Antimicrobial Susceptibility Testing Subcommittee Research Articles

Revision of fluoroquinolone breakpoints used for interpretation of antimicrobial susceptibility testing of canine bacterial isolates.

The fluoroquinolone antimicrobial agents, enrofloxacin and marbofloxacin, were US Food and Drug Administration (FDA) approved in the United States for use in dogs in 1988 and 1999, respectively. There have been many advances since then concerning the pharmacokinetic-pharmacodynamic (PK-PD) evaluation of fluoroquinolones, and there are data available on the susceptibility of targeted pathogens since the original approval. Using this information, the Clinical and Laboratory Standards Institute (CLSI) Veterinary Antimicrobial Susceptibility Testing Subcommittee (VAST) revised its antimicrobial susceptibility testing breakpoints. The previous breakpoints (used in older editions of CLSI standards) for enrofloxacin in dogs were susceptible (S), ≤ 0.5 µg/mL, intermediate (I) 1-2 µg/mL, and resistant (R) ≥ 4 µg/mL. The new breakpoints are S ≤ 0.06 µg/mL for a dose of 5 mg/kg, 0.12 µg/mL for a dose of 10 mg/kg, 0.25 µg/mL for a high dose of 20 mg/kg, and R ≥ 0.5 µg/mL. The breakpoints of 0.12 and 0.25 µg/mL represent a new susceptible-dose dependent (SDD) category. For marbofloxacin, previous breakpoints were S, ≤ 1 µg/mL, I 2 µg/mL, and R ≥ 4 µg/mL. The new breakpoints are S ≤ 0.12 µg/mL for a dose of 2.8 mg/kg, 0.25 µg/mL for a dose of 5.5 mg/kg (SDD), and R ≥ 0.5 µg/mL. The new breakpoints will be published in the next edition of CLSI-Vet01(S) and deviate considerably from the prior breakpoints. Laboratories are encouraged to revise their testing standards. These changes will likely reduce the unnecessary use of these fluoroquinolones in dogs.

The Clinical and Laboratory Standards Institute Subcommittee on Antimicrobial Susceptibility Testing: Background, Organization, Functions, and Processes

The Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing (AST SC) is a volunteer-led, multidisciplinary consensus body that develops and publishes standards and guidelines (among other products) for antimicrobial susceptibility testing (AST) methods and results interpretation in the United States and internationally. The Subcommittee (SC) meets face-to-face twice yearly, and its working groups (WGs) are active throughout the year via teleconferences. All meetings are open to the public. Participants include clinical microbiologists, infectious disease (ID) pharmacists, and infectious disease physicians representing the health care professions, government, and industry. Individuals who work for a company with a primary financial dependency on drug sales cannot serve as voting members, and well-defined conflict of interest polices are in place. In addition to developing and updating susceptibility breakpoints, the SC develops and validates new testing methods, provides guidance on how results should be interpreted and applied, sets quality control ranges, and educates users through seminars, symposia, and webinars. Based on its work, the SC publishes print and electronic standards and guidelines, including an annual update, the Performance Standards for Antimicrobial Susceptibility Testing (M100). This commentary will describe the background, organization, functions, and operational processes of the AST SC.

EUCAST Technical Note on Candida and micafungin, anidulafungin and fluconazole

The European Committee on Antimicrobial Susceptibility Testing Subcommittee on Antifungal Susceptibility Testing has determined breakpoints for micafungin and revised breakpoints for anidulafungin and fluconazole for Candida spp. This Technical Note is based on the corresponding rationale documents (http://www.eucast.org). The micafungin breakpoints are based on PK data, animal PK/PD data, microbiological data and clinical experience. The anidulafungin breakpoints for C. parapsilosis and fluconazole breakpoints for C. glabrata have been modified to species-specific values that categorise the wild-type as intermediate to accommodate use of these compounds in some clinical situations.

EUCAST Technical Note on Voriconazole and Aspergillus spp.

The European Committee on Antimicrobial Susceptibility Testing Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has determined breakpoints for voriconazole against Aspergillus spp. This Technical Note is based on the EUCAST rationale document for voriconazole (available on the EUCAST website: http://www.eucast.org). Voriconazole breakpoints are based on epidemiological cut-off values, pharmacokinetic/pharmacodynamic data and clinical experience. Breakpoints will be reviewed regularly or when new data emerge.

EUCAST technical note on the EUCAST definitive document EDef 7.2: method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST-AFST)

The European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has revised the EDef 7.1 document on the method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for fermentative yeasts. Changes are: dimethylsulphoxide is now the recommended solvent for caspofungin, micafungin and fluconazole; the shelf-life of plates containing the echinocandins prepared from stock solutions in dimethylsulphoxide is extended to 6 months at -80°C; testing of amphotericin and Cryptococcus has been incorporated; and minimum inhibitory concentration ranges for quality control strains and anidulafungin are included.

EUCAST Technical Note on Aspergillus and amphotericin B, itraconazole, and posaconazole

The European Committee on Antimicrobial Susceptibility Testing Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has determined breakpoints for amphotericin B, itraconazole and posaconazole for Aspergillus species. This Technical Note is based on the EUCAST amphotericin B, itraconazole and posaconazole rationale documents (available on the EUCAST website: http://www.eucast.org/antifungal_susceptibility_testing_afst/rationale_documents_for_antifungals/). The amphotericin B and itraconazole breakpoints are based on epidemiological cut-off values and clinical experience. The posaconazole breakpoints are also based on pharmacokinetic and pharmacodynamic data. Breakpoints will be reviewed regularly or when new data emerge.

EUCAST Technical note on Amphotericin B

The European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has determined breakpoints for amphotericin B for Candida spp. This Technical Note is based on the EUCAST amphotericin B rationale document (available on the EUCAST website: http://www.eucast.org). Species-specific breakpoints for C. albicans, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis are S: MIC ≤1 mg/L, R: MIC > 1 mg/L. There are insufficient data to set breakpoints for other species. The breakpoints are based upon pharmacokinetic data, epidemiological cut-ff values and clinical experience. Breakpoints will be reviewed regularly.

EUCAST technical note on posaconazole

The European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has determined breakpoints for posaconazole for Candida spp. This Technical Note is based on the EUCAST posaconazole rationale document (available on the EUCAST website: http://www.eucast.org). Species-specific breakpoints for C. albicans, C. parapsilosis and C. tropicalis are S: MIC ≤0.06 mg/L, R: MIC >0.06 mg/L. There are insufficient data to set breakpoints for C. glabrata and C. krusei as well as non-species-related breakpoints. The breakpoints are based upon pharmacokinetic data, epidemiological cut-off values and clinical experience. Breakpoints will be reviewed regularly.

EUCAST technical note on anidulafungin

The European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing has determined breakpoints for anidulafungin for Candida spp. This Technical Note is based on the EUCAST anidulafungin rationale document (Available at: http://www.eucast.org). Species-specific breakpoints for C. albicans are S ≤0.03 mg/L and R >0.03 mg/L and for C. glabrata, C. tropicalis and C. krusei S ≤0.06 mg/L and R >0.06 mg/L. C. parapsilosis was not regarded a good target for anidulafungin. There are insufficient data to set breakpoints for other species. The breakpoints are based upon pharmacokinetic data, epidemiological cut-off values and clinical experience. Breakpoints will be reviewed regularly.

EUCAST Definitive Document EDef 7.1: method for the determination of broth dilution MICs of antifungal agents for fermentative yeasts: Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST)∗

INTRODUCTIONAntifungal susceptibility tests are performed onfungi that cause disease, especially if they belongto a species exhibiting resistance to commonlyused antifungal agents. Antifungal susceptibilitytesting is also important for resistance surveil-lance, for epidemiological studies and forcomparing the in-vitro activity of new and exist-ing agents.Dilution methods are used to establish theMICs of antimicrobial agents. These are thereference methods for antimicrobial susceptibilitytesting, and are used mainly to establish theactivity of a new antifungal agent, to confirm thesusceptibility of organisms that give equivocalresults in routine tests, and to determine thesusceptibility of fungi where routine dilution testsmay be unreliable. Fungi are tested for theirability to produce visible growth in microdilutionplate wells containing broth culture media andserial dilutions of the antifungal agents (brothmicrodilution). The MIC is defined as the lowestconcentration (in mg⁄L) of an antifungal agentthat inhibits the growth of a fungus. The MICprovides information concerning the susceptibil-ity or resistance of an organism to the antifungalagent and can help in making correct treatmentdecisions.The method described in this document isintended for testing the susceptibility ofyeasts that cause clinically significant infections(primarily Candida spp.). The method encom-passes only those yeasts that are able to fermentglucose. Thus, the susceptibility of non-fermenta-tive yeasts, e.g., Cryptococcus neoformans, cannotbe determined by the current procedure, and themethod is not suitable for testing the yeast formsof dimorphic fungi.SCOPEThe standard method described in this documentprovides a valid method for testing the suscepti-bility of glucose-fermenting yeasts to antifungalagents by determination of the MIC. MICs indi-cate the activity of a given antifungal drug underthe described test conditions, and can be used inmaking decisions concerning patient manage-ment after taking into account other factors, e.g.,pharmacokinetics, pharmacodynamics and resis-tance mechanisms. The MIC also allows fungi tobe categorised as ‘susceptible’ (S), ‘intermediate’(I) or ‘resistant’ (R) to a drug. In addition, MICdistributions can be used to define wild-type ornon-wild-type fungal populations.This method is intended primarily to facilitatean acceptable degree of conformity, i.e., agree-ment within specified ranges among laboratories,in measuring the susceptibility of yeasts to anti-fungal agents. The method is designed to be easyto perform, rapid, economical, and suitable forreading by microdilution plate readers in order toallow direct transfer, storage and manipulation ofthe data by computer. The method is alsointended to yield results that are concordantwith those obtained using the procedure

FDA and the Antibiotic Breakpoint Updating Process

We read with interest the Current Topics article prepared by Jeffrey L. Fox, “FDA reasserts authority over antibiotic breakpoint updating process,” in the May 2007 issue of Microbe (p. 222). We were disappointed that such an important article did not present a more balanced view of all stakeholders, including interviews with additional clinical microbiologists or clinicians including those specializing in infectious diseases. However, Fox accurately points out the key regulatory requirements of the Food and Drug Administration (FDA) and the patient safety benefits of regular updates to breakpoints in the face of emerging bacterial resistance. FDA and Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) share common interests in establishing breakpoints for interpretation of antimicrobial susceptibility tests. CLSI convened a special meeting in January, which included representatives from FDA, CDC, large and small pharmaceutical companies, diagnostic devices companies, clinical microbiologists, and infectious diseases clinicians from the United States and abroad, to discuss how CLSI and FDA can best work together on the issue of antibiotic susceptibility test interpretive breakpoints. The group recommended that CLSI publish the initial FDA breakpoints of newly approved antimicrobial agents following a brief presentation to CLSI by the drug's sponsor. If the CLSI Subcommittee on Antimicrobial Susceptibility Testing (AST) does not agree with the FDA breakpoints, the drug will not be included in the CLSI susceptibility testing publications. Further, CLSI would not propose any alterations of the FDA breakpoints for at least two years following approval of the official drug label. If, after that time, emerging resistance or other issues arise that suggest the need to revise the breakpoints, the subcommittee would review all relevant data to determine if a change would be warranted. These recommendations have been endorsed by CLSI's AST Subcommittee, Area Committee on Microbiology, and Board of Directors. This new approach should assuage the concerns of pharmaceutical companies that the FDA and CLSI breakpoints might differ for their new drugs. It should also better serve clinical microbiology laboratories and diagnostic device manufacturers because there will not be conflicting breakpoint recommendations for new drugs in the United States.

Antibiotic susceptibility testing of bacteria isolated from animals

The responsible use of veterinary antimicrobial agents requires that the most appropriate antibiotic be selected for use in the treatment of animal diseases. The American Veterinary Medical Association has developed judicious-use guidelines for dogs, cats, horses, cattle, swine, poultry, and fish so that the veterinarian can maintain product effectiveness, use antimicrobials safely, and minimize selection of resistant pathogens ( http://www.avma.org/scienact/jtua/default.asp ). One recommendation is that susceptibility testing be performed on bacterial pathogens isolated from animals to guide the selection of the appropriate antimicrobial agent. The key role of the veterinary diagnostic laboratory is predicated on the capability to conduct standardized antimicrobial susceptibility testing and, using validated interpretive criteria, to guide the veterinary practitioner in the selection of the most appropriate antibiotic. The Clinical and Laboratory Standards Institute (formerly NCCLS) Veterinary Antimicrobial Susceptibility Testing subcommittee has developed these laboratory methods and interpretive criteria.

Updates from the NCCLS microbiology area committee

To many microbiologists, the NCCLS represents the main source of up-to-date information regarding antimicrobial susceptibility testing. Indeed, the Antimicrobial Susceptibility Testing Subcommittee is the largest subcommittee of the NCCLS and produces the most widely distributed publications of the NCCLS. However, the Microbiology Area Committee oversees the activities of 11 other important microbiology subcommittees that focus on a diverse array of topics pertinent to clinical microbiologists. The purpose of this article is to highlight the recent activities of all 12 subcommittees and to describe the publications emanating from each group. All NCCLS microbiology documents are designated by a ‘M’ and the number of the publication itself,.as will become evident below. Prior to an in-depth review of the NCCLS’ activities in microbiology, it is important to describe briefly the history and objectives of the NCCLS. The National Committee for Clinical Laboratory Standards was founded in 1968 with the purpose of developing voluntary consensus standards for clinical laboratory testing in the United States. The organization is based upon an equal partnership of clinical laboratory professionals, federal and state government agencies, and the pharmaceutical and diagnostic products industry. It provides a mechanism for bringing together the necessary expertise to develop and publish authoritative and practical consensus documents and to establish specifications for reference materials that deal with all areas of patient testing, not just microbiology. The Area Committees of the NCCLS include Alternate Site Testing, Laboratory Automation, Clinical Chemistry and Toxicology, Evaluation Protocols, General Laboratory Practices, Hematology, Immunology and Ligand Assay, Molecular Methods, and Microbiology. The current members of the Microbiology Area Committee are listed in Table 1. In the pursuit of development of voluntary consensus, the NCCLS assembles experts from the three constituencies described above into area committees, subcommittees, and working groups that develop patient care standards in an open, public forum. The resulting NCCLS publications are widely promulgated, and specific published standards have been adopted as the American National Standards by the American National Standards Institute. However, the strength of the NCCLS consensus process has led to adoption and use of NCCLS guidelines and standards in a variety of other countries. Indeed, the real product of the NCCLS may be the consensus process itself which allows divergent points of view to be heard publicly, and those views to be melded into laboratory standards that can be widely embraced. Standards and guidelines are first published as “proposed” standards, then following extensive review and comments by NCCLS members, they are subsequently published when appropriate as “tentative” or “approved’ standards. An approved standard is a mature document that has gone through several years of refinement through the NCCLS consensus process. The most recent microbiology documents and their dates of publication are summarized in Table 2, and will be described briefly below.