over two dozen diagnostic tests that contain a gene target that is expected to have significantly reduced sensitivity due to a mutation in the SAS-CoV-2 Omicron variant1. Additionally, one of the U.S. Centers for Disease Control and Prevention (CDC) Emergency Use Authorization (EUA) targets for COVID-19 tests, 2019-nCoV_N1, overlaps an Omicron mutation within the sequence targeted by the fluorescent probe. This target from the CDC has been used in many other EUA assays. Using in vitro transcribed (IVT) N gene RNA representing the wild-type (GenBank/GISAID ID MN908947.3) and Omicron variant (BA.1, GISAID ID EPI_ISL_6752027), we evaluated the performance of two different amplification protocols, both of which include the CDC 2019-nCoV_N1 primer-probe set. Both assays were able to detect the mutant N1 sequence as efficiently as the wild-type sequence. Consequently, these data suggest that diagnostic assays that use the 2019-nCoV-N1 primer-probe set are unlikely to be impacted by currently circulating Omicron lineage viruses.

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Aspiration Revisited: Prospective Evaluation of a Physiologically Pressurized Model With Animal Correlation and Broader Applicability to Filler Complications

Aesthetic Surgery Journal ◽

10.1093/asj/sjab194 ◽

2021 ◽

Author(s):

Hyoung-Jin Moon ◽

Won Lee ◽

Ji-Soo Kim ◽

Eun-Jung Yang ◽

Hema Sundaram

Keyword(s):

Normal Saline ◽

False Negative ◽

Vascular Complications ◽

Filler Injection ◽

Negative Results ◽

Needle Position ◽

False Negative Results ◽

In Vivo Testing

Abstract Background Aspiration testing before filler injection is controversial. Some believe that aspiration can help prevent inadvertent intravascular injection, while others cite false-negative results and question its value given that the needle position always changes somewhat during injection procedures. Objectives To test the relation of false-negative results to the viscosity of the material within the needle lumen and determine whether a less viscous material within the needle lumen could decrease the incidence of false-negative results. Methods In vitro aspiration tests were performed using 30-G and 27-G needle gauges, two cross-linked hyaluronic acid fillers, normal saline bags pressurized at 140 and 10 mmHg to mimic human arterial and venous pressures, and three needle lumen conditions (normal saline, air, and filler). Testing was repeated three times under each study condition (72 tests in total). For in vivo correlation, aspiration tests were performed on femoral arteries and central auricular veins in three rabbits (4–5 aspirations per site, 48 tests in total). Results In vitro and in vivo testing using 30-G needles containing filler both showed false-negative results on aspiration testing. In vitro and in vivo testing using needles containing saline or air showed positive findings. Conclusions False-negative results from aspiration testing may be reduced by pre-filling the needle lumen with saline rather than a filler. The pressurized system may help overcome challenges of animal models with intravascular pressures significantly different from those of humans. The adaptability of this system to mimic various vessel pressures may facilitate physiologically relevant studies of vascular complications.

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Most but not all laboratories can detect the recently emerged Neisseria gonorrhoeae porA mutants – results from the QCMD 2013 N. gonorrhoeae external quality assessment programme

Eurosurveillance ◽

10.2807/1560-7917.es2014.19.8.20711 ◽

2014 ◽

Vol 19 (8) ◽

Cited By ~ 5

Author(s):

D Luijt ◽

C Di Lorenzo ◽

A M van Loon ◽

M Unemo

Keyword(s):

Neisseria Gonorrhoeae ◽

Quality Assessment ◽

External Quality Assessment ◽

False Negative ◽

Molecular Diagnostic ◽

External Quality ◽

Diagnostic Assays ◽

Assessment Programme ◽

Negative Results ◽

False Negative Results

We describe the results of the Quality Control for Molecular Diagnostics 2013 Neisseria gonorrhoeae external quality assessment programme that included an N. gonorrhoeae strain harbouring an N. meningitidis porA gene which causes false-negative results in molecular diagnostic assays targeting the gonococcal porA pseudogene. Enhanced awareness of the international transmission of such gonococcal strains is needed to avoid false-negative results in both in-house and commercial molecular diagnostic assays used in laboratories worldwide, but particularly in Europe.

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False-negative results for urinary phenothiazines and imipramine in Forrest's qualitative assays.

Clinical Chemistry ◽

10.1093/clinchem/26.2.345 ◽

1980 ◽

Vol 26 (2) ◽

pp. 345-347 ◽

Cited By ~ 1

Author(s):

G P James ◽

M H DJang ◽

H H Hamilton

Keyword(s):

Ascorbic Acid ◽

Ion Exchange ◽

Exchange Resin ◽

Anion Exchange Resin ◽

False Negative ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Negative Results ◽

False Negative Results

Abstract When a series of patients' urine samples supplemented in vitro with chlorpromazine or imipramine was assayed with the Forrest qualitative assays, we observed an occasional false-negative result, which we found was attributable to interference by ascorbic acid. It interferes with the reagent, not with the analytes, in both assays. We easily eliminated this interference with the phenothiazine test by using an anion-exchange resin. Eliminating the interference with the assay for imipramine, however, is more difficult; false-negative results can be obtained even after ion-exchange chromatography if the imipramine concentration is less than 50 mg/L.

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Towards Multitarget Testing in Molecular Microbiology

International Journal of Microbiology ◽

10.1155/2013/121057 ◽

2013 ◽

Vol 2013 ◽

pp. 1-6 ◽

Cited By ~ 9

Author(s):

Deborah Steensels ◽

Anne Vankeerberghen ◽

Hans De Beenhouwer

Keyword(s):

False Negative ◽

Lessons Learned ◽

Patient Treatment ◽

Culture Methods ◽

Diagnostic Assays ◽

Genome Variability ◽

Negative Results ◽

Conventional Culture ◽

False Negative Results ◽

Single Target

Advantages of PCR assays over more conventional culture-based diagnostics include significantly higher sensitivities and shorter turnaround times. They are particularly useful when patient treatment has already been initiated or for specimens that may contain microorganisms that are slow-growing, difficult to culture, or for which culture methods do not exist. However, due to genome variability, single target testing might lead to false-negative results. This paper focuses on examples from our own experiences and the literature to provide insight into the limitations of single target testing in molecular biology. Lessons learned from these experiences include the careful design of diagnostic assays, preferably multitargeted, the importance of investigating the incidence and epidemiology of infection in detail, the frequent participation in appropriate quality assurance schemes, and the importance of continuous attentiveness by investigators when confronted with inconsistent results. In conclusion, multitargeted testing in microbiological molecular assays should be a rule.

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Validation of Aztreonam-Avibactam Susceptibility Testing Using Digitally Dispensed Custom Panels

Journal of Clinical Microbiology ◽

10.1128/jcm.01944-19 ◽

2020 ◽

Vol 58 (4) ◽

Author(s):

Eric Ransom ◽

Amelia Bhatnagar ◽

Jean B. Patel ◽

Maria-Jose Machado ◽

Sandra Boyd ◽

...

Keyword(s):

Susceptibility Testing ◽

Internal Validation ◽

Serious Infections ◽

Control And Prevention ◽

Approved Drugs ◽

Multiple Experts ◽

Fda Approved Drugs ◽

The U.S ◽

Complex Drug

ABSTRACT Aztreonam-avibactam is a combination antimicrobial agent with activity against carbapenemase-producing Enterobacteriaceae (CPE) with metallo-β-lactamases (MβLs). Although aztreonam-avibactam is not yet approved by the U.S. Food and Drug Administration (FDA), clinicians can administer this combination by using two FDA-approved drugs: aztreonam and ceftazidime-avibactam. This combination of drugs is recommended by multiple experts for treatment of serious infections caused by MβL-producing CPE. At present, in vitro antimicrobial susceptibility testing (AST) of aztreonam-avibactam is not commercially available; thus, most clinicians receive no laboratory-based guidance that can support consideration of aztreonam-avibactam for serious CPE infections. Here, we report our internal validation for aztreonam-avibactam AST by reference broth microdilution (BMD) according to Clinical and Laboratory Standards Institute (CLSI) guidelines. The validation was performed using custom frozen reference BMD panels prepared in-house at the Centers for Disease Control and Prevention (CDC). In addition, we took this opportunity to evaluate a new panel-making method using a digital dispenser, the Hewlett Packard (HP) D300e. Our studies demonstrate that the performance characteristics of digitally dispensed panels were equivalent to those of conventionally prepared frozen reference BMD panels for a number of drugs, including aztreonam-avibactam. We found the HP D300e digital dispenser to be easy to use and to provide the capacity to prepare complex drug panels. Our findings will help other clinical and public health laboratories implement susceptibility testing for aztreonam-avibactam.

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Exocytosis induction in Paramecium tetraurelia cells by exogenous phosphoprotein phosphatase in vivo and in vitro: possible involvement of calcineurin in exocytotic membrane fusion.

The Journal of Cell Biology ◽

10.1083/jcb.105.1.181 ◽

1987 ◽

Vol 105 (1) ◽

pp. 181-189 ◽

Cited By ~ 67

Author(s):

M Momayezi ◽

C J Lumpert ◽

H Kersken ◽

U Gras ◽

H Plattner ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Membrane Fusion ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Current Finding ◽

Phosphoprotein Phosphatase ◽

Negative Results ◽

Molecular Components

Since it had been previously shown that in Paramecium cells exocytosis involves the dephosphorylation of a 65-kD phosphoprotein (PP), we tried to induce exocytotic membrane fusion by exogenous phosphatases (alkaline phosphatase or calcineurin [CaN]). The occurrence of calmodulin (CaM) at preformed exocytosis sites (Momayezi, M., H. Kersken, U. Gras, J. Vilmart-Seuwen, and H. Plattner, 1986, J. Histochem. Cytochem., 34:1621-1638) and the current finding of the presence of the 65-kD PP and of a CaN-like protein in cell surface fragments ("cortices") isolated from Paramecium cells led us to also test the effect of antibodies (Ab) against CaM or CaN on exocytosis performance. Microinjected anti-CaN Ab strongly inhibit exocytosis. (Negative results with microinjected anti-CaM Ab can easily be explained by the abundance of CaM.) Alternatively, microinjection of a Ca2+-CaM-CaN complex triggers exocytosis. The same occurs with alkaline phosphatase. All these effects can also be mimicked in vitro with isolated cortices. In vitro exocytosis triggered by adding Ca2+-CaM-CaN or alkaline phosphatase is paralleled by dephosphorylation of the 65-kD PP. Exocytosis can also be inhibited in cortices by anti-CaM Ab or anti-CaN Ab. In wild-type cells, compounds that inhibit phosphatase activity, but none that inhibit kinases or proteases, are able to inhibit exocytosis. Exocytosis cannot be induced by phosphatase injection in a membrane-fusion-deficient mutant strain (nd9-28 degrees C) characterized by a defective organization of exocytosis sites (Beisson, J., M. Lefort-Tran, M. Pouphile, M. Rossignol, and B. Satir, 1976, J. Cell Biol., 69:126-143). We conclude that exocytotic membrane fusion requires an adequate assembly of molecular components to allow for the dephosphorylation of a 65-kD PP and that this step is crucial for the induction of exocytotic membrane fusion in Paramecium cells. In vivo this probably involves a Ca2+-CaM-stimulated CaN-like PP phosphatase.

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Mycobacterium tuberculosis Dihydrofolate Reductase Is Not a Target Relevant to the Antitubercular Activity of Isoniazid

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00453-10 ◽

2010 ◽

Vol 54 (9) ◽

pp. 3776-3782 ◽

Cited By ~ 45

Author(s):

Feng Wang ◽

Paras Jain ◽

Gulcin Gulten ◽

Zhen Liu ◽

Yicheng Feng ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Dihydrofolate Reductase ◽

Fold Increase ◽

Antitubercular Activity ◽

Experimental Sample ◽

Wild Type ◽

Negative Results ◽

Sequencing Studies ◽

Type Strains

ABSTRACT Mycobacterium tuberculosis enoyl-acyl-ACP reductase (InhA) has been demonstrated to be the primary target of isoniazid (INH). Recently, it was postulated that M. tuberculosis dihydrofolate reductase (DHFR) is also a target of INH, based on the findings that a 4R-INH-NADP adduct synthesized from INH by a nonenzymatic approach showed strong inhibition of DHFR in vitro, and overexpression of M. tuberculosis dfrA in M. smegmatis conferred a 2-fold increase of resistance to INH. In the present study, a plasmid expressing M. tuberculosis dfrA was transformed into M. smegmatis and M. tuberculosis strains, respectively. The transformant strains were tested for their resistance to INH. Compared to the wild-type strains, overexpression of dfrA in M. smegmatis and M. tuberculosis did not confer any resistance to INH based on the MIC values. Similar negative results were obtained with 14 other overexpressed proteins that have been proposed to bind some form of INH-NAD(P) adduct. An Escherichia coli cell-based system was designed that allowed coexpression of both M. tuberculosis katG and dfrA genes in the presence of INH. The DHFR protein isolated from the experimental sample was not found bound with any INH-NADP adduct by enzyme inhibition assay and mass spectroscopic analysis. We also used whole-genome sequencing to determine whether polymorphisms in dfrA could be detected in six INH-resistant clinical isolates known to lack mutations in inhA and katG, but no such mutations were found. The dfrA overexpression experiments, together with the biochemical and sequencing studies, conclusively demonstrate that DHFR is not a target relevant to the antitubercular activity of INH.

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Five days of fever and myocardial inflammation

SAGE Open Medical Case Reports ◽

10.1177/2050313x211050891 ◽

2021 ◽

Vol 9 ◽

pp. 2050313X2110508

Author(s):

Keli D Coleman ◽

Paul Benz ◽

Nirzar S Parikh ◽

Danny G Thomas ◽

David Segar ◽

...

Keyword(s):

False Negative ◽

Clinical Signs ◽

Myocardial Inflammation ◽

Negative Results ◽

Pediatric Illness ◽

Multisystem Involvement ◽

False Negative Results ◽

Control And Prevention ◽

Evidence Based Guidelines ◽

Inflammatory Syndrome

Multisystem inflammatory syndrome in children is an emerging pediatric illness associated with severe acute respiratory syndrome coronavirus 2 infection. The syndrome is rare, and evidence-based guidelines are lacking. This report reviews a patient who presented for medical care multiple times early in the course of his illness, thus offering near-daily documentation of symptoms and laboratory abnormalities. The patient did not have thrombocytopenia, anemia, or myocardial inflammation until the fifth day of fever. These laboratory abnormalities coincided with the onset of rash, conjunctival injection, vomiting, and diarrhea: clinical signs that could serve as indicators for when to obtain blood tests. The timing of this patient’s onset of multisystem involvement suggests that testing for multisystem inflammatory syndrome in children after only 24 h of fever, as the Centers for Disease Control and Prevention recommends, may yield false-negative results. Testing for multisystem inflammatory syndrome in children after 4 days of fever may be more reliable.

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Normal Human Lymphocytes Treated in Vitro With the Sulfhydryl Compound AET: Relationship to the Lymphocytes of Paroxysmal Nocturnal Hemoglobinuria

Blood ◽

10.1182/blood.v37.5.563.563 ◽

1971 ◽

Vol 37 (5) ◽

pp. 563-567 ◽

Cited By ~ 12

Author(s):

G. SIRCHIA ◽

S. FERRONE

Keyword(s):

Paroxysmal Nocturnal Hemoglobinuria ◽

Human Lymphocytes ◽

False Negative ◽

Cytotoxicity Test ◽

Lytic Action ◽

Sulfhydryl Compound ◽

Clear Cut ◽

Negative Results ◽

Monospecific Antisera

Abstract Because in vitro treatment with the sulfhydryl compound AET is known to alter normal red cells in such a way that they become highly susceptible to the lytic action of complement, an investigation was carried out to evaluate whether the same substance could modify normal lymphocytes in a similar way. The lymphocytes from paroxysmal nocturnal hemoglobinuria were also studied as a comparison. AET lymphocytes appeared to be more sensitive than the same untreated cells to the lytic action of antibody and C, as indicated both by the percentage of stained cells and by the titers displayed by the antisera used in a dye exclusion cytotoxicity test. Two HL-A monospecific antisera that gave false negative results with untreated lymphocytes gave clear-cut positive results with the same cells treated with AET. AET-lymphocytes could be employed for the detection of leukocyte antigens and antibodies.

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31Phosphorus Magnetic Resonance Spectroscopy Characterization of Muscular Metabolic Anomalies in Patients with Malignant Hyperthermia

Anesthesiology ◽

10.1097/00000542-199801000-00017 ◽

1998 ◽

Vol 88 (1) ◽

pp. 96-107 ◽

Cited By ~ 45

Author(s):

David Bendahan ◽

Genevieve Kozak-Ribbens ◽

Laurence Rodet ◽

Sylviane Confort-Gouny ◽

Patrick J. Cozzone

Keyword(s):

Magnetic Resonance ◽

Magnetic Resonance Spectroscopy ◽

Malignant Hyperthermia ◽

False Negative ◽

Resonance Spectroscopy ◽

Diagnostic Strategy ◽

Negative Results ◽

Ph Decrease

Background Metabolic anomalies are known in skeletal muscles of patients with malignant hyperthermia (MH). Methods The authors used 31-phosphorus (31P) magnetic resonance spectroscopy (MRS) to compare metabolic changes of the finger flexor muscles recorded throughout two rest-exercise-recovery protocols (each including aerobic or ischemic exercise) in 26 healthy persons and in 13 MH-susceptible (MHS) persons who were unequivocally diagnosed by in vitro halothane-caffeine contracture tests on muscle biopsies. Results No abnormality was observed at rest and during recovery periods. A larger phosphocreatine decrease associated with an early drop of pH was noted during the first minute of both exercise periods for MHS patients compared with controls. The early pH decrease indicated a disorder affecting glycolytic activation, probably reflecting defects of Ca2+ cycling, and provided a sensitivity of 77% for MHS diagnosis. A diagnostic strategy based on the retrospective analysis of 19 selected MR parameters was developed. An MRS score, corresponding to the number of abnormal values among the 19 parameters, was calculated and provided sensitivity and specificity rates of 100%; that is, no false-positive or false-negative results were found. A prospective analysis of 10 new participants further confirmed these findings. Conclusions These results (1) further confirm that MH is associated with the preexistence of latent muscular disorders; (2) enhance the potential diagnostic capacity of MRS, although it should be tested prospectively on a larger group of participants; and (3) allows the characterization of several abnormal metabolic profiles, in persons with MHS, reflecting the recently described polymorphism of MH.

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