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Dwivedi H.P.,North Carolina State University | Dwivedi H.P.,Biomerieux Inc. | Smiley R.D.,North Carolina State University | Smiley R.D.,U.S. Food and Drug Administration | Jaykus L.-A.,North Carolina State University
Applied Microbiology and Biotechnology | Year: 2010

The need for pre-analytical sample processing prior to the application of rapid molecular-based detection of pathogens in food and environmental samples is well established. Although immunocapture has been applied in this regard, alternative ligands such as nucleic acid aptamers have advantages over antibodies such as low cost, ease of production and modification, and comparable stability. To identify DNA aptamers demonstrating binding specificity to Campylobacter jejuni cells, a whole-cell Systemic Evolution of Ligands by EXponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules. FAM-labeled aptamer sequences with high binding affinity to C. jejuni A9a as determined by flow cytometric analysis were identified. Aptamer ONS-23, which showed particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K d value) of 292.8∈±∈53.1 nM with 47.27∈±∈5.58% cells fluorescent (bound) in a 1.48-μM aptamer solution. Binding assays to assess the specificity of aptamer ONS-23 showed high binding affinity (25-36%) for all other C. jejuni strains screened (inclusivity) and low apparent binding affinity (1-5%) with non-C. jejuni strains (exclusivity). Whole-cell SELEX is a promising technique to design aptamer-based molecular probes for microbial pathogens without tedious isolation and purification of complex markers or targets. © 2010 Springer-Verlag.


Fothergill A.,Emory University | Kasinathan V.,Emory University | Hyman J.,Biomerieux Inc. | Walsh J.,Biomerieux Inc. | And 3 more authors.
Journal of Clinical Microbiology | Year: 2013

Rapid identification of microorganisms causing bloodstream infections directly from a positive blood culture would decrease the time to directed antimicrobial therapy and greatly improve patient care. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a fast and reliable method for identifying microorganisms from positive culture. This study evaluates the performance of a novel filtration-based method for processing positive-blood-culture broth for immediate identification of microorganisms by MALDI-TOF with a Vitek MS research-use-only system (VMS). BacT/Alert non-charcoal- based blood culture bottles that were flagged positive by the BacT/Alert 3D system were included. An aliquot of positiveblood- culture broth was incubated with lysis buffer for 2 to 4 min at room temperature, the resulting lysate was filtered through a membrane, and harvested microorganisms were identified by VMS. Of the 259 bottles included in the study, VMS identified the organisms in 189 (73%) cultures to the species level and 51 (19.7%) gave no identification (ID), while 6 (2.3%) gave identifications that were considered incorrect. Among 131 monomicrobic isolates from positive-blood-culture bottles with one spot having a score of 99.9%, the IDs for 131 (100%) were correct to the species level. In 202 bottles where VMS was able to generate an ID, the IDs for 189 (93.6%) were correct to the species level, whereas the IDs provided for 7 isolates (3.5%) were incorrect. In conclusion, this method does not require centrifugation and produces a clean spectrum for VMS analysis in less than 15 min. This study demonstrates the effectiveness of the new lysis-filtration method for identifying microorganisms directly from positive- blood-culture bottles in a clinical setting. Copyright © 2013, American Society for Microbiology. All Rights Reserved.


Van Belkum A.,bioMerieux | Durand G.,bioMerieux | Peyret M.,bioMerieux | Chatellier S.,bioMerieux | And 5 more authors.
Annals of Laboratory Medicine | Year: 2013

Clinical microbiology has always been a slowly evolving and conservative science. The sub-field of bacteriology has been and still is dominated for over a century by culture-based technologies. The integration of serological and molecular methodologies during the seventies and eighties of the previous century took place relatively slowly and in a cumbersome fashion. When nucleic acid amplification technologies became available in the early nineties, the predicted revolution was again slow but in the end a real paradigm shift did take place. Several of the culture-based technologies were successfully replaced by tests aimed at nucleic acid detection. More recently a second revolution occurred. Mass spectrometry was introduced and broadly accepted as a new diagnostic gold standard for microbial species identification. Apparently, the diagnostic landscape is changing, albeit slowly, and the combination of newly identified infectious etiologies and the availability of innovative technologies has now opened new avenues for modernizing clinical microbiology. However, the improvement of microbial antibiotic susceptibility testing is still lagging behind. In this review we aim to sketch the most recent developments in laboratory-based clinical bacteriology and to provide an overview of emerging novel diagnostic approaches. © The Korean Society for Laboratory Medicine.


Spinali S.,bioMerieux | Van Belkum A.,bioMerieux | Goering R.V.,Creighton University | Girard V.,bioMerieux | And 5 more authors.
Journal of Clinical Microbiology | Year: 2015

The integration of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology has revolutionized species identification of bacteria, yeasts, and molds. However, beyond straightforward identification, the method has also been suggested to have the potential for subspecies-level or even type-level epidemiological analyses. This minireview explores MALDI-TOF MS-based typing, which has already been performed on many clinically relevant species. We discuss the limits of the method's resolution and we suggest interpretative criteria allowing valid comparison of strain-specific data. We conclude that guidelines for MALDI-TOF MS-based typing can be developed along the same lines as those used for the interpretation of data from pulsed-field gel electrophoresis (PFGE). Copyright © 2015, American Society for Microbiology. All Rights Reserved.


Marko D.C.,University of Iowa | Saffert R.T.,Mayo Medical School | Cunningham S.A.,Mayo Medical School | Hyman J.,Biomerieux Inc. | And 9 more authors.
Journal of Clinical Microbiology | Year: 2012

The Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) instruments were evaluated for the identification of nonfermenting Gram-negative bacilli (NFGNB) by a blinded comparison to conventional biochemical or molecular methods. Two hundred NFGNB that were recovered from cultures from cystic fibrosis patients in the University of Iowa Health Care (UIHC) Microbiology Laboratory between 1 January 2006 and 31 October 2010 were sent to Mayo Clinic for analysis with the Bruker Biotyper (software version 3.0) and to bioMérieux for testing with Vitek MS (SARAMIS database version 3.62). If two attempts at direct colony testing failed to provide an acceptable MALDI-TOF identification, an extraction procedure was performed. The MS identifications from both of these systems were provided to UIHC for comparison to the biochemical or molecular identification that had been reported in the patient record. Isolates with discordant results were analyzed by 16S rRNA gene sequencing at UIHC. After discrepancy testing, the Bruker Biotyper result agreed with the biochemical or molecular method, with 72.5% of isolates to the species level, 5.5% to the complex level, and 19% to the genus level (3% not identified). The level of agreement for Vitek MS was 80% species, 3.5% complex, 6% genus, and 3.5% family (7% not identified). Both MS systems provided rapid (≤3 min per isolate) and reliable identifications. The agreement of combined species/complex/genus-level identification with the reference method was higher for the Bruker Biotyper (97% versus 89.5%, P=0.004) but required an extraction step more often. Species-level agreement with the reference method was similar for both MS systems (72.5% and 80%, P=0.099). Copyright © 2012, American Society for Microbiology. All Rights Reserved.


Walton A.H.,University of Washington | Muenzer J.T.,University of Washington | Rasche D.,University of Washington | Boomer J.S.,University of Washington | And 9 more authors.
PLoS ONE | Year: 2014

A current controversy is whether patients with sepsis progress to an immunosuppressed state. We hypothesized that reactivation of latent viruses occurred with prolonged sepsis thereby providing evidence of clinically-relevant immunosuppression and potentially providing a means to serially-monitor patients' immune status. Secondly, if viral loads are markedly elevated, they may contribute to morbidity and mortality. This study determined if reactivation of herpesviruses, polyomaviruses, and the anellovirus TTV occurred in sepsis and correlated with severity. Serial whole blood and plasma samples from 560 critically-ill septic, 161 critically-ill non-septic, and 164 healthy age-matched patients were analyzed by quantitative-polymerase-chain-reaction for cytomegalovirus (CMV), Epstein-Barr (EBV), herpes-simplex (HSV), human herpes virus-6 (HHV-6), and TTV. Polyomaviruses BK and JC were quantitated in urine. Detectable virus was analyzed with respect to secondary fungal and opportunistic bacterial infections, ICU duration, severity of illness, and survival. Patients with protracted sepsis had markedly increased frequency of detectable virus. Cumulative viral DNA detection rates in blood were: CMV (24.2%), EBV (53.2%), HSV (14.1%), HHV-6 (10.4%), and TTV (77.5%). 42.7% of septic patients had presence of two or more viruses. The 50% detection rate for herpesviruses was 5-8 days after sepsis onset. A small subgroup of septic patients had markedly elevated viral loads (>104-106 DNA copies/ml blood) for CMV, EBV, and HSV. Excluding TTV, DNAemia was uncommon in critically-ill non-septic patients and in age-matched healthy controls. Compared to septic patients without DNAemia, septic patients with viremia had increased fungal and opportunistic bacterial infections. Patients with detectable CMV in plasma had higher 90-day mortality compared to CMV-negative patients; p<0.05. Reactivation of latent viruses is common with prolonged sepsis, with frequencies similar to those occurring in transplant patients on immunosuppressive therapy and consistent with development of an immunosuppressive state. Whether reactivated latent viruses contribute to morbidity and mortality in sepsis remains unknown. © 2014 Walton et al.


The present invention is directed to a method for separating, characterizing and/or identifying microorganisms in a test sample. The method of the invention comprises an optional lysis step for lysing non-microorganism cells that may be present in a test sample, followed by a subsequent separation step. The method may be useful for the separation, characterization and/or identification of microorganisms from complex samples such as blood-containing culture media. The method may also be useful for the physical separation and/or enrichment of two or more different or individual microorganism species contained in a mixed test sample. The invention further provides for spectroscopic interrogation of the separated microorganism sample(s) to produce measurements of the microorganism and characterizing and/or identifying the microorganism(s) in the sample using said spectroscopic measurements.


Trademark
Biomerieux Inc. | Date: 2016-12-09

Chemical reagent, namely, calibration kits for use in the scientific, laboratory, agri-food, cosmetic, pharmaceutical and industrial fields. Chemical reagent, namely, calibration kits for use in the medical and veterinary fields. Laboratory apparatus and instrument to measure the optical density of a microorganism suspension, not for medical purposes. Laboratory apparatus and instrument to measure the optical density of a microorganism suspension, for medical, pharmaceutical and veterinary purposes.


The present invention is directed to a method for separating, characterizing and/or identifying microorganisms in a test sample. The method of the invention comprises an optional lysis step for lysing non-microorganism cells that may be present in a test sample, followed by a subsequent separation step. The method may be useful for the separation, characterization and/or identification of microorganisms from complex samples such as blood-containing culture media. The invention further provides for spectroscopic interrogation of the separated microorganism sample to produce measurements of the microorganism and characterizing and/or identifying the microorganism in the sample using said spectroscopic measurements.


The present invention is directed to a method for inactivation and/or extraction of fungus samples (e.g., mold or yeast samples), the method comprising the following sequential steps: (a) acquiring a test sample known to contain or that may contain fungus and suspending the test sample in a container containing water and/or suspension medium; (b) adding ethanol to the suspension; (c) centrifuging the container to pellet the fungus and removing and discarding the supernatant; (d) resuspending the fungus in formic acid; (e) adding acetonitrile to the sample; (f) centrifuging the sample; and (g) recovering the supernatant. In accordance with the present invention, the recovered supernatant can be subjected to mass spectrometry analysis for characterization and/or identification of the unknown fungus.

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