Influence of variations in test methods on susceptibility of Haemophilus influenzae to ampicillin, azithromycin, clarithromycin, and telithromycin

The National Committee for Clinical Laboratory Standards standard broth microdilution method for testing the susceptibility of Haemophilus influenzae to ampicillin, azithromycin, clarithromycin, and telithromycin was evaluated by altering one variable at a time. Variables that were tested included age of colony for inoculum preparation, inoculum density, test medium, incubation atmosphere, and incubation time. For the macrolide, azalide, and ketolide agents, incubation in 5 to 7% CO(2) most significantly affected the MICs, producing nearly twofold increases for clarithromycin and telithromycin and a greater than threefold increase for azithromycin. For ampicillin, a 10-fold increase in inoculum density increased the geometric mean MICs for beta-lactamase-negative strains from 1. 50 to 2.45 microg/ml. In addition, 206 H. influenzae strains were tested for their susceptibilities to the same drugs by the broth microdilution tests in two media, as well as by agar dilution tests, disk diffusion tests, and Etests, on six different agar media. The three standard methods with Haemophilus test medium (HTM) compared favorably with each other except for a high minor discrepancy rate (27%) by the disk diffusion test with ampicillin and clarithromycin. Agar dilution test MICs on the five comparative media were generally higher than those on HTM agar but were only rarely more than one twofold concentration higher. Etest MICs of azithromycin and telithromycin were more than twofold higher than agar dilution and broth microdilution MICs on HTM; ampicillin Etest MICs were nearly twofold lower. The use of media other than HTM agar appears to have a minimal effect on susceptibility test results for the ketolide, azalide, or macrolide drugs that we tested against H. influenzae.     

Antimicrobial susceptibility testing of less commonly isolated Haemophilus species using Haemophilus test medium.

Haemophilus test medium (HTM) was developed recently for dilution and disk diffusion antimicrobial agent susceptibility testing of Haemophilus influenzae. The application of HTM to the testing of other, less frequently encountered Haemophilus species recovered from humans was evaluated in this study by using commercially prepared HTM (BBL Microbiology Systems, Cockeysville, Md.) in broth microdilution and agar disk diffusion susceptibility tests with 18 antimicrobial agents. A total of 93.3% of 90 isolates belonging to six Haemophilus species provided acceptable growth in HTM agar disk diffusion tests, while only 63.3% (57 of 90) provided acceptable growth in the broth microdilution tests. However, HTM agar dilution testing provided an alternative method for those strains (primarily H. haemolyticus) which failed to grow adequately in broth. Based on the latest National Committee for Clinical Laboratory Standards guidelines (standard M2-T4) for interpretation of HTM disk tests of H. influenzae, the overall very major, major, and minor errors for all 18 drugs and six species tested were 0.2, 0.7, and 3.4%, respectively. Thus, the use of HTM in agar or broth susceptibility tests can be recommended for testing the less commonly encountered Haemophilus species by using the same test conditions and interpretive guidelines developed for H. influenzae.     

Quantitative antimicrobial susceptibility testing of Haemophilus influenzae and Streptococcus pneumoniae by using the E-test.

The E-test (PDM Epsilometer; AB Biodisk, Solna, Sweden) is an antimicrobial agent gradient-coated plastic test strip which allows MIC determinations on agar media. The test is performed in a manner similar to the agar disk diffusion procedure. A collection of Haemophilus influenzae and Streptococcus pneumoniae strains possessing various resistance mechanisms was used to evaluate the E-test method. H. influenzae strains were tested with both Haemophilus test medium (HTM) and PDM ASM II chocolate agar, while the S. pneumoniae strains were tested on Mueller-Hinton sheep blood agar. E-test MICs for a total of 10 antimicrobial agents were compared with broth microdilution MICs determined according to National Committee for Clinical Laboratory Standards methods. In general, E-test MICs for both species were quickly and easily interpreted and agreed within one log2 MIC increment in 89.8% of tests with H. influenzae and in 80.4% of pneumococcal tests. The majority of disagreements between the E-test and conventional MICs occurred with trimethoprim-sulfamethoxazole because of trailing and diffuse E-test MIC endpoints with both species. Ampicillin MICs for beta-lactamase-producing H. influenzae determined by the E-test differed at times from those determined by conventional testing because of the vagaries of interpreting colonies growing within the E-test inhibition ellipses. E-test penicillin MICs for pneumococci tended to be 1 to 2 log2 dilutions lower than those determined by using Mueller-Hinton broth supplemented with lysed horse blood. Nevertheless, strains of both species with documented resistance to the study drugs were detected by E-tests, i.e., 0.7% of the tests had very major errors with H. influenzae and 0.8% had very major errors with S. pneumoniae. Thus, the E-test represents a potential alternative method for antimicrobial susceptibility testing of these two fastidious bacterial species.     

Use of Haemophilus test medium for broth microdilution antimicrobial susceptibility testing of Streptococcus pneumoniae.

A recently described medium (Haemophilus test medium [HTM]) for antimicrobial susceptibility testing of Haemophilus influenzae was evaluated in this study for broth microdilution testing of Streptococcus pneumoniae. A total of 137 clinical isolates was tested against 11 antimicrobial agents, using Mueller-Hinton broth supplemented with 3% lysed horse blood in parallel with HTM. Inocula of 5 X 10(5) CFU/ml and incubation for 20 to 24 h were used with both media. All isolates of S. pneumoniae produced acceptable growth in both media, and MICs determined in HTM agreed closely with those determined in lysed horse blood. Drugs which provided a MIC within 1 log2 concentration difference in both media included penicillin (100%), ampicillin (98.0%), amoxicillin-clavulanate (100%), ampicillin-sulbactam (100%), cephalexin (98.9%), cefaclor (96.8%), cefuroxime (99.0%), chloramphenicol (96.2%), tetracycline (96.2%), and erythromycin (100%). HTM MICs with trimethoprim-sulfamethoxazole were 1 to 2 log2 concentration increments higher in 92.0% of isolates than MICs determined in lysed horse blood. Based on the results of this study, HTM appears to represent a promising alternative medium for broth microdilution susceptibility testing of S. pneumoniae.     

Variability of clarithromycin and erythromycin susceptibility tests with Haemophilus influenzae in four different broth media and correlation with the standard disk diffusion test.

Four separate laboratories performed antimicrobial susceptibility tests with 40 Haemophilus influenzae isolates, each tested in triplicate. Erythromycin and a new macrolide, clarithromycin (A-56268; TE-031), were tested by the disk diffusion method, by the agar dilution procedure in two different media, and by broth microdilution tests in four different media. Erythromycin MICs for 90% of the strains were 16 micrograms/ml in Mueller-Hinton broth with 3% lysed horse blood and NAD, 4.0 micrograms/ml in hemophilus test medium, and 2.0 micrograms/ml in supplemented Schaedler broth or in the fastidious broth medium from Beckman Instruments, Inc. Clarithromycin MICs were generally 1 doubling dilution greater than erythromycin MICs in each of the media. Erythromycin disk tests corresponded best with MICs determined in the fastidious broth medium. In that same medium, clarithromycin MICs were about 1 doubling dilution greater than what would be expected from the results of disk tests. Because there were fewer growth failures, hemophilus test medium is recommended for microdilution tests with H. influenzae. Incubation of all tests for a full 24 h without an increased CO2 atmosphere was needed to achieve maximal precision of the tests. Interlaboratory and intralaboratory reproducibility of all tests was satisfactory.     

Standardization of Broth Microdilution and Disk Diffusion Susceptibility Tests for Actinobacillus pleuropneumoniae and Haemophilus somnus: Quality Control Standards for Ceftiofur, Enrofloxacin, Florfenicol, Gentamicin, Penicillin, Tetracycline, Tilmicosin, and Trimethoprim-Sulfamethoxazole

Quality control (QC) standards for the in vitro antimicrobial susceptibility testing of two fastidious veterinary pathogens, Actinobacillus pleuropneumoniae and Haemophilus somnus, were developed in a multilaboratory study according to procedures established by the National Committee for Clinical Laboratory Standards for broth microdilution and disk diffusion testing. The medium recommended for the broth microdilution testing is cation-adjusted Mueller-Hinton broth supplemented with 2% lysed horse blood, 2% yeast extract, and 2% supplement C. This medium has been designated veterinary fastidious medium. The medium recommended for the disk diffusion testing is chocolate Mueller-Hinton agar. The recommended QC organisms are A. pleuropneumoniae ATCC 27090 and H. somnus ATCC 700025. The QC MICs of ceftiofur, enrofloxacin, florfenicol, gentamicin, penicillin, tetracycline, tilmicosin, and trimethoprim-sulfamethoxazole were determined for each isolate, as were the zone size ranges. Of the results from the participating laboratories, 94.0% of the zone diameter results and 97.0% of the MIC results fell within the suggested QC ranges for all compounds. These QC guidelines should allow greater accuracy in interpreting results when testing these antimicrobial agents against fastidious pathogens.     

Investigation of ampicillin-intermediate strains of Haemophilus influenzae by using the disk diffusion procedure and current National Committee for Clinical Laboratory Standards guidelines.

It was noted in our laboratory that certain strains of Haemophilus influenzae yielded zone sizes interpreted as resistant to the ampicillin (AMP) disk on chocolate-Mueller-Hinton agar (CMH) but showed no evidence of beta-lactamase (beta-Lac) activity. Although it is known that a second mechanism of AMP resistance exists, strains with this mechanism are uncommon. To investigate this apparent discrepancy, a study of 100 consecutive clinical isolates of H. influenzae collected over a 6-month period was performed. Isolates were simultaneously tested against five antibiotics (AMP, chloramphenicol, cefotaxime, ciprofloxacin, and AMP-sulbactam) on CMH and on two brands of Haemophilus test medium (HTM) by using the disk diffusion procedure and National Committee for Clinical Laboratory Standards (NCCLS) standards. By using CMH and NCCLS standard M2-A3-S2, strains of H. influenzae showing zone sizes of greater than or equal to 20 mm with AMP were considered sensitive. By using HTM and NCCLS standard M2-A4, strains showing zone sizes of greater than or equal to 25 mm to AMP on HTM were considered sensitive. Intermediate strains had zone sizes of 22 to 24 mm. The majority of isolates (68%) were sensitive to all antibiotics. Two percent of the isolates were resistant to chloramphenicol. Seventeen percent of the isolates were AMP-resistant, beta-Lac-producing strains of H. influenzae. Thirteen percent of the isolates gave at least one intermediate or resistant zone for AMP but were beta-Lac negative. MIC determinations with NCCLS standard M7-A2 were performed with resistant and intermediate strains. MICs for beta-Lac-producing strains of H. influenzae were >/= 8.0 microgram/ml. MICs for beta-Lac-negative strains were 相似文献   

Influence of growth medium and supplement on growth of Haemophilus influenzae and on antibacterial activity of several antibiotics.

In the present study, five non-beta-lactamase- and five beta-lactamase-producing strains of Haemophilus influenzae were used to determine whether three different growth media, Mueller-Hinton broth and agar, brain heart infusion broth and agar, and tryptic soy broth and agar, and their added supplements (0.2% hemin-0.1% IsoVitaleX, 1% hemin-1% IsoVitaleX, 2% sheep blood, 10% Fildes enrichment, 5% Fildes enrichment, 1% supplement B, 5% horse erythrocytes, and 2% hemoglobin-1% IsoVitaleX) would influence the growth rate of this microorganism and the antibacterial activity of eight antibiotics, including ampicillin, tetracycline, chloramphenicol, gentamicin, cefamandole, erythromycin, trimethoprim-sulfamethoxazole (TMP-SMX), and cefoperazone. The growth curve studies were carried out with an initial inoculum of 10(4) bacteria per ml, and MICs were determined with an inoculum of 5 X 10(5) microorganisms. Mueller-Hinton broth, brain heart infusion broth, and tryptic soy broth enriched with 5% Fildes resulted in a maximal growth of more than 10(8) CFU/ml at 24 h. When 10% Fildes or 2% sheep blood was added as enrichment to Mueller-Hinton broth, a considerable reduction in the growth rate of H. influenzae strains resulted (P less than 0.01). Significant variations in MICs (P less than 0.01) were observed with chloramphenicol, TMP-SMX, erythromycin, and cefoperazone when brain heart infusion agar, Mueller-Hinton agar, or tryptic soy agar was used. Chloramphenicol, gentamicin, erythromycin, and TMP-SMX were all affected by the different enrichments added to Mueller-Hinton agar. MICs were in general higher with 5% Fildes enrichment and lower with 1% supplement B. Cefoperazone was the only drug which exhibited a lower MIC in 5% Fildes enrichment for ampicillin-resistant H. influenzae strains.     

Interpretive criteria for susceptibilities of Haemophilus influenzae to ampicillin, amoxicillin, and amoxicillin-clavulanic acid.

One hundred fifty isolates of Haemophilus influenzae (including 30 beta-lactamase-positive strains and 23 beta-lactamase-negative, ampicillin-resistant strains) were tested for susceptibilities to ampicillin, amoxicillin, and amoxicillin-clavulanic acid (A/C) by the broth microdilution method in Haemophilus test medium (HTM) and in Mueller-Hinton medium with lysed horse blood and by the disk diffusion method on HTM agar. Our results support the use of HTM for susceptibility testing of H. influenzae but raise a number of questions regarding the interpretive criteria currently in use, particularly with respect to the fourfold difference in MIC susceptibility breakpoints for ampicillin and A/C and a resulting high proportion of A/C-susceptible beta-lactamase-negative, ampicillin-resistant strains.     

Activity of Gatifloxacin against Haemophilus influenzae and Moraxella catarrhalis, Including Susceptibility Test Development, E-Test Comparisons, and Quality Control Guidelines for H. influenzae

In vitro antimicrobial activity and susceptibility testing interpretation criteria and quality control were studied for gatifloxacin, a new 8-methoxy fluoroquinolone, tested against Haemophilus influenzae. Moraxella catarrhalis (600 strains) and H. influenzae (1,400 strains) from the SENTRY Antimicrobial Surveillance Program in North America (Canada and the United States) were also tested against gatifloxacin and 12 other antimicrobial agents. Gatifloxacin (MIC at which 90% of the isolates are inhibited [MIC90], /=18 mm) was also suggested for H. influenzae testing. No interpretive errors were observed. Quality control guidelines for H. influenzae ATCC 49247 were determined by using the NCCLS M23-T3 (1998) study design. The results from the nine-laboratory protocol suggested the following control ranges: for broth microdilution tests, 0.004 to 0.03 microg/ml; for disk diffusion testing, 33 to 41 mm. Gatifloxacin appears to be a potent anti-Haemophilus fluoroquinolone compound with in vitro testing interpretive criteria that will produce accurate results (disk diffusion, broth microdilution, and E-test).     

Effects of various test media on the activities of 21 antimicrobial agents against Haemophilus influenzae

As considerable variation in the antimicrobial susceptibility of Haemophilus influenzae has been reported, the effects of various test media on the susceptibility of H. influenzae were studied. MICs were determined by three laboratories for 21 antimicrobial agents against a panel of 100 selected isolates. Testing was performed using a reference NCCLS frozen broth microdilution method with Haemophilus test medium (HTM) broth and dried commercial MIC trays rehydrated with the following media: in-house and commercially prepared HTM broth, Mueller-Hinton broth with 2% lysed horse blood and NAD, IsoSensitest broth with 2% lysed horse blood and NAD, and IsoSensitest broth-based HTM. Overall, all results were very reproducible, with the MIC at which 50% of the isolates tested are inhibited (MIC(50)), MIC(90), and geometric mean MIC being within one doubling dilution by all six methods and at all three testing centers for 15 of the 21 agents tested. Interlaboratory differences were more marked than intralaboratory differences or differences among media. Cefprozil, cefaclor, and trimethoprim-sulfamethoxazole results differed the most, while results for ampicillin, amoxicillin-clavulanic acid, cefdinir, cefixime, ceftriaxone, and clarithromycin were the most reproducible. However, these variations in results caused considerable differences in susceptibility rates for agents for which NCCLS susceptible breakpoints were close to the geometric mean MIC, particularly for cefaclor and cefprozil. This was much less of a problem when pharmacokinetic-pharmacodynamic breakpoints were used. Reproducible susceptibility results were obtained for a wide range of agents against H. influenzae in three laboratories using a variety of media that support the growth of this fastidious species.     

Broth microdilution testing of Haemophilus influenzae with haemophilus test medium versus lysed horse blood broth. Canadian Haemophilus Study Group.

Broth microdilution testing of 702 community-acquired isolates of Haemophilus influenzae from across Canada was performed with both Mueller-Hinton broth supplemented with 3% lysed horse blood broth (LHB) (BBL Microbiology Systems, Cockeysville, Md.) and haemophilus test medium (HTM). The prevalence of beta-lactamase production was found to be 26% with no regional variation. MICs determined with LHB tended to be higher than those with HTM, but interpretive errors due to these differences were observed only rarely with trimethoprim-sulfamethoxazole (n = 5), cefaclor (n = 8), and cefamandole (n = 3). The interobserver variability in MIC determinations was found to be greater when LHB was used than when HTM was used. There was no difference in intraobserver variability between the two medium formulations. beta-Lactamase-positive isolates developed false resistance to amoxicillin-clavulanate 2 weeks after microdilution panels of both types of medium were stored at -20 degrees C but not when panels were stored at -70 degrees C. In conclusion, this study supports the use of HTM rather than LHB for sensitivity testing of H. influenzae because of its lower rate of interobserver variability and its ability to support the growth of these organisms, which is comparable to that of LHB.     

Development of revised quality control limits for disk diffusion susceptibility tests of selected cephem antibiotics with Haemophilus influenzae and description of a new control strain.

Inconsistent quality control results in disk diffusion testing of cefaclor, cefamandole, cefonicid, and cefuroxime with Haemophilus influenzae ATCC 49247 and Haemophilus test medium (HTM) prompted a search for an alternative control strain that would provide more reliable results. A five-laboratory study was conducted to evaluate two candidate H. influenzae strains as possible alternatives to the aforementioned strain. Repetitive testing of the candidate strains and H. influenzae ATCC 49247 over several days with a total of six different lots of HTM documented consistent performance of the two candidate strains and confirmed inconsistent results for some of the antibiotics with H. influenzae ATCC 49247. In particular, certain lots of HTM failed to yield cefaclor and cefamandole zone sizes within the quality control range advocated by the National Committee for Clinical Laboratory Standards. Because of the greater consistency offered by the new strains, one was selected (now designated H. influenzae ATCC 49766) to be recommended for routine quality control testing of cefaclor, cefamandole, cefonicid, cefuroxime, and the related carbacephem loracarbef. The new control strain and zone size ranges proposed here have been approved by the National Committee for Clinical Laboratory Standards in place of the previously recommended strain and zone size limits for testing of these five cephem antibiotics.     

Disk diffusion versus broth microdilution susceptibility testing of Haemophilus species and Moraxella catarrhalis using seven oral antimicrobial agents: application of updated susceptibility guidelines of the National Committee for Clinical Laboratory Standards.

Susceptibility testing of Haemophilus species and Moraxella catarrhalis is medium and inoculum dependent. Seven oral agents, ampicillin, amoxicillin-clavulanic acid, cefaclor, loracarbef, cefuroxime-axetil, cefixime, and erythromycin, were tested against 400 beta-lactamase-positive and -negative clinically significant respiratory strains of Haemophilus species and 100 strains of M. catarrhalis. Sources of the strains included teaching and regional hospitals and a private laboratory. All strains were tested by broth microdilution and disk diffusion in haemophilus test medium for Haemophilus species and Mueller-Hinton broth and agar for M. catarrhalis. Appropriate National Committee for Clinical Laboratory Standards (NCCLS) standards were followed. For Haemophilus species, by disk diffusion and broth microdilution, respectively, 27 and 27% of strains were resistant to ampicillin, 37 and 5% were resistant to erythromycin, 3 and 0.5% were resistant to cefaclor, 2 and 0.5% were resistant to loracarbef, and 0% were resistant to cefuroxime-axetil, cefixime, and amoxicillin-clavulanic acid. beta-Lactamase-negative ampicillin-resistant strains were not observed. Of M. catarrhalis strains, 56% were resistant to ampicillin by disk diffusion and 95% were resistant by broth microdilution. This species was susceptible to all other agents tested by either method. The disagreements between disk diffusion results and MICs for cefaclor, ampicillin, cefuroxime, and loracarbef that occurred with use of the 1990 NCCLS tables were resolved when the 1992 NCCLS tables were used.     

Haemophilus test medium versus Mueller-Hinton broth with lysed horse blood for antimicrobial susceptibility testing of four bacterial species

Studies were undertaken to determine whether broth microdilution susceptibility tests could be standardized by using a single medium for testing fastidious respiratory pathogens. Mueller-Hinton broth with lysed horse blood and the broth version of Haemophilus Test Medium (HTM) were directly compared. Ten orally administered agents were found to give essentially identical results in both media but minor differences were noted. Because the tests are easier to read when HTM broth is used, that medium is to be preferred for routine testing ofHaemophilus influenzae, Streptococcus pneumoniae, Streptococcus pyogenes andMoraxella catarrhalis isolates by the microdilution procedure.     

Evaluation of the Etest and disk diffusion methods for determining susceptibilities of 235 bloodstream isolates of Candida glabrata to fluconazole and voriconazole

The performances of the Etest and the disk diffusion methods for testing of the susceptibilities of 235 Candida glabrata isolates to fluconazole and voriconazole were compared with that of the National Committee for Clinical Laboratory Standards (NCCLS) approved standard broth microdilution (BMD) method. The NCCLS method used RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. Etest MICs were determined with RPMI 1640 agar containing 2% glucose (RPG agar) and with Mueller-Hinton agar containing 2% glucose and 0.5 microg of methylene blue per ml (MBE agar) and were read after incubation for 48 h at 35 degrees C. Disk diffusion testing was performed with MBE agar, 25-microg fluconazole disks, and 1- microg voriconazole disks and by incubation at 35 degrees C for 24 h. Overall agreements between the Etest and the BMD MICs obtained with RPG and MBE agars were 91 and 96%, respectively, for fluconazole and 93 and 95%, respectively, for voriconazole. Categorical agreements between the agar-based methods and BMD were 52.3 to 64.7% with fluconazole and 94.8 to 97.4% with voriconazole. The vast majority of the discrepancies by the disk diffusion and Etest methods with fluconazole were minor errors. The agar-based methods performed well in identifying isolates with resistance to fluconazole and decreased susceptibility to voriconazole.     

Disk diffusion method for determining susceptibilities of Candida spp. to MK-0991

We have developed an agar-based methodology for testing susceptibilities of Candida spp. to the new antifungal agent MK-0991, a glucan synthase inhibitor. Results obtained with this method correlated well with the results obtained by the National Committee for Clinical Laboratory Standards M27-A broth microdilution reference method. However, as noted with prior comparisons of broth- and agar-based systems, some isolates yielded inhibition zones which were not consistent with the MICs obtained for them. Understanding the implications of these differences will require testing in an in vivo system.     

Stability of viable-bacterium counts in liquid media used for preparation of inocula and subsequent impact on antimicrobial susceptibility test results.

We evaluated the consequences of prolonging the time between initial bacterial inoculum suspension preparation and susceptibility test inoculation. Extending the current National Committee for Clinical Laboratory Standards-recommended time of 15 min between suspension preparation and test inoculation should allow laboratories more flexibility to optimize efficiency from the standpoint of workflow. We assessed the length of time for which viable-bacterium counts remain stable in three liquid media at room temperature. Fifty isolates were examined in water, saline, and cation-supplemented Mueller-Hinton broth (CSMHB). Disk diffusion and microdilution MIC tests were performed on nine of these. Our results suggest that directly prepared inoculum suspensions of members of the family Enterobacteriaceae, Pseudomonas aeruginosa, staphylococci, and enterococci can be held for up to 6 h in water or saline prior to inoculation of disk diffusion and MIC tests without compromising test accuracy. The same organisms can be held for at least 1 h in CSMHB. Viridans group streptococci can be held for up to 6 h in saline and CSMHB and for up to 3 h in water. Similarly, Haemophilus influenzae and Streptococcus pneumoniae isolates may be held in CSMHB for up to 3 h. Because of an early decrease in viable-bacterium counts in water and saline with some H. influenzae and S. pneumoniae isolates, we recommend that National Committee for Clinical Laboratory Standards recommendations be followed for these species.     

Problems with the disk diffusion test for detection of vancomycin resistance in enterococci.

A total of 53 strains of enterococci, including recently isolated strains with high-level resistance to vancomycin, were tested for vancomycin susceptibility by broth microdilution and disk diffusion using Mueller-Hinton media with and without supplementation with 5% blood. By using currently published parameters of the National Committee for Clinical Laboratory Standards for the disk diffusion test, we found that strains for which MICs were 8 to 32 micrograms/ml were incorrectly placed in the susceptible or intermediate category, which caused both very major (1.9%) and minor (11.5%) errors. When we used newer, recently proposed breakpoints for vancomycin, we found 13.5% minor errors but no very major errors. Changing disk diffusion breakpoints to less than or equal to 14 mm for resistant [corrected] and greater than or equal to 15 mm for susceptible [corrected] would eliminate the problem for the strains with MICs of 32 micrograms/ml but not for those with MICs of 8 micrograms/ml. For those strains, it is necessary to perform an MIC test to differentiate them from strains with MICs of less than or equal to 4 micrograms/ml.     

Interpretive criteria and quality control parameters for testing of susceptibilities of Haemophilus influenzae and Streptococcus pneumoniae to trimethoprim and trimethoprim-sulfamethoxazole. The Antimicrobial Susceptibility Testing OC Group.

Two hundred twenty-eight strains of Haemophilus influenzae and 234 strains of Streptococcus pneumoniae were tested by broth microdilution and disk diffusion methods for susceptibility to trimethoprim (TMP) and TMP-sulfamethoxazole (SMX) to evaluate proposed criteria. Data are presented to support the proposed TMP MIC breakpoints of < or = 2.0 micrograms/ml for susceptibility and > or = 4.0 micrograms/ml for resistance for both species and TMP-SMX MIC breakpoints of < or = 2.0-38 micrograms/ml for susceptibility and > or = 4.0-76 micrograms/ml for resistance. Corresponding zone diameter breakpoints for H. influenzae for both drugs are proposed: < or = 10 mm = resistant; > or = 16 mm = susceptible. A 10-laboratory study documented reproducibility of such tests with standard control strains. The following control limits are proposed for tests of H. influenzae ATCC 49247 against TMP; MIC, 0.12 to 0.5 microgram/ml; zone diameter, 27 to 33 mm. The current limits for TMP-SMX were confirmed. For tests of S. pneumoniae ATCC 49619, MICs of TMP were 1.0 to 4.0 micrograms/ml and the current TMP-SMX MIC range was confirmed. Disk susceptibility tests of either drug against pneumococci were not reproducible, and consequently neither quality control limits nor interpretive criteria could be established. Endpoint interpretation and lot-to-lot variability in Mueller-Hinton agars were significant factors leading to interlaboratory variability.