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Evaluation of Rapid Immunochromatographic Tests for the Direct Detection of Extended Spectrum Beta-Lactamases and Carbapenemases in Enterobacterales Isolated from Positive Blood Cultures.

ABSTRACT: NG-Test CTX-M MULTI and NG-Test Carba 5 (NG Biotech) are two rapid in vitro immunochromatographic assays that are widely used for the detection of the most common extended spectrum beta-lactamases (ESBL) and carbapenemases in Enterobacterales. ESBL and carbapenemases are leading causes of morbidity and mortality worldwide and their rapid detection from positive blood cultures is crucial for early initiation of effective antimicrobial therapy in bloodstream infections (BSI) involving antibiotic-resistant organisms. In this study, we developed a rapid workflow for positive blood cultures for direct identification of Enterobacterales by MALDI-TOF mass-spectrometry, followed by detection of ESBL and carbapenemases using NG-Test CTX-M MULTI and NG-Test Carba 5 (NG Biotech). The workflow was evaluated using Enterobacterales isolates (n = 114), primarily Klebsiella species (n = 50) and Escherichia coli (n = 40). Compared to the standard testing approach in our institution using BD Phoenix, our new testing approach demonstrates 100% sensitivity and specificity for organism identification and detection of ESBL and carbapenemases. Implementation of a rapid workflow in diagnostic microbiology laboratories will enable more effective antimicrobial management of patients with BSI due to ESBL- and carbapenemase-producing Enterobacterales. IMPORTANCE The incidence of bloodstream infections (BSI) with extended spectrum beta-lactamase (ESBL) producing and carbapenemase producing Enterobacterales (CPE) is increasing at an alarming rate, for which only limited therapeutic options remain available. Rapid identification of these bacteria along with their antibiotic resistance mechanisms in positive blood cultures with Gram-negative bacteria will allow for early initiation of effective therapy and limit the overuse of broad-spectrum antibiotics in BSI (1). In this study we evaluated a combined approach of testing positive blood cultures directly, using MALDI-TOF MS followed by rapid immunochromatographic tests, for the detection of ESBLs and CPEs. Our approach demonstrates 100% sensitivity and specificity for the identification of Enterobacterales and detection of ESBLs and CPEs in positive blood culture with a turnaround time (TAT) of ≤60 min compared to a TAT of 48 h required by conventional culture and susceptibility testing methods.

SUBMITTER: Keshta AS

PROVIDER: S-EPMC8653814 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundFluoroquinolones have been, and continue to be, routinely used for treatment of many bacterial infections. In recent years, most parts of the world have reported an increasing trend of fluoroquinolone resistant (FQR) Gram-negative bacteria.MethodsA cross-sectional study was conducted between March 2017 and July 2018 among children admitted due to fever to referral hospitals in Dar es Salaam, Tanzania. Rectal swabs were used to screen for carriage of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE). ESBL-PE isolates were tested for quinolone resistance by disk diffusion method. Randomly selected fluroquinolone resistant isolates were characterized by using whole genome sequencing.ResultsA total of 142 ESBL-PE archived isolates were tested for fluoroquinolone resistance. Overall phenotypic resistance to ciprofloxacin, levofloxacin and moxifloxacin was found in 68% (97/142). The highest resistance rate was seen among Citrobacter spp. (100%, 5/5), followed by Klebsiella. pneumoniae (76.1%; 35/46), Escherichia coli (65.6%; 42/64) and Enterobacter spp. (31.9%; 15/47). Whole genome sequencing (WGS) was performed on 42 fluoroquinolone resistant-ESBL producing isolates and revealed that 38/42; or 90.5%, of the isolates carried one or more plasmid mediated quinolone resistance (PMQR) genes. The most frequent PMQR genes were aac(6')-lb-cr (74%; 31/42), followed by qnrB1 (40%; 17/42), oqx, qnrB6 and qnS1. Chromosomal mutations in gyrA, parC and parE were detected among 19/42 isolates, and all were in E. coli. Most of the E. coli isolates (17/20) had high MIC values of > 32 µg/ml for fluoroquinolones. In these strains, multiple chromosomal mutations were detected, and all except three strains had additional PMQR genes. Sequence types, ST131 and ST617 predominated among E. coli isolates, while ST607 was more common out of 12 sequence types detected among the K. pneumoniae. Fluoroquinolone resistance genes were mostly associated with the IncF plasmids.ConclusionThe ESBL-PE isolates showed high rates of phenotypic resistance towards fluoroquinolones likely mediated by both chromosomal mutations and PMQR genes. Chromosomal mutations with or without the presence of PMQR were associated with high MIC values in these bacteria strains. We also found a diversity of PMQR genes, sequence types, virulence genes, and plasmid located antimicrobial resistance (AMR) genes towards other antimicrobial agents.

Project description:Introduction: Antibiotic resistant bacteria are a growing concern worldwide. Extended-spectrum β-lactamases (ESBL) represent the most common resistance mechanism of Gram-negative bacteria against β-lactams, underlining the need for adequate diagnostic methods that provide reliable information in the shortest time possible. AmpC, a less prevalent but increasingly relevant class of β-lactamases, pose an additional challenge as their detection is complex. Here, we present an ESBL and AmpC screening panel employing the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA). Materials and Methods: Four reference strains recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used to develop the panel, which was further validated on 50 clinical Enterobacterales isolates resistant to third generation cephalosporins. The panel relies on the synergistic effect between ESBL and/or AmpC β-lactamase inhibitors and cephalosporins, which indicates β-lactamase production. Microdroplets containing the tested microorganism, cephalosporins in different concentrations and inhibitors were pipetted onto an MBT Biotarget and incubated for 3 or 4 h at 35 ± 1°C. Afterward, the liquid medium was removed and the material adhered to the spots was analyzed by MALDI-TOF MS. Synergy was detected by determining and comparing the minimum inhibitory concentrations of the tested cephalosporins with and without β-lactamase inhibitors. Data were interpreted following a diagnostic algorithm proposed by EUCAST in order to establish a final diagnosis. In comparison, PCR, broth microdilution (BMD) and combination disk tests (CDT) were performed. Results: Compared to the PCR results, the following positive and negative percent agreement values (PPA/NPA) were obtained for each resistance mechanism: ESBL, 94.44/100%; AmpC, 94.44/93.75% and ESBL+AmpC, 100/100%. These results, obtained after 4 h of incubation, were comparable with those of BMD and showed a higher accuracy than CDT. Discussion: We propose a novel phenotypic method for detection of ESBL and AmpC β-lactamases in Enterobacterales that provides reliable results in a short time, representing a promising alternative to the diagnostic techniques currently available. This easy-to-perform approach has potential for being implemented in routine laboratories, contributing to the further diversification of mass spectrometry technology into other fields such as antibiotic resistance testing.

Dataset Information

Evaluation of Rapid Immunochromatographic Tests for the Direct Detection of Extended Spectrum Beta-Lactamases and Carbapenemases in Enterobacterales Isolated from Positive Blood Cultures.

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