Identification by mass spectrometry and automated susceptibility testing from positive bottles: a simple, rapid, and standardized approach to reduce the turnaround time in the management of blood cultures

Rapid identification and antimicrobial susceptibility results of organisms causing bloodstream infections (BSIs) are an absolute priority for the Microbiology Laboratory. Blood culture is the gold standard method for the diagnosis of BSIs, and it is included among the early activities in the Surviving Sepsis Campaign guidelines [1]. However, it is time-consuming and usually takes on average 24–48 h for microbial growth in blood culture bottles, and further 48 h for identification and antimicrobial susceptibility tests after growth in solid culture media [2]. Speeding up these procedures is of foremost importance in the management of bloodstream infections, leading to rapid administration of adequate antimicrobial therapy or adjusting ongoing treatment, and so improving outcome in patients with bacteremia [3]. Reporting Gram stain results of positive blood cultures is useful to guide empiric antimicrobial therapy, but does not provide information about microbial identification at species level [4]. Molecular methods such as real-time polymerase chain reaction (RT-PCR), fluorescent in-situ hybridization (FISH), DNA microarray and nested multiplex PCR have shown to be efficient for the identification of specific microorganisms [5, 6]. However, molecular methods and related target-based multiplex assays can identify a limited range of microorganisms and antimicrobial resistance genes. Furthermore, molecular methods require additional hands-on processing time and costs [7, 8].

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) technology has been introduced as a way to quickly and accurately identify bacteria and yeast [9, 10]. Compared to standard phenotypic identification, this technology is rapid, inexpensive (after initial purchase of the instrument), and can identify bacteria grown on solid media to the species level [11‐13]. Subsequent to the introduction of MALDI-TOF MS for routine diagnostic microbiology, several applications have been developed to further improve and accelerate pathogen identification, especially for bloodstream infections [14, 15]. Particularly, several authors investigated the possibility of direct identification of bacteria from positive blood cultures with MALDI-TOF MS [7, 8, 16‐20] while fewer investigated the combined use of MALDI-TOF MS and automated antimicrobial susceptibility testing (AST) instruments [3, 21, 22]. Also, direct identification and AST performed on positive blood culture using mass-spectrometry and disk diffusion methods, respectively, can provide results about 24 h earlier than routine standard methods. However, disk diffusion for direct susceptibility testing has not been fully standardized, because it is not related to a specific quantification of the bacterial concentration of the inoculum.

At our Institution, a number of specific approaches have been used to reduce the time of result and improve patient outcome: delivery of blood cultures to the laboratory within one hour, immediate incubation of bottles in the automated instrument and continuous management of blood cultures (365 days/year), communication of preliminary results based on Gram stain (performed at the time of positivity), identification from overnight cultures by MALDI-TOF MS, and direct AST by agar diffusion (using selected antimicrobials as indicators of resistance).

This paper describes a standardized, easy-to-perform and practical approach in order to obtain rapid identification with MALDI-TOF MS and to decrease the time needed to obtain final reports using a standardized bacterial concentration and an automated instrument for definitive susceptibility results. The turnaround time (TAT) was also compared to the standard method to better quantify the impact in the laboratory practice. The aim of the study was to compare these data with those obtained with the previously used workflow of blood cultures.

Part of this work has been presented at the 25th ECCMID meeting, Copenhagen (Denmark), 2015 (ePoster EP-054).

The present study describes a procedure based on a very short-term incubation time on solid medium from positive blood cultures followed by MALDI-TOF MS identification and automated AST (starting from a standardized bacterial concentration) in order to obtain rapid final results.

An early basis for decision making is urgently required in the case of life-threatening conditions such as bloodstream infections [26]. Of note, the International Guidelines for Management of Sepsis and Septic Shock recommend that: i) antimicrobial therapy should be assessed daily for de-escalation; ii) the combination therapy, when used empirically in patients with sepsis, should not be routinely used for ongoing treatment.

Empiric antimicrobial therapy should be narrowed once pathogen identification and sensitivities are established and/or adequate clinical improvement is noted [1]. Molecular methods have shown to be efficient for the rapid identification of specific microorganisms but can identify a limited range of microorganisms and antimicrobial resistance genes, thus partially responding to the above recommendations. Furthermore, molecular methods are not available in many laboratories, require additional hands-on processing time and costs. Application of MALDI-TOF MS for identification of bacterial colonies from solid media has considerably improved and accelerated routine microbiological diagnostics [11‐13]. Particularly, direct MALDI-TOF MS pathogen identification from positive blood culture has been demonstrated to have a positive impact on antimicrobial treatment in septic patients [27, 28]. Most of procedures with this direct approach, however, require additional hands-on processing time. For example, Romero-Gómez et al. used a series of centrifugation, washes and an extraction procedure before identification and AST [3]. Machen et al. used a combined lysis-filtration method [21] whereas Prod’hom et al. used a series of centrifugation, washes and lysis procedure [29]. One major advantage of our combined method is its simplified workflow. Our procedure can be easily integrated in the laboratory routine since it needs only a tube with gel separator (as additional consumable) and a centrifugation step. Identification by MALDI-TOF MS and suspension for AST are performed following a common standard procedure, only earlier than usually (i.e., 3 h versus overnight incubation).

With respect to rapid identification, our results demonstrate that the performance of the presented method is very high and satisfactory for both Gram-positive and Gram-negative isolates. These data confirm the capacity of the MALDI-TOF MS technique to identify both Gram-positive and Gram-negative bacteria from positive blood cultures after a very short-term incubation time, as previously reported [18].

AST also showed excellent results. Percentages of errors (minor, major and very major) were overall low enough to fulfill the performance criteria considered acceptable by the International Standard ISO 20776–2 (ME ≤3%; VME ≤ 3%). According to previous observations, CoNS exhibited the majority of errors among Gram-positives [3, 21]. These strains, however, are common contaminants of blood cultures that often do not require an antimicrobial treatment. Consequently, the clinical impact of these errors is very low. Among Gram-negative isolates, E. coli had the highest rate of errors but no very major errors occurred. The large number of isolates could have contributed to this result. Similarly, our results indicate a high concordance with regard to detection and/or suggestion of known resistance mechanisms (e.g., production of ESBLs or carbapenemases, methicillin-resistance and ICR). Taken together, these data demonstrate that the rapid method is reliable not only for wild-type isolates (as previously suggested) [3] but also for multidrug-resistant microorganisms.

Finally, in addition to high performance rates for identification and AST, the new procedure exhibited TATs significantly lower than those obtained using the standard method. It is worth noting that the time to positivity was comparable in the two study periods (thus eliminating a confounding variable between the two groups). The processing time was substantially reduced using the new procedure, leading to a final result approximately one day earlier than usual. In this regard, the reduction of TAT was mainly influenced by the automated AST, performed the same day of bottle positivity, even though the application of MALDI-TOF MS technique was crucial to render it possible.

The new procedure, based on rapid identification by MALDI-TOF MS and automated susceptibility testing performed following short-term incubation cultures on solid agar plates inoculated from positive blood cultures, represents a simple, standardized, and workflow-friendly approach that allows faster and reliable results essentially without additional hands-on processing time and costs. The most relevant conclusion from our experience is that the new procedure permits at least a one day reduction in TAT in the management of bloodstream infections, especially when the most frequently isolated pathogens are involved. The introduction of this method in the microbiology laboratory might facilitate an appropriate species-specific therapy with potential downstream impact on the development of resistance and improved patient outcomes.