Rapid Detection and Identification of Candidemia by Direct Blood Culturing on Solid

Transcription

1 JCM Accepted Manuscript Posted Online 19 October 2016 J. Clin. Microbiol. doi: /jcm Copyright 2016 American Society for Microbiology. 1 2 Rapid Detection and Identification of Candidemia by Direct Blood Culturing on Solid Medium Using Lysis-Centrifugation Method Combined with MALDI-TOF MS Evgeny A. Idelevich, Barbara Grünastel, Karsten Becker# Running title: Direct blood culturing for candidemia diagnostics Affiliation: Institute of Medical Microbiology, University Hospital Münster, Münster, Germany # Address correspondence to Karsten Becker, kbecker@uni-muenster.de (This work was presented in part at the 25th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Copenhagen, Denmark, 25 th to 28 th April 2015 [P1077]) 1

2 22 Abstract Candida sepsis is a life-threatening condition with increasing prevalence. In this study, direct blood culturing on solid medium using lysis-centrifugation procedure allowed successful Candida species identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry on average 3.8 hours (Sabouraud agar) or 7.4 hours (chocolate agar) prior to the positivity signal for control samples in BACTEC Mycosis-IC/F or BACTEC Plus Aerobic/F bottles, respectively. Direct culturing on solid medium accelerates candidemia diagnostics compared to automated broth-based systems. Downloaded from on January 7, 2019 by guest 2

3 Introduction Candidemia is associated with particularly high mortality rate increasingly observed especially among immunocompromised and critically ill patients (1-3). While early and appropriate treatment is vital (3), slow growth of yeasts delays timely diagnosis (4). The current diagnostic standard includes inoculation of patient s blood into special bottles with liquid medium followed by incubation in an automated blood culture (BC) instrument (5). Upon growth detection, Gram stain is performed, and broth is sub-cultivated onto solid medium to grow colonies for identification and antimicrobial susceptibility testing (AST) (4). Lysis-centrifugation blood culture (LC-BC) method is an alternative approach, which has been available for decades, but drifted out of use in the course of blood culture automation (6-9). LC-BC implies selective lysis of blood cells with subsequent centrifugation and culturing of sediment directly on agar plates (6). A recent study has demonstrated that this approach provides a dramatic time reduction in the BC diagnostics for bacteria, when combined with the modern methods of identification and susceptibility testing (10). In this study, we aimed to investigate whether the direct cultivation on solid medium using LC-BC method, combined with the identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), will result in more rapid detection and differentiation of yeasts from blood as compared to the currently used liquid medium-based automated BC instruments. We also investigated impact of specific fungal or general media used with each method. Materials and Methods Four clinical strains of different yeast species were used in the study. Those species included Candida albicans, Candida glabrata, Candida krusei, and Candida tropicalis, as 3

4 confirmed by yeast internal transcribed spacer (ITS) sequencing (11). The strains were subcultured on Sabouraud agar overnight prior to the experiment. Each strain was mixed with 10 ml of human whole blood to produce concentrations 1, 10, and 100 cfu/ml. The exact volume ( µl) of yeast suspension added to the blood was based on the cfu calculations performed in the preliminary experiments for each strain. The actual yeast concentration in the study was confirmed by vital cell count of serial dilutions. 10 ml of inoculated blood was added to Isolator 10 tubes (Wampole Laboratories, Princeton, USA) containing lysis and anticoagulant reagents, followed by centrifugation at 3000g for 30 min. After removal of supernatant, 1.5 ml sediment was vortexed and distributed onto five pre-warmed 150-mm Sabouraud agar plates or five prewarmed 150-mm chocolate agar plates. This processing after centrifugation required approximately several minutes hands-on time per sample. All plates were incubated at 36 C in air with 5% CO 2 to ensure equal atmospheric conditions, which are commonly used when sepsis pathogen is unknown and are reportedly not inhibitory to fungi (12). Visual evaluation of plates was performed hourly beginning from the time of expected appearance of growth as defined in preliminary experiments. Identification by MALDI-TOF MS (Microflex LT, Bruker Daltonics, Bremen, Germany) with on-plate 70% formic acid extraction (13) was performed as soon as microcolonies appeared. The score 1.7 (13) for at least one of three smeared spots was used as criterion for successful species identification. The cultivation time needed for successful identification from microcolonies was recorded. Agar plates were further incubated for a total of 24 hours and MALDI-TOF MS was performed from mature cultures for control. For comparison, 10 ml of inoculated blood were introduced into fungal (BACTEC Mycosis-IC/F, BD Diagnostics, Heidelberg, Germany) and 10 ml into aerobic (BACTEC Plus Aerobic/F, BD Diagnostics) BC bottles, and incubated in the BACTEC 9240 automated BC system (BD Diagnostics). The time to positivity of the automated system was documented. 4

5 Results and discussion The incubation time necessary for species identification by LC-BC method and the time to positivity of the automated BC system are presented in the Table 1, stratified for the different inoculum concentrations as well as for the fungal or general media used. Overall, MALDI-TOF MS identification following the direct blood culturing on Sabouraud agar was achieved before the growth detection in fungal BACTEC bottles for all strains at all yeast concentrations (in average 3.8 h earlier). Also applying general media (chocolate agar and aerobic BACTEC bottle), identification after direct blood culturing was always achieved prior to the growth detection with aerobic BACTEC bottles (in average 7.4 h earlier). Interestingly, the difference between time to successful identification following direct blood culturing on solid medium and time to growth detection by the automated BC system was particularly pronounced for the lowest yeast concentration (1 cfu/ml) (Table 1). This is a relevant finding because the quantitative burden of Candida is very low in the most positive blood cultures (9). Pfeiffer et al. have demonstrated that over half of initial Candida blood cultures have a CFU/ml of 1 (9). There was a pronounced delay of growth detection with C. glabrata by the automated system using aerobic bottle, which occurred for 1 cfu/ml concentration only 26.6 hours after the successful species identification from directly inoculated chocolate agar (Table 1). The delayed detection of C. glabrata using BACTEC Plus Aerobic/F bottle has been previously reported (5, 14). Using the score 1.7 as a criterion for successful identification on species level (13) provided reliable results without misidentifications as compared to ITS sequencing. The growth detection by automated BC systems allows only Gram stain and subcultivation onto agar plates at this time point. Even though direct procedures are available for identification of microorganisms from positive blood culture bottles, they are costly and/or 5

6 laborious and particularly challenging for yeasts (4). Therefore, definitive identification of yeasts in the routine laboratory is still only performed after subsequent incubation of yeasts on agar from grown colonies. In contrast, direct culturing on solid medium provides colonies for immediate testing (15). This means a time advantage of at least one day by using the direct culturing. While Gram stain indicating yeast cells provides important information for initiation of antifungal therapy, species identification and AST results are critical for the administration of appropriate targeted treatment (16). The prevalence of non-albicans Candida spp., particularly C. glabrata, as well as antifungal resistance have been increasing over time (17-19). Occurrence of resistant yeasts including multidrug-resistant strains (20) makes rapid identification and susceptibility testing of fungi isolated from blood essential (18). The availability of colonies on agar is a requirement for definitive testing. While this study didn t additionally investigate the cultivation time needed for initiation of yeast AST, it is obvious that colonies on solid medium could also be used for AST as it has been shown in the previous study with bacteria (10). This study demonstrates that leaving out the incubation of blood samples in liquid medium is beneficial for diagnostics of candidemia as it has been recently demonstrated for bacteremia (10). While the advantages of direct blood culturing on solid medium for detection and identification of yeasts have been pointed out in early publications (6, 7, 21), they are becoming even more obvious in the current era of rapid microbiology. However, technical improvement of the procedure is necessary to ensure contamination-free and easy workflow. In conclusion, direct culturing of blood samples on solid medium allows acceleration of candidemia diagnostics compared to currently applied automated broth-based systems. 6

7 Acknowledgements We are thankful to Katrin Blaschke for expert technical assistance. EAI and KB are inventors of the patent application Apparatus and method for processing of body fluids, owned by the University of Münster. BG declares no conflicts of interest Downloaded from on January 7, 2019 by guest 7

8 133 Table 1. Time to MALDI-TOF MS identification result using lysis-centrifugation direct blood culturing method, in comparison to time to 134 positivity signal of the automated system, hours Fungal media General media Organism Candida albicans Candida glabrata Candida krusei Candida tropicalis Time to species Concentration, identification after cfu/ml direct blood culturing (Sabouraud agar) Time to species Time to positivity Time to positivity identification after signal of automated signal of automated direct blood culturing system (aerobic bottle) system (fungal bottle) (chocolate agar)

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