These authors contributed equally.
Evaluation of a diagnostic flow chart for detection and confirmation of extended spectrum β-lactamases (ESBL) in Enterobacteriaceae
Article first published online: 20 JAN 2012
© 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases
Clinical Microbiology and Infection
Volume 18, Issue 12, pages 1194–1204, December 2012
How to Cite
Polsfuss, S., Bloemberg, G. V., Giger, J., Meyer, V., Böttger, E. C. and Hombach, M. (2012), Evaluation of a diagnostic flow chart for detection and confirmation of extended spectrum β-lactamases (ESBL) in Enterobacteriaceae. Clinical Microbiology and Infection, 18: 1194–1204. doi: 10.1111/j.1469-0691.2011.03737.x
- Issue published online: 13 NOV 2012
- Article first published online: 20 JAN 2012
- Accepted manuscript online: 26 NOV 2011 01:26AM EST
- Original Submission: 21 July 2011; Revised Submission: 21 November 2011; Accepted: 21 November 2011 Editor: F. AllerbergerArticle published online: 26 November 2011
- flow chart;
- Top of page
- Transparency Declarations
Clin Microbiol Infect 2012; 18: 1194–1204
This study aimed to develop a modular, diagnostic algorithm for extended spectrum β-lactamase (ESBL) detection in Enterobacteriaceae. Clinical Enterobacteriaceae strains (n = 2518) were screened for ESBL production using Clinical and Laboratory Standards Institute (CLSI) breakpoints for third-generation cephalosporins and by synergy image detection (clavulanic acid/extended-spectrum cephalosporins). Isolates screening positive for ESBL (n = 242, 108 by critical CLSI diameters alone, five by double disk synergy test (DDST) alone, and 129 by both critical diameters and DDST) and 138 ESBL screening negative isolates (control group) were investigated by molecular methods considered to be the reference standard (multiplex CTX-M type PCR, TEM and SHV type sequence characterization). One hundred and twenty-four out of 242 Enterobacteriaceae isolates screening positive for ESBL were confirmed to be ESBL positive by the reference standard, the majority of them in E. coli, K. pneumoniae and E. cloacae (94, 17 and nine isolates, respectively). Prevalence of ESBL production ranged from <1% for P. mirabilis to 4.7%, 5.1% and 6.6%, for K. pneumoniae, E. cloacae and E. coli, respectively. Combining CLSI ceftriaxone and cefpodoxime critical ESBL diameters was found to be the most sensitive phenotypic screening method (sensitivity 99.2%). Combining critical diameters of cefpodoxime and ceftriaxone with DDST for cefpodoxime resulted in a sensitivity of 100%. For phenotypic confirmation, combining the CLSI recommended combined disk test (CDT) for ceftazidime and cefotaxime amended with a cefepime CDT was highly sensitive (100%) and specific (97.5%). With respect to the studied population, the diagnostic ESBL algorithm developed would have resulted in sensitivity and specificity of 100%. The corresponding flow chart is simple, easy to use, inexpensive and applicable in the routine diagnostic laboratory.
- Top of page
- Transparency Declarations
Increasing prevalence of multidrug-resistant Gram-negative bacteria has continuously been reported over the past years, in particular Enterobacteriaceae producing extended spectrum β-lactamases (ESBLs). ESBLs have the ability to hydrolyse penicillins, first- second- and third-generation cephalosporins and aztreonam (but not cephamycins or carbapenems), and their activity is decreased by inhibitors such as clavulanic acid . ESBL-producing organisms may be responsible for life-threatening infections, leading to increased morbidity, mortality and healthcare-associated costs [2–5].
Klebsiella pneumoniae and Escherichia coli are the most frequently isolated ESBL-producing strains worldwide; however, ESBLs have been identified in other members of the Enterobacteriaceae family . Most ESBLs can be classified in TEM, SHV and CTX-M types . To date, over 100 SHV- and 150 TEM-ESBL types and approximately 90 CTX-M variants (clustered in five groups, CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25) have been identified (for actual update see http://www.lahey.org/studies). In recent years CTX-M type β-lactamase-producing E. coli have emerged and now represent the most prevalent ESBL type in Europe and North America [8,9].
Until 2009 the Clinical and Laboratory Standards Institute (CLSI) recommended adjusting the results of in vitro drug susceptibility testing for penicillins, cephalosporins, cefepime and monobactams and reporting these compounds as resistant to ESBL-producing isolates of E. coli, K. pneumoniae, Klebsiella oxytoca and Proteus mirabilis . In 2010, the CLSI released new cephalosporin susceptibility breakpoints for Enterobacteriaceae. Subsequent adjustment of in vitro susceptibility test results for β-lactams due to ESBL production is no longer recommended, but screening for ESBL-producing isolates is still considered useful and should be continued for epidemiological reasons . The new CLSI strategy is widely in agreement with the guidelines of the European Committee for Antimicrobial Susceptibility Testing (EUCAST), which considers screening for ESBL-producing isolates mandatory for epidemiological purposes .
Different phenotypic tests for ESBL detection have been evaluated and described in the literature. However, little information is available on the performance parameters of standardized approaches combining single methods to identify ESBL-producing Enterobacteriaceae spp. [13–15]. The aim of this study was to develop and prospectively evaluate a standardized, inexpensive and simple approach that is able to detect ESBL-producing Enterobacteriaceae isolates in a routine clinical laboratory.
- Top of page
- Transparency Declarations
In this prospective study all non-duplicate clinical Enterobacteriaceae isolates (n = 2518) isolated over a period of 7 months from October 2009 until April 2010 were systematically screened for ESBL production. Only isolates that were considered clinically relevant were included. Table 1 shows the study design, the numbers of studied clinical isolates and the overview of ESBL detection results. Two hundred and forty-two of the 2518 clinical isolates were selected for further characterization on the basis of (i) positive CLSI screening cut-off values for ESBL for at least one third-generation cephalosporin tested, and/or (ii) observation of a synergy zone between amoxicillin-clavulanic acid and cefpodoxime and/or ceftazidime, and/or ceftriaxone and/or cefotaxime. Of the 242 ESBL suspicious isolates, 108 were selected by critical CLSI diameters alone, five by double disk synergy test (DDST) alone, and 129 by both critical diameters and DDST. Ninety-one of the 242 ESBL suspicious isolates additionally produced an AmpC enzyme.
|Enterobacteriaceae isolates n = 2518|
|Critical ESBL diameters (CLSI) positive for CPD and/or CAZ and/or CTX and/or CRO and/or Synergy CAZ/AMC and/or CDP/AMC and/or synergy CTX/TZP and/or FEP/TZP|
|ESBL screening negative n = 2276||Putative ESBL producersa n = 242|
|ESBL negative control group (representative sample) n = 138|
|AmpC negative n = 113||AmpC positive n = 19||K. oxytoca n = 6||AmpC negative n = 134||AmpC positive n = 91||K. oxytoca n = 17|
|Negative CDT: CAZ and/or CTX and/or FEP n = 113||Positive CDT: CAZ and/or CTX and/or FEP n = 0||Negative CDT: CAZ and/or CTX and/or FEP n = 19||Positive CDT: CAZ and/or CTX and/or FEP n = 0||Negative CDT: CAZ n = 6||Positive CDT: CAZ n = 0||Negative CDT: CAZ and/or CTX and/or FEP n = 25||Positive CDT: CAZ and/or CTX and/or FEP n = 109||Negative CDT: CAZ and/or CTX and/or FEP n = 76||Positive CDT: CAZ and/or CTX and/or FEP n = 13 Borderline n = 2||Negative CDT: CAZ n = 16||Positive CDT: CAZ n = 1|
|Negative CDT: CTX and/or FEP n = 6||Positive CDT: CTX and/or FEP n = 0||Negative CDT: CTX and/or FEP n = 14||Positive CDT: CTX and/or FEP n = 3|
|ESBL genetic analysis (CTX-M multiplex PCR and SHV and TEM sequence characterization) n = 380|
|ESBL negative n = 113||ESBL positive n = 0||ESBL negative n = 19||ESBL positive n = 0||ESBL negative n = 6||ESBL positive n = 0||ESBL negative n = 25||ESBLb positive n = 109||ESBL negative n = 78||ESBL positive n =13||ESBL negative n = 15||ESBL positive n = 2|
To assess sensitivity of the initial screening procedure a negative control group (138 negative in the ESBL screening) was selected, in which species distribution resembled that of the 2518 study isolates (representative sample). The 138 control isolates showed: (i) negative ESBL CLSI screening cut-off values for all third-generation cephalosporins tested; and (ii) no visible synergy zone between a β-lactamase inhibitor disk and a third-generation cephalosporin disk. To develop and evaluate the ESBL detection algorithm the 380 isolates (242 positive isolates screening positive for ESBL and 138 negative control isolates) were characterized for ESBL production by a combination of phenotypic screening and confirmation methods, with genetic methods (CTX-M-Multiplex PCR, TEM and SHV sequencing) serving as the reference standard (Table 1).
For susceptibility testing the disk diffusion method according to Kirby-Bauer was used. Antibiotic disks (Becton Dickinson, Franklin Lakes, NJ, USA) were selected and results were interpreted according to the guidelines of the CLSI . The following ESBL CLSI screening cut-off values were used for third-generation cephalosporins: cefpodoxime (10 μg/disk) ≤ 17 mm, ceftazidime (30 μg/disk) ≤ 22 mm, cefotaxime (30 μg/disk) ≤ 27 mm, and ceftriaxone (30 μg/disk) ≤ 25 mm. Susceptibility testing was carried out on Mueller-Hinton agar (bioMérieux, Marcy L’Etoile, France) using McFarland 0.5 from overnight cultures followed by incubation at 35°C for 16–18 h.
AmpC-β-lactamase (AmpC) detection
All isolates included in this study were systematically characterized for the production of an AmpC type β-lactamase as described . In brief, a multiplex PCR was used for detection of plasmid-mediated ampCβ-lactamase genes, which detects the six plasmid-mediated ampC families. The ampC promoter mutation analysis of E. coli isolates was carried out as described previously .
Double disk synergy test (DDST)
The double disk synergy test was conducted as described . Synergy images were recorded between amoxicillin-clavulanic acid disks (20/10 μg/disk) and cefpodoxime, ceftazidime, ceftriaxone or cefotaxime disks. Antibiotic disks were placed 27 mm apart, centre-to-centre. β-lactam inhibitor-mediated enhancement of the third-generation cephalosporin inhibition zone was interpreted as synergy positive.
Combined disk test (CDT)
The CLSI recommended combined disk test (CDT) was performed with ceftazidime and cefotaxime disks with and without clavulanic acid . Additionally, disks containing 30 μg cefepime and 30 μg cefepime/10 μg clavulanic acid were produced for this study (Liofilchem, Roseto degli Abruzzi, Italy) because this combination was not available from Becton Dickinson. AmpC non-producing strains were inoculated on Mueller-Hinton agar using McFarland 0.5 followed by incubation at 35°C for 16–18 h. For AmpC-producing isolates CDT was compared on Mueller-Hinton agar and Mueller-Hinton agar containing 250 mg/L of cloxacillin (AES CHEMUNEX, Bruz Cedex, France). A difference in the inhibition zone diameters of ≥5 mm for any third-generation cephalosporin/clavulanic acid combination vs. the corresponding third-generation cephalosporin alone was considered indicative of ESBL production.
TEM and SHV ESBL identification
DNA was extracted from colonies grown on sheep blood agar medium using the InstaGene Matrix (Bio-Rad, Reinach, Switzerland) following the manufacturer’s instructions. Molecular TEM and SHV ESBL detection was carried out as described elsewhere . TEM and SHV β-lactamase sequences were compared with E. coliAF427133.1 TEM-1 and E. coliAF148850 SHV-1 using the publicly available database at http://www.lahey.org/studies. Each sequence electropherogram was screened for the presence of double peak signals, which would indicate the presence of multiple SHV or TEM variants.
CTX-M β-lactamase gene identification
For the detection of CTX-M β-lactamase genes a multiplex PCR was performed as described by Pitout et al. . This multiplex PCR detects the CTX-M group 1, CTX-M group 2, CTX-M group 8 (former group III) and CTX-M group 9 (former group IV) genes. In the case of K. oxytoca, the CTX-M primers for group 8 (former group III) also target similar DNA sequences in the chromosomal K1 gene. To discriminate K1 from CTX-M group 8 amplification, CTX-M group 8 (former group III) genes in K. oxytoca were confirmed in a second step by amplification of a 666 bp fragment using primers ‘group 8 forward’ (5′-TCGCGTTAAGCGGATGATGC-3′) and ‘group 8/25 reverse’ (5′-AACCCACGATGTGGGTAGC-3′) as published by Woodford et al. .
Molecular methods were considered to be the reference standard for calculation of performance parameters.
- Top of page
- Transparency Declarations
Screening and confirmation of Enterobacteriaceae isolates for ESBL production
One hundred and twenty-four initial isolates screening positive for ESBL were confirmed as ESBL producers by phenotypic and molecular methods (Table 2). The majority of ESBL producers were found in E. coli, K. pneumoniae and E. cloacae (94, 17 and 9 isolates, respectively). Prevalence of ESBL production ranged from <1% for P. mirabilis to 4.7%, 5.1% and 6.6% for K. pneumonia, E. cloacae and E. coli, respectively. The mean prevalence of ESBL production in the 2518 Enterobacteriaceae isolates was 4.9% (Table 2). The highest proportion of false-positive ESBL screening tests was observed with E. cloacae and K. oxytoca isolates (Table 2).
|Isolates (n, %)||ESBL screening positives (n, % of isolates)||Confirmed ESBLs (n, % of isolates)||CTX-M types (N, % of all ESBL)||SHV ESBL type (n, % of all ESBL)||TEM ESBL type (n, % of all ESBL)|
|Group 1||Group 8||Group 9|
|All species||2518 (100.0)||242 (9.5)||124 (4.9)||86 (69.4)||1 (0.8)||23 (18.5)||11 (8.9)||3 (2.4)|
|Escherichia coli||1435 (57.0)||136 (9.5)||94 (6.6)||66||1||17||8||2|
|Klebsiella pneumoniae||360 (14.2)||31 (8.6)||17 (4.7)||14||0||2||1||0|
|Enterobacter cloacae||178 (7.1)||34 (19.1)||9 (5.1)||6||0||3||0||0|
|Proteus mirabilis||131 (5.2)||2 (1.5)||1 (0.8)||0||0||0||0||1|
|Klebsiella oxytoca||99 (3.9)||17 (17.2)||2 (2.0)||0||0||1||1||0|
|Citrobacter. spp.||1 (0.1)||1 (100.0)||1 (100.0)||0||0||0||1||0|
|Othersa||314 (12.5)||14 (4.5)||0|
CTX-M type genes were predominantly detected among ESBL strains (88.7% of all ESBL), with CTX-M group 1 as the most frequently identified subtype (69.4%). The second most frequently isolated subtype was CTX-M group 9 (18.5%); SHV and TEM ESBL types were observed in 8.9% and 2.4% of all ESBL-producing isolates, respectively (Table 2).
Enterobacteriaceae species were isolated from different clinical materials (Table 3). The majority of ESBL isolates was isolated from urine (51.6%), groin (12.9%), respiratory tract (12.1%) and wound (9.7%) specimens.
|Sample origin||Enterobacteriaceae isolates (n, %)||ESBL isolates (n, %)|
|Urine||1002 (39.8)||64 (51.6)|
|Wound||332 (13.2)||12 (9.7)|
|Respiratory tracta||264 (10.5)||15 (12.1)|
|Groin||81 (3.2)||16 (12.9)|
|Blood culture||209 (8.3)||2 (1.6)|
|Vagina||40 (1.6)||2 (1.6)|
|Miscellaneous||546 (21.7)||4 (3.2)|
|Unknownb||43 (1.7)||9 (7.3)|
|Total||2518 (100)||124 (100)|
All 138 isolates of the negative control group (initial ESBL screening negative) were confirmed ESBL negative by molecular methods. This result was extrapolated to all 2276 isolates negative in initial screening for ESBL and performance parameters of all tests and the algorithm including positive predictive value (PPV) and negative predictive value (NPV) were recalculated (Table 4).
|Method/Method combination||Isolates (n)||TP (n)||FP (n)||TN (n)||FN (n)||Sensitivity (%)||Specificity (%)||PPV (%)||NPV (%)|
|CRO and CPD||2518||123||99||2295||1||99.2||95.9||55.4||99.9|
|CRO and CAZ||2518||122||89||2305||2||98.4||96.3||57.8||99.9|
|CRO and CTX||2518||122||99||2295||2||98.4||95.9||55.2||99.9|
|CPD and CTX||2518||122||104||2290||2||98.4||95.7||54.0||99.9|
|CPD and CAZ||2518||121||91||2297||3||97.6||96.2||57.1||99.9|
|CTX and CAZ||2518||122||97||2303||2||98.4||96.0||55.7||99.9|
|CRO and CPD and CTX||2518||123||110||2284||1||99.2||95.4||52.8||99.9|
|Synergy AMC CPD||2518||109||16||2378||15||87.9||99.3||87.2||99.4|
|Synergy AMC CAZ||2518||105||10||2384||19||84.7||99.6||91.3||99.2|
|Synergy AMC CTX||2518||109||12||2385||12||90.1||99.5||90.1||99.5|
|Synergy AMC CRO||2518||115||6||2389||8||93.5||99.7||95.0||99.7|
|Synergy AMC CPD and/or CAZ||2518||113||18||2376||11||91.1||99.2||86.3||99.5|
|Critical diameters plus DDST|
|CRO and CPD plus synergy AMC CPD||2518||123||108||2287||0||100.0||95.5||53.2||100.0|
|CAZ CTX FEP plus/minus CLA combined||2518||123||3||2391||0||100.0||99.9||97.6||100.0|
Comparison of primary screening markers for ESBL production
Considering critical diameters as the primary screening marker for ESBL detection, ceftriaxone showed the highest sensitivity (98.4%). One ESBL-producing isolate was detected exclusively by the critical diameter of cefpodoxime; this isolate was also positive with the cefpodoxime and ceftazidime DDST. Ceftazidime diameters alone showed the lowest sensitivity for ESBL detection (72.6%). The most sensitive combination was ceftriaxone with cefpodoxime (sensitivity 99.2%); adding cefotaxime to these two substances did not result in a further increase of sensitivity (Table 5). One ESBL-positive isolate was exclusively detected by the DDST with cefpodoxime and/or ceftazidime compared with both critical diameters and the other DDSTs.
|Method/Method combination||Isolates (n)||TP (n)||FP (n)||TN (n)||FN (n)||Sensitivity (%)||Specificity (%)|
|CRO and CPD||242||123||99||19||1||99.2||16.1|
|CRO and CAZ||242||122||89||29||2||98.4||24.6|
|CRO and CTX||242||122||99||19||2||98.4||16.1|
|CPD and CTX||242||122||104||14||2||98.4||11.9|
|CPD and CAZ||242||121||91||27||3||97.6||22.9|
|CTX and CAZ||242||122||97||21||2||98.4||17.8|
|CRO and CPD and CTX||242||123||110||8||1||99.2||6.8|
|Synergy AMC CPD||242||109||16||102||15||87.9||86.4|
|Synergy AMC CAZ||242||105||10||108||19||84.7||91.5|
|Synergy AMC CTX||242||109||12||109||12||90.1||90.1|
|Synergy AMC CRO||242||115||6||113||8||93.5||95.0|
|Synergy AMC CPD and/or CAZ||242||113||18||100||11||91.1||84.7|
|Critical diameters plus DDST|
|CRO and CPD plus synergy AMC CPD||242||123||108||11||0||100.0||9.2|
|CAZ CTX FEP plus/minus CLA combined||242||123||3||115||0||100.0||97.5|
Combining critical diameters for ceftriaxone and cefpodoxime with the cefpodoxime DDST resulted in a sensitivity of 100% for the potentially ESBL-producing clinical strains used in this study (Table 5). Of 16 false-positive cefpodoxime DDST, one was observed in E. cloacae, one in K. pneumoniae, six in E. coli and eight were seen with K. oxytoca. Of 10 false-positive ceftazidime DDST, four occurred with E. coli, four with K. oxytoca, and two with K. pneumoniae. Of 12 false-positive cefotaxime DDST, five were observed with E. coli and seven were seen in K. oxytoca. Of six false-positive ceftriaxone DDST, two were seen in E. coli and four in K. oxytoca. Two of the E. coli isolates listed above showed false-positive DDSTs for all screening agents (cefpodoxime, ceftazidime, cefotaxime and cefepime). One of these isolates produced a CIT type AmpC. Of the 124 confirmed ESBL producers, 10 were detected by critical CLSI diameters alone, one by the DDST alone, and 113 by both methods.
Evaluation of confirmation assays for ESBL production
The CLSI recommended CDTs with ceftazidime and/or cefotaxime and clavulanic acid as inhibitors were compared as phenotypic confirmation tests using molecular methods as the reference standard. Additionally, a cefepime CDT with clavulanic acid as inhibitor was evaluated. All third-generation cephalosporin-clavulanic acid combinations showed a specificity of >98.3%. For the ceftazidime CDT a sensitivity of 82.9% was observed; cefotaxime and cefepime CDTs showed a sensitivity of 99.2% and 91.1%, respectively (Table 5). Four ESBL-positive isolates were detected by the cefotaxime CDT alone; one ESBL was detected by the cefepime CDT alone. In contrast, the ceftazidime CDT alone did not identify any ESBL-producing isolates. If all CDTs were combined, sensitivity and specificity were 100% and 97.5%, respectively. Extrapolated to the 2518 study isolates, the sensitivity, specificity, PPV and NPV of all CDTs combined were 100%, 99.9%, 97.6% and 100%, respectively (Table 4).
Using 91 AmpC-positive ESBL screening positive isolates (Table 1), the three CDTs were compared on Mueller-Hinton agar and Mueller-Hinton agar supplemented with cloxacillin. A significantly increased overall sensitivity of the combined three CDTs was observed when using cloxacillin containing agar (sensitivity 100% and 84.6% for Mueller-Hinton agar with and without cloxacillin, respectively; see Table 6). Overall specificity was equal for both media types (97.4%). Two of the 13 ESBL-positive isolates in AmpC producers would have been missed by all CDTs using conventional Mueller-Hinton agar, while CDTs on Mueller-Hinton agar with cloxacillin detected all 13 ESBLs.
|Method||Isolates (n)||TP (n)||FP (n)||TN (n)||FN (n)||Sensitivity (%)||Specificity (%)|
|MH with cloxacillin|
|CAZ CTX FEP plus/minus CLA combined||91||13||2||76||0||100||97.4|
|MH without cloxacillin|
|CAZ CTX FEP plus/minus CLA combined||91||11||2||76||2||84.6||97.4|
Two isolates were considered as ‘borderline results’ with the CDT combination, because merely one CDT showed a diameter difference of ≤7 mm. In these two isolates TEM-, SHV- and CTX-M type ESBLs could not be detected by molecular methods (Table 1).
In total, 23 K. oxytoca isolates were analysed: 17 were putative ESBL producers, six served as a negative control group (Table 1). Of the 17 isolates positive in the ESBL screening, one isolate was correctly identified as SHV type ESBL by the ceftazidime, cefotaxime and cefepime CDTs. A second K. oxytoca isolate with a negative result in the ceftazidime CDT and positive results in the cefotaxime and cefepime CDTs was identified as CTX-M group 1 type ESBL by PCR (false-negative ceftazidime CDT). In a third K. oxytoca isolate with positive results in the cefotaxime and cefepime CDTs but a negative result in the ceftazidime CDT the presence of an ESBL could not be confirmed by molecular methods (false-positive cefotaxime and cefepime CDTs). The remaining 20 isolates showed congruent results between phenotypic and genotypic tests. All 23 K. oxytoca isolates would have been correctly assigned as ESBL positive (two isolates) and ESBL negative (21 isolates) by the proposed flow chart (see below).
Proposed flow chart for ESBL detection in Enterobacteriaceae
Evaluating the results of the various screening and confirmation assays we aimed to use this information to propose a flow chart for reliable and standardized ESBL detection in Enterobacteriaceae. The proposed procedure is based on: (i) cefpodoxime and ceftriaxone diameters combined with the clavulanic acid DDST as the primary screening markers; (ii) CDTs using ceftazidime, cefotaxime and cefepime combined as phenotypic confirmation tests; (iii) ceftazidime CDT for K. oxytoca in combination with a CTX-M type PCR added in case of a negative result; and (iv) molecular methods added in the case of borderline results with CDTs. For ESBL detection the sensitivity and specificity of the phenotypic algorithm alone would have been 100% and 97.5%, respectively, for the isolates investigated in this study (Fig. 1 and Table 5). Adding molecular methods (required for resolution of borderline phenotypic confirmation tests) would increase specificity to 100%. Extrapolated to the 2518 study isolates (both initial ESBL screening positives and negatives) the ESBL detection algorithm would have displayed a sensitivity, specificity, PPV and NPV each of 100% (see Table 4).
- Top of page
- Transparency Declarations
Detection of ESBL is potentially critical to ensure adequate therapy and to guide epidemiological and hospital hygiene measures [13,20,21]. Laboratory methods for screening and confirmation of ESBL should be accurate, simple and rapid. During the past decades several phenotypic and molecular tests have been evaluated for ESBL detection [13,14,22], though a comprehensive algorithm is lacking. Molecular methods have been suggested as screening methods for ESBL [23–25]. However, in routine clinical diagnostic laboratories implementation of molecular methods is often hampered by the complexity of these assays, personnel needs and associated costs. Hence, an integrative approach combining phenotypic and molecular methods seems to be best suited for optimal and cost-efficient ESBL detection. This study aimed to develop an integrated phenotypic and molecular algorithm for the detection of ESBL production in Enterobacteriaceae. Automated phenotypic methods were not considered in our study because these methods: (i) reportedly show weaknesses in sensitivity and specificity; and (ii) include E. coli and Klebsiella spp. in their expert systems only [14,26–31].
Detection of ESBL is based on a combination of sensitive screening and confirmation assays. First, we evaluated the use of third-generation cephalosporin critical diameters and DDSTs as primary ESBL screening tests. Ceftriaxone critical diameters demonstrated highest sensitivity (98.4%) as a single substance (Table 5). One ESBL isolate, however, was detected exclusively by cefpodoxime. The combination of ceftriaxone and cefpodoxime was the most sensitive screening parameter (99.2%). Addition of cefotaxime as a third substance did not improve sensitivity, but resulted in a decrease of specificity (see Table 5). We do not consider ceftazidime as a primary screening marker on its own because sensitivity was low (72.6%) and no ESBL-producing isolate was exclusively detected by ceftazidime. This finding is most probably related to the higher hydrolytic activity of CTX-M type enzymes on cefotaxime compared with ceftazidime . Sensitivity of ceftazidime in a population with an increased proportion of SHV-type ESBLs may possibly be higher. However, predominance of CTX-M type ESBLs in our study population closely resembles the epidemiological situation in Europe and North America [33–35]. Our results are in agreement with other studies, which showed: (i) cefpodoxime critical diameters to be highly sensitive for ESBL detection, but poorly specific; and (ii) combinations of third-generation cephalosporin critical diameters to result in a higher specificity [31,36]. Critical diameters of third-generation cephalosporins alone or in combination, however, were not able to detect all ESBLs, but combining critical diameters of cefpodoxime and ceftriaxone with DDST for cefpodoxime resulted in a sensitivity of 100% for ESBL screening (Table 5). Thus, a combination of three disks was required for initial and sensitive screening: cefpodoxime, ceftriaxone and amoxicillin-clavulanic acid. In particular, for AmpC β-lactamase-producing species, such as E. cloacae, our results parallel the data reported previously [14,22].
After determination of the most sensitive screening procedure, we evaluated the CDT as an inexpensive and simple phenotypic confirmation method. The Etest ESBL (bioMérieux) is widely used, but was not evaluated in this study for several reasons. The test: (i) shows low specificity for ESBL production in Enterobacteriaceae isolates other than E. coli; (ii) is significantly more expensive than the CDT; and (iii) was shown to be unreliable if used with isolates producing AmpC β-lactamases [14,37]. ESBLs may be missed by phenotypic confirmation tests if an AmpC enzyme is present. For example, in this study all false-negative results with the ceftazidime CDT occurred in E. cloacae isolates overexpressing AmpC . Thus, two modifications of the CLSI recommended CDT were evaluated: (i) performing CDT for AmpC-producing isolates on Mueller-Hinton agar containing cloxacillin that suppresses AmpC activity; and (ii) addition of a cefepime CDT . Our results for the CDT comparing Mueller-Hinton agar with and without cloxacillin show a significantly enhanced sensitivity for the cloxacillin-containing plates (see Table 6). Cefepime is poorly hydrolysed by AmpC enzymes. Addition of a cefepime CDT can, thus, improve the performance of the CLSI recommended cefotaxime and ceftazidime CDTs in the present epidemiological setting with ESBLs frequently present in species producing AmpC β-lactamases. None of the three CDTs applied alone resulted in a sensitivity of 100% (Table 5). In contrast, combining three CDT substances including cefepime proved to be highly sensitive and specific (sensitivity and specificity of 100% and 97.5%, respectively). If performance parameters were calculated for AmpC-producing isolates alone the cefepime CDT displayed sensitivity and specificity both of 100% using Mueller-Hinton agar with cloxacillin (Table 6). All false-negative results in AmpC-producing species listed in Table 6 were seen in E. cloacae isolates overexpressing AmpC using Mueller-Hinton agar without cloxacillin. Our results for the three CDTs are in agreement with data from other studies [14,39]. The ceftazidime CDT did not exclusively detect any ESBL-positive isolate. Omitting ceftazidime CDT as a confirmation test, however, cannot be recommended because ceftazidime is recommended for ESBL confirmation in K. oxytoca by EUCAST to avoid false-positive results with cefotaxime . According to EUCAST rules, one ceftazidime CDT negative, cefotaxime and cefepime CDT positive K. oxytoca isolate overexpressing K1-β-lactamase was correctly identified as ESBL negative in our study. For K. oxytoca the ceftazidime CDT alone should, therefore, be considered as a phenotypic confirmation test to avoid false-positive results. If the ceftazidime CDT gives a negative result a CTX-M type PCR should be carried out for isolates screening positive for ESBL because a ceftazidime CDT negative isolate may have a CTX-M type ESBL (one isolate in this study, Table 1).
Combining three CDTs facilitated correct assignment of borderline CDT results to the ESBL positive or negative category. We considered results borderline or inconclusive if merely one of the three CDTs was marginally positive (Fig. 1) Usually, such isolates would be classified as ESBL positive because one positive CDT alone is considered indicative of ESBL . The two isolates considered ‘borderline’ were subjected to molecular confirmation methods and were shown to be ESBL negative. To define and resolve ‘borderline’ criteria further studies with higher numbers of borderline CDT results are needed.
One possible concern is that the increasing prevalence of carbapenemases will interfere with this diagnostic algorithm. In the case of metallo-carbapenemase producers like NDM-1, CDTs with clavulanic acid would most likely give negative or inconclusive test results because those enzymes are characteristically not inhibited by clavulanic acid . Carbapenemases of Ambler class A (e.g. Klebsiella pneumoniae carbapenemase, KPC) that are inhibited by clavulanic acid could produce false-positive CDTs. Isolates harbouring carbapenemases are, however, readily detected by the use of carbapenems as screening markers . All 2518 isolates included in this study were categorized as susceptible to imipenem, meropenem and ertapenem according to CLSI 2009 guidelines . All ESBL suspicious, but ESBL confirmation test negative, isolates in this study have been additionally checked for the presence of KPC by PCR and were found to be KPC negative (data not shown).
In summary, the proposed diagnostic flow chart (Fig. 1) would have resulted in a sensitivity and specificity of 100% for ESBL detection in the isolates tested. This algorithm is accurate, simple, easy to use, inexpensive and, therefore, applicable in the standard routine clinical microbiology laboratory. In the case of inconclusive results, which will rarely (<1%) occur, molecular methods are recommended. Such a diagnostic flow chart may be automatized in an open expert system as described by Winstanley et al. .
- Top of page
- Transparency Declarations
We thank Professor Reinhard Zbinden for critical discussions and Claudia Merkofer for expert technical assistance.
- Top of page
- Transparency Declarations
This work was supported by the University of Zurich. There are no conflicts of interest to declare.
- Top of page
- Transparency Declarations
- 10Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; nineteenth informational supplement. CLSI document M 100-S 19. Wayne, PA, USA: CLSI, 2009.
- 11Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; twentieth informational supplement. CLSI document M 100-S 20. Wayne, PA, USA: CLSI, 2010.
- 12European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and Zone diameters. Version 1.3. http://www.eucast.org/clinical_breakpoints. Accessed 3 November 2011.
- 25Molecular diagnostics could help in coping with hidden beta-lactamases. Microbe2010; 5: 333–339..
- 29Evaluation of the capability of the Vitek 2 system to detect extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates, in particular with the coproduction of AmpC enzymes. Eur J Clin Microbiol Infect Dis2009; 28: 871–874., , , , .
- 30Evaluation of the Vitek-2 extended-spectrum beta-lactamase test against non-duplicate strains of Enterobacteriaceae producing a broad diversity of well-characterised beta-lactamases. Clin Microbiol Infect2008; 14: 148–154., , , .
- 33Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. Euro Surveill2008; 13: 1–11., , .
- 40European Committee on Antimicrobial Susceptibility Testing (EUCAST). Expert rules in antimicrobial susceptibility testing. Version 1 http://www.eucast.org/expert_rules. Accessed 3 November 2011.