scholarly journals Multiplex Immunochromatographic Detection of OXA-48, KPC, and NDM Carbapenemases: Impact of Inoculum, Antibiotics, and Agar

2018 ◽  
Vol 56 (5) ◽  
Author(s):  
Ahmad Saleh ◽  
Stephan Göttig ◽  
Axel G. Hamprecht
Keyword(s):  
False Negative ◽  
Rapid Test ◽  
Zinc Content ◽  
Inhibition Zone ◽  
Content Type ◽  
Negative Results ◽  
Mueller Hinton ◽  
Mueller Hinton Agar ◽  
Lateral Flow Tests ◽  
The Impact

ABSTRACT For the rapid detection of carbapenemase-producing Enterobacteriaceae (CPE), immunochromatographic lateral flow tests (ICT) have recently been developed. The aim of this study was to assess the new multiplex ICT Resist-3 O.K.N. and to investigate if it can be performed directly from susceptibility testing plates. Additionally, the impact of the inoculum and carbapenem disks on sensitivity and specificity was evaluated. The new ICT was challenged using 63 carbapenem-resistant Enterobacteriaceae (CRE) isolates, including 51 carbapenemase producers. It was assessed under five different conditions directly from Mueller-Hinton agar (MHA): 1 μl or 10 μl of inoculum harvested in the absence of antibiotic pressure or 1 μl taken from the inhibition zone of either an ertapenem, imipenem, or meropenem disk. The sensitivity of the ICT was 100% for OXA-48-like and KPC carbapenemases and 94.4% for the NDM carbapenemase with the 1-μl inoculum. When harvested adjacent to a carbapenem disk, the sensitivity increased to 100%. Additionally, with zinc-supplemented MHA, both the sensitivity increased and the NDM band became visible faster (mean time, 8 ± 3.9 min for MHA compared to 1.9 ± 1.5 min for MHA plus zinc; P = 0.0016). The specificity of the ICT was 100%. The Resist-3 O.K.N. ICT is a sensitive and rapid test for the detection of three highly prevalent carbapenemases. However, false-negative results for NDM can occur. We recommend an inoculum of 1 μl that is harvested adjacent to an ertapenem or meropenem disk and the use of agars with sufficient zinc content to achieve the best performance.

scholarly journals Evaluation of CARBA PAcE, a novel rapid test for detection of carbapenemase-producing Enterobacterales

2020 ◽  
Author(s):  
Janko Sattler ◽  
Anne Brunke ◽  
Axel Hamprecht
Keyword(s):  
False Negative ◽  
Rapid Test ◽  
Content Type ◽  
Columbia Blood Agar ◽  
Fast Detection ◽  
The Poor ◽  
Colorimetric Test ◽  
Mueller Hinton ◽  
Mueller Hinton Agar ◽  
Moderate Sensitivity

Introduction. Carbapenemase-producing Enterobacterales (CPE) are an increasing threat to global health. Fast detection is crucial for patient management and outbreak control. Hypothesis/Gap statement. Recently, a new commercial colorimetric test, CARBA PAcE, was released that has not yet been scientifically evaluated. Aim. Our goals were to evaluate the performance of CARBA PAcE using a large variety of different CPE. Methodology. CARBA PAcE was challenged with 107 molecularly characterized CPE and 53 non-CPE controls. Isolates were grown on Mueller-Hinton agar (MHA); in the case of a false-negative result, isolates were additionally inoculated on Columbia blood agar (CBA) and CARBA PAcE was repeated. The test was performed according to the manufacturer’s protocol. Results. CARBA PAcE showed an overall sensitivity and specificity of 72 % [confidence interval (CI) 62–80 %] and 91 % (CI 79–97 %), respectively, when isolates were grown on MHA. With growth on CBA, detection improved (especially of metallo-β-lactamases), resulting in an extrapolated sensitivity of 89 % (CI 81–94 %) for all carbapenemases and 96 % (CI 89–99 %) for the four major carbapenemases (NDM, OXA-48-like, KPC, VIM). Conclusion. CARBA PAcE is a simple and very rapid test for the detection of CPE which performs well for the major carbapenemases when isolates are grown on CBA. Laboratories should be aware of the limitations of this assay, such as moderate sensitivity when isolates are grown on more challenging agars such as MHA and the poor detection of some rare carbapenemases (e.g. IMI, OXA-58).

scholarly journals 1457. Serial Passage of Enterobacteriaceae to Explore Development of Carbapenem Resistance

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S731-S731
Author(s):  
Yosef Nissim ◽  
Douglas Slain ◽  
P Rocco LaSala
Keyword(s):  
Susceptibility Testing ◽  
Klebsiella Oxytoca ◽  
Carbapenem Resistance ◽  
Serial Passage ◽  
Resistant Isolate ◽  
Mueller Hinton ◽  
The Us ◽  
Zones Of Inhibition ◽  
Mueller Hinton Agar ◽  
The Impact

Abstract Background Carbapenems are broad-spectrum antibacterials that have seen increased usage for the Enterobacteriales family in recent years. While carbapenem usage has been associated with increased antibacterial resistance, there is currently a lack of data comparing the risk of reduced susceptibility selection by the two most commonly used carbapenems in the US, ertapenem (ERT) and meropenem (MER). We conducted a novel serial passage experiment with clinical isolates of Enterobacteriales to assess the impact of repeated exposure to ERT or MER on phenotypic susceptibility patterns. Methods Non-duplicate clinical Enterobacteriales isolates were selected randomly for inclusion. Antimicrobial susceptibility testing was performed by CLSI disc diffusion methods. Standardized suspensions of isolates were plated on Mueller-Hinton agar, and ERT (10mcg) and MER (10mcg) discs applied. Zones of inhibition were measured and recorded after 16-18 hours incubation. Growth from the innermost zone of inhibition around each disc was used to prepare subsequent suspensions for serial susceptibility testing. This process would be repeated daily for 10 days. Each subsequent serially-passaged isolated was tested against both ERT and MER. Daily zones of inhibition were measured and interpreted. Baseline & final susceptibilities were determined by automated methods (Vitek 2). Results Seventeen Enterobacteriaceae isolates were selected, including: Klebsiella pneumoniae (n=11), Klebsiella oxytoca (n=2), Escherichia coli (n=1), Morganella morganii (n=1), and Enterobacter cloacae (n=2). Despite a greater degree of reductions in zones of inhibition with repeated ERT exposure (vs MER), the overall 10 day trends were not found to be significant different (P=0.529). Resistance developed to ERT in six isolates compared to one MER-resistant isolate (P = 0.053). E. cloacae was the only species to show a significant change between drugs (P=0.010). Two of three isolates that developed reduced zone changes > 10mm to MER were initially exposed to ERT on an earlier plate. Conclusion This novel experiment identified the development of some nonsignificant reductions in susceptibility with ERT after serial exposure. Results from this pilot study should encourage larger well-designed studies in this area. Disclosures All Authors: No reported disclosures

scholarly journals Evaluation of the Carba NP Test for Rapid Detection of Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa

2013 ◽  
Vol 57 (9) ◽  
pp. 4578-4580 ◽  
Author(s):  
Nathalie Tijet ◽  
David Boyd ◽  
Samir N. Patel ◽  
Michael R. Mulvey ◽  
Roberto G. Melano
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Positive Predictive Value ◽  
Negative Predictive Value ◽  
Predictive Value ◽  
Rapid Detection ◽  
False Negative ◽  
Bacterial Extract ◽  
Content Type ◽  
Negative Results ◽  
False Negative Results

ABSTRACTThe Carba NP test was evaluated against a panel of 244 carbapenemase- and non-carbapenemase-producingEnterobacteriaceaeandPseudomonas aeruginosaisolates. We confirmed the 100% specificity and positive predictive value of the test, but the sensitivity and negative predictive value were 72.5% and 69.2%, respectively, and increased to 80% and 77.3%, respectively, using a more concentrated bacterial extract. False-negative results were associated with mucoid strains or linked to enzymes with low carbapenemase activity, particularly OXA-48-like, which has emerged globally in enterobacteria.

scholarly journals Evaluation of Recombinant Proteins of Burkholderia mallei for Serodiagnosis of Glanders

2012 ◽  
Vol 19 (8) ◽  
pp. 1193-1198 ◽  
Author(s):  
Vijai Pal ◽  
Subodh Kumar ◽  
Praveen Malik ◽  
Ganga Prasad Rai
Keyword(s):  
Sensitivity And Specificity ◽  
Recombinant Proteins ◽  
False Negative ◽  
Enzyme Linked Immunosorbent Assay ◽  
Burkholderia Mallei ◽  
Content Type ◽  
Whole Cells ◽  
Negative Results ◽  
Elisa Format ◽  
Low Sensitivity

ABSTRACTGlanders is a contagious disease caused by the Gram-negative bacillusBurkholderia mallei. The number of equine glanders outbreaks has increased steadily during the last decade. The disease must be reported to the Office International des Epizooties, Paris, France. Glanders serodiagnosis is hampered by the considerable number of false positives and negatives of the internationally prescribed tests. The major problem leading to the low sensitivity and specificity of the complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e., crude preparations of whole cells. False-positive results obtained from other diagnostic tests utilizing crude antigens lead to financial losses to animal owners, and false-negative results can turn a risk into a possible threat. In this study, we report on the identification of diagnostic targets using bioinformatics tools for serodiagnosis of glanders. The identified gene sequences were cloned and expressed as recombinant proteins. The purified recombinant proteins ofB. malleiwere used in an indirect ELISA format for serodiagnosis of glanders. Two recombinant proteins, 0375H and 0375TH, exhibited 100% sensitivity and specificity for glanders diagnosis. The proteins also did not cross-react with sera from patients with the closely related disease melioidosis. The results of this investigation highlight the potential of recombinant 0375H and 0375TH proteins in specific and sensitive diagnosis of glanders.

scholarly journals The 28 + 28 day design is an effective sampling time for analyzing mutant frequencies in rapidly proliferating tissues of MutaMouse animals

2021 ◽  
Vol 95 (3) ◽  
pp. 1103-1116
Author(s):  
Francesco Marchetti ◽  
Gu Zhou ◽  
Danielle LeBlanc ◽  
Paul A. White ◽  
Andrew Williams ◽  
...  
Keyword(s):  
Germ Cells ◽  
False Negative ◽  
Sampling Time ◽  
Male Germ Cells ◽  
Negative Results ◽  
False Negative Results ◽  
Detection Of Mutations ◽  
The Impact ◽  
Sampling Times

AbstractThe Organisation for Economic Co-Operation and Development Test Guideline 488 (TG 488) uses transgenic rodent models to generate in vivo mutagenesis data for regulatory submission. The recommended design in TG 488, 28 consecutive daily exposures with tissue sampling three days later (28 + 3d), is optimized for rapidly proliferating tissues such as bone marrow (BM). A sampling time of 28 days (28 + 28d) is considered more appropriate for slowly proliferating tissues (e.g., liver) and male germ cells. We evaluated the impact of the sampling time on mutant frequencies (MF) in the BM of MutaMouse males exposed for 28 days to benzo[a]pyrene (BaP), procarbazine (PRC), isopropyl methanesulfonate (iPMS), or triethylenemelamine (TEM) in dose–response studies. BM samples were collected + 3d, + 28d, + 42d or + 70d post exposure and MF quantified using the lacZ assay. All chemicals significantly increased MF with maximum fold increases at 28 + 3d of 162.9, 6.6, 4.7 and 2.8 for BaP, PRC, iPMS and TEM, respectively. MF were relatively stable over the time period investigated, although they were significantly increased only at 28 + 3d and 28 + 28d for TEM. Benchmark dose (BMD) modelling generated overlapping BMD confidence intervals among the four sampling times for each chemical. These results demonstrate that the sampling time does not affect the detection of mutations for strong mutagens. However, for mutagens that produce small increases in MF, sampling times greater than 28 days may produce false-negative results. Thus, the 28 + 28d protocol represents a unifying protocol for simultaneously assessing mutations in rapidly and slowly proliferating somatic tissues and male germ cells.

Routine intraoperative angiography during aneurysm surgery

2002 ◽  
Vol 96 (6) ◽  
pp. 988-992 ◽  
Author(s):  
Veronica L. Chiang ◽  
Phillipe Gailloud ◽  
Kieran J. Murphy ◽  
Daniele Rigamonti ◽  
Rafael J. Tamargo
Keyword(s):  
Surgical Treatment ◽  
False Negative ◽  
Anterior Communicating Artery ◽  
Parent Vessel ◽  
Vessel Occlusion ◽  
Intraoperative Angiography ◽  
Content Type ◽  
Negative Results ◽  
Parent Vessel Occlusion ◽  
Fine Print

Object. The routine use of intraoperative angiography as an aid in the surgical treatment of aneurysms is uncommon. The advantages of the ability to visualize residual aneurysm or unintended occlusion of parent vessels intraoperatively must be weighed against the complications associated with repeated angiography and prolonged vascular access. The authors reviewed the results of their routine use of intraoperative angiography to determine its safety and efficacy. Methods. Prospectively gathered data from all aneurysm cases treated surgically between January 1996 and June 2000 were reviewed. A total of 303 operations were performed in 284 patients with aneurysms; 24 patients also underwent postoperative angiography. Findings on intraoperative angiographic studies prompted reexploration and clip readjustment in 37 (11%) of the 337 aneurysms clipped. Angiography revealed parent vessel occlusion in 10 cases (3%), residual aneurysm in 22 cases (6.5%), and both residual lesion and parent vessel occlusion in five cases (1.5%). When compared with subsequent postoperative imaging, false-negative results were found on two intraoperative angiograms (8.3%) and a false-positive result was found on one (4.2%). Postoperative angiograms obtained in both false-negative cases revealed residual anterior communicating artery aneurysms. Both of these aneurysms also subsequently rebled, requiring reoperation. In the group that underwent intraoperative angiography, in 303 operations eight patients (2.6%) suffered complications, of which only one was neurological. Conclusions. In the surgical treatment of intracranial aneurysms, the use of routine intraoperative angiography is safe and helpful in a significant number of cases, although it does not replace careful intraoperative inspection of the surgical field.

scholarly journals Establishment and Validation of RNA-Based Predictive Models for Understanding Survival of Vibrio parahaemolyticus in Oysters Stored at Low Temperatures

2017 ◽  
Vol 83 (6) ◽  
Author(s):  
Chao Liao ◽  
Yong Zhao ◽  
Luxin Wang
Keyword(s):  
Real Time ◽  
Predictive Models ◽  
Vibrio Parahaemolyticus ◽  
False Negative ◽  
Bacterial Inactivation ◽  
Rt Pcr ◽  
Content Type ◽  
Low Temperature Storage ◽  
Reverse Transcription Pcr ◽  
Negative Results

ABSTRACT This study developed RNA-based predictive models describing the survival of Vibrio parahaemolyticus in Eastern oysters (Crassostrea virginica) during storage at 0, 4, and 10°C. Postharvested oysters were inoculated with a cocktail of five V. parahaemolyticus strains and were then stored at 0, 4, and 10°C for 21 or 11 days. A real-time reverse transcription-PCR (RT-PCR) assay targeting expression of the tlh gene was used to evaluate the number of surviving V. parahaemolyticus cells, which was then used to establish primary molecular models (MMs). Before construction of the MMs, consistent expression levels of the tlh gene at 0, 4, and 10°C were confirmed, and this gene was used to monitor the survival of the total V. parahaemolyticus cells. In addition, the tdh and trh genes were used for monitoring the survival of virulent V. parahaemolyticus. Traditional models (TMs) were built based on data collected using a plate counting method. From the MMs, V. parahaemolyticus populations had decreased 0.493, 0.362, and 0.238 log10 CFU/g by the end of storage at 0, 4, and 10°C, respectively. Rates of reduction of V. parahaemolyticus shown in the TMs were 2.109, 1.579, and 0.894 log10 CFU/g for storage at 0, 4, and 10°C, respectively. Bacterial inactivation rates (IRs) estimated with the TMs (−0.245, −0.152, and −0.121 log10 CFU/day, respectively) were higher than those estimated with the MMs (−0.134, −0.0887, and −0.0732 log10 CFU/day, respectively) for storage at 0, 4, and 10°C. Higher viable V. parahaemolyticus numbers were predicted using the MMs than using the TMs. On the basis of this study, RNA-based predictive MMs are the more accurate and reliable models and can prevent false-negative results compared to TMs. IMPORTANCE One important method for validating postharvest techniques and for monitoring the behavior of V. parahaemolyticus is to establish predictive models. Unfortunately, previous predictive models established based on plate counting methods or on DNA-based PCR can underestimate or overestimate the number of surviving cells. This study developed and validated RNA-based molecular predictive models to describe the survival of V. parahaemolyticus in oysters during low-temperature storage (0, 4, and 10°C). The RNA-based predictive models show the advantage of being able to count all of the culturable, nonculturable, and stressed cells. By using primers targeting the tlh gene and pathogenesis-associated genes (tdh and trh), real-time RT-PCR can evaluate the total surviving V. parahaemolyticus population as well as differentiate the pathogenic ones from the total population. Reliable and accurate predictive models are very important for conducting risk assessment and management of pathogens in food.

scholarly journals Correlation between Mutations inliaFSRof Enterococcus faecium and MIC of Daptomycin: Revisiting Daptomycin Breakpoints

2012 ◽  
Vol 56 (8) ◽  
pp. 4354-4359 ◽  
Author(s):  
Jose M. Munita ◽  
Diana Panesso ◽  
Lorena Diaz ◽  
Truc T. Tran ◽  
Jinnethe Reyes ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Cell Envelope ◽  
Strong Association ◽  
Brain Heart Infusion ◽  
Regulatory System ◽  
Content Type ◽  
Initial Event ◽  
Mueller Hinton ◽  
Envelope Stress ◽  
Mueller Hinton Agar

ABSTRACTMutations inliaFSR, a three-component regulatory system controlling cell-envelope stress response, were recently linked with the emergence of daptomycin (DAP) resistance in enterococci. Our previous work showed that aliaFmutation increased the DAP MIC of a vancomycin-resistantEnterococcus faecalisstrain from 1 to 3 μg/ml (the DAP breakpoint is 4 μg/ml), suggesting that mutations in theliaFSRsystem could be a pivotal initial event in the development of DAP resistance. With the hypothesis that clinical enterococcal isolates with DAP MICs between 3 and 4 μg/ml might harbor mutations inliaFSR, we studied 38Enterococcus faeciumbloodstream isolates, of which 8 had DAP MICs between 3 and 4 μg/ml by Etest in Mueller-Hinton agar. Interestingly, 6 of these 8 isolates had predicted amino acid changes in the LiaFSR system. Moreover, we previously showed that among 6 DAP-resistantE. faeciumisolates (MICs of >4 μg/ml), 5 had mutations inliaFSR. In contrast, none of 16E. faeciumisolates with a DAP MIC of ≤2 μg/ml harbored mutations in this system (P< 0.0001). All but one isolate withliaFSRchanges exhibited DAP MICs of ≥16 μg/ml by Etest using brain heart infusion agar (BHIA), a medium that better supports enterococcal growth. Our findings provide a strong association between DAP MICs within the upper susceptibility range and mutations in theliaFSRsystem. Concomitant susceptibility testing on BHIA may be useful for identifying theseE. faeciumfirst-step mutants. Our results also suggest that the current DAP breakpoint forE. faeciummay need to be reevaluated.

scholarly journals OptimizedIn VitroAntibiotic Susceptibility Testing Method for Small-Colony Variant Staphylococcus aureus

2016 ◽  
Vol 60 (3) ◽  
pp. 1725-1735 ◽  
Author(s):  
Mimi R. Precit ◽  
Daniel J. Wolter ◽  
Adam Griffith ◽  
Julia Emerson ◽  
Jane L. Burns ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Susceptibility Testing ◽  
Brain Heart Infusion ◽  
Chronic Infections ◽  
Poor Growth ◽  
Content Type ◽  
Treatment History ◽  
Mueller Hinton ◽  
Mueller Hinton Agar ◽  
Small Colony

Staphylococcus aureussmall-colony variants (SCVs) emerge frequently during chronic infections and are often associated with worse disease outcomes. There are no standardized methods for SCV antibiotic susceptibility testing (AST) due to poor growth and reversion to normal-colony (NC) phenotypes on standard media. We sought to identify reproducible methods for AST ofS. aureusSCVs and to determine whether SCV susceptibilities can be predicted on the basis of treatment history, SCV biochemical type (auxotrophy), or the susceptibilities of isogenic NC coisolates. We tested the growth and stability of SCV isolates on 11 agar media, selecting for AST 2 media that yielded optimal SCV growth and the lowest rates of reversion to NC phenotypes. We then performed disk diffusion AST on 86S. aureusSCVs and 28 isogenic NCs and Etest for a subset of 26 SCVs and 24 isogenic NCs. Growth and reversion were optimal on brain heart infusion agar and Mueller-Hinton agar supplemented with compounds for which most clinical SCVs are auxotrophic: hemin, menadione, and thymidine. SCVs were typically nonsusceptible to either trimethoprim-sulfamethoxazole or aminoglycosides, in accordance with the auxotrophy type. In contrast, SCVs were variably nonsusceptible to fluoroquinolones, macrolides, lincosamides, fusidic acid, and rifampin;mecA-positive SCVs were invariably resistant to cefoxitin. All isolates (both SCVs and NCs) were susceptible to quinupristin-dalfopristin, vancomycin, minocycline, linezolid, chloramphenicol, and tigecycline. Analysis of SCV auxotrophy type, isogenic NC antibiograms, and antibiotic treatment history had limited utility in predicting SCV susceptibilities. With clinical correlation, this AST method and these results may prove useful in directing treatment for SCV infections.

scholarly journals Performance of SARS-CoV-2 Serology tests: Are they good enough?

2020 ◽  
Author(s):  
Isabelle Piec ◽  
Emma English ◽  
M Annette Thomas ◽  
Samir Dervisevic ◽  
William D Fraser ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Respiratory Infections ◽  
Point Of Care ◽  
False Negative ◽  
Rapid Test ◽  
Diagnostic Sensitivity ◽  
Medical Community ◽  
Antibody Detection ◽  
Serological Tests ◽  
Negative Results

AbstractBackgroundIn the emergency of the SARS-CoV-2 pandemic, great efforts were made to quickly provide serology testing to the medical community however, these methods have been introduced into clinical practice without the complete validation usually required by the regulatory organizations.MethodsSARS-CoV-2 patient samples (n=43) were analysed alongside pre-pandemic control specimen (n=50), confirmed respiratory infections (n=50), inflammatory polyarthritis (n=22) and positive for thyroid stimulating immunoglobulin (n=30). Imprecision, diagnostic sensitivity and specificity and concordance were evaluated on IgG serologic assays from EuroImmun, Epitope Diagnostics (EDI), Abbott Diagnostics and DiaSorin and a rapid IgG/IgM test from Healgen.ResultsEDI and EuroImmun imprecision was 0.02-14.0% CV. Abbott and DiaSorin imprecision (CV) ranged from 5.2% - 8.1% and 8.2% - 9.6% respectively. Diagnostic sensitivity of the assays were 100% (CI: 80-100%) for Abbott, EDI and EuroImmun and 95% (CI: 73-100%) for DiaSorin at ≥14 days post PCR. Only the Abbott assay had a diagnostic specificity of 100% (CI: 91-100%). EuroImmun cross-reacted in 3 non-SARS-CoV-2 respiratory infections and 2 controls. The DiaSorin displayed more false negative results and cross-reacted in six cases across all conditions tested. EDI had one cross-reactive sample. The Healgen rapid test showed excellent sensitivity and specificity. Overall, concordance of the assays ranged from 76.1% to 97.9%.ConclusionsSerological tests for SARS-CoV-2 showed good analytical performance. The head-to-head analysis of samples revealed differences in results that may be linked to the use of nucleocapsid or spike proteins. The point of care device tested demonstrated adequate performance for antibody detection.

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