Identification of
            <i>Staphylococcus</i>
            Species Directly from Positive Blood Culture Broth by Use of Molecular and Conventional Methods

Mehta, Maitry S.; Paule, Suzanne M.; Thomson, Richard B.; Kaul, Karen L.; Peterson, Lance R.

doi:10.1128/jcm.01850-08

Cited by 9 publications

(7 citation statements)

References 19 publications

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“…In addition, automatic rapid systems require 3.5 h for bacterial identification, versus only 20 min for MALDI-TOF-MS. PCR-based techniques have been used for bacterial identification directly from blood culture broth. Some methods require the use of specific targets (8,19,20,24). Despite the fact that these techniques are sensitive, they remain expensive and are specific for one or a few pathogens.…”

Section: Discussionmentioning

confidence: 99%

Real-Time Identification of Bacteria and Candida Species in Positive Blood Culture Broths by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

et al. 2010

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Delays in the identification of microorganisms are a barrier to the establishment of adequate empirical antibiotic therapy of bacteremia. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) allows the identification of microorganisms directly from colonies within minutes. In this study, we have adapted and tested this technology for use with blood culture broths, thus allowing identification in less than 30 min once the blood culture is detected as positive. Our method is based on the selective recovery of bacteria by adding a detergent that solubilizes blood cells but not microbial membranes. Microorganisms are then extracted by centrifugation and analyzed by MALDI-TOF-MS. This strategy was first tested by inoculating various bacterial and fungal species into negative blood culture bottles. We then tested positive patient blood or fluid samples grown in blood culture bottles, and the results obtained by MALDI-TOF-MS were compared with those obtained using conventional strategies. Three hundred twelve spiked bottles and 434 positive cultures from patients were analyzed. Among monomicrobial fluids, MALDI-TOF-MS allowed a reliable identification at the species, group, and genus/ family level in 91%, 5%, and 2% of cases, respectively, in 20 min. In only 2% of these samples, MALDI-TOF MS did not yield any result. When blood cultures were multibacterial, identification was improved by using specific databases based on the Gram staining results. MALDI-TOF-MS is currently the fastest technique to accurately identify microorganisms grown in positive blood culture broths.Blood cultures in liquid medium are the gold standard for the diagnosis of bloodstream infections. Species identification of bacteria that have grown in this biological fluid first requires an overnight subculture on solid agar medium, thus delaying the precise identification of the bacteria by 24 to 48 h. For bacteremic patients, this requirement prevents the rapid prescription of an adequate empirical anti-infective therapy prior to obtaining the results of the antibiotic sensitivity testing. This empirical therapy may be roughly adjusted on the basis of the Gram staining. However, these microscopic results are not accurate enough to reduce the patient's exposure to ineffective antibiotic therapy. In order to reduce the time required for the identification of microorganisms in blood cultures, various methods have been proposed, including identification using automated systems into which fluids from positive blood cultures are directly inoculated, fluorescent in situ hybridization (FISH), and PCR followed by sequencing, hybridization, pyrosequencing, or single-stranded conformation polymorphism. All these methods are expensive and require several hours (2, 4, 7-9, 12-15, 17-24, 26, 28, 29).Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) allows rapid identification of bacteria grown on solid media by the identification of species-specific profiles obtained from isolated ...

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Section: Discussionmentioning

confidence: 99%

Real-Time Identification of Bacteria and Candida Species in Positive Blood Culture Broths by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

et al. 2010

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“…Pathogens with fastidious growth requirements and those difficult to identify by phenotypic methods require more time for identification. Rapid nucleic acid amplification methods such as real-time PCR using melting curve analysis, multiplex PCR, fluorescence in situ hybridization (FISH) and peptide nucleic acid-FISH (PNA-FISH) have been used to detect pathogens in blood cultures including Staphylococcus aureus , Enterococcus faecalis and Candida albicans [6] , [7] , [8] . These assays, however, only target specific organisms; require technical expertise; and specimens are usually processed in batches.…”

Section: Introductionmentioning

confidence: 99%

Identification of Bacteria in Blood Culture Broths Using Matrix-Assisted Laser Desorption-Ionization Sepsityper™ and Time of Flight Mass Spectrometry

et al. 2011

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Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a novel method for the direct identification of bacteria from blood culture broths. We evaluate for the first time, the performance of the MALDI Sepsityper™ Kit and MS for the identification of bacteria compared to standard phenotypic methods using the manufacturer's specified bacterial identification criteria (spectral scores ≥1.700–1.999 and ≥2.000 indicated identification to genus and species level, respectively). Five hundred and seven positive blood culture broths were prospectively examined, of which 379 (74.8%; 358 monomicrobial, 21 polymicrobial) were identified by MALDI-TOF MS; 195 (100%) and 132 (67.7%) of 195 gram-positive; and 163 (100%) and 149 (91.4%) of 163 gram-negative organisms from monomicrobial blood cultures were correctly identified to genus and species level, respectively. Spectral scores <1.700 (no identification) were obtained in 128/507 (25.2%) positive blood culture broths, including 31.6% and 32.3% of gram-positive and polymicrobial blood cultures, respectively. Significantly more gram-negative organisms were identified compared to gram-positive organisms at species level (p<0.0001). Five blood cultures were misidentified, but at species level only; including four monomicrobial blood cultures with Streptococcus oralis/mitis that were misidentified as Streptococcus pneumoniae. Positive predictive values for the direct identification of both gram-positive and gram-negative bacteria from monomicrobial blood culture broths to genus level were 100%. A diagnostic algorithm for positive blood culture broths that incorporates gram staining and MALDI-TOF MS should identify the majority of pathogens, particularly to genus level.

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“…The fastest possible identification of Staphylococcus aureus and its differentiation from coagulase-negative staphylococci (CoNS) are essential for optimal treatment of infected patients (Ruimy et al, 2008;Weinstein, 2003). Conventional phenotypic tests are not sufficiently reliable when applied directly to blood cultures (Chapin & Musgnug, 2003;Mehta et al, 2009;Speers et al, 1998). A wide variety of molecular methods are available, but most are rather complex (Mehta et al, 2009;Ruimy et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

“…Conventional phenotypic tests are not sufficiently reliable when applied directly to blood cultures (Chapin & Musgnug, 2003;Mehta et al, 2009;Speers et al, 1998). A wide variety of molecular methods are available, but most are rather complex (Mehta et al, 2009;Ruimy et al, 2008). A relatively easy and costeffective molecular method for the identification of Staphylococcus aureus from blood cultures is fluorescence in situ hybridization (FISH) with either oligonucleotides of DNA (Gescher et al, 2008;Jansen et al, 2000;Kempf et al, 2000;Peters et al, 2006;Tavares et al, 2008) or peptide nucleic acid (PNA) (Chapin & Musgnug, 2003;Forrest et al, 2006;González et al, 2004;Hartmann et al, 2005;Hensley et al, 2009;Ly et al, 2008;Oliveira et al, 2002Oliveira et al, , 2003.…”

Section: Introductionmentioning

confidence: 99%

Accelerated identification of Staphylococcus aureus from blood cultures by a modified fluorescence in situ hybridization procedure

Poppert

Riecker

Wellinghausen

et al. 2010

Journal of Medical Microbiology

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This study evaluated fluorescence in situ hybridization (FISH) for rapid identification of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) directly from blood cultures. Initially, 360 blood cultures containing Gram-positive cocci were investigated by a previously described microwave-FISH procedure: 44/49 (89.8 %) S. aureus and 298/299 (99.7 %) CoNS were correctly identified. Because FISH proved useful and reliable but handling was found to be inconvenient, the method was modified by employing a recently developed slide chamber. This reduced the time required from 60 to 30 min. The simplified execution allowed integration of the method into the workflow of a routine laboratory without difficulty. The modified method proved to be highly reliable, identifying 37/37 (100 %) S. aureus and 169/172 (98.2 %) CoNS directly from blood cultures.

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Identification of Staphylococcus Species Directly from Positive Blood Culture Broth by Use of Molecular and Conventional Methods

Cited by 9 publications

References 19 publications

Real-Time Identification of Bacteria and Candida Species in Positive Blood Culture Broths by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Real-Time Identification of Bacteria and Candida Species in Positive Blood Culture Broths by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Identification of Bacteria in Blood Culture Broths Using Matrix-Assisted Laser Desorption-Ionization Sepsityper™ and Time of Flight Mass Spectrometry

Accelerated identification of Staphylococcus aureus from blood cultures by a modified fluorescence in situ hybridization procedure

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