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False determinations of resistance to imipenem by a microdilution testing system have been attributed to degradation of imipenem in the testing panels after their prolonged storage (2, 8, 9). The quality-control measures recommended by the National Committee for Clinical Laboratory Standards (NCCLS) are believed to prevent false determinations of resistance (3, 5). In our institution during the 2-year period from September 1994 to August 1996, 12% of all Pseudomonas aeruginosa isolates were resistant to imipenem (7). On the basis of data from a relational clinical database (6), this rate increased to 23% starting in September of 1996 and prompted an investigation. We report here results showing an outbreak of P. aeruginosa with false resistance to imipenem despite adherence to NCCLS quality-control guidelines.

P. aeruginosa was identified from clinical specimens submitted to the microbiology laboratory by using gram-negative type II identification panels (Dade International Inc., West Sacramento, Calif.). Susceptibility to imipenem was determined by microdilution broth testing (MicroScan; Dade International Inc.). Testing was performed according to the manufacturer's instructions. Inocula were prepared by using the Prompt Inoculation System (MicroScan).

Some isolates were also tested for susceptibility to imipenem by a standard Kirby-Bauer agar disk diffusion method according to NCCLS guidelines (4).

A case-control study was implemented to investigate the apparent outbreak of imipenem-resistant P. aeruginosa. By searching the microbiology laboratory database, every patient identified between 15 September and 31 October 1996 was considered to be a case patient if the P. aeruginosa isolate was resistant or intermediate to imipenem (MIC, 8 µg/ml) or a control patient if the isolate was susceptible to imipenem (MIC, <8 µg/ml). Variables considered in the case-control study included admission service, ward of hospitalization, whether a patient shared a room with a case patient or with a patient who shared a room with a case patient, stay in an intensive care unit, dates and types of procedures performed in the operating room, type of procedure performed, surgeon performing the procedure, transfer from another hospital or chronic care facility, and previous imipenem exposure. Isolates from four case patients were available and were studied by pulsed-field gel electrophoresis (PFGE) as previously described (1).

During the outbreak period, a specific lot of an MIC testing panel was used (dried, gram negative, urine panel type 9, lot 8/17/97, MicroScan; Dade International). To examine the possibility of false resistance, imipenem MIC test results for P. aeruginosa and other gram-negative rods for two periods were compared. Time B (15 September to 28 December 1996) consisted of the period during which the specific lot of the MIC testing panel described above was used. Time A (15 September 1995 to 14 September 1996) consisted of the year before the outbreak, during which 18 different MIC testing panel lots were used.

Fourteen isolates for which imipenem MICs were found to be 4 µg/ml when they were tested with the outbreak lot were retested with another MIC testing panel lot (dried, gram negative, combo panel type 20, lot 12/18/97, MicroScan; Dade International). Imipenem MIC weekly quality-control results for P. aeruginosa (ATCC 27853) and for Escherichia coli (ATCC 25922) for the different MIC testing panel lots used during time A and time B were compared. Fresh American Type Culture Collection strains were used during both periods.

The concentration of imipenem at each dilution in the MIC panels from five different lots was determined by high-pressure liquid chromatography (HPLC). The content of each well was reconstituted with phosphate buffer (pH 6.8), diluted to 1 ml, and assayed for imipenem by HPLC (TSPC 1000) after 1 h of incubation at room temperature. The assay results were determined against an imipenem reference standard in a pH 6.8 buffer matrix. Both the area count method and the peak-height-calculation method were used to determine the results.

All of the statistical analyses were performed with Stata Corp. (College Station, Tex.) version 5 software. Fisher's exact est was used for discrete variables. The logarithms of the MICs during the two time periods were compared by a nonparametric test (Kruskal-Wallis). The differences between pairs of MIC results (the same isolate tested with two different panel lots) were compared by the paired Student t test. Analysis of variance was used to examine the variability of the MIC for the quality control strain across the different lots used during the year prior to the outbreak period. All P values calculated were two-tailed.

Case-control study. During the 6-week period (15 September to 31 October 1996) investigated in the case-control study, 16 cases and 49 controls were identified. Ten of the case patients were inpatients, and six were outpatients. No common source or epidemiological link was identified between the cases. Case patients were not exposed to imipenem more often than control patients. P. aeruginosa isolates from four case patients were studied by PFGE (Fig. 1). Each of these isolates had a distinct PFGE pattern. We examined the possibility of false resistance being related to a specific lot of an MIC panel (dried, gram negative, urine panel type 9, lot 8/17/97, MicroScan; Dade International).

View larger version (90K): [in this window] [in a new window]   FIG. 1.   PFGE of P. aeruginosa isolates from four case patients. Each of the isolates has a distinct pattern.

Imipenem MIC distribution. The distributions of imipenem MIC results for P. aeruginosa, and for other gram-negative rods were compared between the two periods: time B (the period during which the implicated lot was used) and time A (the year before this lot was used). The results of this comparison are displayed in Table 1. The imipenem MIC distributions differed significantly between time A and time B, both for P. aeruginosa and for other gram-negative rods (P < 0.0001, Kruskal-Wallis test).

Comparison of results of susceptibility tests. In our laboratory, imipenem resistance as measured by MicroScan is routinely confirmed by Kirby-Bauer disc diffusion for Enterobacteriaceae and since 15 November 1996 also for P. aeruginosa. Discrepant results between the microdilution and the disc diffusion results for Enterobacteriaceae were found for 27 of 37 (73%) isolates in time A and for 42 of 46 (91%) isolates in time B (odds ratio, 3.9; P = 0.04). Fourteen P. aeruginosa isolates were found to be imipenem resistant by the implicated lot after 15 November 1996; all of these isolates tested susceptible by disc diffusion.

Quality-control results. Weekly imipenem MIC results for the quality-control P. aeruginosa strain (ATCC 27853) were within the recommended range (1 to 4 µg/ml) both in time A and in time B, but the 25th, 50th, and 75th percentiles of MICs were 1, 2, and 2 µg/ml, respectively, in time A and 4, 4, and 4 µg/ml, respectively, in time B (P < 0.001). The imipenem MIC results for the quality-control E. coli strain (ATCC 25922) were 1 µg/ml in the two periods.

Imipenem concentration. The concentrations of imipenem as measured by HPLC are shown in Table 2. Analysis revealed that for each imipenem MIC testing dilution examined, the implicated lot had concentrations two- to threefold lower than those of the other four panels tested.