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ALERT blood culture bottles using matrix-assisted laser desorption ionization–time-of-flight mass spectrometry
Diagnostic Microbiology and Infectious Disease 80 (2014) 193–196
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Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Direct identification of bacteria from positive BacT/ALERT blood culture bottles using matrix-assisted laser desorption ionization–time-of-flight mass spectrometry Javier Mestas a, Susanna Felsenstein b, Jennifer Dien Bard c,⁎ a b c
Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles, CA, USA Division of Infectious Diseases, Department of Pediatrics, Children's Hospital Los Angeles, Keck School of Medicine of University of Southern California, Los Angeles, CA, USA Department of Pathology and Laboratory Medicine, Keck School of Medicine, University of Southern California and Children's Hospital Los Angeles, Los Angeles, CA, USA
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Article history: Received 19 March 2014 Received in revised form 1 July 2014 Accepted 23 July 2014 Available online 31 July 2014 Keywords: Blood culture bottles Bacteria Identification MALDI-TOF
a b s t r a c t Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a fast and robust method for the identification of bacteria. In this study, we evaluate the performance of a laboratory-developed lysis method (LDT) for the rapid identification of bacteria from positive BacT/ALERT blood culture bottles. Of the 168 positive bottles tested, 159 were monomicrobial, the majority of which were Gram-positive organisms (61.0% versus 39.0%). Using a cut-off score of ≥1.7, 80.4% of the organisms were correctly identified to the species level, and the identification rate of Gram-negative organisms (90.3%) was found to be significantly greater than that of Gram-positive organisms (78.4%). The simplicity and cost-effectiveness of the LDT enable it to be fully integrated into the routine workflow of the clinical microbiology laboratory, allowing for rapid identification of Gram-positive and Gram-negative bacteria within an hour of blood culture positivity. © 2014 Elsevier Inc. All rights reserved.
Introduction Bloodstream infection is an important cause of morbidity and mortality, and prompt initiation of appropriate antimicrobial therapy is the mainstay of treatment (Ibrahim et al., 2000; Seifert, 2009). The application of matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a routine diagnostic tool for the identification of microorganisms in the microbiology laboratory has been proven to be rapid and accurate (Bizzini et al., 2010; Neville et al., 2011; Seng et al., 2009; Stevenson et al., 2010a, 2010b; Tan et al., 2012; van Veen et al., 2010). Furthermore, various commercial and in-house methods have been developed to rapidly identify bacteria and yeast directly from positive blood cultures, and a wide range in sensitivity of 46–98% has been reported and appears to be dependent on the methodology, organism, and blood culture bottle tested (Chen et al., 2013; Kok et al., 2011; March-Rossello et al., 2013; Schmidt et al., 2012; Wuppenhorst et al., 2012). A recent study using Bactec™ blood culture bottles (Becton Dickinson, Sparks, MD, USA) compared the Sepsityper™ kit (Bruker Daltonik GmbH, Bremen, Germany) to an in-house extraction method and reported species-level identification rates of 81.5% and 80.4%, respectively (Chen et al., 2013). Previous studies have evaluated the ability of MALDI-TOF MS to accurately identify bacteria directly from positive BacT/ALERT blood
⁎ Corresponding author. Tel.: +1-323-361-5443; fax: +1-323-361-8039. E-mail address: [email protected] (J. Dien Bard). http://dx.doi.org/10.1016/j.diagmicrobio.2014.07.008 0732-8893/© 2014 Elsevier Inc. All rights reserved.
culture bottles (bioMérieux, Durham, NC, USA), with or without the presence of charcoal, and the sensitivities reported have been suboptimal, ranging from 23% to 77% (Fothergill et al., 2013; Haigh et al., 2013; Loonen et al., 2012; Meex et al., 2012; Schmidt et al., 2012; Szabados et al., 2011; Wuppenhorst et al., 2012) when compared to the Bactec™ blood culture bottles (Romero-Gomez and Mingorance, 2011; Schmidt et al., 2012; Szabados et al., 2011). The use of commercial methods, such as the Sepsityper™ kit, can also increase the identification rate from BacT/ALERT standard aerobic and anaerobic bottles with 1 study reporting an increased in sensitivity of 13% and 26% when compared to an in-house centrifugation/ washing method and the MolYsis Basic method (Molzym GmbH, Bremen, Germany), respectively (Loonen et al., 2012). However, these methods are laborious and require multiple steps. In this study, we developed a simple, lysis-based, laboratory-developed test (LDT) to directly identify microorganisms from positive BacT/ALERT standard aerobic (SA), non-charcoal bottles using the MALDI-TOF MS Biotyper (BioTyper 3.0; Bruker Daltonik GmbH, Billerica, MA, USA).
2. Materials and methods One hundred sixty-eight positive BacT/Alert SA blood cultures from pediatric patients submitted for routine work-up to the Clinical Microbiology Laboratory at Children's Hospital Los Angeles (CHLA) were tested on MALDI-TOF MS using the LDT. To remove cellular components, 300 μL of blood culture broth was resuspended in 1 mL of
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Table 1 MALDI-TOF MS identification rate by method. Organism (n)
Acinetobacter baumannii (1) Bacillus pumilus (1) Bacillus spp. (3) Corynebacterium jeikeium (2) Corynebacterium spp. (3) Enterobacter cloacae complex (14) Enterococcus faecium (5) Enterococcus faecalis (5) Enterococcus hirae (2) Escherichia coli (17) Granulicatella adiacens (2) Klebsiella oxytoca (1) Klebsiella pneumoniae (9) Lactococcus lactis (1) Microbacterium spp. (1) Micrococcus spp. (2) Moraxella nonliquifaciens (1) Pseudomonas aeruginosa (14) Pseudomonas oryzihabitans (1) Pseudomonas putida (1) Serratia marcescens (1) Staphylococcus aureus (17) Staphylococcus epidermidis (28) Streptococcus agalactiae (2) Streptococcus mitis/oralis (7) Streptococcus parasanguinis (1) Streptococcus pneumoniae (1) Streptococcus pyogenes (1) Streptococcus viridans group (2) Weisella spp. (1) Coagulase-negative Staphylococcus (12) Polymicrobial (9) Total (168)
Direct-LDT
Extracted-LDT
Identified (score ≥1.7)
Correctly identified n (%)
Identified (score ≥1.7)
Correctly identified n (%)
0 0 3 0 1 12 0 1 0 16 0 1 8 0 0 2 0 13 1 1 1 8 11 0 0 0 0 0 0 0 6 5 90 (53.6)
0 0 3 0 1 12 0 1 0 16 0 1 8 0 0 2 0 13 1 1 1 8 11 0 0 0 0 0 0 0 6 5 90
0 1 3 1 2 12 5 3 2 17 0 1 9 1 0 2 0 14 1 1 1 17 23 2 1 1 0 1 0 0 11 5 137 (81.5)
0 (0) 1 (100) 3 (100) 1 (50) 1 (33.3) 12 (85.7) 5 (100) 3 (60) 2 (100) 17 (100) 0 (0) 1 (100) 9 (100) 1 (100) 0 (0) 2 (100) (0) 14 (100) 1 (100) 1 (100) 1 (100) 17 (100) 23 (82.1) 2 (100) 0 (0) 1 (100) 0 (0) 1 (100) 0 (0) 0 (0) 11 (91.7) 5 (55.6) 135 (80.4)
erythrocyte lysis buffer (ACK lysing buffer; Life Technologies, Grand Island, NY, USA), followed by centrifugation at 16000g for 2 minutes and removal of supernatant. The cell pellet is resuspended in 500 μL of distilled water, and the centrifugation and removal of supernatant step are repeated. The cell pellet is allowed to air dry and then smeared directly onto target plate (direct-LDT), followed by addition of 1 μL of 10 ng/μL α-cyano-4-hydroxycinnamic-acid matrix (Bruker Daltonik GmbH) to each spot. A direct plate extraction method (extracted-LDT) was also performed for each sample, with overlay of 1 μL of 70% formic acid, prior to the addition of α-cyano-4hydroxycinnamic-acid. All MALDI-TOF MS testing were performed in duplicate, and acquisition and analysis of mass spectra was performed using the Microflex LT mass spectrometer and flexControl 3.0 software (Bruker Daltonik GmbH). MALDI-TOF MS results were compared to conventional identification methods; blood cultures were aseptically inoculated onto blood agar, MacConkey, and chocolate agar followed by incubation for 18–24 hours at 37 °C and 5% CO2. Isolates were identified using routine identification methods such as Vitek II (bioMérieux) identification, and other biochemical tests and discordant results were confirmed by 16S rRNA sequencing. Blood volume was analyzed to determine significance between low blood volume and identification rate. Paired and unpaired t test were used to calculate the statistical significant differences demonstrated in the study. 3. Results Of the 168 positive blood cultures tested, 159 were monomicrobial and 9 were polymicrobial. Of 159 monomicrobial bottles, 97 (61.0%) grew Gram-positive organisms, and 62/159 (39.0%) grew Gram-negative organisms. The performance characteristics for all blood cultures using LDT compared to conventional methods are shown in Table 1. Using the cut-off score of ≥1.7, 53.6% were identified to genus level and species level using the direct-LDT method, whereas the extracted-LDT method was
(0) (0) (100.0) (0) (33.3) (85.7) (0) (20) (0) (94.1) (0) (100.0) (88.9) (0) (0) (100.0) (0) (92.9) (100) (100) (100) (47.1) (39.3) (0) (0) (0) (0) (0) (0) (0) (50) (55.6) (53.6)
able to identify 81.5% and 80.4% to genus level and species level, respectively. There was a dramatic difference between the Gram-positive bacteria and Gram-negative bacteria, as 33.0% and 78.4% Gram-positive bacteria were identified by direct-LDT and extractedLDT, respectively, compared to 85.5% and 90.3% identification of Gram-negative bacteria. The addition of the direct-plate extraction step (extracted-LDT) was far superior to the direct-LDT, particularly for the Gram-positive organisms. The identification rate of Gram-positive organisms increased by 45.4%, compared to a 4.8% increase in identification rate for Gram-negative organisms (P= 0.0001). Thus, further analysis throughout the study focused on the extracted-LDT. Numerous studies have extensively evaluated the performance of the Bruker MALDI-TOF MS Biotyper and confirm that this system can reliably identify 95% of all isolates cultured on media in the clinical laboratory using Bruker's recommended confidence scoring of ≥2.0 for species-level and 1.7–1.99 for genus-level identification (Carbonnelle et al., 2012; Eigner et al., 2009; Saffert et al., 2011; Tan et al., 2012). We selected a cut-off score of ≥1.7 for species-level identification directly from positive blood cultures on the basis of previous studies that demonstrated accurate species-level identification, ranging from 82% to 94%, using this score (Buchan et al., 2012; LagaceWiens et al., 2012; Saffert et al., 2012). Of the 137/168 bottles that yielded spectra scores of ≥1.7 using extracted-LDT, 98.5% were correctly identified by MALDI-TOF MS. The benefits of modifying the score for species-level identification from ≥2.0 to ≥1.7 was dramatic, particularly for the Gram-positive organisms (Table 2), where a difference of 19.6% and 29.9% identifications were noted for direct-LDT and extracted-LDT, respectively. Of the 159 monomicrobial blood cultures, 130 (81.8%) bacterial isolates were successfully identified by the extracted-LDT. Polymicrobial bacteremias have been previously reported to account for 5–20% of all bloodstream infections (Cooper et al., 1990; Martin et al., 2003; Roselle and Watanakunakorn, 1979; Saarinen et al., 1995), and we have recently reported a rate of 7.9% for polymicrobial bacteremias due to Gram-positive organisms culture from BacT/ ALERT PF and SA bottles combined (Mestas et al., 2013). Similarly, we report a 5.4% (9/ 168) rate of Gram-positive and Gram-negative polymicrobial bacteremias in this study; 3/9 bottles grew 2 Gram-negative organisms, 4/9 bottles grew 1 Gram-positive and 1 Gram-negative organism, 1/9 bottles grew 2 Gram-positive and 1 Gram-negative organism, and 1/9 bottles grew 1 Gram-positive organism and 1 yeast isolate. The extracted-LDT was able to correctly identify 1 pathogen in 55.6% (5/9) of cases. However, this method was not able to successfully identify both pathogens directly from polymicrobial blood culture bottles. Of the 4 bottles that were unsuccessful by extracted-LDT, 1 was mixed with Escherichia coli and Klebsiella pneumoniae, the second was mixed with Enterococcus faecalis and Candida parapsilosis, the third was mixed with viridans group Streptococcus spp. and Moraxella catarrhalis, and the final bottle
J. Mestas et al. / Diagnostic Microbiology and Infectious Disease 80 (2014) 193–196 Table 2 Spectral score distribution by method. Organism
Spectra Score
Direct
Extraction
Gram-negative organism (N = 62)
≥2.0 ≥1.9 ≥1.8 ≥1.7 b1.7 ≥2.0 ≥1.9 ≥1.8 ≥1.7 b1.7 ≥2.0 ≥1.9 ≥1.8 ≥1.7 b1.7
46 (74.2%) 51 (82.3%) 51 (82.3%) 53 (85.5%) 9 (14.5%) 13 (13.4%) 19 (19.6%) 24 (24.7%) 32 (33.0%) 65 (67.0%) 3 (33.3%) 3 (33.3%) 4 (44.4%) 5 (55.6%) 4 (44.4%)
51 (82.3%) 52 (83.9%) 55 (88.7%) 56 (90.3%) 6 (9.7%) 47 (48.5%) 61 (62.9%) 69 (71.1%) 76 (78.4%) 21 (21.6%) 5 (55.6%) 5 (55.6%) 5 (55.6%) 5 (55.6%) 4 (44.4%)
Gram-positive organism (N = 97)
Polymicrobial (N = 9)
was mixed with Rhizobium radiobacter and Pseudomonas fluorescens. Detection of Gram-negative bacteria were predominant over Gram-positive bacteria or yeast, as indicated by the 3 bottles mixed with Gram-negative and Gram-positive organism that identified as E. coli, K. pneumoniae, and Pantoea agglomerans. The performance of the MALDI-TOF MS for direct identification of bacteria from blood cultures appears to be independent of the actual blood volume drawn into each blood culture bottle. Blood volume data were available for 113 blood cultures and 67 Gram-positive and 46 Gram-negative organisms. MALDI-TOF MS identification was not obtained on 50 (44.2%) and 17 (15.0%) of the blood cultures using direct-LDT and extracted-LDT, respectively. The overall blood volume of the 113 blood cultures ranged from 0.6 to 11.6 mL, with a mean of 4.4 mL. There was no significant correlation between blood volumes and spectra scores for both methods. Specifically, using directLDT, blood cultures with scores of ≤1.7 had blood volumes ranging from 0.8 to 11.6 mL compared to 0.6 to 10.6 mL (P= 0.3386) in blood cultures with scores of ≥1.7.
4. Discussion In this study, we developed a robust, 4-step LDT that had a combined identification rate of 83.0% for monomicrobial bloodstream infections (132/159). Furthermore, due to its simplicity, the LDT method can be performed immediately after a bottle flags positive from the automated blood culture system, a similar approach to other sample-to-answer molecular blood culture assays (Mestas et al., 2013), allowing for rapid resulting from time to positivity. In fact, our goal at CHLA is to report out identification using the LDT method within 1 hour of blood culture positivity. The sensitivity of direct MALDI-TOF MS identification using noncharcoal or charcoal BacT/ALERT blood culture bottles has been reported to range from 23% to 77% (Fothergill et al., 2013; Haigh et al., 2013; Loonen et al., 2012; Meex et al., 2012; Schmidt et al., 2012; Szabados et al., 2011; Wuppenhorst et al., 2012). In particular, using a minimum cut-off of 800 match points (SuperSpectrum™) for correct identification, a detection rate of 23% was demonstrated from BacT/ ALERT bottles containing charcoal (Schmidt et al., 2012). In contrast, significantly higher identification rate of 77.8% from charcoal bottles was recently reported. Of note, the cut-off score for species-level identification was lowered to ≥1.4 (Wuppenhorst et al., 2012). Recently, using a cut-off score of ≥1.7 for species-level identification, 74%, 77%, and 86% identification rates were reported on BacT/ALERT bottles, using the Sepsityper™ kit, an enrichment method, and a direct plating method, respectively (Haigh et al., 2013). However, the enrichment method was laborious, requiring the addition of several steps, including sonication for bottles with charcoal harboring presumptive staphylococci and centrifugation over cesium chloride. The direct plating method included a 1- to 4-hour plate culture step, which significantly increased processing time. In addition, all 3 methods required a formic acid/acetonitrile tube extraction final step. Our study yielded similar results of 78.4% and 90.3% identification rate for Gram-positive and Gram-negative organisms, respectively, using a
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simple 4-step method versus a minimum of 5 steps for bottles without charcoal (Haigh et al., 2013). In our institution, the LDT (with direct-plate extraction) proved to be a robust method for direct identification from positive blood cultures, particularly with the Gram-negative organisms. Similar to other reports, identification of the viridans group Streptococcus spp. in this study by the Bruker MALDI-TOF MS was sub-optimal (Kok et al., 2011; Stevenson et al., 2010a). Due to the genetic similarity between the 2 groups, other molecular assays have described similar findings (Altun et al., 2013; Biendo et al., 2013; Mestas et al., 2013; Sullivan et al., 2013; Wojewoda et al., 2013). With regards to MALDI-TOF MS technology, this shortcoming appears to be unique to the Bruker MALDI-TOF MS system, as the Vitek MS system can successfully differentiate between viridans group Streptococcus spp. and S. pneumoniae (Dubois et al., 2013; Karpanoja et al., 2014; Martiny et al., 2012). This drawback with the Bruker system is currently being addressed by the company and should cease to exist with future software upgrades. Furthermore, differentiation between viridans group Streptococcus spp. and S. pneumoniae by MALDI-TOF MS has been addressed through optimization of spectral analysis software and scoring thresholds (Buchan et al., 2012; Ikryannikova et al., 2013; Werno et al., 2012). In addition to the high species-level identification rates demonstrated using our LDT method, time and cost savings were significant over conventional methods of bacterial identification from blood culture. Similar to other studies (Chen et al., 2013), the overall cost per sample by LDT was approximately $0.50, compared to $15 using the Sepsityper™ kit and $5–10 for conventional techniques. Furthermore, identification of organisms using the LDT was possible within 1 hour of blood culture positivity, with a total of 15 minutes (5 minutes hands-on time) required per sample for lysis, washing, spotting, plate extraction, and identification. Compared to the 20–46 hours required to subculture, incubate, and perform confirmatory biochemical tests, the LDT method is a simplified alternative to providing rapid identification directly from positive blood cultures. This study describes a robust 4-step lysis method for the direct identification of Gram-positive and Gram-negative pathogens directly from positive BacT/ALERT SA blood cultures obtained from pediatric patients that is independent of blood volume collected in each bottle. The simplicity of the method warrants the immediate testing once bottles flag positive, allowing for rapid identification and reporting of significant bloodstream pathogens to clinicians. Acknowledgments We thank the Clinical Microbiology Laboratory at Children's Hospital Los Angeles for technical support. References Altun O, Almuhayawi M, Ullberg M, Ozenci V. Clinical evaluation of FilmArray(R) blood culture ID panel in identification of bacteria and yeast from positive blood culture bottles. J Clin Microbiol 2013;51:4130–6. Biendo M, Mammeri H, Pluquet E, Guillon H, Rousseau F, Canarelli B, et al. Value of Xpert MRSA/SA blood culture assay on the Gene Xpert(R) Dx System for rapid detection of Staphylococcus aureus and coagulase-negative staphylococci in patients with staphylococcal bacteremia. Diagn Microbiol Infect Dis 2013;75:139–43. Bizzini A, Durussel C, Bille J, Greub G, Prod'hom G. Performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of bacterial strains routinely isolated in a clinical microbiology laboratory. J Clin Microbiol 2010;48:1549–54. Buchan BW, Riebe KM, Ledeboer NA. Comparison of the MALDI Biotyper system using Sepsityper specimen processing to routine microbiological methods for identification of bacteria from positive blood culture bottles. J Clin Microbiol 2012;50: 346–52. Carbonnelle E, Grohs P, Jacquier H, Day N, Tenza S, Dewailly A, et al. Robustness of two MALDI-TOF mass spectrometry systems for bacterial identification. J Microbiol Methods 2012;89:133–6. Chen JH, Ho PL, Kwan GS, She KK, Siu GK, Cheng VC, et al. Direct bacterial identification in positive blood cultures by use of two commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry systems. J Clin Microbiol 2013;51:1733–9.
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Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Direct identification of bacteria from positive BacT/ALERT blood culture bottles using matrix-assisted laser desorption ionization–time-of-flight mass spectrometry Javier Mestas a, Susanna Felsenstein b, Jennifer Dien Bard c,⁎ a b c
Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles, CA, USA Division of Infectious Diseases, Department of Pediatrics, Children's Hospital Los Angeles, Keck School of Medicine of University of Southern California, Los Angeles, CA, USA Department of Pathology and Laboratory Medicine, Keck School of Medicine, University of Southern California and Children's Hospital Los Angeles, Los Angeles, CA, USA
a r t i c l e
i n f o
Article history: Received 19 March 2014 Received in revised form 1 July 2014 Accepted 23 July 2014 Available online 31 July 2014 Keywords: Blood culture bottles Bacteria Identification MALDI-TOF
a b s t r a c t Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a fast and robust method for the identification of bacteria. In this study, we evaluate the performance of a laboratory-developed lysis method (LDT) for the rapid identification of bacteria from positive BacT/ALERT blood culture bottles. Of the 168 positive bottles tested, 159 were monomicrobial, the majority of which were Gram-positive organisms (61.0% versus 39.0%). Using a cut-off score of ≥1.7, 80.4% of the organisms were correctly identified to the species level, and the identification rate of Gram-negative organisms (90.3%) was found to be significantly greater than that of Gram-positive organisms (78.4%). The simplicity and cost-effectiveness of the LDT enable it to be fully integrated into the routine workflow of the clinical microbiology laboratory, allowing for rapid identification of Gram-positive and Gram-negative bacteria within an hour of blood culture positivity. © 2014 Elsevier Inc. All rights reserved.
Introduction Bloodstream infection is an important cause of morbidity and mortality, and prompt initiation of appropriate antimicrobial therapy is the mainstay of treatment (Ibrahim et al., 2000; Seifert, 2009). The application of matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a routine diagnostic tool for the identification of microorganisms in the microbiology laboratory has been proven to be rapid and accurate (Bizzini et al., 2010; Neville et al., 2011; Seng et al., 2009; Stevenson et al., 2010a, 2010b; Tan et al., 2012; van Veen et al., 2010). Furthermore, various commercial and in-house methods have been developed to rapidly identify bacteria and yeast directly from positive blood cultures, and a wide range in sensitivity of 46–98% has been reported and appears to be dependent on the methodology, organism, and blood culture bottle tested (Chen et al., 2013; Kok et al., 2011; March-Rossello et al., 2013; Schmidt et al., 2012; Wuppenhorst et al., 2012). A recent study using Bactec™ blood culture bottles (Becton Dickinson, Sparks, MD, USA) compared the Sepsityper™ kit (Bruker Daltonik GmbH, Bremen, Germany) to an in-house extraction method and reported species-level identification rates of 81.5% and 80.4%, respectively (Chen et al., 2013). Previous studies have evaluated the ability of MALDI-TOF MS to accurately identify bacteria directly from positive BacT/ALERT blood
⁎ Corresponding author. Tel.: +1-323-361-5443; fax: +1-323-361-8039. E-mail address: [email protected] (J. Dien Bard). http://dx.doi.org/10.1016/j.diagmicrobio.2014.07.008 0732-8893/© 2014 Elsevier Inc. All rights reserved.
culture bottles (bioMérieux, Durham, NC, USA), with or without the presence of charcoal, and the sensitivities reported have been suboptimal, ranging from 23% to 77% (Fothergill et al., 2013; Haigh et al., 2013; Loonen et al., 2012; Meex et al., 2012; Schmidt et al., 2012; Szabados et al., 2011; Wuppenhorst et al., 2012) when compared to the Bactec™ blood culture bottles (Romero-Gomez and Mingorance, 2011; Schmidt et al., 2012; Szabados et al., 2011). The use of commercial methods, such as the Sepsityper™ kit, can also increase the identification rate from BacT/ALERT standard aerobic and anaerobic bottles with 1 study reporting an increased in sensitivity of 13% and 26% when compared to an in-house centrifugation/ washing method and the MolYsis Basic method (Molzym GmbH, Bremen, Germany), respectively (Loonen et al., 2012). However, these methods are laborious and require multiple steps. In this study, we developed a simple, lysis-based, laboratory-developed test (LDT) to directly identify microorganisms from positive BacT/ALERT standard aerobic (SA), non-charcoal bottles using the MALDI-TOF MS Biotyper (BioTyper 3.0; Bruker Daltonik GmbH, Billerica, MA, USA).
2. Materials and methods One hundred sixty-eight positive BacT/Alert SA blood cultures from pediatric patients submitted for routine work-up to the Clinical Microbiology Laboratory at Children's Hospital Los Angeles (CHLA) were tested on MALDI-TOF MS using the LDT. To remove cellular components, 300 μL of blood culture broth was resuspended in 1 mL of
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Table 1 MALDI-TOF MS identification rate by method. Organism (n)
Acinetobacter baumannii (1) Bacillus pumilus (1) Bacillus spp. (3) Corynebacterium jeikeium (2) Corynebacterium spp. (3) Enterobacter cloacae complex (14) Enterococcus faecium (5) Enterococcus faecalis (5) Enterococcus hirae (2) Escherichia coli (17) Granulicatella adiacens (2) Klebsiella oxytoca (1) Klebsiella pneumoniae (9) Lactococcus lactis (1) Microbacterium spp. (1) Micrococcus spp. (2) Moraxella nonliquifaciens (1) Pseudomonas aeruginosa (14) Pseudomonas oryzihabitans (1) Pseudomonas putida (1) Serratia marcescens (1) Staphylococcus aureus (17) Staphylococcus epidermidis (28) Streptococcus agalactiae (2) Streptococcus mitis/oralis (7) Streptococcus parasanguinis (1) Streptococcus pneumoniae (1) Streptococcus pyogenes (1) Streptococcus viridans group (2) Weisella spp. (1) Coagulase-negative Staphylococcus (12) Polymicrobial (9) Total (168)
Direct-LDT
Extracted-LDT
Identified (score ≥1.7)
Correctly identified n (%)
Identified (score ≥1.7)
Correctly identified n (%)
0 0 3 0 1 12 0 1 0 16 0 1 8 0 0 2 0 13 1 1 1 8 11 0 0 0 0 0 0 0 6 5 90 (53.6)
0 0 3 0 1 12 0 1 0 16 0 1 8 0 0 2 0 13 1 1 1 8 11 0 0 0 0 0 0 0 6 5 90
0 1 3 1 2 12 5 3 2 17 0 1 9 1 0 2 0 14 1 1 1 17 23 2 1 1 0 1 0 0 11 5 137 (81.5)
0 (0) 1 (100) 3 (100) 1 (50) 1 (33.3) 12 (85.7) 5 (100) 3 (60) 2 (100) 17 (100) 0 (0) 1 (100) 9 (100) 1 (100) 0 (0) 2 (100) (0) 14 (100) 1 (100) 1 (100) 1 (100) 17 (100) 23 (82.1) 2 (100) 0 (0) 1 (100) 0 (0) 1 (100) 0 (0) 0 (0) 11 (91.7) 5 (55.6) 135 (80.4)
erythrocyte lysis buffer (ACK lysing buffer; Life Technologies, Grand Island, NY, USA), followed by centrifugation at 16000g for 2 minutes and removal of supernatant. The cell pellet is resuspended in 500 μL of distilled water, and the centrifugation and removal of supernatant step are repeated. The cell pellet is allowed to air dry and then smeared directly onto target plate (direct-LDT), followed by addition of 1 μL of 10 ng/μL α-cyano-4-hydroxycinnamic-acid matrix (Bruker Daltonik GmbH) to each spot. A direct plate extraction method (extracted-LDT) was also performed for each sample, with overlay of 1 μL of 70% formic acid, prior to the addition of α-cyano-4hydroxycinnamic-acid. All MALDI-TOF MS testing were performed in duplicate, and acquisition and analysis of mass spectra was performed using the Microflex LT mass spectrometer and flexControl 3.0 software (Bruker Daltonik GmbH). MALDI-TOF MS results were compared to conventional identification methods; blood cultures were aseptically inoculated onto blood agar, MacConkey, and chocolate agar followed by incubation for 18–24 hours at 37 °C and 5% CO2. Isolates were identified using routine identification methods such as Vitek II (bioMérieux) identification, and other biochemical tests and discordant results were confirmed by 16S rRNA sequencing. Blood volume was analyzed to determine significance between low blood volume and identification rate. Paired and unpaired t test were used to calculate the statistical significant differences demonstrated in the study. 3. Results Of the 168 positive blood cultures tested, 159 were monomicrobial and 9 were polymicrobial. Of 159 monomicrobial bottles, 97 (61.0%) grew Gram-positive organisms, and 62/159 (39.0%) grew Gram-negative organisms. The performance characteristics for all blood cultures using LDT compared to conventional methods are shown in Table 1. Using the cut-off score of ≥1.7, 53.6% were identified to genus level and species level using the direct-LDT method, whereas the extracted-LDT method was
(0) (0) (100.0) (0) (33.3) (85.7) (0) (20) (0) (94.1) (0) (100.0) (88.9) (0) (0) (100.0) (0) (92.9) (100) (100) (100) (47.1) (39.3) (0) (0) (0) (0) (0) (0) (0) (50) (55.6) (53.6)
able to identify 81.5% and 80.4% to genus level and species level, respectively. There was a dramatic difference between the Gram-positive bacteria and Gram-negative bacteria, as 33.0% and 78.4% Gram-positive bacteria were identified by direct-LDT and extractedLDT, respectively, compared to 85.5% and 90.3% identification of Gram-negative bacteria. The addition of the direct-plate extraction step (extracted-LDT) was far superior to the direct-LDT, particularly for the Gram-positive organisms. The identification rate of Gram-positive organisms increased by 45.4%, compared to a 4.8% increase in identification rate for Gram-negative organisms (P= 0.0001). Thus, further analysis throughout the study focused on the extracted-LDT. Numerous studies have extensively evaluated the performance of the Bruker MALDI-TOF MS Biotyper and confirm that this system can reliably identify 95% of all isolates cultured on media in the clinical laboratory using Bruker's recommended confidence scoring of ≥2.0 for species-level and 1.7–1.99 for genus-level identification (Carbonnelle et al., 2012; Eigner et al., 2009; Saffert et al., 2011; Tan et al., 2012). We selected a cut-off score of ≥1.7 for species-level identification directly from positive blood cultures on the basis of previous studies that demonstrated accurate species-level identification, ranging from 82% to 94%, using this score (Buchan et al., 2012; LagaceWiens et al., 2012; Saffert et al., 2012). Of the 137/168 bottles that yielded spectra scores of ≥1.7 using extracted-LDT, 98.5% were correctly identified by MALDI-TOF MS. The benefits of modifying the score for species-level identification from ≥2.0 to ≥1.7 was dramatic, particularly for the Gram-positive organisms (Table 2), where a difference of 19.6% and 29.9% identifications were noted for direct-LDT and extracted-LDT, respectively. Of the 159 monomicrobial blood cultures, 130 (81.8%) bacterial isolates were successfully identified by the extracted-LDT. Polymicrobial bacteremias have been previously reported to account for 5–20% of all bloodstream infections (Cooper et al., 1990; Martin et al., 2003; Roselle and Watanakunakorn, 1979; Saarinen et al., 1995), and we have recently reported a rate of 7.9% for polymicrobial bacteremias due to Gram-positive organisms culture from BacT/ ALERT PF and SA bottles combined (Mestas et al., 2013). Similarly, we report a 5.4% (9/ 168) rate of Gram-positive and Gram-negative polymicrobial bacteremias in this study; 3/9 bottles grew 2 Gram-negative organisms, 4/9 bottles grew 1 Gram-positive and 1 Gram-negative organism, 1/9 bottles grew 2 Gram-positive and 1 Gram-negative organism, and 1/9 bottles grew 1 Gram-positive organism and 1 yeast isolate. The extracted-LDT was able to correctly identify 1 pathogen in 55.6% (5/9) of cases. However, this method was not able to successfully identify both pathogens directly from polymicrobial blood culture bottles. Of the 4 bottles that were unsuccessful by extracted-LDT, 1 was mixed with Escherichia coli and Klebsiella pneumoniae, the second was mixed with Enterococcus faecalis and Candida parapsilosis, the third was mixed with viridans group Streptococcus spp. and Moraxella catarrhalis, and the final bottle
J. Mestas et al. / Diagnostic Microbiology and Infectious Disease 80 (2014) 193–196 Table 2 Spectral score distribution by method. Organism
Spectra Score
Direct
Extraction
Gram-negative organism (N = 62)
≥2.0 ≥1.9 ≥1.8 ≥1.7 b1.7 ≥2.0 ≥1.9 ≥1.8 ≥1.7 b1.7 ≥2.0 ≥1.9 ≥1.8 ≥1.7 b1.7
46 (74.2%) 51 (82.3%) 51 (82.3%) 53 (85.5%) 9 (14.5%) 13 (13.4%) 19 (19.6%) 24 (24.7%) 32 (33.0%) 65 (67.0%) 3 (33.3%) 3 (33.3%) 4 (44.4%) 5 (55.6%) 4 (44.4%)
51 (82.3%) 52 (83.9%) 55 (88.7%) 56 (90.3%) 6 (9.7%) 47 (48.5%) 61 (62.9%) 69 (71.1%) 76 (78.4%) 21 (21.6%) 5 (55.6%) 5 (55.6%) 5 (55.6%) 5 (55.6%) 4 (44.4%)
Gram-positive organism (N = 97)
Polymicrobial (N = 9)
was mixed with Rhizobium radiobacter and Pseudomonas fluorescens. Detection of Gram-negative bacteria were predominant over Gram-positive bacteria or yeast, as indicated by the 3 bottles mixed with Gram-negative and Gram-positive organism that identified as E. coli, K. pneumoniae, and Pantoea agglomerans. The performance of the MALDI-TOF MS for direct identification of bacteria from blood cultures appears to be independent of the actual blood volume drawn into each blood culture bottle. Blood volume data were available for 113 blood cultures and 67 Gram-positive and 46 Gram-negative organisms. MALDI-TOF MS identification was not obtained on 50 (44.2%) and 17 (15.0%) of the blood cultures using direct-LDT and extracted-LDT, respectively. The overall blood volume of the 113 blood cultures ranged from 0.6 to 11.6 mL, with a mean of 4.4 mL. There was no significant correlation between blood volumes and spectra scores for both methods. Specifically, using directLDT, blood cultures with scores of ≤1.7 had blood volumes ranging from 0.8 to 11.6 mL compared to 0.6 to 10.6 mL (P= 0.3386) in blood cultures with scores of ≥1.7.
4. Discussion In this study, we developed a robust, 4-step LDT that had a combined identification rate of 83.0% for monomicrobial bloodstream infections (132/159). Furthermore, due to its simplicity, the LDT method can be performed immediately after a bottle flags positive from the automated blood culture system, a similar approach to other sample-to-answer molecular blood culture assays (Mestas et al., 2013), allowing for rapid resulting from time to positivity. In fact, our goal at CHLA is to report out identification using the LDT method within 1 hour of blood culture positivity. The sensitivity of direct MALDI-TOF MS identification using noncharcoal or charcoal BacT/ALERT blood culture bottles has been reported to range from 23% to 77% (Fothergill et al., 2013; Haigh et al., 2013; Loonen et al., 2012; Meex et al., 2012; Schmidt et al., 2012; Szabados et al., 2011; Wuppenhorst et al., 2012). In particular, using a minimum cut-off of 800 match points (SuperSpectrum™) for correct identification, a detection rate of 23% was demonstrated from BacT/ ALERT bottles containing charcoal (Schmidt et al., 2012). In contrast, significantly higher identification rate of 77.8% from charcoal bottles was recently reported. Of note, the cut-off score for species-level identification was lowered to ≥1.4 (Wuppenhorst et al., 2012). Recently, using a cut-off score of ≥1.7 for species-level identification, 74%, 77%, and 86% identification rates were reported on BacT/ALERT bottles, using the Sepsityper™ kit, an enrichment method, and a direct plating method, respectively (Haigh et al., 2013). However, the enrichment method was laborious, requiring the addition of several steps, including sonication for bottles with charcoal harboring presumptive staphylococci and centrifugation over cesium chloride. The direct plating method included a 1- to 4-hour plate culture step, which significantly increased processing time. In addition, all 3 methods required a formic acid/acetonitrile tube extraction final step. Our study yielded similar results of 78.4% and 90.3% identification rate for Gram-positive and Gram-negative organisms, respectively, using a
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simple 4-step method versus a minimum of 5 steps for bottles without charcoal (Haigh et al., 2013). In our institution, the LDT (with direct-plate extraction) proved to be a robust method for direct identification from positive blood cultures, particularly with the Gram-negative organisms. Similar to other reports, identification of the viridans group Streptococcus spp. in this study by the Bruker MALDI-TOF MS was sub-optimal (Kok et al., 2011; Stevenson et al., 2010a). Due to the genetic similarity between the 2 groups, other molecular assays have described similar findings (Altun et al., 2013; Biendo et al., 2013; Mestas et al., 2013; Sullivan et al., 2013; Wojewoda et al., 2013). With regards to MALDI-TOF MS technology, this shortcoming appears to be unique to the Bruker MALDI-TOF MS system, as the Vitek MS system can successfully differentiate between viridans group Streptococcus spp. and S. pneumoniae (Dubois et al., 2013; Karpanoja et al., 2014; Martiny et al., 2012). This drawback with the Bruker system is currently being addressed by the company and should cease to exist with future software upgrades. Furthermore, differentiation between viridans group Streptococcus spp. and S. pneumoniae by MALDI-TOF MS has been addressed through optimization of spectral analysis software and scoring thresholds (Buchan et al., 2012; Ikryannikova et al., 2013; Werno et al., 2012). In addition to the high species-level identification rates demonstrated using our LDT method, time and cost savings were significant over conventional methods of bacterial identification from blood culture. Similar to other studies (Chen et al., 2013), the overall cost per sample by LDT was approximately $0.50, compared to $15 using the Sepsityper™ kit and $5–10 for conventional techniques. Furthermore, identification of organisms using the LDT was possible within 1 hour of blood culture positivity, with a total of 15 minutes (5 minutes hands-on time) required per sample for lysis, washing, spotting, plate extraction, and identification. Compared to the 20–46 hours required to subculture, incubate, and perform confirmatory biochemical tests, the LDT method is a simplified alternative to providing rapid identification directly from positive blood cultures. This study describes a robust 4-step lysis method for the direct identification of Gram-positive and Gram-negative pathogens directly from positive BacT/ALERT SA blood cultures obtained from pediatric patients that is independent of blood volume collected in each bottle. The simplicity of the method warrants the immediate testing once bottles flag positive, allowing for rapid identification and reporting of significant bloodstream pathogens to clinicians. Acknowledgments We thank the Clinical Microbiology Laboratory at Children's Hospital Los Angeles for technical support. References Altun O, Almuhayawi M, Ullberg M, Ozenci V. Clinical evaluation of FilmArray(R) blood culture ID panel in identification of bacteria and yeast from positive blood culture bottles. J Clin Microbiol 2013;51:4130–6. Biendo M, Mammeri H, Pluquet E, Guillon H, Rousseau F, Canarelli B, et al. Value of Xpert MRSA/SA blood culture assay on the Gene Xpert(R) Dx System for rapid detection of Staphylococcus aureus and coagulase-negative staphylococci in patients with staphylococcal bacteremia. Diagn Microbiol Infect Dis 2013;75:139–43. Bizzini A, Durussel C, Bille J, Greub G, Prod'hom G. Performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of bacterial strains routinely isolated in a clinical microbiology laboratory. J Clin Microbiol 2010;48:1549–54. Buchan BW, Riebe KM, Ledeboer NA. Comparison of the MALDI Biotyper system using Sepsityper specimen processing to routine microbiological methods for identification of bacteria from positive blood culture bottles. J Clin Microbiol 2012;50: 346–52. Carbonnelle E, Grohs P, Jacquier H, Day N, Tenza S, Dewailly A, et al. Robustness of two MALDI-TOF mass spectrometry systems for bacterial identification. J Microbiol Methods 2012;89:133–6. Chen JH, Ho PL, Kwan GS, She KK, Siu GK, Cheng VC, et al. Direct bacterial identification in positive blood cultures by use of two commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry systems. J Clin Microbiol 2013;51:1733–9.
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