Swedish Reference Group For Antibiotics Research Articles

Aminoglycoside use in urinary tract infections

To the Editor,The article “Rational use of aminoglycosides — Review and recommendations by the Swedish Reference Group for Antibiotics (SRGA)” by Hanberger et al. [1] raises important questions abo...

Rational use of aminoglycosides—Review and recommendations by the Swedish Reference Group for Antibiotics (SRGA)

The Swedish Reference Group for Antibiotics (SRGA) has carried out a risk-benefit analysis of aminoglycoside treatment based on clinical efficacy, antibacterial spectrum, and synergistic effect with beta-lactam antibiotics, endotoxin release, toxicity, and side effects. In addition, SRGA has considered optimal dosage schedules and advice on serum concentration monitoring, with respect to variability in volume of drug distribution and renal clearance. SRGA recommends that aminoglycoside therapy should be considered in the following situations: (1) progressive severe sepsis and septic shock, in combination with broad-spectrum beta-lactam antibiotics, (2) sepsis without shock, in combination with broad-spectrum beta-lactam antibiotics if the infection is suspected to be caused by multi-resistant Gram-negative pathogens, (3) pyelonephritis, in combination with a beta-lactam or quinolone until culture and susceptibility results are obtained, or as monotherapy if a serious allergy to beta-lactam or quinolone antibiotics exists, (4) serious infections caused by multi-resistant Gram-negative bacteria when other alternatives are lacking, and (5) endocarditis caused by difficult-to-treat pathogens when monotherapy with beta-lactam antibiotics is not sufficient. Amikacin is generally more active against extended-spectrum beta-lactamase (ESBL)-producing and quinolone-resistant Escherichia coli than other aminoglycosides, making it a better option in cases of suspected infection caused by multidrug-resistant Enterobacteriaceae. Based on their resistance data, local drug committees should decide on the choice of first-line aminoglycoside. Unfortunately, aminoglycoside use is rarely followed up with audiometry, and in Sweden we currently have no systematic surveillance of adverse events after aminoglycoside treatment. We recommend routine assessment of adverse effects, including hearing loss and impairment of renal function, if possible at the start and after treatment with aminoglycosides, and that these data should be included in hospital patient safety surveillance and national quality registries.

Evaluation of disk diffusion methods to detect low-level β-lactamase-negative ampicillin-resistant Haemophilus influenzae

We evaluated the efficacy of disk diffusion methods for detection of low-level β-lactamase-negative ampicillin-resistant (low-BLNAR) Haemophilus influenzae. Four hundred and seventy unselected, recent clinical isolates were tested with ampicillin (10 μg), cefaclor (30 μg) and cefuroxime (30 μg) on iso-Sensitest agar enriched with nicotinamide adenine dinucleotide (NAD) and horse blood [ST agar; Swedish Reference Group for Antibiotics (SRGA) guidelines], and on chocolate agar (in-house guidelines). Selected isolates (n = 147) were subjected to partial sequencing of the ftsI gene. Forty-seven strains (10.0%) were genotypically identified as low-BLNAR, which was confirmed by determination of minimal inhibitory concentration (MIC) using microbroth dilution method: only low level resistance to ampicillin was detected [MIC ≤1 μg/mL; MIC(50) = 0.5 μg/mL, implying susceptibility by Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antibiotic Susceptibility Testing (EUCAST) interpretative criteria]. The MIC of cefuroxime varied between 1 and 4 μg/mL (MIC(50) = 2 μg/mL), indicating susceptibility to cefuroxime by CLSI but not by EUCAST guidelines. Disk diffusion methods were able to discriminate low-BLNAR H. influenzae from the wild-type population with sensitivities ranging from 87% to 98% and specificities from 96% to 99%. Cefaclor was found to be superior to cefuroxime and ampicillin. Cefaclor zone diameter breakpoints of 30/29 and 23/22 mm are suggested for ST agar and chocolate agar, respectively.

Putative Link between the Virulence-Associated fluA Gene and Fluoroquinolone Resistance in Uropathogenic Escherichia coli

Resistance to fluoroquinolones is a major problem in the treatment of various infectious diseases, including urinary tract infections (UTI) caused by Escherichia coli. It has been recognized that fluoroquinolone-resistant E. coli strains generally harbor fewer virulence genes than their susceptible counterparts (2, 4, 5, 7, 8). We observed an increased prevalence of the virulence-associated fluA gene, which encodes the autotransporter protein Ag43a (9), in E. coli isolated in 2006 to 2008 compared to that in isolates collected prior to fluoroquinolone use (1983 to 1986). This increase was seen only in E. coli isolated from patients with urosepticemia, displaying a fluoroquinolone resistance rate of 26%, but not among isolates from patients with UTI, all of which were sensitive to these agents. Interestingly, 11 of the 12 resistant isolates carried fluA. We therefore chose to study a set of resistant and sensitive strains from patients with urosepticemia for a possible association between fluA and fluoroquinolone resistance. A total of 34 sensitive and 57 resistant E. coli isolates collected from patients with urosepticemia between 2000 and 2009 at the Karolinska University Hospital Solna, Stockholm, Sweden, were investigated. Fluoroquinolone resistance was determined according to guidelines of the Swedish Reference Group for Antibiotics (www.srga.org). Isolates sensitive to ciprofloxacin but resistant to nalidixic acid were considered resistant after confirmation of target alteration by sequencing (10). The presence of fluA was determined by PCR (9), and isolates were assigned to phylogenetic group A, B1, B2, or D by multiplex PCR (1). Prevalence data were compared by using Fisher's exact test or the chi-square test, as appropriate. Differences with P values of <0.05 were considered statistically significant. The frequency of phylogenetic groups B2 and D differed significantly between the resistant and sensitive populations (Table ​(Table1).1). While the sensitive isolates mostly belonged to group B2, the resistant ones were associated with group D. This distribution is in line with previous studies (4, 5, 7, 8). TABLE 1. Distribution of phylogenetic groups and fluA in relation to susceptibility to fluoroquinolones As indicated by our initial observation, the prevalence of fluA was significantly higher in fluoroquinolone-resistant isolates than in fluoroquinolone-sensitive isolates (42 of 57 [74%] and 16 of 34 [47%], respectively; Table ​Table1).1). Also, the prevalence of fluA differed depending on the phylogenetic origin (P < 0.001), with fluA significantly associated with group D (83% versus 45% in the remaining groups, P = 0.0002). Further analysis revealed equal distribution of fluA in sensitive and resistant isolates belonging to group D (Table ​(Table1).1). In contrast, fluA was associated with resistance in isolates of group B2. This again is in agreement with other studies suggesting a relationship between the presence or absence of certain virulence genes and antimicrobial resistance only in group B2 (3, 7, 8) but not in group D (6). In conclusion, we report here that the virulence-associated fluA gene is positively linked to fluoroquinolone resistance, particularly in E. coli belonging to phylogenetic group B2. To our knowledge, fluA has not been previously described in this connection and might provide another factor to understand the complex relationship between fluoroquinolone resistance and virulence in E. coli.

Low antibiotic resistance rates in Staphylococcus aureus, Escherichia coli and Klebsiella spp but not in Enterobacter spp and Pseudomonas aeruginosa: a prospective observational study in 14 Swedish ICUs over a 5‐year period

Intensive care units (ICUs) are hot zones for emergence and spread of antibiotic resistance because of frequent invasive procedures, antibiotic usage and transmission of bacteria. We report prospective data on antibiotic use and bacterial resistance from 14 academic and non-academic ICUs, participating in the ICU-STRAMA programme 1999-2003. The quantity of antibiotics delivered to each ICU was calculated as defined daily doses per 1,000 occupied bed days (DDD(1,000)). Specimens for culture were taken on clinical indications and only initial isolates were considered. Species-related breakpoints according to the Swedish Reference Group for Antibiotics were used. Antibiotic resistance was defined as the sum of intermediate and resistant strains. Mean antibiotic use increased from 1,245 DDD(1,000) in 1999 to 1,510 DDD(1,000) in 2003 (P = 0.11 for trend). Of Staphylococcus aureus, 0-1.8% were methicillin resistant (MRSA). A presumptive extended spectrum beta-lactamase (ESBL) phenotype was found in <2.4% of Escherichia coli, based on cefotaxime susceptibility, except a peak in 2002 (4.6%). Cefotaxime resistance was found in 2.6-4.9% of Klebsiella spp. Rates of resistance among Enterobacter spp. to cefotaxime (20-33%) and among Pseudomonas aeruginosa to imipenem (22-33%) and ciprofloxacin (5-21%) showed no time trend. MRSA and cefotaxime-resistant E. coli and Klebsiella spp strains were few despite high total antibiotic consumption. This may be the result of a slow introduction of resistant strains into the ICUs, and good infection control. The cause of imipenem and ciprofloxacin resistance in P. aeruginosa could reflect the increased consumption of these agents plus spread of resistant clones.

Detection of low-level oxacillin resistance in mecA-positive Staphylococcus aureus

Detection of low-level oxacillin-resistant Staphylococcus aureus is a problem that needs special attention, particularly in relation to methicillin-resistant S. aureus (MRSA) strains in the community that belong to clonal lineage ST80. This study compared different phenotypic methods for the detection of 74 low-level oxacillin-resistant S. aureus strains (oxacillin MIC <or=1 mg/L), 46 MRSA strains (oxacillin MIC >or=2 mg/L) and 117 methicillin-susceptible S. aureus strains. Determination of microbroth dilution MICs for oxacillin was wholly unsatisfactory, and gave a limited specificity for cefoxitin. The sensitivity of disk-diffusion performed according to CLSI recommendations was 92% with an oxacillin 1-microg disk, and 96% with a cefoxitin 30-microg disk; use of a 10-microg cefoxitin disk and a semi-confluent inoculum (breakpoint for resistance <18 mm zone diameter) gave a sensitivity of 97%. When disk-diffusion was performed on IsoSensitest agar with a zone diameter breakpoint for resistance of <22 mm (as recommended by the Swedish Reference Group for Antibiotics), the sensitivity was 95%.

Comparison of daptomycin MIC results by DIN, NCCLS, SFM, and SRGA methods for 297 Gram-positive organisms

Daptomycin is a novel lipopeptide antibiotic with potent in vitro antibacterial activity against Gram-positive pathogens. For daptomycin minimal inhibitory concentration (MIC) testing, National Committee for Clinical Laboratory Standards (NCCLS) recommends the use of broth containing physiological levels of calcium (50 μg/ml). The daptomycin susceptibility of 297 organisms was determined by NCCLS (Mueller–Hinton (MH) broth), Deutsches Institut für Normung (DIN; isotonic broth), Société Française de Microbiologie (SFM; three batches MH agar), and Swedish Reference Group for Antibiotics (SRGA; PDM agar). All media were supplemented to 50 μg/ml Ca 2+. There was good correlation between DIN and SFM methods (for staphylococci) with NCCLS results. Enterococci MICs using SFM methods were one to three dilutions lower and pneumococci results were one dilution higher than NCCLS. SRGA results were higher than NCCLS by one to four dilutions. Use of isotonic agar is an accepted alternative to isosensitest agar for the DIN method.

Evaluation of a cefoxitin 30 microg disc on Iso-Sensitest agar for detection of methicillin-resistant Staphylococcus aureus.

To evaluate the performance of a cefoxitin 30 microg disc on Iso-Sensitest agar, using a semi-confluent inoculum and overnight incubation at 35-36 degrees C, for detection of methicillin-resistant Staphylococcus aureus (MRSA). A total of 457 S. aureus, including 190 MRSA of several defined PFGE types and a number of low-level resistant isolates, were tested with a cefoxitin 30 microg disc on Iso-Sensitest agar, using a semi-confluent inoculum and overnight incubation at 35-36 degrees C. This method was compared with the standard SRGA (Swedish Reference Group for Antibiotics) method (oxacillin 1 microg disc on Iso-Sensitest agar supplemented with 5% defibrinated horse blood, confluent growth and 24 h incubation in ambient air at 30 degrees C). The cefoxitin method was excellent, with a sensitivity of 100% and a specificity of 99% using an interpretative zone diameter of S > or = 29 mm and R < 29 mm. Its performance was much better than the SRGA method, which with this collection of difficult strains had a sensitivity of only 78% using the current breakpoint of S > or = 12 mm. We suggest that the cefoxitin method should replace that currently recommended by the SRGA for the detection of MRSA, and that it would fit well into BSAC methodology.

A new method for normalized interpretation of antimicrobial resistance from disk test results for comparative purposes

To evaluate a calibration method for disk diffusion antibiotic susceptibility tests, using zone diameter values generated in the individual laboratory as the internal calibrator for combinations of antibiotic and bacterial species. The high-zone side of zone histogram distributions was first analyzed by moving averages to determine the peak position of the susceptible population. The accumulated percentages of isolates for the high zone diameter values were calculated and converted into probit values. The normal distribution of the ideal population of susceptible strains was then determined by using the least-squares method for probit values against zone diameters, and the ideal population was thereby defined, including mean and standard deviation. Zone diameter values were obtained from laboratories at the Karolinska Hospital (KS) and Växjö Hospital (VX), and from two laboratories (LabA, LabB) in Argentina. The method relies on well standardized disk tests, but is independent of differences in MIC limits and zone breakpoints, and does not require the use of reference strains. Resistance was tentatively set at below 3 SD from the calculated, ideal mean zone diameter of the susceptible population. The method, called normalized interpretation of antimicrobial resistance, was tested on results from the KS and VX clinical microbiology laboratories, using the disk diffusion method for antimicrobial susceptibility tests, and for two bacterial species, Staphylococcus aureus and Escherichia coli. In total, 114 217 test results were included for the clinical isolates, and 3582 test results for control strains. The methodology at KS and VX followed the standard of the Swedish Reference Group for Antibiotics (SRGA). Zone diameter histograms for control strains were first analyzed to validate the procedure, and a comparison of actual means with the calculated means showed a correlation coefficient of r = 0.998. Results for clinical isolates at the two laboratories showed an excellent agreement for 54 of 57 combinations of antibiotic and bacterial species between normalized interpretations and the interpretations given by the laboratories. There were difficulties with E. coli and mecillinam, and S. aureus and tetracycline and rifampicin. The method was also tested on results from two laboratories using the NCCLS standard, and preliminary results showed very good agreement with quality-controlled laboratory interpretations. The normalized resistance interpretation offers a new approach to comparative surveillance studies whereby the inhibition zone diameter results from disk tests in clinical laboratories can be used for calibration of the test.

SWEDRES 2001 - report on Swedish antibiotic use and resistance in human medicine

Antimicrobial resistance is an increasing threat to human health. Although the prevalence of resistant bacteria in Sweden is low in comparison to many other countries, international trends are alarming and resistance genes do not respect any borders. Recently, the first yearly report on surveillance and control systems for both antibiotic use and resistance was published and made available on the Internet (1). The report is a joint collaboration between the Swedish Institute for Infectious Disease Control (SMI, &amp;lt;a href=&amp;quot;http://www.smittskyddsinstitutet.se&amp;quot;&amp;gt; http://www.smittskyddsinstitutet.se&amp;lt;/a&amp;gt;) and the Swedish Strategic Programme for the Rational Use of Antimicrobial Agents (STRAMA, &amp;lt;a href=&amp;quot;http://www.strama.org&amp;quot;&amp;gt; http://www.strama.org&amp;lt;/a&amp;gt;). This first yearly report covers data from 2001 and from previous years. Most resistance data have been routinely collected from the local laboratories by the SMI and the Swedish Reference Group for Antibiotics and its subcommittee on methodology (SRGA-M, &amp;lt;a href=&amp;quot;http://www.srga.org/&amp;quot;&amp;gt; http://www.srga.org/&amp;lt;/a&amp;gt;), but data on specific organisms for the SWEDRES Report were also sent from individual contributing laboratories.&amp;nbsp;

Calibration of fusidic acid disk diffusion susceptibility testing of Staphylococcus aureus

Single strain regression analysis, SRA, was used to calibrate disk diffusion fusidic acid susceptibility testing of Staphylococcus aureus in two laboratories using different standard methods but the same interpretative MIC limits. SRA equation constants were calculated using five different fusidic acid disk contents (1.5, 5, 15, 50, 150 microg). These disks were tested on five separate occasions against quality control strain S. aureus ATCC 29213. The National Committee for Clinical Laboratory Standards (NCCLS) method was employed in Tartu, Estonia (TE) and the Swedish Reference Group for Antibiotics (SRGA) method in Sweden at the Karolinska Hospital (KS). SRA constants obtained were used for calculating zone breakpoints corresponding to MIC breakpoints recommended by the SRGA (S < or = 0.5 mg/L, R > or = 1 mg/L). Zone diameter histograms from KS, performed with a 50 microg disk, and from TE, using a 10 microg disk, showed a clustering of wild type strains around 41 mm and 30 mm, respectively, reflecting differences in methodology. Zone breakpoints calculated from the equations were validated by comparison with the histograms. Breakpoints were also calculated for a suggested lower disk content in Sweden, 10 microg, and validated in tests of clinical isolates and by histogram analysis.

Antimicrobial susceptibility patterns of enterococci in intensive care units in Sweden evaluated by different MIC breakpoint systems.

Three hundred and twenty-two (322) clinical isolates were collected from patients admitted to intensive care units (ICUs) at eight Swedish hospitals between December 1996 and December 1998. Of the isolates, 244 (76%) were Enterococcus faecalis, 74 (23%) were Enterococcus faecium and four (1%) were other Enterococcus spp. MICs of ampicillin, imipenem, meropenem, piperacillin/tazobactam, ciprofloxacin, trovafloxacin, clinafloxacin, gentamicin, streptomycin, vancomycin, teicoplanin, quinupristin/dalfopristin, linezolid and evernimicin were determined by Etest. Susceptible and resistant isolates were defined according to the species-related MIC breakpoints of the British Society for Antimicrobial Chemotherapy (BSAC), the National Committee for Clinical Laboratory Standards (NCCLS) and the Swedish Reference Group for Antibiotics (SRGA). Tentative breakpoints were applied for new/experimental antibiotics. Multidrug resistance among enterococci in ICUs is not uncommon in Sweden, particularly among E. faecium, and includes ampicillin resistance and concomitant resistance to fluoroquinolones. Almost 20% of E. faecalis isolates showed high-level resistance to gentamicin and concomitant resistance to fluoroquinolones. Vancomycin-resistant enterococci were only found sporadically. Among the new antimicrobial agents, linezolid and evernimicin showed the best activity against all enterococcal isolates. There was good concordance between the BSAC, NCCLS and SRGA breakpoints in detecting resistance. When applying the SRGA breakpoints for susceptibility, isolates were more frequently interpreted as intermediate. This might indicate earlier detection of emerging resistance using the SRGA breakpoint when the native population is considered susceptible, but with the risk that isolates belonging to the native susceptible population will be incorrectly interpreted as intermediate.

MIC determination of fusidic acid and of ciprofloxacin using multidisk diffusion tests

To investigate the possibility of estimating the MICs of fusidic acid and ciprofloxacin for bacterial isolates using series of antibiotic disk concentrations in diffusion tests, so-called M-tests. Thirty Staphylococcus aureus and S. epidermidis strains were tested for fusidic acid susceptibility. Sixty-one clinical isolates of eight bacterial species were tested for ciprofloxacin susceptibility. Disk diffusion was standardized according to the Swedish reference group for antibiotics (SRGA). For fusidic acid, a series of disks (1.5, 5.0, 15, 50 and 150 microg) was used. Ciprofloxacin was applied in four different diffusion sources (1, 3, 10 and 30 microg) on a single strip, the M-strip, and used. True MIC values were determined using the standardized agar dilution method according to the SRGA. Single-strain regression analysis (SRA) was employed to calculate critical concentration equivalents (Qzero). Fusidic acid and ciprofloxacin critical concentrations were determined for the bacterial isolates. The mean conversion factors for Qzero to yield the true MIC were 2.06 (range 0.34-8.9) for fusidic acid and 2.05 (range 0.37-8.1) for ciprofloxacin. There was a correlation between true MIC values (all MICs expressed as 2 log + 9) and the calculated MIC values (Qzero x conversion factor) for both fusidic acid (R = 0.9822) and ciprofloxacin (R = 0.9696). MIC values of clinical isolates can be estimated using SRA calculations on zone measurements in disk tests with several concentrations of the antibiotic in diffusion sources.

Apparent differences in antimicrobial susceptibility as a consequence of national guidelines

Apparent differences in antimicrobial susceptibility as a consequence of national guidelines

Fusidic acid disk diffusion testing of clostridium difficile can be calibrated using single-strain regression analysis.

Single-strain regression analysis (SRA) was employed to calibrate the disk diffusion antibiotic susceptibility test for fusidic acid and Clostridium difficile. MIC determinations of 40 clinical isolates of C. difficile were performed with the E-test. The disk diffusion test was standardized according to the Swedish Reference Group for Antibiotics (SRGA). Disks used for SRA contained 1.5, 5, 15, 50 and 150 microg fusidic acid and the routine disk contained 50 microg fusidic acid. A control strain, ATCC 9689, was also tested. SRA constants A and B of the regression lines were calculated. This permitted the determination of zone breakpoints for C. difficile. When applying the pharmacological MIC S and R limits set by SRGA to the E-test results I strain of C. difficile was interpreted as resistant. Zone breakpoints corresponding to the pharmacological MIC limits and calculated using the mean SRA constants for the 40 clinical isolates lead to all strains being interpreted as susceptible. SRA calculations enable laboratories to set up calibrated disk tests with species-related and laboratory-specific interpretations.

High frequency of antibiotic resistance among Gram-negative isolates in intensive care units at 10 Swedish hospitals.

OBJECTIVE: To investigate the resistance rates among Gram-negative isolates in Swedish intensive care units (ICUs). METHODS: During 1994-95, members of the Swedish Study Group collected, on clinical indication, 502 consecutive initial isolates of Gram-negative bacteria from patients admitted to ICUs at 10 Swedish hospitals and performed minimal inhibitory concentration (MIC) determinations with the Etest. Breakpoints were defined according to the criteria of the Swedish Reference Group for Antibiotics (SRGA). RESULTS: The distribution of bacterial species was: Escherichia coli > Klebsiella spp. > Enterobacter spp. > Pseudomonas aeruginosa > Haemophilus spp. > Proteus spp. > Stenotrophomonas maltophilia > Citrobacter spp. > Acinetobacter > Pseudomonas spp. > Morganella morganii > Serratia spp. Together these constituted 97% of all isolates. The frequencies of resistance for all the initial Gram-negative isolates were: ceftazidime 6.8%, cefotaxime 14.9%, ceftriaxone 18.5%, cefuroxime 44.1%, ciprofloxacin 4.2%, co-trimoxazole 17.8%, gentamicin 5.8%, imipenem 8.6%, piperacillin 20.2%, piperacillin/tazobactam 12.9% and tobramycin 5.8%. CONCLUSIONS: Among Gram-negative isolates in Swedish ICUs, a very high frequency of resistance was seen to cefuroxime, and rather high frequencies of resistance to cefotaxime, ceftriaxone, piperacillin and piperacillin/tazobactam. These drugs cannot be recommended for further use as empirical monotherapy for severe ICU-acquired Gram-negative infections in ICUs in Sweden.

Comparison of 30 μg and 120 μg gentamicin disks for the prediction of gentamicin resistance in Enterococcus faecalis

Single strain regression analysis was performed on PDM II medium for E. faecalis with 10, 30 and 120 micrograms gentamicin disks using E. faecalis, strain ATCC 29212 as the reference. This method permits the calculation of zone diameters corresponding to different MIC values for different disk contents. The lack of discrimination between normal low-level resistant strains and high-level resistance using the 10 micrograms disk was confirmed. However, both the 30 micrograms and 120 micrograms disks seemed to provide a separation of the normal low-level gentamicin-resistant population from strains with increased resistance. Since the 30 micrograms disk is used routinely in some countries, there should be no need for an extra high content disk in these laboratories. This was confirmed when 96 clinical isolates of E. faecalis were analysed and the results of routine disk diffusion tests were compared with the MIC values. Two of the strains showed high-level gentamicin resistance (greater than 2000 mg/l) and produced no zone of inhibition. The other 94 isolates showed gentamicin MIC values between 4-16 mg/l, and 72 of the MIC results were 8 mg/l. The zone diameters for these strains ranged between 15 and 25 mm with a mean of 18.2 and a median value of 18 mm. In order to include statistical considerations of the zone size populations for setting of breakpoints, a study of gentamicin zone size distributions was performed for several bacterial species. Inhibition zone diameter values around the 30 micrograms gentamicin disk for 2079 clinical isolates of E. faecalis, 2268 S. aureus, 3201 E. coli and 547 strains of P. mirabilis from different years were plotted as histograms. Tests for agreement with a Gaussian distribution showed that the histograms were slightly peaked and skewed towards higher zone values. Parametric and non-parametric statistical tests were compared and the results showed that means and medians were very similar and that parametric fractile estimations at the lower end of the histogram populations were conservative and could be used in view of a slightly lower rate of false resistance. The 1% parametric fractile of 12 mm was selected as a suitable breakpoint for the identification of normal, low-level resistant isolates of E. faecalis using the standardized disk test of the Swedish Reference Group for Antibiotics.

Types of interpretive errors in susceptibility testing. Zone breakpoints for norfloxacin disk diffusion testing.

A total of 548 strains of the eleven most common urinary tract pathogens were investigated for possible errors in norfloxacin susceptibility tests comparing MIC determinations with disk diffusion assays. Most strains were found to be sensitive with MIC-90 values below 1.0 for the Enterobacteriaceae while the classical nalidixic acid resistant species, the gram-positive bacteria and Pseudomonas aeruginosa, were less susceptible to norfloxacin with MIC-90 above 1.0 mg/l. MIC-values close to interpretive MIC-limits were recorded for S. faecalis and S. agalactiae using the recommendations of the national Committee for Clinical Laboratory Standards (NCCLS) (susceptible, S less than or equal to 4.0) and for P. aeruginosa and S. aureus using the Swedish Reference Group for Antibiotics (SRGA) standards (S less than or equal to 1.0). Susceptibility interpretations for these species showed a lack of accuracy consistent with methodological problems of reproducibility, an error called type I. The changes in the MIC-limits required for these strains to correct the error would be S less than or equal to 4 for P. aeruginosa and S. aureus, S less than or equal to 8 for S. agalactiae and S less than or equal to 0.5 for S. faecalis. A type II error, occurring when a bacterial species shows a regression line different from the regular line, was also identified for S. saprophyticus. The use of breakpoints derived from single strains regression analysis corrected this error and also reduced the frequency of similar misinterpretations in other species. The term "species-specific MIC-limits" should be introduced along with the established concept of "species-specific interpretive zone breakpoints" to allow for the correction of type I interpretive errors. Type II errors can be corrected by using species-specific interpretive breakpoints, either issued by reference laboratories or derived by calculations from single-strain regression analysis in the individual laboratory.

Laboratory- and species-specific interpretive breakpoints for disk diffusion tests of chloramphenicol susceptibility of Haemophilus influenzae.

A total of 601 clinical isolates of Haemophilus influenzae isolated in six different regions of Sweden were tested for chloramphenicol susceptibility by using agar dilution MIC determinations and disk diffusion tests. For seven strains MICs were 4 micrograms/ml or higher, and for one strain the MIC was 2 micrograms/ml. All eight strains produced chloramphenicol acetyltransferase. For the remaining 593 strains, MICs were less than or equal to 1 microgram/ml, and the MICs for 50% and 90% of the strains were both 0.5 microgram/ml. Disk diffusion tests carried out by using revised interpretive criteria introduced in 1984 by the Swedish Reference Group for Antibiotics correctly identified the 593 strains as susceptible and the 8 strains as resistant. Quality assessments were performed in 29 clinical microbiology laboratories. The revised criteria for chloramphenicol disk diffusion testing gave rise to false resistance results in some laboratories. The interpretive accuracy improved when the interlaboratory variation was compensated for by using adjusted breakpoints. Such revision was possible through peak correction, single-strain regression analysis, and standard curve regression analysis. Peak-corrected breakpoints improved the accuracy from an overall incidence of false-resistant isolates of 4.4% to 2.3%. Single-strain regression analysis and standard curve regression analysis provided laboratory- and species-specific breakpoints which reduced false resistance rates of 0.14% and 0%, respectively.

A revised system for antibiotic sensitivity testing. The Swedish Reference Group for Antibiotics.

A revised system for antibiotic sensitivity testing in Sweden was proposed in 1977. The new SIR system has been in sue in almost all Swedish routine bacteriological laboratories since 1979. The SIR system implied 2 main changes: 3 sensitivity groups instead of previous 4 and new breakpoints for the sensitivity groups. The new system is presented in comparison with 3 standardized systems currently in use (NCCLS, ICS and DIN systems).