Authors: Zhang Yawei, Wang Hui

　　Source: Chinese Journal of Laboratory Medicine

　　Establishing standardized operating procedures for pathogen identification and in vitro antimicrobial susceptibility testing is one of the fundamental requirements for strengthening the capacity of microbiology laboratories. This is of significant practical importance for optimizing clinical drug selection, enhancing the diagnosis and treatment of infectious diseases, and addressing the emergence of drug‑resistant pathogens. The antimicrobial susceptibility testing standards developed by the Clinical and Laboratory Standards Institute (CLSI) serve as a guiding document that laboratories in China are expected to follow. This article interprets the newly introduced “dose‑dependent susceptibility (SDD)” criteria for Enterobacteriaceae in CLSI M100–S24 (2014), while also summarizing other major updates.

　　I. Interpretation of SDD

　　“SDD” is an important category for interpreting results in antifungal susceptibility testing (see CLSI M27‑A4), and in 2014, CLSI extended its use to antimicrobial susceptibility testing of Enterobacteriaceae. This plays a critical role in improving the accuracy of susceptibility reporting, preventing clinicians from overinterpreting “intermediate” results as “resistant,” and promoting the rational use of antimicrobial agents.

　　1. Definition of SDD: “SDD” refers to strain susceptibility that is dependent on the dose administered to the patient. When a strain’s antimicrobial susceptibility result (MIC or disk diffusion) falls within the SDD range, clinicians should optimize the dosing regimen—such as by increasing the dose and/or frequency—to achieve clinical efficacy. Because higher doses are most likely to adequately cover SDD‑defined strains, clinicians should consider using the maximum allowable dose.

　　2. The relationship between “SDD” and “intermediate”: In bacterial susceptibility testing, the term “intermediate” already encompasses the concept of “SDD”; however, clinicians and microbiologists often fail to understand or overlook intermediate susceptibility results. CLSI recommends using “SDD” instead of “intermediate” when reporting cefepime susceptibility for Enterobacteriaceae. Cefepime is approved in multiple dosage regimens; when a strain exhibits a cefepime MIC of 4 or 8 mg/L (or an inhibition zone diameter of 19–24 mm), “SDD” emphasizes the use of a high‑dose regimen to treat the infection.

　　It is worth noting that not all “intermediate” breakpoints will be replaced by “SDD.” The SDD approach may be applied only when sufficient evidence demonstrates that the alternative approved dosing regimen is appropriate for microorganisms whose MIC (or zone diameter) falls between the “susceptible” and “resistant” categories. At present, for antimicrobial agents with multiple dosing options—such as other extended‑spectrum cephalosporins—CLSI also evaluates whether the SDD criteria can be applied.

　　3. Establishing dosing regimens for “sDD”: As the pharmacokinetics and pharmacodynamics of antimicrobial agents continue to evolve, defining their MIC breakpoints has become increasingly important. For adult patients with normal renal function, appropriate use of these breakpoints requires clinicians to administer systemic doses that meet or exceed the anticipated levels at the site of infection in order to achieve clinical efficacy. Dosing regimens based on “susceptible” or “sDD” breakpoints are provided in Table 1.

　　4. How to implement and utilize “sDD” in clinical practice: Laboratories can communicate the contents of CLSI’s “SDD” to relevant clinicians by distributing pertinent educational materials (see www.clsi.org) or through other means, and coordinate with hospital information system personnel to establish an appropriate implementation plan. When the ceftazidime MIC for Enterobacteriaceae is 4 or 8 mg/L, ensure that “sDD” is displayed on the clinical report.

　　In addition, it is necessary to consult with the manufacturers of antimicrobial susceptibility testing instruments to determine how to implement the new breakpoints on their devices. However, since the U.S. Food and Drug Administration (FDA) has not yet revised the breakpoints for cefepime, manufacturers are currently unable to adopt the CLSI‑recommended new breakpoints. Nevertheless, for most susceptibility testing systems, laboratories can manually adjust the breakpoints and conduct breakpoint verification studies.

　　If the laboratory needs to annotate “sDD,” CLSI recommends: “The susceptible breakpoint is based on a dosage regimen of lg/12 h; the SDD breakpoint is based on a higher cefepime dosing regimen [higher dose and/or more frequent administration], but not exceeding the maximum approved dose.”

　　For situations where the new cefepime breakpoints can be applied but “SDD” cannot be reported, it is necessary to consult with the laboratory director, infection control personnel, the pharmacy department, and the infectious diseases department to determine whether “intermediate” can be used in place of “SDD.” Furthermore, reporting either the MIC alone or the diameter of the inhibition zone carries a certain risk of misinterpretation. If the laboratory is unable to report “SDD,” when reporting both the MIC and the inhibition‑zone diameter—never solely the inhibition‑zone diameter—such results should be accompanied by an interpretation.

　　Although “sDD” is applicable to all patients and any specimen type, clinicians must take into account the clinical and physiological characteristics of individual patients before determining how to interpret sDD results. SDD does not require special quality control; standard quality‑control procedures are sufficient.

　　For strains positive for extended-spectrum β-lactamases (ESBLs), there is no need to reclassify “susceptible” or “SDD” as “resistant.” CLSI discontinued routine ESBL testing in 2010. When the new breakpoints for cefepime are applied, routine ESBL testing is also not required in patient reports; however, ESBL testing may still be performed for infection control or epidemiological studies.

　　II. Relevant Updates on Enterobacteriaceae Bacteria

　　M100‑S24 has revised the document and added susceptibility breakpoints for cefepime and ceftazidime, as shown in Table 2. Pseudomonas aeruginosa ATCC 27853 (used for carbapenems) has been incorporated into routine quality control.

　　In addition, the document has added cefazolin to the standard testing and reporting drug group U (with supplementary testing reserved for the urinary tract group), designating it as an alternative agent for uncomplicated urinary tract infections. When treating uncomplicated urinary tract infections caused by Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis, cefazolin can reliably predict the efficacy of the oral agents cefaclor, cefdinir, cefpodoxime, cefprozil, cefuroxime axetil, cephalexin, and ceftazidime.

　　However, since some cefazolin‑resistant strains may be susceptible to cefpodoxime, cefdinir, and cefuroxime axetil, susceptibility testing for these three agents must be performed separately. When evaluating the efficacy of oral cephalosporins for uncomplicated urinary tract infections, cefazolin is superior to cefalotin. For Salmonella and Shigella species, first‑ and second‑generation cephalosporins as well as cephamycins may exhibit in vitro antibacterial activity yet prove clinically ineffective; therefore, they should not be reported as “susceptible.” For Enterobacteriaceae isolates obtained from cerebrospinal fluid, susceptibility testing and reporting for cefotaxime or ceftazidime are recommended as alternatives to cefazolin.

　　It is worth noting that the new breakpoints for cefepime apply only to Enterobacteriaceae and are not applicable to Pseudomonas aeruginosa or other Gram-negative bacteria.

　　III. Recent Updates on the Genus Acinetobacter

　　In 2014, doripenem and minocycline were added to the routine testing and reporting categories, designated as Group A (preferred testing with routine reporting) and Group B (preferred testing with selective reporting), respectively. In addition, susceptibility breakpoints for doripenem, imipenem, and meropenem against Acinetobacter species were updated and newly established, as shown in Table 3. For routine quality control of antimicrobial susceptibility testing, emphasis was placed on using the quality control strain Escherichia coli ATCC 25922 for tetracycline and trimethoprim–sulfamethoxazole.

　　IV. Recent Updates on the Genus Staphylococcus

　　The M100‑S24 document has removed the breakpoints for the vancomycin disk diffusion method in the genus Staphylococcus and noted that vancomycin‑susceptible Staphylococcus aureus may acquire intermediate susceptibility to vancomycin with prolonged therapy. The revised document clarifies that a 30 µg cefoxitin disk may be used as an alternative to oxacillin for susceptibility testing.

　　For β‑lactamase screening assays, the document has added Staphylococcus aureus ATCC 29213 as an additional quality‑control strain, to be used in the penicillin inhibition‑zone assay (a sharp, well‑defined zone edge—resembling a “cliff”—indicates a positive β‑lactamase result). This supplementary quality control may be employed for evaluating new assays, training personnel, and assessing laboratory proficiency; however, routine daily or weekly antimicrobial susceptibility testing need not include this supplementary quality‑control strain.

　　In addition, β-lactamase–producing Staphylococcus lugdunensis is resistant to penicillin (MIC > 0.12 mg/L or a zone of inhibition diameter < 29 mm); therefore, testing for its β-lactamase activity is unnecessary. If the laboratory employs methods other than CLSI disk diffusion or the MIC reference method and is uncertain about the reliability of the penicillin susceptibility results, the strain should be subjected to an induced cephalothin test or another CLSI‑recommended reference method for assessing penicillin susceptibility.

　　At cLSI 2014, revisions were made regarding the need for confirmatory testing of β‑lactamase screening assays. The document clarifies that the penicillin zone‑of‑inhibition assay does not require additional testing or confirmation; concurrently, a chromogenic cephalosporin method for detecting β‑lactamase has been added. If the chromogenic cephalosporin test is positive, β‑lactamase positivity (or penicillin resistance) should be reported; if the test is negative, a penicillin zone‑of‑inhibition assay must be performed before reporting penicillin susceptibility. For coagulase‑negative staphylococci, if the β‑lactamase screening assay is positive, β‑lactamase positivity (or penicillin resistance) should be reported; if the result is negative, a penicillin zone‑of‑inhibition assay must be conducted prior to reporting penicillin susceptibility.

　　For the screening test for methicillin resistance, the new version of the document has added, under “Use of cefoxitin to detect mecA‑mediated oxacillin resistance,” the following: “With the exception of β‑lactam agents active against MRSA, all other β‑lactams must be reported as resistant or their susceptibility should not be reported.”

　　For screening tests to detect clindamycin‑resistant Staphylococcus species, Streptococcus pneumoniae, and β‑hemolytic streptococci, the document not only emphasizes the use of the standard quality‑control strain Streptococcus pneumoniae ATCC 49619 for both the erythromycin and clindamycin disk diffusion methods but also includes Staphylococcus aureus ATCC BAA‑976 (negative) and Staphylococcus aureus ATCC BAA‑977 (positive) in the supplementary quality‑control recommendations for both the disk diffusion and microbroth dilution methods.

　　V. Relevant Updates on the Genus Enterococcus

　　The M100-S24 document emphasizes that “for Enterococcus species with a vancomycin MIC ≥ 8 mg/L, susceptibility testing must include MIC determination on Enterococcus isolates grown on brain–heart infusion agar supplemented with vancomycin.”

　　For screening tests of high-level aminoglycoside‑resistant Enterococcus species, susceptibility testing is not required, as other aminoglycosides exhibit lower activity than gentamicin and streptomycin.

　　For enterococci, ampicillin susceptibility results have varying predictive value for the susceptibility of other antimicrobial agents; they can be used to predict the antibacterial activity of amoxicillin; they may also predict the susceptibility of non–β-lactamase–producing enterococci to amoxicillin–clavulanate, ampicillin–sulbactam, piperacillin, and piperacillin–tazobactam; and they can be employed to predict imipenem’s susceptibility profile against Enterococcus faecalis.

　　VI. Relevant Updates on Other Strains

　　l. Pseudomonas aeruginosa: Remove Escherichia coli ATCC 25922 from the routine quality control strain list.

　　2. Burkholderia cepacia, Stenotrophomonas maltophilia, and other non-Enterobacteriaceae: It is emphasized that the routine quality-control strain Escherichia coli ATCC 25922 should be used for chloramphenicol, minocycline, and trimethoprim-sulfamethoxazole.

　　VII. Drugs with Only “Sensitive” Interpretation Criteria

　　In M100‑S24, CLSI has added interpretive criteria for antimicrobial agents with susceptibility results reported as “Susceptible” only. These agents rarely or never give rise to resistant strains. For example, for drug Z (disk size 10 μg), a result is considered “Susceptible” when the MICs are ≤1 mg/L or the inhibition zone diameter is ≥16 mm. Strains with susceptibility results indicating “Resistant” should undergo further microbial identification and confirmatory susceptibility testing (see Appendix A of M100‑S24).

　　VIII. Updates on Antimicrobial Susceptibility Quality Control

　　The following drugs have been added to the quality-control ranges for certain strains, as determined by the disk diffusion and MIC methods: Ceftolozane–tazobactam, Eravacycline, ceftazidime–avibactam, aztreonam, aztreonam–avibactam, biapenem, colistin (with 0.002% Tween 80), polymyxin B (with 0.002% Tween 80), and sulperazon; in addition, the MIC range for Klebsiella pneumoniae ATCC 700603 has been included as a supplementary quality-control strain. Furthermore, the MIC quality-control ranges for several drugs have been revised; see Table 4.

　　IX. Updates on the Procedure for Antimicrobial Susceptibility Testing

　　In 2014, CLSI recommended that for Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter species, Staphylococcus species, and Enterococcus species, when performing disk diffusion susceptibility testing using 100-mm plates, no more than six disks should be placed (M100‑S23 recommends five), with a center-to-center distance of at least 24 mm between each disk. The diameter of the inhibition zone should be measured accurately, and overlapping zones should be avoided during measurement.

　　Four new methods for preparing stock solutions of antibacterial agents have been added: the dissolution and dilution solutions for biapenem are both 0.85%–0.9% NaCl (w/v) physiological saline; the dissolution and dilution solutions for eravacycline and surotomycin are both water; and the dilution solution for telavancin is dimethyl sulfoxide. For the composite drug ceftazidime–avibactam susceptibility testing, the avibactam concentration must be fixed at 4 mg/L, while ceftazidime is subjected to serial two-fold dilutions.

　　X. Major Changes to the Format

　　M100‑S24 (2014) differs from M100‑S23 (2013) in that certain tables have been renamed, renumbered, and rearranged; see Table 5.

　　XI. Updates to the Appendices and Glossary

　　Appendix A (Confirmation of Susceptibility Results and Microbial Identification) introduces the inclusion of ceftaroline in the phenotypic susceptibility testing for Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, and β-hemolytic streptococci. Appendix B adds information on the intrinsic resistance of certain strains to specific agents, such as the intrinsic resistance of Acinetobacter baumannii/Calcoaceticus, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Burkholderia cepacia to ampicillin and amoxicillin. Appendix C expands the screening assays for selected quality-control strains used in antimicrobial susceptibility testing.

　　Both Table I and Table II have been updated to include aztreonam–avibactam, biapenem, sulbactam, and eravacycline.

　　This article is excerpted from the Chinese Journal of Laboratory Medicine, Volume 37, Issue 4, April 2014, pages 256–260.

　　Article authors: Zhang Yawei, Wang Hui