Variability of Beta-Lactam Broth Microdilution for Pseudomonas aeruginosa.

Antimicrobial susceptibility testing for Pseudomonas aeruginosa is critical to determine suitable treatment options. Commercial susceptibility tests are typically calibrated against the reference method, broth microdilution (BMD). Imprecision of MICs obtained by BMD for the same isolate on repeat testing is known to exist. Factors that impact the extent of variability include concentration of the inoculum, operator effects, contents of the media, inherent strain properties, and the testing process or materials. We evaluated the variability of BMD for antipseudomonal beta-lactams (aztreonam, cefepime, ceftazidime, meropenem, piperacillin-tazobactam, ceftazidime-avibactam, and ceftolozane-tazobactam) tested against a collection of P. aeruginosa isolates. Multiple replicate BMD tests were performed, and MICs were compared to assess reproducibility, including the impact of the inoculum and operator. Overall, essential agreement (EA) was ≥90% for all beta-lactams tested. Absolute agreement (AA) was as low as 70% for some beta-lactams. Variability from the inoculum and operators impacted the reproducibility of MICs. Piperacillin-tazobactam exhibited the highest degree of variability with 74% AA and 94% EA. The implications of MIC variability are extensive, as the MIC is essential for multiple facets of microbiology, such as the development of new compounds and susceptibility tests, dose optimization, and pharmacokinetic/pharmacodynamic (PK/PD) targets for individual patients.

Public Health Efforts Can Impact Adoption of Current Susceptibility Breakpoints, but Closer Attention from Regulatory Bodies Is Needed.

Microbiological testing, including interpretation of antimicrobial susceptibility testing results using current breakpoints, is crucial for clinical care and infection control. Continued use of obsolete Enterobacteriaceae carbapenem breakpoints is common in clinical laboratories. The purposes of this study were (i) to determine why laboratories failed to update breakpoints and (ii) to provide support for breakpoint updates. The Los Angeles County Department of Public Health conducted a 1-year outreach program for 41 hospitals in Los Angeles County that had reported, in a prior survey of California laboratories, using obsolete Enterobacteriaceae carbapenem breakpoints. In-person interviews with hospital stakeholders and customized expert guidance and resources were provided to aid laboratories in updating breakpoints, including support from technical representatives from antimicrobial susceptibility testing device manufacturers. Forty-one hospitals were targeted, 7 of which had updated breakpoints since the prior survey. Of the 34 remaining hospitals, 27 (79%) assumed that their instruments applied current breakpoints, 17 (50%) were uncertain how to change breakpoints, and 10 (29%) lacked resources to perform a validation study for off-label use of the breakpoints on their systems. Only 7 hospitals (21%) were familiar with the FDA/CDC Antibiotic Resistance Isolate Bank. All hospitals launched a breakpoint update process; 16 (47%) successfully updated breakpoints, 12 (35%) received isolates from the CDC in order to validate breakpoints on their systems, and 6 (18%) were planning to update within 1 year. The public health intervention was moderately successful in identifying and overcoming barriers to updating Enterobacteriaceae carbapenem breakpoints in Los Angeles hospitals. However, the majority of targeted hospitals continued to use obsolete breakpoints despite 1 year of effort. These findings have important implications for the quality of patient care and patient safety. Other public health jurisdictions may want to utilize similar resources to bridge the patient safety gap, while manufacturers, the FDA, and others determine how best to address this growing public health issue.

Performance of ceftazidime/avibactam susceptibility testing methods against clinically relevant Gram-negative organisms.

OBJECTIVES: To ensure the accuracy of susceptibility testing methods for ceftazidime/avibactam. METHODS: The performances of the Etest (bioMérieux), 30/20 µg disc (Hardy diagnostics) and 10/4 µg disc (Mast Group) were evaluated against the reference broth microdilution (BMD) method for 102 clinically relevant Gram-negative organisms: 69 ceftazidime- and meropenem-resistant Klebsiella pneumoniae and 33 MDR non-K. pneumoniae. Essential and categorical agreement along with major and very major error rates were determined according to CLSI guidelines. RESULTS: A total of 78% of isolates were susceptible to ceftazidime/avibactam. None of the three methods met the defined equivalency threshold against all 102 organisms. The Etest performed the best, with categorical agreement of 95% and major errors of 6.3%. Against the 69 ceftazidime- and meropenem-resistant K. pneumoniae, only the Etest and the 10/4 µg disc met the equivalency threshold. None of the three methods met equivalency for the 33 MDR isolates. There were no very major errors observed in any analysis. These results were pooled with those from a previous study of 74 carbapenem-resistant Enterobacteriaceae and data from the ceftazidime/avibactam new drug application to define optimal 30/20 µg disc thresholds using the error-rate bound model-based approaches of the diffusion breakpoint estimation testing software. This analysis identified a susceptibility threshold of ≤19 mm as optimal. CONCLUSIONS: Our data indicate that the Etest is a suitable alternative to BMD for testing ceftazidime/avibactam against ceftazidime- and meropenem-resistant K. pneumoniae. The 30/20 µg discs overestimate resistance and may lead to the use of treatment regimens that are more toxic and less effective.

Evaluation of Oxacillin and Cefoxitin Disk Diffusion and MIC Breakpoints Established by the Clinical and Laboratory Standards Institute for Detection of mecA-Mediated Oxacillin Resistance in Staphylococcus schleiferi.

Staphylococcus schleiferi is a beta-hemolytic, coagulase-variable colonizer of small animals that can cause opportunistic infections in humans. In veterinary isolates, the rate of mecA-mediated oxacillin resistance is significant, with reported resistance rates of >39%. The goal of this study was to evaluate oxacillin and cefoxitin disk diffusion (DD) and MIC breakpoints for detection of mecA-mediated oxacillin resistance in 52 human and 38 veterinary isolates of S. schleiferi Isolates were tested on multiple brands of commercial media and according to Clinical and Laboratory Standards Institute (CLSI) methods. Zone diameters and MIC values were interpreted using CLSI breakpoints (CLSI, Performance Standards for Antimicrobial Susceptibility Testing. M100-S27, 2017) for Staphylococcus aureus/Staphylococcus lugdunensis, coagulase-negative staphylococci (CoNS), and Staphylococcus pseudintermedius Results were compared to those of mecA PCR. Twenty-nine of 90 (32%) isolates were mecA positive. Oxacillin inhibition zone sizes and MICs interpreted by S. pseudintermedius breakpoints reliably differentiated mecA-positive and mecA-negative isolates, with a categorical agreement (CA) of 100% and no very major errors (VMEs) or major errors (MEs) for all media. For cefoxitin DD results interpreted using S. aureus/S. lugdunensis and CoNS breakpoints, CA values were 85% and 75%, respectively, and there were 72% and 64% VMEs, respectively, and 0 MEs. For cefoxitin MICs interpreted using S. aureus/S. lugdunensis breakpoints, CA was 81%, and there were 60% VMEs and no MEs. Our data demonstrate that oxacillin DD or MIC testing methods using the current S. pseudintermedius breakpoints reliably identify mecA-mediated oxacillin resistance in S. schleiferi, while cefoxitin DD and MIC testing methods perform poorly.

Real-Time PCR Targeting the penA Mosaic XXXIV Type for Prediction of Extended-Spectrum-Cephalosporin Susceptibility in Clinical Neisseria gonorrhoeae Isolates.

Neisseria gonorrhoeae isolates with decreased susceptibility to extended-spectrum cephalosporins (ESCs) are increasing. We developed an assay to predict N. gonorrhoeae susceptibility to ESCs by targeting penA mosaic XXXIV, an allele prevalent among U.S. isolates with elevated ESC MICs. The assay was 97% sensitive and 100% specific for predicting at least one ESC MIC above the CDC alert value among clinical isolates, and it could be multiplexed with a previously validated gyrA PCR to predict ciprofloxacin susceptibility.

Emerging Resistance, New Antimicrobial Agents     but No Tests! The Challenge of Antimicrobial Susceptibility Testing in the Current US Regulatory Landscape.

Accurate and timely performance of antimicrobial susceptibility testing (AST) by the clinical laboratory is paramount to combating antimicrobial resistance. The ability of laboratories in the United States to effectively perform ASTs is challenged by several factors. Some, such as new resistance mechanisms and the associated evolution of testing recommendations and breakpoints, are inevitable. Others are entirely man-made. These include unnecessarily strict US Food and Drug Administration (FDA) limitations on how commercial AST systems can be used for diagnostic testing, the absence of up-to-date performance data on these systems, and the lack of commercially available FDA-cleared tests for newer antimicrobial agents or for older agents with updated breakpoints. This viewpoint will highlight contemporary AST challenges faced by the clinical laboratory, and propose some solutions.

Performance and Verification of a Real-Time PCR Assay Targeting the gyrA Gene for Prediction of Ciprofloxacin Resistance in Neisseria gonorrhoeae.

In the United States, 19.2% of Neisseria gonorrhoeae isolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive for N. gonorrhoeae by the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions.

Evaluation of Oxacillin and Cefoxitin Disk and MIC Breakpoints for Prediction of Methicillin Resistance in Human and Veterinary Isolates of Staphylococcus intermedius Group.

Staphylococcus pseudintermedius is a coagulase-positive species that colonizes the nares and anal mucosa of healthy dogs and cats. Human infections with S. pseudintermedius range in severity from bite wounds and rhinosinusitis to endocarditis; historically, these infections were thought to be uncommon, but new laboratory methods suggest that their true incidence is underreported. Oxacillin and cefoxitin disk and MIC tests were evaluated for the detection of mecA- or mecC-mediated methicillin resistance in 115 human and animal isolates of the Staphylococcus intermedius group (SIG), including 111 Staphylococcus pseudintermediusand 4 Staphylococcus delphini isolates, 37 of which were mecA positive. The disk and MIC breakpoints evaluated included the Clinical and Laboratory Standards Institute (CLSI) M100-S25 Staphylococcus aureus/Staphylococcus lugdunensis oxacillin MIC breakpoints and cefoxitin disk and MIC breakpoints, the CLSI M100-S25 coagulase-negative Staphylococcus (CoNS) oxacillin MIC breakpoint and cefoxitin disk breakpoint, the CLSI VET01-S2 S. pseudintermedius oxacillin MIC and disk breakpoints, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) S. pseudintermedius cefoxitin disk breakpoint. The oxacillin results interpreted by the VET01-S2 (disk and MIC) and M100-S25 CoNS (MIC) breakpoints agreed with the results of mecA/mecC PCR for all isolates, with the exception of one false-resistant result (1.3% of mecA/mecC PCR-negative isolates). In contrast, cefoxitin tests performed poorly, ranging from 3 to 89% false susceptibility (very major errors) and 0 to 48% false resistance (major errors). BD Phoenix, bioMérieux Vitek 2, and Beckman Coulter MicroScan commercial automated susceptibility test panel oxacillin MIC results were also evaluated and demonstrated >95% categorical agreement with mecA/mecC PCR results if interpreted by using the M100-S25 CoNS breakpoint. The Alere penicillin-binding protein 2a test accurately detected all mecA-positive isolates, although for four isolates, cefoxitin induction was required prior to testing. These data demonstrate that the cefoxitin surrogate test does not reliably detect the presence of mecA in S. pseudintermedius isolates and that laboratories should perform oxacillin disk or MIC tests of these isolates when they are encountered.

Comparison of Illumigene, Simplexa, and AmpliVue Clostridium difficile molecular assays for diagnosis of C. difficile infection.

We compared the performance of the Simplexa Universal Direct (Focus Diagnostics) and AmpliVue (Quidel Corporation) assays to that of the Illumigene assay (Meridian Bioscience, Inc.) for the diagnosis of Clostridium difficile infection. Two hundred deidentified remnant diarrheal stool specimens were tested by the Simplexa, AmpliVue, and Illumigene methods. Specimens with discrepant results among the three assays and a representative number of concordant specimens were further evaluated by toxigenic culture. The sensitivity and specificity were 98 and 100% and 96 and 100% for the Simplexa Universal Direct and AmpliVue assays, respectively. Both assays are easy to perform, with rapid turn-around-times, supporting their utility in the clinical laboratory as routine diagnostic platforms.

Phenotypic and molecular characteristics of carbapenem-resistant Enterobacteriaceae in a health care system in Los Angeles, California, from 2011 to 2013.

Carbapenem-resistant Enterobacteriaceae (CRE) are a concern for health care in the United States but remain relatively uncommon in California. We describe the phenotype, clonality, and carbapenemase-encoding genes present in CRE isolated from patients at a Californian tertiary health care system. CRE for this study were identified by evaluating the antibiograms of Enterobacteriaceae isolated in the UCLA Health System from 2011 to 2013 for isolates that were not susceptible to meropenem and/or imipenem. The identification of these isolates was subsequently confirmed by matrix-associated laser desorption ionization-time of flight, and broth microdilution tests were repeated to confirm the CRE phenotype. Real-time PCR for bla(KPC), bla(SME), bla(IMP), bla(NDM-1), bla(VIM), and bla(OXA-48) was performed. Clonality was assessed by repetitive sequence-based PCR (repPCR) and multilocus sequence typing (MLST). Of 15,839 nonduplicate clinical Enterobacteriaceae isolates, 115 (0.73%) met the study definition for CRE. This number increased from 0.5% (44/8165) in the first half of the study to 0.9% (71/7674) in the second (P = 0.004). The most common CRE species were Klebsiella pneumoniae, Enterobacter aerogenes, and Escherichia coli. A carbapenemase-encoding gene was found in 81.7% (94/115) of CRE and included bla(KPC) (78.3%), bla(NDM-1) (0.9%), and bla(SME) (2.6%). The majority of bla(KPC) genes were in K. pneumoniae isolates, which fell into 14 clonal groups on typing. bla(KPC) was identified in more than one species of CRE cultured from the same patient in four cases. Three bla(SME)-carrying Serratia marcescens isolates and one bla(NDM-1) carrying Providencia rettgeri isolate were detected. CRE are increasing in California, and carbapenemases, particularly KPC, are a common mechanism for carbapenem resistance in this region.

The accuracy of implant master casts constructed from transfer impressions.

The accuracy of master casts fabricated from three impression techniques commonly used with the Brånemark System was measured. Points placed in a metal surrogate model and onto master abutments were compared after abutments were transferred to stone casts using splinted and unsplinted impression coping techniques. The mean values and standard deviations of each of the reference points on 12 total casts were compared with the values for each point from the surrogate model. Values from techniques using splinted and unsplinted squared polymer copings as well as unsplinted tapered hydrocolloid copings were not significantly different from values recorded from the master surrogate model. Tapered hydrocolloid copings yielded a higher correlation to coordinate values on the master than unsplinted squared polymer copings or splinted squared copings.