A novel rapid bioluminescence-based antimicrobial susceptibility testing method based on adenosine triphosphate consumption.

BackgroundLife-threatening syndromic hospital infections including sepsis, ventilator acquired pneumonia, catheter-associated urinary tract infection (CAUTI), and surgical site infections are often caused by multidrug-resistant pathogens. Implementing the targeted narrow-spectrum antimicrobial therapy as rapidly as possible at the onset of infection is critical for lowering morbidity and mortality for these infections. We present the new MultiPath technology for rapid syndromic infection detection, pathogen identification, and phenotypic antimicrobial susceptibility testing (AST). Our feasibility data demonstrate the technology’s potential application as a rapid CAUTI diagnostic.MethodsThe MultiPath technology detects and counts cells in a 30-minute assay using nonmagnified digital imaging. For identification, target pathogen cells are labeled using fluorescent in situ hybridization (FISH) with rRNA-specific probes, tagged with magnetic nanoparticles, deposited on a surface, imaged, and quantified. For AST, samples are mixed with growth medium, incubated for 4 hours in the presence of serial dilutions of antibiotics, FISH-labeled, magnetically selected, and quantified by digital imaging. The MultiPath assays use a dye-cushion layer to optically sequester the sample and unbound fluorescent probes from the imaging surface, eliminating the need for sample preparation and wash steps.ResultsThe MultiPath ID method specifically detected a range of common CAUTI pathogens including E. coli, K. pneumonia, E. faecium, E. faecalis, and P. aeruginosa. The limit of detection for E. coli was 27 CFU in a 100 µL assay in 10% urine. We present data demonstrating target inclusivity, specificity, and dynamic range. Our AST feasibility study results show excellent correlation with the broth micro dilution reference test for 5 antibiotics. Variable inoculum levels had little impact on MICs in the study.ConclusionThe data presented demonstrate the potential of the rapid ID/AST technology to achieve excellent analytical and clinical performance. This, combined with the method’s simplicity, robustness to sample matrix, and ease-of-use may make the method valuable for rapid syndromic infection diagnostics.Disclosures All authors: No reported disclosures.

Assessment of bacterial viability by laser desorption ionization mass spectrometry for antimicrobial susceptibility testing

To assess the expected clinical and financial benefits of rapid reporting of microbiology results, we compared patients whose cultured samples were processed in the normal manner to patients whose samples were processed more rapidly due to a minor change in work flow. For the samples tested in the rapid-reporting time period, the vast majority of bacterial identification and antimicrobial susceptibility testing (AST) results were verified with the Vitek system on the same day that they were available. This time period was called rapid AST (RAST). For RAST, a technologist on the evening shift verified the data that became available during that shift. For the control time period, cultures were processed in the normal manner (normal AST [NAST]), which did not include evening-shift verification. For NAST, the results for approximately half of the cultures were verified on the first day that the result was available. The average turnaround time for the reporting of AST results was 39.2 h for RAST and 44.4 h for NAST (5.2 h faster for RAST [P = 0.001]). Subsequently, physicians were able to initiate appropriate antimicrobial therapy sooner for patients whose samples were tested as part of RAST (P = 0.006). The mortality rates were 7. 9 and 9.6% for patients whose samples were tested as part of RAST and NAST, respectively (P = 0.45). The average length of stay was 10. 7 days per patient for RAST and 12.6 days for NAST, a difference of 2.0 days less for RAST (P = 0.006). The average variable cost was $4, 927 per patient for RAST and $6,677 for NAST, a difference of $1,750 less per patient for RAST (P = 0.001). This results in over $4 million in savings in variable costs per year in our hospital.

Background: Bloodstream infections (BSIs) are medical emergencies associated with high morbidity and mortality, where delays in initiating appropriate antimicrobial therapy adversely affect outcomes. Although automated blood culture systems reduce time to pathogen detection, conventional antimicrobial susceptibility testing (AST) requires additional incubation, leading to delayed targeted therapy. The EUCAST Rapid Antimicrobial Susceptibility Testing (RAST) method was developed to provide early phenotypic susceptibility results directly from positive blood culture bottles. Aim: To evaluate the diagnostic accuracy of the EUCAST RAST method, compare its performance with conventional disc diffusion AST using FDA Class II criteria, and determine the prevalent pathogens and resistance patterns in bloodstream infections. Methods: A prospective laboratory-based observational study was conducted over six months on monomicrobial positive blood cultures. RAST was performed directly from flagged blood culture bottles, with inhibition zones read at 8 hours and interpreted using EUCAST RAST breakpoints. Conventional Kirby–Bauer disc diffusion testing, interpreted as per CLSI guidelines, served as the reference standard. Categorical agreement and error rates were analyzed. Results: EUCAST RAST showed high categorical agreement (>90%) across all tested organisms. Perfect agreement was observed for Enterococcus faecium, Acinetobacter baumannii, and Salmonella enterica. Minor discrepancies were mainly associated with fluoroquinolones among Enterobacterales. Conclusion: EUCAST RAST is a rapid, reliable AST method that significantly reduces turnaround time and supports early targeted therapy and antimicrobial stewardship in BSIs.

“Barcode” cell sensor microfluidic system: Rapid and sample-to-answer antimicrobial susceptibility testing applicable in resource-limited conditions

The rapid administration of optimal antimicrobial treatment is paramount for the treatment of bloodstream infections (BSIs), and rapid antimicrobial susceptibility testing (AST) results are essential. Q-linea has developed the ASTar system, a rapid phenotypic AST device. Here, we report the performance of the ASTar BC G- (Gram-negative) kit when assessed according to the ISO 20776-2:2007 standard for performance evaluation of in vitro diagnostic AST devices. The evaluated ASTar BC G- kit uses a broad panel of 23 antimicrobials for the treatment of BSIs caused by Gram-negative fastidious and nonfastidious bacteria across a range of 6 to 14 2-fold dilutions, including cefoxitin as a screening agent for AmpC-producing Enterobacterales. The ASTar system processes blood culture samples to generate data on MICs and susceptible, intermediate, or resistant (SIR) category. The automated protocol includes concentration determination and concentration adjustment to enable a controlled inoculum, followed by broth microdilution (BMD) and microscopy performed continuously to generate MIC values within approximately 6 h once the test is run on the ASTar system. The performance of the ASTar system was assessed against the ISO 20776-2:2007 standard BMD reference method. Testing was performed across three sites, with results from 412 contrived blood cultures and 74 fresh clinical blood cultures. The ASTar system was also tested for reproducibility, with triplicate testing of 11 strains. The accuracy study comprised 8,650 data points of bacterium-antimicrobial tests. The ASTar system demonstrated an overall essential agreement (EA) of 95.8% (8,283/8,650) and a categorical agreement (CA) of 97.6% (8,433/8,639) compared to the reference BMD method. The overall rate of major discrepancies (MDs) was 0.9% (62/6,845), and that of very major discrepancies (VMDs) was 2.4% (30/1,239). This study shows that the ASTar system delivers reproducible results with overall EA and CA of >95%.

<i>Introduction</i>: The escalating prevalence of multidrug resistance is a global threat to human health particularly in critically ill patients with bloodstream infections (BSIs). Delay in the administration of the appropriate antimicrobial treatment is associated with higher mortality rates and adverse consequences. This study attempted to estimate the rapid antimicrobial susceptibility testing (RAST) breakpoints directly from flagged BacT/Alert blood culture bottles in clinical practice. <i>Material & Methods</i>: A descriptive, cross-sectional study conducted at a tertiary care hospital in Delhi over a period of two months. The RAST was performed directly from the clinical samples for blood cultures received in our laboratory in parallel with the routine antimicrobial testing as per standard CLSI guidelines. Blood cultures were routinely incubated in BacT/Alert 3D. The inhibition zones were read at 4, 6, 8 and 16-20 hour of incubation as per European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The identification of the isolates was confirmed by Vitek-2 compact system. <i>Results</i>: In our study, the area of technical uncertainty (ATU) percentage was initially high at 4 hours but decreased significantly in later incubation periods. At 4 hours, none of the <i>S. aureus</i> isolates showed >90% categorical agreement (CA) for any antimicrobial tested. However, clindamycin achieved the highest CA (100%) at 6 hours and 90% thereafter, with no very major errors (VME) or major error (ME). Cefoxitin required 8 hours to reach >90% CA, with no VME observed at any time point, but up to 75% ME at 8 hours. At 4 hours, most antimicrobials had high (>1.5%) rates of VME among <i>Enterobacteriales</i>. By 6 hours, only Meropenem and Gentamicin had >90% CA, with no VME observed for other antibiotics. <i>Conclusion</i>: The RAST method is relatively easy to implement in clinical microbiology labs, offering cost-effectiveness, simplicity, and rapid results, especially in resource-limited settings. However, reporting RAST results can be complex due to potential challenges with CA, VME, and ME, particularly in the initial hours of incubation and within the ATU.

Standalone self-compartmentalizing microfluidic chip for digital single-cell antimicrobial susceptibility testing.

Urinary tract infection (UTI) is the most common infection for people with neurogenic lower urinary tract dysfunction (NLUTD) due to spinal cord injury/disease (SCI/D). While the current diagnostic pillars include symptoms, inflammation, and bacterial load, each is uniquely problematic when applied to NLUTD-related UTI. Authoritative guidelines have not taken into account the impact of bladder management on symptoms; baseline inflammation in the absence of symptoms limits usefulness of white blood count; and bacteriuria in the absence of symptoms and limitations of standard urine culture (SUC) limits utility of SUC-based determination of bacterial load. Newer approaches are being developed to better guide diagnosis and antibiotic use. These include the Urinary Symptom Questionnaires for Neurogenic Bladder for symptom measurement and decision-making around symptoms, novel inflammatory markers (urine NGAL, IL-1, and IL-8), and culture-independent microbial detection methods. Rapid antimicrobial susceptibility testing (AST) methods have been developed utilizing a number of different approaches, increasing the likelihood of point-of-care application in the future. Advancements in UTI diagnosis will include reconceptualization of "urine is sterile" to "eubiosis and dysbiosis," symptom complexes to aid decision-making, use of novel inflammatory markers, and less reliance on SUC with increased emphasis on culture-independent methods for bacterial identification, AST, and decision-making.

Early diagnosis and effective treatment of bloodstream infections significantly improve prognosis. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) has developed rapid antimicrobial susceptibility testing(RAST) based on the disk diffusion method. In the present study, we compared the data from the RAST study from positive blood cultures in the Autobio BC (Autobio-diagnostics, Zhengzhou, Chinese) blood culture system with those obtained using the standard disk diffusion method. A total of 234 spiked blood culture samples (Klebsiella penumoniae [n= 50], Escherichia coli [n= 50], Acinetobacter baumannii [n= 20], Pseudomonas aeruginosa [n= 13], Staphylococcus aureus [n= 51], and Enterococcus spp. [n= 50]) were collected. The RAST test was performed on positive bottles, and the results were measured at 4, 8, and 20 h. Standard disk diffusion (sDD) was performed, and zone diameters were measured from subculture samples obtained from blood culture bottles. The RAST zone diameters were compared with the sDD zone diameters. The categorical agreement based on the readable zone diameters for the 4th, 8th, and 20th h was K.pneumoniae (99.7 %,99.7 %,99.7 %), E.coli (99.4 %,99.6 %,99.7 %), A.baumannii (98.7 %,98.7 %,98.7 %), P.aeruginosa (100 %,99.3 %,100 %), S. aureus (100 %,100 %,100 %), and Enterococcus spp. (100 %,100 %,100 %). The minor error(mE), major error(ME), and very major error(VME) results obtained in the whole study were determined as 0.9, 0, and 1.3 for 4 h; 1.4, 0, and 1.3 for 8 h; 0.6, 0, and 1.3 for 20 h. The EUCAST RAST study can be performed from positive bottles in the Autobio BC blood culture system, resulting in reliable outcomes. To report earlier antibiotic susceptibility test results, laboratories using Autobio BC can combine RAST with quality control studies.

Bloodstream infections pose significant clinical challenges, requiring rapid and accurate diagnostics to guide timely antimicrobial therapy. Although efforts to reduce turnaround time (TAT) have focused on emerging molecular techniques and rapid antimicrobial susceptibility testing (AST) methods, these innovations currently complement rather than replace standard protocols. To balance innovation with reliability, we investigated the impact of integrating laboratory automation into the blood culture diagnostic workflow while preserving conventional pathogen identification via mass spectrometry and AST. In this single-center, prospective study, we compared key performance indicators between a historical cohort (March-August 2023; 192 episodes, 110 patients) and an intervention cohort (March-August 2024; 156 episodes, 112 patients) utilizing an automated workflow with the BacT/Alert Virtuo BC system, VPlus 50 automated sample processor, and Vitek 2 XL Microbiology Analyzer. The optimized workflow included automated loading and unloading of blood culture bottles, automatic subculturing of positive cultures, expert system-based review of AST results, and automated reporting of negative results. Post-optimization, the time from blood culture collection to final report was significantly reduced from 95.99 h (79.29-117.63) to 60.81 h (47.06-82.38), with a more pronounced reduction for gram-negative bacteria (44.87 h) compared with gram-positive bacteria (27.24 h). Although the 28-day mortality rate remained unchanged, patients in the post-optimization group experienced significantly shorter hospital stays, faster achievement of optimal therapy, and lower costs related to antibiotics, laboratory tests, and overall hospitalization. These findings indicate that integrating automation in blood culture diagnostics enhances laboratory efficiency, improves clinical outcomes, and reduces healthcare costs, supporting its broader implementation in clinical microbiology.IMPORTANCEBloodstream infections require prompt and accurate diagnosis to enable timely, targeted antimicrobial therapy. Traditional blood culture workflows are often hindered by labor-intensive processes and limited after-hours capacity, delaying critical clinical decisions. This study demonstrates that integrating laboratory automation, including automated loading and unloading of blood culture bottles, automatic subculturing of positive cultures, expert system-based review of antimicrobial susceptibility results, and automated reporting of negative results can significantly reduce diagnostic turnaround time without additional staffing. Implemented in a high-volume tertiary hospital, the optimized workflow led to faster initiation of optimal therapy, shorter hospital stays, and reduced healthcare costs, particularly for gram-negative bacteremia. These findings highlight how automation enhances both laboratory efficiency and patient outcomes, supporting its broader adoption in modern clinical microbiology.

EUCAST rapid antibiotic susceptibility testing method for blood cultures – Implementation in a private laboratory setting

Background: Rapid antimicrobial susceptibility testing (RAST) allows early detection of resistance directly from positive blood cultures, potentially improving outcomes in bloodstream infections (BSIs). We evaluated the performance of EUCAST RAST for Gram-negative ESKAPEEc pathogens and characterized carbapenemase genes in carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods: A total of 354 positive blood cultures were screened, including 51 monomicrobial Gram-negative ESKAPEEc isolates. RAST results at 4, 6, 8, and 16–20 h were compared with standard antimicrobial susceptibility testing (AST) obtained using the BD Phoenix™ system. Categorical agreement (CA) and error frequency were calculated. Multiplex PCR and Sanger sequencing were performed on 15 CRKP isolates to identify carbapenemase genes and allelic variants. Results: 51 Gram-negative ESKAPEEc isolates met the inclusion criteria for RAST (15 E. coli, 19 K. pneumoniae, 11 A. baumannii, and 6 P. aeruginosa). Overall performance varied markedly by species and antibiotic. E. coli showed frequent unreadable or ATU zones at early timepoints and wide CA variability (50–100%), with high very major error (VME) rates for AMP, TZP, and CAZ, particularly at 6–8 h. K. pneumoniae displayed consistently high CA (mostly 100%) for carbapenems, CAZ, and TZP. A. baumannii demonstrated excellent agreement (100% for most agents), except for GEN at 6–8 h. P. aeruginosa could be evaluated only at 16–20 h, showing high CA for AMK, CAZ, and CIP; lower CA for MEM (83%); non-calculable CA for IMI due to universal ATU readings; and a CA value of 0% for TZP due to the predominance of the ATU results. VMEs ranged from 0% to 26.1% across species and reading times, but carbapenems did not generate VMEs. Molecular analysis revealed blaKPC in 66.7%, blaNDM in 46.7%, and blaOXA-48 in 33.3% of isolates, with co-occurrence in several strains. Sequencing identified blaKPC-2 and blaNDM-1 as the predominant variants, with one isolate harboring blaNDM-5. Conclusions: EUCAST RAST markedly accelerates susceptibility reporting from positive blood cultures, but its accuracy is species- and time-dependent. Performance was excellent for K. pneumoniae (including CRKP) and A. baumannii and acceptable for P. aeruginosa at 16–20 h. In contrast, E. coli showed frequent ATU results at early timepoints and high ME/VME rates, making readings before 8 h unreliable for clinical decisions. Overall, RAST can effectively support rapid antimicrobial stewardship when species-specific limitations are recognized, and early-timepoint results are interpreted with caution.

Rapid antimicrobial susceptibility testing by stimulated Raman scattering metabolic imaging and morphological deformation of bacteria

Bloodstream infection (BSI) is a serious clinical condition associated with high morbidity and mortality, requiring rapid identification of causative agents and timely antimicrobial susceptibility testing (AST). This study evaluated accelerated bacterial identification from positive blood culture samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) combined with two rapid processing approaches: a serum separation tube-based centrifugation method (SST method) and shortened cultivation on solid media. Rapid identification was followed by accelerated AST, performed either from a bacterial cell pellet (SST method) and from early-grown bacterial biomass (shortened cultivation protocol). The results were compared with those obtained using routine laboratory procedures. A total of 270 positive blood culture samples were analyzed, with 135 samples processed by each protocol. Both approaches achieved an identification success rate of 93.33%. Rapid AST using the SST method showed error rates of 0.51% minor errors, 0.57% major errors, and 0.23% very major errors, with an overall agreement of 98.69%. The shortened cultivation protocol demonstrated lower error rates (0.46% minor errors and 0.23% major errors) and an overall agreement of 99.31%. These findings confirm that MALDI-TOF MS enables reliable early identification of BSI pathogens and rapid AST, supporting timely optimization of antimicrobial therapy and early detection of multidrug-resistant strains.