A novel flow cytometric assay for rapid detection of extended-spectrum beta-lactamases.

The rapid detection of extended-spectrum beta-lactamases (ESBLs) is a challenge for most clinical microbiology laboratories because inaccurate identification of ESBL producers has important clinical implications for both antibiotic treatment and infection control. The aim of our study was to develop a rapid detection assay of ESBL producers based upon flow cytometric analysis. Antimicrobial susceptibility testing followed by molecular characterization of bla(TEM) , bla(SHV) or bla(CTX-M) genes was performed on clinical isolates (41 ESBL positive and 20 ESBL negative) and isolates expressing well-characterized beta-lactamases, including ESBLs (n = 13), plasmid AmpCs (n = 3), oxacillinases (n = 5) and carbapenemases (n = 3). Additionally, two ATCC strains recommended by CLSI for susceptibility testing were used as controls. The flow cytometry analysis protocol involved an incubation of bacterial cells with different concentrations of ceftazidime (1, 2 and 4 mg/L) and cefotaxime (4, 8 and 16 mg/L) for 1 and 2 hours, in the presence and absence of clavulanic acid; subsequently, cells were stained with the fluorescent dye Bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4) (3)], a lipophilic anion able to diffuse across depolarized membranes. Additionally, CFU counts were performed. Susceptible isolates displayed increased fluorescence after 1 hour of incubation; conversely, the increase of the depolarized population was only observed after incubation with clavulanic acid associated with ceftazidime or cefotaxime in ESBL producers. An excellent correlation was obtained between the number of non-depolarized bacteria quantified by flow cytometry and by conventional CFU assays. A novel, accurate and fast flow cytometric assay is available to detect the presence of ESBLs.

[Positive bronchoalveolar lavage and quantitative cultures results in suspected late-onset ventilator associated penumonia evaluation--retrospective study]. / Resultados positivos do lavado broncoalveolar e das culturas quantitativas na suspeita da pneumonia tardia associada ao ventilador--estudo retrospectivo.

INTRODUCTION: Bronchoalveolar lavage (BAL) with quantitative cultures has been used in order to increase ventilator associated pneumonia (VAP) diagnosis specificity, although the accurate technique for this entity diagnosis remains controversial. OBJECTIVES: To evaluate the influence of using positive BAL and quantitative cultures results in microbiologic diagnosis and treatment of patients with suspected late VAP and prior antibiotherapy. MATERIAL AND METHODS: Retrospective analysis of intensive care unit (UCI) patients, during a one year period, with clinical suspicion of late VAP and prior use of antibiotics that presented a growth in BAL cultures. RESULTS: Of 243 BAL performed, there were 71 (29.2%) positive cultures (60 patients, 76.7% male, 54 ± 19 years). BAL was done after 13 days (median) of invasive mechanical ventilation, 11 days of ICU antibiotherapy and in the day in which a new antibiotic for VAP suspicion was started. Colony forming units (CFU)/ml count was performed in 71.8% and endotracheal aspirate (ETA) simultaneously collected for qualitative analysis in 85.9%. Therapeutic approach was changed in 38.0%: correction (16.9%), de-escalation (12.7%) and directed antibiotherapy start (8.4%). Therapeutic changes were made in the presence of CFU > 10(4) in 84.2% and in agreement with ETA in 70.8%. In cases in which antibiotherapy was maintained (62.0%), quantitative cultures would have allowed de-escalation in 9.1%. Changes in prescription were more frequent when CFU was > 10(4) (48.5%), comparing with situations in which counts were lower and BAL analysis was qualitative (28.9%), p = 0.091. There were no significant differences between patients submitted to different therapeutic approaches concerning to ICU mortality or length of stay. CONCLUSION: In late onset VAP, positive BAL and quantitative cultures allowed therapeutic changes, leading to antibiotic adequacy and consumption reduction, which can however be maximised.