Multisite Evaluation of Cepheid Xpert Carba-R Assay for Detection of Carbapenemase-Producing Organisms in Rectal Swabs.

Rapid identification of patients who are colonized with carbapenemase-producing organisms (CPO) is included in multiple national guidelines for containment of these organisms. In a multisite study, we evaluated the performance of the Cepheid Xpert Carba-R assay, a qualitative diagnostic test that was designed for the rapid detection and differentiation of the blaKPC, blaNDM, blaVIM, blaOXA-48, and blaIMP-1 genes from rectal swab specimens. A double rectal swab set was collected from 383 patients admitted at four institutions (2 in the United States, 1 in the United Kingdom, 1 in Spain). One swab was used for reference culture (MacConkey broth containing 1 mg/liter of meropenem and subcultured to a MacConkey agar plate with a 10-µg meropenem disk) and for sequencing of DNA obtained from carbapenem-nonsusceptible isolates for carbapenemase identification. The other swab was used for the Xpert Carba-R assay. In addition to the clinical rectal swabs, 250 contrived specimens (108 well-characterized CPO and 142 negative controls spiked onto negative rectal swabs) were tested. Overall, 149/633 (23.5%) samples were positive by the Xpert Carba-R assay. In 6 samples, multiple targets were detected (4 VIM/OXA-48, 1 IMP-1/NDM, and 1 NDM/KPC). The Xpert Carba-R assay detected 155 targets (26 IMP-1, 30 VIM, 27 NDM, 33 KPC, 39 OXA-48) within a time range of 32 to 48 min. The sensitivity, specificity, and positive and negative predictive values of the Xpert Carba-R assay compared to those of the reference culture and sequencing results were 96.6% (95% confidence interval [CI], 92.2% to 98.9%), 98.6% (95% CI, 97.1% to 99.4%), 95.3%, and 99.0%, respectively. The Cepheid Xpert Carba-R assay is an accurate and rapid test to identify rectal colonization with CPO, which can guide infection control programs to limit the spread of these organisms.

Quality control limits for posaconazole disk susceptibility tests on Mueller-Hinton agar with glucose and methylene blue.

A multilaboratory collaborative study was performed in order to propose quality control limits for posaconazole disk diffusion susceptibility tests on Mueller-Hinton agar supplemented with 2% glucose and 0.5 microg of methylene blue per ml. Replicate tests were performed on three lots of prepared media using 5-microg posaconazole disks in each of eight laboratories to generate data to propose quality control zone diameter ranges for tests of Candida albicans ATCC 90028 (24 to 34 mm), C. parapsilosis ATCC 22019 (25 to 36 mm), C. tropicalis ATCC 750 (23 to 33 mm), and C. krusei ATCC 6258 (23 to 31 mm).

In vitro activity of doripenem against Pseudomonas aeruginosa and Burkholderia cepacia isolates from both cystic fibrosis and non-cystic fibrosis patients.

The in vitro activities of doripenem, imipenem, levofloxacin, piperacillin, ceftazidime, aztreonam, tobramycin, and cefepime were determined for 160 isolates of Pseudomonas aeruginosa (82 from cystic fibrosis [CF] patients) and 34 isolates of Burkholderia cepacia. Doripenem MIC90s were lower than those of all other comparative agents against all isolates combined and against all P. aeruginosa isolates. Doripenem was as active as levofloxacin and 2- to 32-fold more active than the other comparative agents against B. cepacia.

Development of a standardized susceptibility test for campylobacter with quality-control ranges for ciprofloxacin, doxycycline, erythromycin, gentamicin, and meropenem.

A standardized agar dilution susceptibility testing method was developed for Campylobacter that consisted of testing on Mueller-Hinton medium supplemented with 5% defibrinated sheep blood in an atmosphere of 10% CO2, 5% O2, and 85% N2. Campylobacter jejuni ATCC 33560 was identified as a quality-control (QC) strain. Minimal inhibitory concentration (MIC) QC ranges were determined for two incubation time/temperature combinations: 36 degrees C for 48 hr and 42 degrees C for 24 hr. Quality-control ranges were determined for ciprofloxacin, doxycycline, erythromycin, gentamicin, and meropenem. For all antimicrobial agents tested at both temperatures, 95-100% of the QC MIC results fell within recommended QC ranges. Twenty-one Campylobacter clinical isolates, encompassing five species of Campylobacter (C. jejuni, C. coli, C. jejuni, subsp. doylei, C. fetus, and C. lari) were tested in conjunction with the C. jejuni QC strain. While C. jejuni and C. coli could be reliably tested under both test conditions, growth of C. jejuni subsp. doylei, C. fetus, and C. lari isolates was inconsistent when incubated at 42 degrees C. Therefore, it is recommended that these species only be tested at 36 degrees C.

Quality control limits for voriconazole disk susceptibility tests on Mueller-Hinton agar with glucose and methylene blue.

An international collaborative study was performed in order to propose quality control limits for voriconazole disk diffusion tests on Mueller-Hinton agar with 2% glucose and 0.5 micro g of methylene blue per ml. The supplement may be added to the agar before autoclaving, or Mueller-Hinton agar plates may be flooded with a glucose-methylene blue solution. Replicate tests on both types of agar plates with 1- micro g voriconazole disks generated data to propose zone size limits for tests of Candida parapsilosis ATCC 22019 (28 to 37 mm), Candida albicans ATCC 90028 (31 to 42 mm), and Candida krusei ATCC 6258 (16 to 25 mm). Candida tropicalis ATCC 750 was not useful for this purpose.

Preparation of stock solutions of macrolide and ketolide compounds for antimicrobial susceptibility tests.

Stock solutions of telithromycin, ABT-773, azithromycin, clarithromycin, erythromycin, roxithromycin and dirithromycin were each prepared with eight different combinations of solvents and diluents. Broth microdilution trays were then prepared and frozen at -60 degrees C. Standard quality control strains were evaluated periodically during a 12-week storage time. There were no significant changes in MICs with different solvents and diluents. It was concluded that the easiest approach was to dissolve each compound in water with a small volume (< 2.5 microL/mL) of glacial acetic acid added in a dropwise fashion, followed by further dilutions in deionised water.

Quality control limits for fluconazole disk susceptibility tests on Mueller-Hinton agar with glucose and methylene blue.

An international collaborative study was performed in order to propose quality control limits for fluconazole disk diffusion susceptibility tests on Mueller-Hinton agar with 2% glucose and 0.5 micro g of methylene blue per ml. The supplements may be added before autoclaving the agar, or Mueller-Hinton agar plates may be flooded with a glucose-methylene blue solution. Replicate tests on both types of agar plates generated data to propose zone size limits for tests of Candida parapsilosis ATCC 22019 (22 to 33 mm), C. tropicalis ATCC 750 (26 to 37 mm), and C. albicans ATCC 90028 (28 to 39 mm). C. krusei ATCC 6258 was not useful for this purpose.

Rapid automated antimicrobial susceptibility testing of Streptococcus pneumoniae by use of the bioMerieux VITEK 2.

The VITEK 2 is a new automated instrument for rapid organism identification and susceptibility testing. It has the capability of performing rapid susceptibility testing of Streptococcus pneumoniae with specially configured cards that contain enriched growth medium and antimicrobial agents relevant for this organism. The present study compared the results of testing of a group of 53 challenge strains of pneumococci with known resistance properties and a collection of clinical isolates examined in two study phases with a total of 402 and 416 isolates, respectively, with a prototype of the VITEK 2. Testing was conducted in three geographically separate laboratories; the challenge collection was tested by all three laboratories, and the unique clinical isolates were tested separately by the individual laboratories. The VITEK 2 results of tests with 10 antimicrobial agents were compared to the results generated by the National Committee for Clinical Laboratory Standards reference broth microdilution MIC test method. Excellent interlaboratory agreement was observed with the challenge strains. The overall agreement within a single twofold dilution of MICs defined by the VITEK 2 and reference method with the clinical isolates was 96.3%, although there were a number of off-scale MICs that could not be compared. The best agreement with the clinical isolates was achieved with ofloxacin and chloramphenicol (100%), and the lowest level of agreement among those drugs with sufficient on-scale MICs occurred with trimethoprim-sulfamethoxazole (89.7%). Overall there were 1.3% very major, 6.6% minor, and no major interpretive category errors encountered with the clinical isolates, although >80% of the minor interpretive errors involved only a single log(2) dilution difference. The mean time for generation of susceptibility results with the clinical isolates was 8.1 h. The VITEK 2 provided rapid, reliable susceptibility category determinations with both the challenge and clinical isolates examined in this study.

Detection of extended-spectrum-beta-lactamase-producing members of the family Enterobacteriaceae with Vitek ESBL test.

A three-phase analysis of the Vitek ESBL test and a double-disk (2 disk) test was performed to assess their ability to detect extended-spectrum beta-lactamases (ESBLs) in members of the family Enterobacteriaceae. In the first two phases involving detection of ESBLs in 157 stains processing well-characterized beta-lactamases, sensitivity and specificity were found to be 99.5 and 100%, respectively, for the Vitek ESBl test and 98.1 and 99.4%, respectively, for the 2-disk test. In the third phase, in which the ability of each test to detect ESBLs in 295 clinical isolates was assessed, there was only one false positive (Vitek ESBL test). Across all three phases, the Vitek ESBL test was found to be much easier to perform than the 2-disk test. The latter also involved subjective interpretation of results. There were a total of 176 Escherichia coli and 157 Klebsiella pneumoniae isolates and less than 40 isolates of each of 14 other species evaluated. In a supplemental study of Klebsiella oxytoca, an organism possessing a chromosomal beta-lactamase similar to an ESBL, the Vitek ESBL test was found to be capable of detecting hyperproduction of this enzyme in strains of this species as well. These data indicate that the Vitek ESBL test is reliable for the detection of ESBLs in E. coli and K. pneumoniae, the two species in which ESBLs are most common, and of hyperproduction of the K. oxytoca beta-lactamase, a situation which engenders a level of resistance to this species similar to that seen with ESBLs.

Facilitated detection of antibiotic-resistant Pseudomonas in cystic fibrosis sputum using homogenized specimens and antibiotic-containing media.

Sputa from 30 patients with cystic fibrosis (CF) were cultured on routine and selective media plus three Mueller-Hinton antibiotic resistance screening plates containing tobramycin (5 micrograms/ml), azlocillin (100 micrograms/ml), and ticarcillin (100 micrograms/ml). In addition to direct semiquantitative plating, samples were homogenized for semiquantitative and quantitative culture. Blood agar plates from direct semiquantitative and homogenized semiquantitative cultures were then replica plated onto the antibiotic screening plates. Homogenized semiquantitative and quantitative cultures both detected more Pseudomonas aeruginosa strains than direct semiquantitative plating (103 versus 85 strains), including more antibiotic-resistant strains. Antibiotic screening media facilitated isolation of resistant strains and decreased detection time by 24 hr. Of the 103 strains on homogenized semiquantitative and quantitative cultures isolated before replica plating, 13 (13%) were tobramycin-resistant, 67 (65%) were ticarcillin-resistant, and 42 (41%) were azlocillin-resistant; 13 of 30 cultures (43%) had at least one tobramycin-resistant organism before replica plating. Replica plating detected an additional seven tobramycin-resistant and nine ticarcillin- or azlocillin-resistant strains in seven patients. Homogenization, antibiotic screening media, and replica plating enhance recognition of antibiotic-resistant strains in CF sputum.

In vitro activity of penicillin and rifampin against group B streptococci.

Eighty-eight strains of group B streptococci were studied to determine their in vitro susceptibility to penicillin and rifampin alone and in combination. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were determined by broth microdilution; studies of synergy were performed by microdilution checkerboards and time-kill curves. The MIC90 and MBC90 for penicillin were both less than or equal to 0.06 microgram/ml. The MIC90 and MBC90 for rifampin were less than or equal to 0.25 microgram/ml and greater than 32.0 microgram/ml, respectively. Checkerboard MIC determinations revealed synergy for 46 strains (52%), an additive or indifferent effect for 42 strains (48%), and no antagonism. MBC checkerboards showed antagonism for 62 strains (70%); in 45 (73%) of these 62 strains, high concentrations of rifampin increased the MBC of penicillin, whereas low concentrations decreased the MBC of penicillin. Time-kill studies performed on selected strains supported the results of MBC checkerboards, although concentrations of antibiotics that were antagonistic in the checkerboards had an indifferent effect in time-kill experiments. When concentrations of antibiotics were used that are clinically achievable in cerebrospinal fluid, delayed killing was seen even though synergy had been observed in both MIC and MBC checkerboards.

In vitro activity of rifampin in combination with oxacillin against Staphylococcus aureus.

The in vitro activity of rifampin alone and in combination with oxacillin was determined for 75 Staphylococcus aureus strains (64 susceptible and 11 resistant to oxacillin). Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were determined by broth microdilution; antibiotic combinations were evaluated by microdilution checkerboard and time-kill studies. The 90% MIC of rifampin was less than or equal to 0.015 micrograms/ml after both 24 and 48 h of incubation. The 90% MBC of rifampin was less than or equal to 2.0 micrograms/ml on subculture at 24 h of incubation and less than or equal to 0.5 micrograms/ml on subculture at 48 h. MIC checkerboards with oxacillin-susceptible strains revealed an additive or indifferent effect in 35 strains (55%) and antagonism in 29 strains (45%). MBC checkerboards performed by subculture at 24 h demonstrated antagonism for all but one of the oxacillin-susceptible strains, with sub-MBCs of rifampin impairing the bactericidal activity of oxacillin. MBC checkerboards performed by 48-h subculture revealed antagonism with 37 strains (58%); in 26 additional strains (40%), a synergistic, additive, or indifferent effect was observed at low antibiotic concentrations, but antagonism was seen at higher concentrations. Time-kill studies tended to show indifference rather than antagonism with oxacillin plus rifampin. In checkerboards performed with oxacillin-resistant strains, the addition of rifampin did not improve oxacillin inhibitory or bactericidal activity to a clinically significant extent; however, the addition of oxacillin improved the bactericidal activity of rifampin at easily achievable serum concentrations.