An improved protocol for bacteria identification by MALDI-TOF MS directly from positive blood cultures.

FASTinov® developed a rapid antimicrobial susceptibility test that includes the purification of a bacterial suspension directly from positive blood cultures (BC). In order to streamline laboratory workflow, the use of the bacterial suspension obtained through FASTinov® sample prep was tested for identification (ID) by matrix absorption laser deionization-time of flight mass spectrometry (MALDI-TOF MS) (Bruker) in 364 positive BC, and its accuracy assessed comparing with the MALDI-TOF MS ID of the next-day subcultured colonies. FASTinov sample prep was highly reliable for rapid ID directly from BC with proportion of agreement of 94.9% for Gram-positive and 96.3% for Gram-negative bacteria.

Agreement between corneal diameter measurements obtained with an optical biometer and a Placido-based topographer / Acordo de medições do diâmetro da córnea obtidas por um biômetro óptico e um topógrafo com base em Plácido

ABSTRACT Purpose: The purpose of this study was to compare the white-to-white distance measurements of two devices (IOL Master 500 and Atlas corneal topographer) commonly used in clinical practice to determine if they were interchangeable. Providing information on instrument interchangeability could eliminate several unnecessary tests and consequently reduce the economic burden for the patient and society. Methods: In this prospective, comparative case series, the white-to-white distance was measured by independent examiners using the Atlas topographer (Carl Zeiss Meditec) and the IOL Master 500 (Carl Zeiss Meditec). One eye each of 184 patients was tested. Statistical analyses were performed using a paired t-test, Pearson correlation analysis, and Bland-Altman analysis to compare the measurement methods. Results: The mean white-to-white distance measurements with the Atlas topographer and the IOL Master 500 were 12.20 ± 0.44 mm and 12.12 ± 0.41 mm, respectively (p<0.001). The mean white-to-white difference between the two devices was 0.07 mm (95% confidence interval of mean difference: 0.04-0.11 mm). The Pearson correlation coefficient between the two devices was 0.85 (p<0.0001). The 95% limits of agreement between the two devices were -0.38 mm to 0.53 mm. Conclusions: The Atlas topographer and IOL Master 500 can be used interchangeably with respect to white-to-white distance measurements, as the range of differences is unlikely to affect clinical practice and decision making.

RESUMO Objetivo: O objetivo deste estudo é comparar as medições de diâmetro corneano de dois dispositivos normalmente utilizados na prática clínica (IOL Master 500 e Atlas topógrafo corneal) para ver se são permutáveis. O fornecimento de informações sobre a permutabilidade de instrumentos poderia eliminar vários testes desnecessários e, consequentemente, reduzir a carga econômica para o paciente e para a sociedade. Métodos: Nesta série de casos prospectivos e comparativos, a distância do diâmetro corneano foi medida por examinadores independentes utilizando o Topógrafo Atlas (Carl Zeiss Meditec) e o IOL Master 500 (Carl Zeiss Meditec), em um olho de 184 pacientes. A análise estatística foi realizada utilizando o teste t pareado, a correlação Pearson e a análise Bland-Altman para comparar os métodos de medição. Resultados: As medições médias da distância do diâmetro corneano com o topógrafo Atlas e o IOL Master 500 foram de 12,20 ± 0,44 mm e 12,12 ± 0,41 mm, respectivamente (p<0,001). A diferença média de WTW entre os dois dispositivos foi de 0,07 mm (intervalo de confiança de 95% da diferença média: 0,04 - 0,11 mm). O coeficiente de correlação Pearson entre os dois dispositivos foi de 0,85, p<0,0001. Os limites de concordância de 95% entre os dois dispositivos foram de -0,38 mm a 0,53 mm. Conclusões: O Atlas topographer e o IOL Master 500 podem ser utilizados permutavelmente em relação à medição do diâmetro corneano, uma vez que a gama de diferenças encontradas é pouco susceptível de afetar a prática clínica e a tomada de decisões.

Rapid Detection of Plasmid AmpC Beta-Lactamases by a Flow Cytometry Assay.

Plasmidic AmpC (pAmpC) enzymes are responsible for the hydrolysis of extended-spectrum cephalosporins but they are not routinely investigated in many clinical laboratories. Phenotypic assays, currently the reference methods, are cumbersome and culture dependent. These methods compare the activity of cephalosporins with and without class C inhibitors and the results are provided in 24-48 h. Detection by molecular methods is quicker, but several genes should be investigated. A new assay for the rapid phenotypic detection of pAmpC enzymes of the Enterobacterales group-I (not usually AmpC producers) based on flow cytometry technology was developed and validated. The technology was evaluated in two sites: FASTinov, a spin-off of Porto University (Portugal) where the technology was developed, and the Microbiology Department of Ramón y Cajal University Hospital in Madrid (Spain). A total of 100 strains were phenotypically screened by disk diffusion for the pAmpC with the new 2 h assay. Molecular detection of the pAmpC genes was also performed on discrepant results. Forty-two percent of the strains were phenotypically classified as pAmpC producers using disk diffusion. The percentage of agreement of the flow cytometric assay was 93.0%, with 95.5% sensitivity and 91.1% specificity. Our proposed rapid assay based on flow cytometry technology can, in two hours, accurately detect pAmpC enzymes.

Agreement between corneal diameter measurements obtained with an optical biometer and a Placido-based topographer.

PURPOSE: The purpose of this study was to compare the white-to-white distance measurements of two devices (IOL Master 500 and Atlas corneal topographer) commonly used in clinical practice to determine if they were interchangeable. Providing information on instrument interchangeability could eliminate several unnecessary tests and consequently reduce the economic burden for the patient and society. METHODS: In this prospective, comparative case series, the white-to-white distance was measured by independent examiners using the Atlas topographer (Carl Zeiss Meditec) and the IOL Master 500 (Carl Zeiss Meditec). One eye each of 184 patients was tested. Statistical analyses were performed using a paired t-test, Pearson correlation analysis, and Bland-Altman analysis to compare the measurement methods. RESULTS: The mean white-to-white distance measurements with the Atlas topographer and the IOL Master 500 were 12.20 ± 0.44 mm and 12.12 ± 0.41 mm, respectively (p<0.001). The mean white-to-white difference between the two devices was 0.07 mm (95% confidence interval of mean difference: 0.04-0.11 mm). The Pearson correlation coefficient between the two devices was 0.85 (p<0.0001). The 95% limits of agreement between the two devices were -0.38 mm to 0.53 mm. CONCLUSIONS: The Atlas topographer and IOL Master 500 can be used interchangeably with respect to white-to-white distance measurements, as the range of differences is unlikely to affect clinical practice and decision making.

Evaluation of FASTinov Ultrarapid Flow Cytometry Antimicrobial Susceptibility Testing Directly from Positive Blood Cultures.

The FASTinov flow cytometry kit, an ultrarapid antimicrobial susceptibility test, was directly evaluated on positive blood cultures (BC) at two sites: (i) FASTinov, S.A., in Porto, Portugal, using BC spiked with well-characterized bacteria, and (ii) Ramón y Cajal University Hospital in Madrid, Spain, using positive BC from patients. Two kits were evaluated, FASTgramneg (Enterobacterales, Pseudomonas, Acinetobacter) and FASTgrampos (Staphylococcus, Enterococcus). Dedicated software for cytometric data analysis and interpretative reporting, including both CLSI and EUCAST criteria, was used. The FASTgramneg kit also provides information about the presence of resistant mechanisms, including extended-spectrum beta-lactamases (ESBLs) and carbapenemases. After 1 h of incubation at 37°C, bacteria were analyzed using a CytoFLEX cytometer (Beckman, CA). Disk diffusion was performed as the reference susceptibility method. Overall, 447 positive BC were included, 100 from hospitalized patients. Categorical agreement values for the FASTgramneg panel were 96.8% based on EUCAST criteria and 96.4% based on CLSI criteria. For the FASTgrampos panel, categorical agreement was 98.6% when using both criteria. When EUCAST criteria were used, the percentages of errors for the FASTgramneg panel were 2.1% minor errors (mE), 1.3% major errors (ME), and 0.6% very major errors (VME). When CLSI criteria were used, 2.9% mE, 0.9% ME, and 0.4% VME were found. VME were mainly observed with amoxicillin-clavulanate, cefotaxime, ceftazidime, and gentamicin. The FASTgrampos panel showed 0.3% mE, 1.4% ME, and 0.4% VME when EUCAST criteria were used (VME with respect to gentamicin and Staphylococcus) and 0.4% mE, 1.4% ME, and no VME when CLSI criteria were used. The FASTinov flow cytometry kits represent a rapid alternative for direct antimicrobial susceptibility testing from positive BC, showing time to results of <2 h, and can be used to personalize antibiotic and stewardship practices.

A Rapid Flow Cytometric Antimicrobial Susceptibility Assay (FASTvet) for Veterinary Use - Preliminary Data.

A rapid flow cytometric antimicrobial susceptibility test for bacteria isolated from companion animals - the FASTvet assay, developed by FASTinov®, was evaluated. Bacterial strains isolated from different biological samples of companion animals with infectious diseases in progress were obtained from several veterinary clinical laboratories across the country. A total of 115 strains, comprising 65 Gram-negative and 50 Gram positive isolates, were incubated with 13 antimicrobial drugs (ampicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, cefpodoxime, imipenem, enrofloxacin, gentamicin, amikacin for Gram-negative; penicillin, cefoxitin, enrofloxacin, vancomycin and ampicillin for Gram-positive) at breakpoint concentrations following CLSI protocol (CLSI Vet 01, 2018) for 1 h and analyzed by flow cytometry. The overall categorical agreement was 95.6% in case of Gram-negative and of 96.7% in Gram-positive isolates when compared to microdilution. FASTvet kits contribute to reduce the turnaround time (2 vs. 24 h) with early determination of the antimicrobial susceptibility profile. The correct and rapid choice of the target antibiotic therapy, will have a positive impact on animal care, contributing for preventing antimicrobial resistance. In conclusion, FASTinov® vet kits showed an excellent performance, both for Gram-negative and Gram-positive isolates encouraging us to enlarge the sample size and planning multicentric studies.

Ultra-rapid flow cytometry assay for colistin MIC determination in Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii.

OBJECTIVES: Both EUCAST and CLSI recommend broth microdilution for antimicrobial susceptibility testing of colistin, but this method is cumbersome and takes 16-24 h to give results. Our objective was to evaluate a rapid quantitative colistin MIC susceptibility assay based on flow cytometry analysis (FASTcolistin MIC) in comparison with standard broth microdilution assay. METHODS: One hundred and sixteen Gram-negative bacilli (78 Enterobacterales, 28 Pseudomonas aeruginosa and 10 Acinetobacter baumannii) were studied in parallel using standard broth microdilution following EUCAST recommendations and FASTcolistin MIC kit. In the last one, a bacteria suspension (0.5 MacFarland) was prepared, diluted in Muller-Hinton broth, incubated in the susceptibility panel containing different colistin concentrations (range 0.125-64 mg/L) with a fluorescent probe and incubated 1 h at 35ºC. After that, a flow cytometry analysis using CytoFLEX (Beckmam) was performed. Using a dedicated software (BioFAST) an automated MIC result was obtained after 1.5 h. Performance evaluation was performed according to the ISO standard 20776-2. Reproducibility and repeatability, categorical (CA) and essential agreement (EA), and lot-to-lot variation and operator-to-operator variability, as well as time to results were determined. RESULTS: Overall, 100% CA (CI 97-100%) and 95.7% EA (CI 90-98%) was obtained with high repeatability (100%; CI 80-100%)and reproducibility (97%; (CI 83-99%)). Absence of lot-to-lot variations or differences in the operators' performance was observed. CONCLUSIONS: FASTcolistin MIC is an accurate, reliable and ultra-rapid method (1 h incubation versus 24 h) for susceptibility testing of colistin of common Gram-negative bacilli recovered in clinical laboratories.

Evaluation of ultra-rapid susceptibility testing of ceftolozane-tazobactam by a flow cytometry assay directly from positive blood cultures.

The urgent need for rapid antimicrobial susceptibility is broadly apparent from government reports to the lay press. Accordingly, we developed a flow-cytometry assay (FCM) for evaluating ceftolozane-tazobactam (C/T) susceptibility directly on blood cultures (BC) requiring < 2 h from flag positivity to report. The protocol was optimized with C/T-susceptible and C/T-resistant gram-negative bacilli inoculated in BC aerobic bottles (Becton-Dickinson, USA), and afterward optimized for different C/T concentrations (1/4, 2/4, 4/4, and 8/4 mg/L) for 1 h incubation (37 °C), followed by FCM and software analysis. Fluorescent membrane permeability and membrane potential dyes were comparatively used to detect early cell lesions using the CytoFLEX cytometer (Beckman-Coulter, USA). Repeatability, reproducibility, and stability of the assay up to 48 h after BC positivity were determined. Internal validation was performed in spiked BC bottles with 130 Enterobacterales and 32 Pseudomonas aeruginosa isolates from Porto University (Portugal), including 13 ATCC isolates. Additionally, 64 gram-negative bacilli recovered from positive BC at Ramon y Cajal Hospital (Madrid, Spain) were tested. Categorical agreement (CA) and analytical errors were calculated comparing FCM with broth microdilution results. Only the membrane potential dyes clearly distinguished CT-susceptible and CT-resistant isolates. Excellent repeatability, reproducibility, and inter-method concordance was observed. Overall, CA was 99.1% using EUCAST criteria with 2 major errors and 98.7% with CLSI criteria with 2 major and 1 minor errors. A new, accurate, and ultra-rapid FCM (< 2 h) for testing C/T susceptibility gave accurate results and would expand current FCM antimicrobial susceptibility assay.