Tuesday, September 13, 2011

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

Bloodstream infections (BSIs) are a significant cause of morbidity and mortality in hospitals. In the United States, septicemia has consistently featured in the top 10 causes of death, accounting for 35,587 deaths (11.6 per 105 population) in 2009 alone [1]. In Australia, in-hospital mortality for patients presenting with septic shock ranges from 23.1–27.6% [2], [3]. In the first six hours of septic shock, each hour of delay in initiating effective antimicrobial therapy following the onset of hypotension is associated with a reduction in average survival by 7.6% [4]. Rapid, accurate identification of the etiologic pathogen is critical for guiding effective antimicrobial therapy and improving patient outcomes, and for reducing length of hospitalization and hospital costs [5].

Traditional phenotypic based diagnostic methods for BSIs require the detection of bacterial growth in blood culture broths, followed by species identification and antimicrobial susceptibility testing (turnaround time 24–48 hours after initial growth). 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. Turnaround times are up to 6 hours.

The matrix assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) is a novel method for the direct identification of pathogens in blood culture broths, with results available within 2 hours. Although it does not provide antimicrobial susceptibility data (with the exception of methicillin resistant Staphylococcus aureus [MRSA]), it has good potential to guide empirical antimicrobial choice in the treatment of BSIs, yet there remain technical variables that may affect test performance.

Different methods for the preparation of blood culture broths prior to MS analysis have been utilized. Some investigators have employed an intact cell method, while others have extracted bacterial proteins using different solutions, including home-made ammonium chloride, trifluoroacetic acid (TFA), formic acid and acetonitrile; one study noted improved bacterial identification when formic acid instead of TFA was used [9], [10], [11]. There is also significant variation in the number of washing/centrifugation steps [9]. Different interpretive criteria have also been applied for bacterial identification, with some investigators accepting identification when consecutive spectral scores were greater than a pre-defined threshold on repeated analysis, despite the threshold being lower than the manufacturer's recommendations [11]. The lack of protocol standardization may contribute to reported differences in the performance of MALDI-TOF MS in the direct identification of pathogens from blood culture broths.

More recently, the MALDI Sepsityper™ Kit (Bruker Daltonics Inc., Billerica, MA) has been introduced to standardize the processing of blood culture broths prior to MS identification. This system outlines a specific protocol and provides all the reagents needed. Herein, we evaluate the performance of the MALDI Sepsityper™ Kit and MS for the direct identification of blood culture broths known to contain bacteria.
article published by Jen Kok1,2*, Lee C. Thomas1, Thomas Olma1, Sharon C. A. Chen1, Jonathan R. Iredell

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