Abstract Background Aztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility testing (AST) methods for evaluating the combination in clinical laboratories is lacking. We aimed to evaluate different ATM-AVI AST approaches. Methods 96 characterized carbapenem-resistant clinical isolates belonging to 9 Enterobacterales (EB; n = 80) and P. aeruginosa (PA; n = 16) species, including 90 carbapenemase producers and 72 strains resistant to both CAZ-AVI and ATM, were tested. Paper disk elution (DE; Bio-Rad) and E-test gradient strips stacking (SS; bioMérieux) were performed for the ATM + CAZ-AVI combination. MIC Test Strip (MTS; Liofilchem) was evaluated for ATM-AVI MIC determination. Results were interpreted applying ATM clinical breakpoints of the EUCAST guidelines and compared to the broth microdilution method (Sensititre, Thermofisher). Results According to broth microdilution method, 93% of EB and 69% of PA were tested susceptible to ATM-AVI. The synergistic effect of ATM-AVI was of 95% for EB, but of only 17% for PA. The MTS method yielded higher categorical and essential agreement (CA/EA) rates for both EB (89%/91%) and PA (94%/94%) compared to SS, where the rates were 87%/83% for EB and 81%/81% for PA. MTS and SS yielded 2 and 3 major discrepancies, respectively, while 3 very major discrepancies each were observed for both methods. Concerning the DE method, CA reached 91% for EB and 81% for PA, but high number of very major discrepancies were observed for EB (n = 6; 8%) and for PA (n = 3; 19%). Conclusions The ATM-AVI association displayed excellent in vitro activity against highly resistant clinical Enterobacterales strains. MTS method offers accurate ATM-AVI AST results, while the SS method might serve as better alternative then DE method in assessing the efficacy of ATM + CAZ-AVI combination. However, further investigation is needed to confirm the methods' ability to detect ATM-AVI resistance.

Hypervirulent Klebsiella pneumoniae (hvKp) raised concern worldwide. We studied 22 hvKp clinical invasive isolates referred to the Belgian national reference laboratory between 2014 and 2020. Sixty-four percent of the isolates expressed K2 capsular serotype and belonged to 7 different MLST lineages, while 32% expressed K1 (all belonging to ST23) and were associated with liver abscesses. Primary extra-hepatic infections were reported in 36% and sepsis for 95% of the patients with 30% of deaths. Improved clinical and microbiological diagnostics are required as hvKp may represent an underestimated cause of community-acquired invasive infections in Belgium.

The current reliable recommended test for coronavirus disease 2019 (COVID‐19) diagnosis is quantitative reverse‐transcription polymerase chain reaction (RT‐qPCR). Rapid antigen test devices could be useful as they are less expensive, faster without the need of specialized laboratories to perform the test. We report the performances of two rapid immunochromatographic antigen testing devices compared with RT‐qPCR for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) detection in nasopharyngeal samples. We carried out a lateral‐flow tests study on 401 nasopharyngeal swab samples from nonduplicated suspected COVID‐19 subjects. An equal volume of universal transport medium tubes‐containing samples (dilution ratio = 1:15) were added to the manufacturer's extraction buffer solution (dilution ratio = 1:2) and analyzed on BioSpeedia COVID19Speed‐Antigen Test and on Abbott Panbio™ COVID‐19 Ag Rapid Test, devices. Qualitative results were compared to those obtained by the RT‐qPCR (Allplex™ SARS‐CoV‐2 Assay Seegene). Based on our data, the overall sensitivity for BioSpeedia and Panbio devices was estimated at 65.5% and 75.0%, respectively. The sensitivity was greater for cycle threshold values less than 25 achieving 90.4 and 96.8 for BioSpeedia and Panbio devices, respectively. A perfect specificity of 100.0% was observed for both devices.

Objectives: Two commercially available lateral flow immunochromatographic assays (ICAs) for detection of the major carbapenemases were prospectively assessed for the detection of carbapenemases in Enterobacterales: RESIST-4 O.K.N.V. (Coris BioConcept) and NG-Test CARBA 5 (NG Biotech). Methods: These two assays were performed prospectively on consecutive Enterobacterales suspected of producing a carbapenemase that were referred to the Belgian National Reference Center for Monitoring Antimicrobial Resistance in Gram-Negative Bacteria between March and June 2018. The intensity of the band corresponding to a carbapenemase for each test was compared using ImageJ software. Results: Of the 161 isolates tested, a carbapenemase was detected in 91 (60 OXA-48-like, 15 VIM, 9 KPC, 5 NDM, 1 IMP and 1 IMP+ OXA-48); in the remaining 70, no carbapenemases were detected. For both tests, the results were 100% concordant with the results of the PCR-sequencing reference method. Two IMP producers were only detected by NG-Test CARBA 5 as IMP is not targeted by RESIST-4 O.K.N.V. The mean intensity of the OXA-48, VIM and NDM bands displayed by NG-Test CARBA 5 was 3 to 3.7 times higher than for RESIST-4 O.K.N.V., while the KPC band was on average 1.7 times more intense with RESIST-4 O.K.N.V. Conclusions: RESIST-4 O.K.N.V. and NG-Test CARBA 5 are two efficient assays for identification of the major carbapenemases. NG-Test CARBA 5 offers the advantage of detecting IMP, which remains rare inWestern countries.

OBJECTIVES:Accurate and fast identification of carbapenemase producers is essential for optimal patient management. Here, a new lateral flow immunochromatographic RESIST-4 K-SeT assay was assessed for the detection of carbapenemases in Enterobacteriaceae and non-fermenters.METHODS:The RESIST-4 K-SeT assay targets OXA-48-like, KPC, VIM and NDM, but not IMP carbapenemases. The assay was first evaluated using a collection of isolates with well-characterized resistance mechanisms to β-lactams (n = 134) and against an international external quality assessment carbapenemase panel (n = 8). The assay was then challenged prospectively using 345 consecutive, non-duplicate isolates including 279 Enterobacteriaceae and 66 non-fermenters (mostly Pseudomonas spp.) that were sent to the Belgian National Reference Centre for identification of the mechanisms related to carbapenem resistance.RESULTS:Globally, for the collection of retrospective and prospective clinical isolates (n = 479), the assay showed a sensitivity ranging from 99% for the detection of VIM to 100% for the detection of OXA-48-like, KPC and NDM carbapenemase-producing strains. The specificity was 100% for each carbapenemase and a perfect match in results was observed for the external quality assessment for the carbapenemases targeted by the assay.CONCLUSIONS:The RESIST-4 K-SeT assay is a valuable alternative for detection and identification of carbapenemases from culture isolates compared with the more costly molecular assays, which may also further require skilled staff and dedicated facilities.

Rapid identification and susceptibility testing results are of importance for the early appropriate therapy of bloodstream infections. The ePlex (GenMark Diagnostics) blood culture identification (BCID) panels are fully automated PCR-based assays designed to identify Gram-positive and Gram-negative bacteria, fungi, and bacterial resistance genes within 1.5 h from positive blood culture.

Objectives: To describe a novel plasmid‐borne class D carbapenemase (CHDL) named OXA‐427 identified in several Enterobacteriaceae clinical isolates from nine patients in one Belgian hospital. Methods: OXA‐427‐producing isolates were analysed by an electrochemical imipenem hydrolysis method (BYG Carba test), Carba NP test, conventional phenotypic assays and by molecular methods (PCR, whole sequencing of the OXA‐427‐encoding plasmid and cloning). The antimicrobial resistance profile of OXA‐427 was analysed by expression of the cloned gene in Escherichia coli DH10B and J53. Results: Eleven OXA‐427‐producing Enterobacteriaceae isolates of various species were identified from clinical specimens of nine patients between March 2012 and June 2014. OXA‐427 shares only 22%‐29% amino acid identity with OXA‐48‐like enzymes and other acquired CHDL (e.g. OXA‐23, ‐24/40 and ‐58 of Acinetobacter spp.). Conversely, it appeared closely related to the chromosomal class D &bgr;‐lactamase of Aeromonas media, Aeromonas hydrophila and Aeromonas sobria (99%, 89% and 77% of identity, respectively). When expressed in E. coli, OXA‐427 hydrolysed imipenem and conferred resistance to extended‐spectrum cephalosporins (mostly ceftazidime), penicillins including temocillin, and reduced susceptibility to carbapenems. The blaOXA‐427 gene was located in a 45 kb resistance island on a 177 kb IncA/C plasmid. Conclusions: OXA‐427 is a novel CHDL most closely related to chromosomal class D &bgr;‐lactamase of A. media WS. It confers resistance to penicillins, ceftazidime and aztreonam and in some instances to carbapenems. OXA‐427, which is not detectable by classical molecular tests, caused a protracted outbreak in one university hospital over a 2 year period.

Abstract Objectives: There is an urgent need for accurate and fast diagnostic tests capable of identifying carbapenemase producers. Here, we assessed the performance of a new multiplex lateral flow assay (OKN K-SeT) for the rapid detection of OXA-48-like, KPC and NDM carbapenemase-producing Enterobacteriaceae from culture colonies. Methods: Two hundred collection isolates with characterized β-lactamase content and 183 non-duplicate consecutive isolates referred to two National Reference Centres over a 2 month period in 2016 were used to evaluate the OKN K-SeT assay. Results: The assay correctly detected all 42 OXA-48-like-, 27 KPC- and 30 NDM-producing isolates from the collection panel, including 7 isolates that co-produced NDM and OXA-181 carbapenemases. No cross-reactivity was observed with non-targeted carbapenemases (n = 41) or with non-carbapenemase producers (n = 60). Prospectively, all OXA-48-like (n = 69), KPC (n = 9) and NDM (n = 19) carbapenemase-producing Enterobacteriaceae isolates were correctly detected, while 11 carbapenemase producers not targeted by the assay went undetected [VIM (n = 8) and OXA-23/OXA-58-like (n = 3)]. Overall, the sensitivity and specificity of the assay were 100%. Conclusions: The OKN assay is efficient, rapid and easy to implement in the workflow of a clinical microbiology laboratory for the confirmation of OXA-48, NDM and KPC carbapenemases. This test represents a powerful diagnostic tool as it enables the rapid detection of the most clinically important carbapenemases without the need for more costly and less frequently available molecular assays.

Rapid detection and confirmation of carbapenemases remains very challenging for diagnostic laboratories. The objective of this study was to assess the performance of two new immunochromatographic (IC) commercial assays for the rapid detection of OXA-48-producing and KPC-producing Enterobacteriaceae in pure bacterial isolates. A panel of 92 bacterial isolates predominantly including carbapenem-non-susceptible Enterobacteriaceae with previously defined carbapenem resistance mechanisms was tested. Then, 342 consecutive carbapenem-non-susceptible Enterobacteriaceae isolates referred to the reference laboratory were investigated prospectively in parallel with other phenotypic tests and with multiplex PCR and sequencing as the gold standard. In the collection panel, each of the two IC assays correctly detected all 30 OXA-48-like-producing isolates and 25 KPC-producing isolates, whatever the species, their association with other beta-lactamases and the level of resistance to carbapenems. All other carbapenemase producers and all non-carbapenemase-producing isolates yielded negative results with both tests. In the prospective evaluation, all OXA-48-like-producing Enterobacteriaceae isolates (naEuroS=aEuroS130) and KPC-producing Enterobacteriaceae isolates (naEuroS=aEuroS33) were correctly detected by the individual IC assays, while 179 non-OXA-48-like-producing and non-KPC-producing strains (137 non-carbapenemase producers and 42 isolates belonging to other carbapenemase family types) yielded negative results. Thus, each assay yielded 100% sensitivity and 100% specificity for the detection of OXA-48-like or KPC enzymes, respectively, at 15 min. The two IC assays allow rapid and reliable direct confirmation of OXA-48 and KPC carbapenemases from culture colonies and appear to be very useful additions to the existing tests, obviating the need for more costly characterization by molecular amplification methods.

•A DNA array targeting the most relevant β-lactamases in Enterobacteriaceae and non-fermenters.•Makes the distinction between ESBL and non-ESBL variants of TEM and SHV.•Makes the distinction between ESBL and carbapenemase variants of GES.•Also detects CTX-M, BEL, VEB, PER and carbapenemases of KPC, OXA-48, VIM, NDM, IMP, GIM, SPM, and OXA-23, -24 and -58 types.•Very adapted tool for epidemiological studies on clinical isolates.