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Disk Diffusion Susceptibility Testing Following is the text of a brochure (B-88:
Neisseria gonorrhoeae Reference Strains for Antimicrobial
Susceptibility Testing
[Requires Adobe Acrobat or Adobe Reader]) provided to laboratories susceptibility
testing of Neisseria gonorrhoeae by either the disk diffusion or
Etest procedures. Testing is performed according to Clinical and
Laboratory Standards Institute (CLSI)*-recommended procedures.
Laboratories performing susceptibility testing by non-CLSI procedures
should use quality control and interpretive criteria appropriate for the
specific method used. Protocol
NEISSERIA GONORRHOEAE REFERENCE STRAINS FOR ANTIMICROBIAL SUSCEPTIBILITY TESTING Reference strains listed in this brochure are quality control (QC)
strains for the agar-dilution, disk diffusion, and Etest susceptibility
procedures used to determine the antimicrobial susceptibilities of
isolates of Neisseria gonorrhoeae. These strains may be used to
assess the quality of susceptibilities of N. gonorrhoeae isolates
to penicillin, tetracycline, spectinomycin, ceftriaxone, cefixime,
ciprofloxacin, ofloxacin, levofloxacin, and azithromycin. It is intended
that these strains be used in conjunction with strain ATCC 49226 as
recommended by the Clinical and Laboratory Standards Institute (CLSI)* for
QC of susceptibility testing of gonococcal isolates (1,
2). ANTIMICROBIAL RESISTANCE IN N. GONORRHOEAE N. gonorrhoeae usually develops resistance to antimicrobial agents within a few years of their introduction for gonorrhea therapy. Antimicrobial resistance in N. gonorrhoeae occurs as chromosomally mediated resistance to a variety of antimicrobial agents, including penicillin, tetracycline, spectinomycin, and fluoroquinolones (3, 4), and high-level, plasmid-mediated resistance to penicillin and tetracycline (3). Sentinel surveillance to monitor antimicrobial resistance in N. gonorrhoeae in the United States is performed by the Gonococcal Isolate Surveillance Project (GISP), sponsored by the Centers for Disease Control and Prevention (CDC) in collaboration with local health departments. In GISP, antimicrobial susceptibilities of N. gonorrhoeae isolated from men in approximately 26 cities in the United States are determined (5); annual reports for GISP may be obtained electronically from the internet (6). Because of the spread of gonococcal isolates resistant to penicillin and tetracycline, CDC has recommended that extended-spectrum cephalosporins (ceftriaxone, cefixime) and fluoroquinolones (ciprofloxacin, ofloxacin, levofloxacin) be used as primary therapies to treat uncomplicated gonorrhea (7). At this time, no failures of gonococcal infections to respond to cephalosporins have been confirmed. Failures of gonococcal infections to respond to fluoroquinolones (ciprofloxacin, ofloxacin) have been reported. Fluoroquinolone-resistant isolates are very prevalent in Asia and have been isolated with increasing frequency in the United States. In response to this increase, many states have revised recommendations for the treatment of gonorrhea (8). Agar dilution susceptibility testing is the "gold standard" for susceptibility testing of N. gonorrhoeae. However, when performed correctly, the disk-diffusion and Etest susceptibility tests can be used to identify isolates of N. gonorrhoeae that exhibit decreased susceptibility, intermediate resistance, and resistance to antimicrobial agents. This brochure contains current CLSI-recommended procedures and interpretive criteria for agents used for the treatment of uncomplicated gonorrhea and for routine surveillance of antimicrobial resistance in N. gonorrhoeae (1, 2). The CLSI may revise interpretive criteria contained in this brochure. Consumers in the United States are advised always to consult current CLSI publications and to use the recommended methods and interpretive criteria contained in current CLSI documents if they differ from those in this brochure to ensure compliance with the requirements of the Clinical Laboratory Improvement Amendments (CLIA). Consumers are also advised that results obtained with procedures, media, etc. other than those described in CLSI documents may not give results comparable to those obtained with the CLSI-recommended procedures for QC isolate ATCC 49226 or for CDC-recommended QC strains. If results do not agree with those in CLSI documents (1, 2), it is not appropriate to interpret such results using CLSI-recommended interpretive criteria. In the disk-diffusion susceptibility test, disks containing known amounts of an antimicrobial agent are placed on the surface of an agar plate containing a nonselective medium that has been inoculated with a suspension of a strain of N. gonorrhoeae to give a confluent lawn of growth. The antimicrobial agent diffuses into the medium, causing a zone of inhibition of growth of the strain around the disk corresponding to the susceptibility of the strain to the agent. Interpretative inhibition zone diameters have been established for susceptibility test results to permit classification of an isolate as being susceptible, intermediate (or exhibiting decreased susceptibility), or resistant to an antimicrobial agent (Table 1; 1, 2). Note: The Etest is performed using a procedure similar to that described in this brochure. The Etest is a strip containing a known gradient of an antimicrobial agent and calibrated to give results as minimal inhibitory concentrations (MICs) of the agents. Etest strips are placed on the surface of a 150-mm plate (containing a nonselective medium inoculated as in the disk diffusion method) in a radial pattern with the lowest concentration of the agent toward the center of the plate and the highest concentration of the agent toward the edge of the plate). If MICs for ATCC 49226 correspond with CLSI-designated QC values, MICs of clinical isolates may be interpreted according to CLSI interpretive criteria. Consult Etest product literature for complete instructions. (See also: http://www.abbiodisk.com and http://www.srga.org/RAFMETOD/BASMET.HTM for additional helpful information.) Gonococcal antimicrobial susceptibility reference strains include strain ATCC 49226, which is the CLSI-designated QC strain for antimicrobial susceptibility testing of N. gonorrhoeae. Additional QC strains are used at CDC to control for resistance to penicillin, tetracycline, and spectinomycin; intermediate resistance and resistance to ciprofloxacin and ofloxacin; high MICs of azithromycin and levofloxacin (MICs ≥1.0 µg/ml); and decreased susceptibility to cefixime. It is intended that these strains be used in conjunction with the CLSI-recommended strain ATCC 49226 when determining the susceptibilities of gonococcal isolates (1, 2). Strains have been grown in large volumes, dispensed in 0.5-ml volumes, lyophilized, and stored at 4 C. The strains exhibit the MICs and zone inhibition diameters or modal MICs shown in Table 2a and Table 2b. RECOVERY, MAINTENANCE, AND STORAGE OF REFERENCE STRAINS
CLSI recommends a range of values for the susceptibility of N. gonorrhoeae strain ATCC 49226 to a variety of antimicrobial agents (1). The values given for ATCC 49226 for azithromycin and for all antimicrobial agents for other reference strains in this protocol were obtained when strains were tested on GC II agar base supplemented with 1% IsoVitaleX (Table 2a and Table 2b) and determined in the Neisseria Reference Laboratory (NRL) at CDC and at five GISP regional laboratories located at Emory University, Atlanta, Georgia; University of Washington, Seattle, Washington; University of Alabama, Birmingham, Alabama; University of Colorado, Denver, Colorado; The Cleveland Clinic Foundation, Cleveland, Ohio (5). Values may vary slightly when strains are tested on other media. If the results for reference strain ATCC 49226 meet those recommended by NCCLS and those listed in Table 2a or Table 2b for additional QC strains, the results for the test strains may be interpreted according to the criteria listed in Table 1. The MICs and inhibition zone sizes given in this protocol for ATCC 49226 are recommended by CLSI, MIC results for additional QC isolates are derived from testing performed at the NRL, CDC and in the GISP regional laboratories, and disk diffusion inhibition zone diameters were determined at the NRL, CDC; all values were determined on GC II agar base medium supplemented with 1% IsoVitaleX. These values are provided as a general guideline for the interpretation of disk-diffusion and Etest susceptibility results. The MICs and zone inhibition diameters may vary depending on many factors, including medium base, humidity, and age of the medium. At this time, the NCCLS has not published criteria for interpretation of susceptibilities of N. gonorrhoeae isolates to levofloxacin or azithromycin (1, 2, 9). Levofloxacin (250 mg, single dose, oral) is recommended by CDC for the treatment of uncomplicated gonorrhea in the United States (7). However, based on the pharmacokinetics of levofloxacin, it is estimated that gonococcal isolates with MICs ≥1.0 µg/ml of levofloxacin may be resistant to the CDC-recommended dose of this agent. It is recommended that, until CLSI establishes criteria for the interpretation of susceptibilities of gonococcal isolates to levofloxacin, that laboratories determine susceptibilities to either ciprofloxacin or ofloxacin and that isolates determined to be intermediate or resistant to either ciprofloxacin or ofloxacin be considered to exhibit corresponding levels of resistance to levofloxacin. Although CDC does not recommend azithromycin (2 g, single dose, oral) for the treatment of gonorrhea (8), this dose may be used to treat gonorrhea in areas of the United States where cefixime is no longer available and where fluoroquinolones can no longer be used because of the increase in prevalence of fluoroquinolone-resistance gonococcal strains. Currently, only limited data are available for assessing the clinical outcome of treatment of gonococcal infections with a 2g dose of azithromycin (10). More extensive assessments of treatment outcome associated with this regimen are required. Until interpretive criteria for susceptibilities of gonococcal isolates to these agents are available, the NRL at CDC recommends that MICs ≥1.0 µg/ml of levofloxacin or azithromycin be considered critical MICs which require additional evaluation. When gonococcal infections have been treated with the CDC-recommended dose of levofloxacin (250 mg, single dose, oral) or with azithromycin (2 g, single dose, oral), the isolation of gonococcal isolates with critical MICs of ≥1.0 µg/ml of levofloxacin or azithromycin, respectively, from such infections should prompt further investigation to determine that such infections have been treated successfully. It should be noted that the critical MIC of ≥1.0 µg/ml of azithromycin for the 2 g dose is not valid when evaluating susceptibilities of gonococcal isolates from infections treated with 1 g of azithromycin (which is not recommended by CDC for the treatment of uncomplicated gonorrhea but is recommended for the treatment of Chlamydia trachomatis infections). Evaluation of clinical treatment outcomes has indicated that gonococcal infections caused by strains with MICs of ≥0.125 µg/ml may fail to respond to treatment with the 1-g dose of azithromycin (11). RECOMMENDED TESTING AND CONFIRMATORY TESTING It is recommended that antimicrobial susceptibility testing be performed against agents that are being used as primary therapies against uncomplicated gonorrhea and against agents that would be used as alternative therapies should the primary agent(s) become ineffective for treating uncomplicated gonorrhea. In the United States, reports of apparent failures of infections to respond to treatment with CDC recommended therapies should be reported to the GISP coordinator (gispinfo@cdc.gov; 404-639-8373). Epidemiology and Surveillance Branch, Division of STD Prevention, Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Mailstop E02, Atlanta, GA 30333. In the United States, it is also recommended that isolates from certain infections be submitted to the NRL at CDC for confirmation; these infections comprise those that fail to respond to therapy with CDC-recommended therapies and isolates determined to exhibit intermediate resistance or resistance to fluoroquinolones (MIC >0.06 µg/ml or inhibition zone diameters ≤40 mm of ciprofloxacin; MIC >0.25 µg/ml or inhibition zone diameters ≤30 mm of ofloxacin) or decreased susceptibility to cefixime (MIC >0.25 µg/ml; inhibition zone diameter ≤35 mm) or isolates with MICs ≥1.0 µg/ml (or inhibition zone diameters ≤30 mm) of azithromycin (In the United States, contact David Trees, Ph.D. (DTrees@cdc.gov; 404-639- 2134) or Joan S. Knapp, Ph.D. (JKnapp@cdc.gov; 404-639-3470), Neisseria Reference Laboratory, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop C13, Bldg 1 South, Room B260, Atlanta, Georgia 30333). In addition, please notify the GISP coordinator at the above address if such isolates are sent to CDC. Outside the United States, it is recommended that laboratories contact their local reference laboratory to ascertain criteria for interpreting antimicrobial susceptibilities and criteria for submitting isolates for confirmation of antimicrobial resistance.
*Information on the most recent CLSI publications is available at: http://www.clsi.org/ (select "Shop" menu followed by "Microbiology" menu) or by contacting CLSI. CLSI, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898. Phone: 610 688 0100; fax: 610 688 0700; e-mail: exoffice@nccls.org . Note that performance standards are published each year. Table 1. Critical zone inhibi2aion diameters (mm) and MICs (µg/ml) for the interpretation of susceptibilities of strains of Neisseria gonorrhoeae to selected antimicrobial agents. (Criteria for all agents except levofloxacin and azithromycin are those recommended by CLSI; criteria for interpretation of susceptibilities are those recommended by the NRL.)
Abbreviations: MIC, minimal inhibitory concentration; R, resistant; I, intermediate; S, susceptible. *Criteria for intermediate and resistant not available; strains have not been confirmed to cause clinical treatment failure. However, four isolates with MICs exhibiting decreased susceptibility to cefixime (MIC, range 0.25 - 1.0 µg/ml; modal MIC, 0.5 µg/ml) were identified among gonococcal isolates from Hawaii in 2001 (12). **Preliminary MIC data suggest that isolates exhibiting zone inhibition diameters of ciprofloxacin of 35 to 41 mm are susceptible to ciprofloxacin (MIC ≤0.06 µg/ml). It is recommended that susceptibilities of isolates exhibiting zone inhibition diameters of 35 to 41 mm be confirmed by agar dilution susceptibility testing before isolates are categorized as being intermediate to ciprofloxacin. ***In the absence of CLSI-established criteria, the NRL recommends the use of critical MICs ≥1.0 µg/ml to interpret susceptibilities of gonococcal isolates to these agents until more extensive assessments of clinical treatment outcome to these agents are available (see Special Notes). When gonococcal infections have been treated with the CDC-recommended dose of levofloxacin (250 mg, single dose, oral) or with azithromycin (2 g, single dose, oral), the isolation of gonococcal isolates with critical MICs of ≥1.0 µg/ml of levofloxacin or azithromycin, respectively, from such infections should prompt further investigation to determine that such infections have been treated successfully. The NRL would appreciate receiving isolates with MICs of ≥1.0 µg/ml of levofloxacin or azithromycin, or zone inhibition diameters of ≤30 mm of azithromycin for confirmation of susceptibilities. Table 2a. Ranges of zone inhibition diameters (mm)* and equivalent MIC (µg/ml) range for reference strains of Neisseria gonorrhoeae
Abbreviations: MIC, minimal inhibitory concentration (µg/ml) ; SpcR, spectinomycin resistant (MIC >128.0 µg/ml); PP-TR, penicillinase-producing with TetMconjugative plasmid; CipI, intermediate to ciprofloxacin (MIC 0.125-0.5 µg/ml); CipR, ciprofloxacin-resistant (MIC ≥1.0 µg/ml); AznC, isolates exhibiting MICs of ≥1.0 µg/ml of azithromycin (see Special Notes in text); CfxDS, decreased susceptibility to cefixime (MIC >0.25 µg/ml of cefixime); [ND], not determined; +, ß-lactamase positive; -, ß-lactamase negative. a ATCC 49226 is the NCCLS-recommended quality control strain.
MICs and inhibition zone diameters for ATCC 49226 are those recommended by
the NCCLS except for MICs for azithromycin which were derived from
interlaboratory testing by six laboratories. Table 2b. Quality control values for antimicrobial susceptibilities of reference strains of Neisseria gonorrhoeae determined by agar dilution or Etest
Abbreviations: MIC, minimal inhibitory concentration (µg/ml) ; SpcR, spectinomycin resistant (MIC >128.0 µg/ml); PP-TR, penicillinase-producing with TetMconjugative plasmid; CipI, intermediate to ciprofloxacin (MIC 0.125-0.5 µg/ml); CipR, resistant to ciprofloxacin (MIC ≥1.0 µg/ml); AznC, isolates with MICs of 1.0 µg/ml of azithromycin (see Footnote c); CfxDS, decreased susceptibility to cefixime (MIC >0.25 µg/ml of cefixime); [ND], not determined; +, ß-lactamase positive; -, ß-lactamase negative. a ATCC 49226 is the NCCLS-recommended
quality control strain. MICs for ATCC 49226 are those recommended by
the NCCLS; MICs of
azithromycin were determined by interlaboratory testing by six
laboratories in the United States.
Preparation of McFarland Turbidity Standard An opacity standard is prepared by mixing barium chloride (BaCl2) and sulfuric acid (H2SO4). The resultant precipitate, barium sulfate (BaSO4), is very insoluble. A new standard should be prepared every six months.
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