MALDI-TOF mass spectrometry following short incubation on a solid medium is a valuable tool for rapid pathogen identification from positive blood cultures

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Abstract

Introduction

Rapid identification of the causative microorganism is a key element in appropriate antimicrobial therapy of bloodstream infections. Whereas traditional analysis of positive blood cultures requires subculture over at least 16–24 h prior to pathogen identification by, e.g. matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), sample preparation procedures enabling direct MALDI-TOF MS, i.e. without preceding subculture, are associated with additional effort and costs. Hence, we integrated an alternative MALDI-TOF MS approach in diagnostic routine using a short incubation on a solid medium.

Materials and methods

Positive blood cultures were routinely plated on chocolate agar plates and incubated for 4 h (37 °C, 5% CO2). Subsequently, MALDI-TOF MS using a Microflex LT instrument (Bruker Daltonics) and direct smear method was performed once per sample. For successful identification of bacteria at species level, score cut-off values were used as proposed by the manufacturer (≥2.0) and in a modified form (≥1.5 for MALDI-TOF MS results referring to Gram-positive cocci and ≥1.7 for MALDI-TOF MS results referring to bacteria other than Gram-positive cocci). Further data analysis also included an assessment of the clinical impact of the MALDI-TOF MS result.

Results

Applying the modified score cut-off values, our approach led to an overall correct species identification in 69.5% with misidentification in 3.4% (original cut-offs: 49.2% and 1.8%, respectively); for Gram-positive cocci, correct identification in 68.4% (100% for Staphylococcus aureus and enterococci, 80% for beta-hemolytic streptococci), for Gram-negative bacteria, correct identification in 97.6%. In polymicrobial blood cultures, in 72.7% one of the pathogens was correctly identified. Results were not reliable for Gram-positive rods and yeasts. The approach was easy to implement in diagnostic routine. In cases with available clinical data and successful pathogen identification, in 51.1% our approach allowed an optimized treatment recommendation.

Conclusion

MALDI-TOF MS following 4 h pre-culture is a valuable tool for rapid pathogen identification from positive blood cultures, allowing easy integration in diagnostic routine and the opportunity of considerably earlier treatment adaptation.

Introduction

Bloodstream infections are associated with a high mortality rate estimated to be 20–60% among hospitalized patients (Bearman and Wenzel, 2005). Early and appropriate antimicrobial treatment is critical to a favorable patient outcome (Kumar et al., 2006, Paul et al., 2010). For initial empirical treatment of infections, choosing broad-spectrum antibiotics renders coverage of the etiological pathogen more probable, but might promote subsequent antibiotic resistance development (Livermore, 2005), entail additional side effects and higher costs, and be less effective compared to a high-dose narrow-spectrum regimen. Thus, rapid identification of the causative microorganism and its resistance pattern is important. However, blood culture diagnostics as the gold standard is hampered by slow turn-around time because of time delay from the start of incubation to a positive reading, subsequent subculture on solid media for at least 16 to 24 h, and species identification and susceptibility testing requiring up to 48 h, or even longer for fastidious organisms, using traditional methods (Paolucci et al., 2010). Thus, final results are often obtained too late to influence treatment decisions.

The introduction of the matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of microorganisms following traditional subculture was a major advance in clinical microbiology laboratories comparing favorably with conventional automated biochemical differentiation methods in terms of time to result, ease of use, accuracy, and cost-effectiveness (Bizzini et al., 2010, Cherkaoui et al., 2010, Martiny et al., 2012a, Seng et al., 2009). In bloodstream infection, the use of MALDI-TOF MS after an overnight subculture on solid media substantially decreased time to organism identification and time to effective antibiotic therapy (Huang et al., 2013).

In order to further accelerate pathogen identification in blood culture diagnostics, various sample preparation procedures including a commercially available kit (Sepsityper, Bruker Daltonics, Bremen, Germany) were developed to perform MALDI-TOF MS directly, i.e. without preceding subculture, from positive blood culture bottles (Buchan et al., 2012, Chen et al., 2013, Christner et al., 2010, Ferreira et al., 2011, Haigh et al., 2013, Klein et al., 2012, Kok et al., 2011, La Scola and Raoult, 2009, Lagace-Wiens et al., 2012, Leli et al., 2013, Loonen et al., 2012, Martinez et al., 2014, Martiny et al., 2012b, Meex et al., 2012, Moussaoui et al., 2010, Prod’hom et al., 2010, Saffert et al., 2012, Schubert et al., 2011, Spanu et al., 2012, Stevenson et al., 2010, Yan et al., 2011). As the blood culture broth contains macromolecules from blood and growth media which have to be removed prior to MALDI-TOF MS analysis to reveal species-specific protein spectra of bacteria and yeasts, all procedures for direct MALDI-TOF MS are associated with additional expenditure of consumables, costs and hand-on processing time. Nevertheless, they may reduce time to organism identification by a day or more and result in earlier modification of antimicrobial treatment (Clerc et al., 2013, Martiny et al., 2013, Vlek et al., 2012), even though antimicrobial susceptibility testing is not provided.

The aim of the present study was to evaluate an alternative protocol for earlier pathogen identification avoiding additional costs and effort. To that end, we performed MALDI-TOF MS after a short incubation (4 h) of positive blood culture broths on a solid medium, an approach which has been described by only few recent reports so far (Bhatti et al., 2014, Idelevich et al., 2014, Verroken et al., 2015). Our data confirm and extend those studies. In addition, we assessed the impact the MALDI-TOF MS result gained by our protocol had on empiric therapy whereas an evaluation of the clinical impact has not been included in those previous studies.

Section snippets

Study design

Non-selected positive blood cultures, collected between February and May 2014 in 13 hospitals including the University Hospital of Ruhr-University Bochum representing both urban and rural settings from the Bochum area in Germany, were included in this study. Owing to an integrated 24 h active consultation service, positive blood cultures processed in our laboratory are routinely judged and reported by a clinical microbiologist whereupon treatment options are advised to the treating clinicians.

Specimens

Results

After exclusion of false-positive blood cultures, 598 positive blood cultures, prospectively collected from 319 patients, were included in the study. 325 isolates were derived from monomicrobial blood cultures (n = 553) with a species distribution of 72.9% Gram-positive bacteria, 25.8% Gram-negative bacteria and 1.5% yeasts. In addition, 52 pathogens were merely isolated from polymicrobial blood cultures (n = 45). In total, 319 bacteremia cases were analyzed with respect to clinical parameters.

Discussion

The aim of the present study was to evaluate an alternative protocol for earlier MALDI-TOF MS-based identification that – in comparison to MALDI-TOF MS performed directly from positive blood cultures – maintains the advantage of accelerated pathogen identification but avoids additional costs and hands-on time. As our study is addressed to routine clinical microbiology laboratories, we applied only one simple protocol feasible for routine practice: Positive blood cultures were subcultured on a

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    This work was presented, in part, at the 66th Annual Conference of the Society of Hygiene and Microbiology (DGHM), Dresden, Germany, 5–8 October 2014 [DVP04].

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    Contributed equally.

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