On the theory of the disk diffusion test. Evidence for a non-linear relationship between critical concentration and MIC, and its practical implications for susceptibility testing of Haemophilus influenzae.

The procedure for disk diffusion susceptibility testing has been worked out for rapidly growing non-fastidious bacteria. Using the general zone diameter breakpoints for interpretation of susceptibility, it was found that clinical isolates of H. influenzae were assigned to the wrong SIR category in fifty per cent of the strains for erythromycin, and ten per cent for doxycycline. New species- and laboratory-specific interpretive zone diameter breakpoints corresponding to the recommended MIC limits were therefore worked out. The Standard Curve regression Analysis (SCA) method used for this purpose is based on the correlation between zone size and disk content, using two reference strains with different MICs. In its original version (the Single strain Regression Analysis, SRA) only one reference strain was used. This equation was found not to be generally valid since the relationship between MIC and the critical concentration is not constant, as was originally assumed. The slope and intercept of the regression line obtained by SCA is species related, while a general regression line based on results from many different species assumes that there is the same relation between the zone size and MIC for all species. Breakpoints for erythromycin and doxycycline calculated by the SCA equation gave more accurate results in routine susceptibility testing of H. influenzae and reduced the error rate from fifty three and ten per cent to four and three per cent for the two antibiotics.

Antibiotic disk diffusion testing revisited. Single strain regression analysis. Review article.

The standardized (NCCLS, ICS, DIN etc.) disk diffusion method is the most widespread technique for antibiotic susceptibility testing. Interpretive zone breakpoints are calculated from the regular regression line between minimum inhibitory concentrations (MIC) of bacterial isolates and the corresponding inhibition zone diameters around the disk containing the antibiotic. Studies of the regression line has revealed marked differences between different bacterial species. A newly described equation, the single strain regression analysis (SRA) equation, can be used to determine the regression line constants for individual strains. This method was applied to ciprofloxacin and S. aureus, E. faecalis, E. coli, P. mirabilis, P. aeruginosa, and P. maltophilia. The slope and intercept constants were determined for all 40 strains and showed a strong similarity within each species. A close similarity was also observed between the two Pseudomonas species and between S. aureus and E. faecalis. When the regression lines calculated by SRA for individual strains were extrapolated towards higher MIC values, the lines obtained for the more susceptible strains predicted the zones of more resistant strains within the species. The applications of SRA to several other antibiotics and bacterial species in earlier studies were reviewed. One exception to the predictive power of SRA has been detected earlier, H. influenzae and erythromycin. This led to the formulation of the standard curve regression analysis (SCA) equation which requires the use of two or more strains. Methodological aspects of SRA/SCA applications were presented. Three areas are particularly well suited for the use of SRA/SCA: 1. Calculation of interpretive zone breakpoints corresponding to recommended MIC limits in the individual laboratory. 2. Analysis of the effects of various disk contents of antibiotic on the resulting inhibition zones for various bacteria when new antibiotics are introduced. 3. Analytical tool as part of external quality control programmes.

Viridans streptococci in blood cultures. Can we see any patterns of species related to patient category? Review of 229 cases of positive cultures with viridans streptococci.

We present a review of 229 blood cultures with viridans streptococci collected during a period of eight and a half years from 1986 to 1994 at a teaching hospital in Sweden. The clinical significance of the growth of viridans streptococci is always uncertain, since these bacteria can be contaminants from the skin flora. Growth in more than one culture bottle strengthens the clinical value of the finding. The question was whether species identification might also help in the assessment of the clinical relevance of the finding. The results show that Streptococcus mitis occurs significantly more frequently in blood cultures from the departments dealing with cancer patients. Even with polymicrobial growth in blood cultures, S. mitis should be considered a pathogen of clinical relevance, not a contaminant.

Routine semiquantitative cultures and central venous catheter-related bacteremia.

Semiquantitative cultures were compared with blood cultures during one year in order to see if the routine use of a semiquantitative catheter culture method (SQC) in unselected patients can detect or predict infection associated with central venous catheters. Catheter infection, i.e. greater than or equal to 15 colony forming units (cfu) per plate, occurred in 137 of 542 catheter tips (25%), mainly with coagulase-negative staphylococci. Catheter-associated bacteremia occurred in 17 of 93 cases (18%) where blood cultures had been drawn. In 15 of these, the catheter tip grew greater than or equal to 15 cfu. The predictive value for bacteremia of a positive SQC was only 21%. SQC is not a suitable method for the detection of catheter-associated bacteremia, but may be an indicator of the standard of central venous catheter hygiene.

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.

Comparison of 30 microgram and 120 microgram 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.

Etiology of pneumonia, sepsis and meningitis in infants younger than three months of age in Ethiopia.

METHODS: Within a multicenter study coordinated by WHO, an investigation of the etiologic agents of pneumonia, sepsis and meningitis was performed among infants younger than 3 months of age seen at the Ethio-Swedish Children's Hospital in Addis Ababa for a period of 2 years. Of the 816 infants enrolled 405 had clinical indications for investigation. RESULTS: There were a total of 41 isolates from blood cultures from 40 infants. The study showed that the traditionally known acute respiratory infection pathogen Streptococcus pneumoniae was most common in this extended neonatal age group, found in 10 of 41 blood isolates. Streptococcus pyogenes was a common pathogen in this setting (9 of 41 blood isolates), whereas Salmonella group B was found in 5 of 41 isolates. Streptococcus agalactiae, which is a common pathogen in developed countries, was absent. A study of the susceptibility pattern of these organisms suggests that a combination of ampicillin with an aminoglycoside is adequate for initial treatment of these serious bacterial infections, but the combination is not optimal for the treatment of Salmonella infections. Among 202 infants on whom immunofluorescent antibody studies for viruses were performed based on nasopharyngeal aspirates, respiratory syncytial virus was found in 57 (28%) infants, and Chlamydia trachomatis was isolated in 32 (15.8%) of 203 infants.

Nosocomial Staphylococcus epidermidis septicaemia among very low birth weight neonates in an intensive care unit.

A cluster of cases of septicaemia caused by coagulase-negative staphylococci was observed among the infants at the Neonatal Intensive Care Unit (NICU) of the Karolinska Hospital in May 1987. The presence of a unique antibiogram among the blood culture isolates prompted an investigation to determine whether an epidemic strain existed or not, using antibiogram, biotyping and plasmid profiles as epidemiological markers. All 14 isolates with a unique antibiogram were investigated, and 22 isolates without the unique antibiogram served as controls. Of the 14 isolates, 11 were Staphylococcus epidermidis and had similar plasmid patterns. Of the 22 control isolates, 15 were S. epidermidis, and none had the special plasmid pattern nor any other recurring plasmid patterns. The use of plasmid profile analysis together with antibiograms thus identified a possible epidemic strain of S. epidermidis which may have been responsible for the upsurge of septicaemia cases at the NICU. The presence of an epidemic strain implies that hospital cross infection control could be important in preventing neonatal septicaemia caused by S. epidermidis.

Change of central venous catheter dressings twice a week is superior to once a week in patients with haematological malignancies.

Thirty-two consecutive patients with haematological disorders, in need of a permanent central venous catheter (CVC) were randomly allocated to have their CVC bandages (Tegaderm) changed once (OAW, n = 20) or twice (TAW, n = 19) a week. The two randomization arms were balanced in respect of age, sex, and underlying disease. The exit site of the CVC was inspected daily through the transparent bandage and erythema was noted. If severe erythema occurred, daily wet gauze dressings were applied. Samples for bacterial cultures were taken from the exit site of the CVC at every change of bandages. There was no difference in complications leading to removal of the CVC between the two groups (7/20 OAW vs. 7/19 TAW) or in CVC survival-time (P = 0.4). However, the OAW group had more positive CVC tip cultures (OAW 11/14 vs. TAW 2/9; P < 0.05) and a tendency to: (i) more extra dressings (P = 0.08); (ii) more cultures from the exit skin site showing high numbers of colony forming units (P = 0.07); (iii) shorter time to first exit site infection (P = 0.09); and (iv) more Gram-positive septicaemias (P = 0.08). Both clinical and bacteriological data in this study indicate that changing transparent polyurethane CVC bandages twice a week is superior to once a week.

Clostridium difficile infections related to antibiotic use and infection control facilities in two university hospitals.

We investigated whether a reduction in antibiotic use at the Aker University Hospital (Aker) led to a reduction in Clostridium difficile-associated diarrhoea (CDAD). We compared the incidence of CDAD in Aker and Tromsoe University Hospitals (Tromsoe) and related it to antibiotic use and facilities for infection control between 1993-2001. For this purpose we also performed point prevalence studies. Total antibiotic use was the same in the two hospitals. In spite of a reduction in the use of broad-spectrum antibiotics in Aker the incidence of CDAD increased during 1993-1999. In Tromsoe the use of broad-spectrum antibiotics and clindamycin was two to three times higher than in Aker, but until 1999 the incidence of CDAD remained constant and only half that of Aker. After 1999 the incidence of CDAD was halved in Aker, and increased three-fold in Tromsoe. Point prevalence studies in 2001 revealed an equal prevalence of antibiotic-associated diarrhoea. The facilities for infection control were better in Tromsoe. The percentage of single rooms were 8% in Aker and 14% in Tromsoe, and the percentage of single rooms with a WC was 6% in Aker and 12% in Tromsoe. The bed occupancy was much higher in Aker than in Tromsoe. Lack of facilities for infection control and higher bed occupancy could have contributed to the higher incidence of CDAD in Aker in spite of decreased use of broad-spectrum antibiotics and clindamycin. To limit CDAD in hospitals the focus must be on both rational antibiotic use and infection control.

Colonization with coagulase-negative staphylococci in two neonatal units.

Episodes of septicaemia due to coagulase-negative staphylococci (CNS) were more frequent in a level III than in a level II neonatal unit in Stockholm, Sweden. Colonization with CNS during the first 2 weeks of life was investigated in 10 infants from each unit. As the use of antibiotics differed between the two units, the aim was to correlate colonization and antimicrobial resistance patterns to antibiotic usage. Antimicrobial susceptibility of CNS to isoxazolylpenicillins, co-trimoxazole, erythromycin, clindamycin, chloramphenicol and gentamicin was determined. Selected isolates were typed with restriction endonuclease analysis of plasmid DNA and of genomic DNA. Infants were frequently colonized with multiple strains and species of CNS, and transmission of strains from patient to patient occurred within the unit. Qualitative and quantitative differences in antibiotic use were not correlated with colonization. The prevalence of resistant isolates, mostly of Staphylococcus haemolyticus, was higher in the level II unit with lower use of antibiotics. Staphylococcus epidermidis, which is generally more virulent, prevailed in the level III unit, where there were more severely ill children and invasive procedures were more frequently performed.

A three-year survey of nosocomial and community-acquired infections, antibiotic treatment and re-hospitalization in a Norwegian health region.

In Norway, hospital-acquired infections (HAI) were analysed by repeated point prevalence studies (four each year) performed simultaneously at 14 hospitals in a health region (860,000 inhabitants) during the period 1996-1998. The study included 3200 beds and 121,000 discharged patients each year, and was initiated by and co-ordinated from the regional university hospital; Ullevål University Hospital (UHH). An overall prevalence rate of HAI of 6.5% (interhospital variation 1.4-11.7%) was found for the 32,248 patients studied. The rate of HAI was reduced from 7.7% in 1996 to 5. 9% in 1998. Smaller hospitals (<200 beds) generally had lower rates of HAI, community acquired infections (CAI), postoperative infections and use of antibacterial agents, than the large regional hospital (1200 beds). HAI was reduced in non-operated patients from 5.8% in 1996 to 4.4% in 1998 and in operated patients from 13.2% in 1996 to 10.5% in 1998. The risk of developing HAI was twice as high after surgery. From 1996 to 1998 there was a reduction in: urinary tract infections from 2.4% to 1.7%, lower respiratory tract infections from 1.5% to 0.8% and postoperative wound infections from 5.7% to 4.3%, while septicaemia (from 0.5% to 0.4%) remained unchanged. Re-hospitalization because of HAI was registered in 0.6% (interhospital variation 0.3-1.1%) of patients. The CAI rate in hospitals increased from 8.3% in 1996 to 10.8% in 1998. Approximately 16% (variation:14.4-20.6%) of the patients had an infection. The total use of antibacterial agents was 19.2% in 1996, 16.6% in 1997 and 17.8% in 1998 (variation: 14.9-23%).

Pseudomonas aeruginosa in otitis externa. A particular variety of the bacteria?

BACKGROUND: Pseudomonas aeruginosa rarely affects the epithelium in healthy persons except for the external ear canal, raising the possibility that P aeruginosa in otitis externa is a specific variety that displays particular characteristics. DESIGN: A cohort study was designed to outline distinct characteristics of P aeruginosa in otitis externa compared with P aeruginosa in other infections. The study period was October 1, 1994, to March 27, 1995. PATIENTS: Isolates of P aeruginosa from nonhospitalized patients were collected at the bacteriological laboratory at Karolinska Hospital, Stockholm, Sweden; there were 53 strains of P aeruginosa isolated from otitis externa and 59 strains of P aeruginosa from varicose ulcers and urinary tract infections. METHODS: Pseudomonas aeruginosa was characterized by pigmentation, growth habits, production of mucoid, and biochemical characteristics. RESULTS: Pseudomonas aeruginosa in otitis externa produced less pyocyanin and less urease and exhibited no mucoid-producing strains. CONCLUSIONS: Pseudomonas aeruginosa in otitis externa displayed fewer of the usual biochemical features of the species than did the strains isolated from other infections. Some of these features, such as the production of pyocyanin, are influenced by nutritional factors; strains found in otitis externa probably represent the type of strains present in the natural habitat in water, as opposed to the strains that have adapted to the environment of other human infections. Increased knowledge of the characteristics of the strains found in otitis externa is important in understanding the pathogenesis of the disease and why P aeruginosa is the dominant infectious agent in otitis externa.

Imipenem/cilastatin monotherapy as salvage treatment in febrile neutropenic patients.

Imipenem/cilastatin (I/C) monotherapy was used as salvage treatment in 55 neutropenic patients (58 fever episodes) after treatment failure on first-line antibiotic therapy. Successful antibiotic treatment was defined as eradication of all signs, symptoms and microbiologic evidence of infection on I/C monotherapy alone. Twenty-five out of the 58 episodes (43%) were classified as success, 6 episodes (10%) as initial response but the regimen had to be modified (amphotericin B was added) and 27 episodes (47%) as failures. In episodes with documented infections 9 out of 23 (39%) were classified as success. All patients survived during the first 72 hours after change to I/C therapy. One patient had to discontinue I/C due to a skin rash. In conclusion, the use of a treatment algorithm with I/C monotherapy as second-line treatment was safe and effective. Other antimicrobial agents, most often vancomycin and/or amphotericin B, had to be added in half of the patients.

In vitro susceptibility to tetracycline and doxycycline in clinical isolates of Haemophilus influenzae.

The most common indication for the use of tetracyclines in Sweden is respiratory tract infections. Among the tetracyclines, doxycycline dominates with about 75% of the consumption. It is therefore used for routine susceptibility testing, while tetracycline is the test drug in most other countries. Six hundred strains of Haemophilus influenzae isolated from different parts of Sweden were tested for susceptibility to doxycycline and tetracycline. The results were compared with those from earlier reports on resistance rates in Sweden and other countries. The MIC50's of the strains were slightly lower for tetracycline than for doxycycline, but some strains with reduced susceptibility to tetracycline were fully susceptible to doxycycline. The level of resistance to doxycycline in H. influenzae was very low (less than 1%) and has not changed significantly over the past ten years, making doxycycline a suitable antibiotic for instance in the treatment of chronic bronchitis when H. influenzae is involved.

Increased use of erythromycin causes resistance in Haemophilus influenzae.

The use of erythromycin in Sweden has increased since 1975. An increasing prevalence of Haemophilus influenzae (H.i.) with reduced susceptibility to erythromycin has been noted in our laboratory during the years 1980 to 1985. The largest consumers of erythromycin were found to be children less than 4 years of age. Variations in resistance level of H.i. isolated from patients within this age group and variations in consumption of erythromycin were analysed for each year. The highest prevalence of resistant strains was found during spring and summer. The consumption of the antibiotic was highest during the first quarter of the year, gradually decreasing, and increasing sharply again during the fourth quarter of the year. Judging also from deviations from this pattern there seems to be an association between consumption and resistance. The different levels of resistance reported in the literature may reflect different populations of patients with different consumption of erythromycin, and different levels at different time of the year. Considering the present high level of H.i. with decreased susceptibility to erythromycin the drug is not likely to have clinical effect in H.i. infections.

Neonatal septicemia in Stockholm.

In a prospective study to determine the incidence and etiology of neonatal septicemia at Karolinska Hospital, Stockholm, all cases of neonatal septicemia in infants born at the hospital from 1981 to 1985 were reviewed. There were 4.0 cases of neonatal septicemia per 1,000 live births during the study period. During the five year period the incidence of septicemia tended to increase, mainly due to the increasing incidence of septicemia in infants with a birth weight below 1,500 grams. The single most common causative organism was group B streptococci with an incidence of 1.4 per 1,000 live births, Staphylococcus aureus accounting for 1.1 cases, Staphylococcus epidermidis for 0.7 cases, and gram-negative rods for only 0.4 cases per 1,000 live births. In a retrospective study of the incidence of neonatal septicemia in a non-selected patient population, all cases in the Stockholm area in 1983 were reviewed. In that year there were 17,586 live births in the area and the incidence of neonatal septicemia was 2.6 per 1,000 live births. Group B streptococci, Staphylococcus aureus, Staphylococcus epidermidis and gram-negative rods were equally common as causative agents.

Species-specific interpretive breakpoints for ciprofloxacin disk diffusion susceptibility testing.

When ciprofloxacin was introduced on the Swedish market the recommended interpretive standards for disk diffusion susceptibility testing using a 10 microgram disk were: Sensitive greater than or equal to 24 mm (MIC less than or equal to 1 mg/l) and Resistant less than 18 mm (MIC greater than 4 mg/l), except for enterococci where S greater than or equal to 18 mm and R less than 14 mm were recommended. A quality control study revealed that the accuracy of the routine susceptibility testing of ciprofloxacin was low. 50% of the strains of Staphylococcus aureus and 90% of Streptococcus agalactiae strains were falsely assigned to the intermediate instead of the sensitive category, and the test missed to detect true resistance in Pseudomonas maltophilia. New species-specific breakpoints were determined by the SRA method. Separate breakpoints were required only for the pseudomonas species. Streptococcus faecalis was found to belong mainly to the intermediate group and it is suggested that these strains are not categorized as sensitive. The breakpoints that gave acceptable accuracy in routine susceptibility testing of ciprofloxacin were S greater than or equal to 20 mm, R less than 13 mm for all relevant bacteria except Pseudomonas aeruginosa for which S greater than or equal to 27 mm, R less than 18 mm is proposed, and Pseudomonas maltophilia where S greater than or equal to 30 mm, R less than 23 mm are adequate breakpoints. The 10 microgram disk gave acceptable inhibition zones for all strains with MICs within the clinically interesting MIC range.

Disk diffusion method for susceptibility testing of Neisseria gonorrhoeae.

The standard medium for disk diffusion and MIC testing of Neisseria gonorrhoeae (that of the National Committee for Clinical Laboratory Standards) was tested to establish zone correlations for the MIC breakpoints currently used in Sweden. Eight gonococcal control strains representing both susceptible and resistant strains and 50 clinical isolates were tested. The standard medium did not support the growth of two control strains and three clinical isolates when the standardized inoculum was used in the disk diffusion test. The same medium with the addition of hemoglobin was introduced. This medium supported the growth of all strains. The correlations between the MICs and the zones of inhibition were calculated for penicillin, ampicillin, cefuroxime, erythromycin, tetracycline, doxycycline, ciprofloxacin, and spectinomycin. The range of MICs for the clinical isolates were broad, without bimodal distribution, for all antibiotics, except ciprofloxacin and spectinomycin. With the susceptibility distribution of MICs and zones near the current susceptible and intermediate or intermediate and resistant limits, a low reproducibility of tests and a high frequency of minor interpretive errors can be expected. A revision of MIC breakpoints seems warranted but can only be done after renewed clinical evaluation of different treatment regimens.