Comparison of direct and standardized disk diffusion susceptibility testing of urine cultures

A comparison between direct and standardized disk diffusion tests was made on a total of 300 urine specimens containing >/=10(5) organisms/ml. Of these, 246 represented pure cultures and 54 represented mixed cultures. The number of major discrepancies per organism tested in pure culture was 18 (7.3%) and in mixed cultures it was 23 (42.6%). The percentage of major discrepancies per total number of antimicrobial drug comparisons made was 1.4%. Although this procedure may be of value in selected cases with pure cultures of organisms present in quantities >/=10(5)/ml, its use on a routine basis is not recommended.     

Etest for routine clinical antimicrobial susceptibility testing of rapid-growing mycobacteria isolates

Tumors depend on their blood supply for growth. The blood supply to metastatic neoplasia of lung is usually from the pulmonary circulation or both the pulmonary and systemic circulation. The antineoplastic effect of pulmonary artery occlusion was investigated in a rat model of methylcholanthrene-induced metastatic pulmonary sarcoma. Left pulmonary artery ligation was performed on day 7 after tumor inoculation, and animals were sacrificed on day 14. The tumor burden of the left lung decreased 44% when compared with the control group. The survival of non-tumor-bearing rats undergoing left pulmonary artery ligation for 24 hours followed by right pneumonectomy after 2 weeks was also studied. No significant lung damage after a period of left pulmonary artery ligation was seen, as evidenced by both survival after contralateral right pneumonectomy and histology. Balloon occlusion of pulmonary artery, together with regional chemotherapy for patients with lung metastases, may warrant investigation.     

Simple, direct broth-disk method for antibiotic susceptibility testing of Ureaplasma urealyticum

A simple, direct broth-disk test, utilizing urine sediment as the inoculum and impregnated paper disks as the source of antibiotic, was developed and used to test the susceptibility of 54 isolates of Ureaplasma urealyticum (T-strain mycoplasmas) to minocycline, doxycycline, demeclocycline, tetracycline, and erythromycin. The concentration of each antibiotic was calculated to approximate the attainable blood level. Resistance or susceptibility to each antibiotic was determined by growth, indicated by a color change of the medium in each tube, comparable to that of a control culture without antibiotic. Of the 54 T-mycoplasmas tested, 46 (85.2%) were inhibited by 1 mug of minocycline per ml, 45 (83.3%) were inhibited by 1 mug of doxycycline per ml, 38 (72.2%) were inhibited by 1 mug of demeclocycline per ml, 18 (33.3%) were inhibited by 1 mug of tetracycline per ml, and only 2 (3.7%) were inhibited by 3 mug of erythromycin per ml. Seven (13%) of the 54 T-mycoplasmas tested were resistant to all five antibiotics. There was good correlation between results obtained by this direct broth-disk method and minimal inhibitory concentrations obtained by the direct broth dilution method.     

Vitek system antimicrobial susceptibility testing of O1, O139, and non-O1 Vibrio cholerae

Vibrio cholerae causes epidemic diarrhea throughout the world. Fluid replacement is the primary therapy for cholera; however, high mortality rates often necessitate the use of antibiotics. V. cholerae, like most bacteria, has developed resistance to some antibiotics. In the early 1990s a new serotype strain, Bengal 0139, began a new wave of cholera epidemics. Bengal isolates showed unique trends in antimicrobial resistance. Many clinical laboratories use automated antibiotic susceptibility testing for V. cholerae. It is important to know if automated susceptibility test results for V. cholerae coincide with reported trends in antibiotic susceptibility. In the present study, we used the Vitek automated susceptibility system to determine the susceptibilities of 79 V. cholerae O1 isolates, 100 O139 isolates, and 112 non-O1 isolates. Vitek susceptibilities for V. cholerae showed a good correlation with preestablished epidemiological data. Although the new O139 serogroup showed a trend of increased resistance to trimethoprim-sulfamethoxazole and nitrofurantoin, it was more susceptible to ampicillin than previous serogroup O1 and non-O1 strains. Regardless of serogroup, > or = 98% of the V. cholerae isolates tested were susceptible to most antibiotics tested by us. It is important to continue susceptibility testing of all new isolates of V. cholerae because of emerging resistant strains. However, V. cholerae remains susceptible to most of the available antibiotics.     

Evaluation of the MICUR system for quantitative antimicrobial susceptibility testing: a multiphasic comparison with reference methods

Four laboratories participated in a three-phase study to evaluate the MICUR antimicrobial broth microdilution system (Boehringer Mannheim Diagnostics, Inc., Houston, Tex.). The dried-antimicrobial agent MICUR system was compared with a reference broth microdilution method (National Committee for Clinical Laboratory Standards) by using 304 recently isolated clinical strains and two collections of stock or challenge organisms. Of 7,092 minimum inhibitory concentration (MIC) datum pairs derived from the clinical isolates, 96.6% were within an acceptable (+/- 1 log2 dilution) range. MICUR MICs agreed with the reference broth microdilution method MICs in 95.3% of 6,840 MIC pair determinations performed on stock or challenge cultures. The MICUR intralaboratory reproducibility within +/- 1 log2 dilution step for the clinical isolates was 98.4%. The MICUR intralaboratory and interlaboratory reproducibilities for 26 stock cultures were 98.4 and 95.1%, respectively. For 180 challenge cultures (4,199 MIC pairs) which were included in the MICUR testing to provide a wide variety of antimicrobial susceptibility and resistance patterns, the results for 92.5% were in close agreement with the reference broth microdilution results. No specific resistance mechanism went unrecognized by this new commercial system. The MICUR system gives comparable MIC results when evaluated against the reference broth microdilution method, and it would be acceptable for use in clinical microbiology laboratories.     

Evaluation of the E test for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from patients with long-term bladder catheterization

The E test was evaluated in comparison with reference agar methods (National Committee for Clinical Laboratory Standards) for the susceptibility testing of 248 Pseudomonas aeruginosa isolates from bladder-catheterized patients against nine antibiotics. The E-test MICs correlated well with those determined by the agar dilution and disk diffusion reference methods (88 and 92.5% within 1 log2 dilution step, respectively), confirming that the E test is a reliable method for the determination of MICs of antibiotics for catheterization-associated P. aeruginosa isolates.     

Direct susceptibility testing with positive BacT/Alert blood cultures by using MicroScan overnight and rapid panels

Studies were conducted on a method of direct inoculation of MicroScan dried overnight and of rapid panels with positive aerobic blood cultures obtained from the BacT/Alert to determine antimicrobial susceptibilities. Inocula were limited to specimens that appeared unimicrobic on Gram stain. Results were compared to those obtained from panels inoculated following subculture. For 133 gram-negative bacilli, there were 94.7 and 93.5% categorical agreements between direct and standard methods for all drugs tested with overnight and rapid panels, respectively. For 104 gram-positive cocci, there were 93.2 and 93.1% categorical agreements for overnight and rapid panels, respectively. The major error (false resistance) rate for gram negatives was 1.4% for overnight versus 0.7% for rapid panels. The very major error (false susceptibility) rate was 2.7% for overnight versus 8.1% for rapid panels. The total error rates were 1.6% for overnight panels and 1.5% for rapid panels. The major error rates for gram-positive direct susceptibility tests were 2.6% for overnight and 2.5% for rapid panels. The very major error rates were 8.8 and 7.2% for overnight and rapid panels, respectively. Total error rates were 3.6% for overnight and rapid gram-positive panels. These findings suggest that susceptibility results obtained from directly inoculated gram-negative overnight panels have the greatest correlation to those obtained by standard methods. When discrepant results occur with direct-susceptibility testing, they are more likely to show false susceptibility than false resistance.     

Utility of urine and blood cultures in pyelonephritis

OBJECTIVE: To determine how often the results of urine and blood cultures led to changes in antibiotic therapy for patients discharged from the hospital with the diagnosis of pyelonephritis. METHODS: A retrospective chart review was performed of consecutively admitted patients, 10-90 years old, with an ICD-9 discharge diagnosis of acute pyelonephritis. All patients were admitted to a university-based, tertiary care center and a large HMO medical center from 1993 to 1994. The association of urine and blood culture results with a change in antibiotic therapy was assessed. RESULTS: Of the 194 patients who met inclusion criteria, 189 (97%) had urine cultures obtained at the time of admission and 139 (71%) had blood cultures obtained. Ampicillin, gentamicin, or both were given as initial antibiotics 81% of the time, and isolated organisms from urine or blood were sensitive to the empiric antibiotics 95% of the time. Most (171/189; 90%) urine cultures were positive, but only 9 (5%) of these led to a change in antibiotic therapy. 80% of the urinary pathogens were Escherichia coli, 5% Enterococcus, 5% Proteus, and 4% Klebsiella. Only 40 (29%) of the 139 blood cultures were positive; none prompted a change in antibiotics. There were no cases in which blood and urine cultures grew different pathogens. CONCLUSIONS: Urine cultures are useful in directing antibiotic therapy in patients with the discharge diagnosis of acute pyelonephritis and support a change in therapy in 5% of cases. Among the patients in this study, blood cultures results did not lead to changes in antibiotic therapy. These findings warrant prospective, multicenter evaluation.     

Microbiologic assay for detection of antimicrobial agents in urine

Antimicrobial therapy can be a confounding factor in the diagnosis of urinary tract infection and is not always reported to laboratories by physicians. We developed a microbiologic assay for screening urine specimens for antimicrobial agents. Bacillus stearothermophilus was used as the indicator bacteria. A total of 1,921 urine specimens from three hospitals in Taiwan were screened using this assay. Of the samples assayed, 1,293 were positive for antimicrobial agents. Agreement between information provided by physicians and laboratory findings was 68.5% (419/612). In the presence of antimicrobial agents in the urine samples, the isolation of yeasts and Pseudomonas aeruginosa increased dramatically, from 4.5 to 19.5% and 4.2 to 13.2%, respectively. Additionally, Escherichia coli was more resistant to gentamicin (75.3% vs 48.7%, p < 0.0001), norfloxacin (85.2% vs 64.6%, p = 0.0006) and co-trimoxazole (58.5% vs 35.5%, p = 0.0018). In view of the high rate of occurrence of antimicrobial agents in urine specimens and the lack of information provided by most physicians to laboratories, a screening method to detect the presence (or absence) of antimicrobials in urine specimens may be a useful tool particularly in areas such as Taiwan where antimicrobial agents are commonly abused.     

E-test for susceptibility testing of Mycobacterium tuberculosis

SETTING: Initial isolates should be tested for drug susceptibility to confirm the anticipated effectiveness of chemotherapy. OBJECTIVE: To evaluate E-test strips for susceptibility testing of Mycobacterium tuberculosis. DESIGN: A proportion method using Lowenstein-Jensen medium and the Bactec radiometric system were compared with the E-test (isoniazid [INH], rifampicin [RMP], ethambutol [EMB] and streptomycin [SM]). RESULTS: For 73 of the 81 M. tuberculosis isolates (90.1%) the proportion and E-test methods yielded concordant susceptibility results against all four antimicrobial agents tested. Of these 73 strains, 69 were fully susceptible; the four isolates showing resistance to antimicrobial drugs by both methods were also resistant when tested by Bactec 460TB. While the proportion method indicated susceptibility for the eight remaining strains, E-test results showed mono EMB resistance in five strains, INH resistance for two isolates (including one isolate resistant to EMB plus INH), and for one strain E-test yielded resistance to EMB and SM. Using Bactec as the reference method, the E-test resulted in false resistance in eight strains and no false susceptibility. CONCLUSION: Due to a substantial rate of false resistance, this method cannot be recommended at present for practical use in clinical laboratories.     

Synthesis and antimicrobial testing of novel oxadiazolylbenzimidazole derivatives

OBJECTIVE: To chart the subtle neurological abnormalities in patients with asbestosis relative to possible development of cancer. METHODS: In 1979-81 a standardised neurological examination was made of 115 patients with asbestosis who carried a high risk of occupational cancer and their cancer morbidity was analysed 15 years later. RESULTS: Slight disturbances of unknown aetiology were found in the central nervous system (CNS) of 33 and in the peripheral nervous system (PNS) of 41 patients. Of these 17 had disturbances of both the CNS and PNS. This cohort was followed up to the end of 1994. During this time 47 of the patients developed cancer. Statistical analyses showed that disturbances of the CNS such as psycho-organic syndrome, cerebellar dysfunction, and motor disturbances of unknown origin were significantly associated with cancer, whereas no such association was found for peripheral neuropathy. Interaction between the radiological progression of asbestosis and disturbances of the CNS was an even stronger predictor of cancer. CONCLUSIONS: It seems that slight disturbances of the CNS are predictors of development of cancer. Whether or not these disturbances are manifestations of involvement of a paraneoplastic nervous system or some factor associated with progression of asbestosis remains open.     

Effect of inoculum size on antimicrobial susceptibility of Helicobacter pylori

An agar dilution assay was used to assess the effect of inoculum size and culture period on the susceptibility of 15 clinical isolates of Helicobacter pylori to ampicillin, erythromycin, tetracycline, chloramphenicol, metronidazole and tinidazole. The mean MIC of the isolates increased 2.2- to 21.2-fold as the inoculum size progressed from 10(3) to 10(7) cfu/spot. Identical results were noted when isolates were maintained for two or four days prior to testing. Inoculum size should be carefully controlled when assessing the in vitro susceptibility of Helicobacter pylori.     

Fluconazole disk diffusion susceptibility testing of Candida species

We describe a simple procedure for detecting fluconazole-resistant yeasts by a disk diffusion method. Forty clinical Candida sp. isolates were tested on RPMI-glucose agar with either 25- or 50-microgram fluconazole disks. With 25-microgram disks, zones of inhibition of >/=20 mm at 24 h accurately identified 29 of 29 isolates for which MICs were /=27 mm identified 28 of 29 such isolates. All 11 isolates for which MICs were >8 microgram/ml were identified by using either disk. Disk diffusion may be a useful screening method for clinical microbiology laboratories.     

Disk diffusion susceptibility testing of dermatophytes with imidazoles

Flupirtine belongs to the class of triaminopyridines and is successfully applied clinically as a non-opiate analgesic drug with additional muscle relaxant properties. Recently it was reported that flupirtine acts like an antagonist of the N-methyl-D-aspartate (NMDA) receptor complex in neuronal cells both in vitro and in vivo. Here we have used primary cortical cells from rat embryos to demonstrate that this compound is also neuroprotective against the toxic effects caused by the prion agent PrPSc and lead acetate (Pb). These two agents display pleiotropic effects on neurons, which include activation of the NMDA receptor complex. At concentrations above 30 microM the toxic-peptide fragment of PrPSc causes apoptotic fragmentation of DNA and is consequently neurotoxic. Pb is neurotoxic at concentrations above 10 microM. Co-administration of flupirtine (10 microM) with either of these agents resulted in reduced neurotoxicity. These data indicate that the cytoprotective effect of flupirtine is measurable in vitro against these noxious agents which show their effects, including modulation of the NMDA receptor complex, pleiotropically.     

The antimicrobial susceptibility of Mycobacterium chelonae isolated from corneal ulcer

PURPOSE: To determine the in vitro susceptibility of Mycobacterium chelonae isolates from corneal ulcers to various traditional and newly-developed antimicrobial agents, alone or in combination. METHODS: Fifteen strains of M. chelonae isolated from corneal ulcers were collected at the National Taiwan University Hospital from 1989 to 1993. Susceptibility to antimicrobial agents was tested by the broth microdilution method to determine the minimum inhibitory concentration (MIC). The antimicrobial effects of combinations of antimicrobial agents were assessed by the checkerboard titration method to determine the fractional inhibitory concentration (FIC) index. RESULTS: The MIC results showed that traditional antituberculous drugs had poor activity against M. chelonae. In the aminoglycoside group, tobramycin and amikacin had better activity than gentamicin. Among macrolides, clarithromycin was especially effective, with an MIC ranging from 0.125 to 1 microgram/ml. Among various beta-lactam antibiotics, imipenem was the only one to demonstrate good anti-mycobacterial activity. Of the quinolone group, ciprofloxacin was the most effective, with an MIC ranging from 0.5 to 16 micrograms/ml. Combination of an aminoglycoside with imipenem, ciprofloxacin or clarithromycin all showed antagonistic effect. CONCLUSIONS: The results suggested that amikacin, clarithromyicn, imipenem and ciprofloxacin had good in vitro antimicrobial activity against M. chelonae. However, no synergistic effect could be demonstrated for combinations of an aminoglycoside with other effective drugs.     

Interpretive criteria for testing susceptibility of staphylococci to mupirocin

To determine appropriate mupirocin susceptibility testing interpretive criteria, 177 staphylococci were tested by agar dilution, disk diffusion, and E test. E test was found to be a reliable method for determining susceptibility of staphylococci to mupirocin. The agar dilution and disk diffusion results were plotted on a scattergram, and error rates were calculated. No errors were found with susceptibility breakpoints of > or = 4 microg/ml (MIC) and > or = 14 mm (zone diameter).     

Clinical issues associated with urine testing of substances of abuse

Several factors may affect the validity and outcome of urine testing for abused drugs such as amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine, ethanol, opiates, and phencyclidine. Urine is used for large-scale testing because acquisition of the sample is noninvasive and because most abused drugs can be detected in urine for a reasonable duration after ingestion. Urine testing for drugs of abuse is a two-step process. In the first step, screening assays are used to identify presumably positive specimens. Common screening tests are radioimmunoassays, enzyme immunoassays, fluorescence polarization immunoassay, and thin layer chromatography. Since they may be subject to cross-reactivity, once a possible positive sample has been identified by a preliminary test, a second more specific methodology, gas chromatography with mass spectrometry, is done to confirm the results. Knowledge of the pharmacology and pharmacokinetics of abused drugs affects selection and interpretation of test results.     

Automated analysis of phencyclidine in urine by probability based matching GC/MS

A novel form of selected ion recording mass spectrometry using a microcomputer-managed mass spectrometer was employed to automatically identify and quantitate phencyclidine (PCP) in cyclohexane extracts of urine by Probability Based Matching. Seventy urine samples from known abusers were assayed for PCP content. The positively identified PCP concentrations ranged from 0.01 to 10.5 mug/ml for 65 samples, 26% of which fell in the 0.35-1.0 mug/ml range and 30% in the 1.0-3.4 mug/ml range. Five specimens had no detectable PCP (less than 10 mug/ml). Cyclohexane extraction efficiency for PCP in urine exceeded 95%. Selected ion monitoring was found to be necessary in order to avoid gas chromatographic interferences produced by co-elution of contaminants at the same retention time as PCP.