CME/SAM. Clinical Laboratory Detection of AmpC β-lactamase Does It Affect Patient Outcome?

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1 Microbiology and Infectious Disease / Laboratory Detection of AmpC β-lactamase Clinical Laboratory Detection of AmpC β-lactamase Does It Affect Patient Outcome? Kenneth H. Rand, MD, 1 Bradley Turner, MD, 1 Hilary Seifert, 1 Christine Hansen, PharmD, 2 Judith A. Johnson, PhD, 1,3 and Andrea Zimmer, MD 4 Key Words: AmpC β-lactamase; Modified Hodge test; EDTA disk test; Bacteremia outcome DOI: /AJCP7VD0NMAMQCWA CME/SAM Upon completion of this activity you will be able to: list the major mechanisms of overproduction of AmpC β-lactamases. describe laboratory methods for measurement of AmpC β-lactamases. discuss the relationship between laboratory testing and clinical outcome. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this educational activity for a maximum of 1 AMA PRA Category 1 Credit per article. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 644. Exam is located at Abstract Plasmid-mediated AmpC-producing Escherichia coli and Klebsiella pneumoniae have been associated with poor clinical outcomes, but they are not readily identified in hospital microbiology laboratories. We tested 753 gram-negative bloodstream isolates for AmpC by using the EDTA disk test and the modified Hodge test (n = 172) and the modified Hodge test alone (n = 581). The 30-day mortality for the AmpC group was 9% (2/23) and was 6% (3/51) for the control group. The clinical response was similar: afebrile on day 2 (AmpC group, 16/23 [70%]; control group, 32/45 [71%]) and on day 4 (AmpC group, 19/22 [86%]; control group, 37/44 [84%]). Patients with isolates in the AmpC group were more likely to be in an intensive care unit at the time of the positive blood culture (P =.01) and more likely to be intubated (P =.05) than patients with isolates in the control group. Effective antibiotic treatment within the first 48 hours was given to 47 (92%) of 51 patients with isolates in the control group but to only 14 (61%) of 23 patients with isolates in the AmpC group (P =.001).The modified Hodge test and the EDTA disk test did not identify patients at risk for a poor outcome from AmpC-producing bacterial infections. Escherichia coli and Klebsiella pneumoniae containing plasmid-mediated AmpC β-lactamases have been associated with treatment failure in a case-control study of bacteremia compared with organisms without plasmid-mediated AmpC β-lactamases. 1 Pai et al 2 reported a treatment failure rate of almost 52% for AmpC-containing K pneumoniae bloodstream isolates at 72 hours. Unfortunately, plasmid-mediated AmpC β-lactamases are not reliably detected by standard susceptibility testing methods in the clinical microbiology laboratory. Black et al 3 described the EDTA disk test, and Yong et al 4 described the modified Hodge test for detecting the presence of AmpC β-lactamases that could be carried out routinely in a busy clinical laboratory. A similar test using boronic acid was described by Coudron. 5 However, none of these tests can distinguish plasmid-mediated hyperproduction of AmpC from derepressed chromosomal or any other mechanism of overproduction of an AmpC β-lactamase. Intuitively, we believe the mechanism of AmpC overproduction should not matter for clinical outcome because, either way, such organisms should be resistant to all extended-spectrum cephalosporins, although perhaps not to the advanced-spectrum cephalosporins, cefepime and cefpirome. We compared the clinical outcome for 26 (23 evaluable) patients with bacteremic gram-negative isolates with overproduction of AmpC as measured by the modified Hodge test with 52 (51 evaluable) control patients whose isolates did not produce AmpC. Materials and Methods Bacterial Isolates Between January 2007 and June 2008, we tested 753 consecutive gram-negative bloodstream isolates (excluding 572 Am J Clin Pathol 2011;135: DOI: /AJCP7VD0NMAMQCWA

2 Microbiology and Infectious Disease / Original Article Pseudomonas and Stenotrophomonas) for AmpC in the Shands at the University of Florida Hospital (Gainesville) clinical microbiology laboratory. All isolates were identified and tested for antibiotic susceptibility by standard microbiological procedures using MicroScan (Siemens HealthCare, Deerfield Park, IL). All isolates were tested by the modified Hodge test. 4 As described by Yong et al, 4 3 mm or more of diagonal growth in the cloverleaf pattern was positive for AmpC production Image 1. Isolates that had no or minimal distortion of the cefoxitin zone were considered negative. We also tested 172 isolates by the EDTA disk test 3 with Image 1 Example of a positive and negative EDTA disk test (BLC disk) and positive and negative modified Hodge test (vertical streaks). A cefoxitin-susceptible Escherichia coli indicator strain (American Type Culture Collection 25922) is plated and the cefoxitin disk placed. In the case of the EDTA disk test, the EDTA disks are then placed adjacent to the cefoxitin disk as shown. The organisms to be tested for AmpC are inoculated onto the EDTA disks in 20 μl of saline as described. 3 If the organism expresses AmpC, cefoxitin is hydrolyzed and will give a positive EDTA disk test as shown by growth of the indicator E coli toward the EDTA disk (right side), while a negative test is shown by the lack of growth toward the disk (left side). For the modified Hodge test, the test organism is streaked toward the cefoxitin disk. If the test organism expresses AmpC, it hydrolyzes the cefoxitin and shows growth along the intersection of the streak and the zone of inhibition from the cefoxitin disk. The image shows a positive modified Hodge test (top), indicated by the 3.6-mm diagonal area of growth of the indicator E coli toward the test organism streak. A negative Hodge test is shown on the streak at the bottom, with no diagonal growth into the cefoxitin zone. overall agreement of 97.1% (κ = 0.905; 95% confidence interval, ). Patient Selection The AmpC patient group (n = 26) was selected based on the degree of AmpC positivity on the modified Hodge test, ie, growth of the indicator strain along the test isolate streak of 3 mm or more as described by Yong et al. 4 These isolates were considered positive for AmpC production. The control group was selected from patients with isolates that had no (or only minimal, 0.5 mm) distortion of the circular zone of inhibition surrounding the cefoxitin disk. These non-ampc producers were matched in a 2:1 ratio by bacterial species (n = 52). This matching had the desired ratio for E coli, Acinetobacter species, and Serratia species, but there were insufficient Enterobacter species lacking AmpC, so Klebsiella species were substituted. The organisms included in the AmpC and control groups are shown in Table 1. Because the groups were selected on the basis of AmpC testing in the laboratory, it was only after review of the patient records that 1 patient was found to have a mixed blood culture with an AmpC-producing Klebsiella oxytoca and an AmpC-producing Enterobacter cloacae. The data for this patient were, therefore, counted only once in the AmpC group. Another patient in the AmpC group had 2 bacteremic episodes 6 months apart with an AmpC-producing E cloacae in both instances. Because the patient had multiple negative blood cultures in the interval (as well as a successful liver transplant), the patient was counted as having 2 separate episodes. This left the AmpC group at 25 patient bacteremic episodes. Data Collection The electronic medical records and paper charts were reviewed by 3 of us (K.H.R., B.T., and A.Z.) for the following data Table 2 : peak daily temperature, starting with the day of the positive blood culture (day 1) through day 7; clinical Table 1 AmpC-Producing and Control Bacterial Species AmpC Enterobacter cloacae 12 9 Enterobacter agglomerans 0 3 Enterobacter amnigenus 0 1 Enterobacter aerogenes 2 0 Serratia marcescens 2 6 Escherichia coli 3 6 Klebsiella pneumoniae 0 12 Klebsiella oxytoca 1 7 Citrobacter freundii 1 0 Acinetobacter baumannii 4 8 Providencia stuartii 1 0 Control The patient had AmpC-producing K oxytoca and E cloacae in the same positive culture. Am J Clin Pathol 2011;135: DOI: /AJCP7VD0NMAMQCWA 573

3 Rand et al / Laboratory Detection of AmpC β-lactamase assessment of response during the same period; antibiotics used to treat the patient in the first 48 hours and the in vitro susceptibility results; patient survival; associated illnesses; patient location at the time of the positive culture, eg, intensive care unit (ICU), other inpatient, outpatient; duration of stay in the hospital before and after the positive blood culture; ventilator status; and patient demographics, ie, age and sex. Comorbidities and severity of illness were assessed using the McCabe score and the simplified acute physiology score (SAPS) II. Fever was defined as a peak daily temperature of 38 C or more. Effective antibiotic treatment was defined as the use of at least 1 antibiotic to which the patient s isolate was susceptible by MicroScan using Clinical and Laboratory Standards Institute susceptibility criteria. Results Two patients in the AmpC group and one in the control group died within 24 hours of the positive blood culture because of underlying terminal illness and withdrawal of life support. Of the evaluable cases, 2 (9%) of 23 patients in the AmpC group and 3 (6%) of 51 in the control group died within 30 days. There was no difference in the response to treatment between the 2 groups, as shown by the defervescence of fever Figure 1 and the percentage who were afebrile on days 2 and 4 after the day of the positive blood culture Table 3. One patient in the AmpC group had blood cultures positive for the original organism (Serratia marcescens) on days 1, 3, and 4; and 1 control patient had a subsequent positive blood culture for the original organism on day 2. In both patients, infections cleared without a change in antibiotic therapy. Table 2 Patient Characteristics AmpC Group Control Group (n = 23) (n = 51) P Mean ± SD age (y) 42.4 ± ± 23.1 NS In intensive care 13 (57) 13 (25).01 Intubated 7 (30) 6 (12).05 Effective antibiotic treatment 14 (61) 47 (92).001 in first 48 h Hospital acquired 15 (65) 24 (47) NS Length of stay before positive 25.4 ± ± culture (d) McCabe score (rapidly 5 (22) 16 (31) NS fatal/total) Mean ± SD SAPS II score All patients 37.2 ± ± 14.8 NS 1 y 40.8 ± ± y 39.6 ± ± 15.1 NS NS, not significant; SAPS, simplified acute physiology score. Data are given as number (percentage) unless otherwise indicated. Calculated by the χ 2 test, except for the length of stay, for which the t test was used. Blood culture positive >72 h after admission. SAPS II calculator. The AmpC and control groups did not differ significantly in age (Table 2) or sex distribution (male, AmpC group, 12/23 [52%]; control group, 28/51 [55%]). Table 2 shows that patients in the AmpC group were significantly more likely to be in an ICU (P =.01) and to have been intubated (P =.05) than patients in the control group at the time of the positive blood culture. Moreover, patients in the AmpC group were significantly less likely to have received an effective antibiotic within the first 48 hours after the blood culture was obtained than were patients in the control group (14/23 [61%] vs 47/51 [92%]; P =.001, respectively). Although there was no difference in the percentage of each group that acquired the AmpC Control Temperature ( C) Days After Positive Blood Culture 7 Figure 1 Mean ± SD of the peak daily temperature for all patients in each group who remained in the hospital. Day 1 represents the day of positive blood culture. In the AmpC group, the isolates produced AmpC, whereas in the control group, they did not. 574 Am J Clin Pathol 2011;135: DOI: /AJCP7VD0NMAMQCWA

4 Microbiology and Infectious Disease / Original Article Table 3 Outcome of Bacteremia infection in the hospital, patients in the AmpC group were in the hospital longer than patients in the control group before acquiring the infection (mean ± SD, 25.4 ± 48 vs 10 ± 16 days; P =.047; Table 2). The 2 groups were not statistically different in intensity of illness as measured by the SAPS II score on admission, although patients in the AmpC group tended to have higher scores. Indeed, if infants younger than 1 year were omitted, the SAPS II score was significantly higher for the AmpC group, but restricting the scoring system to adults (18 years or older, as originally intended) showed no significant difference. Likewise, the McCabe index based on estimated survival in relation to the underlying comorbid illness was not significantly different between the groups (Table 2). Patients in the AmpC group remained in the hospital significantly longer than did patients in the control group following resolution of the bacteremic episode (Table 3). Discussion AmpC Group Control Group (n = 25) (n = 52) Mean ± SD length of stay 34.8 ± ± 22 after positive culture (d) Survival, died within 24 h 2 1 Subsequent 30-d mortality 2/23 (9) 3/51 (6) Fever response Afebrile day 2 16/23 (70) 32/45 (71) Afebrile day 4 19/22 (86) 37/44 (84) Data are given as number (percentage) unless otherwise indicated. Denominator of original group. P =.009. Denominators of evaluable cases. Fever defined as temperature 38 C for patients remaining in the hospital. We studied the outcome of patients with gram-negative bacteremia caused by AmpC-producing organisms, as determined by the modified Hodge test, and the outcome in an appropriate control group. Compared with the control group, significantly more patients in the AmpC group were intubated, in an ICU, and received inadequate antibiotic treatment within the first 48 hours after the positive culture was obtained. Despite this bias of the AmpC group toward greater morbidity, the clinical response to antibiotics was identical in the 2 groups as measured by the rate of defervescence of fever following the bacteremia and by 30-day mortality. Although patients in the AmpC group remained in the hospital longer than did patients in the control group after resolution of the bacteremia, they were also in the hospital more than twice as long before becoming bacteremic, suggesting that the length of stay after the bacteremia reflected the generally higher level of morbidity of patients in the AmpC group rather than an effect of the bacteremia itself. Indeed, the SAPS II score was higher for the AmpC group than the control group, although this difference did not reach statistical significance. The AmpC group was obtained from the blood culture isolates demonstrating the highest degree of positivity by the modified Hodge test using cefoxitin as described by Yong et al. 4 As illustrated in Image 1, it is possible to quantitate the growth of the indicator E coli from the expected circular zone of inhibition toward the test organism streak, although this assessment is subjective and only semiquantitative. In the study by Yong et al, 4 the best cutoff distinguishing between plasmid-associated AmpC production and non plasmid-mediated AmpC production in E coli and Klebsiella species was 3 mm. At that level, about 5% of the positive tests were falsepositives, whereas at 4 mm, none were false-positive, but the sensitivity decreased. In our study, there were 10 patients with an AmpC diagonal distance of 4 mm or more, and their rate of defervescence was identical to that of the AmpC and control groups as a whole (70% [7/10] afebrile by day 2 and 80% [8/10] by day 4). The modified Hodge test has been compared with other methods of detecting AmpC expression such as the boronic acid test and the EDTA disk test. 6 The modified Hodge test was, if anything, less sensitive than the boronic acid test. Although we found a very good correlation between the EDTA disk test and the modified Hodge test in a subset of our isolates, the correlation is not perfect and the EDTA disk test identified more positives (data not shown). Thus, it is possible that different methods would have identified a more accurate group of patients with AmpC; however, in our hands and in the studies of other investigators, the ability to detect the strongest positive AmpC-producing strains is virtually 100% among the different methods. Thus, it seems unlikely we missed any significant number of strong AmpC producers, and in the subgroup of the strongest AmpC producers we did identify, there was no difference in outcome compared with the entire AmpC or control group. In a study of 27 plasmid-mediated AmpC-producing bloodstream isolates of Klebsiella, Pai et al 2 found a clinical treatment failure rate of 51.9%, which was similar to that of bacteremic extended-spectrum β-lactamase (ESBL)- producing Klebsiella isolates. Park et al 1 compared the clinical response of 30 patients infected with E coli and Klebsiella isolates carrying AmpC-producing plasmids with control isolates and found a higher treatment failure rate at 72 hours and 7 days in the AmpC group, although the response rate at 30 days was similar (87% in the AmpC group and 97% in the control group). They noted that 70% of the Klebsiella isolates (and 1 E coli isolate) carried AmpC and ESBL plasmids but did not state what percentage of the control isolates produced ESBL. Thus, if their control patients did not have very many Am J Clin Pathol 2011;135: DOI: /AJCP7VD0NMAMQCWA 575

5 Rand et al / Laboratory Detection of AmpC β-lactamase isolates with ESBLs, their interpretation of the relationship between clinical failure and AmpC could have been related to the presence of ESBLs, as they state in their discussion. In our institution, we see few ESBL-containing E coli and Klebsiella isolates (<2%), and there were no ESBL-producing E coli or Klebsiella isolates in the AmpC group or control group. The modified Hodge test, EDTA disk test, and boronic acid test all fail to distinguish between plasmid-mediated AmpC production and derepressed hyperproduction of chromosomal AmpC. Characterization of the AmpC-producing plasmid status is complex and was beyond the scope of the study. If organisms with AmpC production due to a derepressed chromosomal mechanism produced less AmpC than organisms with AmpC-producing plasmids, it is possible that our results reflect the clinical outcome of gram-negative bacteremia by organisms with derepressed chromosomal AmpC production, while the studies of Pai et al 2 and Park et al 1 reflect the outcome of organisms with plasmid-mediated AmpC production. Several groups have noted that Enterobacteriaceae may test susceptible to extended-spectrum cephalosporins (eg, ceftazidime and cefotaxime), despite the presence of AmpCproducing plasmids and, thus, potentially may be reported as falsely susceptible. In fact, 9 (36%) and 14 (56%) of our 25 AmpC-producing isolates were reported as susceptible or intermediate to cefotaxime and ceftazidime, respectively, by standard broth dilution semiautomated methods (MicroScan). Although nearly 40% of our AmpC patient group did not receive an effective antibiotic within the first 48 hours (compared with 8% of the control group), 18 (78%) of 23 received a full treatment course with cefepime or meropenem. The remaining patients received various combinations that most often included a fluoroquinolone and an aminoglycoside. Thus, in our institution, false susceptibility to extended-spectrum cephalosporins might not have the same adverse effect on patient outcome as it otherwise could at institutions where extended-spectrum cephalosporin treatment was continued, particularly if ESBL-producing plasmids were common. Despite the fact that patients in the AmpC group tended to be more ill (more likely to be intubated, in the ICU, and not to have received an effective antibiotic within the first 48 hours), the clinical response and overall outcome of the AmpC and control bacteremic episodes were essentially identical. The modified Hodge test and the EDTA disk test did not seem to identify a subset of patients at risk for a poor outcome from AmpC with the antibiotic use patterns in our hospital. From the Departments of 1 Pathology, Immunology, and Laboratory Medicine and 4 Medicine and the 3 Emerging Pathogens Institute, University of Florida; and 2 Shands at the University of Florida Hospital, Gainesville. Supported in part by the Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, College of Medicine. Address reprint requests to Dr Rand: Dept of Pathology, Immunology, and Laboratory Medicine, PO Box , University of Florida, College of Medicine, Gainesville, FL References 1. Park YS, Yoo S, Seo MR, et al. Risk factors and clinical features of infections caused by plasmid-mediated AmpC beta-lactamase-producing Enterobacteriaceae. Int J Antimicrob Agents. 2009;34: Pai H, Kang CI, Byeon JH, et al. Epidemiology and clinical features of bloodstream infections caused by AmpC-typebeta-lactamase-producing Klebsiella pneumoniae. Antimicrob Agents Chemother. 2004;48: Black JA, Moland ES, Thomson KS. AmpC disk test for detection of plasmid-mediated AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal AmpC betalactamases. J Clin Microbiol. 2005;43: Yong D, Park R, Yum JH, et al. Further modification of the Hodge test to screen AmpC beta-lactamase (CMY-1)- producing strains of Escherichia coli and Klebsiella pneumoniae. J Microbiol Methods. 2009;34: Coudron PE. Inhibitor-based methods for detection of plasmid-mediated AmpC beta-lactamases in Klebsiella spp, Escherichia coli and Proteus mirabilis. J Clin Microbiol. 2005;43: Lee W, Jung B, Hong SG, et al. Comparison of 3 phenotypic-detection methods for identifying plasmidmediated AmpC beta-lactamase-producing Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis strains. Korean J Lab Med. 2004;29: Am J Clin Pathol 2011;135: DOI: /AJCP7VD0NMAMQCWA