Diagnostic stewardship in sepsis: minding the gap from the microbiology lab to ward

The sepsis challenge and diagnostic delay

According to the international Surviving Sepsis Campaign, a joint initiative of the Society of Critical Care Medicine (SCCM) and the European Society of Intensive Care Medicine (ESICM), sepsis affects millions of people worldwide and remains a leading cause of death in intensive care units (ICUs) [1]. Multiple studies have demonstrated that timely diagnosis and rapid response significantly improve survival rates. This involves not only the early identification of septic patients but also the prompt initiation of appropriate antimicrobial therapy. For optimal diagnosis and treatment, extraction of blood cultures and their prompt incubation is relevant to allow the identification of the pathogen involved when positive and determine its susceptibility to antimicrobial agents. This can be better achieved with continuous availability of the clinical microbiology laboratory [2]. However, in most hospitals, clinical microbiology departments operate with restricted hours, leading to diagnostic delays that can negatively impact patient management by postponing the administration of optimal treatment. This was demonstrated in a retrospective study in a hospital analyzing 50,955 blood cultures collected over a 30-month period from 7,035 adult patients with sepsis with a clinical microbiology laboratory that only accepts blood cultures during restricted opening hours (Monday to Friday, 8:00 a.m. to 4:00 p.m., and Saturdays, 8:00 a.m. to 2:00 p.m) [3]. They compared the results of those collected during laboratory opening hours that were immediately processed with those collected during closing hours and stored at room temperature in clinical wards until transported and processed during opening hours. In all cases, blood cultures were incubated using automated systems. The analysis showed that each hour of delay from blood collection to incubation was associated with a 0.3% decrease (P<0.001) in the likelihood of obtaining a positive result. Furthermore, the prevalence of positive cultures was significantly lower when the laboratory was closed (11% vs. 13%, P<0.001), and the probability of a positive result decreased by 16% under these conditions (P<0.01). These findings underscore the necessity of continuous laboratory operation, so-called 24/7 availability, to optimize sepsis diagnosis and treatment. A recent cross-sectional study across European hospitals further revealed that, unfortunately, only 10.4% offered continuous microbiology services, including 24/7 blood culture incubation and reporting [4]. This limitation is associated with lower blood culture positivity rates and delayed report of the results, directly affecting clinical decision-making and patient outcomes.

The impact of timeliness: from culture to cure

Because timely initiation of appropriate antimicrobial therapy is a critical determinant of clinical outcomes in sepsis management, numerous studies have investigated the impact of treatment delays on patient mortality. A landmark study by Kumar et al. [5] demonstrated that, in patients with septic shock, each hour of delay in administering effective antibiotics was associated with a 7.6% decrease in survival. Subsequent research has consistently confirmed these findings, highlighting that the interval to appropriate therapy is a modifiable factor influencing sepsis mortality [1]. In a more recent retrospective study, 10,628 routine medical records from 9,192 patients with bacteraemia were analysed to assess the association between the timing of appropriate treatment, adjusted according to antimicrobial susceptibility results, and mortality [6]. Logistic regression analyses were performed for different initiation times. The crude 30-day mortality rate was approximately 14% among patients with bacteraemia, increasing to 25% in those who developed septic shock. Time-to-treatment analysis revealed that delays exceeding appropriate antimicrobial 12 hours after blood cultures were obtained were significantly associated with higher mortality, underscoring a critical window of opportunity in which blood culture results, including pathogen identification, can guide life-saving interventions. Many diagnostic efforts in microbiology laboratories are therefore directed at reducing response times, being part of broader diagnostic stewardship strategies aimed at improving patient outcomes in sepsis [2].

Diagnostic stewardship programs and sepsis

Diagnostic stewardship is a coordinated approach aimed at maximizing the clinical and economic value of microbiological testing. It ensures that the right test is performed for the right patient at the right time, optimizing laboratory resources to guide effective therapy [7]. These programs emphasize timely sample collection and processing, minimize pre-analytical and post-analytical delays, integrate diagnostic activities with antimicrobial stewardship, and enable rapid feedback to clinical teams. This transforms the laboratory from a passive service into an active participant in clinical decision-making, effectively bridging the gap between diagnosis and treatment. In many institutions, antimicrobial stewardship programs incorporate a “sepsis code” to rapidly identify septic patients and predict adverse outcomes using validated scoring systems such as the Sequential Organ Failure Assessment (SOFA), quick Sepsis-related Organ Failure Assessment (qSOFA), and National Early Warning Score (NEWS) [1,8]. Once a patient is recognized as septic, antimicrobials should be administered immediately—ideally within one hour of diagnosis [1]. Clinical biochemistry laboratories can provide rapid results for biomarkers such as C-reactive protein (CRP) and procalcitonin, aiding in early diagnosis. In contrast, clinical microbiology workflows still depend on blood culture growth, which inherently delays definitive results. Nonetheless, several initiatives have been implemented to reduce time-to-results and support timely, appropriate antimicrobial use [2]. In the context of sepsis, the implementation of diagnostic stewardship in clinical microbiology laboratories has led to substantial reductions in the time required to report blood culture results. This, in turn, allows antimicrobial stewardship teams to provide recommendations sooner, potentially improving patient outcomes, reducing hospital length of stay, and lowering the overall cost of care. Within the diagnostic stewardship background, most of the efforts have been focused in the analytical part of the blood culture, but they can also be applied to the pre-analytical and post-analytical parts of the workflow. A recent review and meta-analysis highlighted that all of these efforts are more efficient if they are aligned with specific actions with the antimicrobial stewardship teams [9].  Preanalytic factors affecting sepsis diagnosis. In the pre-analytical workflows involving diagnostic stewardship strategies, various interventions have aimed to reduce blood culture contamination rates below the commonly accepted threshold of 3% and to optimize blood bottle fill volumes, which are linked to higher positivity rates [10].  A randomized controlled trial showed that modifying blood culture collection techniques significantly reduced contamination rates. Similarly, a systematic review reported that contaminated blood cultures lead to substantial increases in healthcare costs [11]. Across the studies analyzed (n = 49), overall contamination rates ranged from 0.9% to 41%. Most contaminants were gram-positive microorganisms, predominantly coagulase-negative staphylococci, resulting in up to 60% of patients receiving unnecessary parenteral vancomycin. This inappropriate treatment generated additional pharmacy charges of $210–$12,611 per patient, laboratory charges of $2,397–$11,152 per patient, and prolonged hospital stays by 1 to 22 days. In another randomized controlled trial, contamination rates and costs were reduced using an initial-specimen diversion technique. This involved changing the order of blood sampling with regular vacuum specimen tubes instead of commercially available sterile diversion devices. The intervention achieved a 66% reduction in contamination (5% with the standard method vs. 1.6% with the new technique), without compromising the detection of true bloodstream infections and at no additional cost. Notably, hospital-wide implementation was associated with a 1.1% reduction in hospitalization days [12]. Regarding blood bottle fill volumes, a multi-center study involving 10 hospitals and three laboratories found that underfilled bottles were extremely common. However, coordinated operational and educational strategies led to sustained improvements, even in a complex hospital network. Increased fill volumes enhanced pathogen detection, with significant implications for the management of bloodstream infections and sepsis. Strategies included education, standardized data collection, innovative and transparent feedback systems, targeted communication, and bottle volume markings for phlebotomists, a specific team for blood culture sampling. Fill volumes were monitored automatically using an electronic tracking system [13]. Analytic procedures and diagnosis of sepsis in the microbiology laboratory. The concept of “microbiologistics” has been applied to the management of samples and procedures in clinical microbiology laboratories to enable the timely diagnosis of infectious diseases and particularly sepsis [2]. This approach involves streamlining every step of the microbiological diagnostic workflow, ultimately influencing positivity rates and reducing time-to-report. Key processes for sepsis include blood sampling, inoculation into culture bottles, incubation in automatic systems (e.g., BACTEC-FX, VIRTUO), Gram staining and preliminary reporting, rapid identification, currently using MALDI-TOF MS, antimicrobial susceptibility testing (AST), and final report delivery. Within these processes, major efforts have focused on the direct identification of bacteria from positive blood cultures using MALDI-TOF MS, as well as on direct AST once blood cultures flag positive and Gram staining/identification have been performed. The advantages of these strategies have been extensively reviewed, including their impact on patient management, the support they provide to antimicrobial stewardship teams, and their potential economic implications [14-19]. MALDI-TOF MS enables rapid pathogen identification directly from positive blood cultures, reducing time-to-result by 10 to 28 hours. Clinical studies have shown that its use is associated with earlier initiation of targeted therapy, decreased mortality, and lower healthcare costs [15]. However, despite more than 15 years since its introduction, access to this technology remains largely restricted in low and middle-income countries [20,21]. Moreover, traditional AST requires 24–48 hours to generate results. In contrast, emerging methods for AST now provide results within 6–8 hours or less. Among them, disk diffusion protocols applied directly to positive blood cultures (RAST) have been standardized by both EUCAST and CLSI [22-24]. These protocols enable phenotypic susceptibility and resistance detection within 4–8 hours, can be implemented in most laboratories with minimal effort, and are mainly limited by the need for additional personnel to read inhibition zones outside of standard shifts [17]. This can be easily performed in institutions that are open 24/7 and have staff available to perform rapid AST on second and third shifts. Nevertheless, a retrospective modelling study demonstrated that there is a clinical value of rapid AST even in centers that process positive blood cultures during a single shift [25].  Rapid AST automated systems such as QuickMIC (Gradientech), QMAC-dRAST (QuantaMatrix), VITEK Reveal (bioMérieux), ASTar (Q-linea) or PhenoTest BC (Accelerate Diagnostics) further allow MIC-based AST directly from positive cultures within 4-7 hours, with accuracy and performance validated in different studies [18]. Other innovative approaches include flow cytometry, which can provide AST results in as little as 2 hours [26] and novel techniques such as cantilever-based detection of microbial movement, which show promising results [27]. Interestingly, some of these rapid systems also allow the detection of resistance mechanisms on top of phenotypic susceptibility testing and clinical categorization of the results [18].  However, special attention should also be given to the incubation process of blood culture bottles. This step is typically carried out in continuously monitored automated systems located in the microbiology laboratory, which means that transportation to the laboratory is required before the incubation can begin. Ideally, incubation should start immediately upon arrival at the laboratory, as several studies have shown that delays in placing blood culture bottles into incubators can significantly reduce positivity rates [3,28-30]. Nevertheless, in some hospitals, the absence of technical staff during evening and night shifts leads to unavoidable delays. To address this issue, certain institutions have decentralized the incubation process by installing satellite blood culture incubators in emergency departments or intensive care units. This strategy, often referred to as the use of “point-of-care blood culture incubators,” enables immediate incubation at the site of collection. Once a bottle is flagged as positive, it can then be transported to the microbiology laboratory for further processing [3,28-30]. A similar model may also be advantageous in settings where microbiology laboratories are centralized outside the hospital.  Incubation process decentralization was evaluated in an emergency department where an additional blood culture instrument was installed and compared against a historical control group. This modification led to a significant reduction in Gram stain reporting time, from 21.5 hours (IQR 15.5–26.7) to 13.5 hours (IQR 11.2–20.9). Interestingly, the length of hospital stay was also reduced by up to one day [31]. A more complex scenario was reported in which blood cultures collected in the emergency department were first transported by pneumatic tube system to a central medical laboratory with 24/7 operations for initial incubation in decentralized incubators. Subsequently, both negative and positive bottles were transported three times per day to a microbiology laboratory (not operating 24/7) located 20 minutes away. Incubation continued until bottles flagged positive, after which processing resumed during working hours. A comparison of pre-analytical and analytical parameters over a 3-month period before and after implementation showed that, despite this complex workflow, decentralization of incubators reduced median time to positivity (21.5 h vs. 16.2 h, p<0.05), median time to Gram stain reporting by phone (28.9 h vs. 22.5 h, p=0.001) and median time to medical validation (29.3 h vs. 23.1 h, p<0.005). Interestingly, the proportion of positive cultures increased from 1.1% to 3.2%, although this difference did not reach statistical significance [32]. The authors concluded that decentralization improved reporting times, although the benefit was limited by the lack of a fully operational 24/7 microbiology laboratory. Unfortunately, a survey performed in 1,023 hospitals in 69 countries in Europe and published in 2025 revealed that microbiological service was limited in time in nearly 90% of the hospitals, lacking the possibility to incubate blood cultures, to identify pathogens, and to communicate results outside the main working hours or on weekends and holidays [4].  Molecular testing and syndromic panels. The current standard of care for the detection of pathogens in bacteremia and sepsis in the clinical microbiology laboratory is the blood culture in broth media. As previously described, once a blood culture bottle flags positive, a Gram stain is performed, followed by pathogen identification and AST. In recent years, alternative methods independent of blood culture have been developed for the simultaneous detection of pathogens and resistance genes directly from positive blood cultures [18,33,34]. These so-called syndromic panels are based on molecular techniques (Table 1). One of the most widely used systems is the FilmArray platform (bioMérieux). It is a fully automated system that integrates nucleic acid extraction, real-time PCR, and high-resolution melting analysis. It can detect nearly 45 pathogens—including Gram-positive and Gram-negative bacteria and yeasts, as well as major resistance genes, such as those encoding ESBLs, carbapenemases, colistin resistance, methicillin resistance, and vancomycin resistance. Reported overall sensitivity and specificity for pathogen identification are 91–96% and 98–100%, respectively, while detection of resistance markers reaches 100% for both parameters. Key advantages include a rapid turnaround time (<1 h) and minimal hands-on time for laboratory personnel (2–5 min) [35,36]. The main limitation is the higher per-test cost compared with conventional methods. However, cost-effectiveness studies indicate that syndromic panels may reduce overall healthcare costs by enabling earlier targeted antimicrobial therapy and shortening hospital stays [36,37]. These panels have been evaluated in critically ill patients, including those admitted to intensive care units, where their clinical impact has been compared against standard diagnostic workflows [33]. It is important to note that syndromic panels are applied directly to blood culture bottles after positivity is detected; they are not suitable for direct testing of whole blood samples. This limitation arises from the very low microbial load in bloodstream infections (<1×10² CFU/ml), which results in insufficient analytical sensitivity. Nevertheless, alternative platforms have been designed to detect microorganisms and resistance genes directly from blood. Among these, LightCycler Septifast, IRIDICA BAC BSI Assay, and T2Bacteria were notable. LightCycler Septifast, now discontinued, was pioneer detecting microorganisms directly in blood. It could detect main pathogens involved in sepsis, had higher specificity than sensitivity, and did not include resistance markers, precluding effectively introduction in the management of patients with sepsis [38,39]. The IRIDICA system, also discontinued, combined nucleic acid amplification with latter electrospray ionization mass spectrometry [39,40]. Its main drawbacks were the high per-test cost and relatively long detection time (approximately 7 h). Its discontinuation was also influenced by the widespread adoption of MALDI-TOF MS in clinical microbiology laboratories at the time of its launch. The T2 system, also no longer marketed, employed nucleic acid amplification to detect pathogens and resistance markers, followed by hybridization with paramagnetic nanoparticles, detected via probes [41,42]. This platform offered a very low limit of detection (1 CFU/ml), with sensitivity around 90% and specificity of 96-98% for pathogen detection, as well as good concordance for resistance marker identification. The turnaround time ranged from 3 to 5 hours. A couple of solutions for detecting pathogens and resistance mechanisms with short development times have recently been presented. One of them, already CE marked, uses 0.4 ml of blood and is capable of detecting up to 26 pathogens, bacteria, or yeasts in 3 hours, using real-time qPCR (https://microbio.com.au/product/). The other solution, which is not yet commercially available, uses multiplex PCR starting with 8 ml of blood and detects up to 32 pathogens and 9 resistance markers in less than an hour. It is associated with a random access platform for up to 8 samples (https://deepull.com/). In both cases, the publication of their analytical and clinical validation is pending.

Clinical integration of microbiology laboratory: from laboratory to bedside

The synergy between diagnostic stewardship and antimicrobial stewardship programs is crucial, as demonstrated by the integration of rapid diagnostic tests in clinical microbiology laboratories. Evidence shows that even in hospitals with well-established antimicrobial stewardship procedure, the addition of rapid diagnostic testing improves patient outcomes by reducing the time to effective therapy, lowering mortality, and decreasing both hospital stay and healthcare costs [9]. Implementation of rapid tests, together with procedures that optimize blood culture yield, consistently provides these benefits, although demonstrating a direct reduction in mortality remains challenging [43]. Early advantages were documented with the introduction of MALDI-TOF MS for bacterial identification directly from positive blood cultures, particularly when combined with ASP interventions in critically ill patients [44]. Beyond improving the adequacy of antimicrobial therapy, MALDI-TOF implementation was associated with shorter hospitalization and, consequently, reduced healthcare costs [45]. An additional, less frequently explored benefit of reduced hospital stays is a potential decrease in adverse events, including healthcare-associated infections. Rapid AST further shortens diagnostic turnaround times. Several clinical trials and retrospective studies evaluating pre- versus post-implementation of rapid AST have confirmed its positive clinical impact, particularly when results are integrated into antimicrobial stewardship programs decision-making workflows [46,47]. These tools enable earlier de-escalation of empiric therapy, promote more judicious antibiotic use, and may ultimately contribute to mitigating antimicrobial resistance. In our experience, a randomized clinical trial comparing conventional AST (results after overnight incubation) with rapid AST (results available within 5 hours and promptly communicated to the antimicrobial stewardship team) demonstrated significantly shorter times to ASP intervention and to appropriate therapy modification, especially in critically ill patients [47]. Similar benefits have been observed with syndromic panels designed to detect pathogens and resistance mechanisms in positive blood cultures. These approaches have been associated with faster initiation of targeted therapy (notably anti-methicillin-resistant Staphylococcus aureus agents), reduced duration of broad-spectrum antibiotic use, and improved 60-day survival in patients with bloodstream infections [48,49]. Finally, the impact of rapid diagnostic methods depends not only on the availability of results but also on their timely translation into medical decisions. Laboratory information must be effectively integrated with antimicrobial stewardship activities to optimize sepsis management. Efforts to reduce reporting times lose clinical value if results are not promptly translated into actions that benefit the patient. In this regard, a recent study demonstrated that real-time communication of microbiology results regarding the diagnosis of sepsis to ASP teams, compared with delayed reporting until the following morning shift, led to improved clinical outcomes, including increased survival [50].

Conclusions: a microbiology-driven model for sepsis care and future directions in innovation

We are currently at a stage where clinical microbiology laboratories have consolidated the use of innovative techniques that enable rapid diagnosis. Among these are proteomics-based methods (such as MALDI-TOF MS) and real-time PCR techniques for the detection of multidrug-resistant microorganisms, including syndromic panels. Many of them are simple, highly adaptable methods to automation, while others are more innovative and disruptive for assessing AST. Also, whole genome sequencing is increasingly transforming several fields within the microbiology laboratory [50]. This integrated model, assisted by artificial intelligence and real-time clinical alerts, represents the future of infectious disease management. At the same time, traditional techniques of great clinical value, such as Gram staining and culture, remain in routine use, while straightforward methods that require minimal training, such as lateral flow or immunochromatography assays, have been efficiently integrated into diagnostic workflows. All of these tools provide clear benefits for the diagnosis of sepsis. However, their implementation in many centers is still constrained to varying degrees, by organizational challenges, resource availability (including trained laboratory staff capable of providing continuous coverage across all shifts), and the presence of multidisciplinary teams dedicated to antimicrobial stewardship and results management. The incorporation of new technologies and the reorganization of laboratory workflows have made it evident that the role of the microbiology laboratory extends well beyond simply generating test results. Through diagnostic stewardship, microbiology becomes an active, clinically integrated partner in the fight against sepsis and antimicrobial resistance. These programs promote timely, accurate, and clinically actionable diagnostics, which in turn enable earlier interventions and improved patient outcomes. As both technology and healthcare systems continue to evolve, the adoption of 24/7 diagnostic models and the integration of microbiologists into antimicrobial stewardship initiatives and sepsis code teams will be essential, not only to optimize patient outcomes, but also to ensure the sustainability and cost-effectiveness of healthcare delivery [7,52]. The introduction of new technologies for sepsis diagnosis that shorten time-to-result in clinical microbiology laboratories and demonstrate tangible clinical benefits is becoming increasingly challenging. The margin for improvement is narrowing, even when diagnostic stewardship and antimicrobial stewardship programs are well aligned, and outcomes often depend heavily on the characteristics of the study population. A clear example is the persistent difficulty in demonstrating a direct impact on the reduction of mortality.  This situation is not new: before the arrival of mass spectrometry in clinical microbiology laboratories, it was equally challenging to show measurable clinical benefits, yet the technology ultimately revolutionized blood culture processing in microbiology laboratories. A similar scenario is now unfolding with rapid AST systems and syndromic panels applied to positive blood culture bottles [17,33,51]. Looking ahead, a true paradigm shift will occur when rapid, highly sensitive and specific technologies capable of detecting any pathogen directly from blood samples, while simultaneously identifying resistance mechanisms or even providing complete genomic sequences, become available in affordable, user-friendly formats (Figure 1). In many cases, these advances will be strengthened by integration with decision-support algorithms powered by artificial intelligence. Conflict of interest RC has participated in educational programs sponsored by BD, BioMérieux and QuantaMatrix and has received research grants from BioMérieux and QuantaMatrix. AVH has participated in educational programs and has received research grants from BioMérieux and QuantaMatrix. Funding Research studies of RC are funded by Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, CIBER de Enfermedades Infecciosas (CIBERINFEC) (CB21/13/00084), Spain Author contributions  R.C.; conceptualization, writing—original draft preparation, A.V.H; writing—review and editing. All authors have read and agreed to the published version of the manuscript.

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