Use of PCR Coupled with Electrospray Ionization Mass Spectrometry for Rapid Identification of Bacterial and Yeast Bloodstream Pathogens from Blood Culture Bottles

Abstract

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...

Detection, Identification, and Distribution of Fungi in Bronchoalveolar Lavage Specimens by Use of Multilocus PCR Coupled with Electrospray Ionization/Mass Spectrometry

“…This method enables detection and identification of causative pathogens directly from clinical specimens or mixtures of organisms with rapid turnaround and high throughput (9,10). This technique has been used to detect bacteria, viruses, yeasts, and other infectious agents (10)(11)(12)(13), but there are no data on its value for the detection of fungal organisms, including yeasts and molds.…”

Mass spectrometry: a revolution in clinical microbiology?

“…Indeed specific primers may be added to the assay to screen the mecA gene for methicillin resistance, the vanA and vanB genes for vancomycin resistance in enterococci, and the bla KPC gene for resistance to carbapenems [56 -58] . Most recently, the method produced highly accurate results when used to identify bacterial and yeast pathogens directly from the clinical specimen (blood culture) [59] , in particular, to detect Erlichia isolates [60] .…”

In vitro diagnosis of sepsis: a review

“…95 Conversely to MALDI-TOF MS, it uses genetic information to identify microorganisms. 96 This permits the ability to detect silent mutations, and to access antibiotic resistance genes, thus providing a mechanism for rapid antibiotic susceptibility testing.…”

“…98 This platform shows high analytical accuracy in comparison to routine subculture of BC bottles. 96,99 A major advantage of this method is the ability to characterize an organism without previous knowledge and to analyze mixtures of microbes directly from raw BC broth without the need for separation of colonies by subculturing.…”

“…However, the T5000 was hampered by a number of significant mechanical malfunctions; thus, to overcome these limitations, a newer model of this instrument, called the PLEX-ID, has been designed to be more robust and is provided with a software interface more suitable for use in the clinical laboratory. 96,99 The PLEX-ID BAC Spectrum BC assay that runs on the PLEX-ID permits identification of .600 bacteria and Candida spp., and it also detects resistance genes for four clinically relevant antibiotic resistance genes mecA, vanA/B, and blaKPC. 97 Further details on the PLEX-ID system for microorganism detection using whole blood are given later in this review.…”