Are Clinical Laboratories in California Accurately Reporting Vancomycin-Resistant Enterococci?

JOURNAL OF CLINICAL ROBIOLOGY, Oct. 1997, p. 2526 2530 Vol. 35, No. 10 0095-1137/97/$04.00 0 Copyright 1997, American Society for Microbiology Are Clinical Laboratories in California Accurately Reporting Vancomycin-Resistant Enterococci? JON ROSENBERG, 1 * FRED C. TENOVER, 2 JANE WONG, 3 WILLIAM JARVIS, 2 AND DUC J. VUGIA 1 Disease Investigation and Surveillance Branch 1 and Microbial Diseases Laboratory, 3 Division of Communicable Disease Control, California Department of Health Services, Berkeley, California 94704, and Hospital Infections Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333 2 Received 3 March 1997/Returned for modification 21 May 1997/Accepted 14 July 1997 In order to determine whether hospital-based clinical laboratories conducting active surveillance for vancomycin-resistant enterococci in three San Francisco Bay area counties (San Francisco, Alameda, and Contra Costa counties) were accurately reporting vancomycin resistance, five vancomycin-resistant enterococcal strains and one vancomycin-susceptible -lactamase-producing enterococcus were sent to 31 of 32 (97%) laboratories conducting surveillance. Each strain was tested by the laboratory s routine antimicrobial susceptibility testing method. An Enterococcus faecium strain with high-level resistance to vancomycin (, 512 g/ml) was correctly reported as resistant by 100% of laboratories; an E. faecium strain with moderate-level resistance (, 64 g/ml) was correctly reported as resistant by 91% of laboratories; two Enterococcus faecalis strains with low-level resistance (s, 32 g/ml) were correctly reported as resistant by 97 and 56% of laboratories, respectively. An Enterococcus gallinarum strain with intrinsic low-level resistance (, 8 g/ml) was correctly reported as intermediate by 50% of laboratories. A -lactamase-producing E. faecalis isolate was correctly identified as susceptible to vancomycin by 100% of laboratories and as resistant to penicillin and ampicillin by 68 and 44% of laboratories, respectively; all 23 (74%) laboratories that tested for -lactamase recognized that it was a -lactamase producer. This survey indicated that for clinically significant enterococcal isolates, laboratories in the San Francisco Bay area have problems in detecting low- to moderate-level but not high-level vancomycin resistance. Increasing accuracy of detection and prompt reporting of these isolates and investigation of cases are the next steps in the battle for control of the spread of vancomycin resistance. Over the past 10 years enterococci have become one of the most important nosocomial pathogens. Intrinsic enterococcal resistance to numerous antimicrobial agents, including -lactams and aminoglycosides, has contributed to their selection as nosocomial pathogens following the introduction of the cephalosporins (19). Subsequently, enterococci have acquired additional resistance through the production of -lactamases (21), altered penicillin-binding proteins (36), and aminoglycoside-modifying enzymes (15). Genetically mediated (33, 37) resistance to the glycopeptide vancomycin is considered one of the most critical examples of enterococcal resistance since (i) additional resistance genes often are present in the same strain, leaving no effective therapeutic agents available to treat infected patients, and (ii) the gene mediating high-level resistance is transferable, raising the potential that vancomycin resistance will spread to other more pathogenic organisms (10). Vancomycin-resistant enterococci were first recovered from clinical isolates in Europe in 1986 (16) and in the United States * Corresponding author. Mailing address: Disease Investigation and Surveillance Branch, Division of Communicable Disease Control, California Department of Health Services, 2151 Berkeley Way, Berkeley, CA 94704. Phone: (510) 540-2566. Fax: (510) 540-2570. E-mail: jrosenbe@hwl.cahwnet.gov. Member of the California Emerging Infections Program, which includes Arthur L. Reingold, School of Public Health, University of California, Berkeley, Calif.; S. Benson Werner, Sharon Abbott, and J. Michael Janda, Division of Communicable Disease Control, California Department of Health Services; and Gretchen Rothrock, Nandeeni Mukerjee, Lisa Gelling, Susan Shallow, Pamela Daily, and Jenny Flood, California Public Health Foundation, Berkeley, Calif. in 1988 (13). Published outbreaks and National Nosocomial Infections Surveillance System data indicate that vancomycin resistance first emerged in the northeastern United States, causing numerous outbreaks (2, 3, 7, 9, 14, 27, 34) and establishing endemicity in a number of locations (18) at a time when little resistance was evident in the western United States. Surveillance is essential to understanding and controlling antimicrobial resistance (8, 11, 17, 20, 25, 32). Surveillance for antimicrobial agent-resistant pathogens should be laboratory based (5, 6, 26) and should include standardized methods for susceptibility testing with quality controls (1, 4). As a component in the California Emerging Infections Program, a collaborative program of the California Department of Health Services, the University of California, Berkeley, School of Public Health, the Centers for Disease Control and Prevention (CDC), and local health departments, active laboratory-based surveillance for vancomycin resistance among enterococcal clinical isolates was initiated in January 1995 at 35 general acute-care hospitals that use 32 microbiology laboratories to perform antimicrobial susceptibility tests in three San Francisco Bay area counties (San Francisco, Alameda, and Contra Costa counties). In January 1995, a survey indicated that all but one of these laboratories had been routinely testing for vancomycin susceptibility of most clinical isolates, including isolates from urine, for at least 1 year; beginning in 1995 all laboratories did so. During 1994, 6 (19%) laboratories reported detecting one or more (range, one to six) vancomycinresistant enterococci. The sensitivity and specificity of the surveillance system for monitoring resistance are dependent upon the abilities of these laboratories to detect vancomycin resistance. Different methods of susceptibility testing have demonstrated variable success 2526

VOL. 35, 1997 REPORTING OF VANCOMYCIN-RESISTANT ENTEROCOCCI 2527 Antibiotic Strain 1 (E. faecium VanA) a Strain 2 (E. faecium VanB-like) TABLE 1. Enterococcal strains used in study Strain 3 (E. faecalis VanB-like) Strain 4 (E. faecalis VanB) Strain 5 (E. gallinarum VanC) Strain 6 (E. faecalis) Vancomycin 512 6 64 6 32 11 16 32 15 8 17 2 18 Penicillin 32 6 256 6 2 16 4 18 2 21 4 b 15 Ampicillin 4 21 256 6 1 25 1 22 1 22 1 b 19 BL c a Disk diffusion (24). b The clinical significance of the penicillin and ampicillin susceptibility divergence of this strain is unclear. c BL, -lactamase test;, negative reaction;, positive reaction. in detecting vancomycin resistance (28, 30, 31, 35, 37, 39). To determine the abilities of the various antimicrobial susceptibility testing methods used at surveillance study laboratories to detect vancomycin, penicillin, and/or ampicillin resistance, we conducted a proficiency study. MATERIALS AND METHODS Enterococcal strains. Six strains of enterococci (three Enterococcus faecalis, two Enterococcus faecium, and one Enterococcus gallinarum strains), including five strains representing the four most common vancomycin resistance phenotypes (strains 1 to 5; Table 1) and a vancomycin-susceptible -lactamase-producing strain (strain 6; Table 1), were obtained from the CDC strain collection, inoculated onto Trypticase soy agar slants (Becton Dickinson Microbiology Systems [BDMS], Cockeysville, Md.), and sent to the 31 participating laboratories. Susceptibility testing. Each laboratory supervisor was instructed to test the six organisms for resistance to ampicillin, penicillin, and vancomycin by using the antimicrobial susceptibility testing method routinely used at the laboratory and to indicate the and interpretation (susceptible, intermediate, or resistant) or zone size and interpretation on the form provided. After completion of the tests, the forms were sent to the California Department of Health Services and CDC for data entry and analysis. The reported results obtained with the MicroScan Touchscan and AutoSCAN systems were combined. Information on the susceptibility panels (MicroScan) and cards (Vitek) used was obtained after the completion of testing. Reference methods. All reference tests were performed at CDC. Broth microdilution was performed with cation-adjusted Mueller-Hinton broth (BDMS) according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) (23), and disk diffusion was performed with Mueller-Hinton agar and disks from BDMS according to NCCLS guidelines (24). Categorical errors were not calculated for the data because of the small sample sizes. Results were considered to be in agreement if they differed by a single dilution above or below the reference value for the s (38). -Lactamase tests were performed by using the colorimetric cephalosporin nitrocefin (Cefinase; BDMS). RESULTS Thirty-one (97%) of 32 laboratories, representing 34 (97%) of 35 hospitals conducting surveillance for vancomycin-resistant enterococci, participated in the study. One laboratory reported two sets of results obtained by two antimicrobial susceptibility testing methods, and these were counted independently. The methods used for antimicrobial susceptibility testing at participating laboratories varied, with 19 (59%) of 32 laboratories using a MicroScan system (Table 2). Overall, 11 (35%) laboratories used a screen plate consisting of brain heart infusion (BHI) agar containing 6 g of vancomycin per ml as a supplemental method of testing for vancomycin susceptibility (28, 30, 34). Those using MicroScan systems as their primary method were less likely to use a screening plate (3 of 19) than those using the Vitek system (3 of 5), the disk diffusion method (2 of 5), or the other systems (3 of 3). Of 19 MicroScan system users, 18 used the conventional MicroScan Positive Breakpoint Combo Type 6 panel, while 1 used the Type 8 panel. All Vitek users used the Gram-Positive Susceptibility card. Overall, the accuracies of the participating laboratories varied from 50 to 100% for vancomycin, 42 to 100% for penicillin, and 44 to 100% for ampicillin (Table 3). The accuracies for vancomycin were related to the level of resistance: 100% for high-level resistance (VanA;, 512 g/ml), 91% (strain 2) and 97% (strain 3) for moderate resistance (VanB-like;, 32 to 64 g/ml), 84% for low-level resistance (VanB;, 16 to 32 g/ml), 50% for the intermediate E. gallinarum strain (VanC;, 8 g/ml), and 100% for vancomycin susceptibility (, 2 g/ml). Users of all test systems reported vancomycin s that were in agreement with the reference s for strains 1 and 6. All MicroScan Touchscan (AutoSCAN) users additionally reported s in agreement with reference s for strains 3, 4, and 5, while all WalkAway users reported s in agreement with reference s for strains 2, 3, and 5. Test s differed from reference s for MicroScan users for strain 2 (reference, 64 g/ml), which two AutoSCAN users classified as intermediate (, 16 g/ml), and strain 4 (reference s, 16 to 32 g/ml), which two MicroScan WalkAway users classified as intermediate (, 8 g/ml) and one WalkAway user classified as susceptible (, 4 g/ml). For strain 5 (E. gallinarum;, 8 g/ml), all MicroScan users reported s of vancomycin within a single dilution of the reference value. All five AutoSCAN users, five of eight Touchscan users, and three of six WalkAway users classified it as intermediate (s, 8 g/ml). Three Touchscan users reported s of 4 g/ml, but one classified the strain as resistant on the basis of screen plate growth. Two WalkAway users classified it as susceptible (, 4 g/ml), while another classified it as resistant (, 8 g/ml) on the basis of screen plate growth. The Vitek system was unable to detect vancomycin resistance in strains 2 and 4. Three laboratories using the Vitek TABLE 2. Methods used by study laboratories for susceptibility testing Primary method a No. (%) of laboratories No. of laboratories using supplemental screen plate MicroScan AutoSCAN (Touchscan) 13 (40.6) 2 WalkAway 6 (18.8) 1 Vitek 5 (15.6) 3 Disk diffusion 5 (15.6) 2 Pasco (Difco) 1 (3.1) 1 Sensititre 1 (3.1) 1 Microbroth dilution 1 (3.1) 1 a One laboratory reported two sets of results by two susceptibility testing methods.

2528 ROSENBERG ET AL. J. CLIN. ROBIOL. TABLE 3. Results of susceptibility testing by classification % of laboratories reporting the Antibiotic and strain no. following classification a : S I R Vancomycin (n 32) 1 0 0 100 2 3 6 91 3 3 0 97 4 16 28 56 5 22 50 28 6 100 0 0 Penicillin b (n 19) 1 58 0 42 2 0 0 100 3 89 11 0 4 95 5 0 5 95 5 0 6 33 0 67 Ampicillin b (n 30) 1 93 0 7 2 0 0 100 3 97 3 0 4 100 0 0 5 100 0 0 6 56 0 44 a S, susceptible; I, intermediate; R, resistant. Boldface type indicates the correct result. b No intermediate category for penicillin and ampicillin is recognized by NC- CLS. system reported vancomycin s of 2 g/ml for strain 2. However, two of these classified it as resistant on the basis of the growth of this strain on screen plates, together with another Vitek user that did not report an. Four of five Vitek users reported s of 2 g/ml for strain 4, although three classified this organism as vancomycin resistant on the basis of growth on the screen plate. All four Vitek users classified strain 5 (E. gallinarum) as resistant to vancomycin (s, 32 g/ml). All disk diffusion users classified strains 1 and 2 (reference zone size, 6 mm for each strain) as resistant to vancomycin; all reported a zone size of 6 mm for strain 1, while for strain 2, zone sizes ranged from 6 to 14 mm. Disk diffusion users reported a range of zone sizes and/or classification errors for vancomycin susceptibility for the remainder of the vancomycin-resistant strains. Strain 3 (reference zone size, 11 mm) was misclassified as susceptible by one participant using disk diffusion, in spite of a zone size of 12 mm. Two disk diffusion users classified strain 4 (reference zone size, 15 mm) as resistant (zone sizes, 12 and 13 mm). One participant reported a zone size of 15 mm but classified the strain as susceptible instead of intermediate. One laboratory classified it as susceptible (zone size, 18 mm). One laboratory classified the strain as intermediate, on the basis of a zone size of 20 mm and screen plate growth. The same laboratory classified strain 6 as susceptible, with a zone size of 19 mm and no growth on the screen plate. Two of six disk diffusion users classified strain 5 (E. gallinarum; reference zone size, 17 mm) as susceptible (zone size, 17 mm); an additional laboratory classified it as susceptible (zone size, 19 mm). Two laboratories classified the isolate as intermediate, one with a zone size of 16 mm and the other with a zone size of 18 mm and growth on the screen plate. One laboratory classified the organism as resistant (zone size, 18 mm) on the basis of screen plate growth. Strain 6 (reference zone size, 18 mm) was correctly classified by all disk diffusion users as susceptible to vancomycin (zone sizes, 17 to 21 mm). BHI screen plate results were provided for each strain by 11 laboratories. Strains 1 through 5 were all reported as showing growth on the screen plate, while strain 6 was reported as showing no growth on the screen plate. Of the 77 sets of strains for which results were reported, 65 (84%) were correctly classified as resistant or susceptible to vancomycin on the basis of s or zone sizes, and the results were corroborated by growth or no growth on the screen plate, including 11 of 11 sets for strains 1, 3, and 6. Eight laboratories modified vancomycin susceptibility classifications for 12 sets of strains on the basis of growth on screen plates. Three Vitek users classified strains 2 and 4 (s, 2 g/ml) as resistant. One disk diffusion user classified strain 4 (zone size, 20 mm) and strain 5 (zone size, 18 mm) as intermediate. Strain 5 was classified as resistant by one MicroScan WalkAway and one broth microdilution user (s, 4 g/ml) and one disk diffusion user (zone size, 18 mm). One MicroScan Touchscan user misclassified strain 5 as resistant (, 8 g/ml). Of the study participants, 8 of 18 (42%) correctly classified strain 1 as resistant to penicillin and 28 of 30 (93%) reported that this strain was susceptible to ampicillin. Of the MicroScan system users, three of eight Touchscan (AutoSCAN) users and one of four WalkAway users classified the strain as resistant to penicillin (s, 8 g/ml). Most of the penicillin or ampicillin susceptibility results for strains 2, 3, 4, and 5 were correct. One disk diffusion user classified strains 3, 4, and 5 as intermediate to penicillin (zone sizes, 16 to 18 mm). All 23 laboratories that routinely tested enterococci for lactamase production (by the nitrocefin disk method) reported that strain 6 was a -lactamase producer; of these, 2 failed to classify the organism as resistant to either -lactam antibiotic. Three disk diffusion users classified the organism as susceptible to ampicillin (zone sizes, 18, 21, and 22 mm, respectively). One laboratory classified the organism as resistant to ampicillin (zone size, 16 mm), while another laboratory classified it as resistant on the basis of -lactamase production and a zone size of 22 mm. Two disk diffusion users classified the organism as resistant to penicillin (zone sizes, 13 and 14 mm, respectively). DISCUSSION Accurate and reproducible results are critical to the success of any laboratory-based study or surveillance system (6). While all the laboratories in our surveillance system were capable of detecting high-level vancomycin resistance mediated by vana, problems in the detection of lesser degrees of vancomycin resistance were identified, providing an opportunity for the correction of these problems. Previous studies have reported greater degrees of difficulty in detecting low-level vancomycin resistance for both automated and nonautomated commercially available systems (28, 30, 31, 35, 37, 39). For example, strain 2 was correctly identified as resistant by 91% of laboratories in the current study, whereas 22 of 76 (29%) clinical laboratories in New Jersey in 1993 identified the organism as resistant (30). This improved performance is partially accounted for by the high proportion of laboratories that use the MicroScan test systems, which were better able to detect resistance in strains 2 and 3 (VanB-like and VanB phenotypes, respectively) than the Vitek system, and to the use of the BHI agar screen. These results are consistent with those of a recent study confirming the ability of the conventional MicroScan panels, which were used exclusively by laboratories in our

VOL. 35, 1997 REPORTING OF VANCOMYCIN-RESISTANT ENTEROCOCCI 2529 study, to detect vancomycin and ampicillin resistance in enterococci (12). With the possible exception of the 13 Touchscan (AutoSCAN) users, the small number of users of any one system in the study suggests that caution must be used when interpreting our data. One-third of laboratories participating in the study routinely used BHI agar containing vancomycin as a supplemental screen for vancomycin resistance. For 85% of the tests in which this was done, the screen plate results provided corroboration of the results obtained by the primary method of testing. For 11 of 12 strains with discrepant results, laboratories used growth on screen plates to change the classification from susceptible to resistant (n 8, including 7 resistant strains) or intermediate (n 3, including 1 intermediate strain), thus preventing inaccurate reporting of resistant or intermediate strains as susceptible. Since the study was conducted, a number of additional laboratories have begun using the screen plate as a supplemental test and, in a few instances, as the initial test for vancomycin resistance. In one instance, a Vitek user experienced an outbreak of vancomycin-resistant enterococci in which the epidemic strain manifested the vanb phenotype; the addition of a supplemental screen plate indicated that 67% of the resistant isolates had not been detected by the Vitek system (unpublished data). The results of our study support the use of a screening process such as one with BHI-vancomycin agar, which has a sensitivity and a specificity of 100 and 96 to 99%, respectively (29), as a cost-effective supplement to susceptibility testing for vancomycin (31, 35), as currently recommended by NCCLS (23, 24). Compliance by laboratories with NCCLS guidelines was incomplete with regard to testing of enterococci for -lactamase production (22), because only 74% of laboratories recognized the -lactamase-producing isolate. A number of laboratories misclassified isolates as intermediate to -lactam antibiotics, a category not included in NCCLS guidelines for test systems that use s. Additionally, some laboratories reported test or zone results in agreement with reference values but failed to use NCCLS-recommended breakpoints and therefore reported incorrect interpretations. Compliance with NCCLS guidelines would have eliminated a large proportion of the reporting problems in this study. This survey provides further evidence of the critical role of the susceptibility testing method in the accuracy of reporting of antimicrobial resistance. There is likely to be geographic and temporal variability in the testing methods used by clinical laboratories. 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