Use of Positive Blood Cultures for Direct Identification and Susceptibility Testing with the Vitek 2 System

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2004, p Vol. 42, No /04/$ DOI: /JCM Copyright 2004, American Society for Microbiology. All Rights Reserved. Use of Positive Blood Cultures for Direct Identification and Susceptibility Testing with the Vitek 2 System Marina de Cueto,* Esther Ceballos, Luis Martinez-Martinez, Evelio J. Perea, and Alvaro Pascual Microbiology Department, Hospital Virgen Macarena, Seville, Spain Received 7 October 2003/Returned for modification 14 January 2004/Accepted 23 February 2004 In order to further decrease the time lapse between initial inoculation of blood culture media and the reporting of results of identification and antimicrobial susceptibility tests for microorganisms causing bacteremia, we performed a prospective study in which specially processed fluid from positive blood culture bottles from Bactec 9240 (Becton Dickinson, Cockeysville, Md.) containing aerobic media were directly inoculated into Vitek 2 system cards (bio-mérieux, France). Organism identification and susceptibility results were compared with those obtained from cards inoculated with a standardized bacterial suspension obtained following subculture to agar; 100 consecutive positive monomicrobic blood cultures, consisting of 50 gram-negative rods and 50 gram-positive cocci, were included in the study. For gram-negative organisms, 31 of the 50 (62%) showed complete agreement with the standard method for species identification, while none of the 50 gram-positive cocci were correctly identified by the direct method. For gram-negative rods, there were 50% categorical agreements between the direct and standard methods for all drugs tested. The very error rate was 2.4%, and the error rate was 0.6%. The overall error rate for gram-negatives was 6.6%. Complete agreement in clinical categories of all antimicrobial agents evaluated was obtained for 19 of 50 (38%) gram-positive cocci evaluated; the overall error rate was 8.4%, with 2.8% minor, 2.4%, and 3.2% very. These findings suggest that the Vitek 2 cards inoculated directly from positive Bactec 9240 bottles do not provide acceptable bacterial identification or susceptibility testing in comparison with corresponding cards tested by a standard method. Detection of bloodstream infections is one of the most important tasks performed by the clinical microbiology laboratory. Rapid identification results and antimicrobial susceptibility tests are essential for guiding clinicians in the selection of the most appropriate treatment for patients with bloodstream infections (5, 22). There are few well-designed studies that have examined the clinical impact of rapid bacterial identification and antimicrobial susceptibility testing, and there is even less information on blood cultures. The available information suggests that early knowledge of these data may be related to lower mortality rates and could affect patient therapy, leading to reductions in health care costs for the pharmacy, laboratory, and other general charges (1, 11, 20, 22). Many remarkable improvements have been made in an attempt to reduce the time required to identify pathogens in blood, and over the last several decades, a variety of automated systems have been developed for detecting or identifying pathogenic microorganisms as well as for determining antimicrobial susceptibility. Continuously monitoring automated blood culture systems have emerged as the new standard in blood culture technology, enabling detection of a positive blood culture within 24 h of initial incubation. Current automated systems for the identification and susceptibility testing of bacteria enable the generation of test results in 2 to 12 h and, in general, have been shown to provide results which are nearly as accurate as those derived from traditional tests (6, 7). * Corresponding author. Mailing address: Departamento de Microbiología, Hospital Universitario Virgen Macarena, Avda. Dr. Fedriani núm. 3, Seville, Spain. Phone: Fax: m.decueto@vodafone.es. An overnight agar medium subculture from positive blood bottles is the initial step in the microbial identification of pathogens causing bacteremia. This conventional culture method is time-consuming, and several days are usually required for microbial recovery, biochemical identification of the bacterial isolate, and determination of antimicrobial susceptibility (19). In order to further decrease the length of time from initial inoculation of blood culture media to reporting the identification and susceptibility results of microorganisms causing bacteremia, different studies have been conducted in which fluid from a positive blood culture is directly inoculated into automated systems (4, 8, 14, 17, 21, 24). However, the results of these studies have varied depending on the system evaluated and the combination used. The Vitek 2 system (bio-mérieux, France) was introduced to the market in 1999, and its ability to identify and determine the susceptibility of both gram-positive cocci and gram-negative rods has been evaluated in several reports (2, 9, 10, 13, 16). However, only a few studies have evaluated the accuracy of identification and the reliability of susceptibility results when cards of the Vitek 2 system are directly inoculated from a positive blood culture (3, 15). In the present report, we carried out a prospective study in which specially processed fluid from positive blood culture bottles from the Bactec 9240 (Becton Dickinson, Cockeysville, Md.) containing aerobic media were directly inoculated into Vitek 2 cards. Organism identification and susceptibility results were compared with those obtained from standard procedures. 3734

2 VOL. 42, 2004 VITEK 2 SYSTEM FOR BLOOD CULTURES 3735 MATERIALS AND METHODS This study was conducted between October 2001 and January 2002 at the University Hospital Virgen Macarena in Seville (Spain), a 1,000-bed tertiary hospital which provides a full range of medical and surgical care. During the study period, blood cultures from all adult patients with suspected bacteremia were screened for growth of microorganisms with the Bactec 9240 system. One blood culture consisted of at least two Bactec Plus Aerobic F bottles and two Bactec Plus Anaerobic F bottles. Positive aerobic blood cultures detected as positive were examined by Gram stain, and a small volume of blood culture fluid was inoculated onto blood agar and chocolate agar plates (Biomedics, Madrid, Spain). Specimens from different patients that appeared to contain the same organism were included in the study. Specimens yielding more than one isolate after subculture were excluded from the study. Blood cultures with gram-negative rods and gram-positive cocci morphology revealed by microscopy were included. Haemophilus spp, Neisseria spp., Streptococcus pneumoniae, the Streptococcus viridans group, yeasts, and anaerobes were excluded because, in the routine work of the laboratory, identification and antimicrobial susceptibility testing of such microorganisms are carried out by standard biochemical tests. Specimens tested. A total of 100 consecutive positive aerobic blood cultures, consisting of 50 gram-negative rods and 50 gram-positive cocci, were included in the present study. All 100 positive blood samples were inoculated by a direct (that is, the organism was taken from the positive blood culture bottle) and by a standardized (taken from the overnight agar medium subculture) method on Vitek cards for identification and antimicrobial susceptibility testing. Blood culture instrumentation and media. Blood culture testing was performed on Bactec 9240 instruments with standard aerobic media. The Bactec Plus F medium consists of soybean-casein digest broth, primary supplements, and two types of resin, a nonionic absorbing resin and a cation exchange resin. Vitek 2 system and Vitek cards. The Vitek 2 system was used for all reading and interpretation of results. The ID GPC and ID GNB cards were used for the identification of gram-positive cocci and gram-negative rods, respectively, according to the manufacturer s instructions. The AST-523, AST-524, and AST-N020 cards were used for antimicrobial susceptibility testing of staphylococci, streptococci, and gram-negative rods, respectively. For the direct method, the AST Vitek cards were chosen according to the Gram stain results: gram-positive cocci with the staphylococcal gram stain (cocci in clusters, pairs, and tetrads), gram-positive cocci with the streptococcal Gram stain (cocci in pairs and chains), or gram-negative rods. Identification and antimicrobial susceptibility testing. (i) Direct inoculation method. For the direct inoculation method, serum separator tubes (Becton Dickinson Vacutainer Systems) were inoculated with a 6-ml sample of the positive blood culture bottle. The Vacutainer tube was centrifuged at 3,000 rpm for 5 min to pellet the blood cells. Then, 3 ml of the supernatant was transferred to a second Vacutainer tube containing 3 ml of saline, and the bacteria were sedimented by centrifugation at 3,000 rpm for 15 min. The bacterial pellet was harvested from the surface of the silicon layer with a cotton swab to make a suspension in 2 ml of 0.45% saline with the Densichek Vitek colorimeter. For gram-negative rods, a McFarland 0.5 standard suspension was performed. For gram-positive cocci, the inoculum suspension was equivalent to the number 1 standard on the McFarland scale. A subculture of serial dilutions from the McFarland suspension was performed on blood agar plates to correlate it with bacterial growth, to control for chance contamination during sample handling and to exclude polymicrobic specimens. Vitek cards for antimicrobial susceptibility testing are automatically filled with an inoculum prepared from the standardized McFarland suspension used with the identification card. The Vitek 2 system automatically processes the antimicrobial susceptibility card until the MICs are obtained. (ii) Standard inoculation method. With the standard method for identification and susceptibility testing, a small volume of blood culture fluid was inoculated onto blood agar and chocolate agar plates (Biomedics, Madrid, Spain) The inoculated plates were incubated at 35 C in5%co 2 to enable bacterial colonies to develop. After overnight incubation, a standardized inoculum was prepared from the agar medium, and the appropriate Vitek ID and AST cards were inoculated following the manufacturer s recommendations. Data analysis. Bacterial identification and susceptibility data from the direct method were compared to those obtained from standard tests. Only isolates correctly identified by the standard method were evaluated. Organisms reported as not identified by the standard method were excluded from the study. After comparison with the results from the standard method, identification results with the direct method were grouped in three different categories: (i) correctly identified, that is, the correct, unambiguous identification at the species level; (ii) misidentified, where the organism was incorrectly identified at either the genus or species level; and (iii) not identified, where no identification was provided at all. All correctly identified, misidentified, and unidentified isolates were evaluated in the antimicrobial susceptibility test in the present study. To compare the results of susceptibility testing by the direct method with those obtained from the standard method, the MICs obtained by both methods were translated into clinical categories (susceptible, intermediate, or resistant) according to the interpretive criteria of the Vitek 2 expert system. The MIC generated by the Vitek 2 system is not dependent on identification, even though the determination of clinical category is based on identification. For unidentified organisms, the system enables an identification to be introduced manually, and when this is done, the clinical categories are automatically determined by the system. In this way, for organisms not identified by the direct method, MICs were translated into clinical categories by the Vitek 2 system after manually introducing identification of the same isolate obtained by the standard method. Agreements and discrepancies between the direct and standard inoculation methods were classified as follows: agreements, very (false susceptibility), (false resistance), or minor (susceptible/resistant versus intermediate susceptibility). For gram-negative organisms, the following 10 antimicrobial agents were compared: ampicillin, cefazolin, cefotaxime, ceftazidime, ciprofloxacin, piperacillin, piperacillin-tazobactam, imipenem, gentamicin, and trimethoprim-sulfamethoxazole. For gram-positive cocci, the antibiotics evaluated were penicillin, erithromycin, gentamicin, rifampin, ofloxacin, trimethoprim-sulfamethoxazole, vancomycin, teicoplanin, tetracycline, and clindamycin. Additional antimicrobial agents tested by the Vitek 2 system were not compared because they are not reported routinely. RESULTS One hundred aerobic monomicrobial positive blood cultures were evaluated. Ninety-five (95%) isolates were correctly identified by the standard method. The unidentified group consisted of five isolates reported as a low level of discrimination, although a correct identification was given among the organisms listed by the Vitek 2 system. After performing a rapid manual test, such as oxidase or indole, all five isolates were identified. Thus, a total of 100 blood culture isolates could be evaluated to assess the accuracy of direct identification and susceptibility testing. Direct identification of positive blood culture from Bactec. Among the 50 isolates of gram-positive cocci obtained, 22 (44%) were Staphylococcus aureus, 16 (32%) were Staphylococcus epidermidis, 11 (22%) corresponded to species of coagulase-negative staphylococci, and 1 was identified as Streptococcus agalactiae. Of the 50 gram-negative rods studied, 41 (82%) corresponded to the family Enterobacteriaceae (24 Escherichia coli,4 Salmonella spp., 3 Klebsiella pneumoniae, 3 Proteus mirabilis, 2 Klebsiella oxytoca, and one each Enterobacter aerogenes, Enterobacter cloacae, Morganella morganii, Citrobacter freundii, and Klebsiella ornythinolytica) and 9 (18%) were identified as nonfermentative gram-negative rods (5 Pseudomonas aeruginosa,3 Acinetobacter baumannii, and 1 Pseudomonas putida). In Table 1, the direct Vitek method is compared with the routine standard method. None of the gram-positive cocci showed concordant identification between the direct and standard methods: 13 isolates were not identified, and 37 were misidentified. Most of the mistaken isolates (17 of 37) (45%) were identified as Kocuria spp. or Micrococcus luteus. Other discrepancies consisted of misidentification between various species of coagulase-negative staphylococci. One isolate of Staphylococcus aureus was not identified due to insufficient growth on the card, although

3 3736 DE CUETO ET AL. J. CLIN. MICROBIOL. TABLE 1. Identification of 100 isolates from positive Bactec blood culture by the direct inoculation method and the standard identification method Species a Correct identification isolates No. identification Misidentification Total E. coli P. aeruginosa Salmonella spp. 4 4 A. baumannii 3 3 K. pneumoniae P. mirabilis Other GNR S. aureus S. epidermidis S. hominis Other CoNS S. agalactiae 1 1 Total a GNR, gram-negative rods; CoNS, coagulase-negative staphylococci. the inoculum control showed a colony count of CFU/ ml. For 16 isolates, a low inoculum was used, with 10 8 CFU/ml in the subculture of the bacterial suspension used to inoculate the ID Vitek card. Of these 16 isolates, 5 were not identified and 11 were misidentified. Of the 50 gram-negative rods studied, only 31 (62%) showed concordant identification between the direct and standard methods, 14 (28%) were reported as not identified organism, and 5 (10%) were misidentified. All the misidentified organisms belonged to the family Enterobacteriaceae: one Proteus mirabilis isolate was incorrectly identified as a Vibrio sp., another Proteus mirabilis isolate and one Citrobacter freundii were reported as nonfermentative gram-negative rods, one Enterobacter cloacae isolate was identified as Serratia plymuthica, and one Klebsiella pneumoniae isolate was identified as Enterobacter cloacae. The group of unidentified organisms included five gramnegative nonfermentative organisms and nine species of Enterobacteriaceae. For seven isolates, cards were inoculated with less than CFU/ml, of which five were reported as unidentified isolates and the remaining two as misidentified. Susceptibility testing. Standardized susceptibility results were available for all 100 isolates tested. The percent error for each drug tested, compared directly with the standard method, is shown in Tables 2 and 3. The 50 isolates of gram-negative rods were assessed with 10 antimicrobial agents, resulting in a total of 500 isolate-antimicrobial agent combinations. Twenty-five of 50 (50%) gramnegative isolates showed complete categorical agreement, that is, no very,, or minor for any drug tested. The error rate was 6.6% overall; 2.4% very, 0.6%, and 3.6% minor. All arose from the results of testing penicillins and cephalosporins. The error rate for the remaining four agents assessed (ciprofloxacin, imipenem, gentamicin, and trimethoprim-sulfamethoxazole) was 0.3%, with no very or. Among the drugs TABLE 2. Antimicrobial susceptibility correlation for gram-negative rods Antimicrobial agent a very included in the gram-negative AST cards, only imipenem and trimethoprim-sulfamethoxazole showed complete categorical agreement for all 50 isolates tested. Among the 25 isolates that showed complete categorical agreement for all drugs tested, 20 (40%) were correctly identified by the direct method, one (2%) was a misidentified isolate, and four (8%) were not identified. Like gram-negative rods, the gram-positive cocci were assessed for 10 other antimicrobial agents. Complete agreement in the clinical categories of all antimicrobial agents evaluated was obtained for 19 of the 50 (38%) gram-positive cocci studied. The overall error rate was 8.4%, with 2.8% minor, 2.4%, and 3.2% very. Among the drugs on the gram-positive AST cards, only vancomycin had no or very. The drugs with the most very were clindamycin, erythromycin, and gentamicin. No difference in error rate was found between misidentified and nonidentified isolates. DISCUSSION Ampicillin Cefalothin Cefotaxime Ceftazidime Ciprofloxacin Piperacillin Piperacillin-tazobactam Imipenem Gentamicin TMP/SMX minor Total (%) 12 (2.4) 3 (0.6) 18 (3.6) a TMP/SMX, trimethoprim-sulfamethoxazole. We evaluated the ability and accuracy of Vitek 2 cards inoculated directly from Bactec 9240 bottles to identify organisms which caused bacteremia and determine their antimicro- Antimicrobial agent a TABLE 3. Antimicrobial susceptibility correlation for gram-positive cocci very Penicillin Erythromycin Gentamicin Rifampin Ofloxacin TMP/SMX a Vancomycin Teicoplanin Tetracycline Clindamycin minor Total (%) 16 (3.2) 12 (2.4) 14 (2.8) a TMP/SMX, trimethoprim-sulfamethoxazole.

4 VOL. 42, 2004 VITEK 2 SYSTEM FOR BLOOD CULTURES 3737 bial susceptibility. We wanted to assess whether this technique could be used to reduce turnaround time and permit the application of appropriate therapy sooner than if it had been determined by the conventional method. As the ability and accuracy of Vitek 2 system have been well studied in comparison with other automated systems and reference methods (2, 6, 9, 10, 13, 16), and because the primary objective of the study was to determine whether direct identification and direct antimicrobial susceptibility testing would provide results comparable to those obtained from an automated system in routine use, a reference test method was not used. In the present study, and with the direct method, only 31% of isolates showed concordant identification with the standard method. All the concordant identification results came from the gram-negative rods, while none of the 50 gram-positive cocci showed concordant identification results. Although comparisons between the results of different studies are difficult because different blood culture systems, identification systems, or both are used, we found that the correlation between identification rates was lower than for other similar studies which showed a correlation rate of between 72 and 96% (8, 12, 18, 23). Previous studies have reported two main sources of error in both bacterial identification and antimicrobial susceptibility testing by direct inoculation, (i) mixed cultures and (ii) nonstandardized inoculum size (8, 23, 24). Current practice dictates that all positive blood cultures be Gram stained and that a subculture to a chocolate or blood agar plate be routinely performed (19). The Gram stain enables the determination of blood cultures that may be polymicrobic, which would then be excluded from processing by a rapid method. However, it has been reported that 6 to 10% of specimens that appeared unimicrobic by Gram stain are later found to be polymicrobic during subculture (18, 23, 24). Because of this possibility, the results obtained from the direct method should be considered preliminary until verification of direct inoculum purity can be made. In the present study, specimens yielding more than one isolate after subculture were excluded from analysis, and our data are based only on monomicrobic blood cultures. In this way we can exclude the first source of error. Another known source of error is inoculum size. In our study, the technique used has been reported previously by several authors, which allowed us to obtain the standard inoculum size easily (18, 23, 24). To obtain a standardized inoculum by the direct inoculation method, we used the Densichek Vitek colorimeter to make the McFarland standard suspension. However, for 23% of isolates, the inoculum size was low, as demonstrated by fewer colonies in the subculture of dilutions from the standardized McFarland suspension. It is possible that blood cells and traces of blood culture broth interfered with the colorimetric measurement. The low correlation between the direct and standard methods compared with earlier reports could be due to the technique used to prepare the inoculum from the blood culture bottle, which may be ineffective in removing substances which may interfere with the fluorescent biochemical reactions taking place in the ID and AST Vitek cards. However, two recent publications by Ling et al. (15) and Bruins et al. (3) which evaluated the direct inoculation of Vitek 2 cards from positive blood culture bottles detected 82 and 93% concordance, respectively, for the direct identification of gram-negative rods. These results are much more favorable than those obtained in our investigation, which was performed in a similar manner. As noted earlier, in our study, the inoculum was low for 23% of the isolates. When these 23 isolates are excluded from the study, the correlation rate for gram-negative rods reaches 72%. It is possible to argue that the inclusion of these isolates in the overall data analysis could be the reason for the lower correlation rate compared with those reported by Bruins et al. and Ling et al. (3, 15) We evaluated the results of susceptibility testing with the Vitek 2 system, taking into account the clinical categories defined by the expert system, in order to simulate, as much as possible, the performance of the direct method in the routine work of a clinical laboratory. It has been reported that an overall category error rate of 10% should be obtained for an acceptable performance of susceptibility testing, including 1.5% very and 3% (7). In our study, the overall error rate was 15%, 6.6% for gram-negative rods and 8.4% for gram-positive cocci. For gram-negative rods, most of the derived from results testing penicillins and cephalosporins, while for the remaining agents assessed, ciprofloxacin, imipenem, gentamicin, and trimethoprim-sulfamethoxazole, the overall error rate was lower (0.3%), with no very or. For gram-positive cocci, however, there were a large number of, and only vancomycin had no very or. It should be noted that all correctly identified, misidentified, and unidentified isolates were evaluated in the antimicrobial susceptibility testing in the present study, unlike most previous reports, in which only correctly identified isolates were evaluated for direct susceptibility testing. It is possible that the high error rates compared with previous reports (3, 15) are due to incorrect identification. This reason may justify excluding misidentified and unidentified organisms from analysis, as the other authors did. However, we chose to compare two methods, that is, a rapid direct method with one that is used on a day-to-day basis in our laboratory, and therefore no attempt was made to exclude misidentified and unidentified isolates from analysis. Our results, which are based on monomicrobic cultures, demonstrate that the Vitek 2 cards inoculated directly from positive Bactec 9240 bottles do not provide either acceptable bacterial identification or susceptibility testing in comparison with corresponding cards tested by a standard method. At present, when using the direct inoculation method, we cannot recommend the direct inoculum of blood cultures from Bactec 9240 onto Vitek 2 cards. The low bacterial inoculum in blood culture bottles compromising inoculum concentration may partly account for the high error rates in direct identification and susceptibility testing reported in this study. Even though automated systems have reduced the time required to detect microorganisms in bloodstream infections, the reliability of direct identification and antimicrobial susceptibility testing with any automated method should be tested by individual laboratories before the method is considered for routine use.

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