Evaluation of disk diffusion method for testing the rifampicin, erythromycin, and tetracycline susceptibility of Clostridioides (prev. Clostridium) difficile.

INTRODUCTION: Antimicrobial resistance (AMR) is one of the greatest threats to animal and public health. Clostridioides (prev. Clostridium) difficile is a major burden to healthcare and a relevant AMR gene reservoir. Despite the known importance of AMR in C. difficile epidemiology and treatment, antimicrobial susceptibility testing for this pathogen is still based on the determination of the minimal inhibitory concentration (MIC) by the agar dilution method, which is technically demanding and labor-intensive. In this study, the disk diffusion method was used to evaluate the susceptibility of C. difficile to erythromycin, rifampicin, and tetracycline. MATERIAL AND METHODS: A total of 155 isolates isolated between 2011 and 2022 from humans and animals in Brazil were simultaneously tested using the disk diffusion method and the epsilometer test (Etest) for these three antimicrobials on Brucella blood agar supplemented with vitamin K and hemin. RESULTS: The results suggest that disk diffusion can be an interesting routine tool to identify erythromycin- and rifampicin-resistant C. difficile isolates (≥20 mm cut-off) and wild type (WT) strains (≥28 mm). However, the disk diffusion protocol tested in this study does not seem suitable for tetracycline because of the common misclassification of resistant strains.

INVESTIGATION OF TIGECYCLINE SUSCEPTIBILITY OF MULTIDRUG-RESISTANT ACINETOBACTER BAUMANNII ISOLATES BY DISC DIFFUSION, AGAR GRADIENT AND BROTH MICRODILUTION TESTS.

The use of tigecycline is becoming increasingly important because of the high levels of antibiotic resistance in Acinetobacter baumannii (A. baumannii) isolates. In this prospective study, multidrug-resistant A. baumannii isolates were obtained from various tissue and fluid samples of patients admitted to or treated at various departments and tested in Laboratory of Microbiology, Duzce University Medical Faculty between January 2013 and December 2015. Tigecycline resistance in multidrug-resistant A. baumannii isolates were analyzed using the disc diffusion test (DDT), agar gradient test (AGT), and gold standard test [broth microdilution test (BMT)]. A. baumannii isolates resistant to multiple drugs were included in the study (N=94). Using the BMT method, 89 (95%), 4 (4%) and 1 (1%) A. baumannii isolates were determined as tigecycline susceptible, intermediate and resistant isolates, respectively. Using the Food and Drug Administration criteria, the rates of major error (ME), minor error (mE) and categorical agreement (CA) for DDT were 26%, 67% and 9%, respectively. In contrast, for AGT, the rates of ME, mE and CA were 0%, 4%, 95%, respectively. Tigecycline resistance as assessed by BMT showed no increase between 2013 and 2015. Accordingly, isolates found to be resistant or intermediate by DDT should be confirmed by BMT. Due to the ease of application, AGT is a safe method of detecting susceptibility.

Power Analysis for the Wald, LR, Score, and Gradient Tests in a Marginal Maximum Likelihood Framework: Applications in IRT.

The Wald, likelihood ratio, score, and the recently proposed gradient statistics can be used to assess a broad range of hypotheses in item response theory models, for instance, to check the overall model fit or to detect differential item functioning. We introduce new methods for power analysis and sample size planning that can be applied when marginal maximum likelihood estimation is used. This allows the application to a variety of IRT models, which are commonly used in practice, e.g., in large-scale educational assessments. An analytical method utilizes the asymptotic distributions of the statistics under alternative hypotheses. We also provide a sampling-based approach for applications where the analytical approach is computationally infeasible. This can be the case with 20 or more items, since the computational load increases exponentially with the number of items. We performed extensive simulation studies in three practically relevant settings, i.e., testing a Rasch model against a 2PL model, testing for differential item functioning, and testing a partial credit model against a generalized partial credit model. The observed distributions of the test statistics and the power of the tests agreed well with the predictions by the proposed methods in sufficiently large samples. We provide an openly accessible R package that implements the methods for user-supplied hypotheses.

Epidemiological cut-off value and antibiotic susceptibility test methods for azithromycin in a collection of multi-country invasive non-typhoidal Salmonella.

OBJECTIVE: Azithromycin is an alternative to treat invasive non-typhoidal Salmonella (iNTS) infections. We determined its epidemiological cut-off (ECOFF) and compared azithromycin susceptibility testing methods for iNTS. METHODS: We used EUCAST ECOFFinder to determine the minimum inhibitory concentrations (MIC; obtained by broth microdilution) ECOFF and corresponding disk zone diameters of 515 iNTS from blood cultures in Democratic Republic of Congo, Burkina Faso, Rwanda, and Cambodia. Transferable resistance mechanisms were determined by polymerase chain reaction. We compared azithromycin susceptibility testing by semi-automated broth microdilution (customized Sensititre panel; reference), agar dilution, gradient tests (bioMérieux, Liofilchem, HiMedia; read at 80% (MIC80%) and 100% inhibition (MIC100%)), and disk diffusion (Rosco, Oxoid, BD, Liofilchem) for 161 wild- and 198 non-wild-type iNTS. RESULTS: Azithromycin MIC ECOFF was 16 mg/L corresponding to a 12 mm zone diameter; mphA was detected in 192/197 non-wild- and 0/47 wild-type iNTS. Categorical agreement was excellent (≥98%) for all methods. Essential agreement was very good for agar dilution (>90%) but moderate for gradient tests (MIC80%: 52% to 71% and MIC100%: 72% to 91%). Repeatability was good for all methods/brands. Interreader agreement was high for broth microdilution and agar dilution (all ≤1 twofold dilution difference) and disk diffusion (>96% ≤3 mm difference) but lower for gradient tests (MIC80% & MIC100%: 83% to 94% ≤1 twofold dilution difference). DISCUSSION: Azithromycin ECOFF of iNTS was 16 mg/L, i.e. equal to Salmonella Typhi. Disk diffusion is an accurate, precise, and user-friendly alternative for agar dilution and broth microdilution. Reading gradient tests at 100% instead of 80% inhibition improved accuracy and precision.

Reliability of phenotypic methods for detection of colistin resistance among carbapenem-resistant Acinetobacter baumannii clinical isolates from Egypt.

INTRODUCTION: Acinetobacter baumannii is a challenging pathogen responsible for serious nosocomial infections. Colistin resistance in carbapenem-resistant A. baumannii strains is a critical health problem as it limits the available therapeutic options. The current work aimed to study the reliability of several phenotypic methods for the detection of colistin resistance among carbapenem-resistant A. baumannii isolates in Egypt. METHODS: A total of 22 carbapenem-resistant A. baumannii isolates were recovered. Colistin minimum inhibitory concentrations (MICs) were determined using broth microdilution (BMD) and compared to agar dilution (AD), automated system (VITEK-2) and gradient test (E-test) and were analyzed by statistical methods. RESULTS: Phenotypic testing showed that nine of 22 isolates (40.9%) were colistin-resistant by BMD and seven of them were also resistant by AD, with the categorical agreement (CA) of 72.7% and essential agreement (EA) of 90.9%. Colistin MIC results ranged from 1-8 µg/mL and 1-32 µg/mL by both AD and BMD respectively. Detection of colistin resistance by gradient test and automated system showed high very major error (VME) rates (40.9%) compared to BMD with a lack of CA between them. AD gave moderate agreement with BMD by 90.9% EA, 72.7% CA and only 9.1% VME. CONCLUSIONS: In delineating colistin breakpoints BMD followed by AD method are defined as the only reliable phenotypic methods for colistin resistance evaluation. More rapid and reliable tests, other than BMD and AD, are required for the convenient detection of colistin resistance in the routine clinical microbiology laboratory daily workflow.

Performance of polymyxin B Etest in a setting of high prevalence of KPC-producing Klebsiella pneumoniae.

OBJECTIVES: Polymyxin resistance has been increasing in many regions, and appropriate determination of polymyxin susceptibility is now a major challenge worldwide. Many clinical laboratories rely on gradient diffusion methods to assess polymyxin susceptibility, although broth microdilution (BMD) is the only method currently recommended by the CLSI and EUCAST. The aim of this study was to assess the performance of the polymyxin B (PMB) Etest in a setting with a high prevalence of KPC-producing Klebsiella pneumoniae (KPC-KP). METHODS: A commercial Etest susceptibility testing method was evaluated and compared with the reference BMD method, considering isolates with a minimum inhibitory concentration (MIC) ≤2 mg/L for PMB as susceptible to this drug. A total of 310 clinical KPC-KP isolates were evaluated. RESULTS: Susceptibility was significantly higher by Etest compared with BMD (82.6% vs. 75.8%). The MIC50, MIC90 and modal MICs for PMB were 0.25, 32 and 0.25 mg/L (27.1%) by BMD and 0.5, 16 and 0.5 mg/L (49.7%) by Etest, respectively. Although categorical agreement was 90.0%, there was poor essential agreement (50.6%). A high rate (34.7%) of very major errors (VMEs) and a relatively low rate (2.1%) of major errors were found. CONCLUSION: The considerable number of resistant isolates in this study allowed an accurate estimation of VME rates and, consequently, a more comprehensive assessment of susceptibility testing for polymyxins. Etest did not meet fully the acceptance criteria for US FDA requirements. These data do not support the use of this commercial method for determining PMB MICs in carbapenem-resistant Enterobacterales populations.