Use of Ancillary Carbapenemase Tests to Improve Specificity of Phenotypic Definitions for

23 Pages·2017·0.5 MB·English

JCM Accepted Manuscript Posted Online 29 March 2017
J. Clin. Microbiol. doi:10.1128/JCM.00157-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
1 Use of Ancillary Carbapenemase Tests to Improve Specificity of Phenotypic Definitions for
2 Carbapenemase Producing Enterobacteriaceae
3
4 Shelley A. Miller, Janet A. Hindler, Angelo Chengcuenca, Romney M. Humphries*
5
6 UCLA Pathology & Laboratory Medicine
7 * Corresponding author, [email protected]
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11 Abstract
12 Carbapenemase producing Enterobacteriaceae (CPE) are a significant threat to public
13 health. In 2015, CDC revised the surveillance definition for CPE to include all Enterobacteriaceae
14 resistant to any carbapenem tested. However, this definition is associated with poor specificity.
15 We evaluated the performance of this definition for a collection of 125 Enterobacteriaceae, as
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16 compared to carbapenemase PCR. We also investigated the impact of ancillary testing for
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17 carbapenemase of isolates that met the CDC CPE surveillance definition. The two ancillary tests d
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18 evaluated were the Xpert Carba-R Assay, a molecular test, and the carbapenem inactivation f
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19 method (CIM). Two variables were evaluated for the CIM: suspension of organism in ddH2O vs.
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20 TSB to incubate disks, and incubation of plates for 6 h vs. 18 – 20 h. Sensitivity and specificity of :
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21 the Carba-R were 100% as compared to in-house PCR. Sensitivity of the CIM performed with
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22 TSB and read at 6 h was 94.6%, with TSB and read at 18-20 h was 97.7%, with ddH O and read
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23 at 6 h was 88.0% and with ddH2O incubated for 18-20 h was 93.0%. Specificity was 100% for all /
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24 variables tested. Without ancillary testing, the sensitivity of the CDC definition was 98.9% for A
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25 CPE and specificity was 6.1%. Testing isolates that were screen positive by the CDC definition by il
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26 the Xpert Carba-R did not change sensitivity, and improved specificity to 100%. Similarly, use of 0
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27 the CIM (TSB and 18-20 h incubation) to confirm screen-positive isolates resulted in a sensitivity b
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28 of 95.6% and specificity of 100%. e
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29 Introduction
30 Carbapenem-resistant Enterobacteriaceae (CRE), particularly those that are resistant
31 due to carbapenemase production, are a significant threat to public health. Infections caused
32 by CRE are associated with a high attributable mortality (1), and U.S. surveillance data have
33 demonstrated a steady increase in the burden of disease caused by CRE since 2000 (2). The U.S.
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34 CRE epidemic is driven, in part, by Klebsiella pneumoniae isolates of sequence type (ST) 258
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35 that express the K. pneumoniae carbapenemase (KPC) (3). Transfer of patients colonized or d
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36 infected with this organism between healthcare institutions is thought to have led to f
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37 dissemination of ST 258 K. pneumoniae across the country. One particularly well documented
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38 outbreak described by the Centers for Disease Control and Prevention Epicenters program :
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39 demonstrated patient transfers between 26 health care facilities across four counties lead to
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40 spread of KPC-producing CRE to 40 patients over a 1-year period (4). Subsequent studies in this
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41 region documented nearly 1 in 3 residents of long-term acute care facilities were colonized with /

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42 KPC-producing CRE (5). CRE harboring other carbapenem-hydrolyzing enzymes, such as NDM, A
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43 VIM, IMP or the OXA-48-type, common in other areas of the world (6), are also spreading in the il
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44 US (7-9). Introduction of isolates expressing these carbapenemases to a single institution by a 0
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45 patient traveling from an endemic area can lead to rapid spread, as occurred in our own b
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46 institution when an OXA-232 producing K. pneumoniae was introduced to our facility by a e
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47 colonized patient, leading to transmission of CRE to 14 patients via reprocessed duodenoscopes
48 (10). A similar outbreak, caused by a NDM-producing K. pneumoniae in the Chicago area, was
49 also recently documented as associated with use of reprocessed duodenoscopes (11). In both
50 outbreaks, rapid recognition of an unusual CRE type led to the epidemiological investigations
51 that ultimately identified the source of the outbreak.
52 Early identification of patients colonized with carbapenemase-producing (CP-) CRE and
53 implementation of infection prevention strategies that include strict isolation practices have
54 the potential to significantly slow the trajectory of CP-CRE dissemination in the U.S. (12-14).
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55 However, such interventions critically depend on accurate and rapid identification of patients
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56 colonized or infected by CRE through laboratory testing. Unfortunately, detection of CRE, let d
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57 alone CP-CRE remains a challenge for most clinical microbiology laboratories in the U.S. (6). For f
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58 instance, updated carbapenem breakpoints for the Enterobacteriaceae published by the Clinical
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59 and Laboratory Standards Institute (CLSI) in 2010 have been incompletely adopted by clinical :
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60 laboratories (15, 16), because not all manufacturers of antimicrobial susceptibility test (AST)
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61 systems have obtained U.S. Food and Drug Administration (FDA) clearance for these new
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62 breakpoints. Yet, use of the 2010 breakpoints is imperative to accurately identify CRE (15). /

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63 Bartsch and colleagues demonstrated through computer simulations that delayed A
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64 implementation of updated carbapenem breakpoints may be the reason for a staggering 8,497 il
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65 additional patients becoming colonized with CRE in a single region over the past 5 years (17). 0
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66 In 2015, the CDC published a new surveillance definition for CRE in an attempt to help b
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67 laboratories and epidemiologists to identify CP-CRE (18). This definition includes all members of e
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68 the Enterobacteriaceae that are resistant to any carbapenem tested (ertapenem, ≥2 µg/ml;
69 meropenem, ≥4 µg/ml; imipenem, ≥4 µg/ml; and/or doripenem, ≥4 µg/ml) and any isolate that
70 harbors a carbapenemase gene or has a positive phenotypic test for carbapenemase. Some
71 have concerns that this definition may overestimate CP-CRE because isolates resistant to
72 ertapenem but not imipenem, meropenem, or doripenem often result from the presence of
73 extended-spectrum beta-lactamases (ESBL) or AmpC enzymes, which have some hydrolyzing
74 activity against ertapenem, usually in combination with permeability defects. Similarly, some
75 isolates without carbapenemases are resistant to imipenem, meropenem and/or doripenem
76 due to changes in porin proteins, often in conjunction with low level AmpC or ESBL activities,
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77 albeit such isolates are less common (19). For instance, one study using the CDC definition
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78 found 55% of isolates labeled as CP-CRE did not harbor carbapenemase genes (18). The authors d
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79 suggested use of a second phenotypic test for carbapenemase, such as the Modified Hodge test f
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80 (MHT), improved specificity for identification of CP-CRE. However, this was most effective for
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81 isolates of K. pneumoniae and E. coli, whereas 26% of Enterobacter spp. isolates demonstrated :
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82 false-positive MHT result (18). The MHT is also associated with false-negative results for NDM-
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83 producing CRE (6). While at the time, the MHT was the only CLSI-endorsed carbapenemase test
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84 that was readily performed by clinical laboratories, the CLSI voted in January 2017 to retire the /

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85 MHT as a confirmatory test for CP-CRE and will remove this procedure in the M100S 28th Ed. A
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86 Standard, leaving laboratories with few practical options for testing CP-CRE. il
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87 Recently, two new carbapenemase-detection methods have become available to clinical 0
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88 laboratories; one a commercial assay for carbapenem resistance genes and the other a b
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89 phenotypic test for carbapenemase activity that utilizes simple materials readily available in e
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90 most clinical laboratories. A third method, the Carba-NP assay has also been described but this
91 method requires preparation of several reagents in house and is not practical for clinical
92 laboratories. The FDA-cleared Xpert Carba-R (Cepheid, Sunnydale, CA) is a multiplex PCR that
93 detects the genes encoding the 5 most common carbapenemases, KPC, NDM, VIM, IMP and
94 OXA-48-like enzymes, directly from bacterial colonies. This test is also FDA-cleared for screening
95 for patient colonization by rectal swab testing. The carbapenem inactivation method (CIM) was
96 originally described by van der Zwaluw et al. to detect carbapenemase activity in gram-negative
97 bacilli using a cost effective and sensitive approach (17). Additionally, a modified carbapenem
98 inactivation method (mCIM) was recently evaluated by the CLSI and is described in detail in the
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99 new CLSI M100S 27th ed. Standard. At the time our study was performed, the mCIM method w
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100 had not been evaluated by CLSI and as such, the present study evaluates the performance of d
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101 the Xpert Carba-R colony test, as well the original CIM with slight modifications, to enable rapid f
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102 performance of the test by clinical laboratories. The overall purpose was to evaluate the
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103 sensitivity and specificity of using both CDC’s and UCLA’s definition to identify CP-CRE with and :
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104 without the use of CIM and Xpert ancillary assays for detection of carbapenemase production,
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105 in a laboratory that encounters a diverse population of CP-CRE.
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106 /

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107 Methods A
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108 Bacterial Isolates il
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109 A total of one hundred and twenty-five isolates of Enterobacteriaceae isolated from 125 0
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110 patients was evaluated. Isolates were selected using the UCLA CRE definition, i.e., not b
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111 susceptible (intermediate or resistant, ≥ 2 µg/ml) to meropenem and/or imipenem, as defined e
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112 by the current CLSI M100S 27th ed. Standard (21). Isolates of Proteus, Providencia, and
113 Morganella that were not susceptible to imipenem alone were excluded. Of the 125 isolates, 8
114 were prospective isolates that met the CDC CRE definition but not the UCLA definition (i.e.,
115 resistant to ertapenem but susceptible to meropenem and imipenem). Susceptibility testing
116 was performed according to the CLSI reference broth microdilution method (BMD) on panels
117 prepared in-house. The isolates were recovered from the urine (n=40), blood (n=23),
118 respiratory secretions (n=29), wounds/tissue (n=9) and aspirates/fluids (n=16) from UCLA
119 patients between 2014 - 2016. Eight isolates (all positive for bla -like genes) were provided
OXA-48
120 by The Medicines Company (Parsippany, NJ). All isolates were stored at -70°C in Brucella broth
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121 + 15% glycerol (Hardy Diagnostics, Valencia CA) and were subcultured twice on blood agar
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122 plates (BD, BBL Sparks MD) prior to testing. d
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123 CIM f
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124 The CIM method was performed according to the methods described by van der Zwaluw
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125 and colleagues (20), with some modifications. Briefly, 500 μL of sterile double distilled water or :/
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126 tryptic soy broth (TSB, BD, Sparks MD) was added to a 1.5 mL sterile microfuge tube. For each .
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127 CRE isolate, a heaping 1 μL loopful of organism from an 18-24 h culture on a blood agar plate
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128 was added to the broth or water and vortexed to obtain a homogenous suspension. To this, a /

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129 10 μg meropenem disk (BD) was added, ensuring the disk was immersed in the suspension. The A
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130 suspension was then incubated at 35°C for 2 h in an ambient air incubator. After incubation, il 1
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131 the disk was carefully removed from the suspension. It was then applied to a lawn of 1
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132 Escherichia coli ATCC 25922 on a Mueller Hinton agar plate (MHA, BD) that had been inoculated y

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133 as for a routine disk diffusion test using a suspension equivalent to a 0.5 McFarland. Plates were e
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134 incubated at 35°C in ambient air for 6 h, after which time the zones of growth inhibition
135 surrounding the meropenem disk were measured using reflected light. Within 15 minutes
136 plates were re-incubated for an additional 12-14 h (total 18-20 h incubation), and zone sizes
137 were measured again. A zone of ≤15 mm was considered positive for carbapenemse production
138 and a clear zone of ≥19 mm was considered negative. Zones 16-18mm or ≥19 mm with colonies
139 present within the zone were considered indeterminate (Figure).
140 Quality control was performed by testing a carbapenemase positive (K. pneumoniae
141 ATCC BAA-1705) and a carbapenemase negative (K. pneumoniae ATCC BAA-1706) control strain
142 on each day of testing. For additional controls, meropenem disks were incubated in TSB or
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143 water alone (no organism) for 2 h at 35°C. These, and a dry meropenem disk, were also applied w
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144 to the E. coli lawn, and zones were evaluated at 18-20 h incubation to ensure they fell within d
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145 CLSI QC ranges for meropenem and E. coli ATCC 25922 (21). f
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146 Detection of Carbapenem resistance genes
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147 All isolates were tested using in-house developed Taqman PCRs for the detection of :/
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148 bla , bla , bla , bla , bla and bla (19, 22). Isolates positive for bla were
KPC NDM VIM IMP SME OXA-48 OXA-48 .a
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149 subtyped using a high resolution melt assay, as described previously (22). Quality control
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150 included testing an isolate positive and negative for each target each day of testing. /

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151 Additionally, a 16S rRNA gene internal control was included in each PCR reaction. Isolates were A
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152 tested by the Xpert Carba-R assay (Cepheid) according to the manufacturer’s instructions. il 1
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153 Quality control was performed per the Xpert package insert. 0
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154 Study design and data analysis b
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155 CIM and Xpert Carba-R tests were performed on the same day using the same e
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156 suspension of organism. Sensitivity and specificity and were calculated for the CDC CP-CRE
157 definition and for the UCLA CRE definition using results previously obtained from an in-house
158 developed carbapenem resistance gene PCR as the gold standard. Results for isolates classified
159 as CPE by MIC interpretations were considered false positive if the isolate was negative for
160 carbapenem resistance genes by the in-house PCR assay. Similarly, results were false negative
161 if the isolate produced a carbapenemase or carried a carbapenem resistance gene but did not
162 fit the CRE definition based on MIC result interpretive results. Sensitivity and specificity were
163 recalculated following inclusion of results from the CIM and Xpert assays. Discrepant results
164 were repeated and if errors corrected, they were not included in the final data analysis.
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165
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166 Results d
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167 A summary of the 125 isolates evaluated in this study is provided in Table 1. There were f
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168 a total of 92 isolates positive for the presence of a carbapenemase gene, including 2 positive for
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169 bla , 77 for bla , 2 for bla , 11 for bla -like genes, and 33 isolates had no carbapenem :
IMP KPC NDM OXA-48 //
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170 resistance genes detected. Among the bla -like containing isolates, 1 was subtyped as
OXA-48 .
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171 bla , 1 bla , 1 bla and 8 bla .
OXA-162 OXA-181 OXA-232 OXA-48 .
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172 The CDC CRE surveillance definition was 98.9% sensitive (95% confidence interval (CI), /

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173 93.2 – 99.9%) and 6.1% specific (95% CI, 1.1 – 21.6%) for carbapenem resistant gene presence A
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174 among this select collection of isolates, as compared to the in-house PCRs. In contrast, the il
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175 UCLA definition was 100% sensitive (95% CI, 95.0 – 100%) and 24.2% specific (95% CI, 11.7 – 0
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176 42.6%). (Table 3) One K. pneumoniae isolate that tested positive for bla did not meet the CDC b
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177 surveillance definition criteria. This isolate had an ertapenem MIC of 1 µg/mL (susceptible), e
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178 imipenem of 2 µg/mL (intermediate) and meropenem 1 µg/mL (susceptible). bla presence
KPC
179 was confirmed by repeat testing by in-house PCRs and MICs were confirmed by repeat BMD.
180 This isolate was positive by both the Xpert Carba-R for bla and the CIM tests for the presence
KPC
181 of a carbapenemase, which were also performed in duplicate.
182 Both CDC and UCLA CRE definitions resulted in several false-positive results, 28 by the
183 CDC definition and 25 by the UCLA definition. The CDC definition alone classified 5 isolates as
184 CRE that were resistant to ertapenem but susceptible to imipenem and meropenem: one E.
185 coli, three E. cloacae and one K. pneumoniae. In contrast, the UCLA definition alone classified 2
186 E. aerogenes isolates as CRE that had intermediate imipenem MICs of 2 µg/ml but that were
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187 susceptible to meropenem and ertapenem. There were 23 isolates that were categorized as
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188 CRE using both CDC and UCLA definitions that were negative for carbapenem resistance genes d
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189 by PCR and carbapenemase activity by CIM (not shown). f
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190 Performance of the Xpert Carba-R assay
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191 One isolate (negative for carbapenem resistance genes) gave an invalid result (0.8% of :
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192 all isolates tested) on initial testing, but yielded the correct negative result on repeat testing.
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193 After resolution, the Xpert Carba-R test was 100% sensitive and 100% specific for detection of
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194 targeted carbapenem resistance genes in this select collection of isolates as compared to in- /

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195 house PCR. A
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196 CIM test performed with incubation of organism and disks in TSB il
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197 On initial testing, 2 false negative and 1 weakly positive (18 mm) results were observed 0
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198 with TSB after 6 h incubation. The 2 false negatives (both bla positive) and 1 weak positive b
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199 (blaKPC positive) were all indeterminate at the 18-20 h reads, with microcolonies within the e
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200 zones (Figure 1). All 3 isolates were resistant to meropenem by BMD (Table 2). Following
201 resolution, the CIM assay with TSB suspensions of organisms and 6 h incubation was 94.6%
202 sensitive (95% CI, 87.2 – 98.0%) and 100% specific (95% CI, 87 – 100%), Table 2. At 18-20 h
203 incubation, sensitivity was 97.7% (95% CI, 91.4 – 99.6%) and specificity was 100% (95% CI, 87 –