Evaluation of the Reveal® AST (SPECIFIC) for Antimicrobial Susceptibility Testing from Positive Blood Culture Spiked with Carbapenem-Resistant Isolates.

As bloodstream infections and associated septic shock are common causes of mortality in hospitals, rapid antibiotic susceptibility testing (AST) performed directly on positive blood cultures is needed to implement an efficient therapy in clinical settings. We evaluated the Reveal® rapid AST system on a collection of 197 fully characterized carbapenem-resistant Enterobacterales, including 177 carbapenemase producers (CPE) spiked in blood culture bottles. The clinical categorization based on the Minimal Inhibitory Concentration (MIC) determination of eighteen antimicrobial molecules was compared to the clinical categorization based on the disk diffusion assay as a reference. The Reveal AST system provided results within a mean time to result of 5 h. Overall, the categorical agreement (CA) between the two techniques was 94.1%. The rates of very major errors (VMEs), major errors (MEs) and minor errors (mEs) were 3.8%, 3.7% and 5.6%, respectively. Imipenem was the antimicrobial with the lowest CA rate (78.7%), with rates of 15% VMEs and 10.7% MEs, but the performances were better when considering only the non-CPE category (CA of 89%). On this resistant collection of Enterobacterales with numerous acquired ß-lactamases, the Specific Reveal assay proved to be useful for a rapid determination of AST compatible with a quick adaptation of the patient's antimicrobial treatment.

Susceptibility Testing by Volatile Organic Compound Detection Direct from Positive Blood Cultures: A Proof-of-Principle Laboratory Study.

BACKGROUND: Bacteria produce volatile organic compounds (VOCs) during growth, which can be detected by colorimetric sensor arrays (CSAs). The SpecifAST® system (Specific Diagnostics) employs this technique to enable antibiotic susceptibility testing (AST) directly from blood cultures without prior subculture of isolates. The aim of this study was to compare the SpecifAST® AST results and analysis time to the VITEK®2 (bioMérieux) system. METHODS: In a 12-month single site prospective study, remnants of clinical positive monomicrobial blood cultures were combined with a series of antibiotic concentrations. Volatile emission was monitored at 37 °C via CSAs. Minimal Inhibitory Concentrations (MICs) of seven antimicrobial agents for Enterobacterales, Staphylococcus, and Enterococcus spp. were compared to VITEK®2 AST results. MICs were interpreted according to EUCAST clinical breakpoints. Performance was assessed by calculating agreement and discrepancy rates. RESULTS: In total, 96 positive blood cultures containing Enterobacterales, Staphylococcus, and Enterococcus spp. were tested (269 bug-drug combinations). The categorical agreement of the SpecifAST® system compared to the VITEK®2 system was 100% and 91% for Gram-negatives and Gram-positives, respectively. Errors among Gram-positives were from coagulase-negative staphylococci. Overall results were available in 3.1 h (±0.9 h) after growth detection without the need for subculture steps. CONCLUSION: The AST results based on VOC detection are promising and warrant further evaluation in studies with a larger sample of bacterial species and antimicrobials.

Performance of the Reveal Rapid Antibiotic Susceptibility Testing System on Gram-Negative Blood Cultures at a Large Urban Hospital.

Timely and effective antibiotic treatment is vital for sepsis, with increasing incidence of antimicrobial-resistant bacteremia driving interest in rapid phenotypic susceptibility testing. To enable the widespread adoption needed to make an impact, antibiotic susceptibility testing (AST) systems need to be accurate, enable rapid intervention, have a broad antimicrobial menu and be easy to use and affordable. We evaluated the Specific Reveal (Specific Diagnostics, San Jose, CA) rapid AST system on positive blood cultures with Gram-negative organisms in a relatively resistant population in a large urban hospital to assess its potential for routine clinical use. One hundred four randomly selected positive blood cultures (Virtuo; bioMérieux) were Gram stained, diluted 1:1,000 in Pluronic water, inoculated into 96-well antibiotic plates, sealed with the Reveal sensor panel, and placed in the Reveal instrument for incubation and reading. The MIC and susceptible/intermediate/resistant category was determined and compared to results from Vitek 2 (bioMérieux) for the 17 antimicrobials available and to Sensititre (Thermo Fisher) for 24 antimicrobials. Performance was also assessed with contrived blood cultures with 33 highly resistant strains. Reveal was in 98.0% essential agreement (EA) and 96.3% categorical agreement (CA) with Sensititre, with just 1.3% very major error (VME) and 97.0%/96.2%/1.3% EA/CA/VME versus Vitek 2. Reveal results for contrived highly resistant strains were equivalent, with EA/CA/VME of 97.7%/95.2%/1.0% with CDC/FDA Antibiotic Resistance Isolate Bank references. Average time to result (TTR) for Reveal was 4.6 h. Sample preparation was relatively low skill and averaged 3 min. We conclude that the Reveal system enables accurate and rapid susceptibility testing of Gram-negative blood cultures.

How to: ECOFFs-the why, the how, and the don'ts of EUCAST epidemiological cutoff values.

BACKGROUND: Identifying the MIC wild-type distribution and its delineation of species targeted for receiving antimicrobial agent breakpoints is an important first step for determining clinical breakpoints. Having the main responsibility in the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for characterizing the wild-type distributions and setting epidemiological cut-off values (ECOFFs), we explain the why, the how, and frequent misconceptions of wild-type MIC distributions and ECOFFs. OBJECTIVES: To clarify how wild-type MIC distributions and ECOFFs for agents and important target organisms are defined and determined and why these are important tools in microbiology, as well as to point to common misunderstandings and inappropriate use. SOURCES: The EUCAST database of >40 000 MIC distributions; publications addressing the definition of wild-type MIC distributions, and ECOFFs in bacteria and fungi; and the EUCAST Standard Operating Procedure 10 Documents published by the European Centre for Disease Control and the European Food Safety Agency. CONTENT: The rationale for defining wild-type distributions and ECOFFs is explained. Setting breakpoints that bisect wild-type MIC distributions leads to poor methodological reproducibility and poor correlation between clinical outcome and susceptibility testing results. The methods applied by EUCAST to select distributions for aggregation and website display are described, highlighting the importance of incorporating data from multiple sources and methods. The methods used by EUCAST to estimate ECOFFs are outlined. Finally, the common misunderstandings of these processes are addressed. IMPLICATIONS: The international community needs to agree on the phenotypic definitions of wild-type distributions. Systematic methods for developing and applying ECOFFs are essential to the conduct of phenotypic antimicrobial susceptibility testing and interpretation, which will remain the dominant laboratory method for the foreseeable future.

A Direct Rapid Phenotypic Antimicrobial Susceptibility Test Enables Early Selection of Optimal Antibiotics to Treat Bacteremia in COVID-19 Patients.

BACKGROUND: Co-infection with bacteria and severe acute respiratory syndrome coronavirus 2 may result in greater use of healthcare resources and a poor prognosis. Therefore, early selection and use of optimal antibiotics are essential. The direct rapid antibiotic susceptibility test (dRAST) can detect antibiotic resistance within 6 h of a Gram smear result. This study aimed to assess the effectiveness of dRAST for improving early selection of appropriate antibiotics for coronavirus disease 2019 (COVID-19) patients with bacteremia. MATERIALS AND METHODS: This retrospective study included 96 blood culture-positive COVID-19 patients. Bacterial isolates and antimicrobial resistance profiles of each case were evaluated. Cases were divided into two groups based on whether they underwent conventional antibiotic susceptibility test (AST) or dRAST. The time to optimal targeted treatment for the two groups was investigated and compared. In addition, we examined the proportion of cases for which appropriate antibiotics were selected and broad spectrum antibiotics were administered at 72 h from blood sample collection. RESULTS: The mean time to optimal targeted antibiotic treatment was shorter for the dRAST group [55.7; standard deviation (SD), 28.7 vs. 92.3; SD, 51.1 h; P = 0.041]. The proportion of cases receiving optimal targeted antibiotics 72 h after blood collection for culture was higher [6/10 (60.0%) vs. 10/25 (40.0%)] and the percentage receiving broad spectrum antibiotics at 72 h was lower [6/10 (60.0%) vs. 19/25 (76.0%)] in the dRAST group than in the conventional AST group. In terms of microbiology profile, the contamination rate was high (35.5%) and multidrug-resistant strains were common (63.2%) in COVID-19 patients with bacteremia. CONCLUSION: Application of dRAST for selection of antibiotics to treat bacteremia in COVID-19 patients may enable earlier and optimal treatment. The high incidence of contamination and resistant organisms in blood cultures from COVID-19 patients suggest that dRAST may speed up appropriate targeted treatment.

Enhanced antimicrobial stewardship based on rapid phenotypic antimicrobial susceptibility testing for bacteraemia in patients with haematological malignancies: a randomized controlled trial.

OBJECTIVES: Recently, rapid phenotypic antimicrobial susceptibility testing (AST) based on microscopic imaging analysis has been developed. The aim of this study was to determine whether implementation of antimicrobial stewardship programmes (ASP) based on rapid phenotypic AST can increase the proportion of patients with haematological malignancies who receive optimal targeted antibiotics during early periods of bacteraemia. METHODS: This randomized controlled trial enrolled patients with haematological malignancies and at least one positive blood culture. Patients were randomly assigned 1:1 to conventional (n = 60) or rapid phenotypic (n = 56) AST. The primary outcome was the proportion of patients receiving optimal targeted antibiotics 72 hr after blood collection for culture. RESULTS: The percentage receiving optimal targeted antibiotics at 72 hr was significantly higher in the rapid phenotypic AST group (45/56, 80.4%) than in conventional AST group (34/60, 56.7%) (relative risk (RR) 1.42, 95% confidence interval (CI) 1.09-1.83). The percentage receiving unnecessary broad-spectrum antibiotics at 72 hr was significantly lower (7/26, 12.5% vs 18/60, 30.0%; RR 0.42, 95% CI 0.19-0.92) and the mean time to optimal targeted antibiotic treatment was significantly shorter (38.1, standard deviation (SD) 38.2 vs 72.8, SD 93.0 hr; p < 0.001) in the rapid phenotypic AST group. The mean time from blood collection to the AST result was significantly shorter in the rapid phenotypic AST group (48.3, SD 17.6 vs 83.1, SD 22.2 hr). DISCUSSION: ASP based on rapid phenotypic AST can rapidly optimize antibiotic treatment for bacteraemia in patients with haematological malignancy. Rapid phenotypic AST can improve antimicrobial stewardship in immunocompromised patients.

Rapid microbial identification and phenotypic antimicrobial susceptibility testing directly from positive blood cultures: a new platform compared to routine laboratory methods.

The Accelerate Pheno™ System (APS), a new platform that combines rapid identification (ID) of bacteria and yeasts and phenotypic antimicrobial susceptibility testing (AST) in a single assay, has been evaluated directly from positive blood cultures in comparison to routine laboratory methods. The APS ID results showed an overall sensitivity and specificity of 92.6% and 99.6%, respectively. With regard to AST results, 31 discrepancies (8 single errors and 23 combined errors) were observed, including 13 major errors (3.3%) and 18 minor errors (4.6%) mainly involving Pseudomonas aeruginosa. No very major error was observed. The APS ID results were obtained in 1.5 h and the AST results were available in 7 h, on average 34.1 h before routine laboratory methods. This reduction in AST time-to-result represents one of the main advantages of this technology, reducing the time to provide to the physician the microbiological report.

Direct-qPCR Assay for Coupled Identification and Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae.

Multidrug-resistant gonorrhea has become an urgent issue for global public health. As the causative agent of gonorrhea, Neisseria gonorrhoeae, has been progressively developing resistance to nearly all prescribed antimicrobial drugs, monitoring its antimicrobial resistance on a broader scale has become a crucial agenda for effective antibiotic stewardship. Unfortunately, gold standard antimicrobial susceptibility testing (AST) relies on time and labor-intensive phenotypic assays, which lag behind the current diagnostic workflow for N. gonorrhoeae identification based on nucleic acid amplification tests (NAAT). Newer assay technologies based on NAAT can rapidly identify N. gonorrhoeae from clinical specimen but fundamentally lack the capacity to provide phenotypic AST information. Herein, we propose a direct-quantitative PCR (direct-qPCR) assay that enables pathogen-specific identification and phenotypic AST via quantitative measurement of N. gonorrhoeae growth directly from a liquid medium without any sample preprocessing. The assay has an analytical sensitivity of 102 CFU/mL and is highly specific to N. gonorrhoeae in the presence of urogenital flora and clinical swab eluent. We tested seven N. gonorrhoeae strains against three antibiotic agents, penicillin, tetracycline, and ciprofloxacin, and achieved 95.2% category agreement and 85.7% essential agreement with the FDA-approved E-test. The assay presented in this work has the unique ability to identify N. gonorrhoeae and provide phenotypic AST directly from the liquid medium with cell densities as low as 102 CFU/mL, demonstrating an accelerated, sensitive, and scalable workflow for performing both identification and AST of  N. gonorrhoeae.