Commercial MIC Research Articles

Candida auris MIC testing by EUCAST and CLSI broth microdilution, and gradient diffusion strips; to be or not to be amphotericin B resistant?

ObjectivesReported amphotericin B resistance rates for Candida auris vary considerably. This may reflect clinically relevant differences in susceptibility, technical issues with testing or adoption of a clinical breakpoint (BP) that bisects the wild-type population. We compared reference methods and two gradient diffusion strips using a shared C. auris strain collection. MethodsForty C. auris strains from nine US states and ≥3 clades were included. Fourteen MIC data sets were generated using EUCAST E.Def 7.4, CLSI M27Ed4, Etest and MTS (Liofilchem) strip MICs. MICs ≤1 mg/L were classified as susceptible. ResultsEUCAST and CLSI amphotericin B MIC testing were robust across included method variables. The modal MIC was 1 mg/L, distributions unimodal and narrow with similar GM-MICs (0.745-1.072); however, susceptibility classification varied (0-28% resistance). Gradient diffusion strip testing resulted in wider and bimodal distributions for 8/9 data sets. If adopting, per manufacturer’s protocol, double inoculation for the Etest method, the modal MIC increased to 2-4 mg/L and resistance rates to 45-63% versus 25-30% with the single inoculation. The EUCAST, CLSI, Etest and MTS strip MICs correlated to the OD of drug-free control EUCAST wells suggesting that some isolates grew better than others and that this was associated with MIC. ConclusionsThe EUCAST and CLSI MIC results were in close agreement, whereas the strip test showed wider and bimodal distributions with reader to reader and centre to centre variation. Our study adds to the concern for commercial MIC testing of amphotericin B against C. auris and suggest the current breakpoint leads to random susceptibility classification.

Evaluation of the Reveal® rapid AST system to assess the susceptibility of Pseudomonas aeruginosa from blood cultures

This study was set up to assess the performance of the Reveal® rapid AST system to determine the drug susceptibility of Pseudomonas aeruginosa strains directly from blood cultures. Two hundred fully sequenced clinical P. aeruginosa strains were selected for the evaluation, of which 26.5% (n = 53) produced transferable β-lactamases, and 2.0 to 33.0% had susceptibility levels close to the EUCAST 2021 breakpoints of 11 commonly used antipseudomonal antibiotics. The Reveal® AST system was run with a commercial MIC microplate designed for fast-growing Gram-negative bacilli (Microscan Neg MDR MIC 1), and was compared to the manually operated GN6F MIC microdilution panel from Thermo Fisher, as a comparator method. The Reveal® AST system provided MIC results for the 11 antipseudomonal antibiotics tested within a mean time to result of 6h 22min. By comparison with the GN6F panel, the overall rates of categorical agreement (CA), very major errors (VME), major errors (ME), and minor errors (mE for meropenem only) were 96.1%, 1.6%, 4.2%, and 0.6%, respectively. The Specific Reveal® AST system appears to be a reliable and fast technology to determine the susceptibility of P. aeruginosa to antibiotics, including those with resistance levels near categorical breakpoints, directly from blood cultures.

Comparison between EUCAST Broth Microdilution and MIC Strip Test in Defining Isavuconazole In Vitro Susceptibility against Candida and Rare Yeast Clinical Isolates.

Isavuconazole is a new broad-spectrum triazole, with significant in vitro activity against yeasts. Isavuconazole in vitro susceptibility can be evaluated through broth microdilution as a reference method. Considering difficulties in equipping such methods in a laboratory routine, a commercial MIC Strip test has been designed. This study aims to implement data about isavuconazole in vitro activity and compare EUCAST broth microdilution and MIC Strip test in defining yeast isavuconazole susceptibility. The study involved 629 isolates from positive blood cultures (January 2017-December 2021). The identified species were C. albicans (283), C. glabrata (53), C. krusei (23), C. tropicalis (68), C. parapsilosis complex (151), C. guilliermondii (12), C. famata (6), S. cerevisiae (12), C. neoformans (5), S. capitata (12), and Rhodotorula species (4). All the isolates were tested with EUCAST microdilution and MIC Strip methods. The total essential agreement between these two methods was 99.3%. As a result, we can consider that both methods are useful in testing isavuconazole susceptibility. Proposed cut-off values (P-ECOFF) were calculated using ECOFFinder software. Further studies could lead to either definitive E-COFF or clinical breakpoints, which represent the most important categorization tool of the laboratory data, allowing a better insertion of an antimicrobial drug in clinical practice.

‘Breakpoint broth microdilution plate’ for susceptibility testing of Gram negative bacilli against colistin sulfate

The MIC method applicable to Gram negative bacilli including Acinetobacter spp. is broth microdilution (BMD). Cost and/or availability issues limit the use of commercial MIC panels in resource limited settings. ObjectivesTo design and implement an in-house breakpoint BMD panel (BBMD) for colistin against Gram negative bacilli. DesignBBMD panel was prepared in 96-well plate. MIC concentrations of 1, 2, & 4 ​μg/mL for test, and 0.25, 0.5, 1, 2 & 4 ​μg/mL for control strains were selected to accommodate 19 test and 3 quality control strains per plate. Plates were frozen at −80 ​°C until testing. Validation was performed using strains from a previously published study and compared with freshly prepared MIC panel of 16–0.03 ​μg/mL. ResultsValidation showed 100% agreement with the reference method and BBMD was introduced into routine laboratory practice for colistin susceptibility of carbapenem resistant Enterobacterales (CRE), Acinetobacter baumannii complex and Pseudomonas aeruginosa. From 2nd July-16th September 2018, a total of 1294 (mean 16.8 ​± ​5.5 isolates/day) clinical isolates were tested; 1157/1294 were reported (MIC ≤2 ​μg/mL) within 24-h, whereas 133 required resistance confirmation by full-range BMD. Resistance was confirmed for all but 24 isolates. These discrepancies were mostly due to contamination with bacterial genera inherently resistant to colistin. ConclusionThis BBMD plate is a high through-put and practical method that could reliably be utilized in a routine microbiology laboratory for colistin susceptibility testing of CRE, A. bauamanii complex and P. aeruginosa.

Antimicrobial susceptibility testing of colistin – evaluation of seven commercial MIC products against standard broth microdilution for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter spp.

ObjectiveBoth EUCAST and CLSI recommend broth microdilution (BMD) for antimicrobial susceptibility testing of colistin, but BMD is rarely used in routine microbiology laboratories. The objective of this study was to evaluate five commercially available BMD products and two brands of gradient tests for colistin MIC determination using BMD according to ISO standard 20776-1 as reference. MethodsColistin MIC determination was performed according to the manufacturer's instructions on five commercially available BMD products (Sensititre, MICRONAUT-S, MICRONAUT MIC-Strip, SensiTest, and UMIC) and two gradient tests (Etest and MIC Test Strip). Colistin reference MICs were determined using frozen panels according to ISO standard 20776-1. An international collection of Gram-negative bacteria (n=75) with varying levels of colistin susceptibility was tested. ResultsThe colistin BMD products correlated well with reference tests, in particular for Sensititre and the two MICRONAUT products (essential agreement ≥96%: 66/69 (96%, CI 88–99%), 72/75 (96%, CI 88–99%) and 74/75 (99%, CI 92–100%)). The results were somewhat poorer for the BMD products SensiTest and UMIC: EA 88% (51/58, CI 77–95%) and 82% (61/74, CI 72–89%), respectively), and considerably poorer for the gradient tests (EA 43–71% depending on gradient test and Mueller-Hinton agar manufacturer). The gradient tests generally underestimated colistin MICs, resulting in a significant number of false susceptible results (9–18 of total 75 tests, compared with 1–3 for the BMD products). ConclusionsBased on the results of this study, we advise laboratories not to trust gradient tests for colistin susceptibility testing and to use broth microdilution methods for this purpose. There are several commercial broth microdilution tests available and in principle they perform well.

Influencia de la correcta identificación en la interpretación de las pruebas de sensibilidad en aislados de Aeromonas spp. productoras de bacteriemia

ObjectiveTo assess the relevance of correct identification and interpretation of susceptibility testing of Aeromonas spp. bacteremia isolates using newly developed molecular methods in comparison to previous conventional methods. Material and methodsThe study included 22 patients with bacteremia due to Aeromonas hydrophila group, microbiologically characterized using the MicroScan system. Further identification to species level was performed by mass spectrometry, and confirmed by sequencing the rpoB gene.The MIC of imipenem, cefotaxime, piperacillin-tazobactam, ciprofloxacin and cotrimoxazole was studied using a commercial broth microdilution and antibiotic gradient strips with low and high inocula. Detection of carbapenemase production was performed using the modified Hodge test, and was confirmed by amplifying the cphA gene by PCR. ResultsA total of 9 (40.9%) isolates were identified as Aeromonas hydrophila, 8 (36.4%) as Aeromonas veronii, and the remaining 5 (22.7%) isolates as Aeromonas caviae.Resistance to beta-lactams according to both the commercial microdilution and MIC gradient strips methods was: 36%-50% to imipenem; 4%-56% to cefotaxime, and 27%-56% to piperacillin/tazobactam.The agreement between results generated by the automated system and the diffusion antibiotic gradient strip was, for all 3 species, 68% for imipenem, 50% to cefotaxime, and 46% to piperacillin/tazobactam.No resistance to cotrimoxazole and ciprofloxacin was found by either of the two methods, although 22.7% of the strains were resistant to nalidixic acid. ConclusionsIt is essential to identify the isolates of Aeromonas spp. at the species level, due to the fact that beta-lactam resistance is species- and method-dependent. The high rate of resistance to beta-lactam and quinolones reduce their application as empiric treatments for invasive infection by Aeromonas ssp.

Evaluation of Vancomycin Susceptibility Testing for Methicillin-Resistant Staphylococcus aureus: Comparison of Etest and Three Automated Testing Methods

We evaluated the ability of four commercial MIC testing systems (MicroScan, Vitek 2, Phoenix, and Etest) to detect vancomycin MIC values of ≤1 to ≥2 in 200 methicillin-resistant Staphylococcus aureus (MRSA) strains compared to the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference methods. Compared to the BMD method, absolute agreement (0 ± dilution) was highest for the Phoenix system (66.2%) and the MicroScan turbidity method (61.8%), followed by the Vitek 2 system (54.3%). The Etest produced MIC values 1 to 2 dilutions higher than those produced by the BMD method (36.7% agreement). Of interest, the MicroScan system (prompt method) was more likely to overcall an MIC value of 1 mg/liter (74.1%), whereas the Phoenix (76%) and Vitek 2 (20%) systems had a tendency to undercall an MIC of 2 mg/liter. The ability to correctly identify vancomycin MIC values of 1 and 2 has clinical implications and requires further evaluation.

Advances in Antifungal Susceptibility Testing of Candida, 2010–2012

Antifungal susceptibility testing of Candida has been standardized and refined and now may play an important role in managing Candida infections. Important new developments include the establishment of species-specific epidemiological cutoff values (ECVs) for the systemically active antifungal agents and both common and uncommon species of Candida. The clinical breakpoints (CBPs) for fluconazole, voriconazole, and the echinocandins have been revised to provide species-specific interpretive criteria for the six most common species that not only are predictive of clinical outcome but also provide a more sensitive means of identifying those strains with acquired or mutational resistance mechanisms. Collaborative work by the CLSI and EUCAST organizations has made major advances in the harmonization of these two international standards. The impact of the recent changes in the CBPs on commercial MIC methods does not appear to be major but additional studies with well defined resistant populations are necessary to confirm the ability of these systems to detect emerging resistance.

Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus

We compared the results obtained with six commercial MIC test systems (Etest, MicroScan, Phoenix, Sensititre, Vitek Legacy, and Vitek 2 systems) and three reference methods (agar dilution, disk diffusion, and vancomycin [VA] agar screen [VScr]) with the results obtained by the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference method for the detection of VA-intermediate Staphylococcus aureus (VISA). A total of 129 S. aureus isolates (VA MICs by previous BMD tests, <or=1 microg/ml [n = 60 strains], 2 microg/ml [n = 24], 4 microg/ml [n = 36], or 8 microg/ml [n = 9]) were selected from the Centers for Disease Control and Prevention strain collection. The results of BMD with Difco Mueller-Hinton broth were used as the standard for data analysis. Essential agreement (percent +/-1 dilution) ranged from 98 to 100% for all methods except the method with the Vitek Legacy system, for which it was 90.6%. Of the six commercial MIC systems tested, the Sensititre, Vitek Legacy, and Vitek 2 systems tended to categorize VISA strains as susceptible (i.e., they undercalled resistance); the MicroScan and Phoenix systems and Etest tended to categorize susceptible strains as VISA; and the Vitek Legacy system tended to categorize VISA strains as resistant (i.e., it overcalled resistance). Disk diffusion categorized all VISA strains as susceptible. No susceptible strains (MICs <or= 2 microg/ml) grew on the VScr, but all strains for which the VA MICs were 8 microg/ml grew on the VScr. Only 12 (33.3%) strains for which the VA MICs were 4 microg/ml grew on VScr. The differentiation of isolates for which the VA MICs were 2 or 4 microg/ml was difficult for most systems and methods, including the reference methods.

Validation of commercial dry-form broth microdilution panels and test reproducibility for susceptibility testing of dalbavancin, a new very long-acting glycopeptide

Results from dalbavancin dry-form commercial broth microdilution MIC panels (Sensititre, TREK Diagnostics) were compared with reference frozen-form MIC values to assure the validity and reproducibility of the extended shelf-life product. A collection of 402 organisms from four major organism groups were used in the validation trial and 10 strains for reproducibility replicate tests. A total of 98.6% commercial dalbavancin MIC results were within ±1 log 2 dilution of reference values (76.2% were identical) and reproducibility trials produced identical MIC results in 88.9–92.2% of dalbavancin MIC comparisons. These dalbavancin MIC results demonstrated the acceptable accuracy of commercially-prepared broth microdilution products for use in subsequent clinical trials.

Commercial broth microdilution panel validation and reproducibility trials for garenoxacin (BMS-284756), a novel desfluoroquinolone.

Results from garenoxacin dry-form broth microdilution MIC panels prepared commercially (Sensititre, TREK Diagnostics) were compared to reference frozen-form MICs to ensure the validity of the longer-shelf-life product. A total of 1078 organisms from seven major organism groups were used in this trial. All commercial MIC results were within +/- one log(2) dilution of reference garenoxacin values, and reproducibility trials produced identical MIC results for 90.5 to 92.1% of garenoxacin MIC comparisons. Control quinolones (ciprofloxacin and gatifloxacin) also performed at a similarly high level of accuracy.

Validation of commercial dry-form broth microdilution panels for susceptibility testing of AZD2563, a new long-acting oxazolidinone

The MIC results using a dry-form broth microdilution panel (TREK Diagnostic/Sensititre, Westlake, OH, USA) were validated for AZD2563, a novel oxazolidinone compound. In comparision studies against reference frozen-form panels, the commercial MIC results were the same as the reference calue for 82.7% of organisms and all results were within +/- one log2 dilution. Using 462 organisms, most from three genus groups (enterococci, staphylococci, streptococci), test results indicate that Sensititre MIC values were comparable to the reference test and can be utilized in clinical trials of for routine laboratory use when testing AZD2563 and linezolid, the drug class comparator.