clsi, eucast - antimicrobial agents and...
TRANSCRIPT
Validation of antibiotic susceptibility testing guidelines in a routine clinical 1
microbiology laboratory exemplifies general key challenges in setting clinical 2
breakpoints 3
4
Michael Hombach1‡, Patrice Courvalin2, Erik C. Böttger1 5
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1) Institut für Medizinische Mikrobiologie, Universität Zürich, 8006 Zürich, Schweiz 7
2) Unite des Agents Antibactériens, Institut Pasteur, 75724 Paris, France 8
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Short title: Irregularities with EUCAST clinical breakpoints 10
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Keywords: ECOFF, clinical breakpoints, diameter distributions, AST, disk diffusion, 12
CLSI, EUCAST 13
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‡Corresponding author: 15
Michael Hombach, M.D. 16
Institut für Medizinische Mikrobiologie 17
Universität Zürich 18
Gloriastr. 30/32 19
8006 Zürich 20
Switzerland 21
Phone: 0041 44 634 27 00 22
Fax: 0041 44 634 49 06 23
Email: [email protected] 24
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AAC Accepts, published online ahead of print on 28 April 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.02489-13Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Abstract 28
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This study critically evaluated the new European Committee for Antimicrobial 30
Susceptibility Testing (EUCAST) antibiotic susceptibility testing guidelines on the basis of 31
a large set of disk diffusion diameters determined for clinical isolates. We report several 32
paradigmatic problems illustrating key issues in the selection of clinical susceptibility 33
breakpoints, which are of general importance not only for EUCAST but for all guidelines 34
systems, i.e. i) the need for species-specific determination of clinical 35
breakpoints/epidemiological cut-offs (ECOFFs), ii) problems arising from pooling data 36
from various sources, and iii) the importance of the antibiotic disk content for separating 37
non-wild type and wild-type populations. 38
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Introduction 57
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Since 2010 the European Committee for Antimicrobial Susceptibility Testing 59
(EUCAST) has issued regularly updated antibiotic susceptibility testing (AST) guidelines 60
including clinical susceptibility breakpoints (CBPs), which are determined mainly on the 61
basis of epidemiological cut-offs (ECOFFs), pharmacokinetic/pharmacodynamic 62
parameters (PK/PD), and, in part, clinical outcome data (1-3). EUCAST aims at full 63
transparency in the still complex, mostly consensus driven, process of CBP determination 64
by open source publishing of documents on diameter/MIC distributions, and ECOFF data 65
(4). Diameter and MIC distributions may readily be retrieved from the EUCAST webpage 66
under http://www.eucast.org/zone_diameter_distributions. The Clinical and Laboratory 67
Standards Institute (CLSI) uses a similar process for establishing clinical breakpoints (5). 68
In 2010, the Institute of Medical Microbiology (IMM), University of Zurich, changed 69
its guidelines from CLSI to EUCAST. During this shift we have established a database 70
containing non-duplicate disk diffusion diameters of > 30.000 clinical isolates with the 71
view to validate EUCAST AST guidelines as part of the quality control management in our 72
clinical microbiology laboratory (6, 7). Despite our data being largely consistent with 73
EUCAST, we encountered some problems and inconsistencies in EUCAST AST 74
guidelines exemplifying several important key issues in setting CBPs, that, incidentally, 75
also apply to other systems such as CLSI (8): i) the need for species-specific determination 76
of CBPs/ECOFFs, ii) problems arising from pooling data from various sources, and iii) the 77
importance of the antibiotic disk content for the separation of resistant populations from 78
the wild-type population. 79
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Methods 83
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Clinical isolates. 85
All non-duplicate clinical isolates included in this study were isolated over a 3-year-86
period from 2010 until 2012 in the clinical microbiology laboratory of the Institute for 87
Medical Microbiology, University of Zurich, mainly serving a 750-bed tertiary-care 88
hospital (University Hospital of Zurich). Isolates of the same species were considered 89
duplicate(s) if they i) originated from the same patient, and ii) showed one major AND two 90
minor differences in AST interpretation at maximum. All duplicates were excluded from 91
the analysis resulting in a reduction of included isolates by 33% in average (species 92
specific reduction: Staphylococcus aureus 39%, coagulase-negative staphylococci (CoNS) 93
32%, Enterococcus faecalis 23%, Enterobacter cloacae 31%, and Klebsiella pneumoniae 94
30%). Only isolates considered clinically relevant were included. The absolute numbers of 95
species/drug combinations for Staphylococcus aureus, CoNS, Enterococcus faecalis, 96
Enterobacter cloacae, and Klebsiella pneumoniae are available from Figures 1 and 2. 97
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Susceptibility testing. 99
For susceptibility testing the disk (i2a, Montpellier, France) diffusion method was 100
carried out using Mueller-Hinton agar (Becton Dickinson, Franklin Lakes, NJ, USA) and a 101
McFarland 0.5 dilution from overnight cultures as an inoculum followed by incubation at 102
35°C for 16-18 h according to EUCAST recommendations (9). Inhibition zone diameters 103
were determined and recorded using the semi-automated Sirweb/Sirscan system (i2a). 104
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ECOFF determination 106
ECOFFs were determined by visual inspection of diameter distributions (consensus of 107
five experienced persons, i.e. the ”eyeball method”) (8). EUCAST does not recommend a 108
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method for ECOFF determination nor indicates the method used to determine EUCAST 109
ECOFFs. 110
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Software. 112
Calculations were done using Microsoft Excel 2010 software (Microsoft Corporation, 113
Redmond, WA, USA). 114
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Results and Discussion 136
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Reliable CBPs/ECOFFs require species-specific determination 138
Inhibition zone diameter and MIC distributions are species specific in terms of ranges, 139
means, and standard deviations (8). Thus, ECOFFs and CBPs, by definition, need to be 140
determined species-specific; a principle acknowledged by EUCAST and CLSI (10, 11). To 141
avoid increasing guideline complexity group-specific CBPs have been suggested, e.g. the 142
majority of staphylococcal CBPs are defined at the genus level, or Enterobacteriaceae 143
CBPs that apply at the group level (3, 5). However, it is agreed that CBPs must never split 144
the wild-type population due to biological variation within the wild-type isolates (11). A 145
practical solution to reduce the highly complex process of CBP setting for a large number 146
of drugs, is the use of drug class representatives with AST extrapolation to the other class 147
members (3, 5). 148
For sufficient statistical power, an analysis should include a minimum of 30 isolates to 149
form a Gaussian distribution suitable for ECOFF determination (central limit theorem) 150
(12). The 21,434 Enterobacteriaceae isolates tested for antibiotic susceptibility to 27 151
individual drugs in the IMM clinical laboratory from 2010 to 2012 comprised 11597 (54%) 152
E. coli, 3016 (14%) K. pneumoniae, 1685 (8%) Enterobacter cloacae, 1089 (5%) Proteus 153
mirabilis, 905 (4.2%) Klebsiella oxytoca, 735 (3.4%) Serratia marcescens, 535 (2.5%) 154
Citrobacter koseri, 481 (2.2%) Enterobacter aerogenes, 454 (2.1%) Citrobacter freundii, 155
404 (1.9%) Morganella morganii, 276 (1.3%) Proteus vulgaris, 121 (0.6%) Hafnia alvei, 156
50 (0.23%) Providencia rettgeri, 37 (0.17%) Serratia liquefaciens, and 33 (0.15%) 157
Pantoea agglomerans. For all other Enterobacteriaceae species the IMM database of 158
contained less than 30 isolates. The species listed above accounted for >99.5% of all 159
Enterobacteriaceae isolated in our clinical laboratory. Given these numbers that may serve 160
as an approximation for other laboratories, sufficient AST data would be available for 161
>99.5% of clinical Enterobacteriaceae isolates. E. coli, K. pneumoniae, and E. cloacae 162
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alone, as the three most frequently isolated species, accounted for 76.1% of all isolates. 163
Automated diameter reading and documentation systems can facilitate the collection of 164
comparably high numbers of diameter values. 165
Evaluating our clinical isolate database we observed that genus and group-specific 166
CBPs result in several problems including potential interpretation errors. This situation is 167
best exemplified by fluoroquinolones and staphylococci, and ertapenem and E. cloacae 168
and K. pneumoniae: 169
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The ciprofloxacin EUCAST ECOFFs for S. aureus (20 mm) and for CoNS (24 mm) are 171
consistent with both the EUCAST and IMM diameter distributions (Figure 1A and 1B) 172
(13). Likewise, the EUCAST levofloxacin ECOFF of 24 mm for CoNS is consistent with 173
the IMM diameter distribution (Figure 1D). EUCAST does not define an S. aureus 174
levofloxacin ECOFF, most likely due to the low number of available data points (n=107). 175
Despite different S. aureus and CoNS ciprofloxacin ECOFFs, and a missing levofloxacin 176
ECOFF for S. aureus, both EUCAST and CLSI CBP tables define uniform staphylococcal 177
CBPs for fluoroquinolones, which do not differentiate S. aureus from non-S. aureus 178
staphylococci, e.g. uniform staphylococcal ciprofloxacin CBPs for susceptibility of ≥ 20 179
mm and resistance of < 20 mm (EUCAST), or ≥ 21 mm for susceptibility and resistance of 180
≤ 15 mm (CLSI), and uniform staphylococcal levofloxacin CBPs for susceptibility of 181
≥ 22 mm, and resistance of < 19 mm (EUCAST), or ≥ 19 mm for susceptibility and 182
resistance of ≤ 15 mm (CLSI) (3, 5). The IMM S. aureus ciprofloxacin ECOFF (20 mm, n 183
= 5492 isolates, Figure 1A) correlates to EUCAST and CLSI susceptible breakpoints. 184
However, both EUCAST and CLSI susceptible CBPs applied to CoNS split the “out of 185
ECOFF” population (usually considered as “non-wild-type”) to different interpretative 186
categories (Figure 1B). Similarly, the uniform staphylococcal EUCAST and CLSI 187
levofloxacin CBPs split a homogeneously distributed “out of ECOFF” (non-wild type) 188
CoNS population (see IMM distribution, n = 3832 isolates tested with levofloxacin, Figure 189
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1D) between all three interpretative categories (susceptible, intermediate, and resistant). In 190
contrast, the susceptible EUCAST levofloxacin CBP (≥ 22 mm) and the susceptible CLSI 191
CBP (≥ 19 mm) are largely adequate for S. aureus assigning only the wild-type population 192
to the susceptible category (Figure 1C). We assume that the uniform EUCAST 193
staphylococcal ciprofloxacin and levofloxacin CBPs were extrapolated from S. aureus 194
data, a procedure apparently inappropriate for CoNS. Consequently, fluoroquinolone CBPs 195
for staphylococci must be either defined in a species-specific fashion (e.g. S. aureus), or at 196
the group-level (coagulase-negative staphylococci) as done by EUCAST and CLSI for 197
cefoxitin S. aureus, and Staphylococcus lugdunensis or, most recently, by EUCAST for 198
Staphylococcus saprophyticus and S. pseudointermedius (3, 5). 199
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EUCAST does not define an ertapenem ECOFF for E. cloacae. Most probably, this is 201
due to a shoulder in the population caused by a resistant sub-population overlapping the 202
wild type, which becomes clearly visible in the E. cloacae/ertapenem IMM diameter 203
distribution as compared to available EUCAST data (n = 1089 isolates IMM, see 204
Figure 1 E, versus n = 109 isolates in the EUCAST distribution) (13). Despite lack of an 205
ECOFF for ertapenem and E. cloacae, EUCAST recommends uniform susceptible (≥ 25 206
mm) /resistant (< 22 mm) ertapenem CBPs for all Enterobacteriaceae species as does 207
CLSI (susceptible and resistant CBPs of ≥22 mm, and ≤18 mm, respectively (3, 5). 208
However, testing ertapenem susceptibility in E. cloacae appears to be unreliable in terms 209
of clinical categorisation: Wild-type and resistant populations cannot safely be 210
distinguished by inhibition zone diameter determination alone (Figure 1E). Ertapenem 211
AST for E. cloacae may, consequently, be discouraged, or isolates should be tested for the 212
presence of resistance mechanisms. 213
For K. pneumoniae EUCAST and IMM ertapenem ECOFFs are identical (25 mm, 214
Figure 1F). The ECOFF matches the EUCAST susceptible Enterobacteriaceae CBP of 215
≥ 25 mm, appropriately assigning wild-type isolates to the susceptible category (13). IMM 216
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distributions show a minor non-wild type population, which overlaps the wild-type, 217
although the situation is less pronounced when compared with E. cloacae (compare Figure 218
1F with 1E). The minor K. pneumoniae non-wild-type population is categorised 219
intermediate by EUCAST Enterobacteriaceae CBPs, which seems reasonable. According 220
to the susceptible ertapenem CLSI CBP (≥22 mm) this minor non-wild-type populations is 221
categorized susceptible. Clinical and PK/PD data, and molecular characterization for 222
resistance mechanisms are mandatory to decide if such isolates constitute a distinct 223
population and can be reliably be treated. 224
For both E. cloacae and K. pneumoniae the overlapping non-wild type populations are, 225
most likely, caused by increased beta-lactamase production combined with an outer 226
membrane porin deficiency causing reduced permeability. This combination of resistance 227
mechanisms is commonly found in E. cloacae and K. pneumoniae, but rarely in E. coli 228
(14-16). 229
The above examples indicate that CBPs are preferably set species-specific to prevent 230
erroneous treatment recommendations. Combinations of resistance mechanisms can result 231
in complex population diameter distributions lacking clear discrimination of wild-type and 232
non-wild-type populations. This phenotypical indifference may warrant determination of 233
resistance mechanisms (biochemically or genotypically) rather than mere assessment of 234
MIC/diameter CBPs. The reduced discriminative power of CBPs becomes even more 235
limiting when one considers measurement inaccuracy that further hampers correct 236
assignment of isolates to interpretative clinical categories (17). The unavoidable increase in 237
complexity of species-specific CBPs may prompt the use of software systems for AST 238
categorization, e.g. zone diameter classification (18). 239
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Pooling data from various sources may lower population discrimination 244
The EUCAST E. coli ertapenem ECOFF was set at 29 mm with a minor population 245
between 27 and 29 mm that was obviously considered non-wild-type (13). This decision 246
was made on the basis of 579 data points from 2 sources. The IMM distribution comprises 247
6974 ertapenem diameter values of clinically relevant, non-duplicate E. coli isolates. Our 248
data suggest an ECOFF of 25 mm to appropriately assign wild-type and non-wild-type 249
isolates (Figure 2A) as an ECOFF of 29 mm would split the Gaussian distribution of wild-250
type isolates. The S. aureus amikacin EUCAST ECOFF is 18 mm (13). However, the 251
EUCAST distribution (841 data points from 4 sources) seems to contain a resistant S. 252
aureus sub-population overlapping the wild type, which is split by the amikacin CBPs 253
(S/R= ≥ 18mm, < 16 mm) (13). This overlapping resistant population is missing in the 254
IMM distribution (5702 data points, Figure 2B). 255
Diameter and MIC values are influenced by the method used for their determination 256
(19). EUCAST distribution data represent a compilation from numerous sources (13). This 257
raises some concerns about data consistency and bias due to the various methods used and 258
the differing measurement precision in individual laboratories. Technical variation between 259
individual laboratories may decrease the discriminative power of diameter distributions 260
due to higher spread of distribution curves, and, therefore, could hamper clear 261
differentiation of wild-type and non-wild-type populations. On the other hand, analysing 262
data from multiple sources is required to limit bias due to local epidemiology and to make 263
AST data more robust. The zone diameters presented in this study have been determined 264
by an automated reading system (Sirscan). Reading zone diameters by automats is 265
officially approved by EUCAST and CLSI (5, 9). A weekly quality control is performed in 266
our laboratory. Using EUCAST recommended quality control isolates, median diameter 267
values, standard deviations, and diameter ranges closely matched EUCAST criteria (see 268
Supplemental Figure 1) indicating that a comparison of IMM and EUCAST data is 269
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appropriate. In addition, we have recently shown that diameter readings by the Sirscan 270
zone reader can reliably be compared to the EUCAST recommended manual method (20). 271
Possible reasons for the discrepancies observed include different epidemiology, lower 272
number of data points in the EUCAST distribution, pooling results from various sources, 273
bias due to low number of laboratories contributing data (single source in our data, 2 and 4 274
sources for the EUCAST data), and differences in ECOFF determination. In our study we 275
used the “eyeball”-method for ECOFF determination, i.e. a visual consensus value (8). 276
EUCAST does not officially recommend an ECOFF method nor provides information on 277
how EUCAST ECOFFs were determined. The eyeball method is, however, frequently used 278
and can at least provide a reasonable ECOFF estimation. Next to the visual inspection of 279
data distribution several statistical methods have been described to determine ECOFFs in 280
order to deal with statistical problems such as skewed and/or outlier distributions, but no 281
single method has been commonly accepted as the gold standard to date (21-25). 282
The data used in this study are limited as they originate from a single source (the 283
clinical laboratory of the IMM Zurich) and, thus, represent a local epidemiology. While the 284
problems described for our setting may not be encountered by all clinical laboratories, the 285
CBP inconsistencies we observed are of a more general character. 286
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Antibiotic disk content is critical to delineate distinct non-wild-type and wild-type 288
populations 289
For determination of high level aminoglycoside resistance in enterococci antibiotic 290
disks with high drug loads have long been recommended by CLSI and the Comité de 291
l'Antibiogramme de la Societe Francaise de Microbiologie (CA-SFM), i.e. a load of 292
gentamicin 100 µg/disk by CLSI, and 500 µg/disk by the CA-SFM (5, 26). EUCAST 293
recommends a lower disk content of 30 µg/disk in order to enhance the sensitivity of high 294
level resistance detection (3). This approach can cause practical problems in separating 295
resistant and wild-type populations as illustrated by E. faecalis and gentamicin: The IMM 296
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diameter distribution for the 30 µg disk (Figure 2C) shows a wild type population that is 297
left-shifted close to the resistant population. Reliable assignment of isolates to the resistant 298
or wild-type population is hampered due to measurement inaccuracy as per the low 299
distance between the wild-type and non-wild-type populations. As a consequence, 300
interpretation errors will inevitably occur due to technical measurement inaccuracy (17). It 301
may be advisable to either increase the disk content, thereby increasing the distance / 302
separation between wild-type and high level resistant populations, or to introduce a grey, 303
or “buffer” zone surrounding the ECOFF. Figure 2 D shows distribution data for 304
E. faecalis and gentamicin 500 µg/disk according to the 2013 CA-SFM CBPs. Clearly, the 305
wild-type population (deemed susceptible) can be separated from high-level resistant 306
isolates. Wild-type and high-level resistant populations are separated by a transitional 307
population of uncertain assignment (deemed intermediate). Not the least, to assign such 308
transitional populations to the susceptible or resistant category guideline issuing 309
organisations, such as CLSI and EUCAST, increasingly advocate to use determination of 310
resistance mechanisms to validate the phenotypic interpretive criteria (27). 311
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In contrast to the ECOFF definition, CBP setting cannot be based on microbiological 313
data alone, but needs to consider clinical and PK/PD data as mandatory sources of 314
information. However, microbiological data should not be weighed less than PK/PD data. 315
Preliminary CBPs should preferably be consistent with both systems and should ultimately 316
be validated by clinical outcome studies. In conclusion, our data illustrate some issues of 317
general importance in determining CBPs: i) Reliable CBPs/ECOFFs require species-318
specific criteria, ii) pooling data from different sources may influence diameter 319
distributions, and iii) antibiotic disk content is critical for separating resistant and wild-type 320
populations. 321
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Acknowledgments 325
We are grateful to the laboratory technicians of the Institute of Medical Microbiology, 326
University of Zurich for their dedicated help, and to Reinhard Zbinden, Guido Bloemberg, 327
Vanessa Deggim, Florian Maurer, Vera Rüegger, and Andrea Zbinden for valuable 328
discussions. 329
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Funding 331
This work was supported by the University of Zurich 332
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Transparency declarations 334
There are no conflicts of interest to declare. 335
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Figures
Figure 1: Species specific breakpoints and overlapping populations
Inhibition zone diameter distributions of isolates tested in the clinical laboratory of the Institute of
Medical Microbiology, University of Zurich. Values were determined by the EUCAST recommended
disk diffusion procedure. Categorization according to EUCAST clinical breakpoints: White bars,
susceptible; grey bars, intermediate; black bars, resistant. Black arrow, EUCAST ECOFF as listed in
EUCAST diameter distributions (13); white arrow, IMM ECOFF (present study). Thin arrows, CLSI
clinical breakpoints. Dotted lines, predicted population distributions. CoNS, coagulase-negative
staphylococci.
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S RS R
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Figure 2: Diameter distributions, data pooling from various sources, and importance of antibiotic
disk content
Diameter distributions originate from isolates tested in the clinical laboratory of the Institute of
Medical Microbiology, University of Zurich. Values were determined by the EUCAST recommended
disk diffusion procedure. White bars, EUCAST susceptible category; grey bars, EUCAST intermediate
category; black bars, EUCAST resistant category. Black arrows, EUCAST ECOFFs as listed in
EUCAST diameter distributions (13); white arrows, IMM ECOFFs (present study). The 500 µg
gentamicin disk content is recommended by CA-SFM with according CBPs as shown. CoNS,
coagulase-negative staphylococci.
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