Bacterial identification relies primarily on culture-based methodologies requiring 24 h for isolation and an additional 24 to 48 h for species identification. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an emerging technology newly applied to the problem of bacterial species identification. We evaluated two MALDI-TOF MS systems with 720 consecutively isolated bacterial colonies under routine clinical laboratory conditions. Isolates were analyzed in parallel on both devices, using the manufacturers' default recommendations. We compared MS with conventional biochemical test system identifications. Discordant results were resolved with "gold standard" 16S rRNA gene sequencing. The first MS system (Bruker) gave high-confidence identifications for 680 isolates, of which 674 (99.1%) were correct; the second MS system (Shimadzu) gave high-confidence identifications for 639 isolates, of which 635 (99.4%) were correct. Had MS been used for initial testing and biochemical identification used only in the absence of high-confidence MS identifications, the laboratory would have saved approximately US$5 per isolate in marginal costs and reduced average turnaround time by more than an 8-h shift, with no loss in accuracy. Our data suggest that implementation of MS as a first test strategy for one-step species identification would improve timeliness and reduce isolate identification costs in clinical bacteriology laboratories now.

II.

Sepsis is among the top 10 causes of mortality in the United States. Rapid administration of antibiotics is one of the most important contributors to patient survival, yet only a limited number of methods exist for rapid identification of microbes cultivated from bloodstream infections, which can lead to sepsis. While traditional single-target molecular methods have been shown to greatly improve survival for septic patients by enabling rapid deescalation of broad-spectrum antibiotics, multiplex methods offer even greater possibilities. A novel multiplex method, PCR coupled to electrospray ionization mass spectrometry (PCR/ESI-MS), was used to identify the genus and species of microorganisms found to cause human bloodstream infections. DNA was directly extracted from 234 BacT-Alert blood culture bottles, and results were compared to those obtained by clinical reference standard methods. The study results demonstrated 98.7% and 96.6% concordance at the genus and species levels, respectively. Mixtures of microbes were identified in 29 blood culture bottles, including mixed species of the same genus, as well as mixtures containing Gram-positive and Gram-negative organisms, exemplifying the PCR/ESI-MS capability to identify multiple organisms simultaneously without the need for cultivation. This study demonstrates high analytical accuracy in comparison to routine subculture of blood culture bottles and phenotypic identification of microbes. Without foreknowledge of the microorganisms potentially present, the PCR/ESI-MS methods can deliver accurate results in as little as 5 to 6 h after a positive alarm from the automated blood culture system; however, current batch mode testing limits the method's clinical utility at this time.The ability to rapidly identify the causative agent of bloodstream infections (BSIs) is of paramount importance to clinical microbiology laboratories. Rapid identification significantly reduces the rates of patient mortality, reduces the use of unnecessary antibiotics, and lowers costs to the hospital (6,7,15). If untreated, BSIs can lead to sepsis or progress to severe sepsis, with one or more organ dysfunctions, which can ultimately result in septic shock and/or death.Death due to septic shock is currently the 10th leading cause of mortality in the United States (28). More notably, the rate of sepsis-related death is on the rise. In 2000, sepsis was ranked as the 13th leading cause of death (5), symbolizing a 139% increase in incidence over a 10-year period (8). It is estimated that approximately 215,000 deaths per year are sepsis related, and the costs associated with sepsis are exceedingly high, approximately $17 billion each year and rising, due to the length of hospital stay required (1).It is estimated that the survival increases by 7 to 10% for every hour of earlier administration of targeted antibiotics (34). Ideally, bacteria in the bloodstream should be identified as quickly as possible in order to administer the most appropriate antibiotics, as opposed to use of broad-spectrum antib...

When confronted with a septic patient or dealing with an emerging epidemic, clinicians, infection control specialists and microbiologists have often felt an immense 'need for speed' while waiting for culture results. Various mass spectrometry (MS) applications are about to answer most of their demands. Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS of whole bacterial cells has already greatly shortened the time needed for identification of a positive culture in major diagnostic laboratories in Europe. MS is described in this article, with a special emphasis on the different systems currently commercially available for routine identification. MALDI-TOF MS remains, however, limited by the previous time-consuming culture steps, and is not suited for strain typing in epidemic contexts. These limitations can be overcome by other applications of MS in microbiology. MALDI-resequencing is a rapid method for genotyping, offering comparable results to multilocus sequence typing. New systems of broad-range PCR, associated with analyses of amplicons by electrospray ionization MS, might allow nearly full automation for the direct identification of pathogens in blood, thus bypassing the culture stage. This article describes various applications of MS methods in clinical microbiology, and provides a comparative table of these technologies.

BACKGROUND Emerging technologies for rapid identification of microbes demonstrate a shift from traditional biochemical and molecular testing algorithms toward methods using mass spectrometry (MS) for the semiquantitative analysis of microbial proteins and genetic elements. This study was performed to assess the diagnostic accuracy of 2 such technologies, PCR–electrospray ionization (ESI)/MS and MALDI-TOF/MS, with respect to phenotypic and biochemical profiling as a reference standard method. A positive challenge set of blood culture bottles was used to compare PCR-ESI/MS and MALDI-TOF/MS performance on a matched set of samples. METHODS We performed characterization of bloodstream infections from blood cultures using the Ibis T5000 PCR-ESI/MS and the Bruker MALDI Biotyper 2.0 (MALDI-TOF/MS) platforms for microbial identification. Diagnostic accuracy was determined by independent comparison of each method to phenotypic and biochemical characterization with Vitek2 analysis as the reference standard identification. RESULTS The diagnostic accuracy, represented as positive agreement, at the genus level was 0.965 (0.930–0.984) for PCR-ESI/MS and 0.969 (0.935–0.987) for MALDI-TOF/MS, and at the species level was 0.952 (0.912–0.974) with PCR-ESI/MS and 0.943 (0.902–0.968) for MALDI-TOF/MS. No statistically significant difference was found between PCR-ESI/MS and MALDI-TOF/MS in the ability to rapidly identify microorganisms isolated from blood culture. CONCLUSIONS Our results demonstrate that PCR-ESI/MS and MALDI-TOF/MS are equivalent in their ability to characterize bloodstream infections with respect to the reference standard, and highlight key differences in the methods that allow for each method to have a unique niche as a tool for rapid identification of microbes in blood cultures.

Kingella kingae is an emerging pathogen that is recognized as a causative agent of septic arthritis and osteomyelitis, primarily in infants and children. The bacterium is best detected by rapid inoculation in blood culture systems or by real-time PCR assays. Pathogenesis of the agent was linked recently to the production of a potent cytotoxin, known as RTX, which is toxic to a variety of human cell types. The locus encoding the RTX toxin is thought to be a putative virulence factor, and is, apparently, essential for inducing cytotoxic effects on respiratory epithelial, synovial and macrophage-like cells. Herein, we describe a novel real-time PCR assay that targets the RTX toxin gene and illustrate its use in two clinical cases. The assay exhibited a sensitivity of 30 c.f.u., which is 10-fold more sensitive than a previously published semi-nested broad-range 16S rRNA gene PCR, and showed no cross-reactivity with several related species and common osteoarticular pathogens.

Osteoarticular infections caused by Kingella kingae are characterized by mild-to-moderate clinical and biologic inflammatory signs that are different from those caused by Gram-positive cocci. A combined score was built to find the best model to predict K. kingae osteoarticular infections by using the following 4 variables: body temperature <38°C, serum C-reactive protein <55 mg/L, white blood cell count <14,000/mm, and band forms <150/mm.

Detection of K kingae DNA in oropharyngeal swabs of children with clinical findings of OAI is predictive of K kingae OAI. If these findings are replicated in other settings, detection of K kingae by oropharyngeal swab PCR could improve the recognition of OAI.