INTRODUCTION

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The VITEK 2 system is a new system that automatically performs rapid identification and antimicrobial susceptibility testing on a manually prepared inoculum (1). The Advanced Expert System (AES) is designed to analyze results generated by the VITEK 2 system for biologic validity and then provide comments on the results. One important function of the AES is to look for inconsistencies between the identification of the organism and the antimicrobial susceptibility of the isolate. Another important function is to ascertain the antimicrobial phenotype of the isolate based on results of susceptibility tests. A third function is to deduce the susceptibility of the organism to drugs not tested based on its susceptibility to the antibiotics actually tested.

In a previous study, the ability of the AES to correctly ascertain the -lactam phenotype of isolates of Enterobacteriaceae and Pseudomonas aeruginosa was determined using a panel of 196 strains collected worldwide which had been characterized by biochemical and molecular techniques for their -lactamase content (6). The results of that study showed that the AES correctly identified the -lactam phenotype of 183 (93.4%) of these isolates despite the inclusion of many rare phenotypes in the isolate panel. The study, however, did not assess the potential impact of the VITEK 2 system and AES on the workflow of a clinical laboratory in a typical hospital since the isolates examined had been selected for their known and sometimes quite rare resistance mechanisms. Furthermore, only the accuracy of the AES for determining the -lactam phenotypes was determined in the study.

Therefore, a study was designed to ascertain the potential impact of the VITEK 2 system and AES on a typical clinical laboratory located in a university-based hospital. For this study, consecutive nonduplicate isolates of gram-negative rods and gram-positive cocci isolated and identified from specimens by the laboratory were collected and tested by the VITEK 2 system for identification and antibiotic susceptibility. The AES was used to analyze and interpret results generated by the VITEK 2 system. The results were also analyzed by a human expert (CCS) and compared to those of the AES.

	MATERIALS AND METHODS

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Study design. Consecutive, nonduplicate isolates were collected from St. Joseph Hospital, Omaha, Neb., a 400-bed tertiary-care hospital. The microbiology laboratory services not only the hospital but also a large outreach program that includes physician clinics, large group practices, and approximately 20 nursing homes. During 1999, the laboratory processed approximately 88,780 specimens, performing 5,134 blood cultures, 11,215 urine cultures, 3,434 throat cultures, 4,390 wound cultures, 2,062 respiratory system cultures, 1,074 stool cultures, and 7,562 susceptibility tests.

Identification and susceptibility tests. All identifications and susceptibilities were determined using the VITEK 2 system, as recommended by the manufacturer. VITEK 2 ID-GNB and ID-GPC cards were used for identification of gram-negative and gram-positive bacteria, respectively. The VITEK 2 susceptibility cards used in this study were standard European configurations and contained the following antibiotics and concentration ranges: (i) AST-N009 for Enterobacteriaceaeampicillin 2 to 32 µg/ml, amoxicillin-clavulanate (2:1 ratio) 2 and 1 to 32 and 16 µg/ml, cephalothin 2 to 64 µg/ml, cefoxitin 4 to 64 µg/ml, cefotaxime 1 to 64 µg/ml, ceftazidime 1 to 64 µg/ml, ticarcillin 8 to 128 µg/ml, ticarcillin-clavulanate (clavulanate at 2 µg/ml with ticarcillin a twofold dilution) 8 and 2 to 128 and 2 µg/ml, piperacillin-tazobactam (tazobactam at 4 µg/ml with piperacillin a twofold dilution) 4 and 4 to 128 and 4 µg/ml, imipenem 0.5 to 16 µg/ml, amikacin 2 to 64 µg/ml, gentamicin 1 to 16 µg/ml, netilmicin 1 to 32 µg/ml, tobramycin 1 to 16 µg/ml, nalidixic acid 2 to 32 µg/ml, ciprofloxacin 0.25 to 4 µg/ml, norfloxacin 0.5 to 16 µg/ml, ofloxacin 0.25 to 8 µg/ml, nitrofurantoin 16 to 512 µg/ml, and trimethoprim-sulfamethoxazole 20 to 320 µg/ml; (ii) AST-N008 for P. aeruginosacefepime 1 to 64 µg/ml, ceftazidime 1 to 64 µg/ml, piperacillin 4 to 128 µg/ml, piperacillin-tazobactam (tazobactam at 4 µg/ml with piperacillin a twofold dilution) 4 and 4 to 128 and 4 µg/ml, ticarcillin 8 to 128 µg/ml, ticarcillin-clavulanate (clavulanate at 2 µg/ml with ticarcillin a twofold dilution) 8 and 2 to 128 and 2 µg/ml, imipenem 0.5 to 16 µg/ml, meropenem 0.25 to 16 µg/ml, aztreonam 1 to 64 µg/ml, amikacin 2-64 µg/ml, gentamicin 1 to 16 µg/ml, netilmicin 1 to 32 µg/ml, tobramycin 1 to 16 µg/ml, isepamycin 1 to 64 µg/ml, colistin 0.5 to 16 µg/ml, pefloxacin 0.25 to 16 µg/ml, fosfomycin 4 to 128 µg/ml, and trimethoprim-sulfamethoxazole 20 to 320 µg/ml; (iii) AST-P515 for Staphylococcus spp.benzylpenicillin 0.03 to 0.5 µg/ml, clindamycin 0.25 to 8 µg/ml, erythromycin 0.25 to 8 µg/ml, fosfomycin 8 to 128 µg/ml, fusidic acid 0.5 to 32 µg/ml, gentamicin 0.5 to 16 µg/ml, kanamycin 8 to 32 µg/ml, lincomycin 2 to 16 µg/ml, minocycline 4 to 16 µg/ml, nitrofurantoin 16 to 512 µg/ml, norfloxacin 0.25 to 16 µg/ml, ofloxacin 0.5 to 8 µg/ml, oxacillin (breakpoint test) S to R, oxacillin MIC 0.5 to 8 µg/ml, pristinamycin 0.5 to 8 µg/ml, rifampin 0.5 to 32 µg/ml, teicoplanin 0.5 to 32 µg/ml, tetracycline 1 to 16 µg/ml, tobramycin 4 to 16 µg/ml, trimethoprim-sulfamethoxazole 10 to 320 µg/ml, and vancomycin 1 to 32 µg/ml. Quality control was performed daily during testing, using Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 for gram-negative bacteria and S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 for gram-positive bacteria. -Lactamase determinations were made by the chromogenic cephalosporin assay (4).

AES testing. In the biological validation phase, the AES examines the antimicrobial susceptibility data and determines if the MICs obtained are consistent with the species identification of the organism. If a single error is found, the AES recommends either a change in the identification that will make the outlying MIC consistent or a numeric change in the MIC that will make it consistent with the identification. These recommendations are considered biological corrections because AES presumes that (i) an error has occurred in the data generated by the VITEK 2, (ii) results were atypical due to the strain, (iii) a "falsely" negative result has occurred (e.g. noninduced -lactamase), or (iv) an incorrect result was entered manually by a technologist. A biological correction is recommended by the AES if it detects only a single MIC inconsistency. The AES will recommend retesting the isolate if more than one biological correction would be needed to bring the susceptibility in line with the identification or to match phenotypes (see below).

Assessment of the AES. Once the AES had analyzed results, a hard copy of the VITEK 2 laboratory report along with AES comments was printed and given to a human expert (CCS) for evaluation. The validity of any biological correction or therapeutic correction made during the biological validation phase was assessed. Phenotype assignment was evaluated by the human expert, as was any therapeutic correction made by the AES based on phenotype. Finally, susceptibility to drugs not tested and deduced by the AES was evaluated.

	RESULTS

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Identification. Among the 259 isolates, a definitive species was provided for 245 (94.6%) by the VITEK 2 system. This included 36 strains within the genus Klebsiella for which an indole test had to be performed by a technologist before a final species could be designated. Otherwise, no input from laboratory staff was required for species determination. There were 14 strains for which the VITEK 2 could not provide a definitive identification. These are listed in Table 1. Among the Enterobacteriaceae, the VITEK 2 had low discrimination on six strains identified by the clinical laboratory as Klebsiella pneumoniae, although K. pneumoniae was one of the two possible species listed for each of the strains. One strain with low discrimination by VITEK 2 was identified by the clinical laboratory as E. coli. Although VITEK 2 listed Citrobacter freundii or Enterobacter cloacae as possible species, the strain was susceptible to all -lactam drugs tested including ampicillin, suggesting that E. coli was most likely to be correct. Five additional strains were not identifiable by the VITEK 2 system. Two of these were identified as K. pneumoniae by the clinical laboratory, two were identified as E. cloacae, and one was identified as E. coli (Table 1). Two strains identified as P. aeruginosa by the clinical laboratory had low discrimination by VITEK 2 (Table 1). Each was listed as possibly P. aeruginosa or a highly pathogenic organism by VITEK 2. 

Biological validation of results. Among the 259 strains tested, the AES found the results with 194 (74.9%) to be fully consistent, suggesting no biological corrections or therapeutic corrections (this excludes therapeutic corrections based on phenotype, which are considered below). This included 122 Enterobacteriaceae, 33 P. aeruginosa, and 39 S. aureus. The human expert agreed with the AES on 193 of these strains. The sole exception was Providencia stuartii 613, for which the MICs of ciprofloxacin, ofloxacin, norfloxacin, and nalidixic acid were 4, 8, 4, and 32 µg/ml, respectively. The human expert would have made a biological correction to the norfloxacin MIC to 4 µg/ml.

Phenotypes. The AES was able to identify a phenotype for -lactams, aminoglycosides, and quinolones for 207 of the 211 gram-negative rods tested (Table 3). The four strains for which a phenotype could not be identified had so many inconsistencies in the data that the AES suggested retesting or a making change in the identification. These included C. freundii 456, which appeared susceptible to ampicillin, amoxicillin-clavulanate, cephalothin, and cefoxitin; E. cloacae 490 and 457, which appeared susceptible to ampicillin and amoxicillin-clavulanate; and P. mirabilis 481, which appeared resistant to cefoxitin and imipenem.

Deduced drugs. The use of phenotypes to deduce the susceptibility to drugs not tested led to a problem with cefaclor and E. coli. The MIC of cephalothin for certain strains of E. coli fell in the 1 range, while those of all other -lactams tested were in the S range. This pattern was designated the wild-type phenotype by the AES. Accordingly, all wild-type E. coli strains are considered susceptible to cefaclor by deduction. This created a problem of strains testing I to cephalothin but having S deduced for cefaclora clear violation of current NCCLS recommendations that susceptibility to one indicates susceptibility to both and resistance to one indicates resistance to both (2).

	DISCUSSION

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The results of this study indicated that the VITEK 2 system and AES could be used on the majority of clinical isolates encountered in a typical university-based laboratory for identification and antimicrobial susceptibility testing with little or no input from laboratory staff concerning the interpretation of results. Of the 259 isolates included in this study, 94.6% were definitively identified by VITEK 2 and 74.6% had no inconsistencies between the identification of the strain and the antimicrobial susceptibility results. Of the 65 strains for which a correction was identified by AES, 58.5% required only a therapeutic correction to the susceptibility results. Most of these were due to the failure of the VITEK 2 system to detect -lactam resistance in organisms possessing an intrinsic -lactamasea failure common to test systems that are rapid or involve small inocula (7). The AES has been designed to look for such problems in the data and correct them when found. However, if false susceptibility due to this problem occurs with two or more -lactam drugs in tests with the same strain, the AES must recommend retesting the isolate. This occurred with a number of the strains of Enterobacter spp. and C. freundii listed Table 2 as possibly needing retesting. Also of concern was the false resistance to imipenem among 7 of the 13 isolates of P. mirabilis encountered in this study. Although the AES indicated to the user that this result was probably incorrect, this problem with the VITEK 2 system should ultimately be resolved in the algorithm used to determine susceptibility results. Since imipenem resistance, although rare among Enterobacteriaceae, can occur in P. mirabilis (3), it is imperative to be able to ascertain when resistance is real and when it is a problem with the test system.

There was very good agreement between the human expert and the AES in recognizing inconsistencies in this study. For only 5 (7.7%) of the 65 strains identified by the AES as needing corrections to the data did the human expert disagree with the AES about whether an inconsistency existed or how to correct the inconsistency. The major limitation of the AES noted in the biological validation phase of data analysis was its inability to recognize a single pattern of inconsistency and correct it. For example, false susceptibility to ampicillin, amoxicillin-clavulanate, cephalothin, and/or cefoxitin occurred with a few strains of Enterobacter spp. or C. freundii. To the AES, this was more than one inconsistency and required repeat testing or a change in identification of the organism with or without biologic and therapeutic corrections. To the human expert, this multiple drug inconsistency was due to a single problemfailure to detect intrinsic resistance due to low-level expression of a chromosomal -lactamase. Repeat testing of these strains generated the same inconsistent data and the same AES analysis (data not shown) because the problem was intrinsic to the VITEK 2 system and could not be resolved by repeat testing. It should be noted that the design of the AES prevents it from making corrections if two or more inconsistencies are identified. Thus, it cannot recognize single-source problems that lead to multiple inconsistencies.

Agreement between the human expert and the AES was also very good concerning the identification of antimicrobial phenotypes. Overall agreement across the different drug groups varied from 95.7 to 100%. This is similar to results obtained in an earlier study that reported 97.3% agreement between a human expert and an expert system linked to a rapid antibiotic susceptibility test system (8). The major limitation noted for the AES was its inability to rank in order the various phenotypes among the possible phenotypes when more than one matched the MIC distribution. At times, as many as four different phenotypes would be listed by the AES with no indication of which one or possibly two were the most likely. In certain instances, the AES listed the wild type as well as one or two different resistance phenotypes as possibilities. Clearly, there are instances where this can occur. However, it would be more helpful to the user if in these instances the AES gave a message that a specific phenotype could not be ascertained rather than listing multiple phenotypes.

The ability of the AES to deduce drug susceptibility was not thoroughly analyzed in this study since too few drugs were involved. However, inconsistencies between the NCCLS rules for interpretation of susceptibility to cephalothin and cefaclor appeared to be violated when the AES deduced susceptibility to cefaclor from results obtained with cephalothin. Once this problem was identified in the study, it was corrected, so that the most recent version of the AES allows the user to base deductions strictly on NCCLS recommendations.

For all laboratories, regardless of size and type of staffing, the availability of the VITEK 2 system and AES could have a very positive impact on both the work flow and the quality of the information leaving the laboratory. However, the precise impact of the VITEK 2 system and AES will vary depending on the laboratory involved. For larger, more complex laboratories with a human expert on staff, the VITEK 2 and AES could free up valuable time of the expert. It would no longer be necessary for the expert to spend time identifying minor inconsistencies, since once they were identified by the AES, the expert could readily resolve them by using the choices provided by the AES. In fact, once the expert becomes familiar with the corrections usually suggested by the AES for certain organism-drug combinations, the system can be set to automatically correct the inconsistency, eliminating the need for external expert input in selected situations. Thus, more of the human expert's time could be spent dealing with the problematic issues identified by the AES, i.e., organisms with multiple inconsistencies. Identification of antimicrobial phenotypes by the AES would also be most useful for this type of laboratory. In instances where more than one phenotype is listed as possible by the AES, the human expert can select the single most likely phenotype to include in the final report sent out of the laboratory. Also, additional therapeutic comments based on the most likely phenotype may be custom designed by the human expert for inclusion in the final report to aid physician selection of therapy.

For smaller, less complex laboratories without a human expert, the availability of the VITEK 2 system and AES could significantly improve the quality of the information leaving the laboratory. Minor inconsistencies that could be overlooked or go unrecognized would be automatically identified by the AES, and corrections would be suggested before the final report was printed. Most laboratories have personnel with sufficient background to select the most appropriate correction for this type of data inconsistency, e.g. to make a therapeutic correction of I to R for ampicillin or ticarcillin for K. pneumoniae. However, in the setting of organisms with major inconsistencies, the laboratory staff may not be able to correctly choose between options provided by the AES, i.e., either make a correction in the identification or make a biological correction to an MIC. In this situation and instances where multiple biological inconsistencies are identified by the AES, repeat testing will probably be required. Unfortunately, if the same results are obtained on repeat testing, the recommendations of the AES will remain the same as before, leaving the laboratory personnel no further along in resolving the inconsistencies. The utility of the antimicrobial phenotypes identified by the AES will be more limited for the laboratory without a human expert. In instances where more than one phenotype is listed by the AES, it may not be possible for laboratory personnel to select the most likely phenotype. Thus, the final report sent out of the laboratory could be confusing to physicians if it contains a listing of possible phenotypes. It would be much more useful for this type of laboratory if the AES would rank the phenotypes listed in descending order of their likelihood or merely concede that a specific phenotype could not be ascertained.

In summary, the VITEK 2 system and AES can provide accurate information in tests with the majority of clinical isolates that are encountered in the typical university-based hospital. In addition, these new systems remove the need for human analysis of results for many isolates, freeing personnel for other activities and improving the overall quality of the information generated, especially in laboratories without a human expert. As with any microbiology system, the AES will require constant updating and modification to optimize its utility for the laboratory.