Use of an oxacillin disk screening test for detection of penicillin- and ceftriaxone-resistant pneumococci

In a context of worldwide emergence of resistance among Streptococcus pneumoniae strains, early detection of strains with decreased susceptibility to beta-lactam antibiotics is important for clinicians. If the 1-microgram oxacillin disk diffusion test is used as described by the National Committee for Clinical Laboratory Standards, no interpretation is available for strains showing zone sizes of /=2.0 microgram/ml) to penicillin. For ceftriaxone, among 98 strains with no zone of inhibition in response to oxacillin, 68 had intermediate resistance (MIC, 1.0 microgram/ml), and 22 were resistant (MIC, >/=2.0 microgram/ml). To optimize the use of the disk diffusion method, we propose that the absence of a zone of inhibition around the 1-microgram oxacillin disk be regarded as an indicator of nonsusceptibility to penicillin and ceftriaxone and recommend that such strains be reported as nonsusceptible to these antimicrobial agents, pending the results of a MIC quantitation method.     

Optochin resistance inStreptococcus pneumoniae strains isolated from blood and middle ear fluid

Optochin resistantStreptococcus pneumoniae having typical pneumococcal morphology in culture and Gram stain and giving clear agglutination with antipneumococcal polysaccharide antisera were isolated from primary cultures of blood and middle ear fluid. The isolated pneumococci were either fully sensitive to penicillin and other antimicrobial agents commonly used or relatively resistant to penicillin and resistant to cloxacillin, tetracycline, erythromycin, sulphatrimethoprim and clindamycin.     

Optimal inoculation methods and quality control for the NCCLS oxacillin agar screen test for detection of oxacillin resistance in Staphylococcus aureus

To define more precisely the inoculation methods to be used in the oxacillin screen test for Staphylococcus aureus, we tested agar screen plates prepared in house with 6 microg of oxacillin/ml and 4% NaCl using the four different inoculation methods that would most likely be used by clinical laboratories. The organisms selected for testing were 19 heteroresistant mecA-producing strains and 41 non-mecA-producing strains for which oxacillin MICs were near the susceptible breakpoint. The inoculation method that was preferred by all four readers and that resulted in the best combination of sensitivity and specificity was a 1-microl loopful of a 0.5 McFarland suspension. A second objective of the study was to then use this method to inoculate plates from five different manufacturers of commercially prepared media. Although all commercial media performed with acceptable sensitivity compared to the reference lot, one of the commercial lots demonstrated a lack of specificity. Those lots of oxacillin screen medium that fail to grow heteroresistant strains can be detected by using S. aureus ATCC 43300 as a positive control in the test and by using transmitted light to carefully examine the plates for any growth. However, lack of specificity with commercial lots may be difficult to detect using any of the current quality control organisms.     

Screening for cephalosporin-resistant Streptococcus pneumoniae with the Kirby-Bauer disk susceptibility test.

Kirby-Bauer disk susceptibility tests with five standard cephalosporin disks were performed on 23 penicillin-resistant Streptococcus pneumoniae isolates for which ceftriaxone MICs were 0.125 to 4 micrograms/ml. Cefuroxime disk inhibition zone diameters distinguished clearly isolates for which ceftriaxone MICs were > or = 2 micrograms/ml from more susceptible strains, whereas cephalothin, ceftizoxime, cefotaxime, and ceftriaxone disks distinguished these isolates less clearly than the cefuroxime disk did.     

Evaluation of new Vitek 2 card and disk diffusion method for determining susceptibility of Staphylococcus aureus to oxacillin

Detection of methicillin resistance in Staphylococcus aureus is a challenge, especially low-level resistance, which is often misdiagnosed. The aim of this study was to compare the diagnostic accuracies of the automated Vitek 2 system and disk diffusion tests, using cefoxitin and moxalactam, for the detection of methicillin resistance in S. aureus strains. Four sets of genotypically diverse isolates were selected from a national reference collection, including mecA-negative S. aureus isolates (n = 56), hospital-acquired (n = 88) and community-acquired (n = 40) S. aureus isolates, and heterogeneous methicillin-resistant S. aureus isolates (n = 29). Oxacillin susceptibility was tested by the Vitek 2 system with the AST P549 card and by disk diffusion methods using 10, 30, and 60 microg cefoxitin and 30 microg moxalactam. Oxacillin resistance was confirmed by PCR for the mecA gene. The overall sensitivities for oxacillin resistance detection were 97.5% for the Vitek 2 automated system, 98.7% for 60-microg cefoxitin and moxalactam disk diffusion, and 99.6% for 10- and 30-microg cefoxitin disks, respectively. Methicillin-susceptible S. aureus isolates were correctly reported as susceptible by all methods. The median times for methicillin testing were 7 h for the Vitek 2 system versus 24 h for disk diffusion methods. In conclusion, the cefoxitin and moxalactam disk diffusion methods and the Vitek 2 automated system are highly accurate methods for methicillin resistance detection, including a range of representative Belgian methicillin-resistant S. aureus strains and unusual strains exhibiting cryptic or low-level oxacillin resistance.     

Comparison of the Vitek Gram-Positive Susceptibility 106 card and the MRSA-screen latex agglutination test for determining oxacillin resistance in clinical bloodstream isolates of Staphylococcus aureus

The Vitek automated susceptibility testing system with a modified Gram-Positive Susceptibility (GPS) 106 Card (bioMerieux Vitek, Inc., Hazelwood, Mo.) and a rapid slide latex agglutination test (MRSA-Screen; Denka Seiken Co., Ltd., Tokyo, Japan) were evaluated for their ability to detect oxacillin resistance in Staphylococcus aureus. The oxacillin-salt agar screen (OS) test, the reference broth microdilution method, and the detection of the mecA gene by PCR were compared with the commercial products. A total of 200 contemporary (1999) bloodstream infection isolates were collected from the SENTRY Antimicrobial Surveillance Program, representing diverse geographic areas throughout the world. Among the 99 mecA-positive isolates, 3 isolates were found negative by the MRSA-Screen. Another two isolates did not grow on OS plates and had MICs of 0.5 and 2 microg/ml with the Vitek GPS card. All 101 mecA-negative isolates were also found negative by the MRSA-Screen and were categorized as susceptible by the GPS card. Overall, the MRSA-Screen, GPS card, and OS test had sensitivities of 96.9, 98.0, and 98.0% and specificities of 100.0, 100.0, and 98.0%, respectively. MRSA-Screen was a rapid (相似文献   

The combined oxacillin resistance and coagulase (CORC) test for rapid identification and prediction of oxacillin resistance in Staphylococcus species directly from blood culture

The combined oxacillin resistance and coagulase (CORC) protocol for rapid identification and determination of oxacillin-susceptibility in Staphylococcus spp from blood culture is described. It incorporates a modified direct tube coagulase test (TCT) and a novel 4-hour multiplication-induction step, which increases the expression of staphylococcal PBP2a if present, facilitating detection by a commercial PBP2a latex agglutination kit. The protocol shows excellent sensitivity and specificity for determination of coagulase-positivity in staphylococci from patient blood cultures (96.8% (95% CI 81.5 to 99.8) and 100% (95% CI 75.9 to 100), respectively, n = 47), and for prediction of oxacillin resistance in S aureus directly from patient blood cultures (100% (95% CI 59.8 to 100) and 100% (95% CI 82.2 to 100), respectively (100% accuracy), n = 31) within 5 hours of blood culture positivity.     

Performance of CHROMagar selective medium and oxacillin resistance screening agar base for identifying Staphylococcus aureus and detecting methicillin resistance

Two new selective media, oxacillin resistance screening agar base (ORSAB) and CHROMagar Staph aureus (CSA), were evaluated for identification of Staphylococcus aureus and for screening of methicillin resistance by addition of antimicrobial agents to these media. A well-defined collection consisting of 1,140 staphylococci was used. A total of 624 were S. aureus, of which 358 were methicillin susceptible and 266 were methicillin resistant, and 516 were coagulase-negative staphylococci. The methicillin-resistant S. aureus (MRSA) strains were selected based on the results of phage typing; 247 different types were included in the analysis. For identification of S. aureus, both media performed better after 24 h than after 48 h. The sensitivities at 24 h were comparable (CSA, 98.6%; ORSAB, 97.1%), but the specificity of CSA was significantly higher (CSA, 97.1%; ORSAB, 92.1%). For screening of methicillin resistance, antibiotic supplements were added to both media. The sensitivity was lower after 24 h (CSA, 58.6%; ORSAB, 84.2%) and increased significantly after 48 h (CSA, 77.5%; ORSAB, 91.4%). At both time intervals ORSAB was significantly more sensitive than CSA. However, the specificities of both media were high after 24 h (CSA, 99.1%; ORSAB, 98.3%) and decreased significantly after 48 h of incubation (CSA, 94.7%; ORSAB, 95.5%). In conclusion, for identification of S. aureus, CSA is more accurate than ORSAB because of a significantly higher specificity. For screening of MRSA, ORSAB performs better than CSA, but the usefulness in clinical practice is limited because a significant number of strains are not detected.     

Accuracy of cefoxitin disk testing for characterization of oxacillin resistance mediated by penicillin-binding protein 2a in coagulase-negative staphylococci

The Clinical and Laboratory Standards Institute (CLSI) proposed, beginning in 2004, the use of cefoxitin disks to predict resistance mediated by the mecA gene in all species of coagulase-negative staphylococci (CoNS). The aim of this work was to evaluate the efficiency of the cefoxitin disk and of oxacillin-salt agar screening (MHOX) to characterize the oxacillin resistance mediated by the mecA gene in CoNS. One hundred seven CoNS isolates from different clinical samples were studied. Detection of the mecA gene by PCR was considered the "gold standard." The susceptibility to oxacillin and cefoxitin was detected by the disk diffusion and agar dilution tests, as described by the CLSI. MHOX was also performed with 6 microg/ml of oxacillin and 4% NaCl. The sensitivities of the oxacillin and cefoxitin disks for all CoNS species were 88% and 80%, respectively, whereas the specificities were 63% and 100%, respectively. The sensitivities of the agar dilution test for oxacillin and cefoxitin (for proposed breakpoints of > or =4 microg/ml for resistance and < or =2 microg/ml for susceptibility) were 90% and 85%, respectively, whereas the specificities were 76% and 98%, respectively. MHOX showed a sensitivity of 90% and a specificity of 95% for all CoNS species. Both the MHOX and the cefoxitin disk results indicate that these are appropriate methods for the evaluation of oxacillin resistance mediated by the mecA gene in all CoNS species.     

Coagulase-negative staphylococci: comparison of phenotypic and genotypic oxacillin susceptibility tests and evaluation of the agar screening test by using different concentrations of oxacillin

This study evaluated the oxacillin susceptibilities of 152 coagulase-negative staphylococcal (CoNS) strains of 12 species by disk diffusion; agar dilution; E-test; the slide latex agglutination test (Slidex MRSA Detection test; bioMérieux S/A, Paris, France); the agar screening test with 1, 2, 4, or 6 microg of oxacillin per ml and incubation for 24 or 48 h; and detection of the mecA gene by PCR. The results revealed that the agar screening test with 4 micro g of oxacillin per ml and incubation for 48 h was superior to any single phenotype-based susceptibility assay, presenting a sensitivity and a specificity of 100% each. For the different methods evaluated, the sensitivities and specificities were as follows: for disk diffusion, 94.2 and 91.8%, respectively; for the agar dilution test 100 and 73.5%, respectively; for E-test, 100 and 71.4%, respectively; and for the slide latex agglutination test, 97.1 and 98%, respectively. A good correlation was observed between oxacillin susceptibility testing results and PCR results for Staphylococcus epidermidis, S. haemolyticus, S. hominis subsp. hominis, and all mecA-positive strains. However, at least 60% of the mecA-negative isolates of the species S. saprophyticus, S. cohnii subsp. urealyticum, S. lugdunensis, and S. sciuri were erroneously classified as oxacillin resistant by the agar dilution test. Conversely, the slide latex agglutination test presented a high sensitivity (97.1%) and a high specificity (98%) for all CoNS species. Our results demonstrated the accuracy of the agar screening test with 4 micro g of oxacillin per ml and incubation for 48 h and the slide latex agglutination test for the appropriate detection of the oxacillin susceptibilities of CoNS isolates. Both assays are technically simple and can be easier to perform in routine laboratories than PCR.     

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.     

Spurious oxacillin resistance in Staphylococcus aureus because of defective oxacillin disks.

Clinical isolates of Staphylococcus aureus wee inappropriately categorized as intermediate or resistant to oxacillin on the basis of tests with two lots of oxacillin disks. The potency of one lot was tested and found to be below accepted limits. Routine quality control tests failed to detect the defective disks.     

Performance of a New MicroScan WalkAway PC30 panel and disk diffusion method for detection of oxacillin resistance in Staphylococcus spp

The performance of the MicroScan WalkAway PC30 panel for detection of oxacillin resistance was evaluated by use of a collection of 420 staphylococcus isolates. The addition of a cefoxitin test (4 mg/liter) to the oxacillin MIC determination increased its raw performance for Staphylococcus aureus; additional data were required for coagulase-negative staphylococci.     

Influence of disk separation distance on accuracy of the disk approximation test for detection of inducible clindamycin resistance in Staphylococcus spp

We undertook this study to assess the accuracy of the clindamycin-erythromycin disk approximation test (D-test) for detection of inducible clindamycin resistance in Staphylococcus spp. One hundred sixty-three Staphylococcus aureus and 68 coagulase-negative Staphylococcus (CoNS) spp. which were erythromycin nonsusceptible but clindamycin susceptible were tested using the D-test performed at both 15-mm and 22-mm disk separations and compared with genotyping as the "gold standard." The rate of inducible clindamycin resistance was 96.3% for S. aureus and 33.8% for CoNS spp. The sensitivities of the D-tests performed at 15 mm and 22 mm were 100% and 87.7%, respectively, and specificities were 100% for both. The use of 22-mm disk separation for the D-test to detect inducible clindamycin resistance results in an unacceptably high very major error rate (12.3%). All isolates with false-negative results harbored the ermA gene, and the majority were methicillin-resistant Staphylococcus aureus. False-negative results were associated with smaller clindamycin zone sizes and double-edged zones. We recommend using a disk separation distance of 相似文献   

Evaluation of disk approximation and single-well broth tests for detection of inducible clindamycin resistance in Streptococcus pneumoniae

This study evaluated an agar disk diffusion D-zone test and an erythromycin-clindamycin (ERY + CLI) single-well broth test for inducible CLI resistance in Streptococcus pneumoniae. The standard CLSI disk approximation test and a single-well combination test incorporating 1 plus 0.5 μg/ml ERY + CLI detected >96% of isolates containing the ermB determinant.     

Inclusion fluorescent-antibody test as a screening assay for detection of antibodies to Chlamydia pneumoniae

A study was conducted to determine the ability of the inclusion immunofluorescence assay (inclusion IFA) to act as a screening test to detect samples with antibodies to Chlamydia pneumoniae; microimmunofluorescence (MIF) was used as the "gold standard." In addition, the inclusion IFA was compared using HEp-2 cells infected with either C. pneumoniae CM-1 or Chlamydia trachomatis serovar E. A total of 331 serum samples representing a range of MIF titers were evaluated. The sensitivities of the inclusion IFA for detecting samples with C. pneumoniae MIF titers of > or = 16 were 96.9 and 74.8% with C. pneumoniae- and C. trachomatis-infected cells, respectively. For samples with an elevated C. pneumoniae MIF titer of > or = 512, the sensitivities of the C. pneumoniae- and C. trachomatis-based inclusion IFA were 97.0 and 8.8%, respectively. These results suggest that the inclusion IFA is not a genus-specific test, as evidenced by the failure of the C. trachomatis-infected cells to detect a significant number of samples with C. pneumoniae antibodies. Samples that had elevated C. pneumoniae inclusion IFA and MIF titers but that were found negative (titer, <16) by the C. trachomatis inclusion IFA were further tested by an in vitro neutralization assay for functional antibodies that might not have been detected by the serological assays. The in vitro neutralization results corroborated the serological results in that all seven sera tested had a neutralization titer for C. pneumoniae (range, 20 to 225), while all but one failed to have any effect on the infectivity of C. trachomatis serovar E. While the C. pneumoniae inclusion IFA had a high sensitivity for detecting chlamydial antibodies, depending on whether it was used as a screening test for detecting samples with low (> or = 16) or elevated (> or = 512) MIF titers, its specificity ranged from 53.4 to 77.1%. In conclusion, the inclusion IFA with C. pneumoniae-infected cells was best suited as a sensitive screening test for identifying specimens with elevated MIF titers (those associated with a possible acute infection with C. pneumoniae).