Development of the iCubate Molecular Diagnostic Platform Utilizing Amplicon Rescue Multiplex Polymerase Chain Reaction

2019 ◽  
Vol 15 (7) ◽  
pp. 1598-1608
Author(s):  
Hongna Liu ◽  
Kathryn Heflin ◽  
Jian Han ◽  
Matt Conover ◽  
Leslie Wagner ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Bacterial Species ◽  
Bloodstream Infections ◽  
Cross Reactivity ◽  
Blood Cultures ◽  
Molecular Diagnostic ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
High Level

We utilized Amplicon-Rescue Multiplex PCR (ARM-PCR) and microarray hybridization to develop and validate the iC-GPC Assay, a multiplexed, in vitro diagnostic test that identifies five of the most common gram positive bacteria and three clinically relevant resistance markers associated with bloodstream infections (BSI). The iC-GPC Assay is designed for use with the iC-System™, which automates sample preparation, ARM-PCR, and microarray detection within a closed cassette. Herein, we determined the limit of detection for each of the iC-GPC Assay targets to be between 3.0 × 105–1.7 × 107 CFU/mL, well below clinically relevant bacterial levels for positive blood cultures. Additionally, we tested 106 strains for assay inclusivity and observed a target performance of 99.4%. 95 of 96 non-target organisms tested negative for cross-reactivity, thereby assuring a high level of assay specificity. Overall performance above 99% was observed for iC-GPC Assay reproducibility studies across multiple sites, operators and cassette lots. In conclusion, the iC-GPC Assay is capable of accurately and rapidly identifying bacterial species and resistance determinants present in blood cultures containing gram positive bacteria. Utilizing molecular diagnostics like the iC-GPC Assay will decrease time to treatment, healthcare costs, and BSI-related mortality.

scholarly journals Clinical Evaluation of the iCubate iC-GPC Assay for Detection of Gram-Positive Bacteria and Resistance Markers from Positive Blood Cultures

2018 ◽  
Vol 56 (9) ◽  
Author(s):  
Paul A. Granato ◽  
Melissa M. Unz ◽  
Raymond H. Widen ◽  
Suzane Silbert ◽  
Stephen Young ◽  
...  
Keyword(s):  
Blood Culture ◽  
Staphylococcus Epidermidis ◽  
Bloodstream Infections ◽  
Blood Cultures ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Sequencing Method ◽  
Resistance Markers ◽  
Gram Positive Cocci

ABSTRACT The iC-GPC Assay (iCubate, Huntsville, AL) is a qualitative multiplex test for the detection of five of the most common Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, and Enterococcus faecium) responsible for bacterial bloodstream infections, performed directly from positive blood cultures. The assay also detects the presence of the mecA, vanA, and vanB resistance determinants. This study comparatively evaluated the performance of the iC-GPC Assay against the Verigene Gram-positive blood culture (BC-GP) assay (Luminex Corp., Austin, TX) for 1,134 patient blood culture specimens positive for Gram-positive cocci. The iC-GPC Assay had an overall percent agreement with the BC-GP assay of 95.5%. Discordant specimens were further analyzed by PCR and a bidirectional sequencing method. The results indicate that the iC-GPC Assay together with the iCubate system is an accurate and reliable tool for the detection of the five most common Gram-positive bacteria and their resistance markers responsible for bloodstream infections.

scholarly journals In vitro activity of the trinem sanfetrinem (GV104326) against gram-positive organisms.

1996 ◽  
Vol 40 (9) ◽  
pp. 2142-2146 ◽  
Author(s):  
K V Singh ◽  
T M Coque ◽  
B E Murray
Keyword(s):  
Staphylococcus Aureus ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Gentamicin Resistance ◽  
High Level

The in vitro activity of the trinem sanfetrinem (formerly GV104326) (GV) was compared with that of vancomycin, ampicillin, and/or nafcillin against 287 gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and multiresistant enterococci, by the agar and microbroth dilution methods. GV demonstrated 2 to 16 times more activity than ampicillin and nafcillin against the majority of these organisms. The MIC range of GV was 16 to 64 micrograms/ml for 19 Enterococcus faecium strains that were highly resistant to ampicillin (ampicillin MIC range, 64 to 512 micrograms/ml) and vancomycin resistant and 0.25 to 32 micrograms/ml for resistant Rhodococcus spp. Similar activities (+/-1 dilution) were observed by either the agar or the broth microdilution method. GV demonstrated bactericidal activity against a beta-lactamase-producing Enterococcus faecalis strain and against two methicillin-susceptible Staphylococcus aureus strains in 10(5)-CFU/ml inocula. Synergy between GV and gentamicin was observed against an E. faecalis strain that lacked high-level gentamicin resistance. The activity of GV suggests this compound warrants further study.

scholarly journals In Vitro Activities of RWJ-54428 (MC-02,479) against Multiresistant Gram-Positive Bacteria

2001 ◽  
Vol 45 (5) ◽  
pp. 1422-1430 ◽  
Author(s):  
Suzanne Chamberland ◽  
Johanne Blais ◽  
Monica Hoang ◽  
Cynthia Dinh ◽  
Dylan Cotter ◽  
...  
Keyword(s):  
Multidrug Resistant ◽  
Penicillin G ◽  
Significant Activity ◽  
Coagulase Negative Staphylococci ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Level Of Activity ◽  
Resistant Strains ◽  
High Level

ABSTRACT RWJ-54428 (MC-02,479) is a new cephalosporin with a high level of activity against gram-positive bacteria. In a broth microdilution susceptibility test against methicillin-resistant Staphylococcus aureus (MRSA), RWJ-54428 was as active as vancomycin, with an MIC at which 90% of isolates are inhibited (MIC90) of 2 μg/ml. For coagulase-negative staphylococci, RWJ-54428 was 32 times more active than imipenem, with an MIC90 of 2 μg/ml. RWJ-54428 was active against S. aureus, Staphylococcus epidermidis, and Staphylococcus haemolyticus isolates with reduced susceptibility to glycopeptides (RWJ-54428 MIC range, ≤0.0625 to 1 μg/ml). RWJ-54428 was eight times more potent than methicillin and cefotaxime against methicillin-susceptible S. aureus (MIC90, 0.5 μg/ml). For ampicillin-susceptible Enterococcus faecalis (including vancomycin-resistant and high-level aminoglycoside-resistant strains), RWJ-54428 had an MIC90 of 0.125 μg/ml. RWJ-54428 was also active against Enterococcus faecium, including vancomycin-, gentamicin-, and ciprofloxacin-resistant strains. The potency against enterococci correlated with ampicillin susceptibility; RWJ-54428 MICs ranged between ≤0.0625 and 1 μg/ml for ampicillin-susceptible strains and 0.125 and 8 μg/ml for ampicillin-resistant strains. RWJ-54428 was more active than penicillin G and cefotaxime against penicillin-resistant, -intermediate, and -susceptible strains ofStreptococcus pneumoniae (MIC90s, 0.25, 0.125, and ≤0.0625 μg/ml, respectively). RWJ-54428 was only marginally active against most gram-negative bacteria; however, significant activity was observed against Haemophilus influenzae andMoraxella catarrhalis (MIC90s, 0.25 and 0.5 μg/ml, respectively). This survey of the susceptibilities of more than 1,000 multidrug-resistant gram-positive isolates to RWJ-54428 indicates that this new cephalosporin has the potential to be useful in the treatment of infections due to gram-positive bacteria, including strains resistant to currently available antimicrobials.

Bacteraemia in 66 cats and antimicrobial susceptibility of the isolates (1995–2004)

2007 ◽  
Vol 9 (5) ◽  
pp. 404-410 ◽  
Author(s):  
Martina Greiner ◽  
Georg Wolf ◽  
Katrin Hartmann
Keyword(s):  
Bacterial Species ◽  
Small Animal ◽  
Blood Cultures ◽  
Bacterial Isolates ◽  
Gram Positive ◽  
Gram Negative ◽  
Obligate Anaerobic ◽  
Animal Medicine ◽  
Privately Owned

Bacterial blood culture results of 292 privately owned cats presented to the Clinic for Small Animal Medicine, Ludwig Maximilian University Munich with signs of sepsis were evaluated retrospectively. Of the blood cultures, 23% were positive. In 88%, a single bacterial species was isolated. Of all bacterial isolates, 45% were Gram-positive, 43% were Gram-negative, and 12% were obligate anaerobes. The most frequently isolated bacteria were Enterobacteriaceae, obligate anaerobic species, Staphylococcus species and Streptococcus species. Of the cats with positive blood cultures, 32% were pretreated with antibiotics. Of all bacterial isolates, 77% were susceptible to enrofloxacin, 69% to chloramphenicol, 67% to gentamicin, and 64% to amoxycillin clavulanic acid. Only enrofloxacin reached an in vitro efficacy of more than 70% against Gram-positive and more than 74% against Gram-negative bacteria.

scholarly journals Comparative In Vitro Activity Profiles of Novel Bis-Indole Antibacterials against Gram-Positive and Gram-Negative Clinical Isolates

2010 ◽  
Vol 54 (9) ◽  
pp. 3974-3977 ◽  
Author(s):  
Michelle M. Butler ◽  
John D. Williams ◽  
Norton P. Peet ◽  
Donald T. Moir ◽  
Rekha G. Panchal ◽  
...  
Keyword(s):  
Multidrug Resistant ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Indole Compounds ◽  
Clinical Pathogens ◽  
High Level

ABSTRACT Antimicrobial susceptibilities of 233 Gram-positive and 180 Gram-negative strains to two novel bis-indoles were evaluated. Both compounds were potent inhibitors of Gram-positive bacteria, with MIC90 values of 0.004 to 0.5 μg/ml. One bis-indole, MBX 1162, exhibited potent activity against all Gram-negative strains, with MIC90 values of 0.12 to 4 μg/ml, even against high-level-resistant pathogens, and compared favorably to all comparator antibiotics. The bis-indole compounds show promise for the treatment of multidrug-resistant clinical pathogens.

scholarly journals Value of Time to Positivity of Blood Culture in Children with Bloodstream Infections

2019 ◽  
Vol 2019 ◽  
pp. 1-6 ◽  
Author(s):  
Fen Pan ◽  
Wantong Zhao ◽  
Hong Zhang
Keyword(s):  
Bacterial Species ◽  
Bloodstream Infections ◽  
Blood Cultures ◽  
Drug Resistant ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Microbiological Characteristics ◽  
The Mean ◽  
The Relationship

Objective. This study was to investigate the microbiological characteristics and the relationship between the time to positivity (TTP) of blood cultures and different bacterial species and to assess the clinical value of TTP in children with bloodstream infections (BSIs). Methods. The TTP of all the blood cultures from children with suspected BSIs was retrospectively collected in 2016. The microbiological characteristics and the relationship between the TTP of blood cultures and different bacterial species were also analyzed. Results. A total of 808 strains were isolated from 15835 blood cultures collected, and 145 (17.9%) were Gram-negative, 636 (78.7%) were Gram-positive, and 27 (3.3%) were fungi. The bacteria were divided into definite pathogens (174), possible pathogens (592), fungi (27), and contaminants (15). The average TTP of all positive blood cultures was 30.97 and ranged from 3.23 h to 92.73 h. The TTP of Gram-negative strains was significantly shorter than that of Gram-positive strains (P<0.001) and fungi (P = 0.032). The mean TTP for E. coli (15.60 h) was shortest within the group of Gram-negative isolates, and the mean TTP for Streptococcus (17.34 h) within the group of Gram-positive isolates. Significant difference of the TTP was detected in methicillin-resistant vs methicillin-susceptible S. aureus, extended-spectrum beta-lactamases (ESBLs) positive vs negative Enterobacteriaceae, and extensive drug-resistant and non-XDR A. baumannii. The median TTP in patients with BSI was significantly shorter than in those without it (P<0.001). ROC curve analysis indicated that the TTP cutoff value of CoNS, S. aureus, E. coli, and K. pneumoniae was 22.72 h, 19.6 h, 18.58 h, and 16.43 h, respectively, with most sensitive and specific predictor of BSIs. Conclusions. Our data acknowledged that TTP is a valuable index for the early prognosis of BSIs. TTP not only provides additional utility as a general predictor of bacteria with smear result but also provides the implication of drug-resistant organisms.

scholarly journals Effective Small Molecule Antibacterials from a Novel Anti-Protein Secretion Screen

2021 ◽  
Vol 9 (3) ◽  
pp. 592
Author(s):  
Mohamed Belal Hamed ◽  
Ewa Burchacka ◽  
Liselotte Angus ◽  
Arnaud Marchand ◽  
Jozefien De Geyter ◽  
...  
Keyword(s):  
Protein Secretion ◽  
Small Molecule ◽  
Bacterial Resistance ◽  
Bacterial Species ◽  
Attractive Potential ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Gram Positive Bacteria

The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. Protein export pathways are attractive potential targets. The Sec pathway is essential for bacterial viability and includes components that are absent from eukaryotes. Here, we used a new high-throughput in vivo screen based on the secretion and activity of alkaline phosphatase (PhoA), a Sec-dependent secreted enzyme that becomes active in the periplasm. The assay was optimized for a luminescence-based substrate and was used to screen a ~240K small molecule compound library. After hit confirmation and analoging, 14 HTS secretion inhibitors (HSI), belonging to eight structural classes, were identified with IC50 < 60 µM. The inhibitors were evaluated as antibacterials against 19 Gram-negative and Gram-positive bacterial species (including those from the WHO’s top pathogens list). Seven of them—HSI#6, 9; HSI#1, 5, 10; and HSI#12, 14—representing three structural families, were bacteriocidal. HSI#6 was the most potent hit against 13 species of both Gram-negative and Gram-positive bacteria with IC50 of 0.4 to 8.7 μM. HSI#1, 5, 9 and 10 inhibited the viability of Gram-positive bacteria with IC50 ~6.9–77.8 μM. HSI#9, 12, and 14 inhibited the viability of E. coli strains with IC50 < 65 μM. Moreover, HSI#1, 5 and 10 inhibited the viability of an E. coli strain missing TolC to improve permeability with IC50 4 to 14 μM, indicating their inability to penetrate the outer membrane. The antimicrobial activity was not related to the inhibition of the SecA component of the translocase in vitro, and hence, HSI molecules may target new unknown components that directly or indirectly affect protein secretion. The results provided proof of the principle that the new broad HTS approach can yield attractive nanomolar inhibitors that have potential as new starting compounds for optimization to derive potential antibiotics.

scholarly journals Comparing BioFire FilmArray BCID2 and BCID panels for direct detection of bacterial pathogens and antimicrobial resistance genes from positive blood cultures

2021 ◽  
Author(s):  
Venere Cortazzo ◽  
Tiziana D’Inzeo ◽  
Liliana Giordano ◽  
Giulia Menchinelli ◽  
Flora Marzia Liotti ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Yeast Species ◽  
Bacterial Species ◽  
Blood Cultures ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Antimicrobial Resistance Genes

Among molecular assays currently developed for detection and identification of pathogens (and their antimicrobial resistance genes) in positive blood cultures (BCs) (1), the BioFire FilmArray blood culture identification (BCID) panel (bioMérieux, Marcy l’Étoile, France)—a multiplex PCR assay with less than 2 minutes of hands-on time and a ∼1-hour turnaround time—allows syndromic diagnosis of bloodstream infection (BSI) (2, 3). Previously, the panel could identify 24 etiological agents of BSI (11 Gram-negative bacteria, 8 Gram-positive bacteria, and 5 yeast species), as well as three antimicrobial resistance genes (mecA, vanA/B, and blaKPC, which encodes Klebsiella pneumoniae carbapenemase). Now, the BioFire FilmArray BCID2 panel encompasses 43 molecular targets associated with BSI, including 15 Gram-negative bacteria, 11 Gram-positive bacteria, 7 yeast species, and 10 antimicrobial resistance genes (https://www.biomerieux-diagnostics.com/biofire-bcid-panel). The last targets include genes encoding for carbapenemases (IMP, KPC, OXA-48-like, NDM, and VIM), colistin resistance (mcr-1), ESBL (CTX-M), methicillin-resistance (mecA/C and, specifically for methicillin-resistant Staphylococcus aureus [MRSA], mecA/C and MREJ [mec right-extremity junction]), or vancomycin resistance (vanA/B). Unlike BCID, no published studies to date reported on the BCID2 performance. This study evaluated and compared the accuracy of BCID2 with that of BCID to identify bacterial species and relative antimicrobial resistance genes directly from positive BCs.

Rapid demonstration of septic or infectious arthritis due to Gram-negative bacteria

1992 ◽  
Vol 50 (1) ◽  
pp. 686-687
Author(s):  
Jacob S. Hanker ◽  
Paul R. Gross ◽  
Beverly L. Giammara
Keyword(s):  
Chronic Arthritis ◽  
Blood Cultures ◽  
Gram Negative Bacteria ◽  
Infectious Arthritis ◽  
Bacterial Arthritis ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria

Blood cultures are positive in approximately only 50 per cent of the patients with nongonococcal bacterial infectious arthritis and about 20 per cent of those with gonococcal arthritis. But the concept that gram-negative bacteria could be involved even in chronic arthritis is well-supported. Gram stains are more definitive in staphylococcal arthritis caused by gram-positive bacteria than in bacterial arthritis due to gram-negative bacteria. In the latter situation where gram-negative bacilli are the problem, Gram stains are helpful for 50% of the patients; they are only helpful for 25% of the patients, however, where gram-negative gonococci are the problem. In arthritis due to gram-positive Staphylococci. Gramstained smears are positive for 75% of the patients.

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