Categorical Agreement Rates Research Articles

Comprehensive pathogen identification and antimicrobial resistance prediction from positive blood cultures using nanopore sequencing technology

BackgroundBlood cultures are essential for diagnosing bloodstream infections, but current phenotypic tests for antimicrobial resistance (AMR) provide limited information. Oxford Nanopore Technologies introduces nanopore sequencing with adaptive sampling, capable of real-time host genome depletion, yet its application directly from blood cultures remains unexplored. This study aimed to identify pathogens and predict AMR using nanopore sequencing.MethodsIn this cross-sectional genomic study, 458 positive blood cultures from bloodstream infection patients in central Taiwan were analyzed. Parallel experiments involved routine microbiologic tests and nanopore sequencing with a 15-h run. A bioinformatic pipeline was proposed to analyze the real-time sequencing reads. Subsequently, a comparative analysis was performed to evaluate the performance of species identification and AMR prediction.ResultsThe pipeline identified 76 species, with 88 Escherichia coli, 74 Klebsiella pneumoniae, 43 Staphylococcus aureus, and 9 Candida samples. Novel species were also discovered. Notably, precise species identification was achieved not only for monomicrobial infections but also for polymicrobial infections, which was detected in 23 samples and further confirmed by full-length 16S rRNA amplicon sequencing. Using a modified ResFinder database, AMR predictions showed a categorical agreement rate exceeding 90% (3799/4195) for monomicrobial infections, with minimal very major errors observed for K. pneumoniae (2/186, 1.1%) and S. aureus (1/90, 1.1%).ConclusionsNanopore sequencing with adaptive sampling can directly analyze positive blood cultures, facilitating pathogen detection, AMR prediction, and outbreak investigation. Integrating nanopore sequencing into clinical practices signifies a revolutionary advancement in managing bloodstream infections, offering an effective antimicrobial stewardship strategy, and improving patient outcomes.

Comparative evaluation of eravacycline susceptibility testing methods in 587 clinical carbapenem-resistant Acinetobacter baumannii isolates: broth microdilution, MIC test strip and disc diffusion.

The study aimed to evaluate the accuracy of different methods for determining carbapenem-resistant Acinetobacter baumannii (CRAB) susceptibility to eravacycline. We collected 587 CRAB strains from Huashan Hospital affiliated to Fudan University between 2019 and 2023. The broth microdilution (BMD) method served as the reference standard. The susceptibility results were evaluated using the clinical breakpoints established by the Chinese Committee on Antimicrobial Susceptibility Testing (ChinaCAST) (susceptible MIC, ≤ 1 mg/L; inhibition zone diameter, ≥ 15 mm). The study compared the reliability of the MIC test strip (MTS) and disc diffusion (DD) methods in detecting CRAB susceptibility to eravacycline. The MICs required to inhibit 50% and 90% of CRAB growth were as follows: BMD, 0.5/1 mg/L; MTS, 0.38/0.75 mg/L. According to the ChinaCAST breakpoints, the BMD method demonstrated a 98.13% (576/587) susceptibility rate, whereas the MTS and DD methods showed susceptibility rates of 97.96% (575/587) and 97.61% (573/587), respectively. The essential agreement rate between the MTS and BMD methods was 94.55%. Categorical agreement (CA) rates for the MTS and DD methods were 99.83% and 99.49%, respectively. The major error (ME) rate for MTS was 0.17%, with no very major errors (VMEs) observed. For the DD method, the ME rate was 0.51%, also with no VMEs. The MTS and DD methods demonstrated strong consistency with the BMD reference method, with CA, ME and VME rates meeting methodological evaluation criteria. Both MTS and DD methods are reliable alternatives for assessing the antibacterial activity of eravacycline in clinical microbiology laboratories.

Evaluation of several routine methods for fosfomycin and mecillinam susceptibility testing of Enterobacterales urine isolates.

Performance evaluation of routine laboratory methods to determine the susceptibility of Enterobacterales urinary isolates to fosfomycin (oral administration) and mecillinam. We collected 347 Enterobacterales isolates from monomicrobial midstream urine samples from women with significant bacteriuria and leukocyturia. Mostly non-Escherichia coli isolates (i.e. Klebsiella spp., Citrobacter koseri, Enterobacter cloacae complex and Proteus mirabilis) were included (n = 298). Performance of VITEK®2, ETEST®, and disc diffusion to determine fosfomycin and mecillinam susceptibility was evaluated following International Organization for Standardization (ISO) 20776-2:2021 (or 20776-2:2007 for disc diffusion) in comparison with the agar dilution reference method. For fosfomycin testing, VITEK®2 and ETEST® were close to reaching ISO requirements (essential agreement ≥ 90%; bias ±30%) for C. koseri, E. coli and P. mirabilis. Categorical agreement (CA) and major error rates were acceptable for disc diffusion. Fosfomycin displayed lower activity against E. cloacae complex and Klebsiella spp., with MIC50 (minimum inhibitory concentration required to inhibit the growth of 50% of tested isolates) equal to the E. coli EUCAST breakpoint (8 mg/L). For these species, the three alternative techniques overestimated MICs and resistance, and did not meet performance criteria. For mecillinam testing of Enterobacterales isolates, apart from P. mirabilis, ETEST® nearly fulfilled ISO requirements, and CA rates were acceptable for disc diffusion. ISO criteria were reached for C. koseri and E. coli testing with VITEK®2, apart from too high rates of very major errors. For P. mirabilis, performances were unacceptable, whatever the routine method used. Commercially available tests may serve as alternatives to agar dilution to assess fosfomycin (oral) and mecillinam susceptibility of Enterobacterales urinary isolates, with important interspecies variabilities. Additional studies comprising more fosfomycin- and mecillinam-resistant isolates are needed to strengthen our conclusions.

Investigation of antifungal susceptibility of Aspergillus species isolated from systemic clinical specimens by different methods

PurposeDue to the potential for Aspergillus species to cause lethal infections and the rising rates of antifungal resistance, the significance of antifungal susceptibility tests has increased. We aimed to assess the sensitivities of Aspergillus species to amphotericin B (AMB), voriconazole (VOR), itraconazole (ITZ), and caspofungin (CAS) using disk diffusion (DD) and gradient diffusion (GD) methods and compare them with broth microdilution (BMD) as the reference susceptibility method. MethodsThe study involved 62 Aspergillus fumigatus, 28 Aspergillus flavus, and 16 Aspergillus terreus isolates, totaling 106 Aspergillus isolates. BMD and DD methods were performed in accordance with CLSI M38-A2 and CLSI M51-A documents, respectively. The GD method utilized nonsupplemented Mueller Hinton agar (MHA) as the medium. ResultsIn the BMD method, the lowest minimal inhibitory concentration (MIC)90 or minimal effective concentration (MEC)90 values were observed for VOR and CAS (0.5 μg/mL and 0.06 μg/mL, respectively). AMB and ITZ MIC90 values were both 2 μg/mL. In our comparison of the GD method with the BMD method at ±2 dilution, we observed essential agreement rates of 91.6%, 99.1%, 100%, and 38.6% for AMB, VOR, ITZ, and CAS, respectively. When comparing DD and BMD methods, we found categorical agreement rates of 65.1%, 99.1%, 77.3%, and 100% for AMB, VOR, ITZ, and CAS, respectively. For GD and BMD methods, these rates were 79.2%, 99.1%, 87.8%, and 100%. ConclusionsGiven the high essential and categorical agreement rates, we posit that the GD method is a viable alternative to the BMD method for AMB, ITZ and VOR but not for CAS. In addition, the use of nonsupplemented MHA in the GD method proves advantageous due to its cost-effectiveness and widespread availability compared to other growth media.

Comparison of testing methods assessing the in vitro efficacy of the combination of aztreonam with avibactam on multidrug-resistant Gram-negative bacilli

BackgroundAztreonam-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.Methods96 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).ResultsAccording 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%).ConclusionsThe 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.

Assessment of the gradient diffusion method for fosfomycin susceptibility testing in Staphylococcus spp. and Enterococcus spp. isolated from the urine of companion dogs in Thailand.

The agar dilution method is the approved method for determining the minimum inhibitory concentration (MIC) in fosfomycin susceptibility testing, whereas the broth dilution method is not recommended. This study aimed to investigate the potential of the gradient diffusion method as a more convenient alternative to agar dilution method for MIC evaluation, particularly for the susceptibility testing of Staphylococcus spp. and Enterococcus spp. to fosfomycin. A total of 194 isolates of Staphylococcus spp. and Enterococcus spp. were collected from urine samples of dogs diagnosed with bacterial cystitis. Bacterial identification and susceptibility to multiple antibiotics were tested using the Vitek 2 automated system. The susceptibility to fosfomycin was compared between agar dilution (reference method) and the gradient diffusion method. We assessed the agreement rates and errors between the two approaches by analyzing the MIC data. Staphylococcus pseudintermedius (98.7%) and Enterococcus faecalis (80.0%) exhibited high fosfomycin susceptibility rates, whereas Enterococcus faecium exhibited a lower susceptibility rate (38.5%). The gradient diffusion method demonstrated unacceptably low essential agreement (EA) rates (>90%) but acceptable categorical agreement (CA) rates (≥ 90%) for S. pseudintermedius (83.54% EA and 97.47% CA) and coagulase-negative staphylococci (CoNS) such as Staphylococcus chromogenes, Staphylococcus hominis, and Staphylococcus simulans (85.00% EA and 95.00% CA). Enterococcus spp. had an acceptable EA of 93.75%, but an unacceptably low CA rate of 82.81%, with a minor error rate of 17.19%. No significant errors were observed for Staphylococcus and Enterococcus spp. The gradient diffusion method reliably determines MICs and interpretative breakpoints (S, I, R) for S. pseudintermedius. However, its applicability to CoNS and enterococci may be limited due to unacceptable errors.

In-vitro Susceptibility Testing Methods for Ceftazidime-avibactam against Carbapenem-resistant Enterobacterales: Comparison with Reference Broth Microdilution Method.

β-lactam antibiotics, mainly cephalosporins, and carbapenems, have been the mainstay of treatment for infections caused by Enterobacterales. However, their role in treating clinical infections has become limited because of the increase in resistance. There is a need to have cost-effective and rapid methods for antimicrobial susceptibility testing methods for newer antibiotics like ceftazidime-avibactam against carbapenem-resistant Enterobacterales (CRE), which can be applied in routine clinical microbiology laboratories. With this aim, the present study was conducted to compare the disk diffusion and gradient diffusion, i.e., the E-test method with the reference broth microdilution (BMD) method for in-vitro testing of ceftazidime-avibactam against CRE. A total of 111 CRE isolates from various clinical samples were included. Conventional PCR (Polymerase Chain Reaction) was done for the detection of genes encoding carbapenemases and to see their expression, modified carbapenem inactivation method (mCIM) along with EDTA (Ethylenediaminetetraacetic acid) carbapenem inactivation method (eCIM) was done. 42.3% (47/111) isolates were resistant to ceftazidime-avibactam by the standard broth microdilution method; however, 45.9% (51/111) were resistant by both disk diffusion and E-test. In 5.4% of isolates (similar in both methods), microbroth dilution method results did not match with E-strip and disk diffusion. Very major errors (VME) by both disk diffusion and E-test were found in 2.1% (1/47), and major errors (ME) were found in 7.8% (5/64) isolates (similar isolates in both methods). The overall categorical agreement (CA) rate was 94.6% for both E-test and disk diffusion, and the essential agreement (EA) rate was 90.1% (100/111) for E-test. 98% (109/111) of CRE harbored carbapenemase genes either singly (30.3%) or in combination with others (69.7%). In conclusion, for CRE, E-test and the disk diffusion method for ceftazidimeavibactam depicted an acceptable performance as an alternative to the reference broth microdilution method.

Short incubation of disc diffusion for Streptococcus pneumoniae and Haemophilus influenzae to reduce time to susceptibility report.

Rapidly instituted antimicrobial therapy is important in severe infections, and reduced time to the antimicrobial susceptibility testing (AST) report is thus of importance. Disc diffusion (DD) is a cheap, rapidly adaptable, flexible and comprehensive method for phenotypic AST. Previous studies have shown that early reading of inhibition zones for non-fastidious species is possible. To evaluate zone reading after short incubation of DD in Haemophilus influenzae (n = 73) and Streptococcus pneumoniae (n = 112). The readability was evaluated and susceptibility interpretation (SIR) was performed, using the EUCAST 18 ± 2 h incubation breakpoint table (version 12.0), after 6 and 8 h of incubation. Categorical agreement (CA) and error rates were calculated using standard DD and broth microdilution as reference. The proportion of readable zones in H. influenzae was 19% (6 h) and 89% (8 h). The CA was 98% after 8 h. The corresponding readability in S. pneumoniae was 63%/98% and CA was 95%/97% after 6 and 8 h, respectively. Early reading of the screening discs (benzylpenicillin 1 unit in H. influenzae and oxacillin 1 µg in S. pneumoniae) correctly identified 18/22 of the H. influenzae isolates and all the readable S. pneumoniae isolates with reduced β-lactam susceptibility. For non-β-lactam agents, very major errors were most common for quinolones in S. pneumoniae. Introduction of areas of technical uncertainty (ATUs) reduced the error rate to ≤1.1%. We conclude that shortened incubation is feasible for H. influenzae and S. pneumoniae. To reduce the risk of false categorization a buffer zone (i.e. ATU) near the breakpoints must be used.

Performance of the Disc Diffusion Method, MTS Gradient Tests and Two Commercially Available Microdilution Tests for the Determination of Cefiderocol Susceptibility in Acinetobacter spp.

Cefiderocol is a siderophore-conjugated cephalosporin with potent activity against multidrug-resistant Gram-negative pathogens including Acinetobacter baumannii. The aim of this study was to evaluate cefiderocol testing methods on a relevant collection of 97 Acinetobacter spp. isolates. Commercialized broth microdilution methods (ComASP®, Liofilchem and UMIC®, Bruker), MIC test strips (Liofilchem) and disc diffusion using discs of three different brands (Mast Diagnostic, Liofilchem and Oxoid-Thermo Fisher Scientific) were compared with the broth microdilution reference method. None of the methods tested fulfilled acceptable criteria (essential agreement [EA] ≥ 90%; bias = ±30%) but both BMD methods achieved acceptable categorical agreement rates (CA = 95.9% [93/97, 95% CI 89.9-98.4] and CA = 93.8% [91/97, 95% CI 87.2-97.1] for ComASP® and UMIC®, respectively) and bias < 30% (-7.2% and -25.2% for ComASP® and UMIC®, respectively). The use of MIC gradient testing is strongly discouraged due to misclassification of 55% (n = 23/42) of resistant strains. Finally, the disc diffusion method could be used to rapidly screen for susceptible strains by setting a critical diameter of 22 mm.

Low performance of Policimbac® broth microdilution in determining polymyxin B MIC for Klebsiella pneumoniae.

Klebsiella pneumoniae is a global threat to healthcare, and despite the availability of new drugs, polymyxins are still an important therapeutic option for this and other resistant gram-negative pathogens. Broth microdilution is the only method that is recommended for polymyxins. In this study, we evaluated the accuracy of a commercial Policimbac® plate in determining the polymyxin B MIC for K. pneumoniae clinical isolates. The results were compared with those of the broth microdilution method according to ISO 16782. The Policimbac® plate had an excellent 98.04% categorical agreement, but unacceptable 31.37% essential agreement rates. Almost 2% of major errors as observed. Additionally, 52.94% of the strains overestimated the MIC at 1 µg/mL. Three isolates were excluded from the analysis due to the drying of the Policimbac® plate. To avoid dryness, we included wet gauze for the test, obtaining a 100% of categorical agreement rate; however, a low essential agreement was maintained (25.49%). In conclusion, the Policimbac® plate was unable to correctly determine the polymyxin B MIC for K. pneumoniae isolates. This low performance may interfere with the clinical use of the drug and, thus, with the result of the patient's treatment.

Multicenter evaluation of rapid antimicrobial susceptibility testing by VITEK®2 directly from positive blood culture

Study objectiveTo compare the antimicrobial susceptibility testing (AST) performance of positive blood cultures (PBC) VITEK®2 off-label use (D0) and traditional VITEK®2 workflow using isolated colonies after overnight (D1). MethodsPatient samples with monomicrobial Gram-negative rod or Gram-positive cocci in clusters bacteremia were tested on D0 and compared to D1 AST results in 7 laboratories in France. ResultsOverall, categorical and essential agreement rates were 98.4% and 96.7%, respectively. Very major discrepancy and major discrepancy rates for Enterobacterales and Staphylococci satisfied the NF EN ISO 20776-2 (2007) criteria for sepsis-relevant drugs. Very major discrepancies were >3% for amoxicillin-clavulanate (4.9%, 6/122), piperacillin-tazobactam (7.5%, 4/53) and meropenem (33%,1/3) for Enterobacterales and gentamicin for Staphylococci (4.6%, 4/87). ConclusionDirect AST from PBC broths by VITEK®2 for Enterobacterales and Staphylococci is reliable and fast and may positively influence antimicrobial stewardship.

Comparison of disk diffusion and broth microdilution methods for antimicrobial susceptibility testing of Campylobacter isolates of meat origin

The aim of this study was to compare the disk diffusion (DD) and the broth microdilution (BMD) methods in determining the antimicrobial susceptibility of 36 Campylobacter isolates of meat-origin to six antibacterial drugs (erythromycin, ciprofloxacin, tetracycline, streptomycin, gentamicin and nalidixic acid). All the available zone diameter and minimum inhibitory concentration (MIC) breakpoints of C. jejuni and C. coli as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were utilized. In addition, the zone diameter breakpoints of Enterobacterales for nalidixic acid, gentamicin, and streptomycin, as recommended by the Clinical and Laboratory Standards Institute (CLSI), were applied. All Campylobacter isolates were categorised as susceptible to erythromycin and gentamicin by both methods indicating completely concordant classification results. The overall highest ‘Very major error’ (VME) and ‘Major error’ (ME) rates were detected for nalidixic acid (13.3%) and tetracycline (26.3%), respectively, whereas a ‘Minor error’ (mE) rate was detected only for ciprofloxacin (60.1%). However, the Cohen's kappa statistic indicated a substantial concordance between the DD and BMD classification results for tetracycline and streptomycin, and almost perfect agreement for nalidixic acid, with corresponding categorical agreement rates of over 86% and approximately up to 92%. The correlation between the complementary inhibition zones and MIC breakpoints was strong and statistically highly significant (p < 0.001) for ciprofloxacin, tetracycline, streptomycin, and nalidixic acid.

Detection of colistin resistance via four methods in multidrug-resistant Gram-negative rods isolated from blood cultures.

The broth microdilution (BMD) method recommended for the detection of colistin resistance is labor-intensive, time-consuming, and difficult to apply in routine laboratories. Thus, various methods, such as disk elution, commercial microdilution, and rapid polymyxin-NP tests have been developed for the detection of colistin resistance. In this study, a total of 102 multi-resistant Gram-negative bacteria isolated from blood cultures were evaluated by four different methods for the detection of colistin resistance, and compared with the reference method. For the detection of the compatibility of these methods with the reference method, categorical and essential agreements, very major, major, and minor error rates were determined. Colistin-tigecycline and colistin-meropenem combinations were investigated in colistin-resistant isolates. Of the isolates, 15 (15%) [K. pneumoniae (n = 12), A. baumannii (n = 2), E. coli (n = 1)] were resistant to colistin with reference BMD method. MIC50 and MIC90 values of all isolates were ≤ 0.25 μg/mL and 16 μg/mL, respectively. The categorical agreement rates were 100% for commercial microdilution, disk elution, and RPNP test. The essential agreement rates of commercial microdilution, disk elution, and broth macrodilution were 78.4%, 86.3%, and 100%, respectively. Although there were no major errors in these methods, the macrodilution (12%) and commercial microdilution (20.6%) methods showed the most minor errors. Colistin-meropenem combination showed a 100% synergistic effect, but the colistin-tigecycline combination showed an 80% synergistic effect and 20% indifference effect. Disk elution and RPNP tests are suitable for routine use because they are the most efficient, easiest, low-cost, and good performance tests in detecting colistin resistance.

A prospective study to reduce turnaround time of microbiologically positive blood cultures in patients with sepsis in intensive care unit

PurposeTo determine the accuracy of direct microbial identification (dID) and antimicrobial susceptibility testing by EUCAST rapid antimicrobial susceptibility testing (rAST) methodology from positively flagged blood culture bottles (BCBs) as well as reduction in turnaround time (TAT) compared to standard methodology. MethodsIt was a hospital based, prospective cohort study conducted over a period of 21 months from March 2020 to November 2021 in which positively flagged blood culture bottles were simultaneously processed by dID ​+ ​rAST and by VITEK®-2 Compact system or Kirby-Bauer disk diffusion method. TAT was calculated as the time (hours) taken from receipt of sample in bacteriology laboratory to release of clinical reports with complete identification and susceptibility testing results in both methods. ResultsOf 301 dID ​+ ​rAST performed in study, 125 (41.5%) BCBs were identified as having one of the 8 reportable micro-organisms by EUCAST rAST standard. Amongst VITEK concordant BCBs with gram-negatives, mean reduction in TAT by dID ​+ ​rAST methodology was 23 ​± ​1.4 ​h. Amongst VITEK concordant gram-negatives, Categorical Agreement (CA) rates for any drug-bug combination were 94.4%, 94.5% and 93.6% and Very Major Error (VME) rates were 3.1%, 3.4% and 3.9% at 4-, 6- and 8-h reading time, respectively. ConclusionsEUCAST rAST methodology can generate susceptibility testing reports a day earlier if incorporated into the laboratory workflow. For resource-limited settings, implementing EUCAST rAST approach can be used effectively in early reporting, which can reduce antimicrobial use and improve patient outcomes by promoting timely escalation or de-escalation of empirical antibiotic therapy.

The Use of Resapolymyxin NP Test in Determining the Sensitivity of Colistin in Escherichia coli, and Klebsiella pneumoniae Isolates

Colistin is used as the last choice of drug in multidrug resistant gram-negative bacilli infections; therefore, accurate detection of colistin susceptibility has gained critical importance. Unfortunately, many of the widely used and practical methods in the clinical laboratory have various limitations for the determination of colistin susceptibility. This situation has led researchers to search for new methods to determine colistin susceptibility. In this study, the performance of the ResaPolymyxin NP test, which was developed for the rapid detection of colistin susceptibility of Pseudomonas aeruginosa and Acinetobacter baumannii isolates, was evaluated for various Gram-negative bacteria for the determination of colistin susceptibility. For this purpose, the colistin MIC values of 105 Escherichia coli, and 196 Klebsiella pneumoniae isolates were determined by broth microdilution (BMD) using cation-adjusted Mueller-Hinton broth and then ResaPolymyxin NP test was applied for each isolate. While 242 (%80.4) of the isolates included in the study were found to be susceptible to colistin with BMD, 214 (71.1%) of the isolates were found as sensitive to colistin with the ResaPolymyxin NP test. The categorical agreement rate for the ResaPolymyxin NP test was 85.7% for E.coli isolates, and 92.3% for K.pneumoniae isolates. The major error rate was 14.7% for E.coli, 10.8% for K.pneumoniae, whereas the very major error rate was 1.8% for K.pneumoniae. For ResaPolymyxin NP test, sensitivity, specificity, positive and negative predictive values were %98,3; %88.0; %66.7; and %99.5. In contrast to the available data about the ResaPolymyxin NP test, both the categorical agreement rate with BMD, and the very major and major error rates varied according to the isolate type, and it was concluded that the test performed relatively better in E.coli and K.pneumoniae isolates. Since the data obtained in this study are quite different from the previously published data, more comprehensive and multicenter studies are needed to evaluate the test effectiveness in order to recommend the use of the ResaPolymyxin NP test in clinical microbiology laboratories.

Evaluation of three different methods for susceptibility testing of gentamicin in carbapenem resistant Enterobacterales.

Carbapenem-resistant Enterobacterales (CRE) have become a growing problem worldwide in recent years. Options for the treatment of CRE are limited and one of these options is gentamicin. For this reason, gentamicin susceptibility should be properly determined. In a recently reported study, it is recommended to review the results of automated systems for assessing gentamicin susceptibility in carbapenem-resistant isolates. In this study, we aimed to determine gentamicin susceptibility using three different methods and compare the methods. The study included 107 CRE isolates from different samples. Gentamicin susceptibility was determined using Vitek 2 Compact (bioMérieux, France), Microscan Walkaway Plus (Beckman Coulter, USA) automatic systems, and disk diffusion (DD) method. The broth microdilution method (BMD) was used as reference method. Minor, major, and very major errors and categorical agreement rates were determined for each method. Aminoglycoside-modifying enzymes (aac(6')Ib and aph(2″)Ia) were assayed in discrepant isolates. According to BMD results, 90.7%, 1,8 %, and 7.5 % of the isolates were determined as susceptible, intermediate, and resistant to gentamicin, respectively. Compared to the results of the BMD for detecting gentamicin susceptibility, disk diffusion method showed the highest categorical agreement (98.1%), and Vitek 2 Compact showed the lowest categorical agreement (90.6%). The very major error rates were determined 7.5%, 0.9%, and 0.9% for Vitek 2 Compact, Microscan Walkaway Plus, and DD method, respectively. In addition, aac(6')Ib and aph(2″)Ia genes were detected in 8 discrepant isolates. For gentamicin susceptibility, the DD showed the most compatible results. The DD can be used as a reliable method for determining gentamicin susceptibility. Compatibility of automated systems with BMD was acceptable, although lower than DD. The discrepancies detected in the Vitek 2 Compact results could be due to the presence of aac(6')Ib and/or aph(2″)Ia aminoglycoside-modifying enzymes.

Multilaboratory Comparison of Omadacycline MIC Test Strip to Broth Microdilution MIC against Gram-Negative, Gram-Positive, and Fastidious Bacteria.

The performance of the Liofilchem omadacycline MIC Test Strip (MTS) was evaluated in a multisite study. Three testing sites collected/tested clinical isolates and one site tested challenge isolates that totaled 175 S. aureus, 70 S. lugdunensis, 121 E. faecalis, 100 E. faecium, 578 Enterobacterales, 142 Haemophilus spp., 181 S. pneumoniae, 45 S. anginosus group, 35 S. pyogenes,and 20 S. agalactiae. MIC testing was performed by CLSI broth microdilution (BMD) and MTS. Fastidious isolates testing included BMD and MTS testing with both CLSI and EUCAST Mueller-Hinton Fastidious (MH-F). In addition, each site performed reproducibility for nonfastidious and fastidious isolates and QC by MTS and BMD. All BMD and MTS results for the QC strains were within expected ranges, with exception of one MTS HTM result for H. influenzae ATCC 49247. Among reproducibility isolates, omadacycline MTS results were within one dilution of the modal MIC for 95.2% of nonfastidious Gram-positive, 100% of Gram-negative, 99.3% and 98.5% of fastidious isolates tested on CLSI and EUCAST media, respectively. MTS results for all study isolates were within one doubling dilution of the CLSI BMD MIC for 98.9% of S. aureus, 100% of S. lugdunensis, 98.3% of E. faecalis, 100% of E. faecium, and 99.6% of Enterobacterales. Essential agreement rates for CLSI and EUCAST MH-F agar compared to CLSI BMD were 98.2% and 98.2%, for H. influenzae, 91.1% and 73.6%, for S. pneumoniae and 100% and 85-91.7% for other streptococcus species, respectively. Based on CLSI media, all categorical errors were minor errors and categorical agreement rates were >90% with exception of C. freundii, S. lugdunensis, E. faecalis, S. anginosus and S. constellatus.

Comparative Analysis of Gradient Diffusion and Disk Diffusion with Agar Dilution for Susceptibility Testing of Elizabethkingia anophelis

Elizabethkingia anophelis has recently emerged as a cause of life-threatening infections. This study compared the results of antimicrobial susceptibility testing (AST) conducted for E. anophelis through different methods. E. anophelis isolates collected between January 2005 and June 2019 were examined for their susceptibility to 14 antimicrobial agents by using disk diffusion, gradient diffusion (Etest; bioMérieux S.A., Marcy l’Etoile, France), and agar dilution methods. The agar dilution method was the reference assay. According to the agar dilution method, the isolates exhibited the highest susceptibility to minocycline (100%), doxycycline (97.6%), rifampin (95.2%), and levofloxacin (78.6%). A very major error rate of >1.5% was observed for nine antibiotics tested using the disk diffusion method. The overall categorical agreement rate between the disk diffusion and agar dilution methods was 74.8%, and ceftazidime, minocycline, levofloxacin, and rifampin met the minimum requirements for discrepancy and agreement rates. The Etest method tended to produce lower log2 minimum inhibitory concentrations for the antibiotics, except for trimethoprim–sulfamethoxazole and rifampin; the method resulted in very major errors for nine antibiotics. The overall essential and categorical agreement rates between the Etest and agar dilution methods were 67.3% and 76.1%, respectively. The Etest method demonstrated acceptable discrepancy and agreement rates for ceftazidime, minocycline, doxycycline, levofloxacin, and rifampin. AST results obtained through the disk diffusion and Etest methods for multiple antibiotics differed significantly from those obtained using the agar dilution method. These two assays should not be a routine alternative for AST for E. anophelis.

652. Comparison of fosfomycin (FOF) activity and prevalence of subpopulations between Escherichia coli (EC) and Klebsiella pneumoniae (KP) during susceptibility testing

BackgroundIn the United States, interpretive criteria for FOF are established only for EC, yet those criteria are often extrapolated to KP. Recent studies have highlighted both inferior clinical outcomes after FOF treatment and difficulties in interpretation of inner colony subpopulations, the presence of which may affect clinical efficacy. We sought to compare FOF activity against EC and KP and to determine the prevalence of inner colony subpopulations following disk diffusion (DD) testing of the two species.MethodsA convenience collection of 73 KP and 42 EC isolates from 3 U.S. institutions were included. Minimal inhibitory concentration (MIC) testing was performed in duplicate on separate days using agar dilution (AD) and DD as recommended by the Clinical and Laboratory Standards Institute guidelines, with application of EC susceptibility (≤ 64mg/L) breakpoints. The frequency and counts of inner colonies observed during DD testing was calculated, and colonies were subcultured for use in future studies.ResultsMIC50/90 values were 1/16 mg/L and 32/256 mg/L for EC and KP respectively. All EC isolates were considered susceptible and therefore categorical agreement was 100%. The majority of KP isolates were considered susceptible (83.6% with AD and 86.3% with DD) and categorical agreement between the methods was 84.9%. Inner colonies were observed during DD testing in 88.1% of EC isolates and 80.8% of KP isolates during at least one replicate, with 47.6% of EC isolates and 39.7% of KP isolates showing inner colony growth during both DD test replicates. More than 10 inner colonies were observed in 50% of EC isolates compared to 12.3% of KP isolates.ConclusionKP isolates demonstrated considerably higher FOF MIC values compared to EC, as evidenced by MIC50/90 values 4-5 dilutions higher than those for EC. The categorical agreement rate was higher among EC than KP, highlighting concerns regarding the practice of extrapolating FOF susceptibility breakpoints for EC to KP. The high frequency of inner colonies observed in DD for both species necessitates further studies to determine best practices for interpreting their relevance, fitness, and resistance in order to identify potential impacts to clinical efficacy of FOF.Disclosures Elizabeth B. Hirsch, PharmD, Merck (Grant/Research Support)Nabriva Therapeutics (Advisor or Review Panel member)

Comprehensive Statistical Evaluation of Etest®, UMIC®, MicroScan and Disc Diffusion versus Standard Broth Microdilution: Workflow for an Accurate Detection of Colistin-Resistant and Mcr-Positive E. coli.

Four colistin susceptibility testing methods were compared with the standard broth microdilution (BMD) in a collection of 75 colistin-susceptible and 75 mcr-positive E. coli, including ST131 isolates. Taking BMD as reference, all methods showed similar categorical agreement rates (CA) of circa 90%, and a low number of very major errors (VME) (0% for the MicroScan system and Etest®, 0.7% for UMIC®), except for the disc diffusion assay (breakpoint ≤ 11 mm), which yielded false-susceptible results for 8% of isolates. Of note is the number of mcr-positive isolates (17.3%) categorized as susceptible (≤2 mg/L) by the BMD method, but as resistant by the MicroScan system. ST131 mcr-positive E. coli were identified as colistin-resistant by all MIC-based methods. Our results show that applying the current clinical cut-off (>2 mg/L), many mcr-positive E. coli remain undetected, while applying a threshold of >1 mg/L the sensitivity of detection increases significantly without loss of specificity. We propose two possible workflows, both starting with the MicroScan system, since it is automated and, importantly, it categorized all mcr-positive isolates as colistin-resistant. MicroScan should be followed by either BMD or MIC-based commercial methods for colistin resistance detection; or, alternatively, MicroScan, followed by PCR for the mcr screening.