Four-Hour Immunochromatographic Detection of Intestinal Carriage of Carbapenemase-Producing Enterobacteriaceae: a Validation Study.

The increasing incidence of carbapenemase-producing Gram-negative bacilli (C-PGNB) represents a major public health challenge. Rapid detection of digestive colonization with C-PGNB is fundamental to control their spread. We performed the validation of a rapid protocol for C-PGNB detection directly on rectal swabs. We developed a protocol combining enrichment by a rapid selective subculture of the rectal swab medium and realization of a Resist-4 O.K.N.V. K-SeT test on the bacterial pellet obtained. The limit of detection and performances of this protocol were validated in vitro on 52 C-PGNB strains spiked on a calibrated sample suspension and confirmed in clinical settings on 144 rectal swabs sampled from patients with C-PGNB digestive colonization (n = 48) and controls (patients with extended-spectrum beta-lactamase [ESBL] colonization [n = 48] and without carbapenemase/ESBL [n = 48]). The protocol detected, with 100% sensitivity, the presence of the 15 OXA-48-, 14 KPC-, 13 NDM-, and 10 VIM-producing GNB from 103 CFU/ml. The limit of detection was 2 × 102 CFU/ml. Among the 48 C-PGNB-containing rectal swabs of the validation cohort, 46 were accurately detected. False negative were observed for 1 NDM-producing Acinetobacter baumannii strain and 1 OXA-48-producing Escherichia coli strain. The 96 control swabs were negative. Sensitivity and specificity for C-PGNB detection were 97.7% (95% confidence interval [CI], 87.7 to 100) and 100% (95% CI, 96.2 to 100). The negative likelihood ratio was 0.04 (95% CI, 0.01 to 0.16). Considering a C-PGNB digestive colonization prevalence between 0.01% and 0.1%, positive and negative predictive values were 100%. Our protocol is a rapid and low-cost method detecting accurately the digestive colonization with carbapenemase-producing Enterobacteriaceae in 4 h without any requirement for specific equipment.

Emergence of Plasmid-Mediated Fosfomycin-Resistance Genes among Escherichia coli Isolates, France.

FosA, a glutathione S-transferase that inactivates fosfomycin, has been reported as the cause of enzymatic resistance to fosfomycin. We show that multiple lineages of FosA-producing extended spectrum ß-lactamase Escherichia coli have circulated in France since 2012, potentially reducing the efficacy of fosfomycin in treating infections with antimicrobial drug-resistant gram-negative bacilli.

Evaluation of early antimicrobial therapy adaptation guided by the BetaLACTA® test: a case-control study.

BACKGROUND: Rapid diagnostic tests detecting microbial resistance are needed for limiting the duration of inappropriateness of empirical antimicrobial therapy (EAT) in intensive care unit patients, besides reducing the use of broad-spectrum antibiotics. We hypothesized that the betaLACTA® test (BLT) could lead to early increase in the adequacy of antimicrobial therapy. METHODS: This was a case-control study. Sixty-one patients with BLT-guided adaptation of EAT were prospectively included, and then matched with 61 "controls" having similar infection characteristics (community or hospital-acquired, and source of infection), in whom EAT was conventionally adapted to antibiogram results. Endpoints were to compare the proportion of appropriate (primary endpoint) and optimal (secondary endpoint) antimicrobial therapies with each of the two strategies, once microbiological sample culture results were available. RESULTS: Characteristics of patients, infections and EAT at inclusion were similar between groups. Nine early escalations of EAT occurred in the BLT-guided adaptation group, reaching 98% appropriateness vs. 77% in the conventional adaptation group (p < 0.01). The BLT reduced the time until escalation of an inappropriate EAT from 50.5 (48-73) to 27 (24-28) hours (p < 0.01). Seventeen early de-escalations occurred in the BLT-guided adaptation group, compared to one in the conventional adaptation group, reducing patients' exposure to broad-spectrum beta-lactam such as carbapenems. In multivariate analysis, use of the BLT was strongly associated with early appropriate (OR = 18 (3.4-333.8), p = 0.006) and optimal (OR = 35.5 (9.6-231.9), p < 0.001) antimicrobial therapies. Safety parameters were similar between groups. CONCLUSIONS: Our study suggests that a BLT-guided adaptation strategy may allow early beta-lactam adaptation from the first 24 hours following the beginning of sepsis management.

The ß-Lacta test for direct detection of extended-spectrum-ß-lactamase-producing Enterobacteriaceae in urine.

With the ß-Lacta test, production of extended-spectrum ß-lactamases (ESBLs) was assayed in 200 urine samples showing Gram-negative bacilli during direct microscopic examination. While 168 samples tested negative, all samples yielding ESBL-producing Enterobacteriaceae after culture gave positive (n = 30) or uninterpretable (n = 2) results. The sensitivity and specificity of ESBL detection were 94% and 100%, respectively.

Five-Hour Detection of Intestinal Colonization with Extended-Spectrum-ß-Lactamase-Producing Enterobacteriaceae Using the ß-Lacta Phenotypic Test: the BLESSED Study.

Extended-spectrum-ß-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-PE) intestinal colonization is of particular concern as it negatively impacts morbidity and is the main source of external cross-contamination in hospitalized patients. Contact isolation strategies may be caught out due to the turnaround time needed by laboratories to report intestinal colonization, during which patients may be inappropriately isolated or not isolated. Here, we developed a protocol combining enrichment by a rapid selective subculture of rectal swab medium and realization of a ß-Lacta test on the obtained bacterial pellet (named the BLESSED protocol). The performances of this protocol were validated in vitro on 12 ESBL-PE strains spiked into calibrated sample suspensions and confirmed in clinical settings using 155 rectal swabs, of which 23 (reference method) and 31 (postenrichment broth culture) came from ESBL-PE carriers. In vitro, the protocol detected, with 100% sensitivity, the presence of the 12 ESBL-PE strains from 104 CFU/mL. In the clinical validation cohort, 22 out of the 23 (reference method) and 28 out of the 31 (postenrichment broth culture) ESBL-PE-positive rectal samples were accurately detected. The diagnostic performances for ESBL-PE detection, considering all ESBL-PE carriers, were 90% sensitivity, 98% specificity, an 87% positive predictive value, and a 98% negative predictive value. Our protocol is a rapid and low-cost method that can detect intestinal colonization with ESBL-PE in less than 5 h more accurately than the reference method, opening the field for further studies assessing a rapid and targeted isolation strategy applied only to patients with a positive BLESSED protocol result. IMPORTANCE To both improve the efficiency of contact isolation among ESBL-PE carriers and avoid the unnecessary isolation of noncolonized patients, we should reduce the turnaround time of ESBL screening in laboratories and improve the sensitivity of diagnostic methods. The development of rapid and low-cost methods that satisfy these two goals is a promising approach. In this study, we developed such a technique and report its good diagnostic performance, opening the door for further studies assessing a rapid and targeted isolation strategy applied in a few hours only for patients truly colonized with ESBL-producing bacteria.

Differential Performance of the FilmArray Meningitis/Encephalitis Assay To Detect Bacterial and Viral Pathogens in Both Pediatric and Adult Populations.

Meningitis/encephalitis (ME) syndromic diagnostic assays can be applied for the rapid one-step detection of the most common pathogens in cerebrospinal fluid (CSF). However, the comprehensive performance of multiplex assays is still under evaluation. In our multisite university hospital of eastern Paris, France, ME syndromic testing has been gradually implemented since 2017 for patients with neurological symptoms presenting to an adult or pediatric emergency unit. We analyzed the results from the BioFire FilmArray ME panel versus standard routine bacteriology and virology techniques, together with CSF cytology and clinical data, over a 2.5-year period to compare the diagnostic accuracy of the FilmArray ME panel to that of the reference methods. In total, 1,744 CSF samples from 1,334 pediatric and 336 adult patients were analyzed. False-positive (mostly bacterial) and false-negative (mostly viral) cases were deciphered with the help of clinical data. The performance of the FilmArray ME panel in our study was better for bacterial detection (specificity >99%, sensitivity 100%) than viral detection (specificity >99%, sensitivity 75% for herpes simplex virus 1 [HSV-1] and 89% for enterovirus), our study being one of the largest, to date, concerning enteroviruses. The use of a threshold of 10 leukocytes/mm3 considerably increased the positive agreement between the results of the FilmArray ME panel and the clinical features, especially for bacterial pathogens, for which agreement increased from 58% to 87%, avoiding two-thirds of inappropriate testing. Based on this analysis, we propose an algorithm for the use of both syndromic and specific assays for the optimal management of suspected meningitis/encephalitis in adult and pediatric patients. IMPORTANCE Based on our comparative analysis of performances of the diagnostic assays, we propose an algorithm for the use of both syndromic and specific assays, for an optimal care of the meningitis/encephalitis threat in adult and pediatric patients.

Revisiting Species Identification within the Enterobacter cloacae Complex by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is commonly used by clinical microbiology laboratories to identify pathogens, despite some limitations of the technique. The Enterobacter cloacae complex (ECC) taxonomy has recently been expanded, leading to uncertain identification of some species within the ECC when commercial MALDI-TOF MS is used. This technique is especially unsuited in the case of E. hormaechei, the main species responsible for infections and one of the most prone, within the ECC, to acquire antibiotic resistance. Hence, rapid and reliable identification at the species level could improve patient management. Here, we evaluated the performance of the Bruker Microflex MALDI-TOF MS instrument to identify ECC isolates using two databases and algorithms in comparison to the hsp60 gene sequencing reference method: the Bruker database included in the MALDI Biotyper software and an extensive online database coupled to an original Mass Spectrometric Identification (MSI) algorithm. Among a panel of 94 ECC isolates tested in triplicate, the online database coupled to MSI software allowed the highest rate of identification at the species level (92%) compared to the MALDI Biotyper database (25%), especially for the species E. hormaechei (97% versus 20%). We show that by creating a database of MALDI-TOF reference spectral profiles with a high number of representatives associated with the performant MSI software, we were able to substantially improve the identification of the E. cloacae complex members, with only 8% of isolates misidentified at the species level. This online database is available through a free online MSI application (https://msi.happy-dev.fr/). IMPORTANCE Creation of a database of MALDI-TOF reference spectral profiles with a high number of representatives associated with the performant MSI software enables substantial improvement in identification of E. cloacae complex members. Moreover, this online database is available through a free online MSI application (https://msi.happy-dev.fr/).

Synergistic in vitro activity between aztreonam and amoxicillin-clavulanate against Enterobacteriaceae-producing class B and/or class D carbapenemases with or without extended-spectrum ß-lactamases.

Introduction. Carbapenemase-producing Enterobacteriaceae have become a major public health concern over the last decade and treatment options are limited.Aims. We evaluated the synergistic activity of the combination of aztreonam (ATM) and clavulanate for 41 ß-lactam-resistant clinical isolates harbouring class B or/and class D carbapenemases combined or not with extended-spectrum ß-lactamases (ESBLs).Methodology. The MICs of ATM, with and without amoxicillin-clavulanate (AMC), were determined. Time-kill assays were performed for three representative strains.Results. The ATM-AMC combination had a synergistic effect on 34/41 (83 %) isolates. The MIC of ATM, in the presence of clavulanate, was ≤1 mg l-1 for 15/41 (37 %) isolates and ≤4 mg l-1 for 29/41 (71 %) isolates. Synergistic activity was observed for 34/37 (92 %) isolates producing ESBLs and carbapenemases, compared to 0/4 (0 %) for ESBL-negative strains. Complete or partial bactericidal activity was obtained when the MIC of the combination was 0.5 mg l-1 and 1.5 mg l-1 or 8 mg l-1, respectively.Conclusion. The combination of ATM and AMC could be an attractive unconventional treatment for infections due to carbapenemase- and ESBL-producing Enterobacteriaceae.

Performance evaluation of the ßLACTA™ Test for rapid detection of ceftazidime resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients.

The chromogenic ßLACTA™ test was evaluated to detect ceftazidime resistance in P. aeruginosa isolates from patients with cystic fibrosis. Best results were obtained after one hour of incubation with low sensitivity (64.1%), high specificity (98.3%), and negative and positive predictive values of 80.3% and 96.2%, respectively.

Multicentre randomised controlled trial to investigate usefulness of the rapid diagnostic ßLACTA test performed directly on bacterial cell pellets from respiratory, urinary or blood samples for the early de-escalation of carbapenems in septic intensive care unit patients: the BLUE-CarbA protocol.

INTRODUCTION: The dramatic increase of the incidence of infections caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) has led to an increase of 50% of carbapenem consumption all around Europe in only 5 years. This favours the spread of carbapenem-resistant Gram-negative bacilli (GNB), causing life-threatening infections. In order to limit use of carbapenems for infections actually due to ESBL-PE, health authorities promote the use of rapid diagnostic tests of bacterial resistance. The objective of this work conducted in the intensive care unit (ICU) is to determine whether an early de-escalation of empirical carbapenems guided by the result of the ßLACTA test is not inferior to the reference strategy of de-escalating carbapenems after the antibiogram result has been rendered. METHODS AND ANALYSIS: This multicentre randomised controlled open-label non-inferiority clinical trial will include patients suffering from respiratory and/or urinary and/or bloodstream infections documented with GNB on direct examination and empirically treated with carbapenems. Empirical carbapenems will be adapted before the second dose depending on the results of the ßLACTA test performed directly on the microbiological sample (intervention group) or after 48-72 hours depending on the definite antibiogram (control group). The primary outcome will combine 90-day mortality and percentage of infection recurrence during the ICU stay. The secondary outcomes will include the number of carbapenems defined daily doses and carbapenem-free days after inclusion, the proportion of new infections during ICU stay, new colonisation of patients' digestive tractus with multidrug-resistant GNB, ICU and hospital length of stay and cost-effectiveness ratio. ETHICS AND DISSEMINATION: This protocol has been approved by the ethics committee of Paris-Ile-de-France IV, and will be carried out according to the principles of the Declaration of Helsinki and the Good Clinical Practice guidelines. The results of this study will be disseminated through presentation at scientific conferences and publication in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT03147807.