Acceptable performance of VITEK 2 system to detect extended-spectrum β-lactamases in clinical isolates of Escherichia coli: a comparative study of phenotypic commercial methods and NCCLS guidelinesommercial methods and NCCLS guidelines

Antimicrobial susceptibility study Acceptable performance of VITEK 2 system to detect extended-spectrum h-lactamases in clinical isolates of Escherichia coli: a comparative study of phenotypic commercial methods and NCCLS guidelines Antonio Sorlo´zano, Jose´ Gutie´rrez*, Gonzalo Pie´drola, Marı´a Jose´ Soto Department of Microbiology, School of Medicine, San Cecilio University Hospital, University of Granada, E-18012 Granada, Spain Received 12 July 2004; accepted 4 November 2004 Abstract The disk approximation method, Etest (AB Biodisk, Solna, Sweden), and VITEK 2 system (bioMe´rieux, Marcy l’Etoile, France) were used to study 399 extended-spectrum h-lactamase (ESBL)-producing (115 strains) and non-ESBL-producing (284 strains) clinical isolates of Escherichia coli after recommended procedures. Comparative study of the phenotypic findings yielded data on the sensitivity, specificity, and positive and negative predictive values and performance of each method. The sensitivity (100% using 2 substrates), specificity (99.3–100%), and predictive values of the disk approximation, Etest, and VITEK 2 methods were similar. D2005 Elsevier Inc. All rights reserved. Keywords: Escherichia coli; ESBL; Etest; VITEK 2 1. Introduction Extended-spectrum h-lactamases (ESBLs) are enzymes produced by Gram-negative bacteria, providing these with resistance to all cephalosporins, penicillins, and aztreonam (Bradford, 2001). Infected subjects commonly have greater morbidity and mortality when treated by antibiotics with inadequate in vitro activity (Paterson et al., 2002). Control of infection by these microorganisms is a major challenge that requires accurate and fast ESBL detection in clinical microbiology laboratories. Rapid characterization of isolates is especially important in the treatment of hospitalized patients. Because of the characteristics of theseenzymes, theisolates that produce them can appear falsely sensitive to many cephalosporins (Paterson and Yu, 1999) and therefore escape identification. The National Committee for Clinical Laboratory Standards (NCCLS, 2004) published norms for detecting ESBLs in vitro in Escherichia coli,Klebsiella pneumoniae, and K. oxytoca, establishing cutoff points for reduction of sensitivity to drugs usually hydrolyzed by these enzymes, with confirmation based on enzyme inhibition by clavulanic acid. Methods evaluated for ESBL detection include disk approximation (Jarlier et al., 1988), Etest (AB Biodisk, Solna, Sweden) (Cormican et al., 1996), combination disk method with cephalosporins alone plus cephalosporins with clavulanic acid (Carter et al., 2000), and automated antibiogram interpretation systems such as VITEK 2 (bioMe´rieux, Marcy-l’Etoile, France) (Sanders et al., 2000). However, these methods have not been compared using the same clinical isolates. The present study was designed to compare the capacity of disk approximation method, Etest, and VITEK 2 to detect ESBLs in clinical isolates of E. coli. 2. Material and methods A study was conducted of 115 ESBL-producing (group 1) and 284 non-ESBL-producing (group 2) clinical isolates of E. coli identified by biochemical tests (Murray et al., 2003). Clinical isolates were obtained during 2002 from patients treated at San Cecilio University Hospital, Granada; 71% of isolates were of outpatient origin, and 63% came from women. The phenotypic detection of ESBLs and the sensitivity to cefoxitin (FOX) and cefepime (FEP) were characterized by NCCLS disk diffusion method (NCCLS 2004). Strains were inoculated onto Mueller-Hinton agar plates (bioMe´rieux) 0732-8893/$ – see front matter D2005 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2004.11.001 * Corresponding author. Fax: +34 958 24 61 19. E-mail address: [email protected] (J. Gutie´rrez). Diagnostic Microbiology and Infectious Disease 51 (2005) 191–193 www.elsevier.com/locate/diagmicrobio and the following disks were used: cefpodoxime (CPD, 10 Ag), CPD plus clavulanic acid (CD01, 10/1 Ag), ceftazidime (CAZ, 30 Ag), CAZ plus clavulanic acid (CD02, 30/10 Ag), cefotaxime (CTX, 30 Ag), CTX plus clavulanic acid (CD03, 30/10 Ag), FOX (30 Ag), and FEP (30 Ag, Oxoid, England). ESBL presence was suspected when inhibition haloes of V17, 22, or 27 mm were obtained for CPD, CAZ, or CTX, respectively. ESBL presence was confirmed by demonstration of synergy between cephalosporin with reduced halo and clavulanic acid (defined by increase of z5 mm in inhibition halo of disks containing cephalosporin plus clavulanic acid versus disks with cephalosporin alone). Molecular typing was not performed. Group 1 included 54 isolates resistant to CTX and CPD and 61 isolates also resistant to CAZ, all showing synergy with clavulanic acid; 5 isolates were also resistant to FOX and 10 to FEP. Group 2 included 24 isolates resistant to CPD but without synergy with clavulanic acid and 36 resistant to FOX; none were resistant to FEP. Reference strains K. pneumoniae ATCC 700603 and E. coli ATCC 25922 were used. Assays were performed in duplicate and intraobserver agreement was tested. 2.1. Disk approximation method The method of Jarlier et al. (1988) was followed, using 30-Ag disks of CTX, CAZ, CPD, and ATM (BBL, Becton Dickinson, Franklin Lakes, NJ) at 30 mm from amoxicillin/ clavulanic acid (20/10 Ag) disk (BBL). Positive synergy and ESBL presence were defined by increase of z5 mm in the inhibition halo of cephalosporin or ATM or appearance of an area of intermediate synergy in presence of clavulanic acid. If no such increase was shown, the test was repeated using a distance of 20 mm to increase the sensitivity. 2.2. Etest Paper strips containing CAZ/CD02 and CTX/CD03 were used. ESBL presence was defined by reduction in CAZ or CTX minimum inhibitory concentration (MIC) of 3 or more dilutions in presence of clavulanic acid (Bradford, 2001). FEP ESBL tests were not used because no isolate was resistant to both cephalosporins and their combination with clavulanic acid. 2.3. VITEK 2 system VITEK 2 is an automated susceptibility testing system enabling rapid determination of MICs by analysis of bacteria growth kinetics with antibiotics in test cards. VITEK cards for susceptibility testing (AST-N020) were inoculated and incubated following manufacturer’s recommendations (Livermore et al., 2002). 2.4. Statistical analysis The diagnostic capacity of each phenotypic method was studied by analyzing the sensitivity, specificity, and positive and negative predictive values. 3. Results Table 1 lists results obtained from the comparative phenotypic study using VITEK 2, Etest, and disk approximation method in the 399 strains. The sensitivity, specificity, and positive and negative predictive values are shown in Table 2. 4. Discussion E. coli is the most frequently isolated enterobacterium in clinical microbiology laboratories, and possible resistances to antibiotics must be accurately detected. Table 3 summarizes the behavior in the laboratory of the studied methods. The Jarlier disk approximation approach is easy to perform and interpret but requires correct placement of the disks. The NCCLS method is also easy to use and interpret but the disks are used exclusively for this task. The Etest is again easy to use and interpret but colonies can appear in Table 1 Distribution of results for determination of ESBL presence by 3 phenotypic methods used ESBL Total Positive Negative VITEK 2 Positive 115 2 117 Negative 0 282 282 CAZ/CD02 a Positive 57 0 57 Negative 58 284 342 CTX/CD03 a Positive 115 0 115 Negative 0 284 284 CAZ/CD02 and CTX/CD03 a Positive 115 0 115 Negative 0 284 284 Disk approximation b Positive 115 0 115 Negative 0 284 284 a Etest results using the following: CAZ, ceftazidime; CD02, CAZ plus clavulanic acid; CTX, cefotaxime; CD03, CTX plus clavulanic acid. b Method as described by Jarlier et al. (1988). Table 2 Sensitivity, specificity, and predictive values of the phenotypic methods used Method Sensitivity (%) Specificity (%) PPV (%) NPV (%) VITEK 2 100 99.3 98.3 100 CAZ/CD02 a 49.6 100 100 83 CTX/CD03 a 100 100 100 100 CAZ/CD02 and CTX/CD03 a 100 100 100 100 Disk approximation b 100 100 100 100 PPV, positive predictive value; NPV, negative predictive value. a Etest results using the following: CAZ, ceftazidime; CD02, CAZ plus clavulanic acid; CTX, cefotaxime; CD03, CTX plus clavulanic acid. b Method as described by Jarlier et al. (1988). A. Sorlo´zano et al. / Diagnostic Microbiology and Infectious Disease 51 (2005) 191–193192 the halo (although not in the present study), increasing the MIC and complicating interpretations. Moreover, the strips are only used for this analysis. Although the CTX/CD03 strip detected all strains in the present study, this does not always occur, and the manufacturer recommends simultaneous use of CTX/CD03 and CAZ/CD02. According to Vercauteren et al. (1997), the CAZ/CD02 Etest detects 81% of ESBL-producing clinical isolates of E. coli, higher than the percentage detected in this study. The above methods are specific procedures that involve a diagnostic delay of 36–48 h. In contrast, VITEK 2 can diagnose ESBL presence 4 h after card inoculation. Furthermore, the behavior of VITEK 2 in this study suggests that confirmation of E. coli results may not be necessary. Sensitivity and specificity values obtained were somewhat better than those reported by Leverstein-van Hall et al. (2002) (100% sensitivity, 87% specificity), Sanders et al. (2000) (91% sensitivity), and Livermore et al. (2002) (93% sensitivity), although these other studies evaluated ESBLpositive strains belonging to various species (and producing different types of ESBL), not only E. coli. Moreover, VITEK 2 is performed as part of routine laboratory work and does not require a specific procedure. In conclusion, VITEK-2 showed an acceptable capacity to detect ESBL-producing E. coli in our setting. Acknowledgments The authors are grateful to Oxoid, bioMe´rieux, and IZASA SA for supplying the reagents used in this study. References Bradford PA (2001) Extended-spectrum h-lactamases in the 21st century: Characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 14:933–951. Carter MW, Oakton KJ, Warner M, Livermore DM (2000) Detection of extended-spectrum h-lactamase in Klebsiellae with Oxoid combination disk method. J Clin Microbiol 38:4228– 4232. Cormican MG, Marshall SA, Jones RN (1996) Detection of extendedspectrum beta-lactamases (ESBL) producing strains by Etest ESBL screen. J Clin Microbiol 34:1880–1884. Jarlier V, Nicolas MH, Fournier G, Philippon A (1988) Extended broadspectrum beta-lactamases conferring transferable resistance to newer beta-lactam agents in Enterobacteriaceae: Hospital prevalence and susceptibility patterns. Rev Infect Dis 10:867–878. Leverstein-van Hall MA, Fluit AC, Paauw A, Box AT, Brisse S, Verhoef J (2002) Evaluation of the Etest ESBL and the BDPhoenix, VITEK 1, and VITEK 2 automated instruments for detection of extendedspectrum h-lactamases in multiresistant Escherichia coli and Klebsiella spp. J Clin Microbiol 40:3703–3711. Livermore DM, Struelens M, Amorim J, et al (2002) Multicentre evaluation of the VITEK 2 Advanced Expert System for interpretive reading of antimicrobial resistance tests. J Antimicrob Chemother 49: 289– 300. Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH (2003) Manual of clinical microbiology. 8th ed. Washington (DC)7ASM. National Committee for Clinical Laboratory Standards (2004) Performance standards for antimicrobial susceptibility testing Approved standard M100-S14. Wayne (PA)7NCCLS. Paterson DL, Yu VL (1999) Extended-spectrum h-lactamases: A call for improved detection and control. Clin Infect Dis 29:1419–1422. Paterson DL, Ko WC, Von Gottberg A, et al (2002) Outcome of cephalosporin treatment for serious infections due to apparently susceptible organisms producing extended-spectrum h-lactamases: Implications for the clinical microbiology laboratory. J Clin Microbiol 39:2206–2212. Sanders CC, Peyret M, Moland ES, et al (2000) Ability of the VITEK 2 Advanced Expert System to identify h-lactam phenotypes in isolates of Enterobacteriaceae and Pseudomonas aeruginosa.J Clin Microbiol 38: 570– 574. Vercauteren E, Descheemaeker P, Ieven M, Sanders CC, Goossens H (1997) Comparison of screening methods for the detection of extendedspectrum h-lactamases and their prevalence among blood isolates of Escherichia coli and Klebsiella spin a Belgian teaching hospital. J Clin Microbiol 35:2191–2197. Table 3 Characteristics of the methods used to detect ESBL Test Is the test for ESBL a specific procedure? Interpretation Delay in obtaining results Benefits Drawbacks VITEK 2 No. It is performed as part of routine laboratory work. Automated by an expert system Minimum 4 h Automated interpretation and rapid result The high price limits its use to hospital isolates. Etest Requires specific reagents Ratio between MICs obtained Minimum 24 h MIC report Precision is required in the correct reading of the MICs. Disk approximation (Jarlier method) Requires correct placement of the disks with antibiotics. Requires demonstration of synergy with clavulanic acid Minimum 24 h Simple performance in the laboratory Precision is required in the correct reading of the inhibition haloes. Disk combination (NCCLS) Requires specific reagents Requires demonstration of synergy with clavulanic acid Minimum 24 h Easy results interpretation. It does not rule out other simultaneous resistance mechanisms A. Sorlo´zano et al. / Diagnostic Microbiology and Infectious Disease 51 (2005) 191–193 193