Published at ASM Journals
ABSTRACT
Bloodstream infections frequently cause sepsis, a condition that can lead to organ dysfunction and death. Rapid antimicrobial susceptibility testing (AST) results are critical for appropriate medical intervention and to either de-escalate or escalate antibiotics to appropriate therapy. We describe the results from a multicenter clinical study evaluating the performance of the eQUANT system (Avails Medical, Inc., Menlo Park, CA). The eQUANT system generates a 0.5 McFarland equivalent suspension, the eMcFarland, directly from a positive blood culture bottle for use with downstream disk diffusion AST, thereby saving up to 24 hours compared to traditional AST workflow. A combination of fresh, prospectively collected clinical (42) and contrived (525) blood cultures were tested, and results for disk diffusion using the Avails eQUANT eMcFarland suspension as the inoculum were compared to disk diffusion results the next day using the standard 0.5 McFarland suspension prepared from plate subculture. Thirteen species of gram-negative rods were evaluated against 12 antibiotics. From the 2,679 pairs of AST results, overall categorical agreement was 95.0%. Overall very major errors, major errors, and minor errors were 0.15%, 0.60%, and 4.48%, respectively. The Avails eQUANT system has the potential to significantly accelerate the standard of care by eliminating the need for subculture.
Importance
Rapid reporting of antimicrobial susceptibility test results for bacterial isolates from blood cultures is critical for timely implementation of optimal antimicrobial therapy and improves outcomes of sepsis patients. In this study, we demonstrate the accuracy and performance characteristics of the eQUANT system, which generates a 0.5 McFarland equivalent suspension, the eMcFarland, directly from a positive blood culture bottle for use with downstream disk diffusion antimicrobial susceptibility testing (AST). Using the eMcFarland suspension allows for accurate and standardized reporting of disk diffusion AST results from positive blood cultures.
INTRODUCTION
Bloodstream infections and sepsis are major causes of morbidity and mortality worldwide (1, 2). In the USA, every year, at least 1.7 million adults develop sepsis, with at least 350,000 deaths annually (3). In the USA, sepsis is the most common cause of in-hospital deaths, and associated costs exceed $24 billion annually (3–5). Blood culture (BC) remains the most important cornerstone of sepsis diagnosis, considering that the identification of the offending bacterial or fungal organism is essential to selecting optimal antimicrobial therapy (6–8). Improvements to continuously monitored BC systems and BC broths during the past decades have improved the ability to recover the causative microorganisms. Antimicrobial susceptibility testing (AST) of blood culture isolates is essential to optimize the patient’s antimicrobial therapy, enabling either de-escalation or escalation of antibiotics to more appropriate or optimal therapy (9, 10). While most microbiology laboratories can use technologies for faster organism identification from positive BCs, traditional AST methods still require between 24 and 96 hours. AST is traditionally performed by broth microdilution (BMD) testing or a BMD-derived method, including automated AST systems or disk diffusion testing. However, BMD and disk diffusion testing have a few technical limitations, in addition to the longer turnaround times (TATs) of results reporting. Such limitations include the need for a bacterium to grow in pure culture on agar plates inoculated from positive BC bottles and the need for a standardized inoculum of the bacterium, measured by a standardized McFarland suspension. The latter is a manual process and fraught with some variability in obtaining an accurate and standardized suspension, partly due to a mix of viable and dead organisms. Despite these limitations, BMD is still considered the gold standard, as AST results have repeatedly been shown to be most predictive of treatment outcome in sepsis patients (11). In addition, modifications of the disk diffusion (DD) testing method have been developed for rapid AST directly from positive blood cultures; such methods have subsequently been approved by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (12, 13). These methods, however, require use of non-standardized inocula, adjustments to laboratory workflow, and use of alternate breakpoints, compared to standard DD methods. There are a few rapid genotypic diagnostic test methods that provide not only organism identification (ID) directly from a positive blood culture (PBC), but also the detection of select antimicrobial resistance genes (14). Results with these instruments, e.g., BioFire (bioMérieux), Verigene (Diasorin), ePlex (Roche Diagnostics), are available within 1.5 to 4 hours after a BC bottle turns positive and a Gram stain is performed. However, these genotypic detection methods can only be considered to be supplemental to the actual determination of full range, phenotypic antimicrobial susceptibility testing, since only a limited set of antimicrobial resistance genes for gram-positive and/or gram-negative bacteria is included on these panels. Therefore, it is desirable to expedite and shorten the time of the procedural steps to perform full-scale phenotypic AST following standard laboratory practices.
In this regard, the eQUANT instrument (Avails Medical, Inc.) is a small (21.6 cm × 11.7 cm × 21.6 cm) bench-top automated instrument which uses potentiometric sensing of changes in oxidation-reduction potential (ORP) from gram-negative or gram-positive pathogen metabolism in cation-adjusted Mueller-Hinton broth (CAMHB) with an antifoaming agent to prepare an organism suspension (eMcFarland) equivalent to a traditional 0.5 McFarland (1–2 × 108 CFU/mL ± 0.6 log or 2.51 × 107 to 7.96 × 108 CFU/mL; CFU: colony-forming unit) directly from PBC broth in 64–72 minutes on average (15, 16). For each organism and blood culture bottle type combination, there exists a mapping of changes in ORP which correlates to changes in the bacterial cell density (15). Thus, by monitoring the ORP changes in real-time, the system can precisely determine the time at which the bacterial cell density reaches the desired count (equivalent to a 0.5 McFarland). Based on the user selections of organism and bottle type, the appropriate algorithm (and algorithm parameters) is applied, and as soon as the desired cell density is reached, the system rapidly cools down the sample to 15°C to limit further growth and starts a countdown timer for 1 hour (15). This eMcFarland suspension of the respective organism can then be used to set up the traditional phenotypic AST using methods available to the respective clinical laboratory. This approach eliminates the need to grow the organism in pure culture, therefore shortening the overall TAT from the time of a positive BC bottle to the final AST result by up to 24 hours. Currently, the eQUANT system is FDA cleared for use with a panel of gram-negative organisms with disk diffusion AST (17). Here, we report the results from the evaluation of the Avails Medical eQUANT System for gram-negative AST using the disk diffusion method at three clinical microbiology laboratories, using prospective clinical blood culture samples positive for gram-negative rods, as well as contrived blood culture samples . The AST results using the eQUANT eMcFarland standard were compared to the AST results obtained the next day using the currently established standard method for disk diffusion AST with the manually prepared McFarland standard from plate subculture.
